Amino Acid Sequences Directed Against Cellular Receptors For Viruses And Bacteria

  *US09005963B2*
  US009005963B2                                 
(12)United States Patent(10)Patent No.: US 9,005,963 B2
 Blanchetot et al. (45) Date of Patent:Apr.  14, 2015

(54)Amino acid sequences directed against cellular receptors for viruses and bacteria 
    
(75)Inventors: Christophe Blanchetot,  Destelbergen (BE); 
  Martine Smit,  Amsterdam (NL); 
  Regorius Leurs,  Amsterdam (NL); 
  Sven Jähnichen,  Amsterdam (NL); 
  Dominique Schols,  Herentals (BE); 
  Michael John Scott Saunders,  Brussels (BE); 
  Johannes Joseph Wilhelmus De Haard,  Oudelande (NL); 
  Peter Vanlandschoot,  Bellem (BE); 
  Peter Verheesen,  Ghent (BE) 
(73)Assignee:Ablynx N.V.,  Zwijnaarde (BE), Type: Foreign Company 
(*)Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 496 days. 
(21)Appl. No.: 13/124,001 
(22)PCT Filed:Oct.  14, 2009 
(86)PCT No.: PCT/EP2009/063411 
 § 371 (c)(1), (2), (4) Date: Sep.  1, 2011  
(87)PCT Pub. No.:WO20/10/043650 
 PCT Pub. Date:Apr.  22, 2010 
(65)Prior Publication Data 
 US 2011/0318347 A1 Dec.  29, 2011 
 Related U.S. Patent Documents 
(60)Provisional application No. 61/105,259, filed on Oct.  14, 2008.
 
Jan.  1, 2013 C 07 K 16 2812 F I Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 16 2866 L I Apr.  14, 2015 US B H C Jan.  1, 2013 A 61 K 2039 505 L A Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 2316 96 L A Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 2317 21 L A Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 2317 22 L A Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 2317 31 L A Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 2317 565 L A Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 2317 569 L A Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 2317 92 L A Apr.  14, 2015 US B H C Jan.  1, 2013 C 07 K 2319 31 L A Apr.  14, 2015 US B H C Jan.  1, 2013 Y 10 S 530 866 L A Apr.  14, 2015 US B H C
(51)Int. Cl. C07K 016/00 (20060101); C07K 016/18 (20060101); C07K 016/28 (20060101); C07H 021/04 (20060101); C12N 005/07 (20100101); C12N 005/10 (20060101); C12N 005/16 (20060101); C12N 005/22 (20060101); A61K 039/00 (20060101); A61K 039/395 (20060101)
(52)U.S. Cl. 435/326; 435/328; 435/334; 530/387.1; 530/387.3; 530/866; 536/23.1; 536/23.5; 536/23.53

 
(56)References Cited

 
 FOREIGN PATENT DOCUMENTS 
 
       EP       0 368 684       A1                5/1990      
       WO       WO 92/05274       A1                4/1992      
       WO       WO 20/05/018629       A1                3/2005      
       WO       WO 20/06/003388       A2                1/2006      
       WO       WO 20/06/030220       A1                3/2006      
       WO       WO 20/07/118670       A1                10/2007      
       WO       WO-2/009138519       A1       *       11/2009      

 OTHER PUBLICATIONS
  
  Forsman et al. Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. J Virol 82(24): 12069-12081, Oct. 8, 2008. *
  Kaufman et al. Blood 94: 3178-3184, 1999. *
  Wang et al. Rapid analysis of gene expression (RAGE) facilitates universal expression profiling. Nucleic Acids Res 27(23): 4609-4618, 1999. *
  Phillips, A., The challenge of gene therapy and DNA delivery. J Pharm Pharmacology 53: 1169-1174, 2001. *
  Brummell et al. (Biochemistry 32:1180-1187 (1993)). *
  Burks et al. (PNAS 94:412-417 (1997)). *
  Brorson et al. (J. Immunol. 163:6694-6701 (1999)). *
  Casset et al. Biochem Biophys Res Comm 307: 198-205, 2003. *
  Chen et al. J Mol Biol 293: 865-881, 1999. *
  Colman (Research in Immunol. 145:33-36 (1994)). *
  De Pascalis et al. J Immunol 169: 3076-3084, 2002. *
  Deschacht et al. J Immunol 184: 5696-5704, 2010. *
  Holm et al. Mol Immunol 44: 1075-1084, 2007. *
  Juengst, E.T. What next for human gene therapy? BMJ 326: 1410-1411, 2003. *
  Kobayashi et al. Protein Engineering 12(10): 879-884, 1999. *
  MacCallum et al. J Mol Biol 262: 732-745, 1996. *
  Paul, William E., Fundamental Immunology, 3rd Edition, Raven Press, New York, Chapt. 8, pp. 292-295 (1993), under the heading “Fv Structure and Diversity in Three Dimensions”. *
  Phillips, A.J. J Pharmacy Pharmacol 53: 1169-1174, 2001. *
  Rubanyi, G.M. The future of human gene therapy. Mol Aspects Med 22: 113-142, 2001. *
  Vajdos et al. J Mol Biol 320: 415-428, 2002. *
  Vincke et al. J Biol Chem 284: 3273-3284, 2009. *
  Wu et al. J Mol Biol 294: 151-162, 1999. *
  Kartikeyan et al., HIV and AIDS: Basic Elements and Priorities. The Netherlands: Springer, 2007, pp. 67-78, 171-181, 27 pages. *
  “MT-4”, NIH AIDS Reagent Program Datasheet, dated Feb. 18, 2014; www.aidsreagent.org/pdfs/ds0120026.pdf; 2 total pages. *
  Jain et al., Engineering antibodies for clinical applications. Trends Biotechnol. Jul. 2007;25(7):307-16. Epub May 21, 2007. Review.
  Mack et al., Preferential targeting of CD4-CCR5 complexes with bifunctional inhibitors: a novel approach to block HIV-1 infection. J Immunol. Dec. 1, 2005;175(11):7586-93.
  Marasco et al., The growth and potential of human antiviral monoclonal antibody therapeutics. Nat Biotechnol. Dec. 2007;25(12):1421-34. Review.
  Rudikoff et al., Single amino acid substitution altering antigen-binding specificity. Proc Natl Acad Sci U S A. Mar. 1982;79(6):1979-83.
  Saerens et al., Single-domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol. Oct. 2008;8(5):600-8. Epub Aug. 22, 2008. Review.
  Schneider-Schaulies, Cellular receptors for viruses: links to tropism and pathogenesis. J Gen Virol. Jun. 2000;81(Pt 6):1413-29. Review.
  Skov et al., HuMax-CD4: a fully human monoclonal anti-CD4 antibody for the treatment of psoriasis vulgaris. Arch Dermatol. Nov. 2003;139(11):1433-9.
  Wark et al., Latest technologies for the enhancement of antibody affinity. Adv Drug Deliv Rev. Aug. 7, 2006;58(5-6):657-70. Epub May 22, 2006. Review.
  Baribaud et al., Antigenically distinct conformations of CXCR4. J Virol. Oct. 2001;75(19):8957-67.
  Garrigues et al., Integrin alphaVbeta3 Binds to the RGD motif of glycoprotein B of Kaposi's sarcoma-associated herpesvirus and functions as an RGD-dependent entry receptor. J Virol. Feb. 2008;82(3):1570-80. Epub Nov. 28, 2007.
  Kielian et al., Virus membrane-fusion proteins: more than one way to make a hairpin. Nat Rev Microbiol. Jan. 2006;4(1):67-76.
  Raman et al., Role of chemokines in tumor growth. Cancer Lett. Oct. 28, 2007;256(2):137-65. Epub Jul. 12, 2007.
  Rassa et al., Murine retroviruses activate B cells via interaction with toll-like receptor 4. Proc Natl Acad Sci U S A. Feb. 19, 2002;99(4):2281-6.
  Stewart et al., Cell integrins: commonly used receptors for diverse viral pathogens. Trends Microbiol. Nov. 2007;15(11):500-7. Epub Nov. 7, 2007.
  Dahmen et al., Expression of olfactory receptors in Xenopus oocytes. J Neurochem. Mar. 1992;58(3):1176-9.
  Halaby et al., The immunoglobulin fold family: sequence analysis and 3D structure comparisons. Protein Eng. Jul. 1999;12(7):563-71.
  Holliger et al., Engineered antibody fragments and the rise of single domains. Nat Biotechnol. Sep. 2005;23(9):1126-36.
  Holt et al., Domain antibodies: proteins for therapy. Trends Biotechnol. Nov. 2003;21(11):484-90.
  Houamed et al., Cloning, expression, and gene structure of a G protein-coupled glutamate receptor from rat brain. Science. May 31, 1991;252(5010):1318-21.
  Kiefer, In vitro folding of alpha-helical membrane proteins. Biochim Biophys Acta. Feb. 17, 2003;1610(1):57-62.
  Ward et al., Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature. Oct. 12, 1989;341(6242):544-6.
 
 
     * cited by examiner
 
     Primary Examiner —Bridget E Bunner
     Art Unit — 1647
     Exemplary claim number — 1
 
(74)Attorney, Agent, or Firm — Wolf, Greenfield & Sacks, P.C.

(57)

Abstract

The present invention relates to amino acid sequences that are directed against (as defined herein) human cellular receptors for viruses and/or bacteria such as e.g. NANOBODIES specifically recognizing hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences. The amino acid sequences may be used to prevent human cell entry of HIV, HCV, adenoviruses, hantavirus, herpesvirus, echo-virus 1 and others.
18 Claims, 24 Drawing Sheets, and 24 Figures


RELATED APPLICATIONS

[0001] This application is a national stage filing under 35 U.S.C. §371 of international application PCT/EP2009/063411, filed Oct. 14, 2009, which was published under PCT Article 21(2) in English, and claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No. 61/105,259, filed Oct. 14, 2008, the disclosures of which are incorporated by reference herein in their entireties.
[0002] The present invention relates to amino acid sequences that are directed against (as defined herein) human cellular receptors for viruses and/or bacteria such as e.g. NANOBODIES specifically recognizing hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences (also referred to herein as “amino acid sequences of the invention”, “compounds of the invention”, and “polypeptides of the invention”, respectively). Said amino acid sequences may be used to prevent human cell entry of HIV, HCV, adenoviruses, hantavirus, herpesvirus, echo-virus 1 and others.
[0003] The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to herein as “nucleic acids of the invention” or “nucleotide sequences of the invention”); to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.
[0004] Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.
[0005] When searching for their favorite host tissues, animal viruses and bacteria frequently attach to cell-surface receptors that have key roles in normal cell physiology. The receptors mentioned herein are examples of physiologically important receptors that have been usurped by nonenveloped and enveloped viruses for attachment, virus-cell fusion and/or virus entry into mammalian cell or partial virus entry into mammalian cell.
[0006] The polypeptides and compositions of the present invention can generally be used to modulate, and in particular inhibit and/or prevent attachment, virus/bacterium-cell fusion and/or virus/bacterium entry into mammalian cell or partial virus/bacterium entry into mammalian cell.
[0007] As such, the polypeptides and compositions of the present invention can be used for the prevention and treatment (as defined herein) of viruses and/or bacteria induced diseases and disorders. Generally, “viruses and/or bacteria induced diseases and disorders” can be defined as diseases and disorders that can be prevented and/or treated, respectively, by suitably administering to a subject in need thereof (i.e. having the disease or disorder or at least one symptom thereof and/or at risk of attracting or developing the disease or disorder) of either a polypeptide or composition of the invention (and in particular, of a pharmaceutically active amount thereof). Examples of such viruses and/or bacteria induced diseases and disorders will be clear to the skilled person based on the disclosure herein.
[0008] Thus, without being limited thereto, the amino acid sequences and polypeptides of the invention can for example be used to prevent and/or to treat all diseases and disorders that are currently being prevented or treated with active principles that can modulate virus/bacterium attachment, virus/bacterium-cell fusion and/or virus/bacterium entry into mammalian cell or partial virus/bacterium entry into mammalian cell. It is also envisaged that the polypeptides of the invention can be used to prevent and/or to treat all diseases and disorders for which treatment with such active principles is currently being developed, has been proposed, or will be proposed or developed in future. In addition, it is envisaged that, because of their favourable properties as further described herein, the polypeptides of the present invention may be used for the prevention and treatment of other diseases and disorders than those for which these known active principles are being used or will be proposed or developed; and/or that the polypeptides of the present invention may provide new methods and regimens for treating the diseases and disorders described herein.
[0009] Generally, it is an object of the invention to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the diagnosis, prevention and/or treatment of viruses and/or bacteria induced diseases and disorders and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or use of such agents and compositions.
[0010] In particular, it is an object of the invention to provide such pharmacologically active agents, compositions and/or methods that have certain advantages compared to the agents, compositions and/or methods that are currently used and/or known in the art. These advantages will become clear from the further description below.
[0011] More in particular, it is an object of the invention to provide therapeutic proteins that can be used as pharmacologically active agents, as well as compositions comprising the same, for the diagnosis, prevention and/or treatment of viruses and/or bacteria induced diseases and disorders and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or the use of such therapeutic proteins and compositions.
[0012] Accordingly, it is a specific object of the present invention to provide amino acid sequences that are directed against (as defined herein) human cellular receptors for viruses and/or bacteria such as e.g. NANOBODIES specifically recognizing hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9 (also referred to herein as “Targets of the invention” or each of these referred to as “one of the Targets of the invention” or “Target of the invention”), in particular against Targets of the invention from a warm-blooded animal, more in particular against Targets of the invention from a mammal, and especially against human Targets of the invention; and to provide proteins and polypeptides comprising or essentially consisting of at least one such amino acid sequence.
[0013] In particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that are suitable for prophylactic, therapeutic and/or diagnostic use in a warm-blooded animal, and in particular in a mammal, and more in particular in a human being.
[0014] More in particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used for the prevention, treatment, alleviation and/or diagnosis of one or more diseases, disorders or conditions associated with Targets of the invention and/or mediated by Targets of the invention (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.
[0015] It is also a specific object of the invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used in the preparation of pharmaceutical or veterinary compositions for the prevention and/or treatment of one or more diseases, disorders or conditions associated with and/or mediated by Targets of the invention (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.
[0016] In the invention, generally, these objects are achieved by the use of the amino acid sequences, proteins, polypeptides and compositions that are described herein.
[0017] In general, the invention provides amino acid sequences that are directed against (as defined herein) and/or can specifically bind (as defined herein) to Targets of the invention; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.
[0018] More in particular, the invention provides amino acid sequences that can bind to Targets of the invention with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.
[0019] In particular, amino acid sequences and polypeptides of the invention are preferably such that they:
[0020] bind to Targets of the invention with a dissociation constant (KD) of 10−5 to 10−12 moles/liter or less, and preferably 10−7 to 10−12 moles/liter or less and more preferably 10−8 to 10−12 moles/liter (i.e. with an association constant (KA) of 105 to 1012 liter/moles or more, and preferably 107 to 1012 liter/moles or more and more preferably 108 to 1012 liter/moles);
and/or such that they:
[0021] bind to Targets of the invention with a kon-rate of between 102 M−1s−1 to about 107 M−1s−1, preferably between 103 M−1s−1 and 107 M−1s−1, more preferably between 104 M−1s−1 and 107 M−1s−1, such as between 105 M−1s−1 and 107 M−1s−1;
and/or such that they:
[0022] bind to Targets of the invention with a koff rate between 1 s−1 (t1/2=0.69 s) and 10−6 s−1 (providing a near irreversible complex with a t1/2 of multiple days), preferably between 10−2 s−1 and 10−6 s−1, more preferably between 10−3 s−1 and 10−6 s−1, such as between 10−4 s−1 and 10−6 s−1.
[0023] Preferably, a monovalent amino acid sequence of the invention (or a polypeptide that contains only one amino acid sequence of the invention) is preferably such that it will bind to Targets of the invention with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 μM.
[0024] Some preferred IC50 values for binding of the amino acid sequences or polypeptides of the invention to Targets of the invention will become clear from the further description and examples herein.
[0025] For binding to Targets of the invention, an amino acid sequence of the invention will usually contain within its amino acid sequence one or more amino acid residues or one or more stretches of amino acid residues (i.e. with each “stretch” comprising two or amino acid residues that are adjacent to each other or in close proximity to each other, i.e. in the primary or tertiary structure of the amino acid sequence) via which the amino acid sequence of the invention can bind to Targets of the invention, which amino acid residues or stretches of amino acid residues thus form the “site” for binding to Targets of the invention (also referred to herein as the “antigen binding site”).
[0026] The amino acid sequences provided by the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more amino acid sequences of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than Targets of the invention), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. Such a protein or polypeptide may also be in essentially isolated form (as defined herein).
[0027] The amino acid sequences and polypeptides of the invention as such preferably essentially consist of a single amino acid chain that is not linked via disulphide bridges to any other amino acid sequence or chain (but that may or may not contain one or more intramolecular disulphide bridges. For example, it is known that NANOBODIES—as described herein—may sometimes contain a disulphide bridge between CDR3 and CDR1 or FR2). However, it should be noted that one or more amino acid sequences of the invention may be linked to each other and/or to other amino acid sequences (e.g. via disulphide bridges) to provide peptide constructs that may also be useful in the invention (for example Fab′ fragments, F(ab′)2 fragments, ScFv constructs, “diabodies” and other multispecific constructs. Reference is for example made to the review by Holliger and Hudson, Nat. Biotechnol. 2005 September; 23(9):1126-36).
[0028] Generally, when an amino acid sequence of the invention (or a compound, construct or polypeptide comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably either an amino acid sequence that does not occur naturally in said subject; or, when it does occur naturally in said subject, in essentially isolated form (as defined herein).
[0029] It will also be clear to the skilled person that for pharmaceutical use, the amino acid sequences of the invention (as well as compounds, constructs and polypeptides comprising the same) are preferably directed against human Targets of the invention; whereas for veterinary purposes, the amino acid sequences and polypeptides of the invention are preferably directed against Targets of the invention from the species to be treated, or at least cross-reactive with Targets of the invention from the species to be treated.
[0030] Furthermore, an amino acid sequence of the invention may optionally, and in addition to the at least one binding site for binding against Targets of the invention, contain one or more further binding sites for binding against other antigens, proteins or targets. The efficacy of the amino acid sequences and polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person, and for example include the expression of the receptors in Xenopus oocytes, after which the coupling of many Targets of the invention to ion channels allows the activation or inhibition of these Targets of the invention to be monitored in oocytes via voltage clamping techniques. Heterologous Targets of the invention can be functionally expressed in the oocyte by injecting exogenous, Targets-encoding mRNA into the oocyte and then allowing the oocyte's endogenous cellular machinery to translate and insert the receptors into the plasma membrane (see, e.g., Houamed et al., Science 252:1318-21, 1991; Dahmen et al., J. Neurochem. 58:1176-79, 1992). Following functional expression of receptors, the ability of ligands to induce transmembrane conductance changes can be observed via a two-electrode voltage clamp system (Dahmen et al., supra), which can detect either a depolarization or hyperpolarization of the membrane potential. Other suitable assays are e.g. described in the example section.
[0031] Also, according to the invention, amino acid sequences and polypeptides that are directed against Targets of the invention from a first species of warm-blooded animal may or may not show cross-reactivity with Targets of the invention from one or more other species of warm-blooded animal. For example, amino acid sequences and polypeptides directed against human Targets of the invention may or may not show cross reactivity with Targets of the invention from one or more other species of primates (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) and/or with Targets of the invention from one or more species of animals that are often used in animal models for diseases (for example mouse, rat, rabbit, pig or dog), and in particular in animal models for diseases and disorders associated with Targets of the invention (such as the species and animal models mentioned herein). In this respect, it will be clear to the skilled person that such cross-reactivity, when present, may have advantages from a drug development point of view, since it allows the amino acid sequences and polypeptides against human Targets of the invention to be tested in such disease models.
[0032] More generally, amino acid sequences and polypeptides of the invention that are cross-reactive with Targets of the invention from multiple species of mammal will usually be advantageous for use in veterinary applications, since it will allow the same amino acid sequence or polypeptide to be used across multiple species. Thus, it is also encompassed within the scope of the invention that amino acid sequences and polypeptides directed against Targets of the invention from one species of animal (such as amino acid sequences and polypeptides against human Targets of the invention) can be used in the treatment of another species of animal, as long as the use of the amino acid sequences and/or polypeptides provide the desired effects in the species to be treated.
[0033] The present invention is in its broadest sense also not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of Targets of the invention against which the amino acid sequences and polypeptides of the invention are directed. For example, amino acid sequences of the invention may be raised by suitably immunizing a mammal (such as a Camelid) with Targets of the invention that have been expressed in a suitable expression system or that has been isolated from a suitable cell or cell fraction. In particular, amino acid sequences of the invention may be raised against Targets of the invention (or suitable parts or fragments thereof) that have been refolded (for example using the techniques described in the review by Kiefer, Biochim. Biophys. Acta, 1610 (2003), 57-62), and amino acid sequences, NANOBODIES and polypeptides that are directed against and/or that have been raised against a refolded Target of the invention form a further aspect of the invention.
[0034] The amino acid sequences and polypeptides of the invention may generally be directed against any desired Target of the invention, and may in particular be directed against Targets of the invention that have at least one extracellular loop or domain. Examples of such Targets of the invention will be clear to the skilled person based on the prior art cited herein.
[0035] According to a specific aspect of the invention, an amino acid sequence or polypeptide of the invention may be directed against (as defined herein) the Targets of the invention that are expressed on the surface of a cell and/or against at least one extracellular region, domain, loop or other extracellular epitope of one of the Targets of the invention. For example, such amino acid sequences may be raised by suitably immunizing a mammal (such as a Camelid) with a cell or cell fraction that has one of the Targets of the invention expressed.
[0036] In particular, according to this aspect, an amino acid sequence or polypeptide of the invention is directed against (as defined herein) at least one extracellular region, domain, loop or other extracellular epitope of one of the Targets of the invention, e.g. hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9, and is preferably further such that said amino acid sequence or polypeptide of the invention is capable of modulating (as defined herein) said Target of the invention, e.g. hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9. More in particular, according to this aspect, an amino acid sequence or polypeptide of the invention is directed against (as defined herein) at least one extracellular region, domain, loop or other extracellular epitope of a Target of the invention, e.g. hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9; and is preferably further such that said amino acid sequence or polypeptide of the invention is capable of (fully or partially) blocking said Targets of the invention, e.g. hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9.
[0037] According to this aspect of the invention, the amino acid sequence or polypeptide of the invention may be directed against any suitable extracellular part, region, domain, loop or other extracellular epitope of one of the Targets of the invention, e.g. hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9, but is preferably directed against one of the extracellular parts of the transmembrane domains or more preferably against one of the extracellular loops that link the transmembrane domains.
[0038] The amino acid sequence of such suitable extracellular parts, regions, domains, loops or epitopes may be derived by Kyte-Doolittle analysis of the amino acid sequence of the pertinent Target of the invention, e.g. hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9; by aligning the Targets of the invention belonging to the same (sub)families and identifying the various transmembrane domains and extracellular parts, regions, domain or loops; by TMAP-analysis; or by any suitable combination thereof. The invention also relates to amino acid sequences and polypeptides (as further defined herein) that are directed against and/or have been raised against such extracellular parts, regions, domains, loops or epitopes (and/or that are directed against and/or have been raised against suitable parts or fragments of such extracellular parts, regions, domains, loops or epitopes and/or against synthetic or semi-synthetic peptides that are derived from or based on such extracellular parts, regions, domains, loops or epitopes).
[0039] In particular, amino acid sequences and polypeptides of the invention are preferably such that they:
[0040] bind to an extracellular part, region, domain, loop or other extracellular epitope of one of the Targets of the invention (as described herein) or against a peptide derived therefrom with a dissociation constant (KD) of 10−5 to 10−12 moles/liter or less, and preferably 10−7 to 10−12 moles/liter or less and more preferably 10−8 to 10−12 moles/liter (i.e. with an association constant (KA) of 105 to 1012 liter/moles or more, and preferably 107 to 1012 liter/moles or more and more preferably 108 to 1012 liter/moles);
and/or such that they:
[0041] bind to an extracellular part, region, domain, loop or other extracellular epitope of one of the Targets of the invention (as described herein) or against a peptide derived therefrom with a kon-rate of between 102 M−1s−1 to about 107 M−1s−1, preferably between 103 M−1s−1 and 107 M−1s−1, more preferably between 104 M−1s−1 and 107 M−1s−1, such as between 105 M−1s−1 and 107 M−1s−1;
and/or such that they:
[0042] bind to an extracellular part, region, domain, loop or other extracellular epitope of one of the Targets of the invention (as described herein) or against a peptide derived therefrom with a koff rate between 1 s−1 (t1/2=0.69 s) and 10−6 s−1 (providing a near irreversible complex with a t1/2 of multiple days), preferably between 10−2 s−1 and 10−6 s−1, more preferably between 10−3 s−1 and 10−6 s−1, such as between 104 s−1 and 10−6 s−1.
[0043] Preferably, a monovalent amino acid sequence of the invention (or a polypeptide that contains only one amino acid sequence of the invention) is preferably such that it will bind to an extracellular part, region, domain, loop or other extracellular epitope of one of the Targets of the invention (as described herein) with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.
[0044] Also, according to this aspect, any multivalent or multispecific (as defined herein) polypeptides of the invention may also be suitably directed against two or more different extracellular parts, regions, domains, loops or other extracellular epitopes on the same antigen, for example against two different extracellular loops, against two different extracellular parts of the transmembrane domains or against one extracellular loops and one extracellular loop. Such multivalent or multispecific polypeptides of the invention may also have (or be engineered and/or selected for) increased avidity and/or improved selectivity for one of the desired Targets of the invention, and/or for any other desired property or combination of desired properties that may be obtained by the use of such multivalent or multispecific polypeptides.
[0045] Generally, it is expected that amino acid sequences and polypeptides of the invention that are directed against an extracellular loop or domain of one of the Targets of the invention (or against a small peptide derived therefrom or based thereon), and/or that have been screened against, selected using and/or raised against an extracellular loop or domain of one of the Targets of the invention (or against a small peptide derived therefrom or based thereon) will also be able to bind (and in particular, to specifically bind, as defined herein) to such an extracellular loop or domain (or peptide derived therefrom) that forms part of one of the Targets of the invention (or at least one subunit thereof) that is present on the surface of a cell. Thus, such (peptides derived from) an extracellular loop or domain may find particular use in methods for generating amino acid sequences and polypeptides of the invention (as defined herein); and such methods and uses form further aspects of the invention; as do amino acid sequences, NANOBODIES and polypeptides of the invention that are directed against or raised against such an extracellular loop, domain or peptide derived therefrom.
[0046] For example, such a method may comprises the following:
[0047] a) a step of suitably immunizing a Camelid with a suitable antigen that comprises the desired extracellular part, region, domain, loop or other extracellular epitope(s), or with a suitable peptide derived therefrom or based thereon, such that an immune response against the desired extracellular part, region, domain, loop or other extracellular epitope(s) is raised. The antigen may be any suitable antigen that is capable of raising an immune response against the desired extracellular part, region, domain, loop or other extracellular epitope(s); such as, for example and without limitation, whole cells that are alive and overexpress the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface in their native confirmation, cell wall fragments thereof or any other suitable preparation derived from such cells, vesicles that have the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface, a subunit or fragment of a subunit of one of the Targets of the invention, that comprises the desired extracellular part, region, domain, loop or other extracellular epitope(s), or a synthetic or semi-synthetic peptide that comprises and/or is based on (the amino acid sequence of) the desired extracellular part, region, domain, loop or other extracellular epitope(s), more preferably, whole cells (e.g. HEK293) that are alive and overexpress the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface in their native confirmation; and
[0048] b) a step of selection for binding for the desired extracellular part, region, domain, loop or other extracellular epitope(s) using cell membranes preparation of different (than the one using in immunization) and several cell types overexpressing one of the Targets of the invention. This may for example be performed by selecting from a set, a collection or a library of cells that express heavy chain antibodies on their surface (e.g. B-cells obtained from a suitably immunized Camelid) and using a cell membranes preparation of e.g. a first type of cells such as e.g. CHO for a first round selection and e.g. a second type of cells such as e.g. COS-7 cells for a second round selection; by selecting from a (naïve or immune) library of VHH sequences or NANOBODY sequences by using a cell membranes preparation of e.g. a first type of cell such as e.g. CHO for a first round selection and e.g. a second type of cell such as e.g. COS-7 cell for a second round selection; or by selecting from a (naïve or immune) library of nucleic acid sequences that encode VHH sequences or NANOBODY sequences by using a cell membranes preparation of e.g. a first type of cell such as e.g. CHO for a first round selection and e.g. a second type of cells such as e.g. COS-7 cell for a second round selection; which may all be performed in a manner known per se; and optionally
[0049] c) washing only mildly with a buffer such as PBS without detergents; and which method may optionally further comprise one or more other suitable steps known per se, such as, for example and without limitation, a step of affinity maturation, a step of expressing the desired amino acid sequence, a step of screening for binding and/or for activity against the desired antigen (in this case, against one of the Targets of the invention), a step of determining the desired amino acid sequence or nucleotide sequence, a step of introducing one or more humanizing substitutions (e.g. as further described herein), a step of formatting in a suitable multivalent and/or multispecific format, a step of screening for the desired biological and/or physiological properties (i.e. using a suitable assay, such as those described herein); and/or any suitable combination of one or more of such steps, in any suitable order.
[0050] Such methods and the amino acid sequences obtained via such methods, as well as proteins and polypeptides comprising or essentially consisting of the same, form further aspects of this invention.
[0051] The amino acid sequences, NANOBODIES and polypeptides of the invention may also bind to one of the Targets of the invention at the same site as the endogenous agonist (i.e. at an orthosteric site).
[0052] The amino acid sequences, NANOBODIES and polypeptides of the invention may also bind to one of the Targets of the invention in such a way that they block adhesion and/or fusion of a virus and/or bacteria.
[0053] The amino acid sequences, NANOBODIES and polypeptides of the invention may also bind to of one of the Targets of the invention in such a way that they mediate allosteric modulation (e.g. bind to one of the Targets of the invention at an allosteric site).
[0054] The amino acid sequences, NANOBODIES and polypeptides of the invention may also bind to one of the Targets of the invention in such a way that they inhibit or enhance the assembly of one of the Targets of the invention functional homodimers or heterodimers and thus prevent adhesion and/or fusion of a virus and/or bacteria.
[0055] More specifically, the amino acid sequences and polypeptides according to the present invention may neutralize a virus (as defined herein) and/or modulate, reduce and/or inhibit the infectivity of a virus (as defined herein). Accordingly, the amino acid sequences and polypeptides of the present invention that neutralize a virus (as defined herein) and/or modulate, reduce and/or inhibit the infectivity of a virus (as defined herein) in the pre-entry phase of viral infection (i.e. before and/or during viral entry in a target host cell has taken place), are said herein to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0056] The invention also relates to the use of an one amino acid sequence, NANOBODY, compound or polypeptide of the invention in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating viral entry and/or for modulating the biological pathways that are mediated by the Target of the invention, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a viral disease.
[0057] In the context of the present invention, “modulating” or “to modulate” generally means either reducing, preventing or inhibiting viral entry and/or reducing, preventing or inhibiting the biological pathways that are mediated by a Target of the invention and/or its ligand, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein). In particular, “modulating” or “to modulate” may mean either reducing, preventing or inhibiting viral entry and/or reducing, preventing or inhibiting the biological pathways that are mediated by the Target of the invention and/or its ligand as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to normal (i.e. naturally occurring) viral entry and/or normal (i.e. naturally occurring) biological pathways that are mediated by the Target of the invention and/or its ligand in the same assay under the same conditions but without the presence of the amino acid sequence, NANOBODY, compound or polypeptide of the invention.
[0058] As will be clear to the skilled person, “modulating” may also involve effecting a change (which may either be an increase or a decrease) in binding specificity and/or selectivity of the Target of the invention for one or more of its binding partners; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of a Target of the invention for one or more conditions in the medium or surroundings in which an Target of the invention is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the amino acid sequence, NANOBODY, compound or polypeptide of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, such as the assays described herein or in the prior art cited herein.
[0059] “Modulating” may also mean effecting a change with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the Target of the invention (or in which its binding partners or pathway(s)) are involved. Again, as will be clear to the skilled person this may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, such as the assays described herein or in the prior art cited herein. In particular, with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the Target of the invention and/or its ligand is involved, effecting a change can mean a change by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological mechanisms, effects, responses, functions, pathways or activities in the same assay under the same conditions but without the presence of the amino acid sequence, NANOBODY, compound or polypeptide of the invention.
[0060] Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.
[0061] Accordingly, the present invention also relates to amino acid sequences and polypeptides that can be used to modulate, and in particular to inhibit and/or to prevent the viral-mediated biological pathways in which a Target of the invention is involved. In particular, the amino acid sequences and polypeptides of the present invention can be used to neutralize a virus (as defined herein) and/or to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein).
[0062] More specifically, the amino acid sequences and polypeptides according to the present invention may neutralize a virus (as defined herein) and/or modulate, reduce and/or inhibit the infectivity of a virus (as defined herein) in the pre-entry phase of viral infection (i.e. before and/or during viral entry in a target host cell has taken place) and/or in the post-entry phase of viral infection (i.e. after viral entry in a target host cell has taken place). Accordingly, the amino acid sequences and polypeptides of the present invention that neutralize a virus (as defined herein) and/or modulate, reduce and/or inhibit the infectivity of a virus (as defined herein) in the pre-entry phase of viral infection (i.e. before and/or during viral entry in a target host cell has taken place), are said herein to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell. The amino acid sequences and polypeptides according to the present invention may neutralize virus by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to virus neutralization under the same conditions but without the presence of the amino acid sequence, NANOBODY, compound or polypeptide of the invention. The neutralization may be increased by at least 2 times, preferably at least 3 times, such as at least 5 times or at least 10 times, for example by at least 15 times, at least 20 times, at least 30 times, at least 50 times, or 100 times or more, compared to virus neutralization under the same conditions but without the presence of the amino acid sequence, NANOBODY, compound or polypeptide of the invention. The amino acid sequences and polypeptides according to the present invention may modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to viral entry in a target host under the same conditions but without the presence of the amino acid sequence, NANOBODY, compound or polypeptide of the invention.
[0063] In a specific aspect, the present invention relates to multivalent (such as bivalent, biparatopic, bispecific, trivalent, triparatopic, trispecific, as further defined herein) amino acid sequences and polypeptides that modulate, and in particular to inhibit and/or to prevent the viral-mediated biological pathways a Target of the invention is involved. In particular, the multivalent (preferably bivalent, more preferably trivalent) amino acid sequences and polypeptides of the present invention can neutralize a virus (as defined herein), modulate, reduce and/or inhibit the infectivity of a virus (as defined herein), and/or modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell. In one aspect, these multivalent (preferably bivalent, more preferably trivalent) amino acid sequences and polypeptides are directed against hCXCR4 and show increased in vitro and/or in vivo neutralization of HIV (as e.g. measured by HIV infection assay such as described herein) compared to the corresponding monovalent amino acid sequence. The neutralization may be increased by at least 2 times, preferably at least 3 times, such as at least 5 times or at least 10 times, for example by at least 15 times, at least 20 times, at least 30 times, at least 50 times, or 100 times or more, compared to the neutralization in the same assay under the same conditions by the corresponding monovalent amino acid sequence. In another aspect, these multivalent (preferably bivalent, more preferably trivalent) amino acid sequences and polypeptides are directed against hCD4 and show increased in vitro and/or in vivo neutralization of HIV (as e.g. measured by a HIV infection assay as described herein) compared to the corresponding monovalent amino acid sequence. The neutralization may be increased by at least 2 times, preferably at least 3 times, such as at least 5 times or at least 10 times, for example by at least 15 times, at least 20 times, at least 30 times, at least 50 times, or 100 times or more, compared to the neutralization in the same assay under the same conditions by the corresponding monovalent amino acid sequence.
[0064] In yet another aspect, these multivalent (preferably bivalent, more preferably trivalent) amino acid sequences and polypeptides are directed against hCXCR4 and show increased competition with CXCL12 for binding hCXCR4 compared to the corresponding monovalent amino acid sequence. The competition may be increased by at least 2 times, preferably at least 3 times, such as at least 5 times or at least 10 times, for example by at least 15 times, at least 20 times, at least 30 times, at least 50 times, or 100 times or more, compared to the competition in the same assay under the same conditions by the corresponding monovalent amino acid sequence.
[0065] In yet another aspect, these multivalent (preferably bivalent, more preferably trivalent) amino acid sequences and polypeptides are directed against hCD4 and show increased competition with gp120 for binding hCD4 compared to the corresponding monovalent amino acid sequence. The competition may be increased by at least 2 times, preferably at least 3 times, such as at least 5 times or at least 10 times, for example by at least 15 times, at least 20 times, at least 30 times, at least 50 times, or 100 times or more, compared to the competition in the same assay under the same conditions by the corresponding monovalent amino acid sequence.
[0066] Accordingly, the amino acid sequences and (multivalent) polypeptides of the present invention can modulate and in particular inhibit and/or prevent viral entry in a target host cell by specifically binding to a Target of the invention; preferably, the amino acid sequences and polypeptides of the present invention can modulate and in particular inhibit and/or prevent viral entry in a target host cell by binding to a Target of the invention, such that virion attachment to a target host cell is modulated, inhibited and/or prevented (for instance by modulating and/or inhibiting and/or preventing the interaction between an envelope protein of a virus and the Target of the invention on a target host cell and/or the interaction between the an envelope protein of a virus and a target host cell or by competing with an envelope protein for binding to the Target of the invention or said target host cell). In a preferred aspect, viral entry in a target host cell is modulated and in particular inhibited and/or prevented by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to prevent viral entry in a target host under the same conditions but without the presence of the amino acid sequence, NANOBODY, compound or polypeptide of the invention.
[0067] A further aspect of the invention relates to a multispecific polypeptide of the invention (as defined herein) that comprises at least one amino acid sequence of the invention (such as a NANOBODY) against one of the Targets of the invention. Such multispecific proteins may further be as described herein.
[0068] In a preferred embodiment, the amino acid sequence or polypeptide of the invention is a “monoclonal” amino acid sequence or polypeptide, by which is meant that at least each of the one or more amino acid sequences directed against the Target of the invention that are present in said protein or polypeptide (and preferably all of the immunoglobulin sequences that are present in said protein or polypeptide) are “monoclonal” as commonly understood by the skilled person. In this respect, it should however be noted that, as further described herein, the present invention explicitly covers multivalent or multispecific proteins that comprise two or more immunoglobulin sequences (and in particular monoclonal immunoglobulin sequences) that are directed against different parts, regions, domains, loops or epitopes of the same Target of the invention, and in particular against different extracellular parts, regions, domains, loops or epitopes of the same Target of the invention.
[0069] It is also within the scope of the invention that, where applicable, an amino acid sequence of the invention can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or confirmations of Targets of the invention. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of Targets of the invention to which the amino acid sequences and/or polypeptides of the invention bind may be essentially the same (for example, if Targets of the invention contain repeated structural motifs or occurs in a multimeric form) or may be different (and in the latter case, the amino acid sequences and polypeptides of the invention may bind to such different antigenic determinants, epitopes, parts, domains, subunits of Targets of the invention with an affinity and/or specificity which may be the same or different). Also, for example, when Targets of the invention exists in an activated conformation and in an inactive conformation, the amino acid sequences and polypeptides of the invention may bind to either one of these confirmation, or may bind to both these confirmations (i.e. with an affinity and/or specificity which may be the same or different). Also, for example, the amino acid sequences and polypeptides of the invention may bind to a conformation of Targets of the invention in which it is bound to a pertinent ligand, may bind to a conformation of Targets of the invention in which it not bound to a pertinent ligand, or may bind to both such conformations (again with an affinity and/or specificity which may be the same or different).
[0070] As further described herein, a polypeptide of the invention may be bivalent and/or multivalent (as defined herein) and contain two or more amino acid sequences of the invention that are directed against a Target of the invention. Generally, such polypeptides will bind to the Target of the invention with increased avidity compared to a single amino acid sequence of the invention. It has also been observed that such polypeptides show (synergistically) increased binding, competition, and/or in vitro and/or in vivo neutralization of the virus.
[0071] Such a polypeptide may for example comprise two amino acid sequences of the invention that are directed against the same antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of a Target of the invention (which may or may not be an interaction site); or such a polypeptide may be biparatopic and/or multiparatopic (as defined herein) and comprise at least one “first” amino acid sequence of the invention that is directed against a first antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of a Target of the invention (which may or may not be an interaction site); and at least one “second” amino acid sequence of the invention that is directed against a second antigenic determinant, epitope, part, domain, subunit or conformation (where applicable) of said Target of the invention, wherein said second antigenic determinant, epitope, part, domain, subunit or conformation is different from the first (and again may or may not be an interaction site). Preferably, in such “bi- and/or multiparatopic” polypeptides of the invention, at least one amino acid sequence of the invention is directed against an interaction site (as defined herein), although the invention in its broadest sense is not limited thereto.
[0072] It is thus also within the scope of the invention that, where applicable, a polypeptide of the invention can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or conformations of a Target of the invention. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of said Target of the invention to which the amino acid sequences and/or polypeptides of the invention bind may be essentially the same (for example, if a Target of the invention contains repeated structural motifs or occurs in a multimeric form) or may be different (and in the latter case, the amino acid sequences and polypeptides of the invention are said to be “bi- and/or multiparatopic” and may bind to such different antigenic determinants, epitopes, parts, domains, subunits of said Target of the invention with an affinity and/or specificity which may be the same or different). Accordingly, bi- or multiparatopic polypeptides of the present invention are directed against and/or specifically bind to at least two epitopes of a Target of the invention, and are for example (but not limited to) polypeptides that are directed against and/or can specifically bind to three or even more epitopes of the same Target of the invention.
[0073] For example, and generally, a bivalent polypeptide of the invention may comprise two amino acid sequences of the invention directed against an antigenic determinant, epitope, part or domain of the Target of the invention which may be suitably linked, for example via a suitable linker as further described herein. Preferably, such a bivalent polypeptide of the invention is further such that, when it binds to the Target of the invention, it is capable of simultaneously binding to both antigenic determinants, epitopes, parts or domains (i.e. via the two amino acid sequences of the invention capable of binding to said antigenic determinants, epitopes, parts or domains). Examples of such bivalent polypeptides of the invention will become clear from the further description herein. Also, a trivalent polypeptide of the invention may comprise three amino acid sequences of the invention directed against an antigenic determinant, epitope, part or domain of the viral envelope protein, and generally multivalent polypeptides of the invention may contain at least two amino acid sequences of the invention directed against an antigenic determinants, epitopes, parts or domains of the viral envelope protein. Generally, such bivalent, trivalent and multivalent polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bivalent, trivalent and multivalent polypeptides of the invention (for example, these bivalent, trivalent and multivalent polypeptides of the invention preferably comprise single variable domains and more preferably NANOBODIES).
[0074] In one aspect of the invention, the amino acid sequences and (in particular) polypeptides of the invention are capable of binding to two or more antigenic determinants, epitopes, parts, domains of an envelope protein of a virus which are essentially the same. In this context, the amino acid sequences and polypeptides of the invention are also referred to as “multivalent (monospecific)” (such as e.g. “bivalent (monospecific)” or “trivalent (monospecific)”, etc.) amino acid sequences and polypeptides. The multivalent amino acid sequences and polypeptides of the invention can be directed against any antigenic determinants, epitopes, parts, and/or domains of the envelope protein of a virus.
[0075] In a preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bivalent and are directed against the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding to hCXCR4.
[0076] Generally, such a bivalent polypeptide of the invention may contain two amino acid sequences of the invention that are capable of binding to the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding to hCXCR4. Generally, such bivalent polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bivalent polypeptides of the invention (for example, these bivalent polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the CXCL12 binding site; and preferably comprise single variable domains and more preferably NANOBODIES).
[0077] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bivalent and are at least capable, upon binding to hCXCR4, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0078] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bivalent and are directed against the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4.
[0079] Generally, such a bivalent polypeptide of the invention will contain two amino acid sequences of the invention that are capable of binding to the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4. Generally, such bivalent polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bivalent polypeptides of the invention (for example, these bivalent polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the gp120 binding site; and preferably comprise single variable domains and more preferably NANOBODIES).
[0080] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bivalent and are at least capable, upon binding to hCD4, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0081] In a preferred aspect, the amino acid sequences and (in particular) polypeptides of the invention are capable of binding to two or more different antigenic determinants, epitopes, parts, domains of a Target of the invention. In this context, the amino acid sequences and polypeptides of the invention are also referred to as “multiparatopic” (such as e.g. “biparatopic” or “triparatopic”, etc.) amino acid sequences and polypeptides. The multiparatopic amino acid sequences and polypeptides of the invention can be directed against any antigenic determinants, epitopes, parts, and/or domains of the Target of the invention.
[0082] For example, and generally, a biparatopic polypeptide of the invention may comprise at least one amino acid sequence of the invention directed against a first antigenic determinant, epitope, part or domain of the Target of the invention and at least one amino acid sequence of the invention directed against a second antigenic determinant, epitope, part or domain of the Target of the invention different from the first antigenic determinant, epitope, part or domain (in which said amino acid sequences may be suitably linked, for example via a suitable linker as further described herein). Preferably, such a biparatopic polypeptide of the invention is further such that, when it binds to the Target of the invention, it is capable of simultaneously binding to the first antigenic determinant, epitope, part or domain (i.e. via the at least one amino acid sequence of the invention capable of binding to said first antigenic determinant, epitope, part or domain) and binding to said second antigenic determinant, epitope, part or domain (i.e. via the at least one amino acid sequence of the invention capable of binding to said second antigenic determinant, epitope, part or domain). Examples of such biparatopic polypeptides of the invention will become clear from the further description herein. Also, a triparatopic polypeptide of the invention may comprise at least one further amino acid sequence of the invention directed against a third antigenic determinant, epitope, part or domain of the Target of the invention (different from both the first and second antigenic determinant, epitope, part or domain), and generally multiparatopic polypeptides of the invention may contain at least two amino acid sequences of the invention directed against at least two different antigenic determinants, epitopes, parts or domains of the Target of the invention. Generally, such biparatopic, triparatopic and multiparatopic polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic, triparatopic and multiparatopic polypeptides of the invention (for example, these biparatopic, triparatopic and multiparatopic polypeptides of the invention preferably comprise single variable domains and more preferably NANOBODIES).
[0083] In a preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are directed against the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding to hCXCR4, as well as against at least one other antigenic determinant, epitope, part or domain on hCXCR4.
[0084] Generally, such a biparatopic (or multiparatopic) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding to hCXCR4, as well as at least one further amino acid sequence of the invention that is capable of binding to at least one other antigenic determinant, epitope, part or domain on hCXCR4. Generally, such biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the CXCL12 binding site and the at least one other antigenic determinant, epitope, part or domain on hCXCR4; and preferably comprise single variable domains and more preferably NANOBODIES).
[0085] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are at least capable, upon binding to hCXCR4, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0086] In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are biparatopic (or multiparatopic) and are directed against the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4, as well as against at least one other antigenic determinant on hCD4.
[0087] Generally, such a biparatopic (or multiparatopic) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4, as well as at least one further amino acid sequence of the invention that is capable of binding to at least one other antigenic determinant, epitope, part or domain on hCD4. Generally, such biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the gp120 binding site and the at least one other antigenic determinant, epitope, part or domain on hCD4; and preferably comprise single variable domains and more preferably NANOBODIES).
[0088] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic (or multiparatopic) and are at least capable, upon binding to hCD4, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0089] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic with both paratopes directed against the CXCL12 binding site on hCXCR4.
[0090] Generally, such a biparatopic (or multiparatopic) polypeptide of the invention will contain two amino acid sequences of the invention that are capable of binding to the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding to hCXCR4. Generally, such biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the CXCL12 binding site on hCXCR4; and preferably comprise single variable domains and more preferably NANOBODIES).
[0091] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are biparatopic with both paratopes directed against the gp120 binding site on hCD4.
[0092] Generally, such a biparatopic (or multiparatopic) polypeptide of the invention will contain two amino acid sequences of the invention that are capable of binding to the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4. Generally, such biparatopic (or multiparatopic) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these biparatopic (or multiparatopic) polypeptides of the invention (for example, these biparatopic and multiparatopic polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind the gp120 binding site on hCD4; and preferably comprise single variable domains and more preferably NANOBODIES).
[0093] Also, the polypeptides of the present invention may also be directed against and/or can specifically bind to at least one particular Target of the invention and at least one further epitope of another Target of the invention, which is different from said at least one particular Target of the invention. Thus, the polypeptides according to the invention may be directed against and/or may specifically bind to at least two (or even more) epitopes of at least two different Targets of the invention. Also, said at least one further epitope may or may not be involved in viral entry in a target host cell.
[0094] Generally, bi-, and multivalent (as defined herein), bi-, and multispecific (as defined herein) and bi-, and multiparatopic (as defined herein) polypeptides according to the invention may be useful for the prevention and/or treatment of viral diseases by specifically binding to at least one epitope of a Target of the invention and at least one further epitope (which may or may not be different from said at least one epitope) of the Target of the invention.
[0095] Preferably, bi-, and multiparatopic polypeptides (as defined herein) according to the invention may be useful for the prevention and/or treatment of viral diseases by specifically binding to at least two (or even more) epitopes (which may be the same or different) on the same Target of the invention.
[0096] Alternatively, the polypeptides of the present invention may be directed against and/or can specifically bind to at least one epitope of a Target of the invention and at least one further epitope of another target, which is different from said Target of the invention and which is for instance a further Target of the invention.
[0097] In a preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are bispecific (or multispecific) and are directed against an antigenic determinant, epitope, part or domain on hCXCR4 as well as against an antigenic determinant, epitope, part or domain on hCD4. Generally, such a bispecific (or multispecific) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCXCR4, as well at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCD4. Generally, such bispecific (or multispecific) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bispecific (or multispecific) polypeptides of the invention (for example, these bispecific and multispecific polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind both binding sites; and preferably comprise single variable domains and more preferably NANOBODIES).
[0098] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bispecific (or multispecific) and are at least capable, upon binding, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0099] In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are bispecific (or multispecific) and are directed against the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding hCXCR4 as well as against an antigenic determinant, epitope, part or domain on hCD4.
[0100] Generally, such a bispecific (or multispecific) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding hCXCR4, as well at least one amino acid sequence of the invention that is capable of binding to an antigenic determinant, epitope, part or domain on hCD4. Generally, such bispecific (or multispecific) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bispecific (or multispecific) polypeptides of the invention (for example, these bispecific and multispecific polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind both binding sites; and preferably comprise single variable domains and more preferably NANOBODIES).
[0101] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bispecific (or multispecific) and are at least capable, upon binding, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0102] In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are bispecific (or multispecific) and are directed against an antigenic determinant, epitope, part or domain on hCXCR4, as well as against the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4.
[0103] Generally, such a bispecific (or multispecific) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to an antigenic determinant, epitope, part or domain on hCXCR4, as well at least one amino acid sequence of the invention that is capable of binding to the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4. Generally, such bispecific (or multispecific) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bispecific (or multispecific) polypeptides of the invention (for example, these bispecific and multispecific polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind both binding sites; and preferably comprise single variable domains and more preferably NANOBODIES).
[0104] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bispecific (or multispecific) and are at least capable, upon binding, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0105] In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are bispecific (or multispecific) and are directed against the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding hCXCR4 as well as against the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4.
[0106] Generally, such a bispecific (or multispecific) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the CXCL12 binding site on hCXCR4 and/or capable of competing with CXCL12 for binding hCXCR4, as well at least one amino acid sequence of the invention that is capable of binding to the gp120 binding site on hCD4 and/or capable of competing with gp120 for binding to hCD4. Generally, such bispecific (or multispecific) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bispecific (or multispecific) polypeptides of the invention (for example, these bispecific and multispecific polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind both binding sites; and preferably comprise single variable domains and more preferably NANOBODIES).
[0107] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bispecific (or multispecific) and are at least capable, upon binding, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0108] In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are bispecific (or multispecific) and are directed against an antigenic determinant, epitope, part or domain on hCXCR4 as well as against an antigenic determinant, epitope, part or domain on hCCR5. Generally, such a bispecific (or multispecific) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCXCR4, as well at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCCR5. Generally, such bispecific (or multispecific) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bispecific (or multispecific) polypeptides of the invention (for example, these bispecific and multispecific polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind both binding sites; and preferably comprise single variable domains and more preferably NANOBODIES).
[0109] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bispecific (or multispecific) and are at least capable, upon binding, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0110] In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are bispecific (or multispecific) and are directed against an antigenic determinant, epitope, part or domain on hCD4 as well as against an antigenic determinant, epitope, part or domain on hCCR5. Generally, such a bispecific (or multispecific) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCD4, as well at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCCR5. Generally, such bispecific (or multispecific) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these bispecific (or multispecific) polypeptides of the invention (for example, these bispecific and multispecific polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind both binding sites; and preferably comprise single variable domains and more preferably NANOBODIES).
[0111] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are bispecific (or multispecific) and are at least capable, upon binding, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0112] In another preferred, but non-limiting aspect, the amino acid sequences and polypeptides of the invention are trispecific (or multispecific) and are directed against an antigenic determinant, epitope, part or domain on hCXCR4, against an antigenic determinant, epitope, part or domain on hCD4, as well as against an antigenic determinant, epitope, part or domain on hCCR5. Generally, such a tripecific (or multispecific) polypeptide of the invention will contain at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCXCR4, at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCD4, as well at least one amino acid sequence of the invention that is capable of binding to the antigenic determinant, epitope, part or domain on hCCR5. Generally, such tripecific (or multispecific) polypeptides of the invention may be as further described herein, and the various preferred aspects of the invention as described herein also apply to these tripecific (or multispecific) polypeptides of the invention (for example, these trispecific and multispecific polypeptides of the invention may comprise suitable linkers; are preferably such that they can simultaneously bind both binding sites; and preferably comprise single variable domains and more preferably NANOBODIES).
[0113] In another preferred, but non-limiting aspect, the amino acid sequences and (in particular) polypeptides of the invention are trispecific (or multispecific) and are at least capable, upon binding, to neutralize a virus (as defined herein); to modulate, reduce and/or inhibit the infectivity of a virus (as defined herein); and/or to modulate and in particular inhibit and/or prevent viral entry (as further defined herein) in a target host cell.
[0114] It is also expected that the amino acid sequences and polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of Targets of the invention; or at least to those analogs, variants, mutants, alleles, parts and fragments of Targets of the invention that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the amino acid sequences and polypeptides of the invention bind in Targets of the invention (e.g. in wild-type Targets of the invention). Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and/or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to (wild-type) Targets of the invention. It is also included within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to some analogs, variants, mutants, alleles, parts and fragments of Targets of the invention, but not to others.
[0115] When Targets of the invention exists in a monomeric form and in one or more multimeric forms, it is within the scope of the invention that the amino acid sequences and polypeptides of the invention only bind to Targets of the invention in monomeric form, only bind to Targets of the invention in multimeric form, or bind to both the monomeric and the multimeric form. Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to the monomeric form with an affinity and/or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to the multimeric form.
[0116] Also, when Targets of the invention can associate with other proteins or polypeptides to form protein complexes (e.g. with multiple subunits), it is within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to Targets of the invention in its non-associated state, bind to Targets of the invention in its associated state, or bind to both. In all these cases, the amino acid sequences and polypeptides of the invention may bind to such multimers or associated protein complexes with an affinity and/or specificity that may be the same as or different from (i.e. higher than or lower than) the affinity and/or specificity with which the amino acid sequences and polypeptides of the invention bind to Targets of the invention in its monomeric and non-associated state.
[0117] Also, as will be clear to the skilled person, proteins or polypeptides that contain two or more amino acid sequences directed against Targets of the invention may bind with higher avidity to Targets of the invention than the corresponding monomeric amino acid sequence(s). For example, and without limitation, proteins or polypeptides that contain two or more amino acid sequences directed against different epitopes of Targets of the invention may (and usually will) bind with higher avidity than each of the different monomers, and proteins or polypeptides that contain two or more amino acid sequences directed against Targets of the invention may (and usually will) bind also with higher avidity to a multimer of Targets of the invention.
[0118] Generally, amino acid sequences and polypeptides of the invention will at least bind to those forms of Targets of the invention (including monomeric, multimeric and associated forms) that are the most relevant from a biological and/or therapeutic point of view, as will be clear to the skilled person.
[0119] It is also within the scope of the invention to use parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the amino acid sequences and polypeptides of the invention, and/or to use proteins or polypeptides comprising or essentially consisting of one or more of such parts, fragments, analogs, mutants, variants, alleles and/or derivatives, as long as these are suitable for the uses envisaged herein. Such parts, fragments, analogs, mutants, variants, alleles and/or derivatives will usually contain (at least part of) a functional antigen-binding site for binding against Targets of the invention; and more preferably will be capable of specific binding to Targets of the invention, and even more preferably capable of binding to Targets of the invention with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein. Some non-limiting examples of such parts, fragments, analogs, mutants, variants, alleles, derivatives, proteins and/or polypeptides will become clear from the further description herein. Additional fragments or polypeptides of the invention may also be provided by suitably combining (i.e. by linking or genetic fusion) one or more (smaller) parts or fragments as described herein.
[0120] In one specific, but non-limiting aspect of the invention, which will be further described herein, such analogs, mutants, variants, alleles, derivatives have an increased half-life in serum (as further described herein) compared to the amino acid sequence from which they have been derived. For example, an amino acid sequence of the invention may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide a derivative of an amino acid sequence of the invention with increased half-life.
[0121] In one specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that comprises an immunoglobulin fold or may be an amino acid sequence that, under suitable conditions (such as physiological conditions) is capable of forming an immunoglobulin fold (i.e. by folding). Reference is inter alia made to the review by Halaby et al., J. (1999) Protein Eng. 12, 563-71. Preferably, when properly folded so as to form an immunoglobulin fold, such an amino acid sequence is capable of specific binding (as defined herein) to Targets of the invention; and more preferably capable of binding to Targets of the invention with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein. Also, parts, fragments, analogs, mutants, variants, alleles and/or derivatives of such amino acid sequences are preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold.
[0122] In particular, but without limitation, the amino acid sequences of the invention may be amino acid sequences that essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR's, as further described herein).
[0123] The amino acid sequences of the invention may in particular be an immunoglobulin sequence or a suitable fragment thereof, and more in particular be an immunoglobulin variable domain sequence or a suitable fragment thereof, such as light chain variable domain sequence (e.g. a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a VH-sequence) or a suitable fragment thereof. When the amino acid sequence of the invention is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a VH sequence that is derived from a human antibody) or be a so-called VHH-sequence (as defined herein) that is derived from a so-called “heavy chain antibody” (as defined herein).
[0124] However, it should be noted that the invention is not limited as to the origin of the amino acid sequence of the invention (or of the nucleotide sequence of the invention used to express it), nor as to the way that the amino acid sequence or nucleotide sequence of the invention is (or has been) generated or obtained. Thus, the amino acid sequences of the invention may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences. In a specific but non-limiting aspect of the invention, the amino acid sequence is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to “humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences or NANOBODIES), “camelized” (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing. Reference is for example made to the standard handbooks, as well as to the further description and prior art mentioned herein.
[0125] Similarly, the nucleotide sequences of the invention may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.
[0126] The amino acid sequence of the invention may in particular be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a “dAb” (or an amino acid sequence that is suitable for use as a dAb) or a NANOBODY (as defined herein, and including but not limited to a VHH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term “dAb's”, reference is for example made to Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003, 21 (11):484-490; as well as to for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called “IgNAR domains”, see for example WO 05/18629).
[0127] In particular, the amino acid sequence of the invention may be a NANOBODY (as defined herein) or a suitable fragment thereof. [Note: NANOBODY®, NANOBODIES® and Nanoclone® are registered trademarks of Ablynx N. V.] Such NANOBODIES directed against Targets of the invention will also be referred to herein as “NANOBODIES of the invention”.
[0128] For a general description of NANOBODIES, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described NANOBODIES of the so-called “VH3 class” (i.e. NANOBODIES with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51 or DP-29), which NANOBODIES form a preferred aspect of this invention. It should however be noted that the invention in its broadest sense generally covers any type of NANOBODY directed against Targets of the invention, and for example also covers the NANOBODIES belonging to the so-called “VH4 class” (i.e. NANOBODIES with a high degree of sequence homology to human germline sequences of the VH4 class such as DP-78), as for example described in WO 07/118,670.
[0129] Generally, NANOBODIES (in particular VHH sequences and partially humanized NANOBODIES) can in particular be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences (again as further described herein).
[0130] Thus, generally, a NANOBODY can be defined as an amino acid sequence with the (general) structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.
[0131] In particular, a NANOBODY can be an amino acid sequence with the (general) structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.
[0132] More in particular, a NANOBODY can be an amino acid sequence with the (general) structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
[0133] i) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 below; and in which:
[0134] ii) said amino acid sequence has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are disregarded.
[0135] In these NANOBODIES, the CDR sequences are generally as further defined herein.
[0136] Thus, the invention also relates to such NANOBODIES that can bind to (as defined herein) and/or are directed against Targets of the invention, to suitable fragments thereof, as well as to polypeptides that comprise or essentially consist of one or more of such NANOBODIES and/or suitable fragments.
[0137] SEQ ID NO's 238 to 253 give the amino acid sequences of a number of VHH sequences that have been raised against human CXCR4 (Table 1).
[0138] In particular, the invention in some specific aspects provides:
[0139] amino acid sequences that are directed against (as defined herein) a hCXCR4 and that have at least 80%, preferably at least 85%, such as 90% or 95% or more sequence identity with at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably 238 to 239. These amino acid sequences may further be such that they neutralize binding of the cognate ligand to hCXCR4; and/or compete with the cognate ligand for binding to hCXCR4; and/or are directed against an interaction site (as defined herein) on hCXCR4 (such as the ligand binding site);
[0140] amino acid sequences that cross-block (as defined herein) the binding of at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably 238 to 239 to hCXCR4 and/or that compete with at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably 238 to 239 for binding to hCXCR4. Again, these amino acid sequences may further be such that they neutralize binding of the cognate ligand to hCXCR4; and/or compete with the cognate ligand for binding to hCXCR4; and/or are directed against an interaction site (as defined herein) on hCXCR4 (such as the ligand binding site);
[0141] which amino acid sequences may be as further described herein (and may for example be NANOBODIES); as well as polypeptides of the invention that comprise one or more of such amino acid sequences (which may be as further described herein, and may for example be bispecific and/or biparatopic polypeptides as described herein), and nucleic acid sequences that encode such amino acid sequences and polypeptides. Such amino acid sequences and polypeptides do not include any naturally occurring ligands.
[0142] SEQ ID NO's 308 to 311 give the amino acid sequences of a number of VHH sequences that have been raised against human CD4 (Table 3).
[0143] In particular, the invention in some specific aspects provides:
[0144] amino acid sequences that are directed against (as defined herein) a hCD4 and that have at least 80%, preferably at least 85%, such as 90% or 95% or more sequence identity with at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably 311. These amino acid sequences may further be such that they neutralize binding of the cognate ligand to hCD4; and/or compete with the cognate ligand for binding to hCD4; and/or are directed against an interaction site (as defined herein) on hCD4 (such as the ligand binding site);
[0145] amino acid sequences that cross-block (as defined herein) the binding of at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably 311 to hCD4 and/or that compete with at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably 311 for binding to hCD4. Again, these amino acid sequences may further be such that they neutralize binding of the cognate ligand to hCD4; and/or compete with the cognate ligand for binding to hCD4; and/or are directed against an interaction site (as defined herein) on hCD4 (such as the ligand binding site);
[0146] which amino acid sequences may be as further described herein (and may for example be NANOBODIES); as well as polypeptides of the invention that comprise one or more of such amino acid sequences (which may be as further described herein, and may for example be bispecific and/or biparatopic polypeptides as described herein), and nucleic acid sequences that encode such amino acid sequences and polypeptides. Such amino acid sequences and polypeptides do not include any naturally occurring ligands.
[0147] 
[00001] [TABLE-US-00001]
  TABLE 1
 
  Nanobodies directed against human CXCR4 (SEQ ID NO: 254)
  SEQ ID    
  NO: X,    
  where X =   Name   Amino acid sequence
 
  238   238C1   EVQLVESGGGLVQTGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIKSSGD
    238D2   STRYAGSVKGRFTISRDNAKNMLYLQMYSLKPEDTAVYYCAKSRVSRTGLYTYDNR
      GQGTQVTVSS
 
  239   238D4   EVQLMESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRAPGKEREFVAAITRSGV
    238G3   RSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRR
      FEYDYSGQGTQVTVSS
 
  240   237B5   KVQLVESGGGLVQPGGSLRLSCAASGFAFSIHTMSWVRQAPGKGPEWVSTIKPSGD
      TTNYANAVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKDYFGTGVRGQGTQV
      TVSS
 
  241   237B6   EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSAISWNGG
    237A5   STDYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAEYYCARDQGPFYSGTYYYTR
    237D2   QYGYRGQGTQVTVSS
    237D3  
    237E4  
    237F4  
    237G2  
    237G4  
    237xH5  
    237F1  
    237C5  
    237G1  
 
  242   238B10   EVQLVESGGGFVQAGGSLRLSCETSGRPLLGYTIAWFRQVPGKEREFVAYHRWSDG
      ANLYADSVKGRFTISGHNAKNTVSLQMNSLKPEDTAVYYCAAARMTTSNDKEYLYW
      GQGTQVTVSS
 
  243   238C5   EVQLMESGGGLVQAGGSLRLACAASGFTFEDYAIGWFRKAPGKEREGVSCISGSDG
    237G2   STTYADSVKGRFTISTDNAKNTVYLEMNSLKPEDTAVYYCAQQYGVGGRVVCPGPY
    237xH5   EYDVWGQSTQVTVSS
    238C3  
    237D6  
    237E6  
 
  244   238F7   EVQLVESGGGFVQAGGSLRLSCETSGRPLLGYTIAWFRQVPGKEREFVAYHRWSDG
      ANLYADSVKGRFTISGHNAKNTVSLQMNSLKPEDTAVYYCAAAWMTTSNDKETLYW
      GQGTQVTVSS
 
  245   238H2   EVQLVESGGGLVQAGGSLRLSCAASGLTFSPSAMAWYRQGPGKERDFVASTIWSRG
      DTYFADSVKGRFTISRDTANYTLYLQMNNEKPEDTAVYYCSLRVRPYGQYDYWGQG
      TQVTVSS
 
  246   237D4   EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSAISWNGG
      SADYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDQGPFYSGTYYYTK
      GYAYWGQGTQVTVSS
 
  247   238F3   EVQLVESGGGLAQAGGSLRLSCAASGRTYAMGWFRQAPGKEREFVTTSRLITDNII
      YADSVKGRFTLTRDNGKNTVYLQMDSLKPDDTAVYFCAARQNYSRSVFGAKDYDYW
      GQGTQVTVSS
 
  248   237A6   EVQLVESGGGLVQAGGSLRLSCAASGSIFSLNAMGWYRQAPGKQRELVAGITSSTS
      TYYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAVYYCNVDCPDYYSDYECPLED
      RGQGTQVTVSS
 
  249   237D1   EVQLVESGGGLAQPGGPLRLTCEASGVIYSVNDMGWYRQAPGKQRELVAVITSGGG
      TNYVDSVKGRFTISGDNRKKTVYLQMNSLKPEDTAVYYCSIYYSSGISTLRSWGQG
      TQVTVSS
 
  250   237E1   EVQLVESGGGLVQPGGSLRLSCEVSGFTRDYYTIGWFRQAPGKEREGVSCISSSDG
      STAYLGSVQGRFTVSRDNAKNTVYLQMNNLKPEDTAVYYCALBSADSRCSIGSIGF
      TWLYNNWGQGTQVTVSS
 
  251   23707   EVQLVESGGGLVQPGGSLRLSCAASSFIGNYHAIVWLRQAPGKELEGVSCITSRDS
      ITYYASFVKGRFTISRDDAKNTVYLQMNNLKPEDTAVYYCAVBTSMTCPTLIVRFN
      YRGQGTQVTVSS
 
  252   23804   EVQLVESGGGLVQAGGSLRLSCKASGGTFNNYAMGWFRRAPGKEREFVAAITRSGV
      RSGVSAIYGDSVKDRFTISRDNVKNTLYLQMNTLKPEDTAVYTCAASAIGSGALRR
      FEYDYSGQGTQVTVSS
 
  253   23701   EVQLVESGGGLVQAGGSLRLSCAASGSFFSINAMGWYRQAP0KQRELVASITSGGS
      TVYADSVKGRFTISRDNYNTVYLQMNSLKPEDTAVYYCNADGVPEWGKVQYPDTYR
      GQGTQVTVSS
 
  CDR sequences of Nanobodies directed against human CXCR4 (SEQ ID NO: 254)
  SEQ ID NO:     CDR X,  
  X, where X =   Name   where X =   Amino acid sequence
 
  142   238C1, 238D2   1   SYAMS
 
  143   238D4, 238G3   1   NYAMG
 
  144   237B5   1   IHTMS
 
  145   237B6, 237A5, 237D2, 237D3,   1   DYADS
    237E4, 237F4, 237G2, 237G4,    
    237xH5, 237F1, 237C5,    
    2387G1    
 
  146   238B10   1   GYTIA
 
  147   238C5, 238G2, 238xH5,   1   DYAIG
    238C3, 238D6, 238E6    
 
  148   238F7   1   GYTIA
 
  149   238H2   1   PSAMA
 
  150   237D4   1   DYADS
 
  151   238F3   1   MG
 
  152   237A6   1   LNAMG
 
  153   237D1   1   VNDMG
 
  154   237E1   1   YYTIG
 
  155   237G7   1   YHAIV
 
  156   238C4   1   NYAMG
 
  157   237C1   1   INAMG
 
  174   238C1, 238D2   2   GIKSSGDSTRYAGSVKG
 
  175   238D4, 238G3   2   AITRSGVRSGVSAIYGDSVKD
 
  176   237B5   2   TIKPSGDTTNYANAVKG
 
  177   237B6, 237A5, 237D2, 237D3,   2   AISWNGGSTDYADSVKG
    237E4, 237F4, 237G2, 237G4,    
    237xH5, 237F1, 237C5, 237G1    
 
  178   238B10   2   YHRWSDGANLYADSVNG
 
  179   238C5, 238G2, 238xH5,   2   CISGSDGSTTYADSVKG
    238C3, 238D6, 238E6    
 
  180   238F7   2   YHRWSDGANLYADSVKG
 
  181   238H2   2   STIWSRGDTYFADSVKG
 
  182   237D4   2   AISWNGGSADYADSVKG
 
  183   238F3   2   TSRLTIDNIIYADSVKG
 
  184   237A6   2   GITSSTSTYYADSVKG
 
  185   237D1   2   VITSGGGTKYVDSVKG
 
  186   237E1   2   CISSSDGSTAYLGSVQG
 
  187   237G7   2   CITSRDSITYYASEVKG
 
  188   238C4   2   AITRSGVRSGVSAIYGDSVKD
 
  189   237C1   2   SITSGGSTVYADSVHG
 
  206   238D1, 238D2   3   SRVSRTGLYTYDN
 
  207   238D4, 238G3   3   SAIGSGALRRFEYDY
 
  208   237B5   3   DYFGTGV
 
  209   237B6, 237A5, 237D2, 237D3,   3   DQGPFYSGTYYYTRWGY
    237E4, 237F4, 237G2, 237G4,    
    237xH5, 237F1, 237C5, 237G1    
 
  210   238B10   3   ARMTTSNDKEYLY
 
  211   238C5,238G2, 238xH5, 238C3,   3   QYGVGGRVVCPGPYEYDV
    238D6, 238E6    
 
  212   238F7   3   AWMTTSNDKEYLY
 
  213   238H2   3   RVRPYGQYDY
 
  214   237D4   3   DQGPFYSCTYYYTKGYAY
 
  215   238F3   3   RQNYSRSVFGAKDYDY
 
  216   237A6   3   DCPDYYSDYECPLED
 
  217   237D1   3   YYSSGISTLRS
 
  218   237E1   3   BSADSRCSIGSIGFTWLYNN
 
  219   237G7   3   BTSMTCPTLIVRFNY
 
  220   238C4   3   SAIGSGALRRFEYDY
 
  221   237C1   3   DGVPEWSKVQYPDTY
 
[0148] 
[00002] [TABLE-US-00002]
  TABLE 3
 
  Nanobodies directed against human CD4 (SEQ ID NO: 268)
  SEQ ID NO: X,    
  where X =   Name   Amino acid sequence
 
  308   01B6   EVQLVESGGGLVQ5GGSLRLSCAASGFTFSGYWMYWVRQAPGKGLE
      WVSAISPGGGSTYYPDSVKGRFTISRDNAKNTLYLQMNSLEPEDTA
      LYYCASSLTATHTYEYDYWGQGTQVTVSS
 
  309   01E2   EVQLVESGGGLVQAGGSLRLSCAASGRTSASYSMGWFRQAPGKERE
      FVAAISWSGDETSYADSVKGRFTIARGNAKNTVYLQMNSLKSEDTA
      IYYCAGDRWWRPAGLQWDYWGQGTQVTVSS
 
  310   01H12   EVQLVESGGGLVQAGGSLKLSCAASRSILDFNAMGWYRQAPGKQRE
      WVTTIARAGATKYADSVKGRFSISRDNAKNTVYLQMSSLKPEDTAT
      YYCNARVFDLPNDYWGQGTQVTVSS
 
  311   03F11   EVQLVESGGGSVQPGGSLTLSCGTSGRTFNVMGWFRQAPGKEREFV
      AAVRWSSTGIYYTQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDT
      AVYYCAADTYNSNPARWDGYDFRGQGTQVTVSS
 
  CDR sequences of Nanobodies directed against human CD4 (SEQ ID NO: 268)
  SEQ ID NO:     CDR X,  
  X, where X =   Name   where X =   Amino acid sequence
 
  284   01B6   1   GYWMY
 
  285   01E2   1   SYSMG
 
  286   01H12   1   FNAMG
 
  287   03F11   1   VMG
 
  292   01B6   2   AISPGGGSTYYPDSVKG
 
  293   01E2   2   AISWSGDETSYADSVKG
 
  294   01H12   2   TIARAGATKYADSVKG
 
  295   03F11   2   AVRWSSTGIYYTQYADSVKS
 
  300   01B6   3   SLTATHTYEYDY
 
  301   01E2   3   DRWWRPAGLQWDY
 
  302   01H12   3   RVFDLPNDY
 
  303   03F11   3   DTYNSNPARWDGYDF
 
[0149] Accordingly, some particularly preferred NANOBODIES of the invention are NANOBODIES which can bind (as further defined herein) to and/or are directed against human CXCR4 and which:
[0150] i) have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded. In this respect, reference is also made to Table A-1, which lists the framework 1 sequences (SEQ ID NO's: 126 to 141), framework 2 sequences (SEQ ID NO's: 158 to 173), framework 3 sequences (SEQ ID NO's: 190 to 205) and framework 4 sequences (SEQ ID NO's: 222 to 237) of the NANOBODIES of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 (with respect to the amino acid residues at positions 1 to 4 and 27 to 30 of the framework 1 sequences, reference is also made to the comments made below. Thus, for determining the degree of amino acid identity, these residues are preferably disregarded);
and in which:
[0151] ii) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 below.
[0152] Accordingly, some particularly preferred NANOBODIES of the invention are NANOBODIES which can bind (as further defined herein) to and/or are directed against human CD4 and which:
[0153] iii) have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded. In this respect, reference is also made to Table A-1, which lists the framework 1 sequences (SEQ ID NO's: 280 to 283), framework 2 sequences (SEQ ID NO's: 288 to 291), framework 3 sequences (SEQ ID NO's: 296 to 299) and framework 4 sequences (SEQ ID NO's: 304 to 307) of the NANOBODIES of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311 (with respect to the amino acid residues at positions 1 to 4 and 27 to 30 of the framework 1 sequences, reference is also made to the comments made below. Thus, for determining the degree of amino acid identity, these residues are preferably disregarded);
and in which:
[0154] preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 below.
[0155] In these NANOBODIES, the CDR sequences are generally as further defined herein.
[0156] Again, such NANOBODIES may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring VHH sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to “humanized” (as defined herein) NANOBODIES, “camelized” (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as NANOBODIES that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. Also, when a NANOBODY comprises a VHH sequence, said NANOBODY may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized NANOBODIES of the invention. Similarly, when a NANOBODY comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence), said NANOBODY may optionally be further suitably humanized, again as described herein, again so as to provide one or more further (partially or fully) humanized NANOBODIES of the invention.
[0157] In particular, humanized NANOBODIES may be amino acid sequences that are as generally defined for NANOBODIES in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanizing substitution (as defined herein). Some preferred, but non-limiting humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a NANOBODY may be partially humanized or fully humanized.
[0158] Some particularly preferred humanized NANOBODIES of the invention are humanized variants of the NANOBODIES of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239.
[0159] Thus, some other preferred NANOBODIES of the invention are NANOBODIES which can bind (as further defined herein) to human CXCR4 and which:
[0160] i) are a humanized variant of one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239; and/or
[0161] ii) have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded;
and in which:
[0162] i) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 below.
[0163] Some other preferred NANOBODIES of the invention are NANOBODIES which can bind (as further defined herein) to human CD4 and which:
[0164] i) are a humanized variant of one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311; and/or
[0165] ii) have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences are disregarded;
and in which:
[0166] i) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 below.
[0167] According to another specific aspect of the invention, the invention provides a number of stretches of amino acid residues (i.e. small peptides) that are particularly suited for binding to Targets of the invention. These stretches of amino acid residues may be present in, and/or may be incorporated into, an amino acid sequence of the invention, in particular in such a way that they form (part of) the antigen binding site of an amino acid sequence of the invention. As these stretches of amino acid residues were first generated as CDR sequences of heavy chain antibodies or VHH sequences that were raised against Targets of the invention (or may be based on and/or derived from such CDR sequences, as further described herein), they will also generally be referred to herein as “CDR sequences” (i.e. as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively). It should however be noted that the invention in its broadest sense is not limited to a specific structural role or function that these stretches of amino acid residues may have in an amino acid sequence of the invention, as long as these stretches of amino acid residues allow the amino acid sequence of the invention to bind to Targets of the invention. Thus, generally, the invention in its broadest sense comprises any amino acid sequence that is capable of binding to Targets of the invention and that comprises one or more CDR sequences as described herein, and in particular a suitable combination of two or more such CDR sequences, that are suitably linked to each other via one or more further amino acid sequences, such that the entire amino acid sequence forms a binding domain and/or binding unit that is capable of binding to Targets of the invention. It should however also be noted that the presence of only one such CDR sequence in an amino acid sequence of the invention may by itself already be sufficient to provide an amino acid sequence of the invention that is capable of binding to Targets of the invention; reference is for example again made to the so-called “Expedite fragments” described in WO 03/050531.
[0168] Thus, in another specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that comprises at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof). In particular, an amino acid sequence of the invention may be an amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof).
[0169] Generally, in this aspect of the invention, the amino acid sequence of the invention may be any amino acid sequence that comprises at least one stretch of amino acid residues, in which said stretch of amino acid residues has an amino acid sequence that corresponds to the sequence of at least one of the CDR sequences described herein. Such an amino acid sequence may or may not comprise an immunoglobulin fold. For example, and without limitation, such an amino acid sequence may be a suitable fragment of an immunoglobulin sequence that comprises at least one such CDR sequence, but that is not large enough to form a (complete) immunoglobulin fold (reference is for example again made to the “Expedite fragments” described in WO 03/050531). Alternatively, such an amino acid sequence may be a suitable “protein scaffold” that comprises least one stretch of amino acid residues that corresponds to such a CDR sequence (i.e. as part of its antigen binding site). Suitable scaffolds for presenting amino acid sequences will be clear to the skilled person, and for example comprise, without limitation, to binding scaffolds based on or derived from immunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived from protein A domains (such as Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 2006 9 (8):619-32).
[0170] Again, any amino acid sequence of the invention that comprises one or more of these CDR sequences is preferably such that it can specifically bind (as defined herein) to Targets of the invention, and more in particular such that it can bind to Targets of the invention with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein), that is as defined herein.
[0171] More in particular, the amino acid sequences according to this aspect of the invention may be any amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least two amino acid sequences that are chosen from the group consisting of the CDR1 sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that (i) when the first amino acid sequence is chosen from the CDR1 sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein or the CDR3 sequences described herein; (ii) when the first amino acid sequence is chosen from the CDR2 sequences described herein, the second amino acid sequence is chosen from the CDR1 sequences described herein or the CDR3 sequences described herein; or (iii) when the first amino acid sequence is chosen from the CDR3 sequences described herein, the second amino acid sequence is chosen from the CDR1 sequences described herein or the CDR3 sequences described herein.
[0172] Even more in particular, the amino acid sequences of the invention may be amino acid sequences that comprise at least one antigen binding site, wherein said antigen binding site comprises at least three amino acid sequences that are chosen from the group consisting of the CDR1 sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that the first amino acid sequence is chosen from the CDR1 sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein, and the third amino acid sequence is chosen from the CDR3 sequences described herein. Preferred combinations of CDR1, CDR2 and CDR3 sequences will become clear from the further description herein. As will be clear to the skilled person, such an amino acid sequence is preferably an immunoglobulin sequence (as further described herein), but it may for example also be any other amino acid sequence that comprises a suitable scaffold for presenting said CDR sequences.
[0173] Thus, in one specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against hCXCR4, that comprises one or more stretches of amino acid residues chosen from the group consisting of:
[0174] a) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0175] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0176] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0177] d) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0178] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0179] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0180] g) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0181] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0182] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207; or any suitable combination thereof.
[0183] When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):
[0184] i) any amino acid substitution in such an amino acid sequence according to b) and/or c) is preferably, and compared to the corresponding amino acid sequence according to a), a conservative amino acid substitution, (as defined herein);
and/or
[0185] ii) the amino acid sequence according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to a);
and/or
[0186] iii) the amino acid sequence according to b) and/or c) may be an amino acid sequence that is derived from an amino acid sequence according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0187] Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):
[0188] i) any amino acid substitution in such an amino acid sequence according to e) and/or f) is preferably, and compared to the corresponding amino acid sequence according to d), a conservative amino acid substitution, (as defined herein);
and/or
[0189] ii) the amino acid sequence according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to d);
and/or
[0190] iii) the amino acid sequence according to e) and/or f) may be an amino acid sequence that is derived from an amino acid sequence according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0191] Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):
[0192] i) any amino acid substitution in such an amino acid sequence according to h) and/or i) is preferably, and compared to the corresponding amino acid sequence according to g), a conservative amino acid substitution, (as defined herein);
and/or
[0193] ii) the amino acid sequence according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to g); and/or
[0194] iii) the amino acid sequence according to h) and/or i) may be an amino acid sequence that is derived from an amino acid sequence according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0195] It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.
[0196] In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:
[0197] i) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0198] ii) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175; and
[0199] iii) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207; or any suitable combination thereof.
[0200] Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against hCXCR4.
[0201] In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against hCXCR4 that comprises two or more stretches of amino acid residues chosen from the group consisting of:
[0202] a) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0203] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0204] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0205] d) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0206] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0207] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0208] g) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0209] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0210] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
such that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b) or c), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e), f), g), h) or i); (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), g), h) or i); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to g), h) or i), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), d), e) or f).
[0211] In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:
[0212] i) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0213] ii) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175; and
[0214] iii) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207; such that, (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175 or of SEQ ID NO's: 206 to 221, more preferably 206 to 207; (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143 or of SEQ ID NO's: 206 to 221, more preferably 206 to 207; or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143 or of SEQ ID NO's: 174 to 189, more preferably 174 to 175.
[0215] Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against hCXCR4.
[0216] In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against hCXCR4, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:
[0217] a) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0218] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0219] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143; the second stretch of amino acid residues is chosen from the group consisting of:
[0220] d) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0221] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0222] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175; and the third stretch of amino acid residues is chosen from the group consisting of:
[0223] g) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0224] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0225] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207.
[0226] Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207.
[0227] Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against hCXCR4.
[0228] Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.
[0229] Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.
[0230] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to hCXCR4; and more in particular bind to hCXCR4 with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0231] When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:
[0232] CDR1 is chosen from the group consisting of:
[0233] a) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0234] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0235] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
and/or
[0236] CDR2 is chosen from the group consisting of:
[0237] d) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0238] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0239] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
and/or
[0240] CDR3 is chosen from the group consisting of:
[0241] g) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0242] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0243] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207.
[0244] In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207.
[0245] In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:
[0246] CDR1 is chosen from the group consisting of:
[0247] a) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0248] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0249] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
and
[0250] CDR2 is chosen from the group consisting of:
[0251] d) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0252] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0253] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
and
[0254] CDR3 is chosen from the group consisting of:
[0255] g) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0256] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0257] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207; or any suitable fragment of such an amino acid sequence
[0258] In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207.
[0259] Again, preferred combinations of CDR sequences will become clear from the further description herein.
[0260] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to hCXCR4; and more in particular bind to hCXCR4 with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0261] In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.
[0262] In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against human CXCR4 able to block CXCL12/SDF1-dependent activation of human CXCR4 (such as a NANOBODY of the invention, as further described herein), that comprises one or more stretches of amino acid residues chosen from the group consisting of:
[0263] a) the amino acid sequences of SEQ ID NO's: 142 to 143;
[0264] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
[0265] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
[0266] d) the amino acid sequences of SEQ ID NO's: 174 to 175;
[0267] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
[0268] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
[0269] g) the amino acid sequences of SEQ ID NO's: 206 to 207;
[0270] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207;
[0271] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207;
or any suitable combination thereof.
[0272] When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):
[0273] i) any amino acid substitution in such an amino acid sequence according to b) and/or c) is preferably, and compared to the corresponding amino acid sequence according to a), a conservative amino acid substitution, (as defined herein);
and/or
[0274] ii) the amino acid sequence according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to a);
and/or
[0275] iii) the amino acid sequence according to b) and/or c) may be an amino acid sequence that is derived from an amino acid sequence according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0276] Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):
[0277] i) any amino acid substitution in such an amino acid sequence according to e) and/or f) is preferably, and compared to the corresponding amino acid sequence according to d), a conservative amino acid substitution, (as defined herein);
and/or
[0278] ii) the amino acid sequence according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to d); and/or
[0279] iii) the amino acid sequence according to e) and/or f) may be an amino acid sequence that is derived from an amino acid sequence according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0280] Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):
[0281] i) any amino acid substitution in such an amino acid sequence according to h) and/or i) is preferably, and compared to the corresponding amino acid sequence according to g), a conservative amino acid substitution, (as defined herein);
and/or
[0282] ii) the amino acid sequence according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to g);
and/or
[0283] iii) the amino acid sequence according to h) and/or i) may be an amino acid sequence that is derived from an amino acid sequence according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0284] It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.
[0285] In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:
[0286] a) the amino acid sequences of SEQ ID NO's: 142 to 143;
[0287] b) the amino acid sequences of SEQ ID NO's: 174 to 175; and
[0288] c) the amino acid sequences of SEQ ID NO's: 206 to 207;
or any suitable combination thereof.
[0289] Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against HUMAN CXCR4.
[0290] In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against human CXCR4, that comprises two or more stretches of amino acid residues chosen from the group consisting of:
[0291] a) the amino acid sequences of SEQ ID NO's: 142 to 143;
[0292] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
[0293] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
[0294] d) the amino acid sequences of SEQ ID NO's: 174 to 175;
[0295] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
[0296] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
[0297] g) the amino acid sequences of SEQ ID NO's: 206 to 207;
[0298] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207;
[0299] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207;
such that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b) or c), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e), f), g), h) or i); (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), g), h) or i); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to g), h) or i), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), d), e) or f).
[0300] In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:
[0301] a) the amino acid sequences of SEQ ID NO's: 142 to 143;
[0302] b) the amino acid sequences of SEQ ID NO's: 174 to 175; and
[0303] c) the amino acid sequences of SEQ ID NO's: 206 to 207; such that, (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 142 to 143, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 174 to 175 or of SEQ ID NO's: 206 to 207; (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 174 to 175, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 142 to 143 or of SEQ ID NO's: 206 to 207; or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 206 to 207, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 142 to 143 or of SEQ ID NO's: 174 to 175.
[0304] Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against human CXCR4.
[0305] In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against human CXCR4, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:
[0306] a) the amino acid sequences of SEQ ID NO's: 142 to 143;
[0307] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
[0308] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143; the second stretch of amino acid residues is chosen from the group consisting of:
[0309] d) the amino acid sequences of SEQ ID NO's: 174 to 175;
[0310] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
[0311] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
and the third stretch of amino acid residues is chosen from the group consisting of:
[0312] g) the amino acid sequences of SEQ ID NO's: 206 to 207;
[0313] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207;
[0314] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207.
[0315] Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 142 to 143; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 174 to 175; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 206 to 207.
[0316] Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against human CXCR4.
[0317] Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.
[0318] Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 238 to 239. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 238 to 239, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.
[0319] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to human CXCR4; and more in particular bind to human CXCR4 with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0320] When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:
[0321] CDR1 is chosen from the group consisting of:
[0322] a) the amino acid sequences of SEQ ID NO's: 142 to 143;
[0323] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
[0324] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
and/or
[0325] CDR2 is chosen from the group consisting of:
[0326] d) the amino acid sequences of SEQ ID NO's: 174 to 175;
[0327] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
[0328] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
and/or
[0329] CDR3 is chosen from the group consisting of:
[0330] g) the amino acid sequences of SEQ ID NO's: 206 to 207;
[0331] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207;
[0332] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207.
[0333] In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 142 to 143; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 174 to 175; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 206 to 207.
[0334] In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:
[0335] CDR1 is chosen from the group consisting of:
[0336] a) the amino acid sequences of SEQ ID NO's: 142 to 143;
[0337] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
[0338] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 143;
and
[0339] CDR2 is chosen from the group consisting of:
[0340] d) the amino acid sequences of SEQ ID NO's: 174 to 175;
[0341] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
[0342] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 175;
and
[0343] CDR3 is chosen from the group consisting of:
[0344] g) the amino acid sequences of SEQ ID NO's: 206 to 207;
[0345] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207;
[0346] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 207; or any suitable fragment of such an amino acid sequence
[0347] In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 142 to 143; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 174 to 175; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 206 to 207.
[0348] Again, preferred combinations of CDR sequences will become clear from the further description herein.
[0349] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to human CXCR4; and more in particular bind to human CXCR4 with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0350] In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 238 to 239. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 238 to 239, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.
[0351] Thus, in another specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against hCD4, that comprises one or more stretches of amino acid residues chosen from the group consisting of:
[0352] a) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0353] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0354] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0355] d) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0356] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0357] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0358] g) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0359] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0360] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
or any suitable combination thereof.
[0361] When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):
[0362] i) any amino acid substitution in such an amino acid sequence according to b) and/or c) is preferably, and compared to the corresponding amino acid sequence according to a), a conservative amino acid substitution, (as defined herein);
and/or
[0363] ii) the amino acid sequence according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to a);
and/or
[0364] iii) the amino acid sequence according to b) and/or c) may be an amino acid sequence that is derived from an amino acid sequence according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0365] Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):
[0366] i) any amino acid substitution in such an amino acid sequence according to e) and/or f) is preferably, and compared to the corresponding amino acid sequence according to d), a conservative amino acid substitution, (as defined herein);
and/or
[0367] ii) the amino acid sequence according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to d);
and/or
[0368] iii) the amino acid sequence according to e) and/or f) may be an amino acid sequence that is derived from an amino acid sequence according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0369] Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):
[0370] i) any amino acid substitution in such an amino acid sequence according to h) and/or i) is preferably, and compared to the corresponding amino acid sequence according to g), a conservative amino acid substitution, (as defined herein);
and/or
[0371] ii) the amino acid sequence according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to g);
and/or
[0372] iii) the amino acid sequence according to h) and/or i) may be an amino acid sequence that is derived from an amino acid sequence according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0373] It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.
[0374] In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:
[0375] i) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0376] ii) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295; and
[0377] iii) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303; or any suitable combination thereof.
[0378] Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against hCD4.
[0379] In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against hCD4 that comprises two or more stretches of amino acid residues chosen from the group consisting of:
[0380] a) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0381] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0382] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0383] d) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0384] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0385] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0386] g) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0387] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0388] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
such that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b) or c), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e), f), g), h) or i); (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), g), h) or i); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to g), h) or i), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), d), e) or f).
[0389] In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:
[0390] i) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0391] ii) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295; and
[0392] iii) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
such that, (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295 or of SEQ ID NO's: 300 to 303, more preferably 303; (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287 or of SEQ ID NO's: 300 to 303, more preferably 303; or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303, the second stretch of amino acid residues corresponds to one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287 or of SEQ ID NO's: 292 to 295, more preferably 295.
[0393] Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against hCD4.
[0394] In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against hCD4, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:
[0395] a) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0396] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0397] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
the second stretch of amino acid residues is chosen from the group consisting of:
[0398] d) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0399] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0400] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
and the third stretch of amino acid residues is chosen from the group consisting of:
[0401] g) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0402] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0403] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303.
[0404] Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303.
[0405] Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against hCD4.
[0406] Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.
[0407] Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.
[0408] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to hCD4; and more in particular bind to hCD4 with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0409] When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:
[0410] CDR1 is chosen from the group consisting of:
[0411] a) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0412] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0413] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
and/or
[0414] CDR2 is chosen from the group consisting of:
[0415] d) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0416] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0417] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
and/or
[0418] CDR3 is chosen from the group consisting of:
[0419] g) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0420] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0421] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303.
[0422] In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303.
[0423] In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:
[0424] CDR1 is chosen from the group consisting of:
[0425] a) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0426] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0427] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
and
[0428] CDR2 is chosen from the group consisting of:
[0429] d) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0430] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0431] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
and
[0432] CDR3 is chosen from the group consisting of:
[0433] g) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0434] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0435] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303; or any suitable fragment of such an amino acid sequence
[0436] In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303.
[0437] Again, preferred combinations of CDR sequences will become clear from the further description herein.
[0438] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to hCD4; and more in particular bind to hCD4 with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0439] In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.
[0440] In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against human CD4 able to block gp120 binding to CD4 (such as a NANOBODY of the invention, as further described herein), that comprises one or more stretches of amino acid residues chosen from the group consisting of:
[0441] a) the amino acid sequence of SEQ ID NO's: 287;
[0442] b) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 287;
[0443] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 287;
[0444] d) the amino acid sequence of SEQ ID NO: 295;
[0445] e) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 295;
[0446] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 295;
[0447] g) the amino acid sequence of SEQ ID NO: 303;
[0448] h) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 303;
[0449] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 303; or any suitable combination thereof.
[0450] When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):
[0451] i) any amino acid substitution in such an amino acid sequence according to b) and/or c) is preferably, and compared to the corresponding amino acid sequence according to a), a conservative amino acid substitution, (as defined herein);
and/or
[0452] ii) the amino acid sequence according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to a);
and/or
[0453] iii) the amino acid sequence according to b) and/or c) may be an amino acid sequence that is derived from an amino acid sequence according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0454] Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):
[0455] i) any amino acid substitution in such an amino acid sequence according to e) and/or f) is preferably, and compared to the corresponding amino acid sequence according to d), a conservative amino acid substitution, (as defined herein);
and/or
[0456] ii) the amino acid sequence according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to d);
and/or
[0457] iii) the amino acid sequence according to e) and/or f) may be an amino acid sequence that is derived from an amino acid sequence according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0458] Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):
[0459] i) any amino acid substitution in such an amino acid sequence according to h) and/or i) is preferably, and compared to the corresponding amino acid sequence according to g), a conservative amino acid substitution, (as defined herein);
and/or
[0460] ii) the amino acid sequence according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding amino acid sequence according to g);
and/or
[0461] iii) the amino acid sequence according to h) and/or i) may be an amino acid sequence that is derived from an amino acid sequence according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0462] It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.
[0463] In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:
[0464] a) the amino acid sequence of SEQ ID NO: 287;
[0465] b) the amino acid sequence of SEQ ID NO: 295; and
[0466] c) the amino acid sequence of SEQ ID NO: 303; or any suitable combination thereof.
[0467] Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against hCD4.
[0468] In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against human CD4, that comprises two or more stretches of amino acid residues chosen from the group consisting of:
[0469] a) the amino acid sequence of SEQ ID NO: 287;
[0470] b) amino acid sequence that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 287;
[0471] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 287;
[0472] d) the amino acid sequences of SEQ ID NO's: 295;
[0473] e) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 295;
[0474] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 295;
[0475] g) the amino acid sequences of SEQ ID NO: 303;
[0476] h) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 303;
[0477] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 303;
such that (i) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b) or c), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e), f), g), h) or i); (ii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to d), e) or f), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), g), h) or i); or (iii) when the first stretch of amino acid residues corresponds to one of the amino acid sequences according to g), h) or i), the second stretch of amino acid residues corresponds to one of the amino acid sequences according to a), b), c), d), e) or f).
[0478] In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:
[0479] d) the amino acid sequence of SEQ ID NO: 287;
[0480] e) the amino acid sequence of SEQ ID NO: 295; and
[0481] f) the amino acid sequence of SEQ ID NO: 303;
such that, (i) when the first stretch of amino acid residues corresponds to the amino acid sequence of SEQ ID NO: 287, the second stretch of amino acid residues corresponds to the amino acid sequence of SEQ ID NO: 295 or of SEQ ID NO: 303; (ii) when the first stretch of amino acid residues corresponds to the amino acid sequence of SEQ ID NO: 295, the second stretch of amino acid residues corresponds to the amino acid sequence of SEQ ID NO: 287 or of SEQ ID NO: 303; or (iii) when the first stretch of amino acid residues corresponds to the amino acid sequence of SEQ ID NO: 303, the second stretch of amino acid residues corresponds to the amino acid sequence of SEQ ID NO: 287 or of SEQ ID NO: 295.
[0482] Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against human CD4.
[0483] In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against human CD4, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:
[0484] a) the amino acid sequence of SEQ ID NO: 287;
[0485] b) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 287;
[0486] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 287;
the second stretch of amino acid residues is chosen from the group consisting of:
[0487] d) the amino acid sequence of SEQ ID NO: 295;
[0488] e) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 295;
[0489] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 295;
and the third stretch of amino acid residues is chosen from the group consisting of:
[0490] g) the amino acid sequence of SEQ ID NO: 303;
[0491] h) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 303;
[0492] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 303.
[0493] Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the amino acid sequence of SEQ ID NO: 287; the second stretch of amino acid residues is chosen from the amino acid sequence of SEQ ID NO: 295; and the third stretch of amino acid residues is chosen from the amino acid sequence of SEQ ID NO: 303.
[0494] Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against human CD4.
[0495] Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.
[0496] Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least the amino acid sequence of SEQ ID NO: 311. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and the sequence of SEQ ID NO: 311, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.
[0497] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to human CD4; and more in particular bind to human CD4 with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0498] When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:
[0499] CDR1 is chosen from the group consisting of:
[0500] a) the amino acid sequence of SEQ ID NO: 287;
[0501] b) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 287;
[0502] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 287;
and/or
[0503] CDR2 is chosen from the group consisting of:
[0504] d) the amino acid sequence of SEQ ID NO: 295;
[0505] e) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 295;
[0506] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 295;
and/or
[0507] CDR3 is chosen from the group consisting of:
[0508] g) the amino acid sequence of SEQ ID NO: 303;
[0509] h) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 303;
[0510] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 303.
[0511] In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the amino acid sequence of SEQ ID NO: 287; and/or CDR2 is chosen from the amino acid sequence of SEQ ID NO: 295; and/or CDR3 is chosen from the amino acid sequence of SEQ ID NO: 303.
[0512] In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions
[0513] (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:
[0514] CDR1 is chosen from the group consisting of:
[0515] a) the amino acid sequence of SEQ ID NO: 287;
[0516] b) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 287;
[0517] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 287;
and
[0518] CDR2 is chosen from the group consisting of:
[0519] d) the amino acid sequence of SEQ ID NO: 295;
[0520] e) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 295;
[0521] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 295;
and
[0522] CDR3 is chosen from the group consisting of:
[0523] g) the amino acid sequence of SEQ ID NO: 303;
[0524] h) amino acid sequences that have at least 80% amino acid identity with at least the amino acid sequence of SEQ ID NO: 303;
[0525] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least the amino acid sequence of SEQ ID NO: 303; or any suitable fragment of such an amino acid sequence
[0526] In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the amino acid sequence of SEQ ID NO: 287; and CDR2 is chosen from the amino acid sequence of SEQ ID NO: 295; and CDR3 is chosen from the amino acid sequence of SEQ ID NO: 303.
[0527] Again, preferred combinations of CDR sequences will become clear from the further description herein.
[0528] Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to human CD4; and more in particular bind to human CD4 with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0529] In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least the amino acid sequence of SEQ ID NO: 311. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and the sequence of SEQ ID NO: 311, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.
[0530] In such an amino acid sequence of the invention, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.
[0531] The framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a VL-sequence) and/or from a heavy chain variable domain (e.g. a VH-sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a VHH-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized (as defined herein). The framework sequences are preferably such that the amino acid sequence of the invention is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); is a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody); is a “dAb” (or an amino acid sequence that is suitable for use as a dAb); or is a NANOBODY (including but not limited to VHH sequence). Again, suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.
[0532] In particular, the framework sequences present in the amino acid sequences of the invention may contain one or more of Hallmark residues (as defined herein), such that the amino acid sequence of the invention is a NANOBODY. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.
[0533] Again, as generally described herein for the amino acid sequences of the invention, it is also possible to use suitable fragments (or combinations of fragments) of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (for example, in the same order as these CDR's and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived). Such fragments may also again be such that they comprise or can form an immunoglobulin fold, or alternatively be such that they do not comprise or cannot form an immunoglobulin fold.
[0534] In one specific aspect, such a fragment comprises a single CDR sequence as described herein (and in particular a CDR3 sequence), that is flanked on each side by (part of) a framework sequence (and in particular, part of the framework sequence(s) that, in the immunoglobulin sequence from which the fragment is derived, are adjacent to said CDR sequence. For example, a CDR3 sequence may be preceded by (part of) a FR3 sequence and followed by (part of) a FR4 sequence). Such a fragment may also contain a disulphide bridge, and in particular a disulphide bridge that links the two framework regions that precede and follow the CDR sequence, respectively (for the purpose of forming such a disulphide bridge, cysteine residues that naturally occur in said framework regions may be used, or alternatively cysteine residues may be synthetically added to or introduced into said framework regions). For a further description of these “Expedite fragments”, reference is again made to WO 03/050531, as well as to as well as to WO 08/068,280.
[0535] In another aspect, the invention relates to a compound or construct, and in particular a protein or polypeptide (also referred to herein as a “compound of the invention” or “polypeptide of the invention”, respectively) that comprises or essentially consists of one or more amino acid sequences of the invention (or suitable fragments thereof), and optionally further comprises one or more other groups, residues, moieties or binding units. As will become clear to the skilled person from the further disclosure herein, such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the amino acid sequence of the invention (and/or to the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence of the invention.
[0536] For example, such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, “dAb”'s, amino acid sequences that are suitable for use as a dAb, or NANOBODIES.
[0537] Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more amino acid sequences of the invention so as to provide a “derivative” of an amino acid sequence or polypeptide of the invention, as further described herein.
[0538] The polypeptides of the invention comprise or essentially consist of at least one NANOBODY of the invention. Some preferred, but non-limiting examples of polypeptides of the invention are given in SEQ ID NO's: 261 to 264, more preferably SEQ ID NO's 263 to 264.
[0539] 
[00003] [TABLE-US-00003]
  TABLE 2
 
  Preferred polypeptide or compound sequences (also referred herein as a
  sequence with a particular name or SEQ ID NO: X, wherein X is a number 
  referring to the relevant amino acid sequence):
  Amino Acid Sequence   Clone name   SEQ ID NO:
 
  EVQLVESGGGLVQTGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL   238D2-10GS-   261
  EWVSGIKSSGDSTRYAGSVKGRFTISRDNAKNMLYLQMYSLKPED   238D2  
  TAVYYCAKSRVSRTGLYTYDNRGQGTQVTVSSGGGGSGGGGSEVQ    
  LVESGGGLVQTGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV    
  SGIKSSGDSTRYAGSVKGRFTISRDNAKNMLYLQMYSLKPEDTAV    
  YYCAKSRVSRTGLYTYDNRGQGTQVTVSS    
 
  EVQLMESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRAPGKER   238D4-20GS-   262
  EFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSL   238D4  
  KPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSSGGGGSGG    
  GGSGGGGSGGGGSEVQLMESGGGLVQAGGSLRLSCAASGRTFNNY    
  AMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRD    
  NAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGT    
  QVTVSS    
 
  EVQLVESGGGLVQTGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL   238D2-15GS-   263
  EWVSGIKSSGDSTRYAGSVKGRFTISRDNAKNMLYLQMYSLKPED   238D4  
  TAVYYCAKSRVSRTGLYTYDNRGQGTQVTVSSGGGGSGGGGSGGG    
  GSEVQLMESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRRAPGK    
  EREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMN    
  SLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSS    
 
  EVQLVESGGGLVQTGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL   238D2-20GS   264
  EWVSGIKSSGDSTRYAGSVKGRFTISRDNAKNMLYLQMYSLKPED   238D4  
  TAVYYCAKSRVSRTGLYTYDNRGQGTQVTVSSGGGGSGGGGSGGG    
  GSGGGGSEVQLMESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFR    
  RAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTL    
  YLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGTQVTVSS
 
[0540] Also within the scope of the present invention are compounds or constructs, that comprises or essentially consists of one or more derivatives as described herein, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are amino acid sequences.
[0541] In the compounds or constructs described above, the one or more amino acid sequences of the invention and the one or more groups, residues, moieties or binding units may be linked directly to each other and/or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be amino acid sequences, so that the resulting compound or construct is a fusion (protein) or fusion (polypeptide).
[0542] The compounds or polypeptides of the invention can generally be prepared by a method which comprises at least one step of suitably linking the one or more amino acid sequences of the invention to the one or more further groups, residues, moieties or binding units, optionally via the one or more suitable linkers, so as to provide the compound or polypeptide of the invention. Polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.
[0543] The process of designing/selecting and/or preparing a compound or polypeptide of the invention, starting from an amino acid sequence of the invention, is also referred to herein as “formatting” said amino acid sequence of the invention; and an amino acid of the invention that is made part of a compound or polypeptide of the invention is said to be “formatted” or to be “in the format of” said compound or polypeptide of the invention. Examples of ways in which an amino acid sequence of the invention can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted amino acid sequences form a further aspect of the invention.
[0544] In one specific aspect of the invention, a compound of the invention or a polypeptide of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin; see for example EP 0 368 684 B1, page 4); or polypeptides of the invention that comprise at least one amino acid sequence of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the amino acid sequence of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, “dAb”'s, amino acid sequences that are suitable for use as a dAb, or NANOBODIES that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin; reference is made to the further description and references mentioned herein); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to WO 08/068,280.
[0545] Generally, the compounds or polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the compounds or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.
[0546] In a preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention have a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.
[0547] In another preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).
[0548] In another aspect, the invention relates to a nucleic acid that encodes an amino acid sequence of the invention or a polypeptide of the invention (or a suitable fragment thereof). Such a nucleic acid will also be referred to herein as a “nucleic acid of the invention” and may for example be in the form of a genetic construct, as further described herein.
[0549] In another aspect, the invention relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) an amino acid sequence of the invention and/or a polypeptide of the invention; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.
[0550] The invention further relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention (or a suitable fragment thereof) and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.
[0551] The invention also relates to the use of an amino acid sequence, NANOBODY or polypeptide of the invention, or of a composition comprising the same, in (methods or compositions for) modulating a Target of the invention, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from Targets of the invention related disease or disorder).
[0552] The invention also relates to methods for modulating a Target of the invention, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from Targets of the invention related disease or disorder), which method comprises at least the step of contacting Targets of the invention with at least one amino acid sequence, NANOBODY or polypeptide of the invention, or with a composition comprising the same, in a manner and in an amount suitable to modulate Targets of the invention, with at least one amino acid sequence, NANOBODY or polypeptide of the invention.
[0553] The invention also relates to the use of an one amino acid sequence, NANOBODY or polypeptide of the invention in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating Targets of the invention, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a Targets of the invention related disease or disorder).
[0554] In the context of the present invention, “modulating” or “to modulate” generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, Targets of the invention, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein). In particular, “modulating” or “to modulate” may mean either reducing or inhibiting the activity of, or alternatively increasing the activity of, Targets of the invention, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of Targets of the invention in the same assay under the same conditions but without the presence of the amino acid sequence, NANOBODY or polypeptide of the invention.
[0555] As will be clear to the skilled person, “modulating” may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of Targets of the invention for one or more of its targets, ligands or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of a Target of the invention for one or more conditions in the medium or surroundings in which the Target of the invention is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the amino acid sequence, NANOBODY or polypeptide of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, such as the assays described herein or in the prior art cited herein.
[0556] “Modulating” may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the Target of the invention and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which a Target of the invention (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, such as the assays described herein or in the prior art cited herein. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the amino acid sequence, NANOBODY or polypeptide of the invention.
[0557] Modulating may for example involve allosteric modulation (see for example George et al., Nat Rev Drug Discov 1:808-820 (2002); Kenakin, Trends Pharmacol Sci 25:186-192 (2002) and Rios et al., Pharmacol Ther 92:71-87 (2001)) and/or reducing or inhibiting the binding of a Target of the invention to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to a Target of the invention. Modulating may also involve activating a Target of the invention or the mechanism or pathway in which it is involved. Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.
[0558] The invention further relates to methods for preparing or generating the amino acid sequences, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.
[0559] Generally, these methods may comprise the steps of:
[0560] a) providing a set, collection or library of amino acid sequences; and
[0561] b) screening said set, collection or library of amino acid sequences for amino acid sequences that can bind to and/or have affinity for Targets of the invention; and
[0562] c) isolating the amino acid sequence(s) that can bind to and/or have affinity for Targets of the invention.
[0563] In particular, in step b) of such a method, the set, collection or library may be screened for amino acid sequences that can bind to and/or have affinity for Targets of the invention that are expressed on the surface of a suitable cell; for amino acid sequences that can bind to and/or have affinity for an extracellular part, region, domain, loop or other extracellular epitope of a Target of the invention (as described herein); and/or for amino acid sequences that can bind to and/or have affinity for a peptide that has been derived from or based on the amino acid sequence of an extracellular part, region, domain, loop or other extracellular epitope of a Target of the invention. This can be performed using methods and techniques known per se, for example those mentioned herein.
[0564] In such a method, the set, collection or library of amino acid sequences may be any suitable set, collection or library of amino acid sequences. For example, the set, collection or library of amino acid sequences may be a set, collection or library of immunoglobulin sequences (as described herein), such as a naïve set, collection or library of immunoglobulin sequences; a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.
[0565] Also, in such a method, the set, collection or library of amino acid sequences may be a set, collection or library of heavy chain variable domains (such as VH domains or VHH domains) or of light chain variable domains. For example, the set, collection or library of amino acid sequences may be a set, collection or library of domain antibodies or single domain antibodies, or may be a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.
[0566] In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of immunoglobulin sequences, for example derived from a mammal that has been suitably immunized with Targets of the invention or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s), or a suitable peptide derived therefrom. Alternatively, as mentioned herein, the set, collection or library of amino acid sequences may be an immune set, collection or library of immunoglobulin sequences, for example derived from a mammal that has been suitably immunized with a refolded Target of the invention or with a cell, or cell fraction or preparation derived from a cell that has a Target of the invention on its surface.
[0567] In the above methods, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[0568] In another aspect, the method for generating amino acid sequences comprises at least the steps of:
[0569] a) providing a collection or sample of cells expressing amino acid sequences;
[0570] b) screening said collection or sample of cells for cells that express an amino acid sequence that can bind to and/or have affinity for Targets of the invention;
and
[0571] c) either (i) isolating said amino acid sequence; or (ii) isolating from said cell a nucleic acid sequence that encodes said amino acid sequence, followed by expressing said amino acid sequence.
[0572] In particular, in step b) of such a method, the set, collection or library may be screened for cells that express amino acid sequences that can bind to and/or have affinity for Targets of the invention that are expressed on the surface of a suitable cell; for cells that express amino acid sequences that can bind to and/or have affinity for an extracellular part, region, domain, loop or other extracellular epitope of a Target of the invention (as described herein); and/or for cells that express amino acid sequences that can bind to and/or have affinity for a peptide that has been derived from or based on the amino acid sequence of an extracellular part, region, domain, loop or other extracellular epitope of a Target of the invention. This can be performed using methods and techniques known per se, for example those mentioned herein.
[0573] For example, when the desired amino acid sequence is an immunoglobulin sequence, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a mammal that has been suitably immunized with Targets of the invention or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s), or a suitable peptide derived therefrom. Alternatively, as mentioned herein, the sample of cells may be derived from a mammal that has been suitably immunized with a refolded Target of the invention or with a cell, or cell fraction or preparation derived from a cell that has a Target of the invention on its surface.
[0574] The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820 (2001).
[0575] In another aspect, the method for generating an amino acid sequence directed against Targets of the invention may comprise at least the steps of:
[0576] a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences;
[0577] b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for Targets of the invention;
and
[0578] c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence.
[0579] In particular, in step b) of such a method, the set, collection or library may be screened for nucleotide sequences that encode amino acid sequences that can bind to and/or have affinity for Targets of the invention that are expressed on the surface of a suitable cell; for nucleotide sequences that encode amino acid sequences that can bind to and/or have affinity for an extracellular part, region, domain, loop or other extracellular epitope of a Target of the invention (as described herein); and/or for nucleotide sequences that encode amino acid sequences that can bind to and/or have affinity for a peptide that has been derived from or based on the amino acid sequence of an extracellular part, region, domain, loop or other extracellular epitope of a Target of the invention. This can be performed using methods and techniques known per se, for example those mentioned herein.
[0580] In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.
[0581] Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as VH domains or VHH domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.
[0582] In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with Targets of the invention or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s), or a suitable peptide derived therefrom. Alternatively, as mentioned herein, the set, collection or library of nucleic acid sequences may be an immune set, collection or library derived from a mammal that has been suitably immunized with a Target of the invention or with a cell, or cell fraction or preparation derived from a cell that has a Target of the invention on its surface.
[0583] The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of VHH sequences.
[0584] In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[0585] In another aspect, the method for generating an amino acid sequence directed against hCXCR4 may comprise at least the steps of:
[0586] a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences;
[0587] b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for hCXCR4 and that is cross-blocked or is cross blocking a NANOBODY of the invention, e.g. SEQ ID NO: 238 to 253 (Table 1), or a polypeptide or construct of the invention, e.g. SEQ ID NO: 261-266 (see Table 2); and
[0588] c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence.
[0589] In yet another aspect, the method for generating an amino acid sequence directed against hF4 may comprise at least the steps of:
[0590] a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences;
[0591] b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode an amino acid sequence that can bind to and/or has affinity for hCD4 and that is cross-blocked or is cross blocking a NANOBODY of the invention, e.g. SEQ ID NO: 308 to 311 (Table 3); and
[0592] c) isolating said nucleic acid sequence, followed by expressing said amino acid sequence.
[0593] The invention also relates to amino acid sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said immunoglobulin sequence; and of expressing or synthesizing said amino acid sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.
[0594] Also, following the steps above, one or more amino acid sequences of the invention may be suitably humanized (or alternatively camelized); and/or the amino acid sequence(s) thus obtained may be linked to each other or to one or more other suitable amino acid sequences (optionally via one or more suitable linkers) so as to provide a polypeptide of the invention. Also, a nucleic acid sequence encoding an amino acid sequence of the invention may be suitably humanized (or alternatively camelized) and suitably expressed; and/or one or more nucleic acid sequences encoding an amino acid sequence of the invention may be linked to each other or to one or more nucleic acid sequences that encode other suitable amino acid sequences (optionally via nucleotide sequences that encode one or more suitable linkers), after which the nucleotide sequence thus obtained may be suitably expressed so as to provide a polypeptide of the invention.
[0595] The invention further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with Targets of the invention. Some preferred but non-limiting applications and uses will become clear from the further description herein.
[0596] The invention also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy.
[0597] In particular, the invention also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of a disease or disorder that can be prevented or treated by administering, to a subject in need thereof, of (a pharmaceutically effective amount of) an amino acid sequence, compound, construct or polypeptide as described herein.
[0598] More in particular, the invention relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of viral and bacterial induced diseases and/or disorders, such as e.g. AIDS, Hepatitis C, Heptatitis B, adenovirus infection, hantavirus infection, herpesvirus infection, echo-virus infection, etc.
[0599] The amino acid sequences, NANOBODIES and polypeptides of the invention may also find use as markers for detecting cells that express the Targets of the invention against which they are directed, for example in vitro (e.g. using Western blot, immunoprecipitation or immunofluorescence techniques) or in vivo (e.g. using suitable imaging techniques). The amino acid sequences, NANOBODIES and polypeptides of the invention may also find use in affinity purification techniques for (cells expressing) the Targets of the invention against which they are directed.
[0600] Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description herein, in which the invention will be described and discussed in more detail with reference to the NANOBODIES of the invention and polypeptides of the invention comprising the same, which form some of the preferred aspects of the invention.
[0601] As will become clear from the further description herein, NANOBODIES generally offer certain advantages (outlined herein) compared to “dAb's” or similar (single) domain antibodies or immunoglobulin sequences, which advantages are also provided by the NANOBODIES of the invention. However, it will be clear to the skilled person that the more general aspects of the teaching below can also be applied (either directly or analogously) to other amino acid sequences of the invention.

FIGURES

[0602] FIG. 1: Schematic representation of the selection procedure. Abbreviations: CHO-CXCR4: membrane of CHO (Chinese Hamster Ovary) cells transiently transfected with human CXCR4; COS7-CXCR4: membrane of COS7 (Monkey cells) cells transiently transfected with human CXCR4; R1 is first round of selection; R2 is the second round of selection; Counterselection means that the selection was done in the presence of CHO-membrane (non expressing CXCR4); ligand is CXCL12/SDF1 (3 ug in 100 ul PBS), antagonist is AMD3100 (50 uM), antibody is 12G5 (5 ug in 100 ul PBS).
[0603] FIG. 2: Results from Phage ELISA (as described in example 1.5), showing that 238C1, 238D2, 238F3 have high specificity toward CXCR4 expressing membrane compared to non-expressing membrane (−).
[0604] FIG. 3: Primary and secondary screen of NANOBODY clones binding to CXCR4. [125I]-CXCL12 competition binding experiments were directly performed with periplasma fractions (1:10) on cell membranes from HEK293T cells transiently expressing CXCR4. All primary hits (FIG. 3A) were confirmed in a second screen (FIG. 3B). Control experiments with AMD3100 (3 μM) or vehicle (−) were performed to show full and no displacement, respectively.
[0605] FIG. 4: Competition binding of monovalent NANOBODIES and reference ligands to CXCR4. FIG. 4A-C) Competition binding experiments with [125I]-CXCL12, [125I]-238D2 or [125I]-238D4 as radioligand were performed on cell membranes from HEK293T cells transiently expressing CXCR4. Control displacement experiments with AMD3100 (3 μM; AMD), CXCL12 (30 nM; XL12) or vehicle (−) were performed. Data are shown as means±S.E.M (n=2-6). FIG. 4D) Total binding (vehicle; −) and competition binding experiments (3 M AMD3100; AMD) with [125I]-238D2 or [125I]-238D4 as radioligand were performed on cell membranes from HEK293T cells transiently expressing CXCR4 or CXCR3. Data are shown as means±S.E.M (n=3).
[0606] FIG. 5: The monovalent antibodies 238D2 and 238D4 are potent CXCR4 antagonists. FIG. 5A) Inositol phosphate (IP) accumulation experiments were performed in HEK293T cells transiently expressing CXCR4 and Gαqi5. Agonism experiments (left graph) show no intrinsic activity for 238D2 and 238D4. Antagonism experiments (right graph) were performed in the presence of CXCL12 (30 nM) following 1 h pre-incubation with 238D2 or 238D4. Control experiments with vehicle (−) or AMD3100 (3 μM; AMD) were performed. Data are shown as means±S.E.M (n=4). FIG. 5B, C) Reporter gene experiments were performed in HEK293T cells transiently transfected with pCRE/β-galactosidase and a plasmid encoding CXCR4. No agonist activity for 238D2 and 238D4 was observed (left graph). Data are shown as means±S.E.M (n=3). Experiments showing competitive antagonism of the CXCL12-induced reporter gene activation were performed by establishing concentration response curves for CXCL12 in the presence of increasing concentrations of 238D2 or 238D4 (right graphs). Schild regression analysis graphs are embedded. Data are shown as means±S.E.M (n=4-6). FIG. 5D) Chemotaxis experiments using ChemoTx™ plates were performed with Jurkat cells endogenously expressing CXCR4. Agonism experiments (left graph) show migration of Jurkat cells from the upper compartment towards CXCL12 but not towards 238D2 and 238D4 in the lower compartment of the chemotaxis plate. Experiments showing the inhibition of migration towards CXCL12 (0.3 nM) were performed in the presence of 238D2 or 238D4 in both compartments. Control experiments with AMD3100 (3 μM; AMD) were performed. Data are shown as means±S.E.M (n=4).
[0607] FIG. 6: Monoclonals were sequenced and grouped based on their similarity to form families (more than 2 sequences). The clones on the same lane are 100% identical. U=unique sequences non related to each others.
[0608] FIG. 7: The monovalent NANOBODIES 238D2 and 238D4 do not act as agonists or antagonists on chemokine CCR5 (FIG. 7c), CCR7 (FIG. 7c), CXCR1 (FIG. 7a), CXCR2 (FIG. 7a), CXCR3 (FIG. 7b), CXCR6 (FIG. 7b), β2 adrenergic (FIG. 7d) and histamine H4 receptors (FIG. 7d). The selectivity screen was performed with two concentrations of 238D2 and 238D4 in the presence of a EC50-EC50 of an agonist and in the absence (for the investigation of β2 adrenoceptors) or the presence of forskolin (3 μM; for the investigation of all other receptors) on HEK293T cells transiently transfected with cDNA encoding the mentioned receptors (or mock for the investigation of endogenously expressed β2 adrenoceptors) using the CRE/β-galactosidase reporter gene assay. Data are shown as means±S.E.M (n=2-3).
[0609] FIG. 8: Bivalent NANOBODIES show increased affinity and inhibitory potency compared to their monovalent counterparts. FIG. 8A) Competition binding experiments with [125I]-CXCL12 were performed on cell membranes from HEK293T cells transiently expressing CXCR4. Control displacement experiments with AMD3100 (3 μM; AMD), CXCL12 (30 nM; XL12) or vehicle (−) were performed. Data are shown as means±S.E.M (n=2-6). FIG. 8B) Chemotaxis experiments using ChemoTx™ plates were performed with Jurkat cells endogenously expressing CXCR4. Experiments showing the inhibition of migration towards CXCL12 (0.3 nM) in the lower compartment were performed in the presence of NANOBODIES in both compartments. Control experiments with AMD3100 (3 μM; AMD) were performed. Data are shown as means±S.E.M (n=3-4).
[0610] FIG. 9: CD4 binding assay for a selection of clones. Negative controls are addition of irrelevant phage and no phage addition.
[0611] FIG. 10: CD4-gp120 blocking assay for selected NANOBODIES. Monoclonal mAb A1 was used as positive control. Negative control was no addition of NANOBODY.
[0612] FIG. 11: FACS experiment showing the binding of NANOBODIES to Jurkat cells expressing CD4. Negative controls-TOPRO, Unstained, anti mouse-PE, anti myc+anti mouse-PE. Positive control-Monoclonal anti CD4 antibody mAb A1.

DETAILED DESCRIPTION OF THE INVENTION

[0613] In some embodiments, targets of the invention are human cellular receptors for viruses and/or bacteria including hCD4, hCXCR4, hCCR5, hTLR4, human alphaV integrin, human beta3 integrin, human beta1 integrin, human alpha2 integrin, hCD81, hSR-BI, hClaudin-1, hClaudin-6 and hClaudin-9.
[0614] Viruses and bacteria target a variety of human cellular receptors. These cellular receptors include receptors that are part of the immunoglobulin superfamily, such as CD4, which is a co-receptor to the T-cell receptor, and can interact with MHC calls II molecules on the surface of the antigen presenting cell. CD4 is thought to be the primary receptor used by HIV-1 to gain entry into host T cells. Other receptors that are involved in entry of HIV-I are the chemokine receptors CCR5 and CXCR4.
[0615] Targets of the invention also include Toll-like receptors, such as hTLR4. Toll like receptors are a class of single membrane-spanning non-catalytic receptors that recognize structurally conserved molecules derived from viruses and bacteria.
[0616] Targets of the invention also include integrins such as human alphaV integrin, human beta3 integrin, human beta1 integrin and human alpha2 integrin. Integrin receptors are membrane receptors involved in cell adhesion and recognition in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metatastatic diffusion of tumor cells. Integrins can act as cellular targets for viruses, such as HIV-1.
[0617] Targets of the invention also include members of the tetraspan family such as hCD81. The receptors mediate signal transduction events that play a role in the regulation of cell development, activation, growth and motility and are known to complex with integrins.
[0618] Targets of the invention also include scavenger receptors such as hSR-BI. hSR-BI facilitates high density lipoprotein cholesterol transport and correlates with protection against atherosclerosis.
[0619] Targets of the invention also include claudins, such as hClaudin-1, hClaudin-6 and hClaudin-9. Claudins are integral membrane proteins that are components of the tight junction strands and that may play a role in organ development.
[0620] Targets of the invention also include G protein coupled receptors (GPCRs) such as CXCR4 and CCR5. GPCRs are also known as seven transmembrane receptors, 7TM receptors, heptahelical receptors, and G protein linked receptors (GPLR), which are a protein family of transmembrane receptors that transduce an extracellular signal (ligand binding) into an intracellular signal (G protein activation). GPCRs are integral membrane proteins that possess seven membrane-spanning domains or transmembrane helices. The extracellular parts of the receptor can be glycosylated. These extracellular loops also contain two highly conserved cysteine residues which build disulfide bonds to stabilize the receptor structure. An overview of GPCRs can be found in standard handbooks, such as the G Protein Coupled Receptors Handbook, L. Devi (Ed.), Humana Press, 2005; as well as from the standard databases, such as GPCR DB.
[0621] In the present description, examples and aspects:
[0622] a) Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of Gene Manipulation: An Introduction to Genetic Engineering”, 2nd edition, University of California Press, Berkeley, Calif. (1981); Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology” (6th Ed.), Garland Science Publishing/Churchill Livingstone, New York (2005), as well as to the general background art cited herein;
[0623] b) Unless indicated otherwise, the term “immunoglobulin sequence”—whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody—is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH/VL domains, respectively). In addition, the term “sequence” as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation. Also, the term “nucleotide sequence” as used herein also encompasses a nucleic acid molecule with said nucleotide sequence, so that the terms “nucleotide sequence” and “nucleic acid” should be considered equivalent and are used interchangeably herein;
[0624] c) Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2 (1): 49-57; Irving et al., J. Immunol. Methods, 2001, 248 (1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., Tumour Biol., 2005, 26 (1), 31-43, which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.
[0625] d) Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code, as mentioned in Table A-2;
[0626] 
[00004] [TABLE-US-00004]
  TABLE A-2
 
  one-letter and three-letter ammo acid code
 
 
    Nonpolar,   Alanine   Ala   A
    uncharged   Valine   Val   V
  (at pH 6.0-7.0)(3)   Leucine   Leu   L
      Isoleucine   Ile   I
      Phenylalanine   Phe   F
    Methionine(1)   Met   M
      Tryptophan   Trp   W
      Proline   Pro   P
    Polar,Glycine(2)   Gly   G
    uncharged   Serine   Ser   S
    (at pH 6.0-7.0)   Threonine   Thr   T
      Cysteine   Cys   C
      Asparagine   Asn   N
      Glutamine   Gln   Q
      Tyrosine   Tyr   Y
    Polar,   Lysine   Lys   K
    charged   Arginine   Arg   R
    (at pH 6.0-7.0)Histidine(4)   His   H
      Aspartate   Asp   D
      Glutamate   Glu   E
 
  Notes:
(1)Sometimes also considered to be a polar uncharged amino acid.
(2)Sometimes also considered to be a nonpolar uncharged amino acid.
(3)As will be clear to the skilled person, the fact that an amino acid residue is referred to in this Table as being either charged or uncharged at pH 6.0 to 7.0 does not reflect in any way on the charge said amino acid residue may have at a pH lower than 6.0 and/or at a pH higher than 7.0; the amino acid residues mentioned in the Table can be either charged and/or uncharged at such a higher or lower pH, as will be clear to the skilled person.
(4)As is known in the art, the charge of a His residue is greatly dependant upon even small shifts in pH, but a His residue can generally be considered essentially uncharged at a pH of about 6.5.
[0627] e) For the purposes of comparing two or more nucleotide sequences, the percentage of “sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated by dividing [the number of nucleotides in the first nucleotide sequence that are identical to the nucleotides at the corresponding positions in the second nucleotide sequence] by [the total number of nucleotides in the first nucleotide sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence—compared to the first nucleotide sequence—is considered as a difference at a single nucleotide (position).
[0628] Alternatively, the degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as NCBI Blast v2.0, using standard settings.
[0629] Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.
[0630] Usually, for the purpose of determining the percentage of “sequence identity” between two nucleotide sequences in accordance with the calculation method outlined hereinabove, the nucleotide sequence with the greatest number of nucleotides will be taken as the “first” nucleotide sequence, and the other nucleotide sequence will be taken as the “second” nucleotide sequence;
[0631] f) For the purposes of comparing two or more amino acid sequences, the percentage of “sequence identity” between a first amino acid sequence and a second amino acid sequence (also referred to herein as “amino acid identity”) may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence—compared to the first amino acid sequence—is considered as a difference at a single amino acid residue (position), i.e. as an “amino acid difference” as defined herein.
[0632] Alternatively, the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.
[0633] Usually, for the purpose of determining the percentage of “sequence identity” between two amino acid sequences in accordance with the calculation method outlined hereinabove, the amino acid sequence with the greatest number of amino acid residues will be taken as the “first” amino acid sequence, and the other amino acid sequence will be taken as the “second” amino acid sequence.
[0634] Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called “conservative” amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 as well as WO 98/49185 and from the further references cited therein.
[0635] Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a)-(e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
[0636] Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.
[0637] Any amino acid substitutions applied to the polypeptides described herein may also be based on the analysis of the frequencies of amino acid variations between homologous proteins of different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicity patterns in proteins developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein in their entirety by reference. Information on the primary, secondary and tertiary structure of NANOBODIES is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a VHH domain from a llama is for example given by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information about some of the amino acid residues that in conventional VH domains form the VH/VL interface and potential camelizing substitutions on these positions can be found in the prior art cited above.
[0638] g) Amino acid sequences and nucleic acid sequences are said to be “exactly the same” if they have 100% sequence identity (as defined herein) over their entire length;
[0639] h) When comparing two amino acid sequences, the term “amino acid difference” refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one, two or more such amino acid differences;
[0640] i) When a nucleotide sequence or amino acid sequence is said to “comprise” another nucleotide sequence or amino acid sequence, respectively, or to “essentially consist of” another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein). By means of a non-limiting example, when a NANOBODY of the invention is said to comprise a CDR sequence, this may mean that said CDR sequence has been incorporated into the NANOBODY of the invention, but more usually this generally means that the NANOBODY of the invention contains within its sequence a stretch of amino acid residues with the same amino acid sequence as said CDR sequence, irrespective of how said NANOBODY of the invention has been generated or obtained. It should also be noted that when the latter amino acid sequence has a specific biological or structural function, it preferably has essentially the same, a similar or an equivalent biological or structural function in the first mentioned amino acid sequence (in other words, the first mentioned amino acid sequence is preferably such that the latter sequence is capable of performing essentially the same, a similar or an equivalent biological or structural function). For example, when a NANOBODY of the invention is said to comprise a CDR sequence or framework sequence, respectively, the CDR sequence and framework are preferably capable, in said NANOBODY, of functioning as a CDR sequence or framework sequence, respectively. Also, when a nucleotide sequence is said to comprise another nucleotide sequence, the first mentioned nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g. a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the first mentioned, larger nucleotide sequence).
[0641] j) A nucleic acid sequence or amino acid sequence is considered to be “(in) essentially isolated (form)”—for example, compared to its native biological source and/or the reaction medium or cultivation medium from which it has been obtained—when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a nucleic acid sequence or amino acid sequence is considered “essentially isolated” when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, and up to 1000-fold or more. A nucleic acid sequence or amino acid sequence that is “in essentially isolated form” is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as polyacrylamide-gel electrophoresis;
[0642] k) The term “domain” as used herein generally refers to a globular region of an amino acid sequence (such as an antibody chain, and in particular to a globular region of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region. Usually, such a domain will comprise peptide loops (for example 3 or 4 peptide loops) stabilized, for example, as a sheet or by disulfide bonds. The term “binding domain” refers to such a domain that is directed against an antigenic determinant (as defined herein);
[0643] l) The term “antigenic determinant” refers to the epitope on the antigen recognized by the antigen-binding molecule (such as a NANOBODY or a polypeptide of the invention) and more in particular by the antigen-binding site of said molecule. The terms “antigenic determinant” and “epitope” may also be used interchangeably herein.
[0644] m) An amino acid sequence (such as a NANOBODY, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be “against” or “directed against” said antigenic determinant, epitope, antigen or protein.
[0645] n) The term “specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen-binding protein (such as a NANOBODY or a polypeptide of the invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD). As will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is the measure of the strength of binding between an antigen-binding molecule (such as a NANOBODY or polypeptide of the invention) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins (such as the amino acid sequences, NANOBODIES and/or polypeptides of the invention) will bind to their antigen with a dissociation constant (KD) of 10−5 to 10−12 moles/liter or less, and preferably 10−7 to 10−12 moles/liter or less and more preferably 10−8 to 10−12 moles/liter (i.e. with an association constant (KA) of 105 to 1012 liter/moles or more, and preferably 107 to 1012 liter/moles or more and more preferably 108 to 1012 liter/moles). Any KD value greater than 104 mol/liter (or any KA value lower than 104 M−1) liters/mol is generally considered to indicate non-specific binding. Preferably, a monovalent immunoglobulin sequence of the invention will bind to the desired antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 μM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
[0646] The dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more then 10−4 moles/liter or 10−3 moles/liter (e.g., of 10−2 moles/liter). Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be calculated on the basis of the (actual or apparent) association constant (KA), by means of the relationship [KD=1/KA].
[0647] The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the KD, or dissociation constant, which has units of mol/liter (or M). The affinity can also be expressed as an association constant, KA, which equals 1/KD and has units of (mol/liter)−1 (or M−1). In the present specification, the stability of the interaction between two molecules (such as an amino acid sequence, NANOBODY or polypeptide of the invention and its intended target) will mainly be expressed in terms of the KD value of their interaction; it being clear to the skilled person that in view of the relation KA=1/KD, specifying the strength of molecular interaction by its KD value can also be used to calculate the corresponding KA value. The KD-value characterizes the strength of a molecular interaction also in a thermodynamic sense as it is related to the free energy (DG) of binding by the well known relation DG=RT·ln(KD) (equivalently DG=−RT·ln(KA)), where R equals the gas constant, T equals the absolute temperature and ln denotes the natural logarithm.
[0648] The KD for biological interactions which are considered meaningful (e.g. specific) are typically in the range of 10−10M (0.1 nM) to 10−5 M (10000 nM). The stronger an interaction is, the lower is its KD.
[0649] The KD can also be expressed as the ratio of the dissociation rate constant of a complex, denoted as koff, to the rate of its association, denoted kon (so that KD=koff/kon and KA=kon/koff). The off-rate koff has units s−1 (where s is the SI unit notation of second). The on-rate kon has units M−1s−1. The on-rate may vary between 102 M−1s−1 to about 107 M−1s−1, approaching the diffusion-limited association rate constant for bimolecular interactions. The off-rate is related to the half-life of a given molecular interaction by the relation t1/2=ln(2)/koff. The off-rate may vary between 10−6 s−1 (near irreversible complex with a t1/2 of multiple days) to 1 s−1 (t1/2=0.69 s).
[0650] The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al., Intern. Immunology, 13, 1551-1559, 2001) where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding kon, koff measurements and hence KD (or KA) values. This can for example be performed using the well-known BIACORE instruments.
[0651] It will also be clear to the skilled person that the measured KD may correspond to the apparent KD if the measuring process somehow influences the intrinsic binding affinity of the implied molecules for example by artefacts related to the coating on the biosensor of one molecule. Also, an apparent KD may be measured if one molecule contains more than one recognition sites for the other molecule. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules.
[0652] Another approach that may be used to assess affinity is the 2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This method establishes a solution phase binding equilibrium measurement and avoids possible artefacts relating to adsorption of one of the molecules on a support such as plastic.
[0653] However, the accurate measurement of KD may be quite labor-intensive and as consequence, often apparent KD values are determined to assess the binding strength of two molecules. It should be noted that as long all measurements are made in a consistent way (e.g. keeping the assay conditions unchanged) apparent KD measurements can be used as an approximation of the true KD and hence in the present document KD and apparent KD should be treated with equal importance or relevance. Finally, it should be noted that in many situations the experienced scientist may judge it to be convenient to determine the binding affinity relative to some reference molecule. For example, to assess the binding strength between molecules A and B, one may e.g. use a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated ELISA detection). Typically, the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B. As a result an IC50 value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved. Provided KD ref, the KD of the reference molecule, is known, as well as the total concentration cref of the reference molecule, the apparent KD for the interaction A-B can be obtained from following formula: KD=IC50/(1+cref/KD ref). Note that if cref<<KD ref, KD≈IC50. Provided the measurement of the IC50 is performed in a consistent way (e.g. keeping cref fixed) for the binders that are compared, the strength or stability of a molecular interaction can be assessed by the IC50 and this measurement is judged as equivalent to KD or to apparent KD throughout this text.
[0654] o) The half-life of an amino acid sequence, compound or polypeptide of the invention can generally be defined as the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering to a warm-blooded animal (i.e. to a human or to another suitable mammal, such as a mouse, rabbit, rat, pig, dog or a primate, for example monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) a suitable dose of the amino acid sequence, compound or polypeptide of the invention; collecting blood samples or other samples from said animal; determining the level or concentration of the amino acid sequence, compound or polypeptide of the invention in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence, compound or polypeptide of the invention has been reduced by 50% compared to the initial level upon dosing. Reference is for example made to the Experimental Part below, as well as to the standard handbooks, such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).
[0655] As will also be clear to the skilled person (see for example pages 6 and 7 of WO 04/003019 and in the further references cited therein), the half-life can be expressed using parameters such as the t1/2-alpha, t1/2-beta and the area under the curve (AUC). In the present specification, an “increase in half-life” refers to an increase in any one of these parameters, such as any two of these parameters, or essentially all three these parameters. As used herein “increase in half-life” or “increased half-life” in particular refers to an increase in the t1/2-beta, either with or without an increase in the t1/2-alpha and/or the AUC or both.
[0656] p) In the context of the present invention, “modulating” or “to modulate” generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay. In particular, “modulating” or “to modulate” may mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of the target or antigen in the same assay under the same conditions but without the presence of the construct of the invention.
[0657] As will be clear to the skilled person, “modulating” may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention. As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, depending on the target or antigen involved.
[0658] “Modulating” may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, depending on the target or antigen involved. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the construct of the invention.
[0659] Modulating may for example also involve allosteric modulation of the target or antigen; and/or reducing or inhibiting the binding of the target or antigen to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target or antigen. Modulating may also involve activating the target or antigen or the mechanism or pathway in which it is involved. Modulating may for example also involve effecting a change in respect of the folding or confirmation of the target or antigen, or in respect of the ability of the target or antigen to fold, to change its confirmation (for example, upon binding of a ligand), to associate with other (sub)units, or to disassociate.
[0660] Modulating may for example also involve effecting a change in the ability of the target or antigen to transport other compounds or to serve as a channel for other compounds (such as ions).
[0661] Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.
[0662] q) In respect of a target or antigen, the term “interaction site” on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerisation (such as homomerization or heterodimerization) of the target or antigen; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen. More generally, an “interaction site” can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the invention can bind such that the target or antigen (and/or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein).
[0663] r) An amino acid sequence or polypeptide is said to be “specific for” a first target or antigen compared to a second target or antigen when is binds to the first antigen with an affinity (as described above, and suitably expressed as a KD value, KA value, Koff rate and/or Kon rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10,000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to the second target or polypeptide. For example, the first antigen may bind to the target or antigen with a KD value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10.000 times less or even less than that, than the KD with which said amino acid sequence or polypeptide binds to the second target or polypeptide. Preferably, when an amino acid sequence or polypeptide is “specific for” a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen.
[0664] s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are used interchangeably herein to mean the ability of an amino acid sequence or other binding agents (such as a NANOBODY, polypeptide or compound or construct of the invention) to interfere with the binding of other amino acid sequences or binding agents of the invention to a given target. The extend to which an amino acid sequence or other binding agents of the invention is able to interfere with the binding of another to [target], and therefore whether it can be said to cross-block according to the invention, can be determined using competition binding assays. One particularly suitable quantitative cross-blocking assay uses a Biacore machine which can measure the extent of interactions using surface plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequences or other binding agents in terms of their binding to the target.
[0665] The following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross-blocking according to the invention. It will be appreciated that the assay can be used with any of the amino acid sequences or other binding agents described herein. The Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations. Thus in one cross-blocking assay, the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target. Typically 200-800 resonance units of the target would be coupled to the chip (an amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used). Two test amino acid sequences (termed A* and B*) to be assessed for their ability to cross-block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture. When calculating the concentrations on a binding site basis the molecular weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on that amino acid sequence. The concentration of each amino acid sequence in the test mix should be high enough to readily saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip. The amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis). Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix. The test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target. Typically this is done by treating the chip with 30 mM HCl for 60 seconds. The solution of A* alone is then passed over the target-coated surface and the amount of binding recorded. The chip is again treated to remove all of the bound amino acid sequences without damaging the chip-bound target. The solution of B* alone is then passed over the target-coated surface and the amount of binding recorded. The maximum theoretical binding of the mixture of A* and B* is next calculated, and is the sum of the binding of each amino acid sequence when passed over the target surface alone. If the actual recorded binding of the mixture is less than this theoretical maximum then the two amino acid sequences are cross-blocking each other. Thus, in general, a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to the target in the above Biacore cross-blocking assay such that, during the assay and in the presence of a second amino acid sequence or other binding agent of the invention, the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination. The Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version. In this particular format, an anti-His amino acid sequence would be coupled to the Biacore chip and then the His-tagged target would be passed over the surface of the chip and captured by the anti-His amino acid sequence. The cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface. In addition to the example given using N-terminal His-tagged target, C-terminal His-tagged target could alternatively be used. Furthermore, various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies; biotin tag with streptavidin).
[0666] The following generally describes an ELISA assay for determining whether an amino acid sequence or other binding agent directed against a target cross-blocks or is capable of cross-blocking as defined herein. It will be appreciated that the assay can be used with any of the amino acid sequences (or other binding agents such as polypeptides of the invention) described herein. The general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules. The plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target. The amount of bound target is then measured using a reagent that is appropriate to detect the target. An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence. In the instance where the first amino acid sequence, e.g. an Ab-X, is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELISA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of the second amino acid sequence, i.e. Ab-Y, is then added to the ELISA plate such that the moles of Ab-Y target binding sites per well are at least 10 fold higher than the moles of Ab-X target binding sites that were used, per well, during the coating of the ELISA plate. Target is then added such that the moles of target added per well are at least 25-fold lower than the moles of Ab-X target binding sites that were used for coating each well. Following a suitable incubation period the ELISA plate is washed and a reagent for detecting the target is added to measure the amount of target specifically bound by the coated anti-target amino acid sequence (in this case Ab-X). The background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence (in this case Ab-Y), target buffer only (i.e. without target) and target detection reagents. The positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X), second solution phase amino acid sequence buffer only (i.e. without second solution phase amino acid sequence), target and target detection reagents. The ELISA assay may be run in such a manner so as to have the positive control signal be at least 6 times the background signal. To avoid any artefacts (e.g. significantly different affinities between Ab-X and Ab-Y for the target) resulting from the choice of which amino acid sequence to use as the coating amino acid sequence and which to use as the second (competitor) amino acid sequence, the cross-blocking assay may to be run in two formats: 1) format 1 is where Ab-X is the amino acid sequence that is coated onto the ELISA plate and Ab-Y is the competitor amino acid sequence that is in solution and 2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the ELISA plate and Ab-X is the competitor amino acid sequence that is in solution. Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal {i.e. the amount of target bound by the coated amino acid sequence) as compared to the target detection signal obtained in the absence of the solution phase anti-target amino acid sequence (i.e. the positive control wells).
[0667] t) An amino acid sequence is said to be “cross-reactive” for two different antigens or antigenic determinants (such as serum albumin from two different species of mammal, such as human serum albumin and cyno serum albumin) if it is specific for (as defined herein) both these different antigens or antigenic determinants.
[0668] u) By binding that is “essentially independent of the pH” is generally meant herein that the association constant (KA) of the amino acid sequence with respect to the serum protein (such as serum albumin) at the pH value(s) that occur in a cell of an animal or human body (as further described herein) is at least 5%, such as at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 60%, such as even more preferably at least 70%, such as at least 80% or 90% or more (or even more than 100%, such as more than 110%, more than 120% or even 130% or more, or even more than 150%, or even more than 200%) of the association constant (KA) of the amino acid sequence with respect to the same serum protein at the pH value(s) that occur outside said cell. Alternatively, by binding that is “essentially independent of the pH” is generally meant herein that the koff rate (measured by Biacore) of the amino acid sequence with respect to the serum protein (such as serum albumin) at the pH value(s) that occur in a cell of an animal or human body (as e.g. further described herein, e.g. pH around 5.5, e.g. 5.3 to 5.7) is at least 5%, such as at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 60%, such as even more preferably at least 70%, such as at least 80% or 90% or more (or even more than 100%, such as more than 110%, more than 120% or even 130% or more, or even more than 150%, or even more than 200%) of the koff rate of the amino acid sequence with respect to the same serum protein at the pH value(s) that occur outside said cell, e.g. pH 7.2 to 7.4. By “the pH value(s) that occur in a cell of an animal or human body” is meant the pH value(s) that may occur inside a cell, and in particular inside a cell that is involved in the recycling of the serum protein. In particular, by “the pH value(s) that occur in a cell of an animal or human body” is meant the pH value(s) that may occur inside a (sub)cellular compartment or vesicle that is involved in recycling of the serum protein (e.g. as a result of pinocytosis, endocytosis, transcytosis, exocytosis and phagocytosis or a similar mechanism of uptake or internalization into said cell), such as an endosome, lysosome or pinosome.
[0669] v) As further described herein, the total number of amino acid residues in a NANOBODY can be in the region of 110-120, is preferably 112-115, and is most preferably 113. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) of a NANOBODY are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein;
[0670] w) The amino acid residues of a NANOBODY are numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195; or referred to herein. According to this numbering, FR1 of a NANOBODY comprises the amino acid residues at positions 1-30, CDR1 of a NANOBODY comprises the amino acid residues at positions 31-35, FR2 of a NANOBODY comprises the amino acids at positions 36-49, CDR2 of a NANOBODY comprises the amino acid residues at positions 50-65, FR3 of a NANOBODY comprises the amino acid residues at positions 66-94, CDR3 of a NANOBODY comprises the amino acid residues at positions 95-102, and FR4 of a NANOBODY comprises the amino acid residues at positions 103-113. [In this respect, it should be noted that—as is well known in the art for VH domains and for VHH domains—the total number of amino acid residues in each of the CDR's may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDR's, position 1 according to the Kabat numbering corresponds to the start of FR1 and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.]. Alternative methods for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains from Camelids and to NANOBODIES, are the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called “AbM definition” and the so-called “contact definition”. However, in the present description, aspects and figures, the numbering according to Kabat as applied to VHH domains by Riechmann and Muyldermans will be followed, unless indicated otherwise; and
[0671] x) The Figures, Sequence Listing and the Experimental Part/Examples are only given to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or of the appended aspects in any way, unless explicitly indicated otherwise herein.
[0672] For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the prior art cited herein, to the review article by Muyldermans in Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (=EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application WO 06/040153, which list and references are incorporated herein by reference.
[0673] In accordance with the terminology used in the art (see the above references), the variable domains present in naturally occurring heavy chain antibodies will also be referred to as “VHH domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “VL domains”).
[0674] As mentioned in the prior art referred to above, VHH domains have a number of unique structural characteristics and functional properties which make isolated VHH domains (as well as NANOBODIES based thereon, which share these structural characteristics and functional properties with the naturally occurring VHH domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, VHH domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and NANOBODIES can function as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a VH domain covalently linked to a VL domain).
[0675] Because of these unique properties, the use of VHH domains and NANOBODIES as single antigen-binding proteins or as antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional VH and VL domains, scFv's or conventional antibody fragments (such as Fab- or F(ab′)-2-fragments):
[0676] only a single domain is required to bind an antigen with high affinity and with high selectivity, so that there is no need to have two separate domains present, nor to assure that these two domains are present in the right spatial conformation and configuration (i.e. through the use of especially designed linkers, as with scFv's);
[0677] VHH domains and NANOBODIES can be expressed from a single gene and require no post-translational folding or modifications;
[0678] VHH domains and NANOBODIES can easily be engineered into multivalent and multispecific formats (as further discussed herein);
[0679] VHH domains and NANOBODIES are highly soluble and do not have a tendency to aggregate (as with the mouse-derived “dAb's” described by Ward et al., Nature, Vol. 341, 1989, p. 544);
[0680] VHH domains and NANOBODIES are highly stable to heat, pH, proteases and other denaturing agents or conditions (see for example Ewert et al, supra);
[0681] VHH domains and NANOBODIES are easy and relatively cheap to prepare, even on a scale required for production. For example, VHH domains, NANOBODIES and proteins/polypeptides containing the same can be produced using microbial fermentation (e.g. as further described below) and do not require the use of mammalian expression systems, as with for example conventional antibody fragments;
[0682] VHH domains and NANOBODIES are relatively small (approximately 15 kDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and antigen-binding fragments thereof, and therefore show high(er) penetration into tissues (including but not limited to solid tumors and other dense tissues) than such conventional 4-chain antibodies and antigen-binding fragments thereof;
[0683] VHH domains and NANOBODIES can show so-called cavity-binding properties (inter alia due to their extended CDR3 loop, compared to conventional VH domains) and can therefore also access targets and epitopes not accessible to conventional 4-chain antibodies and antigen-binding fragments thereof. For example, it has been shown that VHH domains and NANOBODIES can inhibit enzymes (see for example WO 97/49805; Transue et al., Proteins 1998 Sep. 1; 32(4): 515-22; Lauwereys et al., EMBO J. 1998 Jul. 1; 17(13): 3512-20).
[0684] In a specific and preferred aspect, the invention provides NANOBODIES against Targets of the invention, and in particular NANOBODIES against Targets of the invention from a warm-blooded animal, and more in particular NANOBODIES against Targets of the invention from a mammal, and especially NANOBODIES against human Targets of the invention; as well as proteins and/or polypeptides comprising at least one such NANOBODY.
[0685] In particular, the invention provides NANOBODIES against Targets of the invention, and proteins and/or polypeptides comprising the same, that have improved therapeutic and/or pharmacological properties and/or other advantageous properties (such as, for example, improved ease of preparation and/or reduced costs of goods), compared to conventional antibodies against Targets of the invention or fragments thereof, compared to constructs that could be based on such conventional antibodies or antibody fragments (such as Fab′ fragments, F(ab′)2 fragments, ScFv constructs, “diabodies” and other multispecific constructs (see for example the review by Holliger and Hudson, Nat. Biotechnol. 2005 September; 23(9):1126-36)), and also compared to the so-called “dAb's” or similar (single) domain antibodies that may be derived from variable domains of conventional antibodies. These improved and advantageous properties will become clear from the further description herein, and for example include, without limitation, one or more of:
[0686] increased affinity and/or avidity for Targets of the invention, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described hereinbelow);
[0687] better suitability for formatting in a multivalent format (for example in a bivalent format);
[0688] better suitability for formatting in a multispecific format (for example one of the multispecific formats described hereinbelow);
[0689] improved suitability or susceptibility for “humanizing” substitutions (as defined herein);
[0690] less immunogenicity, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described hereinbelow);
[0691] increased stability, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described hereinbelow);
[0692] increased specificity towards Targets of the invention, either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described hereinbelow);
[0693] decreased or where desired increased cross-reactivity with Targets of the invention from different species;
and/or
[0694] one or more other improved properties desirable for pharmaceutical use (including prophylactic use and/or therapeutic use) and/or for diagnostic use (including but not limited to use for imaging purposes), either in a monovalent format, in a multivalent format (for example in a bivalent format) and/or in a multispecific format (for example one of the multispecific formats described hereinbelow).
[0695] As generally described herein for the amino acid sequences of the invention, the NANOBODIES of the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more NANOBODIES of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than Targets of the invention), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. In particular, such a protein or polypeptide may comprise or essentially consist of one or more NANOBODIES of the invention and optionally one or more (other) NANOBODIES (i.e. directed against other targets than Targets of the invention), all optionally linked via one or more suitable linkers, so as to provide a monovalent, multivalent or multispecific NANOBODY construct, respectively, as further described herein. Such proteins or polypeptides may also be in essentially isolated form (as defined herein).
[0696] In a NANOBODY of the invention, the binding site for binding against Targets of the invention is preferably formed by the CDR sequences. Optionally, a NANOBODY of the invention may also, and in addition to the at least one binding site for binding against Targets of the invention, contain one or more further binding sites for binding against other antigens, proteins or targets. For methods and positions for introducing such second binding sites, reference is for example made to Keck and Huston, Biophysical Journal, 71, October 1996, 2002-2011; EP 0 640 130 and WO 06/07260.
[0697] As generally described herein for the amino acid sequences of the invention, when a NANOBODY of the invention (or a polypeptide of the invention comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably directed against human Targets of the invention; whereas for veterinary purposes, it is preferably directed against Targets of the invention from the species to be treated. Also, as with the amino acid sequences of the invention, a NANOBODY of the invention may or may not be cross-reactive (i.e. directed against Targets of the invention from two or more species of mammal, such as against human Targets of the invention and Targets of the invention from at least one of the species of mammal mentioned herein).
[0698] Also, again as generally described herein for the amino acid sequences of the invention, the NANOBODIES of the invention may generally be directed against any antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of Targets of the invention. However, it is generally assumed and preferred that the NANOBODIES of the invention (and polypeptides comprising the same) are directed against and/or have been raised against at least one extracellular region, domain, loop or other extracellular epitope of one of the Targets of the invention (or a suitable peptide derived therefrom).
[0699] As already described herein, the amino acid sequence and structure of a NANOBODY can be considered—without however being limited thereto—to be comprised of four framework regions or “FR's” (or sometimes also referred to as “FW's”), which are referred to in the art and herein as “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”, respectively; which framework regions are interrupted by three complementary determining regions or “CDR's”, which are referred to in the art as “Complementarity Determining Region 1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3”, respectively. Some preferred framework sequences and CDR's (and combinations thereof) that are present in the NANOBODIES of the invention are as described herein. Other suitable CDR sequences can be obtained by the methods described herein.
[0700] According to a non-limiting but preferred aspect of the invention, (the CDR sequences present in) the NANOBODIES of the invention are such that:
[0701] the NANOBODIES can bind to Targets of the invention with a dissociation constant (KD) of 10−5 to 10−12 moles/liter or less, and preferably 10−7 to 10−12 moles/liter or less and more preferably 10−8 to 10−12 moles/liter (i.e. with an association constant (KA) of 105 to 1012 liter/moles or more, and preferably 107 to 1012 liter/moles or more and more preferably 108 to 1012 liter/moles);
and/or such that:
[0702] the NANOBODIES can bind to Targets of the invention with a kon-rate of between 102 M−1s−1 to about 107 M−1s−1, preferably between 103 M−1s−1 and 107 M−1s−1, more preferably between 104 M−1s−1 and 107 M−1s−1, such as between 105 M−1s−1 and 107 M−1s−1;
and/or such that they:
[0703] the NANOBODIES can bind to Targets of the invention with a koff rate between 1 s−1 (t1/2=0.69 s) and 10−6 s−1 (providing a near irreversible complex with a t1/2 of multiple days), preferably between 10−2 s−1 and 10−6 s−1, more preferably between 10−3 s−1 and 10−6 s−1, such as between 10−4 s−1 and 10−6 s−1.
[0704] Preferably, (the CDR sequences present in) the NANOBODIES of the invention are such that: a monovalent NANOBODY of the invention (or a polypeptide that contains only one NANOBODY of the invention) is preferably such that it will bind to Targets of the invention with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 μM.
[0705] The affinity of the NANOBODY of the invention against Targets of the invention can be determined in a manner known per se, for example using the general techniques for measuring KD. KA, koff or kon mentioned herein, as well as some of the specific assays described herein.
[0706] Some preferred IC50 values for binding of the NANOBODIES of the invention (and of polypeptides comprising the same) to Targets of the invention will become clear from the further description and examples herein.
[0707] In a preferred but non-limiting aspect, the invention relates to a NANOBODY (as defined herein) against hCXCR4, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:
[0708] CDR1 is chosen from the group consisting of:
[0709] a) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0710] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0711] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
and/or
[0712] CDR2 is chosen from the group consisting of:
[0713] d) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0714] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0715] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
and/or
[0716] CDR3 is chosen from the group consisting of:
[0717] g) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0718] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0719] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207; or any suitable fragment of such an amino acid sequence.
[0720] In particular, according to this preferred but non-limiting aspect, the invention relates to a NANOBODY (as defined herein) against human CXCR4, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:
[0721] CDR1 is chosen from the group consisting of:
[0722] a) the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0723] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
[0724] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 142 to 157, more preferably 142 to 143;
and
[0725] CDR2 is chosen from the group consisting of:
[0726] d) the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0727] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
[0728] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 174 to 189, more preferably 174 to 175;
and
[0729] CDR3 is chosen from the group consisting of:
[0730] g) the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0731] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
[0732] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 206 to 221, more preferably 206 to 207;
or any suitable fragment of such an amino acid sequences.
[0733] In a preferred but non-limiting aspect, the invention relates to a NANOBODY (as defined herein) against hCD4, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:
[0734] CDR1 is chosen from the group consisting of:
[0735] a) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0736] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0737] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287; and/or
[0738] CDR2 is chosen from the group consisting of:
[0739] d) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0740] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0741] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
and/or
[0742] CDR3 is chosen from the group consisting of:
[0743] g) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0744] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0745] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303; or any suitable fragment of such an amino acid sequence.
[0746] In particular, according to this preferred but non-limiting aspect, the invention relates to a NANOBODY (as defined herein) against human CD4, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:
[0747] CDR1 is chosen from the group consisting of:
[0748] a) the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0749] b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
[0750] c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 284 to 287, more preferably 287;
and
[0751] CDR2 is chosen from the group consisting of:
[0752] d) the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0753] e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295;
[0754] f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 292 to 295, more preferably 295; and
[0755] CDR3 is chosen from the group consisting of:
[0756] g) the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0757] h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303;
[0758] i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO's: 300 to 303, more preferably 303; or any suitable fragment of such an amino acid sequences.
[0759] As generally mentioned herein for the amino acid sequences of the invention, when a NANOBODY of the invention contains one or more CDR1 sequences according to b) and/or c):
[0760] i) any amino acid substitution in such a CDR according to b) and/or c) is preferably, and compared to the corresponding CDR according to a), a conservative amino acid substitution (as defined herein);
and/or
[0761] ii) the CDR according to b) and/or c) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to a);
and/or
[0762] iii) the CDR according to b) and/or c) may be a CDR that is derived from a CDR according to a) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0763] Similarly, when a NANOBODY of the invention contains one or more CDR2 sequences according to e) and/or f):
[0764] i) any amino acid substitution in such a CDR according to e) and/or f) is preferably, and compared to the corresponding CDR according to d), a conservative amino acid substitution (as defined herein); and/or
[0765] ii) the CDR according to e) and/or f) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to d);
and/or
[0766] iii) the CDR according to e) and/or f) may be a CDR that is derived from a CDR according to d) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0767] Also, similarly, when a NANOBODY of the invention contains one or more CDR3 sequences according to h) and/or i):
[0768] i) any amino acid substitution in such a CDR according to h) and/or i) is preferably, and
[0769] compared to the corresponding CDR according to g), a conservative amino acid substitution (as defined herein);

and/or

[0770] ii) the CDR according to h) and/or i) preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR according to g);
and/or
[0771] iii) the CDR according to h) and/or i) may be a CDR that is derived from a CDR according to g) by means of affinity maturation using one or more techniques of affinity maturation known per se.
[0772] It should be understood that the last three paragraphs generally apply to any NANOBODY of the invention that comprises one or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e), f), h) or i), respectively.
[0773] Of the NANOBODIES of the invention, NANOBODIES comprising one or more of the CDR's explicitly listed above are particularly preferred; NANOBODIES comprising two or more of the CDR's explicitly listed above are more particularly preferred; and NANOBODIES comprising three of the CDR's explicitly listed above are most particularly preferred.
[0774] Some particularly preferred, but non-limiting combinations of CDR sequences, as well as preferred combinations of CDR sequences and framework sequences, are mentioned in Table A-1 below, which lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) NANOBODIES of the invention. As will be clear to the skilled person, a combination of CDR1, CDR2 and CDR3 sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the same line in Table A-1) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences mentioned in Table A-1). Also, a combination of CDR sequences and framework sequences that occur in the same clone (i.e. CDR sequences and framework sequences that are mentioned on the same line in Table A-1) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences and framework sequences mentioned in Table A-1, as well as combinations of such CDR sequences and other suitable framework sequences, e.g. as further described herein).
[0775] Also, in the NANOBODIES of the invention that comprise the combinations of CDR's mentioned in Table A-1, each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR's; in which:
[0776] i) any amino acid substitution in such a CDR is preferably, and compared to the corresponding CDR sequence mentioned in Table A-1, a conservative amino acid substitution (as defined herein); and/or
[0777] ii) any such CDR sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the corresponding CDR sequence mentioned in Table A-1; and/or
[0778] iii) any such CDR sequence is a CDR that is derived by means of a technique for affinity maturation known per se, and in particular starting from the corresponding CDR sequence mentioned in Table A-1.
[0779] However, as will be clear to the skilled person, the (combinations of) CDR sequences, as well as (the combinations of) CDR sequences and framework sequences mentioned in Table A-1 will generally be preferred.
[0780] 
[00005] [TABLE-US-00005]
  TABLE A-1
 
  SEQ   FR1   SEQ   CDR1   SEQ   FR2   SEQ   CDR2   SEQ   FR3   SEQ   CDR3   SEQ   FR4
 
 
  (A) CDR's and framework sequences of Nanobodies against human CXCR4
  126   EVQLVESGGG   142   SYAMS   158   WVRQAPG   174   GIKSSGDSTR   190   RFTISRDNAKNML   206   SRVSRTGL   222   RGQGTQ
    LVQTGGSLRL         KGLEWVS     YAGSVKG     YLQMYSLKPEDTA     YTYDN     VTVSS
    SCAASGFTFS                 VYYCAK        
 
  127   EVQLVESGGG   143   NYAMG   159   WFRRAPG   175   AITRSGVRSG   191   RFTISRDNAKNTL   207   SAIGSGAL   223   SGQGTQ
    LVQAGGSLRL         KEREFVA     VSAIYGDSVK     YLQMDSLKPDDTA     RRFEYDY     VTVSS
    SCAASGRTFN             D     VYTCAA        
 
  128   KVQLVESGGG   144   IHTMS   160   WVRQAPG   176   TIKPSGDTTN   192   RFTISRDNAKNTL   208   DYFGTGV   224   RGQGTQ
    LVQPGGSLRL         KCPEWVS     YANAVKG     YLQMNSLKPEDTA         VTVSS
    SCAASGFAFS                 VYYCAK        
 
  129   EVQLVESGGG   145   DYAMS   161   WVRQAPG   177   AISWNGGSTD   193   RFTISRDNAKNTL   209   DQGPEYSG   225   RGQGTQ
    LVQPGGSLRL         KGLEWVS     YADSVKG     YLQMNSLKSEDTA     TYYYTRQY     VTVSS
    SCAASGFTFD                 EYYCAR     GY    
 
  130   EVQLVESGGG   46   GYTIA   162   WFRQVPG   178   YHRWSDGANL   194   RFTISGHNAKNTV   210   APWITSND   226   WGQGTQ
    FVQAGGSLRL         KEREFVA     YADSVKG     SLQMNSLKPEDTA     KEYLY     VTVSS
    SCETSGRPLL                 VYYCAA        
 
  131   EVQLVESGGG   147   DYATG   163   WFRKAPG   179   CISGSDGSTT   195   RPTISTDNAKNIV   211   QYGVGGRV   227   WGQGTQ
    LVQAGGSLRL         KEREGVS     YADSVKG     YLEMNSLKPEDTA     VCPCPYFY     VTVSS
    ACAASGFTFE                 VYYCAQ     DV    
 
  132   EVQLVESGGG   148   GYTIA   164   WFRQVPG   180   YHRWSDGANL   196   RFTISGHNAKNTV   212   AWIATTSND   228   WGQGTQ
    FVQAGGSLRL         KEREFVA     YADSVKG     SLQMNSLKPEDTA     KEYLY     VTVSS
    SCETSGRPLL                 VYYCAA        
 
  133   EVQLVESGGG   149   PSAMA   165   WyRQGPG   181   STIWSRGETY   197   RFTISRDTANYTL   213   RVRPYGQY   229   WGQGTQ
    LVQAGGSLRL         KERDFVA     FADSVKG     YLQMNNLKPEDTA     DY     VTVSS
    SCAASGLTFS                 VYYCSE        
 
  134   EVQLVESGGG   150   DYAMS   166   WVRQAPG   182   AISWNGGSAD   198   RFTISRDNAKNTL   214   DQGPFYSG   230   WGQGTQ
    LVQPGGSLRL         KGLEWVS     YADSVKG     YLQMNSLKSEDTA     TYYYTKGY     VTVSS
    SCAASGFTFD                 VYYCAK         Al
 
  135   EVQLVESGGG   151   MG   167   WVRQAPG   183   TSRLITDNII   199   RFTLTRDNGKNTV   215   RQNYSRSV   231   WGQGTQ
    LAQAGGSLRL         KEREFVT     YADSVKG     YLQMDSLKPDDTA     FGAKDYDY     VTVSS
    SCAASGRTYA                 VYYCAA        
 
  136   EVQLVESGGG   152   LNAMG   168   WVRQAPG   184   GITSSTSTYY   200   RFTISRDNTKNTV   216   DCPDYYSD   232   RGQGTQ
    LVQAGGSLRL         KQRELVA     ADSVKG     YLQMNSLKPEDTA     YECPLED     VTVSS
    SCAASGSIFS                 VYYCNV        
 
  137   EVQLVESGGG   153   VNDMG   169   WYRQAPG   185   VITSGGGTNY   201   RFTISGDNRKKTV   217   YYSSGIST   233   WGQGTQ
    LAQPGGPLRL         KQRELVA     VDSVKG     YLQMNSLKPEDTA     LRS     VTVSS
    TCEASGVIYS                 VYYCSI        
 
  138   EVQLVESGGG   154   YYTIG   170   WYRQAPG   186   CISSSDGSTA   202   RFTVSRDNAKNTV   218   BSADSRCS   234   WGQGTQ
    LVQAGGSLRL         KEREGVS     YLGSVQG     YLQMNNLKPEDTA     IGSIGFTW     VTVSS
    SCEVSGFTRD                 VYYCAL     LYNN    
 
  139   EVQLVESGGG   155   YEAIV   171   WLRQAPG   187   CITSRDSTTY   203   RFTISRDDAKETV   219   BTSMTCPT   235   RGQGTQ
    LVQPGGSLRL         KELEGVS     YASFVKG     YLQMNNLKPEDTA     LIVRFNY     VTVSS
    SCAASSFIGN                 VYYCAV        
 
  140   EVQLVESGGG   156   NYAMG   172   WFRRAPG   188   AITRSGVRSG   204   RFTISRDNVKNTL   220   SAIGSGAL   236   SGQGTQ
    LVQAGGSLRL         KEREFVA     VSAIYGDSVK     YLQMNTLKPEDTA     RRFEYDY     VTVSS
    SCKASGGTFN             D     VYYCAA        
 
  141   EVQLVESGGG   157   INAMG   173   WYRQAPG   189   SITSGGSTVY   205   RFTISRENYNTVY   221   DGVPEWGK   237   RGQGTQ
    LVQAGGSLRL         KQRELVA     ADSVKC     LQMNSLKPEDTAV     VQYPDTY     VTVSS
    SCAASGSFFS                 YYCNA        
  (B) CDR's and framework sequences of Nanobodies against human CD4
  280   EVQLVESGGG   284   GYWMY   288   WVRQAPG   292   AISPCGGSTY   296   RFTISRDNAKNTL   300   SLTATHTY   304   WGQGTQ
    LVQAGGSLRL         KCLEWVS     YPDSVKG     YLQMNSLKPEDTA     EYDY     VTVSS
    SCAASGFTFS                 LYYCAS        
 
  281   EVQLVESGGG   285   SYSMG   289   NFRQAPG   293   AISWSGDETS   297   RFTIARGNAKNTV   301   DRWWRPAG   305   WGQGTQ
    LVQAGGSLRL         KEREFVA     YADSVKG     YLQMNSLKSEDTA     LQWDY     VTVSS
    SCAASGRTSA                 IYYCAG        
 
  282   EVQLVESGGG   286   FNAMG   290   WYRQAPG   294   TIARAGATKY   298   RFSISRDNAKNTV   302   RVFDLPND   306   WGQGTQ
    LVQAGGSLKL         KQREWVT     ADSVKG     YLQMSSLKPODTA     Y     VTVSS
    SCAASRSILD                 TYYCNA        
 
  283   EVQLVESGGG   287   VMG   291   WERQAPG   295   AVRWSSTGIY   299   RFTISRDNAKNTV   303   DTYNSNPA   307   RGQGTQ
    SVQPGGSLTL         KEREFVA     YTQYADSVKS     YLEMNSLKPEDTA     RWDGYDF     VTVSS
    SCGTSGRTFN                 VYYCAA
 
[0781] Thus, in the NANOBODIES of the invention, at least one of the CDR1, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% “sequence identity” (as defined herein) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 “amino acid difference(s)” (as defined herein) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.
[0782] In this context, by “suitably chosen” is meant that, as applicable, a CDR1 sequence is chosen from suitable CDR1 sequences (i.e. as defined herein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as defined herein), respectively. More in particular, the CDR sequences are preferably chosen such that the NANOBODIES of the invention bind to Targets of the invention with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a kof-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.
[0783] In particular, in the NANOBODIES of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-1; and/or from the group consisting of the CDR3 sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR3 sequences listed in Table A-1.
[0784] Preferably, in the NANOBODIES of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 “amino acid difference(s)” with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.
[0785] In particular, in the NANOBODIES of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-1, respectively; and at least one of the CDR1 and CDR2 sequences present is suitably chosen from the group consisting of the CDR1 and CDR2 sequences, respectively, listed in Table A-1 or from the group of CDR1 and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1 and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1.
[0786] Most preferably, in the NANOBODIES of the invention, all three CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1 or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.
[0787] Even more preferably, in the NANOBODIES of the invention, at least one of the CDR1, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1. Preferably, in this aspect, at least one or preferably both of the other two CDR sequences present are suitably chosen from CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences, respectively, listed in Table A-1.
[0788] In particular, in the NANOBODIES of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 listed in Table A-1. Preferably, in this aspect, at least one and preferably both of the CDR1 and CDR2 sequences present are suitably chosen from the groups of CDR1 and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR1 and CDR2 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1 and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1.
[0789] Even more preferably, in the NANOBODIES of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences listed in Table A-1.
[0790] In particular, in the NANOBODIES of the invention, at least the CDR3 sequence is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1, and either the CDR1 sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDR1 and CDR2 sequences, respectively, listed in Table A-1. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the corresponding CDR sequences listed in Table A-1.
[0791] Even more preferably, in the NANOBODIES of the invention, all three CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.
[0792] Also, generally, the combinations of CDR's listed in Table A-1 (i.e. those mentioned on the same line in Table A-1) are preferred. Thus, it is generally preferred that, when a CDR in a NANOBODY of the invention is a CDR sequence mentioned in Table A-1 or is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with a CDR sequence listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with a CDR sequence listed in Table A-1, that at least one and preferably both of the other CDR's are suitably chosen from the CDR sequences that belong to the same combination in Table A-1 (i.e. mentioned on the same line in Table A-1) or are suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR sequence(s) belonging to the same combination and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the CDR sequence(s) belonging to the same combination. The other preferences indicated in the above paragraphs also apply to the combinations of CDR's mentioned in Table A-1.
[0793] Thus, by means of non-limiting examples, a NANOBODY of the invention can for example comprise a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1, a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination), and a CDR3 sequence.
[0794] Some preferred NANOBODIES of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination); and a CDR3 sequence that has more than 80% sequence identity with one of the CDR3 sequences mentioned in Table A-1 (but belonging to a different combination); or (2) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence, and one of the CDR3 sequences listed in Table A-1; or (3) a CDR1 sequence; a CDR2 sequence that has more than 80% sequence identity with one of the CDR2 sequence listed in Table A-1; and a CDR3 sequence that has 3, 2 or 1 amino acid differences with the CDR3 sequence mentioned in Table A-1 that belongs to the same combination as the CDR2 sequence.
[0795] Some particularly preferred NANOBODIES of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence mentioned in Table A-1 that belongs to the same combination; (2) a CDR1 sequence; a CDR2 listed in Table A-1 and a CDR3 sequence listed in Table A-1 (in which the CDR2 sequence and CDR3 sequence may belong to different combinations).
[0796] Some even more preferred NANOBODIES of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; the CDR2 sequence listed in Table A-1 that belongs to the same combination; and a CDR3 sequence mentioned in Table A-1 that belongs to a different combination; or (2) a CDR1 sequence mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid differences with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence listed in Table A-1 that belongs to the same or a different combination.
[0797] Particularly preferred NANOBODIES of the invention may for example comprise a CDR1 sequence mentioned in Table A-1, a CDR2 sequence that has more than 80% sequence identity with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and the CDR3 sequence mentioned in Table A-1 that belongs to the same combination.
[0798] In the most preferred NANOBODIES of the invention, the CDR1, CDR2 and CDR3 sequences present are suitably chosen from one of the combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.
[0799] According to another preferred, but non-limiting aspect of the invention (a) CDR1 has a length of between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, such as 16 and 17 amino acid residues; and/or (c) CDR3 has a length of between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as between 6 and 23 amino acid residues.
[0800] In another preferred, but non-limiting aspect, the invention relates to a NANOBODY in which the CDR sequences (as defined herein) have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239, and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311.
[0801] Generally, NANOBODIES with the above CDR sequences may be as further described herein, and preferably have framework sequences that are also as further described herein. Thus, for example and as mentioned herein, such NANOBODIES may be naturally occurring NANOBODIES (from any suitable species), naturally occurring VHH sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences or NANOBODIES, including but not limited to partially humanized NANOBODIES or VHH sequences, fully humanized NANOBODIES or VHH sequences, camelized heavy chain variable domain sequences, as well as NANOBODIES that have been obtained by the techniques mentioned herein.
[0802] Thus, in one specific, but non-limiting aspect, the invention relates to a humanized NANOBODY, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which CDR1 to CDR3 are as defined herein and in which said humanized NANOBODY comprises at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).
[0803] In another preferred, but non-limiting aspect, the invention relates to a NANOBODY in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said NANOBODY and one or more of the sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239, and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311, in which the amino acid residues that form the framework regions are disregarded. Such NANOBODIES can be as further described herein.
[0804] In another preferred, but non-limiting aspect, the invention relates to a NANOBODY with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311.
[0805] Another preferred, but non-limiting aspect of the invention relates to humanized variants of the NANOBODIES of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311, that comprise, compared to the corresponding native VHH sequence, at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).
[0806] The polypeptides of the invention comprise or essentially consist of at least one NANOBODY of the invention. Thus in another preferred, but non-limiting aspect, the invention relates to a polypeptide comprise or essentially consist of at least one NANOBODY that is chosen from the group consisting of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311 or from the group consisting of amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311. Thus in another preferred, but non-limiting aspect, the invention relates to a polypeptide comprise or essentially consist of amino acid sequences of SEQ ID NO's: 261 to 264, more preferably SEQ ID NO: 263 to 264.
[0807] It will be clear to the skilled person that the NANOBODIES that are mentioned herein as “preferred” (or “more preferred”, “even more preferred”, etc.) are also preferred (or more preferred, or even more preferred, etc.) for use in the polypeptides described herein. Thus, polypeptides that comprise or essentially consist of one or more “preferred” NANOBODIES of the invention will generally be preferred, and polypeptides that comprise or essentially consist of one or more “more preferred” NANOBODIES of the invention will generally be more preferred, etc.
[0808] Generally, proteins or polypeptides that comprise or essentially consist of a single NANOBODY (such as a single NANOBODY of the invention) will be referred to herein as “monovalent” proteins or polypeptides or as “monovalent constructs”. Proteins and polypeptides that comprise or essentially consist of two or more NANOBODIES (such as at least two NANOBODIES of the invention or at least one NANOBODY of the invention and at least one other NANOBODY) will be referred to herein as “multivalent” proteins or polypeptides or as “multivalent constructs”, and these may provide certain advantages compared to the corresponding monovalent NANOBODIES of the invention. Some non-limiting examples of such multivalent constructs will become clear from the further description herein.
[0809] According to one specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least two NANOBODIES of the invention, such as two or three NANOBODIES of the invention. As further described herein, such multivalent constructs can provide certain advantages compared to a protein or polypeptide comprising or essentially consisting of a single NANOBODY of the invention, such as a much improved avidity for Targets of the invention. Such multivalent constructs will be clear to the skilled person based on the disclosure herein, and e.g. are polypeptides that comprise or essentially consist of amino acid sequences of SEQ ID NO: 261 to 266.
[0810] According to another specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least one NANOBODY of the invention and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is preferably also a NANOBODY. Such proteins or polypeptides are also referred to herein as “multispecific” proteins or polypeptides or as “multispecific constructs”, and these may provide certain advantages compared to the corresponding monovalent NANOBODIES of the invention (as will become clear from the further discussion herein of some preferred, but-nonlimiting multispecific constructs). Such multispecific constructs will be clear to the skilled person based on the disclosure herein, and e.g. are polypeptides that comprise or essentially consist of amino acid sequences of SEQ ID NO: 263 to 264.
[0811] According to yet another specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least one NANOBODY of the invention, optionally one or more further NANOBODIES, and at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the NANOBODY of the invention and/or to the resulting fusion protein. Again, such fusion proteins may provide certain advantages compared to the corresponding monovalent NANOBODIES of the invention. Some non-limiting examples of such amino acid sequences and of such fusion constructs will become clear from the further description herein.
[0812] It is also possible to combine two or more of the above aspects, for example to provide a trivalent bispecific construct comprising two NANOBODIES of the invention and one other NANOBODY, and optionally one or more other amino acid sequences. Further non-limiting examples of such constructs, as well as some constructs that are particularly preferred within the context of the present invention, will become clear from the further description herein.
[0813] In the above constructs, the one or more NANOBODIES and/or other amino acid sequences may be directly linked to each other and/or suitably linked to each other via one or more linker sequences. Some suitable but non-limiting examples of such linkers will become clear from the further description herein.
[0814] In one specific aspect of the invention, a NANOBODY of the invention or a compound, construct or polypeptide of the invention comprising at least one NANOBODY of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such NANOBODIES, compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise NANOBODIES sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention that comprise at least one NANOBODY of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the NANOBODY of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more NANOBODIES of the invention are suitable linked to one or more serum proteins or fragments thereof (such as serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, NANOBODIES or (single) domain antibodies that can bind to serum proteins such as serum albumin, serum immunoglobulins such as IgG, or transferrin); polypeptides in which a NANOBODY of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more NANOBODIES of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to WO 08/068,280.
[0815] Again, as will be clear to the skilled person, such NANOBODIES, compounds, constructs or polypeptides may contain one or more additional groups, residues, moieties or binding units, such as one or more further amino acid sequences and in particular one or more additional NANOBODIES (i.e. not directed against Targets of the invention), so as to provide a tri- of multispecific NANOBODY construct.
[0816] Generally, the NANOBODIES of the invention (or compounds, constructs or polypeptides comprising the same) with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the NANOBODIES, compounds, constructs or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.
[0817] In a preferred, but non-limiting aspect of the invention, such NANOBODIES, compound, constructs or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).
[0818] In another one aspect of the invention, a polypeptide of the invention comprises one or more (such as two or preferably one) NANOBODIES of the invention linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that allow the resulting polypeptide of the invention to cross the blood brain barrier. In particular, said one or more amino acid sequences that allow the resulting polypeptides of the invention to cross the blood brain barrier may be one or more (such as two and preferably one) NANOBODIES, such as the NANOBODIES described in WO 02/057445, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.
[0819] In particular, polypeptides comprising one or more NANOBODIES of the invention are preferably such that they:
[0820] bind to Targets of the invention with a dissociation constant (KD) of 10−5 to 10−12 moles/liter or less, and preferably 10−7 to 10−12 moles/liter or less and more preferably 10−8 to 10−12 moles/liter (i.e. with an association constant (KA) of 105 to 1012 liter/moles or more, and preferably 107 to 1012 liter/moles or more and more preferably 108 to 1012 liter/moles);
and/or such that they:
[0821] bind to Targets of the invention with a kon-rate of between 102 M−1s−1 to about 107 M−1s−1, preferably between 103 M−1s−1 and 107 M−1s−1, more preferably between 104 M−1s−1 and 107 M−1s−1, such as between 105 M−1s−1 and 107 M−1s−1;
and/or such that they:
[0822] bind to Targets of the invention with a koff rate between 1 s−1 (t1/2=0.69 s) and 10−6 s−1 (providing a near irreversible complex with a t1/2 of multiple days), preferably between 10−2 s−1 and 10−6 s−1, more preferably between 10−3 s−1 and 10−6 s−1, such as between 10−4 s−1 and 10−6 s−1.
[0823] Preferably, a polypeptide that contains only one amino acid sequence of the invention is preferably such that it will bind to Targets of the invention with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 μM. In this respect, it will be clear to the skilled person that a polypeptide that contains two or more NANOBODIES of the invention may bind to Targets of the invention with an increased avidity, compared to a polypeptide that contains only one amino acid sequence of the invention.
[0824] Some preferred IC50 values for binding of the amino acid sequences or polypeptides of the invention to Targets of the invention will become clear from the further description and examples herein.
[0825] Another aspect of this invention relates to a nucleic acid that encodes an amino acid sequence of the invention (such as a NANOBODY of the invention) or a polypeptide of the invention comprising the same. Again, as generally described herein for the nucleic acids of the invention, such a nucleic acid may be in the form of a genetic construct, as defined herein.
[0826] In another aspect, the invention relates to host or host cell that expresses or that is capable of expressing an amino acid sequence (such as a NANOBODY) of the invention and/or a polypeptide of the invention comprising the same; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.
[0827] Another aspect of the invention relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.
[0828] The invention further relates to methods for preparing or generating the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.
[0829] The invention further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with Targets of the invention. Some preferred but non-limiting applications and uses will become clear from the further description herein.
[0830] Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description hereinbelow. Generally, it should be noted that the term NANOBODY as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the NANOBODIES of the invention can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” (as described herein) of a naturally occurring VHH domain or by expression of a nucleic acid encoding a such humanized VHH domain; (4) by “camelization” (as described herein) of a naturally occurring VH domain from any animal species, and in particular a from species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelisation” of a “domain antibody” or “Dab” as described by Ward et al (supra), or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a NANOBODY using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to the skilled person based on the disclosure herein and for example include the methods and techniques described in more detail herein.
[0831] One preferred class of NANOBODIES corresponds to the VHH domains of naturally occurring heavy chain antibodies directed against Targets of the invention. As further described herein, such VHH sequences can generally be generated or obtained by suitably immunizing a species of Camelid with Targets of the invention (i.e. so as to raise an immune response and/or heavy chain antibodies directed against Targets of the invention), by obtaining a suitable biological sample from said Camelid (such as a blood sample, serum sample or sample of B-cells), and by generating VHH sequences directed against Targets of the invention, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person and/or are further described herein.
[0832] Alternatively, such naturally occurring VHH domains against Targets of the invention, can be obtained from naïve libraries of Camelid VHH sequences, for example by screening such a library using Targets of the invention, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naïve VHH libraries may be used, such as VHH libraries obtained from naïve VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.
[0833] Thus, in another aspect, the invention relates to a method for generating NANOBODIES, that are directed against Targets of the invention. In one aspect, said method at least comprises the steps of:
[0834] a) providing a set, collection or library of NANOBODY sequences; and
[0835] b) screening said set, collection or library of NANOBODY sequences for NANOBODY sequences that can bind to and/or have affinity for Targets of the invention; and
[0836] c) isolating the NANOBODY or NANOBODIES that can bind to and/or have affinity for Targets of the invention.
[0837] In such a method, the set, collection or library of NANOBODY sequences may be a naïve set, collection or library of NANOBODY sequences; a synthetic or semi-synthetic set, collection or library of NANOBODY sequences; and/or a set, collection or library of NANOBODY sequences that have been subjected to affinity maturation.
[0838] In a preferred aspect of this method, the set, collection or library of NANOBODY sequences may be an immune set, collection or library of NANOBODY sequences, and in particular an immune set, collection or library of VHH sequences, that have been derived from a species of Camelid that has been suitably immunized with Targets of the invention or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).
[0839] In the above methods, the set, collection or library of NANOBODY or VHH sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) NANOBODY sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein.
[0840] Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[0841] In another aspect, the method for generating NANOBODY sequences comprises at least the steps of:
[0842] a) providing a collection or sample of cells derived from a species of Camelid that express immunoglobulin sequences;
[0843] b) screening said collection or sample of cells for (i) cells that express an immunoglobulin sequence that can bind to and/or have affinity for Targets of the invention; and (ii) cells that express heavy chain antibodies, in which substeps (i) and (ii) can be performed essentially as a single screening step or in any suitable order as two separate screening steps, so as to provide at least one cell that expresses a heavy chain antibody that can bind to and/or has affinity for Targets of the invention;
and
[0844] c) either (i) isolating from said cell the VHH sequence present in said heavy chain antibody; or (ii) isolating from said cell a nucleic acid sequence that encodes the VHH sequence present in said heavy chain antibody, followed by expressing said VHH domain.
[0845] In another aspect, the method for generating NANOBODY sequences directed against at least one of the Targets of the invention, comprises at least the steps of:
[0846] a. a step of suitably immunizing a Camelid with a suitable antigen that comprises the desired extracellular part, region, domain, loop or other extracellular epitope(s), or with a suitable peptide derived therefrom or based thereon, such that an immune response against the desired extracellular part, region, domain, loop or other extracellular epitope(s) is raised. The antigen may be any suitable antigen that is capable of raising an immune response against the desired extracellular part, region, domain, loop or other extracellular epitope(s); such as, for example and without limitation, whole cells that are alive and overexpress the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface in their native confirmation, cell wall fragments thereof or any other suitable preparation derived from such cells, vesicles that have the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface, a subunit or fragment of a subunit of a Target of the invention, e.g. human CXCR4 or hCD4, that comprises the desired extracellular part, region, domain, loop or other extracellular epitope(s), or a synthetic or semi-synthetic peptide that comprises and/or is based on (the amino acid sequence of) the desired extracellular part, region, domain, loop or other extracellular epitope(s), more preferably, whole cells (e.g. HEK293) that are alive and overexpress the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface in their native confirmation; and
[0847] b. a step of selection for binding for the desired extracellular part, region, domain, loop or other extracellular epitope(s) using cell membranes preparation of different (than the one using in immunization) and several cell types overexpressing said Target of the invention, e.g. human CXCR4. This may for example be performed by selecting from a set, a collection or a library of cells that express heavy chain antibodies on their surface (e.g. B-cells obtained from a suitably immunized Camelid) and using a cell membranes preparation of e.g. a first type of cells such as e.g. CHO for a first round selection and e.g. a second type of cells such as e.g. COS-7 cells for a second round selection, by selecting from a (naïve or immune) library of VHH sequences or NANOBODY sequences by using a cell membranes preparation of e.g. a first type of cell such as e.g. CHO for a first round selection and e.g. a second type of cell such as e.g. COS-7 cell for a second round selection, or by selecting from a (naïve or immune) library of nucleic acid sequences that encode VHH sequences or NANOBODY sequences by using a cell membranes preparation of e.g. a first type of cell such as e.g. CHO for a first round selection and e.g. a second type of cells such as e.g. COS-7 cell for a second round selection; which may all be performed in a manner known per se; and optionally
[0848] c. washing only mildly with a buffer such as PBS without detergents; and which method may optionally further comprise one or more other suitable steps known per se, such as, for example and without limitation, a step of affinity maturation, a step of expressing the desired amino acid sequence, a step of screening for binding and/or for activity against the desired antigen (in this case, the Target of the invention, e.g. human CXCR4 or hCD4), a step of determining the desired amino acid sequence or nucleotide sequence, a step of introducing one or more humanizing substitutions (e.g. as further described herein), a step of formatting in a suitable multivalent and/or multispecific format, a step of screening for the desired biological and/or physiological properties (i.e. using a suitable assay, such as those described herein); and/or any suitable combination of one or more of such steps, in any suitable order.
[0849] In another aspect, the method for generating NANOBODY sequences directed against a transmembranal protein comprises at least the steps of:
[0850] a. a step of suitably immunizing a Camelid with a suitable antigen that comprises the desired extracellular part, region, domain, loop or other extracellular epitope(s), or with a suitable peptide derived therefrom or based thereon, such that an immune response against the desired extracellular part, region, domain, loop or other extracellular epitope(s) is raised. The antigen may be any suitable antigen that is capable of raising an immune response against the desired extracellular part, region, domain, loop or other extracellular epitope(s); such as, for example and without limitation, whole cells that are alive and overexpress the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface in their native confirmation, cell wall fragments thereof or any other suitable preparation derived from such cells, vesicles that have the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface, a subunit or fragment of a subunit of a transmembrane protein, in particular multiple spanning transmembrane protein for which the native conformation cannot be reproduced in other “in vitro” system (at least at time of filing of this application), that comprises the desired extracellular part, region, domain, loop or other extracellular epitope(s), or a synthetic or semi-synthetic peptide that comprises and/or is based on (the amino acid sequence of) the desired extracellular part, region, domain, loop or other extracellular epitope(s), more preferably, whole cells (e.g. HEK293) that are alive and overexpress the desired extracellular part, region, domain, loop or other extracellular epitope(s) on their surface in their native confirmation; and
[0851] b. a step of selection for binding for the desired extracellular part, region, domain, loop or other extracellular epitope(s) using cell membranes preparation of different (than the one using in immunization) and several cell types overexpressing said transmembrane protein, in particular multiple spanning transmembrane protein for which the native conformation cannot be reproduced in other “in vitro” system (at least at time of filing of this application). This may for example be performed by selecting from a set, a collection or a library of cells that express heavy chain antibodies on their surface (e.g. B-cells obtained from a suitably immunized Camelid) and using a cell membranes preparation of e.g. a first type of cells such as e.g. CHO for a first round selection and e.g. a second type of cells such as e.g. COS-7 cells for a second round selection, by selecting from a (naïve or immune) library of VHH sequences or NANOBODY sequences by using a cell membranes preparation of e.g. a first type of cell such as e.g. CHO for a first round selection and e.g. a second type of cell such as e.g. COS-7 cell for a second round selection, or by selecting from a (naïve or immune) library of nucleic acid sequences that encode VHH sequences or NANOBODY sequences by using a cell membranes preparation of e.g. a first type of cell such as e.g. CHO for a first round selection and e.g. a second type of cells such as e.g. COS-7 cell for a second round selection; which may all be performed in a manner known per se; and optionally
[0852] c. washing only mildly with a buffer such as PBS without detergents; and which method may optionally further comprise one or more other suitable steps known per se, such as, for example and without limitation, a step of affinity maturation, a step of expressing the desired amino acid sequence, a step of screening for binding and/or for activity against the desired antigen (in this case, the transmebrane protein), a step of determining the desired amino acid sequence or nucleotide sequence, a step of introducing one or more humanizing substitutions (e.g. as further described herein), a step of formatting in a suitable multivalent and/or multispecific format, a step of screening for the desired biological and/or physiological properties (i.e. using a suitable assay, such as those described herein); and/or any suitable combination of one or more of such steps, in any suitable order.
[0853] In the method according to this aspect, the collection or sample of cells may for example be a collection or sample of B-cells.
[0854] Also, in this method, the sample of cells may be derived from a Camelid that has been suitably immunized with Targets of the invention or a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).
[0855] The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820. Particular reference is made to the so-called “Nanoclone” technique described in International application WO 06/079372 by Ablynx N.V.
[0856] In another aspect, the method for generating an amino acid sequence directed against Targets of the invention may comprise at least the steps of:
[0857] a) providing a set, collection or library of nucleic acid sequences encoding heavy chain antibodies or NANOBODY sequences;
[0858] b) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences that encode a heavy chain antibody or a NANOBODY sequence that can bind to and/or has affinity for Targets of the invention;
and
[0859] c) isolating said nucleic acid sequence, followed by expressing the VHH sequence present in said heavy chain antibody or by expressing said NANOBODY sequence, respectively.
[0860] In such a method, the set, collection or library of nucleic acid sequences encoding heavy chain antibodies or NANOBODY sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of heavy chain antibodies or VHH sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of NANOBODY sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of NANOBODY sequences that have been subjected to affinity maturation.
[0861] In a preferred aspect of this method, the set, collection or library of nucleic acid sequences may be an immune set, collection or library of nucleic acid sequences encoding heavy chain antibodies or VHH sequences derived from a Camelid that has been suitably immunized with Targets of the invention or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).
[0862] In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
[0863] As will be clear to the skilled person, the screening step of the methods described herein can also be performed as a selection step. Accordingly the term “screening” as used in the present description can comprise selection, screening or any suitable combination of selection and/or screening techniques. Also, when a set, collection or library of sequences is used, it may contain any suitable number of sequences, such as 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 104, 105, 106, 107, 108 or more sequences.
[0864] Also, one or more or all of the sequences in the above set, collection or library of amino acid sequences may be obtained or defined by rational, or semi-empirical approaches such as computer modelling techniques or biostatics or datamining techniques.
[0865] Furthermore, such a set, collection or library can comprise one, two or more sequences that are variants from one another (e.g. with designed point mutations or with randomized positions), compromise multiple sequences derived from a diverse set of naturally diversified sequences (e.g. an immune library)), or any other source of diverse sequences (as described for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005 and Binz et al, Nat
[0866] Biotechnol 2005, 23:1247). Such set, collection or library of sequences can be displayed on the surface of a phage particle, a ribosome, a bacterium, a yeast cell, a mammalian cell, and linked to the nucleotide sequence encoding the amino acid sequence within these carriers. This makes such set, collection or library amenable to selection procedures to isolate the desired amino acid sequences of the invention. More generally, when a sequence is displayed on a suitable host or host cell, it is also possible (and customary) to first isolate from said host or host cell a nucleotide sequence that encodes the desired sequence, and then to obtain the desired sequence by suitably expressing said nucleotide sequence in a suitable host organism. Again, this can be performed in any suitable manner known per se, as will be clear to the skilled person.
[0867] Yet another technique for obtaining VHH sequences or NANOBODY sequences directed against Targets of the invention involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and/or heavy chain antibodies directed against Targets of the invention), obtaining a suitable biological sample from said transgenic mammal that contains (nucleic acid sequences encoding) said VHH sequences or NANOBODY sequences (such as a blood sample, serum sample or sample of B-cells), and then generating VHH sequences directed against Targets of the invention, starting from said sample, using any suitable technique known per se (such as any of the methods described herein or a hybridoma technique). For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5 can be used. For example, such heavy chain antibody expressing mice can express heavy chain antibodies with any suitable (single) variable domain, such as (single) variable domains from natural sources (e.g. human (single) variable domains, Camelid (single) variable domains or shark (single) variable domains), as well as for example synthetic or semi-synthetic (single) variable domains.
[0868] The invention also relates to the VHH sequences or NANOBODY sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said VHH sequence or NANOBODY sequence; and of expressing or synthesizing said VHH sequence or NANOBODY sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.
[0869] As mentioned herein, a particularly preferred class of NANOBODIES of the invention comprises NANOBODIES with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art on humanization referred to herein. Again, it should be noted that such humanized NANOBODIES of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material. Another particularly preferred class of NANOBODIES of the invention comprises NANOBODIES with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such “camelizing” substitutions are preferably inserted at amino acid positions that form and/or are present at the VH-VL interface, and/or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996), supra). Preferably, the VH sequence that is used as a starting material or starting point for generating or designing the camelized NANOBODY is preferably a VH sequence from a mammal, more preferably the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized NANOBODIES of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.
[0870] For example, again as further described herein, both “humanization” and “camelization” can be performed by providing a nucleotide sequence that encodes a naturally occurring VHH domain or VH domain, respectively, and then changing, in a manner known per se, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a “humanized” or “camelized” NANOBODY of the invention, respectively. This nucleic acid can then be expressed in a manner known per se, so as to provide the desired NANOBODY of the invention. Alternatively, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized NANOBODY of the invention, respectively, can be designed and then synthesized de novo using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized NANOBODY of the invention, respectively, can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to provide the desired NANOBODY of the invention.
[0871] Other suitable methods and techniques for obtaining the NANOBODIES of the invention and/or nucleic acids encoding the same, starting from naturally occurring VH sequences or preferably VHH sequences, will be clear from the skilled person, and may for example comprise combining one or more parts of one or more naturally occurring VH sequences (such as one or more FR sequences and/or CDR sequences), one or more parts of one or more naturally occurring VHH sequences (such as one or more FR sequences or CDR sequences), and/or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide a NANOBODY of the invention or a nucleotide sequence or nucleic acid encoding the same (which may then be suitably expressed). Nucleotide sequences encoding framework sequences of VHH sequences or NANOBODIES will be clear to the skilled person based on the disclosure herein and/or the further prior art cited herein (and/or may alternatively be obtained by PCR starting from the nucleotide sequences obtained using the methods described herein) and may be suitably combined with nucleotide sequences that encode the desired CDR's (for example, by PCR assembly using overlapping primers), so as to provide a nucleic acid encoding a NANOBODY of the invention.
[0872] As mentioned herein, NANOBODIES may in particular be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences.
[0873] Thus, according to one preferred, but non-limiting aspect of the invention, a NANOBODY in its broadest sense can be generally defined as a polypeptide comprising:
[0874] a) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q;
and/or:
[0875] b) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E; and/or:
[0876] c) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S.
[0877] Thus, in a first preferred, but non-limiting aspect, a NANOBODY of the invention may have the structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which
[0878] a) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which:
[0879] b) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid or a cysteine and the amino acid residue at position 44 according to the Kabat numbering is preferably E;
and/or in which:
[0880] c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S;
and in which:
[0881] d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0882] In particular, a NANOBODY in its broadest sense can be generally defined as a polypeptide comprising:
[0883] a) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q;
and/or:
[0884] b) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R;
and/or:
[0885] c) an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S.
[0886] Thus, according to a preferred, but non-limiting aspect, a NANOBODY of the invention may have the structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which
[0887] a) the amino acid residue at position 108 according to the Kabat numbering is Q;
and/or in which:
[0888] b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R;
and/or in which:
[0889] c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S;
and in which:
[0890] d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0891] In particular, a NANOBODY against Targets of the invention according to the invention may have the structure:
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which
[0892] a) the amino acid residue at position 108 according to the Kabat numbering is Q; and/or in which:
[0893] b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or in which:
[0894] c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S;
and in which:
[0895] d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0896] In particular, according to one preferred, but non-limiting aspect of the invention, a NANOBODY can generally be defined as a polypeptide comprising an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which;
[0897] a-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, G, Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q; and
[0898] a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R; and
[0899] a-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W;
[0900] a-4) the amino acid residue at position 108 according to the Kabat numbering is Q; or in which:
[0901] b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and
[0902] b-2) the amino acid residue at position 45 according to the Kabat numbering is R; and
[0903] b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W;
[0904] b-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q;
or in which:
[0905] c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q; and
[0906] c-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R; and
[0907] c-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and
[0908] c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q; and in which
[0909] d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0910] Thus, in another preferred, but non-limiting aspect, a NANOBODY of the invention may have the structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
[0911] a-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, G, Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q;
and in which:
[0912] a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R;
and in which:
[0913] a-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W;
and in which
[0914] a-4) the amino acid residue at position 108 according to the Kabat numbering is Q; and in which:
[0915] d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0916] In another preferred, but non-limiting aspect, a NANOBODY of the invention may have the structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
[0917] b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q;
and in which:
[0918] b-2) the amino acid residue at position 45 according to the Kabat numbering is R; and in which:
[0919] b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W;
and in which:
[0920] b-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q;
and in which:
[0921] d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0922] In another preferred, but non-limiting aspect, a NANOBODY of the invention may have the structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
[0923] c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q;
and in which:
[0924] c-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R;
and in which:
[0925] c-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S;
and in which:
[0926] c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q;
and in which:
[0927] d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0928] Two particularly preferred, but non-limiting groups of the NANOBODIES of the invention are those according to a) above; according to (a-1) to (a-4) above; according to b) above; according to (b-1) to (b-4) above; according to (c) above; and/or according to (c-1) to (c-4) above, in which either:
[0929] i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as described herein) and the amino acid residue at position 108 is Q;
or in which:
[0930] ii) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-like sequence as described) and the amino acid residue at position 108 is Q or L, and is preferably Q.
[0931] Thus, in another preferred, but non-limiting aspect, a NANOBODY of the invention may have the structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
[0932] i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein) and the amino acid residue at position 108 is Q;
and in which:
[0933] ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0934] In another preferred, but non-limiting aspect, a NANOBODY of the invention may have the structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
[0935] i) the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-like sequence) and the amino acid residue at position 108 is Q or L, and is preferably Q; and in which:
[0936] ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0937] In the NANOBODIES of the invention in which the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE, the amino acid residue at position 37 is most preferably F. In the NANOBODIES of the invention in which the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW, the amino acid residue at position 37 is chosen from the group consisting of Y, H, I, L, V or F, and is most preferably V.
[0938] Thus, without being limited hereto in any way, on the basis of the amino acid residues present on the positions mentioned above, the NANOBODIES of the invention can generally be classified on the basis of the following three groups:
[0939] i) The “GLEW-group”: NANOBODIES with the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering and Q at position 108 according to the Kabat numbering. As further described herein, NANOBODIES within this group usually have a V at position 37, and can have a W, P, R or S at position 103, and preferably have a W at position 103. The GLEW group also comprises some GLEW-like sequences such as those mentioned in Table A-3 below. More generally, and without limitation, NANOBODIES belonging to the GLEW-group can be defined as NANOBODIES with a G at position 44 and/or with a W at position 47, in which position 46 is usually E and in which preferably position 45 is not a charged amino acid residue and not cysteine;
[0940] ii) The “KERE-group”: NANOBODIES with the amino acid sequence KERE or KQRE (or another KERE-like sequence) at positions 43-46 according to the Kabat numbering and Q or L at position 108 according to the Kabat numbering. As further described herein, NANOBODIES within this group usually have a F at position 37, an L or F at position 47; and can have a W, P, R or S at position 103, and preferably have a W at position 103. More generally, and without limitation, NANOBODIES belonging to the KERE-group can be defined as NANOBODIES with a K, Q or R at position 44 (usually K) in which position 45 is a charged amino acid residue or cysteine, and position 47 is as further defined herein;
[0941] iii) The “103 P, R, S-group”: NANOBODIES with a P, R or S at position 103. These NANOBODIES can have either the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering or the amino acid sequence KERE or KQRE at positions 43-46 according to the Kabat numbering, the latter most preferably in combination with an F at position 37 and an L or an F at position 47 (as defined for the KERE-group); and can have Q or L at position 108 according to the Kabat numbering, and preferably have Q.
[0942] Also, where appropriate, NANOBODIES may belong to (i.e. have characteristics of) two or more of these classes. For example, one specifically preferred group of NANOBODIES has GLEW or a GLEW-like sequence at positions 44-47; P, R or S (and in particular R) at position 103; and Q at position 108 (which may be humanized to L).
[0943] More generally, it should be noted that the definitions referred to above describe and apply to NANOBODIES in the form of a native (i.e. non-humanized) VHH sequence, and that humanized variants of these NANOBODIES may contain other amino acid residues than those indicated above (i.e. one or more humanizing substitutions as defined herein). For example, and without limitation, in some humanized NANOBODIES of the GLEW-group or the 103 P, R, S-group, Q at position 108 may be humanized to 108L. As already mentioned herein, other humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a NANOBODY may be partially humanized or fully humanized.
[0944] Thus, in another preferred, but non-limiting aspect, a NANOBODY of the invention may be a NANOBODY belonging to the GLEW-group (as defined herein), and in which CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0945] In another preferred, but non-limiting aspect, a NANOBODY of the invention may be a NANOBODY belonging to the KERE-group (as defined herein), and CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0946] Thus, in another preferred, but non-limiting aspect, a NANOBODY of the invention may be a NANOBODY belonging to the 103 P, R, S-group (as defined herein), and in which CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0947] Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,S residues mentioned above, the NANOBODIES of the invention can contain, at one or more positions that in a conventional VH domain would form (part of) the VH/VL interface, one or more amino acid residues that are more highly charged than the amino acid residues that naturally occur at the same position(s) in the corresponding naturally occurring VH sequence, and in particular one or more charged amino acid residues (as mentioned in Table A-2). Such substitutions include, but are not limited to, the GLEW-like sequences mentioned in Table A-3 below; as well as the substitutions that are described in the International Application WO 00/29004 for so-called “microbodies”, e.g. so as to obtain a NANOBODY with Q at position 108 in combination with KLEW at positions 44-47. Other possible substitutions at these positions will be clear to the skilled person based upon the disclosure herein.
[0948] In one aspect of the NANOBODIES of the invention, the amino acid residue at position 83 is chosen from the group consisting of L, M, S, V and W; and is preferably L.
[0949] Also, in one aspect of the NANOBODIES of the invention, the amino acid residue at position 83 is chosen from the group consisting of R, K, N, E, G, I, T and Q; and is most preferably either K or E (for NANOBODIES corresponding to naturally occurring VHH domains) or R (for “humanized” NANOBODIES, as described herein). The amino acid residue at position 84 is chosen from the group consisting of P, A, R, S, D T, and V in one aspect, and is most preferably P (for NANOBODIES corresponding to naturally occurring VHH domains) or R (for “humanized” NANOBODIES, as described herein).
[0950] Furthermore, in one aspect of the NANOBODIES of the invention, the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G.
[0951] Collectively, the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108, which in the NANOBODIES are as mentioned above, will also be referred to herein as the “Hallmark Residues”. The Hallmark Residues and the amino acid residues at the corresponding positions of the most closely related human VH domain, VH3, are summarized in Table A-3.
[0952] Some especially preferred but non-limiting combinations of these Hallmark Residues as occur in naturally occurring VHH domains are mentioned in Table A-4. For comparison, the corresponding amino acid residues of the human VH3 called DP-47 have been indicated in italics.
[0953] 
[00006] [TABLE-US-00006]
  TABLE A-3
 
  Hallmark Residues in Nanobodies
  PositionHuman VH3   Hallmark Residues
 
  11   L, V;   L, M, S, V, W; preferably L
    predominantly L  
  37   V, I, F; usually VF(1), Y, H, I, L or V, preferably
    F(1) or Y
44(8)   GG(2), E(3), A, D, Q, R, S, L;
    preferably G(2), E(3) or Q;
    most preferably G(2) or E(3)
45(8)   LL(2), R(3), C, I, L, P, Q, V;
    preferably L(2) or R(3)
47(8)   W, YW(2), L(1) or F(1), A, G, I, M, R, S, V
    or Y; preferably W(2), L(1), F(1) or R
  83   R or K; usually RR, K(5), N, E(5), G, I, M, Q or T;
      preferably K or R; most preferably K
  84   A, T, D;P(5), A, L, R, S, T, D, V; preferably P
    predominantly A  
  103    WW(4), P(6), R(6), S; preferably W
  104    G   G or D; preferably G
  108    L, M or T;Q, L(7) or R; preferably Q or L(7)
    predominantly L
 
  Notes:
(1)In particular, but not exclusively, in combination with KERE or KQRE at positions 43-46.
(2)Usually as GLEW at positions 44-47.
(3)Usually as KERE or KQRE at positions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF or KEREG at positions 43-47. Alternatively, also sequences such as TERE (for example TEREL), KECE (for example KECEL or KECER), RERE (for example REREG), QERE (for example QEREG), KGRE (for example KGREG), KDRE (for example KDREV) are possible. Some other possible, but less preferred sequences include for example DECKL and NVCEL.
(4)With both GLEW at positions 44-47 and KERE or KQRE at positions 43-46.
(5)Often as KP or EP at positions 83-84 of naturally occurring VHH domains.
(6)In particular, but not exclusively, in combination with GLEW at positions 44-47.
(7)With the proviso that when positions 44-47 are GLEW, position 108 is always Q in (non-humanized) VHH sequences that also contain a W at 103.
(8)The GLEW group also contains GLEW-like sequences at positions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW.
[0954] 
[00007] [TABLE-US-00007]
  TABLE A-4
 
  Some preferred but non-limiting combinations of Hallmark
  Residues in naturally occurring Nanobodies.
    11   37   44   45   47   83   84   103   104   108
   
  DP-47 (human)   M   V   G   L   W   R   A   W   G   L
  “KERE” group   L   F   E   R   L   K   P   W   G   Q
    L   F   E   R   F   E   P   W   G   Q
    L   F   E   R   F   K   P   W   G   Q
    L   Y   Q   R   L   K   P   W   G   Q
    L   F   L   R   V   K   P   Q   G   Q
    L   F   Q   R   L   K   P   W   G   Q
    L   F   E   R   F   K   P   W   G   Q
  “GLEW” group   L   V   G   L   W   K   S   W   G   Q
    M   V   G   L   W   K   P   R   G   Q
 
  For humanization of these combinations, reference is made to the specification.
[0955] In the NANOBODIES, each amino acid residue at any other position than the Hallmark Residues can be any amino acid residue that naturally occurs at the corresponding position (according to the Kabat numbering) of a naturally occurring VHH domain.
[0956] Such amino acid residues will be clear to the skilled person. Tables A-5 to A-8 mention some non-limiting residues that can be present at each position (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4 of naturally occurring VHH domains. For each position, the amino acid residue that most frequently occurs at each position of a naturally occurring VHH domain (and which is the most preferred amino acid residue for said position in a NANOBODY) is indicated in bold; and other preferred amino acid residues for each position have been underlined (note: the number of amino acid residues that are found at positions 26-30 of naturally occurring VHH domains supports the hypothesis underlying the numbering by Chothia (supra) that the residues at these positions already form part of CDR1).
[0957] In Tables A-5-A-8, some of the non-limiting residues that can be present at each position of a human VH3 domain have also been mentioned. Again, for each position, the amino acid residue that most frequently occurs at each position of a naturally occurring human VH3 domain is indicated in bold; and other preferred amino acid residues have been underlined.
[0958] For reference only, Tables A-5-A-8 also contain data on the VHH entropy (“VHH Ent.”) and VHH variability (“VHH Var.”) at each amino acid position for a representative sample of 1118 VHH sequences (data kindly provided by David Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The values for the VHH entropy and the VHH variability provide a measure for the variability and degree of conservation of amino acid residues between the 1118 VHH sequences analyzed: low values (i.e. <1, such as <0.5) indicate that an amino acid residue is highly conserved between the VHH sequences (i.e. little variability). For example, the G at position 8 and the G at position 9 have values for the VHH entropy of 0.1 and 0 respectively, indicating that these residues are highly conserved and have little variability (and in case of position 9 is G in all 1118 sequences analysed), whereas for residues that form part of the CDR's generally values of 1.5 or more are found (data not shown). Note that (1) the amino acid residues listed in the second column of Tables A-5-A-8 are based on a bigger sample than the 1118 VHH sequences that were analysed for determining the VHH entropy and VHH variability referred to in the last two columns; and (2) the data represented below support the hypothesis that the amino acid residues at positions 27-30 and maybe even also at positions 93 and 94 already form part of the CDR's (although the invention is not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat is used). For a general explanation of sequence entropy, sequence variability and the methodology for determining the same, see Oliveira et al., PROTEINS: Structure, Function and Genetics, 52: 544-552 (2003).
[0959] 
[00008] [TABLE-US-00008]
  TABLE A-5
 
  Non-limiting examples of amino acid residues in FR1
  (for the footnotes, see the footnotes to Table A-3)
    Amino acid residue(s):VHHVHH
  Pos.Human VH3Camelid VHH's   Ent.   Var.
 
  1E, QQ, A, E   —   —
  2VV   0.2   1
  3QQ, K   0.3   2
  4LL   0.1   1
  5V, LQ, E, L, V   0.8   3
  6EE, D, Q, A   0.8   4
  7S, TS, F   0.3   2
  8G, RG   0.1   1
  9GG   0   1
  10G, VG, D, R   0.3   2
  11   Hallmark residue: L, M, S, V, W; preferably L   0.8   2
  12V, IV, A   0.2   2
  13Q, K, RQ, E, K, P, R   0.4   4
  14PA, Q, A, G, P, S, T, V   1   5
  15GG   0   1
  16G, RG, A, E, D   0.4   3
  17SS, F   0.5   2
  18LL, V   0.1   1
  19R, KR, K, L, N, S, T   0.6   4
  20LL, F, I, V   0.5   4
  21SS, A, F, T   0.2   3
  22CC   0   1
  23A, TA, D, E, P, S, T, V   1.3   5
  24AA, I, L, S, T, V   1   6
  25SS, A, F, P, T   0.5   5
  26GG, A, D, E, R, S, T, V   0.7   7
  27F   S, F, R, L, P, G, N,   2.3   13
  28T   N, T, E, D, S, I, R, A, G, R, F, Y   1.7   11
  29F, V   F, L, D, S, I, G, V, A   1.9   11
  30S, D, G   N, S, E, G, A, D, M, T   1.8   11
 
[0960] 
[00009] [TABLE-US-00009]
  TABLE A-6
 
  Non-limiting examples of amino acid residues in FR2
  (for the footnotes, see the footnotes to Table A-3)
    Amino acid residue(s):VHHVHH
  Pos.Human VH3Camelid VHH's   Ent.   Var.
 
  36WW   0.1   1
  37Hallmark residue: F(1), H, I, L, Y   1.1   6
  or V, preferably F(1) or Y    
  38RR   0.2   1
  39QQ, H, P, R   0.3   2
  40AA, F, G, L, P, T, V   0.9   7
  41P, S, TP, A, L, S   0.4   3
  42GG, E   0.2   2
  43KK, D, E, N, Q, R, T, V   0.7   6
  44Hallmark residue: G(2), E(3), A, D, Q,   1.3   5
  R, S, L; preferably G(2), E(3) or    
  Q; most preferably G(2) or E(3).    
  45Hallmark residue: L(2), R(3), C, I, L, P, Q, V;   0.6   4
  preferably L(2) or R(3)    
  46E, VE, D, K, Q, V   0.4   2
  47Hallmark residue: W(2), L(1) or F(1), A, G, I,   1.9   9
  M, R, S, V or Y; preferably W(2), L(1), F(1) or R    
  48VV, I, L   0.4   3
  49S, A, GA, S, G, T, V   0.8   3
 
[0961] 
[00010] [TABLE-US-00010]
  TABLE A-7
 
  Non-limiting examples of amino acid residues in FR3
  (for the footnotes, see the footnotes to Table A-3)
    Amino acid residue(s):VHHVHH
  Pos.Human VH3Camelid VHH's   Ent.   Var.
 
  66RR   0.1   1
  67FF, L, V   0.1   1
  68TT, A, N, S   0.5   4
  69II, L, M, V   0.4   4
  70SS, A, F, T   0.3   4
  71RR, G, H, I, L, K, Q, S, T, W   1.2   8
  72D, ED, E, G, N, V   0.5   4
  73N, D, GN, A, D, F, I, K, L, R, S, T, V, Y   1.2   9
  74A, SA, D, G, N, P, S, T, V   1   7
  75KK, A, E, K, L, N, Q, R   0.9   6
  76N, SN, D, K, R, S, T, Y   0.9   6
  77   S, T, IT, A, E, I, M, P, S   0.8   5
  78L, AV, L, A, F, G, I, M   1.2   5
  79Y, HY, A, D, F, H, N, S, T   1   7
  80LL, F, V   0.1   1
  81QQ, E, I, L, R, T   0.6   5
  82MM, I, L, V   0.2   2
   82aN, GN, D, G, H, S, T   0.8   4
   82bSS, N, D, G, R, T   1   6
   82cLL, P, V   0.1   2
  83Hallmark residue: R, K(5), N, E(5), G, I, M, Q or T;   0.9   7
    preferably K or R; most preferably K    
  84Hallmark residue: P(5), A, D, L, R, S, T, V;   0.7   6
    preferably P    
  85E, GE, D, G, Q   0.5   3
  86DD   0   1
  87T, MT, A, S   0.2   3
  88AA, G, S   0.3   2
  89V, LV, A, D, I, L, M, N, R, T   1.4   6
  90YY, F   0   1
  91Y, HY, D, F, H, L, S, T, V   0.6   4
  92CC   0   1
  93A, K, TA, N, G, H, K, N, R, S, T, V, Y   1.4   10
  94K, R, TA, V, C, F, G, I, K, L, R, S or T   1.6   9
 
[0962] 
[00011] [TABLE-US-00011]
  TABLE A-8
 
  Non-limiting examples of amino acid residues in FR4
  (for the footnotes, see the footnotes to Table A-3)
    Amino acid residue(s):VHHVHH
  Pos.Human VH3Camelid VHH's   Ent.   Var.
 
  103Hallmark residue: W(4), P(6), R(6), S; preferably W   0.4   2
  104   Hallmark residue: G or D; preferably G   0.1   1
  105Q, RQ, E, K, P, R   0.6   4
  106GG   0.1   1
  107TT, A, I   0.3   2
  108Hallmark residue: Q, L(7) or R: preferably Q or L(7)   0.4   3
  109VV   0.1   1
  110TT, I, A   0.2   1
  111VV, A, I   0.3   2
  112SS, F   0.3   1
  113SS, A, L, P, T   0.4   3
 
[0963] Thus, in another preferred, but not limiting aspect, a NANOBODY of the invention can be defined as an amino acid sequence with the (general) structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[0964] in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
[0965] i) one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3;
and in which:
[0966] ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0967] The above NANOBODIES may for example be VHH sequences or may be humanized NANOBODIES. When the above NANOBODY sequences are VHH sequences, they may be suitably humanized, as further described herein. When the NANOBODIES are partially humanized NANOBODIES, they may optionally be further suitably humanized, again as described herein.
[0968] In particular, a NANOBODY of the invention can be an amino acid sequence with the (general) structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
[0969] in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:
[0970] i) (preferably) one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 (it being understood that VHH sequences will contain one or more Hallmark residues; and that partially humanized NANOBODIES will usually, and preferably, [still] contain one or more Hallmark residues [although it is also within the scope of the invention to provide—where suitable in accordance with the invention—partially humanized NANOBODIES in which all Hallmark residues, but not one or more of the other amino acid residues, have been humanized]; and that in fully humanized NANOBODIES, where suitable in accordance with the invention, all amino acid residues at the positions of the Hallmark residues will be amino acid residues that occur in a human VH3 sequence. As will be clear to the skilled person based on the disclosure herein that such VHH sequences, such partially humanized NANOBODIES with at least one Hallmark residue, such partially humanized NANOBODIES without Hallmark residues and such fully humanized NANOBODIES all form aspects of this invention);
and in which:
[0971] ii) said amino acid sequence has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are disregarded;
and in which:
[0972] iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0973] The above NANOBODIES may for example be VHH sequences or may be humanized NANOBODIES. When the above NANOBODY sequences are VHH sequences, they may be suitably humanized, as further described herein. When the NANOBODIES are partially humanized NANOBODIES, they may optionally be further suitably humanized, again as described herein.
[0974] 
[00012] [TABLE-US-00012]
  TABLE A-9
 
  Representative amino acid sequences for Nanobodies of the KERE, GLEW and P, R, S 103 group.
  The CDR's are indicated with XXXX
 
 
  KERE sequence no. 1   SEQ ID NO: 1   EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTI
      SRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS
 
  KERE sequence no. 2   SEQ ID NO: 2   QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREEVAXXXXXRETI
      SRDTASNRGYLFIMNNLTPEDTAVYYCAAXXXXXWGQGTQVIVSS
 
  KERE sequence no. 3   SEQ ID NO: 3   AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTI
      SRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS
 
  KERE sequence no. 4   SEQ ID NO: 4   QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTI
      SRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS
 
  KERE sequence no. 5   SEQ ID NO: 5   AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTI
      SMDYTKQTVYLFIMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS
 
  KERE sequence no. 6   SEQ ID NO: 6   DVKEVESGGGLVQAGGSLRLSCVASGENFDXXXXXWERQAPGKEREEVAXXXXXRFTI
      SSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS
 
  KERE sequence no. 7   SEQ ID NO: 7   QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTI
      ARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVIVSS
 
  KERE sequence no. 8   SEQ ID NO: 8   EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXVVFRQAPGKPREGVSXXXXXRFT
      ISTDNAKNWHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS
 
  KERE sequence no. 9   SEQ ID NO: 9   QVQLVESGGGLVOPGGSLRLSCQASGDISTXXXXXWYROVPGKLREFVAXXXXXRFTI
      SGDNAKRAIYLQMNINLKPDDTAVYYCNRXXXXXVVGQGTQVTVSP
 
  KERE sequence no. 10   SEQ ID NO: 10   QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTI
      SRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS
 
  KERE sequence no. 11   SEQ ID NO: 11   EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTI
      ARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVIVSS
 
  KERE sequence no. 12   SEQ ID NO: 12   AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTI
      SRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS
 
  KERE sequence no 13   SEQ ID NO: 13   AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTI
      SRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTOVTVSS
 
  KERE sequence no. 14   SEQ ID NO: 14   AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTV
      SRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXVVGQGTRVTVSS
 
  KERE sequence no. 15   SEQ ID NO: 15   QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTI
      SRDYAGNTAFLOMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS
 
  KERE sequence no. 16   SEQ ID NO: 16   EVOLVESGGELVQAGGSLKLSCTASGRNEVXXXXXWERRAPGKEREFVAXXXXXRFTV
      SRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS
 
  GLEW sequence no. 1   SEQ ID NO: 17   AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTI
      SRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS
 
  GLEW sequence no. 2   SEQ ID NO: 18   EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKI
      SRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS
 
  GLEW sequence no. 3   SEQ ID NO: 19   EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTI
      SRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS
 
  P, R, S 103 sequence no. 1   SEQ ID NO: 20   AVOLVESGGGLVQAGGSLRLSCAASGRTESXXXXXWERQAPGKEREFVAXXXXXRFTI
      SRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS
 
  P, R, S 103 sequence no. 2   SEQ ID NO: 21   DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFTI
      SRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVIVSS
 
  P, R, S 103 sequence no. 3   SEQ ID NO: 22   EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKI
      SRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS
 
[0975] In particular, a NANOBODY of the invention of the KERE group can be an amino acid sequence with the (general) structure
          FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
in which:
[0976] i) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E;
and in which:
[0977] ii) FR1 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[0978] 
[00013] [TABLE-US-00013]
  TABLE A-10
 
  Representative FW1 sequences for Nanobodies of the KERE-group.
 
 
  KERE FW1 sequence no. 1   SEQ ID NO: 23   QVQRVESGGGLVQAGGSLRLSCAASGRTSS
 
  KERE FW1 sequence no. 2   SEQ ID NO: 24   QVQLVESGGGLVQTGDSLSLSCSASGRTFS
 
  KERE FW1 sequence no. 3   SEQ ID NO: 25   QVKLEESGGGLVQAGDSLRLSCAATGRAFG
 
  KERE FW1 sequence no. 4   SEQ ID NO: 26   AVQLVESGGGLVQPGESLGLSCVASGRDFV
 
  KERE FW1 sequence no. 5   SEQ ID NO: 27   EVQLVESGGGLVQAGGSLRLSCEVLGRTAG
 
  KERE FW1 sequence no. 6   SEQ ID NO: 28   QVQLVESGGGWVQPGGSLRLSCAASETILS
 
  KERE FW1 sequence no. 7   SEQ ID NO: 29   QVQLVESGGGTVQPGGSLNLSCVASGNTFN
 
  KERE FW1 sequence no. 8   SEQ ID NO: 30   EVQLVESGGGLAQPGGSLQLSCSAPGFTLD
 
  KERE FW1 sequence no. 9   SEQ ID NO: 31   AQELEESGGGLVQAGGSLRLSCAASGRTFN
 

and in which:
[0979] iii) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[0980] 
[00014] [TABLE-US-00014]
  TABLE A-11
 
  Representative FW2 sequences for
  Nanobodies of the KERE-group.
 
 
  KERE FW2 sequence no. 1   SEQ ID NO: 41   WERQAPGKEREEVA
 
  KERE FW2 sequence no. 2   SEQ ID NO: 42   WFRQTPGREREFVA
 
  KERE FW2 sequence no. 3   SEQ ID NO: 43   WYRQAPGKQREMVA
 
  KERE FW2 sequence no. 4   SEQ ID NO: 44   WYRQGPGKQRELVA
 
  KERE FW2 sequence no. 5   SEQ ID NO: 45   WIRQAPGKEREGVS
 
  KERE FW2 sequence no. 6   SEQ 1D NO: 46   WFREAPGKEREGIS
 
  KERE FW2 sequence no. 7   SEQ ID N0: 47   WYRQAPGKERDLVA
 
  KERE FW2 sequence no. 8   SEQ ID NO: 48   WFRQAPGKQREEVS
 
  KERE FW2 sequence no. 9   SEQ ID NO: 49   WFRQPPGKVREFVG
 

and in which:
[0981] iv) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[0982] 
[00015] [TABLE-US-00015]
  TABLE A-12
 
  Representative FW3 sequences for Nanobodies of the KERE-group.
 
 
  KERE FW3 sequence no. 1    SEQ ID NO: 50   RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYF
 
  KERE FW3 sequence no. 2    SEQ ID NO: 51   RFAISRDNNKNTGYLOMNSLEPEDTAVYYCAA
 
  KERE FW3 sequence no. 3    SEQ ID NO: 52   RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAA
 
  KERE FW3 sequence no. 4    SEQ ID NO: 53   RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAA
 
  KERE FW3 sequence no. 5    SEQ ID NO: 54   RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAA
 
  KERE FW3 sequence no. 6    SEQ ID NO: 55   RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAA
 
  KERE FW3 sequence no. 7    SEQ ID NO: 56   RFTISKDSGKNTVYLQMTSLKPEDTAVYYCAT
 
  KERE FW3 sequence no. 8    SEQ ID NO: 57   RFTISRDSAKNMMYLQMNNLKPQDTAVYYCAA
 
  KERE FW3 sequence no. 9    SEQ ID NO: 58   RFTISRENDKSTVYLQLNSLKPEDTAVYYCAA
 
  KERE FW3 sequence no. 10   SEQ ID NO: 59   RFTISRDYAGNTAYLQMNSLKPEDTGVYYCAT
 

and in which:
[0983] v) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[0984] 
[00016] [TABLE-US-00016]
  TABLE A-13
 
  Representative FW4 sequences
  for Nanobodies of the KERE-group.
 
 
  KERE FW4 sequence no. 1   SEQ ID NO: 60   WGQGTQVTVSS
 
  KERE FW4 sequence no. 2   SEQ ID NO: 61   WGKGTLVTVSS
 
  KERE FW4 sequence no. 3   SEQ ID NO: 62   RGQGTRVTVSS
 
  KERE FW4 sequence no. 4   SEQ ID NO: 63   WGLGTQVTISS
 

and in which:
[0985] vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0986] In the above NANOBODIES, one or more of the further Hallmark residues are preferably as described herein (for example, when they are VHH sequences or partially humanized NANOBODIES).
[0987] Also, the above NANOBODIES may for example be VHH sequences or may be humanized NANOBODIES. When the above NANOBODY sequences are VHH sequences, they may be suitably humanized, as further described herein. When the NANOBODIES are partially humanized NANOBODIES, they may optionally be further suitably humanized, again as described herein.
[0988] With regard to framework 1, it will be clear to the skilled person that, when an amino acid sequence as outlined above is generated by expression of a nucleotide sequence, the first four amino acid sequences (i.e. amino acid residues 1-4 according to the Kabat numbering) may often be determined by the primer(s) that have been used to generate said nucleic acid. Thus, for determining the degree of amino acid identity, the first four amino acid residues are preferably disregarded.
[0989] Also, with regard to framework 1, and although amino acid positions 27 to 30 are according to the Kabat numbering considered to be part of the framework regions (and not the CDR's), it has been found by analysis of a database of more than 1000 VHH sequences that the positions 27 to 30 have a variability (expressed in terms of VHH entropy and VHH variability—see Tables A-5 to A-8) that is much greater than the variability on positions 1 to 26. Because of this, for determining the degree of amino acid identity, the amino acid residues at positions 27 to 30 are preferably also disregarded.
[0990] In view of this, a NANOBODY of the KERE class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:
[0991] i) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E;
and in which:
[0992] ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:
[0993] 
[00017] [TABLE-US-00017]
  TABLE A-14
 
  Representative FW1 sequences (amino acid residues 5 to 26)
  for Nanobodies of the KERE-group.
 
 
  KERE FW1 sequence no. 10   SEQ ID NO: 32   VESGGGLVQPGGSLRLSCAASG
 
  KERE FW1 sequence no. 11   SEQ ID NO: 33   VDSGGGLVQAGDSLKLSCALTG
 
  KERE FW1 sequence no. 12   SEQ ID NO: 34   VDSGGGLVQAGDSLRLSCAASG
 
  KERE FW1 sequence no. 13   SEQ ID NO: 35   VDSGGGLVEAGGSLRLSCQVSE
 
  KERE FW1 sequence no. 14   SEQ ID NO: 36   QDSGGGSVQAGGSLKLSCAASG
 
  KERE FW1 sequence no. 15   SEQ ID NO: 37   VQSGGRLVQAGDSLRLSCAASE
 
  KERE FW1 sequence no. 16   SEQ ID NO: 38   VESGGTLVQSGDSLKLSCASST
 
  KERE FW1 sequence no. 17   SEQ ID NO: 39   MESGGDSVQSGGSLTLSCVASG
 
  KERE FW1 sequence no. 18   SEQ ID NO: 40   QASGGGLVQAGGSLRLSCSASV
 

and in which:
[0994] iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of NANOBODIES of the KERE-class;
and in which:
[0995] iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[0996] The above NANOBODIES may for example be VHH sequences or may be humanized NANOBODIES. When the above NANOBODY sequences are VHH sequences, they may be suitably humanized, as further described herein. When the NANOBODIES are partially humanized NANOBODIES, they may optionally be further suitably humanized, again as described herein.
[0997] A NANOBODY of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which
[0998] i) preferably, when the NANOBODY of the GLEW-class is a non-humanized NANOBODY, the amino acid residue in position 108 is Q;
[0999] ii) FR1 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1000] 
[00018] [TABLE-US-00018]
  TABLE A-15
 
  Representative FW1 sequences for Nanobodies of the GLEW-group.
 
 
  GLEW FW1 sequence no. 1   SEQ ID NO: 64   QVQLVESGGGLVQPGGSLRLSCAASGFTFS
 
  GLEW FW1 sequence no. 2   SEQ ID NO: 65   EVHLVESGGGLVRPGGSLRLSCAAFGFIFK
 
  GLEW FW1 sequence no. 3   SEQ ID NO: 66   QVKLEESGGGLAQPGGSLRLSCVASGFTFS
 
  GLEW FW1 sequence no. 4   SEQ ID NO: 67   EVQLVESGGGLVQPGGSLRLSCVCVSSGCT
 
  GLEW FW1 sequence no. 5   SEQ ID NO: 68   EVQLVESGGGLALPGGSLTLSCVFSGSTFS
 

and in which:
[1001] iii) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1002] 
[00019] [TABLE-US-00019]
  TABLE A-16
 
  Representative FW2 sequences for
  Nanobodies of the GLEW-group.
 
 
  GLEW FW2 sequence no. 1   SEQ ID NO: 72   WVRQAPGKVLEWVS
 
  GLEW FW2 sequence no. 2   SEQ ID NO: 73   WVRRPPGKGLEWVS
 
  GLEW FW2 sequence no. 3   SEQ ID NO: 74   WVRQAPGMGLEWVS
 
  GLEW FW2 sequence no. 4   SEQ ID NO: 75   WVRQAPGKEPEWVS
 
  GLEW FW2 sequence no. 5   SEQ ID NO: 76   WVRQAPGKDQEWVS
 
  GLEW FW2 sequence no. 6   SEQ ID NO: 77   WVRQAPGKAEEWVS
 
  GLEW FW2 sequence no. 7   SEQ ID NO: 78   WVRQAPGKGLEWVA
 
  GLEW FW2 sequence no. 8   SEQ ID NO: 79   WVRQAPGRATEWVS
 

and in which:
[1003] iv) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1004] 
[00020] [TABLE-US-00020]
  TABLE A-17
 
  Representative FW3 sequences for Nanobodies of the GLEW-group.
 
 
  GLEW FW3 sequence no. 1   SEQ ID NO: 80   RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVK
 
  GLEW FW3 sequence no. 2   SEQ ID NO: 81   RFTISRDNARNTLYLQMDSLIPEDTALYYCAR
 
  GLEW FW3 sequence no. 3   SEQ ID NO: 82   RFTSSRDNAKSTLYLQMNDLKPEDTALYYCAR
 
  GLEW FW3 sequence no. 4   SEQ ID NO: 83   RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQR
 
  GLEW FW3 sequence no. 5   SEQ ID NO: 84   RFTASRDNAKNTLYLQMNSLKSEDTARYYCAR
 
  GLEW FW3 sequence no. 6   SEQ ID NO: 85   RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGR
 

and in which:
[1005] v) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1006] 
[00021] [TABLE-US-00021]
  TABLE A-18
 
  Representative FW4 sequences
  for Nanobodies of the GLEW-group.
 
 
  GLEW FW4 sequence no. 1   SEQ ID NO: 86   GSQGTQVTVSS
 
  GLEW FW4 sequence no. 2   SEQ ID NO: 87   LRGGTQVTVSS
 
  GLEW FW4 sequence no. 3   SEQ ID NO: 88   RGQGTLVTVSS
 
  GLEW FW4 sequence no. 4   SEQ ID NO: 89   RSRGIQVTVSS
 
  GLEW FW4 sequence no. 5   SEQ ID NO: 90   WGKGTQVTVSS
 
  GLEW FW4 sequence no. 6   SEQ ID NO: 91   WGQGTQVTVSS
 

and in which:
[1007] vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[1008] In the above NANOBODIES, one or more of the further Hallmark residues are preferably as described herein (for example, when they are VHH sequences or partially humanized NANOBODIES).
[1009] With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.
[1010] In view of this, a NANOBODY of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:
[1011] i) preferably, when the NANOBODY of the GLEW-class is a non-humanized NANOBODY, the amino acid residue in position 108 is Q;
and in which:
[1012] ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1013] 
[00022] [TABLE-US-00022]
  TABLE A-19
 
  Representative FW1 sequences (amino acid residues 5 to 26) for
  Nanobodies  of the KERE-group.
 
 
  GLEW FW1 sequence no. 6   SEQ ID NO: 69   VESGGGLVQPGGSLRLSCAASG
 
  GLEW FW1 sequence no. 7   SEQ ID NO: 70   EESGGGLAQPGGSLRLSCVASG
 
  GLEW FW1 sequence no. 8   SEQ ID NO: 71   VESGGGLALPGGSLTLSCVFSG
 

and in which:
[1014] iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of NANOBODIES of the GLEW-class; and in which:
[1015] iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[1016] The above NANOBODIES may for example be VHH sequences or may be humanized NANOBODIES. When the above NANOBODY sequences are VHH sequences, they may be suitably humanized, as further described herein. When the NANOBODIES are partially humanized NANOBODIES, they may optionally be further suitably humanized, again as described herein. In the above NANOBODIES, one or more of the further Hallmark residues are preferably as described herein (for example, when they are VHH sequences or partially humanized NANOBODIES).
[1017] A NANOBODY of the P, R, S103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which
[1018] i) the amino acid residue at position 103 according to the Kabat numbering is different from W;
and in which:
[1019] ii) preferably the amino acid residue at position 103 according to the Kabat numbering is P, R or S, and more preferably R;
and in which:
[1020] iii) FR1 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1021] 
[00023] [TABLE-US-00023]
  TABLE A-20
 
  Representative FW1 sequences for Nanobodies of the P, R, S 103-group.
 
 
  P, R, S 103 FW1 sequence no. 1   SEQ ID NO: 92   AVQLVESGGGLVQAGGSLRLSCAASGRTFS
 
  P, R, S 103 FW1 sequence no. 2   SEQ ID NO: 93   QVQLQESGGGMVQPGGSLRLSCAASGFDFG
 
  P, R, S 103 FW1 sequence no. 3   SEQ ID NO: 94   EVHLVESGGGLVRPGGSLRLSCAAFGFIFK
 
  P, R, S 103 FW1 sequence no. 4   SEQ ID NO: 95   QVQLAESGGGLVQPGGSLKLSCAASRTIVS
 
  P, R, S 103 FW1 sequence no. 5   SEQ ID NO: 96   QEHLVESGGGLVDIGGSLRLSCAASERIFS
 
  P, R, S 103 FW1 sequence no. 6   SEQ ID NO: 97   QVKLEESGGGLAQPGGSLRLSCVASGFTFS
 
  P, R, S 103 FW1 sequence no. 7   SEQ ID NO: 98   EVQLVESGGGLVQPGGSLRLSCVCVSSGCT
 
  P, R, S 103 FW1 sequence no. 8   SEQ ID NO: 99   EVQLVESGGGLALPGGSLTLSCVFSGSTFS
 

and in which
[1022] iv) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1023] 
[00024] [TABLE-US-00024]
  TABLE A-21
 
  Representative FW2 sequences for Nanobodies of the
  P, R, S 103-group.
 
 
  P, R, S 103 FW2 sequence no. 1   SEQ ID NO:102   WFRQAPGKEREFVA
 
  P, R, S 103 FW2 sequence no. 2   SEQ ID NO:103   WVRQAPGKVLEWVS
 
  P, R, S 103 FW2 sequence no. 3   SEQ ID NO:104   WVRRPPGKGLEWVS
 
  P, R, S 103 FW2 sequence no. 4   SEQ ID NO:105   WIRQAPGKEREGVS
 
  P, R, S 103 FW2 sequence no. 5   SEQ ID NO:106   WVRQYPGKEPEWVS
 
  P, R, S 103 FW2 sequence no. 6   SEQ ID NO:107   WFRQPPGKEHEFVA
 
  P, R, S 103 FW2 sequence no. 7   SEQ ID NO:108   WYRQAPGKRTELVA
 
  P, R, S 103 FW2 sequence no. 8   SEQ ID NO:109   WLRQAPGQGLEWVS
 
  P, R, S 103 FW2 sequence no. 9   SEQ ID NO:110   WLRQTPGKGLEWVG
 
  P, R, S 103 FW2 sequence no. 10   SEQ ID NO:111   WVRQAPGKAEEFVS
 

and in which:
[1024] v) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1025] 
[00025] [TABLE-US-00025]
  TABLE A-22
 
  Representative FW3 sequences for Nanobodies of the P ,R, S 103-group.
 
 
  P, R, S 103 FW3 sequence no. 1   SEQ ID NO: 112   RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA
 
  P, R, S 103 FW3 sequence no. 2   SEQ ID NO: 113   RFTISRDNARNTLYLQMDSLIPEDTALYYCAR
 
  P, R, S 103 FW3 sequence no. 3   SEQ ID NO: 114   RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA
 
  P, R, S 103 FW3 sequence no. 4   SEQ ID NO: 115   RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAA
 
  P, R, S 103 FW3 sequence no. 5   SEQ ID NO: 116   RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR
 
  P, R, S 103 FW3 sequence no. 6   SEQ ID NO: 117   RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNL
 
  P, R, S 103 FW3 sequence no. 7   SEQ ID NO: 118   RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR
 
  P, R, S 103 FW3 sequence no. 8   SEQ ID NO: 119   RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAV
 

and in which:
[1026] vi) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1027] 
[00026] [TABLE-US-00026]
  TABLE A-23
 
  Representative FW4 sequences for Nanobodies of the P, R, S 103-group.
 
 
  P, R, S 103 FW4 sequence no. 1   SEQ ID NO: 120   RGQGTQVTVSS
 
  P, R, S 103 FW4 sequence no. 2   SEQ ID NO: 121   LRGGTQVTVSS
 
  P, R, S 103 FW4 sequence no. 3   SEQ ID NO: 122   GNKGTLVTVSS
 
  P, R, S 103 FW4 sequence no. 4   SEQ ID NO: 123   SSPGTQVTVSS
 
  P, R, S 103 FW4 sequence no. 5   SEQ ID NO: 124   SSQGTLVTVSS
 
  P, R, S 103 FW4 sequence no. 6   SEQ ID NO: 125   RSRGIQVTVSS
 

and in which:
[1028] vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[1029] In the above NANOBODIES, one or more of the further Hallmark residues are preferably as described herein (for example, when they are VHH sequences or partially humanized NANOBODIES).
[1030] With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.
[1031] In view of this, a NANOBODY of the P,R,S103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:
[1032] i) the amino acid residue at position 103 according to the Kabat numbering is different from W;
and in which:
ii) preferably the amino acid residue at position 103 according to the Kabat numbering is P, R or S, and more preferably R;
and in which:
[1033] iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:
[1034] 
[00027] [TABLE-US-00027]
  TABLE A-24
 
  Representative FW1 sequences (amino acid residues 5 to 26) for
  Nanobodies of the P, R, S 103-group.
 
 
  P, R, S 103 FW1 sequence no. 9   SEQ ID NO: 100   VESGGGLVQAGGSLRLSCAASG
 
  P, R, S 103 FW1 sequence no. 10   SEQ ID NO: 101   AESGGGLVQPGGSLKLSCAASR
 

and in which:
[1035] iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of NANOBODIES of the P,R,S103 class;
and in which:
[1036] v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.
[1037] The above NANOBODIES may for example be VHH sequences or may be humanized NANOBODIES. When the above NANOBODY sequences are VHH sequences, they may be suitably humanized, as further described herein. When the NANOBODIES are partially humanized NANOBODIES, they may optionally be further suitably humanized, again as described herein.
[1038] In the above NANOBODIES, one or more of the further Hallmark residues are preferably as described herein (for example, when they are VHH sequences or partially humanized NANOBODIES).
[1039] In another preferred, but non-limiting aspect, the invention relates to a NANOBODY as described above, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said NANOBODY and one or more of the sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239, in which the amino acid residues that form the framework regions are disregarded. Such NANOBODIES can be as further described herein.
[1040] As already mentioned herein, another preferred but non-limiting aspect of the invention relates to a NANOBODY with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239.
[1041] Also, in the above NANOBODIES:
[1042] i) any amino acid substitution (when it is not a humanizing substitution as defined herein) is preferably, and compared to the corresponding amino acid sequence of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239, a conservative amino acid substitution, (as defined herein);
and/or:
[1043] ii) its amino acid sequence preferably contains either only amino acid substitutions, or otherwise preferably no more than 5, preferably no more than 3, and more preferably only 1 or 2 amino acid deletions or insertions, compared to the corresponding amino acid sequence of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239;
and/or
[1044] iii) the CDR's may be CDR's that are derived by means of affinity maturation, for example starting from the CDR's of to the corresponding amino acid sequence of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239.
[1045] Preferably, the CDR sequences and FR sequences in the NANOBODIES of the invention are such that the NANOBODIES of the invention (and polypeptides of the invention comprising the same):
[1046] bind to hCXCR4 with a dissociation constant (KD) of 10−5 to 10−12 moles/liter or less, and preferably 10−7 to 10−12 moles/liter or less and more preferably 10−8 to 10−12 moles/liter (i.e. with an association constant (KA) of 105 to 1012 liter/moles or more, and preferably 107 to 1012 liter/moles or more and more preferably 108 to 1012 liter/moles);
and/or such that they:
[1047] bind to hCXCR4 with a kon-rate of between 102 M−1s−1 to about 107 M−1s−1, preferably between 103 M−1s−1 and 107 M−1s−1, more preferably between 104 M−1s−1 and 107 M−1s−1, such as between 105 M−1s−1 and 107 M−1s−1;
and/or such that they:
[1048] bind to hCXCR4 with a koff rate between 1 s−1 (t1/2=0.69 s) and 10−6 s−1 (providing a near irreversible complex with a t1/2 of multiple days), preferably between 10−2 s−1 and 10−6 s−1, more preferably between 10−1 s−3 and 10−6 s−1, such as between 104 s−1 and 10−6 s−1.
[1049] Preferably, CDR sequences and FR sequences present in the NANOBODIES of the invention are such that the NANOBODIES of the invention will bind to Targets of the invention with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 μM.
[1050] In another preferred, but non-limiting aspect, the invention relates to a NANOBODY as described above, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said NANOBODY and one or more of the sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311, in which the amino acid residues that form the framework regions are disregarded. Such NANOBODIES can be as further described herein.
[1051] As already mentioned herein, another preferred but non-limiting aspect of the invention relates to a NANOBODY with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311.
[1052] Also, in the above NANOBODIES:
[1053] i) any amino acid substitution (when it is not a humanizing substitution as defined herein) is preferably, and compared to the corresponding amino acid sequence of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311, a conservative amino acid substitution, (as defined herein);
and/or:
[1054] ii) its amino acid sequence preferably contains either only amino acid substitutions, or otherwise preferably no more than 5, preferably no more than 3, and more preferably only 1 or 2 amino acid deletions or insertions, compared to the corresponding amino acid sequence of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311;
and/or
[1055] iii) the CDR's may be CDR's that are derived by means of affinity maturation, for example starting from the CDR's of to the corresponding amino acid sequence of SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311.
[1056] Preferably, the CDR sequences and FR sequences in the NANOBODIES of the invention are such that the NANOBODIES of the invention (and polypeptides of the invention comprising the same):
[1057] bind to hCD4 with a dissociation constant (KD) of 10−5 to 10−12 moles/liter or less, and preferably 10−7 to 10−12 moles/liter or less and more preferably 10−8 to 10−12 moles/liter (i.e. with an association constant (KA) of 105 to 1012 liter/moles or more, and preferably 107 to 1012 liter/moles or more and more preferably 108 to 1012 liter/moles);
and/or such that they:
[1058] bind to hCD4 with a kon-rate of between 102 M−1s−1 to about 107 M−1s−1, preferably between 103 M−1s−1 and 107 M−1s−1, more preferably between 104 M−1s−1 and 107 M−1s−1 such as between 105 M−1s−1 and 107 M−1s−1;
and/or such that they:
[1059] bind to hCD4 with a koff rate between 1 s−1 (t1/2=0.69 s) and 10−6 s−1 (providing a near irreversible complex with a t1/2 of multiple days), preferably between 10−2 s−1 and 10−6 s−1, more preferably between 10−3 s−1 and 10−6 s−1, such as between 104 s−1 and 10−6 s−1,
[1060] Preferably, CDR sequences and FR sequences present in the NANOBODIES of the invention are such that the NANOBODIES of the invention will bind to Targets of the invention with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.
[1061] According to one non-limiting aspect of the invention, a NANOBODY may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring human VH domain, and in particular compared to the corresponding framework region of DP-47. More specifically, according to one non-limiting aspect of the invention, a NANOBODY may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring human VH domain, and in particular compared to the corresponding framework region of DP-47. Usually, a NANOBODY will have at least one such amino acid difference with a naturally occurring VH domain in at least one of FR2 and/or FR4, and in particular at at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).
[1062] Also, a humanized NANOBODY of the invention may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring VHH domain. More specifically, according to one non-limiting aspect of the invention, a humanized NANOBODY may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring VHH domain. Usually, a humanized NANOBODY will have at least one such amino acid difference with a naturally occurring VHH domain in at least one of FR2 and/or FR4, and in particular at at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).
[1063] As will be clear from the disclosure herein, it is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “analogs”) of the NANOBODIES of the invention as defined herein, and in particular analogs of the NANOBODIES of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311. Thus, according to one aspect of the invention, the term “NANOBODY of the invention” in its broadest sense also covers such analogs.
[1064] Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the NANOBODIES of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDR's. When such substitutions, insertions or deletions are made in one or more of the framework regions, they may be made at one or more of the Hallmark residues and/or at one or more of the other positions in the framework residues, although substitutions, insertions or deletions at the Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).
[1065] By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another VHH domain (see Tables A-5 to A-8 for some non-limiting examples of such substitutions), although the invention is generally not limited thereto. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the NANOBODY of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the NANOBODY of the invention (i.e. to the extent that the NANOBODY is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the NANOBODIES thus obtained.
[1066] For example, and depending on the host organism used to express the NANOBODY or polypeptide of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation (again as described herein).
[1067] As can be seen from the data on the VHH entropy and VHH variability given in Tables A-5 to A-8 above, some amino acid residues in the framework regions are more conserved than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions.
[1068] The analogs are preferably such that they can bind to Targets of the invention with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein for the NANOBODIES of the invention.
[1069] The analogs are preferably also such that they retain the favourable properties the NANOBODIES, as described herein.
[1070] Also, according to one preferred aspect, the analogs have a degree of sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95% or 99% or more; and/or preferably have at most 20, preferably at most 10, even more preferably at most 5, such as 4, 3, 2 or only 1 amino acid difference (as defined herein), with one of the NANOBODIES of SEQ ID NOs: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311.
[1071] Also, the framework sequences and CDR's of the analogs are preferably such that they are in accordance with the preferred aspects defined herein. More generally, as described herein, the analogs will have (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103.
[1072] One preferred class of analogs of the NANOBODIES of the invention comprise NANOBODIES that have been humanized (i.e. compared to the sequence of a naturally occurring NANOBODY of the invention). As mentioned in the background art cited herein, such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring VHH with the amino acid residues that occur at the same position in a human VH domain, such as a human VH3 domain. Examples of possible humanizing substitutions or combinations of humanizing substitutions will be clear to the skilled person, for example from the Tables herein, from the possible humanizing substitutions mentioned in the background art cited herein, and/or from a comparison between the sequence of a NANOBODY and the sequence of a naturally occurring human VH domain.
[1073] The humanizing substitutions should be chosen such that the resulting humanized NANOBODIES still retain the favourable properties of NANOBODIES as defined herein, and more preferably such that they are as described for analogs in the preceding paragraphs. A skilled person will generally be able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible humanizing substitutions and determining their influence on the properties of the NANOBODIES thus obtained.
[1074] Generally, as a result of humanization, the NANOBODIES of the invention may become more “human-like”, while still retaining the favorable properties of the NANOBODIES of the invention as described herein. As a result, such humanized NANOBODIES may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. Again, based on the disclosure herein and optionally after a limited degree of routine experimentation, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring VHH domains on the other hand.
[1075] The NANOBODIES of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined herein) or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof.
[1076] One preferred humanizing substitution for NANOBODIES of the “P,R,S-103 group” or the “KERE group” is Q108 into L108. NANOBODIES of the “GLEW class” may also be humanized by a Q108 into L108 substitution, provided at least one of the other Hallmark residues contains a camelid (camelizing) substitution (as defined herein). For example, as mentioned above, one particularly preferred class of humanized NANOBODIES has GLEW or a GLEW-like sequence at positions 44-47; P, R or S (and in particular R) at position 103, and an L at position 108.
[1077] The humanized and other analogs, and nucleic acid sequences encoding the same, can be provided in any manner known per se. For example, the analogs can be obtained by providing a nucleic acid that encodes a naturally occurring VHH domain, changing the codons for the one or more amino acid residues that are to be substituted into the codons for the corresponding desired amino acid residues (e.g. by site-directed mutagenesis or by PCR using suitable mismatch primers), expressing the nucleic acid/nucleotide sequence thus obtained in a suitable host or expression system; and optionally isolating and/or purifying the analog thus obtained to provide said analog in essentially isolated form (e.g. as further described herein). This can generally be performed using methods and techniques known per se, which will be clear to the skilled person, for example from the handbooks and references cited herein, the background art cited herein and/or from the further description herein. Alternatively, a nucleic acid encoding the desired analog can be synthesized in a manner known per se (for example using an automated apparatus for synthesizing nucleic acid sequences with a predefined amino acid sequence) and can then be expressed as described herein. Yet another technique may involve combining one or more naturally occurring and/or synthetic nucleic acid sequences each encoding a part of the desired analog, and then expressing the combined nucleic acid sequence as described herein. Also, the analogs can be provided using chemical synthesis of the pertinent amino acid sequence using techniques for peptide synthesis known per se, such as those mentioned herein.
[1078] In this respect, it will be also be clear to the skilled person that the NANOBODIES of the invention (including their analogs) can be designed and/or prepared starting from human VH sequences (i.e. amino acid sequences or the corresponding nucleotide sequences), such as for example from human VH3 sequences such as DP-47, DP-51 or DP-29, i.e. by introducing one or more camelizing substitutions (i.e. changing one or more amino acid residues in the amino acid sequence of said human VH domain into the amino acid residues that occur at the corresponding position in a VHH domain), so as to provide the sequence of a NANOBODY of the invention and/or so as to confer the favourable properties of a NANOBODY to the sequence thus obtained. Again, this can generally be performed using the various methods and techniques referred to in the previous paragraph, using an amino acid sequence and/or nucleotide sequence for a human VH domain as a starting point.
[1079] Some preferred, but non-limiting camelizing substitutions can be derived from Tables A-5-A-8. It will also be clear that camelizing substitutions at one or more of the Hallmark residues will generally have a greater influence on the desired properties than substitutions at one or more of the other amino acid positions, although both and any suitable combination thereof are included within the scope of the invention. For example, it is possible to introduce one or more camelizing substitutions that already confer at least some the desired properties, and then to introduce further camelizing substitutions that either further improve said properties and/or confer additional favourable properties. Again, the skilled person will generally be able to determine and select suitable camelizing substitutions or suitable combinations of camelizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible camelizing substitutions and determining whether the favourable properties of NANOBODIES are obtained or improved (i.e. compared to the original VH domain).
[1080] Generally, however, such camelizing substitutions are preferably such that the resulting an amino acid sequence at least contains (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably also an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103; and optionally one or more further camelizing substitutions. More preferably, the camelizing substitutions are such that they result in a NANOBODY of the invention and/or in an analog thereof (as defined herein), such as in a humanized analog and/or preferably in an analog that is as defined in the preceding paragraphs.
[1081] As will also be clear from the disclosure herein, it is also within the scope of the invention to use parts or fragments, or combinations of two or more parts or fragments, of the NANOBODIES of the invention as defined herein, and in particular parts or fragments of the NANOBODIES of SEQ ID NO's: 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311. Thus, according to one aspect of the invention, the term “NANOBODY of the invention” in its broadest sense also covers such parts or fragments.
[1082] Generally, such parts or fragments of the NANOBODIES of the invention (including analogs thereof) have amino acid sequences in which, compared to the amino acid sequence of the corresponding full length NANOBODY of the invention (or analog thereof), one or more of the amino acid residues at the N-terminal end, one or more amino acid residues at the C-terminal end, one or more contiguous internal amino acid residues, or any combination thereof, have been deleted and/or removed.
[1083] The parts or fragments are preferably such that they can bind to Targets of the invention with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein for the NANOBODIES of the invention.
[1084] Any part or fragment is preferably such that it comprises at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, more preferably at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues, of the amino acid sequence of the corresponding full length NANOBODY of the invention.
[1085] Also, any part or fragment is such preferably that it comprises at least one of CDR1, CDR2 and/or CDR3 or at least part thereof (and in particular at least CDR3 or at least part thereof). More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least one other CDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connected by suitable framework sequence(s) or at least part thereof. More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least part of the two remaining CDR's, again preferably connected by suitable framework sequence(s) or at least part thereof.
[1086] According to another particularly preferred, but non-limiting aspect, such a part or fragment comprises at least CDR3, such as FR3, CDR3 and FR4 of the corresponding full length NANOBODY of the invention, i.e. as for example described in the International application WO 03/050531 (Lasters et al.).
[1087] As already mentioned above, it is also possible to combine two or more of such parts or fragments (i.e. from the same or different NANOBODIES of the invention), i.e. to provide an analog (as defined herein) and/or to provide further parts or fragments (as defined herein) of a NANOBODY of the invention. It is for example also possible to combine one or more parts or fragments of a NANOBODY of the invention with one or more parts or fragments of a human VH domain.
[1088] According to one preferred aspect, the parts or fragments have a degree of sequence identity of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, such as at least 90%, 95% or 99% or more with one of the NANOBODIES of SEQ ID NOs 238 to 253, more preferably SEQ ID NO: 238 and SEQ ID NO: 239 and SEQ ID NO's: 308 to 311, more preferably SEQ ID NO: 311.
[1089] The parts and fragments, and nucleic acid sequences encoding the same, can be provided and optionally combined in any manner known per se. For example, such parts or fragments can be obtained by inserting a stop codon in a nucleic acid that encodes a full-sized NANOBODY of the invention, and then expressing the nucleic acid thus obtained in a manner known per se (e.g. as described herein). Alternatively, nucleic acids encoding such parts or fragments can be obtained by suitably restricting a nucleic acid that encodes a full-sized NANOBODY of the invention or by synthesizing such a nucleic acid in a manner known per se. Parts or fragments may also be provided using techniques for peptide synthesis known per se.
[1090] The invention in its broadest sense also comprises derivatives of the NANOBODIES of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g. enzymatical) modification, of the NANOBODIES of the invention and/or of one or more of the amino acid residues that form the NANOBODIES of the invention.
[1091] Examples of such modifications, as well as examples of amino acid residues within the NANOBODY sequence that can be modified in such a manner (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person.
[1092] For example, such a modification may involve the introduction (e.g. by covalent linking or in an other suitable manner) of one or more functional groups, residues or moieties into or onto the NANOBODY of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the NANOBODY of the invention. Example of such functional groups will be clear to the skilled person.
[1093] For example, such modification may comprise the introduction (e.g. by covalent binding or in any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or the absorption of the NANOBODY of the invention, that reduce the immunogenicity and/or the toxicity of the NANOBODY of the invention, that eliminate or attenuate any undesirable side effects of the NANOBODY of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties of the NANOBODIES and/or polypeptides of the invention; or any combination of two or more of the foregoing. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv's and single domain antibodies), for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functional groups may for example be linked directly (for example covalently) to a NANOBODY of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.
[1094] One of the most widely used techniques for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv's); reference is made to for example Chapman, Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA.
[1095] Preferably, site-directed pegylation is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a NANOBODY of the invention, a NANOBODY of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of a NANOBODY of the invention, all using techniques of protein engineering known per se to the skilled person.
[1096] Preferably, for the NANOBODIES and proteins of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example in the range of 20,000-80,000.
[1097] Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the NANOBODY or polypeptide of the invention.
[1098] Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled NANOBODY. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as 152Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes (such as 3H, 125I, 32P, 35S, 14C, 51Cr, 36Cl, 57C, 58Co, 59Fe, and 75Se), metals, metal chelates or metallic cations (for example metallic cations such as 99mTc, 123I, 111In, 131I, 97Ru, 67Cu, 67Ga, and 68Ga or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, such as (157Gd, 55Mn, 162Dy, 52Cr, and 56Fe), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy.
[1099] Such labelled NANOBODIES and polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other “sandwich assays”, etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.
[1100] As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, diethyl-enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[1101] Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the NANOBODY of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a NANOBODY of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated NANOBODY may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the NANOBODY of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposomal formulations described by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the NANOBODY of the invention.
[1102] For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the NANOBODIES of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation such a cell, the NANOBODIES of the invention may also be linked to a toxin or to a toxic residue or moiety. Examples of toxic moieties, compounds or residues which can be linked to a NANOBODY of the invention to provide—for example—a cytotoxic compound will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.
[1103] Other potential chemical and enzymatical modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g. to study function-activity relationships). Reference is for example made to Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).
[1104] Preferably, the derivatives are such that they bind to Targets of the invention with an affinity (suitably measured and/or expressed as a KD-value (actual or apparent), a KA-value (actual or apparent), a kon-rate and/or a koff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein for the NANOBODIES of the invention.
[1105] As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of or comprise at least one NANOBODY of the invention. By “essentially consist of” is meant that the amino acid sequence of the polypeptide of the invention either is exactly the same as the amino acid sequence of a NANOBODY of the invention or corresponds to the amino acid sequence of a NANOBODY of the invention which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the amino acid sequence of the NANOBODY.
[1106] Said amino acid residues may or may not change, alter or otherwise influence the (biological) properties of the NANOBODY and may or may not add further functionality to the NANOBODY. For example, such amino acid residues:
[1107] can comprise an N-terminal Met residue, for example as result of expression in a heterologous host cell or host organism.
[1108] may form a signal sequence or leader sequence that directs secretion of the NANOBODY from a host cell upon synthesis. Suitable secretory leader peptides will be clear to the skilled person, and may be as further described herein. Usually, such a leader sequence will be linked to the N-terminus of the NANOBODY, although the invention in its broadest sense is not limited thereto;
[1109] may form a sequence or signal that allows the NANOBODY to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the NANOBODY to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such amino acid sequences will be clear to the skilled person. Some non-limiting examples are the small peptide vectors (“Pep-trans vectors”) described in WO 03/026700 and in Temsamani et al., Expert Opin. Biol. Ther., 1, 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9, 1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131 (2001), and the membrane translocator sequence described by Zhao et al., Apoptosis, 8, 631-637 (2003). C-terminal and N-terminal amino acid sequences for intracellular targeting of antibody fragments are for example described by Cardinale et al., Methods, 34, 171 (2004). Other suitable techniques for intracellular targeting involve the expression and/or use of so-called “intrabodies” comprising a NANOBODY of the invention, as mentioned below;
[1110] may form a “tag”, for example an amino acid sequence or residue that allows or facilitates the purification of the NANOBODY, for example using affinity techniques directed against said sequence or residue. Thereafter, said sequence or residue may be removed (e.g. by chemical or enzymatical cleavage) to provide the NANOBODY sequence (for this purpose, the tag may optionally be linked to the NANOBODY sequence via a cleavable linker sequence or contain a cleavable motif). Some preferred, but non-limiting examples of such residues are multiple histidine residues, glutathione residues and a myc-tag (see for example SEQ ID NO:31 of WO 06/12282).
[1111] may be one or more amino acid residues that have been functionalized and/or that can serve as a site for attachment of functional groups. Suitable amino acid residues and functional groups will be clear to the skilled person and include, but are not limited to, the amino acid residues and functional groups mentioned herein for the derivatives of the NANOBODIES of the invention.
[1112] According to another aspect, a polypeptide of the invention comprises a NANOBODY of the invention, which is fused at its amino terminal end, at its carboxy terminal end, or both at its amino terminal end and at its carboxy terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein comprising said NANOBODY of the invention and the one or more further amino acid sequences. Such a fusion will also be referred to herein as a “NANOBODY fusion”.
[1113] The one or more further amino acid sequence may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the NANOBODY, and may or may not add further functionality to the NANOBODY or the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the NANOBODY or the polypeptide of the invention.
[1114] For example, the further amino acid sequence may also provide a second binding site, which binding site may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including but not limited to the same protein, polypeptide, antigen, antigenic determinant or epitope against which the NANOBODY of the invention is directed, or a different protein, polypeptide, antigen, antigenic determinant or epitope).
[1115] Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv's and single domain antibodies). Reference is for example made to the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).
[1116] For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptides of the invention, compared to the NANOBODY of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).
[1117] In particular, it has been described in the art that linking fragments of immunoglobulins (such as VH domains) to serum albumin or to fragments thereof can be used to increase the half-life. Reference is for made to WO 00/27435 and WO 01/077137). According to the invention, the NANOBODY of the invention is preferably either directly linked to serum albumin (or to a suitable fragment thereof) or via a suitable linker, and in particular via a suitable peptide linked so that the polypeptide of the invention can be expressed as a genetic fusion (protein). According to one specific aspect, the NANOBODY of the invention may be linked to a fragment of serum albumin that at least comprises the domain III of serum albumin or part thereof. Reference is for example made to WO 07/112,940.
[1118] Alternatively, the further amino acid sequence may provide a second binding site or binding unit that is directed against a serum protein (such as, for example, human serum albumin or another serum protein such as IgG), so as to provide increased half-life in serum. Such amino acid sequences for example include the NANOBODIES described below, as well as the small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO 04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, as well as to EP 0 368 684, as well as to WO 08/028,977, WO 08/043,821, WO 08/043,822.
[1119] Such amino acid sequences may in particular be directed against serum albumin (and more in particular human serum albumin) and/or against IgG (and more in particular human IgG). For example, such amino acid sequences may be amino acid sequences that are directed against (human) serum albumin and amino acid sequences that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787) and/or amino acid sequences that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see again for example WO 06/0122787); amino acid sequences that have or can provide an increased half-life (see for example WO 08/028,977); amino acid sequences against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), reference is again made to WO 08/028,977; amino acid sequences that can bind to serum albumin in a pH independent manner (see for example WO 08/043,821) and/or amino acid sequences that are conditional binders (see for example WO 08/043,822).
[1120] According to another aspect, the one or more further amino acid sequences may comprise one or more parts, fragments or domains of conventional 4-chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, although usually less preferred, a NANOBODY of the invention may be linked to a conventional (preferably human) VH or VL domain or to a natural or synthetic analog of a VH or VL domain, again optionally via a linker sequence (including but not limited to other (single) domain antibodies, such as the dAb's described by Ward et al.).
[1121] The at least one NANOBODY may also be linked to one or more (preferably human) CH1, CH2 and/or CH3 domains, optionally via a linker sequence. For instance, a NANOBODY linked to a suitable CH1 domain could for example be used—together with suitable light chains—to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab′)2 fragments, but in which one or (in case of an F(ab′)2 fragment) one or both of the conventional VH domains have been replaced by a NANOBODY of the invention. Also, two NANOBODIES could be linked to a CH3 domain (optionally via a linker) to provide a construct with increased half-life in vivo.
[1122] According to one specific aspect of a polypeptide of the invention, one or more NANOBODIES of the invention may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more CH2 and/or CH3 domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and/or may form (part of) and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 or IgG4), from IgE or from another human Ig such as IgA, IgD or IgM. For example, WO 94/04678 describes heavy chain antibodies comprising a Camelid VHH domain or a humanized derivative thereof (i.e. a NANOBODY), in which the Camelidae CH2 and/or CH3 domain have been replaced by human CH2 and CH3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a NANOBODY and human CH2 and CH3 domains (but no CH1 domain), which immunoglobulin has the effector function provided by the CH2 and CH3 domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the NANOBODIES of the invention so as to provide an effector function will be clear to the skilled person, and may be chosen on the basis of the desired effector function(s). Reference is for example made to WO 04/058820, WO 99/42077, WO 02/056910, WO 09/068,628 and WO 05/017148, as well as the review by Holliger and Hudson, supra. Coupling of a NANOBODY of the invention to an Fc portion may also lead to an increased half-life, compared to the corresponding NANOBODY of the invention. For some applications, the use of an Fc portion and/or of constant domains (i.e. CH2 and/or CH3 domains) that confer increased half-life without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more NANOBODIES and one or more constant domains with increased half-life in vivo will be clear to the skilled person, and may for example comprise two NANOBODIES linked to a CH3 domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weight of more than 50 kD, the cut-off value for renal absorption.
[1123] In another one specific, but non-limiting, aspect, in order to form a polypeptide of the invention, one or more amino acid sequences of the invention may be linked (optionally via a suitable linker or hinge region) to naturally occurring, synthetic or semisynthetic constant domains (or analogs, variants, mutants, parts or fragments thereof) that have a reduced (or essentially no) tendency to self-associate into dimers (i.e. compared to constant domains that naturally occur in conventional 4-chain antibodies). Such monomeric (i.e. not self-associating) Fc chain variants, or fragments thereof, will be clear to the skilled person. For example, Helm et al., J Biol Chem 1996 271 7494, describe monomeric Fce chain variants that can be used in the polypeptide chains of the invention.
[1124] Also, such monomeric Fc chain variants are preferably such that they are still capable of binding to the complement or the relevant Fc receptor(s) (depending on the Fc portion from which they are derived), and/or such that they still have some or all of the effector functions of the Fc portion from which they are derived (or at a reduced level still suitable for the intended use). Alternatively, in such a polypeptide chain of the invention, the monomeric Fc chain may be used to confer increased half-life upon the polypeptide chain, in which case the monomeric Fc chain may also have no or essentially no effector functions.
[1125] Bivalent/multivalent, bispecific/multispecific or biparatopic/multiparatopic polypeptides of the invention may also be linked to Fc portions, in order to provide polypeptide constructs of the type that is described in WO 09/068,630.
[1126] The further amino acid sequences may also form a signal sequence or leader sequence that directs secretion of the NANOBODY or the polypeptide of the invention from a host cell upon synthesis (for example to provide a pre-, pro- or prepro-form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention).
[1127] The further amino acid sequence may also form a sequence or signal that allows the NANOBODY or polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the NANOBODY or polypeptide of the invention to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Suitable examples of such amino acid sequences will be clear to the skilled person, and for example include, but are not limited to, the “Peptrans” vectors mentioned above, the sequences described by Cardinale et al. and the amino acid sequences and antibody fragments known per se that can be used to express or produce the NANOBODIES and polypeptides of the invention as so-called “intrabodies”, for example as described in WO 94/02610, WO 95/22618, U.S. Pat. No. 7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag; and in Kontermann, Methods 34, (2004), 163-170, and the further references described therein.
[1128] For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the NANOBODIES of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation of such a cell, the NANOBODIES of the invention may also be linked to a (cyto)toxic protein or polypeptide. Examples of such toxic proteins and polypeptides which can be linked to a NANOBODY of the invention to provide—for example—a cytotoxic polypeptide of the invention will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.
[1129] According to one preferred, but non-limiting aspect, said one or more further amino acid sequences comprise at least one further NANOBODY, so as to provide a polypeptide of the invention that comprises at least two, such as three, four, five or more NANOBODIES, in which said NANOBODIES may optionally be linked via one or more linker sequences (as defined herein). Polypeptides of the invention that comprise two or more NANOBODIES, of which at least one is a NANOBODY of the invention, will also be referred to herein as “multivalent” polypeptides of the invention, and the NANOBODIES present in such polypeptides will also be referred to herein as being in a “multivalent format”. For example a “bivalent” polypeptide of the invention comprises two NANOBODIES, optionally linked via a linker sequence, whereas a “trivalent” polypeptide of the invention comprises three NANOBODIES, optionally linked via two linker sequences; etc.; in which at least one of the NANOBODIES present in the polypeptide, and up to all of the NANOBODIES present in the polypeptide, is/are a NANOBODY of the invention.
[1130] In a multivalent polypeptide of the invention, the two or more NANOBODIES may be the same or different, and may be directed against the same antigen or antigenic determinant (for example against the same part(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. For example, a bivalent polypeptide of the invention may comprise (a) two identical NANOBODIES; (b) a first NANOBODY directed against a first antigenic determinant of a protein or antigen and a second NANOBODY directed against the same antigenic determinant of said protein or antigen which is different from the first NANOBODY; (c) a first NANOBODY directed against a first antigenic determinant of a protein or antigen and a second NANOBODY directed against another antigenic determinant of said protein or antigen; or (d) a first NANOBODY directed against a first protein or antigen and a second NANOBODY directed against a second protein or antigen (i.e. different from said first antigen). Similarly, a trivalent polypeptide of the invention may, for example and without being limited thereto. comprise (a) three identical NANOBODIES; (b) two identical NANOBODY against a first antigenic determinant of an antigen and a third NANOBODY directed against a different antigenic determinant of the same antigen; (c) two identical NANOBODY against a first antigenic determinant of an antigen and a third NANOBODY directed against a second antigen different from said first antigen; (d) a first NANOBODY directed against a first antigenic determinant of a first antigen, a second NANOBODY directed against a second antigenic determinant of said first antigen and a third NANOBODY directed against a second antigen different from said first antigen; or (e) a first NANOBODY directed against a first antigen, a second NANOBODY directed against a second antigen different from said first antigen, and a third NANOBODY directed against a third antigen different from said first and second antigen.
[1131] Multiparatopic
[1132] Polypeptides of the invention that contain at least two NANOBODIES, in which at least one NANOBODY is directed against a first antigen (i.e. against Targets of the invention,) and at least one NANOBODY is directed against a second antigen (i.e. different from Targets of the invention), will also be referred to as “multispecific” polypeptides of the invention, and the NANOBODIES present in such polypeptides will also be referred to herein as being in a “multispecific format”. Thus, for example, a “bispecific” polypeptide of the invention is a polypeptide that comprises at least one NANOBODY directed against a first antigen (i.e. Targets of the invention,) and at least one further NANOBODY directed against a second antigen (i.e. different from Targets of the invention), whereas a “trispecific” polypeptide of the invention is a polypeptide that comprises at least one NANOBODY directed against a first antigen (i.e. Targets of the invention), at least one further NANOBODY directed against a second antigen (i.e. different from Targets of the invention,) and at least one further NANOBODY directed against a third antigen (i.e. different from both Targets of the invention, and the second antigen); etc.
[1133] Accordingly, in its simplest form, a bispecific polypeptide of the invention is a bivalent polypeptide of the invention (as defined herein), comprising a first NANOBODY directed against Targets of the invention, and a second NANOBODY directed against a second antigen, in which said first and second NANOBODY may optionally be linked via a linker sequence (as defined herein); whereas a trispecific polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined herein), comprising a first NANOBODY directed against Targets of the invention, a second NANOBODY directed against a second antigen and a third NANOBODY directed against a third antigen, in which said first, second and third NANOBODY may optionally be linked via one or more, and in particular one and more, in particular two, linker sequences.
[1134] However, as will be clear from the description hereinabove, the invention is not limited thereto, in the sense that a multispecific polypeptide of the invention may comprise at least one NANOBODY against Targets of the invention, and any number of NANOBODIES directed against one or more antigens different from Targets of the invention.
[1135] Furthermore, although it is encompassed within the scope of the invention that the specific order or arrangement of the various NANOBODIES in the polypeptides of the invention may have some influence on the properties of the final polypeptide of the invention (including but not limited to the affinity, specificity or avidity for Targets of the invention, or against the one or more other antigens), said order or arrangement is usually not critical and may be suitably chosen by the skilled person, optionally after some limited routine experiments based on the disclosure herein. Thus, when reference is made to a specific multivalent or multispecific polypeptide of the invention, it should be noted that this encompasses any order or arrangements of the relevant NANOBODIES, unless explicitly indicated otherwise.
[1136] Finally, it is also within the scope of the invention that the polypeptides of the invention contain two or more NANOBODIES and one or more further amino acid sequences (as mentioned herein).
[1137] For multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to for example WO 96/34103 and WO 99/23221. Some other examples of some specific multispecific and/or multivalent polypeptide of the invention can be found in the applications by Ablynx N.V. referred to herein.
[1138] One preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one NANOBODY of the invention and at least one NANOBODY that provides for an increased half-life. Such NANOBODIES may for example be NANOBODIES that are directed against a serum protein, and in particular a human serum protein, such as human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one of the serum proteins listed in WO 04/003019. Of these, NANOBODIES that can bind to serum albumin (and in particular human serum albumin) or to IgG (and in particular human IgG, see for example NANOBODY VH-1 described in the review by Muyldermans, supra) are particularly preferred (although for example, for experiments in mice or primates, NANOBODIES against or cross-reactive with mouse serum albumin (MSA) or serum albumin from said primate, respectively, can be used. However, for pharmaceutical use, NANOBODIES against human serum albumin or human IgG will usually be preferred). NANOBODIES that provide for increased half-life and that can be used in the polypeptides of the invention include the NANOBODIES directed against serum albumin that are described in WO 04/041865, in WO 06/122787 and in the further patent applications by Ablynx N.V., such as those mentioned above.
[1139] For example, the some preferred NANOBODIES that provide for increased half-life for use in the present invention include NANOBODIES that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787); NANOBODIES that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see for example WO 06/0122787); NANOBODIES that have or can provide an increased half-life (see for example WO 08/028,977); NANOBODIES against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) (see for example WO 08/028,977); NANOBODIES that can bind to serum albumin in a pH independent manner (see for example WO 08/04382) and/or NANOBODIES that are conditional binders (see for example WO 08/043,822).
[1140] Some particularly preferred NANOBODIES that provide for increased half-life and that can be used in the polypeptides of the invention include the NANOBODIES ALB-1 to ALB-10 disclosed in WO 06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) is particularly preferred.
[1141] According to a specific, but non-limiting aspect of the invention, the polypeptides of the invention contain, besides the one or more NANOBODIES of the invention, at least one NANOBODY against human serum albumin.
[1142] Generally, any polypeptides of the invention with increased half-life that contain one or more NANOBODIES of the invention, and any derivatives of NANOBODIES of the invention or of such polypeptides that have an increased half-life, preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding NANOBODY of the invention per se. For example, such a derivative or polypeptides with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding NANOBODY of the invention per se.
[1143] In a preferred, but non-limiting aspect of the invention, such derivatives or polypeptides may exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, such derivatives or polypeptides may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).
[1144] According to one aspect of the invention the polypeptides are capable of binding to one or more molecules which can increase the half-life of the polypeptide in vivo.
[1145] The polypeptides of the invention are stabilised in vivo and their half-life increased by binding to molecules which resist degradation and/or clearance or sequestration. Typically, such molecules are naturally occurring proteins which themselves have a long half-life in vivo.
[1146] Another preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one NANOBODY of the invention and at least one NANOBODY that directs the polypeptide of the invention towards, and/or that allows the polypeptide of the invention to penetrate or to enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the NANOBODY to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such NANOBODIES include NANOBODIES that are directed towards specific cell-surface proteins, markers or epitopes of the desired organ, tissue or cell (for example cell-surface markers associated with tumor cells), and the single-domain brain targeting antibody fragments described in WO 02/057445 and WO 06/040153, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.
[1147] In the polypeptides of the invention, the one or more NANOBODIES and the one or more polypeptides may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.
[1148] Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be clear to the skilled person, and may generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing proteins or polypeptides that are intended for pharmaceutical use.
[1149] Some particularly preferred spacers include the spacers and linkers that are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, its should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent VH and VL domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each NANOBODY by itself forms a complete antigen-binding site).
[1150] For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (glyxsery)z, such as (for example (gly4ser)3 or (gly3ser2)3, as described in WO 99/42077 and the GS30, GS15, GS9 and GS7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/040153 and WO 06/122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678).
[1151] Some other particularly preferred linkers are poly-alanine (such as AAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).
[1152] Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, poly(ethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026.
[1153] It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for Targets of the invention, or for one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
[1154] For example, in multivalent polypeptides of the invention that comprise NANOBODIES directed against a multimeric antigen (such as a multimeric receptor or other protein), the length and flexibility of the linker are preferably such that it allows each NANOBODY of the invention present in the polypeptide to bind to the antigenic determinant on each of the subunits of the multimer. Similarly, in a multispecific polypeptide of the invention that comprises NANOBODIES directed against two or more different antigenic determinants on the same antigen (for example against different epitopes of an antigen and/or against different subunits of a multimeric receptor, channel or protein), the length and flexibility of the linker are preferably such that it allows each NANOBODY to bind to its intended antigenic determinant. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
[1155] It is also within the scope of the invention that the linker(s) used confer one or more other favourable properties or functionality to the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g. as described herein for the derivatives of the NANOBODIES of the invention). For example, linkers containing one or more charged amino acid residues (see Table A-2 above) can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
[1156] Finally, when two or more linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.
[1157] Usually, for easy of expression and production, a polypeptide of the invention will be a linear polypeptide. However, the invention in its broadest sense is not limited thereto. For example, when a polypeptide of the invention comprises three of more NANOBODIES, it is possible to link them by use of a linker with three or more “arms”, which each “arm” being linked to a NANOBODY, so as to provide a “star-shaped” construct. It is also possible, although usually less preferred, to use circular constructs.
[1158] The invention also comprises derivatives of the polypeptides of the invention, which may be essentially analogous to the derivatives of the NANOBODIES of the invention, i.e. as described herein.
[1159] The invention also comprises proteins or polypeptides that “essentially consist” of a polypeptide of the invention (in which the wording “essentially consist of” has essentially the same meaning as indicated hereinabove).
[1160] According to one aspect of the invention, the polypeptide of the invention is in essentially isolated from, as defined herein.
[1161] The amino acid sequences, NANOBODIES, polypeptides and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from the further description herein. For example, the NANOBODIES and polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for preparing the amino acid sequences, NANOBODIES, polypeptides and nucleic acids include the methods and techniques described herein.
[1162] As will be clear to the skilled person, one particularly useful method for preparing an amino acid sequence, NANOBODY and/or a polypeptide of the invention generally comprises the steps of:
[1163] i) the expression, in a suitable host cell or host organism (also referred to herein as a “host of the invention”) or in another suitable expression system of a nucleic acid that encodes said amino acid sequence, NANOBODY or polypeptide of the invention (also referred to herein as a “nucleic acid of the invention”), optionally followed by:
[1164] ii) isolating and/or purifying the amino acid sequence, NANOBODY or polypeptide of the invention thus obtained.
[1165] In particular, such a method may comprise the steps of:
[1166] i) cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one amino acid sequence, NANOBODY and/or polypeptide of the invention; optionally followed by:
[1167] ii) isolating and/or purifying the amino acid sequence, NANOBODY or polypeptide of the invention thus obtained.
[1168] A nucleic acid of the invention can be in the form of single or double stranded DNA or RNA, and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism).
[1169] According to one aspect of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein.
[1170] The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.
[1171] The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. To provide analogs, nucleotide sequences encoding naturally occurring VHH domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analog. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least one nucleotide sequence encoding a NANOBODY and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.
[1172] Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more “mismatched” primers, using for example a sequence of a naturally occurring form of Targets of the invention as a template. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as the Examples below.
[1173] The nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as “genetic constructs of the invention”.
[1174] The genetic constructs of the invention may be DNA or RNA, and are preferably double-stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).
[1175] In a preferred but non-limiting aspect, a genetic construct of the invention comprises
[1176] i) at least one nucleic acid of the invention; operably connected to
[1177] ii) one or more regulatory elements, such as a promoter and optionally a suitable terminator;
and optionally also
[1178] iii) one or more further elements of genetic constructs known per se; in which the terms “regulatory element”, “promoter”, “terminator” and “operably connected” have their usual meaning in the art (as further described herein); and in which said “further elements” present in the genetic constructs may for example be 3′- or 5′-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase (the efficiency of) transformation or integration. These and other suitable elements for such genetic constructs will be clear to the skilled person, and may for instance depend upon the type of construct used, the intended host cell or host organism; the manner in which the nucleotide sequences of the invention of interest are to be expressed (e.g. via constitutive, transient or inducible expression); and/or the transformation technique to be used. For example, regulatory sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) may be used in an essentially analogous manner.
[1179] Preferably, in the genetic constructs of the invention, said at least one nucleic acid of the invention and said regulatory elements, and optionally said one or more further elements, are “operably linked” to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being “under the control of” said promoter). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.
[1180] Preferably, the regulatory and further elements of the genetic constructs of the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism.
[1181] For instance, a promoter, enhancer or terminator should be “operable” in the intended host cell or host organism, by which is meant that (for example) said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence—e.g. a coding sequence—to which it is operably linked (as defined herein).
[1182] Some particularly preferred promoters include, but are not limited to, promoters known per se for the expression in the host cells mentioned herein; and in particular promoters for the expression in the bacterial cells, such as those mentioned herein and/or those used in the Examples.
[1183] A selection marker should be such that it allows—i.e. under appropriate selection conditions—host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.
[1184] A leader sequence should be such that—in the intended host cell or host organism—it allows for the desired post-translational modifications and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell. A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism. Leader sequences may not be required for expression in a bacterial cell. For example, leader sequences known per se for the expression and production of antibodies and antibody fragments (including but not limited to single domain antibodies and ScFv fragments) may be used in an essentially analogous manner.
[1185] An expression marker or reporter gene should be such that—in the host cell or host organism—it allows for detection of the expression of (a gene or nucleotide sequence present on) the genetic construct. An expression marker may optionally also allow for the localisation of the expressed product, e.g. in a specific part or organelle of a cell and/or in (a) specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism. Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP.
[1186] Some preferred, but non-limiting examples of suitable promoters, terminator and further elements include those that can be used for the expression in the host cells mentioned herein; and in particular those that are suitable for expression in bacterial cells, such as those mentioned herein and/or those used in the Examples below. For some (further) non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention—such as terminators, transcriptional and/or translational enhancers and/or integration factors—reference is made to the general handbooks such as Sambrook et al. and Ausubel et al. mentioned above, as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat. No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person. Reference is also made to the general background art cited above and the further references cited herein.
[1187] The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.
[1188] Often, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the Examples below, as well as those mentioned herein.
[1189] The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the amino acid sequence, NANOBODY or polypeptide of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, for example:
[1190] a bacterial strain, including but not limited to gram-negative strains such as strains of Escherichia coli; of Proteus, for example of Proteus mirabilis; of Pseudomonas, for example of Pseudomonas fluorescens; and gram-positive strains such as strains of Bacillus, for example of Bacillus subtilis or of Bacillus brevis; of Streptomyces, for example of Streptomyces lividans; of Staphylococcus, for example of Staphylococcus carnosus; and of Lactococcus, for example of Lactococcus lactis;
[1191] a fungal cell, including but not limited to cells from species of Trichoderma, for example from Trichoderma reesei; of Neurospora, for example from Neurospora crassa; of Sordaria, for example from Sordaria macrospora; of Aspergillus, for example from Aspergillus niger or from Aspergillus sojae; or from other filamentous fungi;
[1192] a yeast cell, including but not limited to cells from species of Saccharomyces, for example of Saccharomyces cerevisiae; of Schizosaccharomyces, for example of Schizosaccharomyces pombe; of Pichia, for example of Pichia pastoris or of Pichia methanolica; of Hansenula, for example of Hansenula polymorpha; of Kluyveromyces, for example of Kluyveromyces lactis; of Arxula, for example of Arxula adeninivorans; of Yarrowia, for example of Yarrowia lipolytica;
[1193] an amphibian cell or cell line, such as Xenopus oocytes;
[1194] an insect-derived cell or cell line, such as cells/cell lines derived from lepidoptera, including but not limited to Spodoptera SF9 and Sf21 cells or cells/cell lines derived from Drosophila, such as Schneider and Kc cells;
[1195] a plant or plant cell, for example in tobacco plants; and/or
[1196] a mammalian cell or cell line, for example a cell or cell line derived from a human, a cell or a cell line from mammals including but not limited to CHO-cells, BHK-cells (for example BHK-21 cells) and human cells or cell lines such as HeLa, COS (for example COS-7) and PER.C6 cells;
as well as all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be clear to the skilled person. Reference is also made to the general background art cited hereinabove, as well as to for example WO 94/29457; WO 96/34103; WO 99/42077; Frenken et al., (1998), supra; Riechmann and Muyldermans, (1999), supra; van der Linden, (2000), supra; Thomassen et al., (2002), supra; Joosten et al., (2003), supra; Joosten et al., (2005), supra; and the further references cited herein.
[1197] The amino acid sequences, NANOBODIES and polypeptides of the invention can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g. as a gene therapy). For this purpose, the nucleotide sequences of the invention may be introduced into the cells or tissues in any suitable way, for example as such (e.g. using liposomes) or after they have been inserted into a suitable gene therapy vector (for example derived from retroviruses such as adenovirus, or parvoviruses such as adeno-associated virus). As will also be clear to the skilled person, such gene therapy may be performed in vivo and/or in situ in the body of a patient by administering a nucleic acid of the invention or a suitable gene therapy vector encoding the same to the patient or to specific cells or a specific tissue or organ of the patient; or suitable cells (often taken from the body of the patient to be treated, such as explanted lymphocytes, bone marrow aspirates or tissue biopsies) may be treated in vitro with a nucleotide sequence of the invention and then be suitably (re-)introduced into the body of the patient. All this can be performed using gene therapy vectors, techniques and delivery systems which are well known to the skilled person, and for example described in Culver, K. W., “Gene Therapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.); Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.: 811 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,895,466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. For example, in situ expression of ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has been described in the art.
[1198] For expression of the NANOBODIES in a cell, they may also be expressed as so-called “intrabodies”, as for example described in WO 94/02610, WO 95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag; and in Kontermann, Methods 34, (2004), 163-170.
[1199] The amino acid sequences, NANOBODIES and polypeptides of the invention can for example also be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep (see for example U.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No. 6,849,992 for general techniques for introducing transgenes into mammals), in plants or parts of plants including but not limited to their leaves, flowers, fruits, seed, roots or tubers (for example in tobacco, maize, soybean or alfalfa) or in for example pupae of the silkworm Bombix mori.
[1200] Furthermore, the amino acid sequences, NANOBODIES and polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear to the skilled person. Some preferred, but non-limiting examples include expression in the wheat germ system; in rabbit reticulocyte lysates; or in the E. coli Zubay system.
[1201] As mentioned above, one of the advantages of the use of NANOBODIES is that the polypeptides based thereon can be prepared through expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, host cells, regulatory elements, etc., will be clear to the skilled person, for example from the references cited above. It should however be noted that the invention in its broadest sense is not limited to expression in bacterial systems.
[1202] Preferably, in the invention, an (in vivo or in vitro) expression system, such as a bacterial expression system, is used that provides the polypeptides of the invention in a form that is suitable for pharmaceutical use, and such expression systems will again be clear to the skilled person. As also will be clear to the skilled person, polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.
[1203] For production on industrial scale, preferred heterologous hosts for the (industrial) production of NANOBODIES or NANOBODY-containing protein therapeutics include strains of E. coli, Pichia pastoris, S. cerevisiae that are suitable for large scale expression/production/fermentation, and in particular for large scale pharmaceutical (i.e. GMP grade) expression/production/fermentation. Suitable examples of such strains will be clear to the skilled person. Such strains and production/expression systems are also made available by companies such as Biovitrum (Uppsala, Sweden).
[1204] Alternatively, mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, can be used for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also made available by some of the companies mentioned above.
[1205] The choice of the specific expression system would depend in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a NANOBODY-containing recombinant protein for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e. the kind, number and position of residues attached) will depend on the cell or cell line that is used for the expression. Preferably, either a human cell or cell line is used (i.e. leading to a protein that essentially has a human glycosylation pattern) or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same as human glycosylation or at least mimics human glycosylation. Generally, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotes such as yeast usually leads to a glycosylation pattern that differs from human glycosylation. Nevertheless, it should be understood that all the foregoing host cells and expression systems can be used in the invention, depending on the desired amino acid sequence, NANOBODY or polypeptide to be obtained.
[1206] Thus, according to one non-limiting aspect of the invention, the amino acid sequence, NANOBODY or polypeptide of the invention is glycosylated. According to another non-limiting aspect of the invention, the amino acid sequence, NANOBODY or polypeptide of the invention is non-glycosylated.
[1207] According to one preferred, but non-limiting aspect of the invention, the amino acid sequence, NANOBODY or polypeptide of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale pharmaceutical production, such as cells of the strains mentioned above.
[1208] According to another preferred, but non-limiting aspect of the invention, the amino acid sequence, NANOBODY or polypeptide of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, such as cells of the species mentioned above.
[1209] According to yet another preferred, but non-limiting aspect of the invention, the amino acid sequence, NANOBODY or polypeptide of the invention is produced in a mammalian cell, in particular in a human cell or in a cell of a human cell line, and more in particular in a human cell or in a cell of a human cell line that is suitable for large scale pharmaceutical production, such as the cell lines mentioned hereinabove.
[1210] When expression in a host cell is used to produce the amino acid sequences, NANOBODIES and the polypeptides of the invention, the amino acid sequences, NANOBODIES and polypeptides of the invention can be produced either intracellullarly (e.g. in the cytosol, in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. When eukaryotic host cells are used, extracellular production is usually preferred since this considerably facilitates the further isolation and downstream processing of the NANOBODIES and proteins obtained. Bacterial cells such as the strains of E. coli mentioned above normally do not secrete proteins extracellularly, except for a few classes of proteins such as toxins and hemolysin, and secretory production in E. coli refers to the translocation of proteins across the inner membrane to the periplasmic space. Periplasmic production provides several advantages over cytosolic production. For example, the N-terminal amino acid sequence of the secreted product can be identical to the natural gene product after cleavage of the secretion signal sequence by a specific signal peptidase. Also, there appears to be much less protease activity in the periplasm than in the cytoplasm. In addition, protein purification is simpler due to fewer contaminating proteins in the periplasm. Another advantage is that correct disulfide bonds may form because the periplasm provides a more oxidative environment than the cytoplasm. Proteins overexpressed in E. coli are often found in insoluble aggregates, so-called inclusion bodies. These inclusion bodies may be located in the cytosol or in the periplasm; the recovery of biologically active proteins from these inclusion bodies requires a denaturation/refolding process. Many recombinant proteins, including therapeutic proteins, are recovered from inclusion bodies. Alternatively, as will be clear to the skilled person, recombinant strains of bacteria that have been genetically modified so as to secrete a desired protein, and in particular an amino acid sequence, NANOBODY or a polypeptide of the invention, can be used. Thus, according to one non-limiting aspect of the invention, the amino acid sequence, NANOBODY or polypeptide of the invention is an amino acid sequence, NANOBODY or polypeptide that has been produced intracellularly and that has been isolated from the host cell, and in particular from a bacterial cell or from an inclusion body in a bacterial cell.
[1211] According to another non-limiting aspect of the invention, the amino acid sequence, NANOBODY or polypeptide of the invention is an amino acid sequence, NANOBODY or polypeptide that has been produced extracellularly, and that has been isolated from the medium in which the host cell is cultivated.
[1212] Some preferred, but non-limiting promoters for use with these host cells include,
[1213] for expression in E. coli: lac promoter (and derivatives thereof such as the lacUV5 promoter); arabinose promoter; left- (PL) and rightward (PR) promoter of phage lambda; promoter of the trp operon; hybrid lac/trp promoters (tac and trc); T7-promoter (more specifically that of T7-phage gene 10) and other T-phage promoters; promoter of the Tn10 tetracycline resistance gene; engineered variants of the above promoters that include one or more copies of an extraneous regulatory operator sequence;
[1214] for expression in S. cerevisiae: constitutive: ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1), GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1 (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated: GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper metallothionein); heterologous: CaMV (cauliflower mosaic virus 35S promoter);
[1215] for expression in Pichia pastoris: the AOX1 promoter (alcohol oxidase I);
[1216] for expression in mammalian cells: human cytomegalovirus (hCMV) immediate early enhancer/promoter; human cytomegalovirus (hCMV) immediate early promoter variant that contains two tetracycline operator sequences such that the promoter can be regulated by the Tet repressor; Herpes Simplex Virus thymidine kinase (TK) promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR) enhancer/promoter; elongation factor 1α (hEF-1α) promoter from human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1 long terminal repeat promoter; β-actin promoter;
[1217] Some preferred, but non-limiting vectors for use with these host cells include:
[1218] vectors for expression in mammalian cells: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565), as well as viral-based expression systems, such as those based on adenovirus;
[1219] vectors for expression in bacterial cells: pET vectors (Novagen) and pQE vectors (Qiagen);
[1220] vectors for expression in yeast or other fungal cells: pYES2 (Invitrogen) and Pichia expression vectors (Invitrogen);
[1221] vectors for expression in insect cells: pBlueBacII (Invitrogen) and other baculovirus vectors
[1222] vectors for expression in plants or plant cells: for example vectors based on cauliflower mosaic virus or tobacco mosaic virus, suitable strains of Agrobacterium, or Ti-plasmid based vectors.
[1223] Some preferred, but non-limiting secretory sequences for use with these host cells include:
[1224] for use in bacterial cells such as E. coli: PelB, Bla, OmpA, OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the like; TAT signal peptide, hemolysin C-terminal secretion signal;
[1225] for use in yeast: a-mating factor prepro-sequence, phosphatase (phol), invertase (Suc), etc.;
[1226] for use in mammalian cells: indigenous signal in case the target protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal peptide; etc.
[1227] Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above.
[1228] After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the amino acid sequence of the invention, e.g. using specific antibodies.
[1229] The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.
[1230] Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g. under suitable conditions), an amino acid sequence, NANOBODY or polypeptide of the invention (and in case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention, that may for instance be obtained by cell division or by sexual or asexual reproduction.
[1231] To produce/obtain expression of the amino acid sequences of the invention, the transformed host cell or transformed host organism may generally be kept, maintained and/or cultured under conditions such that the (desired) amino acid sequence, NANOBODY or polypeptide of the invention is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Ag