Anti-nucleolin Antibody

ANTI-NUCLEOLIN ANTIBODY

BACKGROUND OF INVENTION

Cancer is currently the second leading cause of death worldwide, only slightly surpassed by heart diseases. Surgery, radiotherapy and chemotherapy are the most common prescribed treatment modalities. The majority of cancer subtypes are associated with poor clinical outcomes owing to the ineffectiveness of treatments targeting metastases, the lack of tumor specificity and the development of drug resistance (Torre et al. 2015. A Cancer Journal for Clinicians., 65(2), pp.87-108).

Antibody-based therapy is considered a major breakthrough for better therapeutic outcomes in several types of tumors, from hematological malignancies to solid tumors.

Antibodies combine the capacity to promote cell death by mechanisms ranging from direct cell killing to immune -mediated cell death, with activity against specific targets, thus potentially decreasing the associated side effects (Scott et al. 2012. Nature reviews. Cancer, 12(4), pp.278- 87).

SUMMARY OF INVENTION

The disclosure relates to antibodies targeting the N-terminal domain of nucleolin, including antibodies comprising part or all of the F3 peptide, and having ADCC activity, for example, by including the Fc region of IgGl . The disclosure relates to methods for treating conditions associated with cell-surface expressed nucleolin, e.g., cancer, by administration of said antibodies. The disclosure is based, in part, on the surprising discovery that antibodies targeting the N-terminal domain of nucleolin have cytotoxic activity against nucleolin- overexpressing cells, including cancer cells, and that when said antibodies comprise the Fc region of IgGl, the antibodies have effective ADCC activity against cancer cells.

In one aspect, provided are antibodies or antigen binding fragments thereof that (i) specifically bind the N-terminal domain of nucleolin; and (ii) promote antibody dependent cellular cytotoxicity (ADCC) towards a cell expressing nucleolin. In some embodiments, the antibodies or antigen binding fragments comprise an F3 peptide. In some embodiments, the F3 peptide comprises 8-31 contiguous amino acids of F3. In some embodiments, the F3 peptide comprises residues 5-14 of F3.

In some embodiments, the antibodies or antigen binding fragments comprise a variable domain comprising a CDRl, a CDR2, and a CDR3. In some embodiments, one or more of CDRl, CDR2, and CDR3 comprises the F3 peptide. In some embodiments, one or both of CDRl or CDR3 comprising the F3 peptide. In some embodiments, the variable domain comprises a CDRl, a CDR2, and a CDR3 comprising the F3 peptide. In some embodiments, the variable domain comprises a CDRl, a CDR2, and the F3 peptide substituted for CDR3.

In some embodiments, the N-terminal domain of nucleolin comprises amino acids 1-283 of nucleolin. In some embodiments, the antibodies or antigen binding fragments specifically bind amino acids 1-283 of the N-terminal domain of nucleolin. In some embodiments, the antibodies or antigen binding fragments specifically bind amino acids 43-51 of the N-terminal domain of nucleolin. In some embodiments, the antibodies or antigen binding fragments specifically bind amino acids 221-233 of the N-terminal domain of nucleolin.

In some embodiments, the antibodies or antigen binding fragments are human or humanized monoclonal antibodies. In some embodiments, the antibodies are full length antibodies. In some embodiments, the antibodies comprise an IgGl Fc domain. In some embodiments, the antigen binding fragments are Fab, Fab', F(ab')2, Fv, a VHH, or scFv. In some embodiments, the antigen binding fragments comprise scFv-Fc. In some embodiments, the antigen binding fragments comprise VHH-Fc. In some embodiments, the Fc domain is an IgGl Fc domain.

In some embodiments, the Fc domain comprises one or more modifications to increase ADCC. In some embodiments, the one or more modifications comprises one or more of L234Y, S239D, T256A, K290A, K290Y, Y296W, S298A, A330F, A330L, I332E, E333A, K334A, K334V, A339T, E356K, K392D, D339K and K409D. In some embodiments, the one or more modifications comprises the level of Fc-bound carbohydrate structure, including, but not limited to, alterations at the level of fructose, galactose, mannose, bisecting sugars and/or sialic acid.

In some embodiments, the antibodies are bispecific antibodies, and wherein the antibodies specifically bind a molecule present on the surface of immune cells. In some embodiments, the molecule present on the surface of immune cells comprises MHC class I or MHC class II proteins, T cell receptors, B cell receptors, CD28, ICOS, TLT2, CD27, CD 137, OX40, HVEM, DR3, NKG2D, TIM-1, TIM-2, DN AM- 1 , CRTAM, CTLA-4, PD-1, PD-L1, PD-L2, CXCR4, CD3, B7-1, B7-2, BTLA, CD 160, LAG-3, TIM-3, TIGIT, LAIR-1, CAR, CD40, GITR, BAFF-R, TACI, BCMA, CD72, CD22, CD96, 2B4, NTB-A, CRACC, Siglec- 3.7.9, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, CD94-NKG2A, CEACAM1, Plexin-Al, Plexin-A4, CD300b, CD300e, TREM1, TREM2, TREM3, ILT7, ILT3,4, TLT-1, CD200R, TAM family, CD300a, CD300f, DC-SIGN, Kit, Allergin-1, Pir-B, MAFA, or Gp49B l .

In another aspect, provided herein is an isolated nucleic acid or a set of nucleic acids, which collectively encode any of the anti-nucleolin antibodies or antibody fragments described herein, a vector or vector set (e.g., expression vectors) that comprise the nucleic acid or the set of nucleic acids, and a host cell or host cell set that comprises the vector or vector set.

Exemplary host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, and mammalian cells.

In another aspect, provided herein are pharmaceutical compositions comprising any of the anti-nucleolin antibodies or antibody fragments described herein and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be for use in treating a disease associated with cell-surface localized nucleolin or for treating cancer.

In another aspect, provided herein are methods for treating a disease associated with cell- surface localized nucleolin. The method comprises administering a therapeutically effective amount of any of the anti-nucleolin antibodies or antibody fragments described herein to a subject in need thereof.

In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor forming cancer.

In some embodiments, the subject is administered a treatment for cancer. In some embodiments, the treatment for cancer is a chemotherapy, a radiation therapy, or an

immunotherapy.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawing and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF DRAWINGS

The following drawing forms part of the present specification and is included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

Figure 1 is a schematic representation of a cloning process used to generate the novel anti-nucleolin small antibody formats (VHHs), with indication of the restriction sites of Hindlll and Bglll, engrafted sequence and linkers, and the regions of primer hybridization.

Figures 2A-2J are graphical representations of the parental VHH and the novel anti- nucleolin VHH constructions, highlighting the leader peptide sequence, histidine tag (used for protein purification) and HA tag (used for protein detection) as well as CDR1 and CDR3 regions, in which the nucleolin-binding sequence was grafted. In Figures 2A and 2B, the nucleotide sequence of the parental VHH and its amino acid sequence are presented, respectively. In Figures 2C-2J, the nucleotide and amino acid sequences of each one of the four novel anti-nucleolin VHHs are presented. These were the result of the F3 peptide-derived amino acid sequence grafting onto CDR1 (aNCL-CDRl VHH, Figures 2C (nucleic acid) and 2D (amino acid)) or CDR3 (aNCL-CDR3 VHH, Figures 2E (nucleic acid) and 2F (amino acid)). Also generated were grafts in which the F3 peptide included the SGGGS flanking linker sequences (aNCL-CDRl -L, Figures 2G (nucleic acid) and 2H (amino acid), or aNCL-CDR3-L VHHs, Figures 21 (nucleic acid) and 2 J (amino acid)).

Figures 3A and 3B are graphs showing the binding of 100 pmol of the VHH fragments to human recombinant nucleolin and human recombinant TNF-a, respectively, following 1 h incubation at 37°C, as evaluated by ELISA. The results are from a representative experiment.

Figures 4A-4F illustrate the binding of the generated VHH fragments to nucleolin- overexpressing cells, following incubation for 45 min at 4°C, as evaluated by flow cytometry with an anti-HA-FITC antibody. Figures 4A-4C show the binding of anti-nucleolin VHH (aNCL-CDRl , aNCL-CDRl -L, aNCL-CDR3 and aNCL-CDR3-L) to nucleolin-overexpressing MDA-MB-435S (Figure 4A), MDA-MD-231 (Figure 4B) and 4T1 (Figure 4C) cell lines. Parental VHH without the F3 peptide graft was also tested. Figures 4D-4F show competitive inhibition assays, upon pre-incubation of the nucleolin-overexpressing MDA-MB-435S (Figure 4D), MDA-MD-231 (Figure 4E) and 4T1 (Figure 4F) cell lines with 75 μΜ F3 peptide or 1 μΜ infliximab, for 30 min at 4°C. An additional control, without pre-incubation of a ligand, was also performed. Data represents the mean ± SD of three independent experiments. One-way ANOVA followed by Tukey's Multiple Comparison Test was performed to evaluate the differences in binding among the five VHH fragments (*p<0.05, **p<0.01, ***p<0.001). For each protein, differences in the competitive inhibition assays were evaluated by Student's t-test (*p<0.05, **p<0.01, ***p<0.001).

Figures 5A-5C are graphs showing the cytotoxicity of the VHH fragments against the nucleolin-overexpressing MDA-MB-435S (Figure 5A), MDA-MD-231 (Figure 5B) and 4T1 (Figure 5C) cell lines, upon incubation at 37°C for 72 h, as determined by the 3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Data represents the mean ± SD of at least three independent experiments. One-way ANOVA followed by Tukey's Multiple Comparison Test was performed to evaluate the differences in cytotoxicity among the four anti-nucleolin VHH fragments tested, along with the parental VHH fragment (*p<0.05, **p<0.01, ***p<0.001).

Figures 6A-6B are graphical representations of the nucleic acid (Figure 6A) and amino acid (Figure 6B) sequences of anti-nucleolin VHH-Fc, highlighting the IL2 signaling sequence, the anti-nucleolin VHH, and the Fc regions.

Figures 7A-7C are graphs showing the cytotoxicity of anti-nucleolin and parental VHH- Fc antibody against the nucleolin-overexpressing MDA-MB-435S (Figure 7A), MDA-MD-231 (Figure 7B) and 4T1 (Figure 7C) cell lines, after an incubation at 37°C for 72 h, as determined by the MTT assay. Data represents the mean ± SD of three independent experiments. Student's t-test was performed to evaluate the differences in cytotoxicity between the proteins (*p<0.05, **p<0.01, ***p<0.001).

Figures 8A and 8B are graphs showing the cytotoxic effect of anti-nucleolin and parental VHH-Fc antibody against the nucleolin-overexpressing MDA-MB-435S cell line, with or without effector cells (PBMCs), as evaluated by cell impedance using xCELLigence technology. The cytotoxic effect of the anti-nucleolin VHH-Fc (aNCL VHH-Fc) was assessed relative to the parental VHH-Fc (Figure 8A) or the anti-nucleolin VHH (Figure 8B) constructs. DETAILED DESCRIPTION OF INVENTION

The present disclosure, in one aspect, relates to the surprising discovery that antibodies targeting the N-terminal domain of nucleolin have cytotoxicity against nucleolin-overexpressing cells, including different sub-populations of tumor cells, such as cancer cells, cancer stem cells and endothelial cells from tumor blood vessels. When comprising the Fc region of IgGl, the resulting anti-nucleolin antibody triggers ADCC responses against these nucleolin- overexpressing cells.

Thus, in one aspect, the present disclosure provides antibodies to the N-terminal domain of nucleolin, e.g., antibodies comprising the F3 peptide, and having ADCC activity. In another aspect, the present disclosure provides methods for treating conditions associated with cell- surface expressed nucleolin, e.g., cancer, by administration of antibodies to the N-terminal domain of nucleolin, e.g., antibodies comprising the F3 peptide.

Nucleolin

Nucleolin is a 76.6 kDa protein involved in the synthesis and maturation of ribosomes.

Nucleolin has a 3 -component structure: an N-terminal domain with acidic stretches, which is involved in several protein-protein interactions, and which controls rDNA transcription; a central globular domain, with four RNA-binding domains, which is involved in pre-RNA processing; and a C-terminal domain, constituted by arginine-glycine-glycine repeats, which interacts with ribosomal proteins (Srivastava & Pollard 1999. The FASEB Journal, 13, pp.191 1- 1922).

Nucleolin is located mainly in dense fibrillar regions of the nucleolus. Although nucleolin is typically present in the nucleus, including in the nucleolis, expression increases and nucleolin is translocated to the cell surface in highly proliferating cells, such as cancer cells (Srivastava & Pollard 1999). Importantly, this overexpression at the cell surface is also observed in endothelial cells from angiogenic blood vessels, which play a central role in tumor growth and progression (Christian et al. 2003. The Journal of Cell Biology, 163(4), pp.871- 878). As such, cell-surface localized nucleolin serves as a marker for oncogenesis and potentially as a means of targeting cancerous cells.

One of the main challenges arising from the choice of nucleolin as a target, results from its rapid internalization. In fact, cell surface nucleolin represents less than 10% of nuclear nucleolin (Hovanessian et al. 2010) and undergoes a rapid turnover, with an estimated half-life of less than one hour, compared to more than eight hours in the nucleus (Hovanessian et al. 2010).

The amino acid sequence of nucleolin is as follows:

MVKLAKAGKNQGDPKKMAPPPKEVEEDSEDEEMSEDEEDDSSGEEVVI PQKKGKKAAATSAKKWVSPTK KVAVATPAKKAAVTPGKKAAATPAKKTVTPAKAVTTPGKKGATPGKALVATPGKKGAAI PAKGAKNGKNA KKE DSDEEEDDDSEE DEEDDEDEDEDEDE IEPAAMKAAAAAPASEDEDDEDDEDDEDDDDDEE DDSEEEA METTPAKGKKAAKWPVKAKNVAEDEDEEEDDEDEDDDDDEDDEDDDDEDDEEEEEEEEEEPVKEAPGKR KKEMAKQKAAPEAKKQKVEGTEPTTAFNLFVGNLNFNKSAPELKTGI SDVFAKNDLAVVDVRI GMTRKFG YVDFESAEDLEKALELTGLKVFGNE I KLEKPKGKDSKKERDARTLLAKNLPYKVTQDELKEVFEDAAE IR LVSKDGKSKGIAYIE FKTEADAEKTFEEKQGTE I DGRS I SLYYTGEKGQNQDYRGGKNSTWSGESKTLVL SNLSYSATEETLQEVFEKATFI KVPQNQNGKSKGYAFI EFASFEDAKEALNSCNKREI EGRAI RLELQGP RGS PNARSQPSKTLFVKGLSEDTTEETLKESFDGSVRARIVTDRETGSSKGFGFVDFNSEEDAKAAKEAM EDGEI DGNKVTLDWAKPKGEGGFGGRGGGRGGFGGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGGDH KPQGKKTKFE ( SEQ I D NO : 1 )

Nucleolin has previously been targeted with peptides and pseudopeptides. The nucleolin-binding F3 peptide is a 31 -amino acid peptide that binds to the N-terminal domain of nucleolin (Christian et al. 2003 The Journal of Cell Biology, 163(4), pp.871-878). F3 is able to be internalized and has been used as a targeting ligand of nanoparticles aiming at the delivery of small weight drugs, such as doxorubicin (Moura et al. 2012. Breast Cancer Research and Treatment, 133(1), pp.61-73), paclitaxel (Hu et al. 2013. Biomaterials, 34, pp.1 135-1 145) or cisplatin (Winer et al. 2010. Cancer Research, 70(21), pp.8674-8683) to nucleolin- overexpressing cells. This strategy has also been used for the delivery of radiotherapeutics (Drecoll et al. 2009; Cornelissen et al. 2012. EJNMMI Research, 2(9)) and photodynamic therapy (Reddy et al. 2006. Clinical Cancer Research, 12, pp.6677-6686; Ai et al. 2014. Talanta, 1 18, pp.54-60), as well as nucleic acids (siRNA) (Gomes-Da-Silva et al. 2012. International Journal of Pharmaceutics, 434(1-2), pp.9-19; Gomes-da- Silva et al. 2013. Nanomedicine, 8(9), pp.1397^113). The amino acid sequence of the F3 peptide is as follows:

KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 2)

The nucleolin binding- aptamer AS141 1, has anti-tumorigenic activity (Bates et al. 2009. Experimental and Molecular Pathology, 86(3), pp.151-64), and has also been used as a targeting ligand of nanoparticles containing siRNA (Li et al. 2014. Biomaterials, 35(12), pp.3840-3850) or small weight drugs (Guo et al. 201 1. Biomaterials, 32(31), pp.8010-8020; Z. Li et al. 2012. Biomacromolecules, 13, pp.4257-4263; Song et al. 2013. Molecular Pharmaceutics, 10(10), pp.3555-3563; Latorre et al. 2014. Nanoscale, 6, pp.7436-7442; Ai et al. 2014. Talanta, 1 18, pp.54-60), towards nucleolin-overexpressing cells.

Pseudopeptides HB-19 (Destouches et al. 2008. PloS one, 3(6), p.e2518) and N6L

(Destouches et al. 201 1. Cancer Research, 71(9), pp.3296-3305) presented both anti-tumoral and antiangiogenic effects, in vitro and in vivo. Peritumoral injections of HB-19 inhibited tumor growth in an ectopic MDA-MD-231 breast cancer model (Destouches et al. 2008).

Intraperitoneal administration of N6L led to tumor growth inhibition of orthotopic models of breast and prostate cancer, derived from MDA-MB-231 and PC3 cell lines, respectively, as well as to increased survival in A20- and T29-derived lymphoma models when administered by intravenous injection (Destouches et al. 201 1). Inhibition of angiogenesis, was also

demonstrated for both pseudopeptide HB-19 and N6L in a chick embryo chorioallantoic membrane (CAM) assay, as well as in an animal model for HB- 19 (Destouches et al. 2008; Destouches et al. 201 1). A phase I/IIa clinical trial exploring the use of N6L for therapy of advanced solid tumors (NCT0171 1398) has been completed, although its results are not yet known.

Previously reported nucleolin antibodies, including antibodies that target the RNA- binding domains of the central globular domain of nucleolin or the C-terminal domain, have not demonstrated ADCC activity. (Palmieri, D. et al., 2015. Proceedings of the National Academy of Sciences of the United States of America, 1 12(30), pp.9418-23; WO201 1062997).

A rabbit nucleolin-specific antibody (NCL3), which binds the N-terminal domain of nucleolin (Christian et al. 2003. The Journal of Cell Biology, 163(4), pp.871-878), was developed. No ADCC activity was reported. A nucleolin-targeting single-chain Fragment variable (scFv) antibody, 4LB5, has also been developed, against the central domain of the protein. Once again, no ADCC activity was reported (Palmieri et al. 2015).

In some embodiments, the antibodies presented herein provide for improved anti-cancer cell cytotoxicity by targeting the N-terminal domain of nucleolin and by triggering ADCC activity.

An efficient ADCC response depends on the availability of a target protein at the cell surface. This determines the extent of antibody binding and the level of binding of the antibody to the Fc receptor of NK cells through the Fc domain, which triggers ADCC. This is supported by the demonstration antibodies engineered against the same target having lower internalization rates have the strongest ADCC response (Yang et al. PLoS ONE, 201 1, 6(6); Vasu et al. 2016 Blood, 127(23), pp.2879-2889). In addition, strategies to block antibody internalization resulted in improved ADCC response (WO2014063205 Al ; Roghanian et al. 2015). The N-terminal domain of nucleolin is believed to have greater exposure to the cell surface than the central globular domain of nucleolin or the C-terminal domain. Accordingly, without wishing to be bound by theory, it is believed that anti-nucleolin N-terminal domain antibodies having Fc domains have optimal ADCC activity because of the cell surface exposure.

Antibodies binding to Nucleolin

The present disclosure provides antibodies that bind nucleolin, for example, the N- terminal domain of nucleolin.

As used herein, the term "antibody molecule" refers to a protein comprising at least one immunoglobulin variable domain sequence. The term antibody molecule includes, for example, full-length, mature antibodies and antigen-binding fragments of an antibody. For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab' , F(ab')2, Fc, Fd, Fd', Fv, VHH, scFv-Fc, VHH-Fc, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. The antibodies can be monoclonal or polyclonal. The antibody can also be a human, humanized, CDR-grafted, or in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from, e.g., kappa or lambda. The antibody can be a whole antibody. Examples of antigen-binding fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody; (iv) a VHH. These antibody fragments may be obtained using any suitable method, including several conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as are intact antibodies.

The term "antibody" includes intact molecules as well as functional fragments thereof. Constant regions of the antibodies can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody

glycosylation, the number of cysteine residues, effector cell function, or complement function).

The antibodies disclosed herein can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. According to some aspects, a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are also contemplated. VHH molecules are about 1 Ox smaller than IgG molecules. They are single polypeptides and very stable, resisting extreme pH and temperature conditions. Moreover, they are resistant to the action of proteases which is not the case for conventional antibodies. Furthermore, in vitro expression of VHHs produces high yield, properly folded functional VHHs. In addition, antibodies generated in Camelids will recognize epitopes other than those recognised by antibodies generated in vitro through the use of antibody libraries or via immunisation of mammals other than Camelids (WO 9749805). As such, anti- albumin VHH's may interact in a more efficient way with serum albumin which is known to be a carrier protein. As a carrier protein some of the epitopes of serum albumin may be inaccessible by bound proteins, peptides and small chemical compounds. Since VHH's are known to bind into 'unusual' or non-conventional epitopes such as cavities (WO 97/49805), the affinity of such VHH's to circulating albumin may be increased. In some embodiments, the VHH comprises an Fc domain. In some embodiments, the Fc comprises CH2 and CH3.

The VH and VL regions can be subdivided into regions of hypervariability, termed "complementarity determining regions" (CDR), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer- Verlag, Heidelberg). In some embodiments, the following definitions are used: AbM definition of CDR1 of the heavy chain variable domain and Kabat definitions for the other CDRs. In certain embodiments, Kabat definitions are used for all CDRs. In addition, embodiments described with respect to Kabat or AbM CDRs may also be implemented using Chothia hypervariable loops. Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, an "immunoglobulin variable domain sequence" refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations that are compatible with formation of the protein structure.

The term "antigen-binding region" refers to the part of an antibody molecule that comprises determinants that form an interface that binds to nucleolin, or an epitope thereof. With respect to proteins (or protein mimetics), the antigen-binding region typically includes one or more loops (of at least, e.g., four amino acids or amino acid mimics) that form an interface that binds to nucleolin. Typically, the antigen-binding region of an antibody molecule includes at least one or two CDRs, or more typically at least three, four, five or six CDRs.

The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

The antibodies described herein can be murine, rat, human, or any other origin (including chimeric or humanized antibodies). Such antibodies are non-naturally occurring, i.e., would not be produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof).

Any of the antibodies described herein can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogenous antibody population and a "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the manner in which it is made.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g. murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.

Humanized antibodies may also involve affinity maturation.

In another example, the antibody described herein is a chimeric antibody, which can include a heavy constant region and a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.

In some embodiments, the anti-nucleolin antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof. An antibody that "specifically binds" to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (nucleolin, e.g., the N-terminal domain of nucleolin) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that "specifically binds" to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen. In some embodiments, the antibodies described herein specifically bind to nucleolin. In some embodiments, the antibodies described herein specifically bind to the N-terminal domain of nucleolin, e.g., amino acids 1-283 of nucleolin. In some embodiments, the antibodies described herein specifically bind to amino acids 43-51 of the N-terminal domain of nucleolin. In some embodiments, the antibodies described herein specifically bind to amino acids 221-233 of the N-terminal domain of nucleolin. In general, the antibody binds nucleolin with sufficient specificity such that is can distinguish nucleolin, in vivo, adequately to yield a treatment that is without off-target effects that would undermine its ability to perform as a therapeutic.

In some embodiments, an anti-nucleolin antibody as described herein has a suitable binding affinity for the target antigen {e.g., nucleolin) or antigenic epitopes thereof. As used herein, "binding affinity" refers to the apparent association constant or KA. The KA is the reciprocal of the dissociation constant (KD). The anti-nucleolin antibody described herein may have a binding affinity (KD) of at least 10"5, 10"6, 10"7, 10"8, 10"9, 10 10 M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased KD. In some embodiments, any of the anti-nucleolin antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation:

[Bound] = [Free]/(Kd+[Free]) It is not always necessary to make an exact determination of KA, though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to KA, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

In some embodiments, the anti-nucleolin antibody comprises a F3 peptide.

In some embodiments, the F3 peptide comprises 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 contiguous amino acids of F3. In some embodiments, the F3 peptide comprises 7- 12, 8-1 1, or 9-10 contiguous amino acids of F3. In some embodiments, the F3 peptide comprises residues 3 to 11, 3 to 12, or 3 to 13 of F3. In some embodiments, the F3 peptide comprises residues 4 to 12, 4 to 13, or 4 to 14 of F3. In some embodiments, the F3 peptide comprises residues 5 to 13, 5 to 14, or 5 to 15 of F3. In some embodiments, the F3 peptide comprises residues 6 to 14, 6 to 15, or 6 to 16 of F3. In some embodiments, the F3 peptide comprises residues 7 to 15, 7 to 16, or 7 to 17 of F3. In some embodiments, the F3 peptide comprises residues 5 to 14 of F3. In some embodiments, the F3 peptide comprises the sequence PQRRSARLSA (SEQ ID NO: 7).

In some embodiments, the F3 peptide is 80, 82, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9 or 100% identical to the corresponding sequence of F3 (SEQ ID NO: 2). In some embodiments, the F3 peptide comprises a sequence with 1, 2, or 3 substitutions relative to the corresponding sequence of F3. In some embodiments, the F3 peptide comprises the nucleic acid sequence of SEQ ID NO: 7.

In some embodiments, the F3 peptide further comprises a linker. In some embodiments, the linker comprises the sequence SGGGS (SEQ ID NO: 3). In some embodiments, the linker is N-terminal to the sequence of F3 in the F3 peptide. In some embodiments, the linker is C- terminal to the sequence of F3 in the F3 peptide. In some embodiments, the F3 peptide comprises a linker at both the N-terminus and the C-terminus.

In some embodiments, the F3 peptide comprises the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the F3 peptide is 80, 82, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9 or 100% identical to SEQ ID NO: 8. In some embodiments, the F3 peptide comprises a sequence with 1, 2, or 3 substitutions relative to SEQ ID NO: 8. Any of the anti-nucleolin antibodies described herein may comprise a heavy chain that includes a heavy chain variable region and optionally, a heavy chain constant region. In some embodiments, anti-nucleolin antibodies described herein may comprise a light chain that includes a light chain variable region and optionally, a light chain constant region. In some embodiments, anti-nucleolin antibodies described herein may comprise a heavy chain and a light chain. In some embodiments, anti-nucleolin antibodies described herein may comprise a VHH chain that includes a VHH chain variable region and optionally, a Fc domain.

In some embodiments, the anti-nucleolin antibody comprises a CDRl, a CDR2, and a CDR3. In some embodiments, the anti-nucleolin antibody comprises a heavy chain comprising a heavy chain variable region that comprises a heavy chain CDRl, a heavy chain CDR2, and/or a heavy chain CDR3. In some embodiments, the anti-nucleolin antibody comprises a light chain comprising a light chain variable region that comprises a light chain CDRl, a light chain CDR2, and/or a light chain CDR3. In some embodiments, the anti-nucleolin antibody comprises a VHH comprising a VHH variable region that comprises a VHH CDRl, a VHH CDR2, and/or a VHH CDR3.

In some embodiments, CDRl, CDR2, and/or CDR3 comprises an F3 peptide. In some embodiments, CDRl comprises an F3 peptide. In some embodiments, CDR2 comprises an F3 peptide. In some embodiments, CDR3 comprises an F3 peptide. In some embodiments, CDRl and CDR2 comprises an F3 peptide. In some embodiments, CDRl and CDR3 comprises an F3 peptide. In some embodiments, CDR2 and CDR3 comprises an F3 peptide. In some embodiments, CDRl, CDR2, and CDR3 comprises an F3 peptide.

In some embodiments, CDRl, CDR2, and/or CDR3 are substituted for an F3 peptide. In some embodiments, CDRl is substituted for an F3 peptide. In some embodiments, CDR2 is substituted for an F3 peptide. In some embodiments, CDR3 is substituted for an F3 peptide. In some embodiments, CDRl and CDR2 are substituted for an F3 peptide. In some embodiments, CDRl and CDR3 are substituted for an F3 peptide. In some embodiments, CDR2 and CDR3 are substituted for an F3 peptide. In some embodiments, CDRl, CDR2, and CDR3 are substituted for an F3 peptide. In some embodiments, CDR1, CDR2, and/or CDR3 comprises or are substituted for an F3 peptide and the CDRs that do not comprise F3 or are not substituted for F3 target an antigen other than nucleolin, e.g., TNF-a.

In some embodiments, the anti-nucleolin antibody comprises an F3 peptide flanked by FW (framework) 1 and FW2. In some embodiments, the anti-nucleolin antibody comprises an F3 peptide flanked by FW2 and FW3. In some embodiments, the anti-nucleolin antibody comprises an F3 peptide flanked by FW3 and FW4.

In some instances, the CDR1 region comprises the amino acid sequence of SEQ ID NO: 4, 7, or 8. In some instances, the CDR2 region comprises the amino acid sequence of SEQ ID NO: 5, 7, or 8. In some instances, the CDR3 region comprises the amino acid sequence of SEQ ID NO: 6, 7, or 8. In some specific examples, the variable region of an anti-nucleolin antibody as described herein comprises the amino acid sequence of any of SEQ ID NOs: 15- 18. In some specific examples, the variable region of an anti-nucleolin antibody as described herein comprises the nucleic acid sequence of any of SEQ ID NOs: 1 1-14. In some embodiments, the anti-nucleolin antibody as described herein comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the anti-nucleolin antibody as described herein comprises the nucleic acid sequence of SEQ ID NO: 31.

In some embodiments, the anti-nucleolin antibody comprises chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to any one of the CDR sequence provided by SEQ ID NO 4-8. In some embodiments, the anti-nucleolin antibody comprises a variable region that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the variable region of any of SEQ ID NO: 15-18.

The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403- 10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Also within the scope of the present disclosure are functional variants of any of the exemplary anti-nucleolin antibodies as disclosed herein. In some examples, the anti-nucleolin antibody is a functional variant of an antibody comprising a VHH variable region provided by any one of SEQ ID NO: 15- 18. A functional variant can comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in one or more of the CDR regions of the antibody that comprise the F3 peptide and binds the same epitope of nucleolin with substantially similar affinity (e.g., having a KD value in the same order). In one example, the amino acid residue variations are conservative amino acid residue substitutions. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. The heavy chain constant region is responsible for the prolonged serum half-life of the antibody, upon interacting with the neonatal Fc receptor (FcRn), which transports the antibody within and across cells, thus preventing its degradation. In addition, the heavy chain constant region also plays a central role in mediating different types of cell death, such as antibody- dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) (Brekke & Sandlie 2002. Nature Reviews Drug Discovery, 2(1), pp.52-62).

In some embodiments, the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4. In some embodiments, the anti-nucleolin antibodies as described herein comprise a portion (e.g., CHI , CH2, CH3, or a combination thereof) of a heavy chain constant region. In some embodiments, anti-nucleolin antibodies as described herein comprise Fc, e.g., an IgGl Fc, which is CH2 and CH3 of a heavy chain constant region. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The heavy and light chain constant regions can of any suitable origin, e.g., human, mouse, rat, or rabbit.

The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and/or complement function). In some embodiments the antibody has effector function and can fix complement. In other embodiments the antibody does not recruit effector cells or fix complement.

The antibody constant region is altered in some embodiments. Methods for altering an antibody constant region are known in the art. Antibodies with altered function, e.g. altered affinity for an effector ligand, such as FcR on a cell, or the CI component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388, 151 Al, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents of all of which are hereby incorporated by reference). Amino acid mutations which stabilize antibody structure, such as S228P (EU nomenclature, S241P in Kabat nomenclature) in human IgG4 are also contemplated. Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.

In some examples, the antibody that specifically binds to nucleolin described herein binds an epitope that comprises the following segments of SEQ ID NO: 1 : residues 1-283, residues 43-51, and/or residues 221-233.

It will be understood that the antibodies described herein minimally require nucleolin binding activity and ADCC. Other modifications are contemplated provided they do not interfere with these properties.

The anti-nucleolin antibody molecule can be used alone in unconjugated form, or can be bound to a substance, e.g., a toxin or moiety (e.g., a therapeutic drug; a compound emitting radiation; molecules of plant, fungal, or bacterial origin; or a biological protein or particle. For example, the anti-nucleolin antibody can be coupled to a radioactive isotope such as an α-, β-, or γ-emitter, or a β-and γ-emitter. An antibody molecule can be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a toxin, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

Useful detectable agents with which an anti-nucleolin antibody molecule may be derivatized (or labeled) to include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent emitting metal atoms, e.g., europium (Eu), and other anthanides, and radioactive materials (described below). Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5 dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin and the like. An antibody may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, β- galactosidase, acetylcholinesterase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody molecule may also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin). For example, an antibody may be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of bioluminescent materials include luciferase, luciferin, and aequorin. Labeled antibody molecule can be used, for example, diagnostically and/or experimentally in a number of contexts, including (i) to isolate a predetermined antigen by standard techniques, such as affinity chromatography or immunoprecipitation; (ii) to detect a predetermined antigen (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the protein; (iii) to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.

Some types of derivatized antibody molecule are produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL.

In some embodiments, an antibody or antigen fragment thereof, as is described herein, is bispecific. In some embodiments, the bispecific antibody or antigen fragment specifically binds nucleolin and a second antigen. In some embodiments, the second antigen is a molecule present on the surface of immune cells. In some embodiments, the molecule present on the surface of immune cells comprises MHC class I or MHC class II proteins, T cell receptors, B cell receptors, CD28, ICOS, TLT2, CD27, CD 137, OX40, HVEM, DR3, NKG2D, TIM- 1, TIM-2, DNAM-1, CRTAM, CTLA-4, PD-1, PD-Ll, PD-L2, CXCR4, CD3, B7-1, B7-2, BTLA, CD160, LAG-3, TIM-3, TIGIT, LAIR- 1, CAR, CD40, GITR, BAFF-R, TACI, BCMA, CD72, CD22, CD96, 2B4, NTB-A, CRACC, Siglec-3.7.9, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, CD94-NKG2A, CEACAMl, Plexin-Al, Plexin-A4, CD300b, CD300e, TREM1, TREM2, TREM3, ILT7, ILT3,4, TLT- 1, CD200R, TAM family, CD300a, CD300f, DC-SIGN, Kit, Allergin-1, Pir-B, MAFA, or Gp49B l .

An antibody molecule may be conjugated to another molecular entity, typically a label or a therapeutic (e.g., immunomodulatory, immunostimularoty, cytotoxic, or cytostatic) agent or moiety. Radioactive isotopes can be used in diagnostic or therapeutic applications. Radioactive isotopes that can be coupled to the anti-nucleolin antibodies include, but are not limited to α-, β-, or γ-emitters, or β-and γ-emitters.

ADCC

A key factor in the outcome of antibody-based cancer therapy is the antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is triggered by the simultaneous binding of an antibody to its target protein, through the Fab domains, and to Fc receptor (FcR)-expressing immune effector cells (e.g., Natural Killer, NK, cells and T cells), through the Fc region. This dual binding initiates a signaling pathway in the immune effector cells resulting in the secretion of several toxins towards the cells targeted by the antibody, resulting in cell death (Weiner et al. 2010. Nature reviews. Immunology, 10(5), pp.317-27).

The importance of this cell death mechanism for therapeutic outcomes has been characterized in several studies on polymorphisms in the IgGl Fc receptor-coding FcyRIIA and FcyRIIIA genes. These receptors present different expression patterns in different types of immune cells. FcyRIIa and FcYRIIIa/CD16a are low-affinity activating receptors for IgGl Fc and are expressed by subpopulations of NK cells, macrophages, and T cells. Polymorphisms in these genes augment the affinity of the IgGl Fc region towards the receptor and correlate with better clinical responses, when compared with the responses in the cohort without the polymorphisms. For example, non-Hodgkin lymphoma patients presenting with a mutation at position 158 of FcyRIIIa (where phenylalanine is replaced by valine), show a complete response to rituximab and the Fc receptor has stronger binding to the Fc region relative to wild-type (Cartron et al. 2002. Blood, 99(3), pp.754-758; Paiva et al. 2008. Cancer Genetics and

Cytogenetics, 183(1), pp.35-40). Also, in a cohort of patients with metastatic colorectal cancer, progression free survival was higher in patients with FcyRIIa- 131H/H and FcyRIIIa- 158V/V genotypes, regardless of the KRAS status (Bibeau et al. 2009. Journal of Clinical Oncology, 27(7), pp.1122-1 129). In another setting, treatment efficacy with trastuzumab was increased in patients with metastatic HER2 -positive breast cancer presenting V/V or H/H genotype, which correlated with higher ex-vivo ADCC activity of peripheral blood mononuclear cells (PBMCs) (Musolino et al. 2008. Journal of Clinical Oncology, 26(1 1), pp.1789-1796; Tamura et al. 201 1. Annals of Oncology, 22(6), pp.1302-1307). In this respect, there is evidence suggesting a stronger ADCC component underlying trastuzumab mechanism of action than solely interference at the level of the HER2-associated intracellular signaling pathway. In fact, in patients with HER2+ metastatic breast tumors, with complete or partial remission, upon treatment with preoperative trastuzumab, tumor infiltration of lymphoid cells was identified along with ex-vivo ADCC activity of PMBCs. In these patients, neither HER2 downmodulation nor changes in proliferation (as evaluated by Ki-67 staining) were observed during therapy

(Gennari et al. 2004). Enhanced ADCC responses were observed upon incubation of cetuximab with a squamous cell carcinoma of the head and neck (SCCHN) cell line and patient-derived NK cells, and they were also predictive of increased progression- free survival, further supporting the relevance of ADCC in antibody therapy (Taylor et al. 2015. Cancer Immunol Res, 3(5), pp.567- 574).

Differences in the amino acid composition and structure of the hinge region are the main characteristics for grouping IgGs in four subclasses (from IgGl to IgG4). IgG3 presents the longest hinge, thus being the subclass with the most flexible hinge region between Fabs and Fc region. The flexibility of the hinge region decreases, subsequently, in the following order: IgGl, IgG4 and IgG2. Based on this, IgGl and IgG3 are more prone to trigger immune functions. IgGl has the strongest ADCC activity, and IgG3 has the strongest CDC capacity. However, other factors besides the hinge region flexibility, rule the effectiveness and extent of responses of this nature. CDC effects are regulated by the presence of membrane -bound complement inhibitory proteins, whereas ADCC responses are affected, among other factors, by the antibody affinity and the antigen density (Velders et al. 1998. British Journal of Cancer, 78(4), pp.478^183; Tang et al. 2007. The Journal of Immunology, 179(5), pp.2815-2823; Gancz & Fishelson 2009. Molecular Immunology, 46(14), pp.2794-2800; M. Li et al. 2012. Cellular and Molecular Immunology, 9(1), pp.54-61). In terms of serum stability, IgG3 is the least stable, with a serum half- life of 7 days. The other subclasses present half-lives of about 21 days, which make them more adequate for therapeutic applications. Therefore, IgG2 or IgG4 are the common choices when immune responses arising from release of pro-inflammatory cytokines are undesirable, as in inflammatory and autoimmune disorders. For diseases in which immune functions have a beneficial effect, such as cancer and viral diseases, IgGl is the subclass of preference.

The anti-nucleolin fusion proteins (VHH-Fc) herein generated were based on the F3 peptide, which binds to the N-terminal domain of nucleolin (Christian et al. 2003. The Journal of Cell Biology, 163(4), pp.871-878). Based on the state of the art, it would not have been evident that targeting nucleolin, a fast internalizing protein, could elicit an ADCC response.

Without wishing to be bound by theory, due to the small size and paratope structure of the anti-nucleolin VHH antibodies described herein, the epitope recognized by the structure herein reported may detect nucleolin regions that do not trigger endocytosis of the

immunocomplexes. Such constructs then may be particularly useful. Without wishing to be bound by theory, the absence of ADCC responses of the anti- nucleolin antibodies developed so far could be an effect of the nucleolin domain being targeted. The antibodies herein developed are not only the first human antibody-based structures that target the N-terminal domain of nucleolin, but also the only reported structure with ADCC activity experimentally confirmed.

In some embodiments, the Fc region of the antibody or antibody fragment contains one or more substations selected from L234Y, S239D, T256A, K290A, K290Y, Y296W, S298A, A330F, A330L, I332E, E333A, K334A, K334V, A339T, E356K, K392D, D339K and K409D. In some embodiments, said substitutions increase the ADCC effect.

In some embodiments, the level of Fc-bound carbohydrate chains, including, but not limited to, alterations at the level of fructose, galactose, mannose, bisecting sugars and/or sialic acid is altered. In some embodiments, said alterations increase the ADCC effect.

In some embodiments, the Fc portion of the antibody or antibody fragment is coupled to a cytotoxic drug, e.g., auristatin, maytansinoid, calicheamicin, duocarmycin, amatoxin or pyrrolobenzodiazepine. In some embodiments, said coupling increases the ADCC effect.

In some embodiments, an anti-nucleolin antibody described herein has cytotoxicity towards a cell expressing nucleolin. In some embodiments, an anti-nucleolin antibody described herein has ADCC towards a cell expressing nucleolin.

In some embodiments, an anti-nucleolin antibody described herein has ADCC towards a cell expressing nucleolin sufficient to cause 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% cell death. In some embodiments, an anti-nucleolin antibody described herein has ADCC towards a cell expressing nucleolin sufficient to cause 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% more cell death than a control antibody. In some embodiments, a control antibody an antibody to a target other than nucleolin, e.g., TNF-a. In some embodiments, an anti-nucleolin antibody described herein with an Fc region has ADCC towards a cell expressing nucleolin sufficient to cause 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%, 300%, 400%, or 500% more cell death than the same antibody without an Fc region. Preparation of anti-nucleolin antibodies Antibodies capable of binding nucleolin as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

In some embodiments, antibodies specific to a target antigen (e.g., nucleolin or the N- terminal domain thereof) can be made by the conventional hybridoma technology. The full- length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen. The route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of immunogen, including as described herein.

Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982).

Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif, USA, may be used in the hybridization.

Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After the fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parent cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells may be used to produce the anti-nucleolin monoclonal antibodies described herein. The hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogen activity by conventional

immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay). Hybridomas that may be used as source of antibodies encompass all derivatives, progeny cells of the parent hybridomas that produce monoclonal antibodies capable of interfering with cell-surface localized nucleolin activity. Hybridomas that produce such antibodies may be grown in vitro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesired activity if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired antibodies off the immunogen. Immunization of a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N- hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC1, or R1N=C=NR, where R and Rl are different alkyl groups, can yield a population of antibodies (e.g., monoclonal antibodies).

If desired, an antibody (monoclonal or polyclonal) of interest (e.g. , produced by a hybridoma) may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. In an alternative, the polynucleotide sequence may be used for genetic manipulation to "humanize" the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody. For example, the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity to the target antigen and greater efficacy in inhibiting the activity of cell- surface localized nucleolin. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XenomouseR™ from Amgen, Inc. (Fremont, Calif.) and HuMAb-MouseR™ and TC Mouse™ from Medarex, Inc. (Princeton, N.J.). In another alternative, antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265, 150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455, and . Alternatively, the phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody) can be prepared via routine methods. For example, F(ab')2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments.

Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g., conventional recombinant technology. In one example, DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g. , PCT Publication No. WO 87/04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81 :6851 , or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. In that manner, genetically engineered antibodies, such as "chimeric" or "hybrid" antibodies; can be prepared that have the binding specificity of a target antigen. Techniques developed for the production of "chimeric antibodies" are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10033 (1989). In one example, variable regions of VH and VL of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art. Next, framework amino acid residues predicted to be important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences that are homologous to those of the parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected.

The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the parent non-human antibody or functional variants thereof. When necessary, residues within the framework regions of the parent chain that are predicted to be important in interacting with the CDR regions (see above description) can be used to substitute for the corresponding residues in the human acceptor genes.

A single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage or yeast scFv library and scFv clones specific to a nucleolin can be identified from the library following routine procedures. Positive clones can be subjected to further screening to identify those that inhibit cell surface-localized nucleolin activity.

Antibodies obtained following a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or "epitope mapping." There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide -based assays, as described, for example, in Chapter 1 1 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g. , recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to the gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactive ly labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of the nucleolin polypeptide have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein. By assessing binding of the antibody to the mutant nucleolin, the importance of the particular antigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.

In some examples, an anti-nucleolin antibody is prepared by recombinant technology as exemplified below. Nucleic acids encoding the heavy and light chain of an anti-nucleolin antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct prompter.

Alternatively, the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter. When necessary, an internal ribosomal entry site (IRES) can be inserted between the heavy chain and light chain encoding sequences. In the case of VHH antibodies, nucleic acids encoding the VHH chain of an anti-nucleolin antibody can be cloned into an expression vector under a suitable promoter.

In some examples, the nucleotide sequences encoding the heavy and light chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.

Generally, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.

A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV- 1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and the herpes simplex tk virus promoter.

Regulatable promoters can also be used. Such regulatable promoters include those using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those using the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9: 1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP 16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

Regulatable promoters that include a repressor with the operon can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with the transcription activator (VP 16) to create a tetR-mammalian cell transcription activator fusion protein, tTa (tetR- VP 16), with the tetO-bearing minimal promoter derived from the human cytomegalovirus (hCMV) major immediate-early promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline inducible switch is used. The tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.

Additionally, the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Examples of polyadenylation signals useful to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification. If necessary, polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.

In some embodiments, methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti- nucleolin antibody, or a VHH chain of an anti-nucleolin antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate -mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chain(s) that form the antibody, which can be recovered from the cells or from the culture medium. When the antibody comprises heavy and light chains, when necessary, the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.

In one example, two recombinant expression vectors are provided, one encoding the heavy chain of the anti-nucleolin antibody and the other encoding the light chain of the anti- nucleolin antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate - mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody. When the two expression vectors are introduced into the same host cells, the antibody produced therein can be recovered from the host cells or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody. When the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.

Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

Any of the nucleic acids encoding the heavy chain, the light chain, or both of an anti- nucleolin antibody as described herein, vectors (e.g., expression vectors) containing such; and host cells comprising the vectors are within the scope of the present disclosure.

Pharmaceutical compositions

The antibodies, as well as the encoding nucleic acids or nucleic acid sets, vectors comprising such, or host cells comprising the vectors, as described herein can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;

hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;

cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt- forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some examples, the pharmaceutical composition described herein comprises liposomes containing the antibodies (or the encoding nucleic acids) which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

The antibodies, or the encoding nucleic acid(s), may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene -vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3- hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. , water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface- active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. , soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im, and have a pH in the range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an antibody with

Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

Methods of treatment

Any of the antibodies, as well as the encoding nucleic acids or nucleic acid sets, vectors comprising such, or host cells comprising the vectors, described herein are useful for treating a disease or disorder associated with cell-surface localized nucleolin, including cancer. To practice the method disclosed herein, an effective amount of the antibody described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g. , as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the antibodies as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder. In a preferred embodiment, the antibodies described herein are administered by intravenous administration.

The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having, at risk for, or suspected of having a target disease/disorder, such as cancer. A subject having a target disease or disorder can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. A subject suspected of having any of such target disease/disorder might show one or more symptoms of the disease/disorder. A subject at risk for the disease/disorder can be a subject having one or more of the risk factors for that disease/disorder.

The methods and compositions described herein may be used to treat any disease or disorder associated with cell-surface localized nucleolin. In some embodiments, the target disease is cancer. Cancers include but are not limited to: Oral: buccal cavity, lip, tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: non-small cell lung cancer (NSCLC), small cell lung cancer, bronchogenic carcinoma (squamous cell or epidermoid, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, larynx, adenocarcinoma,

leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or small intestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestines (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), rectal, colon, colon-rectum, colorectal; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:

hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages; Bone: osteogenic sarcoma

(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), head and neck cancer, meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous

cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoll-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal

rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma] hairy cell; lymphoid disorders; Skin:

malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma; and Adrenal glands:

neuroblastoma.

As used herein, "an effective amount" refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is reduced cell-surface localized nucleolin. Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a target disease/disorder. Alternatively, sustained continuous release formulations of an antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one example, dosages for an antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the antagonist. To assess efficacy of the antagonist, an indicator of the disease/disorder can be followed.

For the purpose of the present disclosure, the appropriate dosage of an antibody as described herein will depend on the specific antibody, antibodies, and/or non-antibody peptide (or compositions thereof) employed, the type and severity of the disease/disorder, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antagonist, and the discretion of the attending physician. Typically the clinician will administer an antibody, until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a decrease the severity of cancer. Methods of determining whether a dosage resulted in the desired result would be evident to one of skill in the art. Administration of one or more antibodies can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of an antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.

As used herein, the term "treating" refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease/disorder, or a predisposition toward the disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.

Alleviating a target disease/disorder includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

"Development" or "progression" of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a target disease or disorder includes initial onset and/or recurrence.

In some embodiments, the antibodies described herein are administered to a subject in need of the treatment at an amount sufficient to inhibit the activity of the target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In other embodiments, the antibodies are administered in an amount effective in reducing the activity level of a target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

Injectable compositions may contain various carriers such as vegetable oils,

dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.

In one embodiment, an antibody is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/5321 1 and U.S. Pat. No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisense polynucleotide, expression vector, or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 1 1 :202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621 ; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1 :51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1 : 185; and Kaplitt, Nature Genetics (1994)

6: 148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/1 1230; WO 93/10218; WO 91/02805; U.S. Pat. Nos.

5,219,740 and 4,777, 127; GB Patent No. 2,200,651 ; and EP Patent No. 0 345 242), alphavirus- based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/1 1984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3: 147 can also be employed.

Non- viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264: 16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/1 1092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91 : 1581.

The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history.

In some embodiments, more than one antibody, or a combination of an antibody and another suitable therapeutic agent, may be administered to a subject in need of the treatment. The antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

Treatment efficacy for a target disease/disorder can be assessed by methods well-known in the art.

Combination Therapies

In some embodiments, the anti-nucleolin antibody or antibody fragment described herein is administered in conjunction with additional cancer therapy.

As used herein, "in conjunction with" shall mean that the anti-nucleolin antibody or antibody fragment is administered to a subject concurrently with one or more additional therapies (either simultaneously or separately but in close proximity), prior to, or after administration of one or more additional therapies. In some embodiments, the anti-nucleolin antibody or antibody fragment is conjugated to one or more additional therapies.

In some embodiments, an additional cancer therapy comprises a chemotherapeutic agent. Chemotherapeutic agents include, for example, including alkylating agents, anthracyclines, cytoskeletal disruptors (Taxanes), epothilones, histone deacetylase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, nucleotide analogs and precursor analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids and derivatives thereof. Non-limiting examples include: (i) anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin- 1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT- 1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000)); (ii) a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof; and (iii) chemotherapeutic compounds such as, e.g., pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, epidipodophyllotoxins (etoposide and teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin

(actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes- dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin,

dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, and irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors;

mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

In some embodiments, an additional cancer therapy comprises external radiation therapy and internal radiation therapy (also called brachytherapy). Energy sources for external radiation therapy include x-rays, gamma rays and particle beams, energy sources used in internal radiation include radioactive iodine (iodine 125 or iodinel31), strontium89, or radioisotopes of phosphorous, palladium, cesium, indium, phosphate, or cobalt. Methods of administering radiation therapy are well known to those of skill in the art.

In some embodiments, an additional cancer therapy comprises an immunotherapy. Cancer immunotherapy is the use of the immune system to reject cancer. The main premise is stimulating the subject's immune system to attack the tumor cells that are responsible for the disease. This can be either through immunization of the subject, in which case the subject's own immune system is rendered to recognize tumor cells as targets to be destroyed, or through the administration of therapeutics, such as antibodies, as drugs, in which case the subject's immune system is recruited to destroy tumor cells by the therapeutic agents. Cancer immunotherapy includes an antibody-based therapy and cytokine-based therapy.

A number of therapeutic monoclonal antibodies have been approved by the FDA for use in humans, and more are underway. The FDA-approved monoclonal antibodies for cancer immunotherapy include antibodies against CD52, CD33, CD20, ErbB2, vascular endothelial growth factor and epidermal growth factor receptor. These and other antibodies targeting one or more cancer-associated antigen are thus suitable for use in a combination therapy to be administered in conjunction with an anti-nucleolin antibody or antibody fragment. Examples of monoclonal antibodies approved by the FDA for cancer therapy include, without limitation: Rituximab (available as Rituxan™), Trastuzumab (available as Herceptin™), Alemtuzumab (available as Campath-IH™), Cetuximab (available as Erbitux™), Bevacizumab (available as Avastin™), Panitumumab (available as Vectibix™), Gemtuzumab ozogamicin (available as Mylotarg™), Ibritumomab tiuxetan (available as Zevalin™), Tositumomab (available as Bexxar™), Ipilimumab (available as Yervoy™), Ofatunumab (available as Arzerra™), Daclizumab (available as Zinbryta™), Nivolumab (available as Opdivo™), and Pembrolizumab (available as Keytruda™). Examples of monoclonal antibodies currently undergoing human clinical testing for cancer therapy in the United States include, without limitation: WX-G250 (available as Rencarex™), Zanolimumab (available as HuMax-CD4), chl4.18, Zalutumumab (available as HuMax-EGFr), Oregovomab (available as B43.13, OvalRex™), Edrecolomab (available as IGN-101, Panorex™), 131I-chTNT-I/B (available as Cotara™), Pemtumomab (available as R-1549, Theragyn™), Lintuzumab (available as SGN-33), Labetuzumab (available as hMN14, CEAcide™), Catumaxomab (available as Removab™), CNTO 328 (available as cCLB8), 3F8, 177Lu-J591 , Nimotuzumab, SGN-30, Ticilimumab (available as CP-675206), Epratuzumab (available as hLL2, LymphoCide™), 90Y-Epratuzumab, Galiximab (available as IDEC- 1 14), MDX-060, CT-011, CS-1008, SGN-40, Mapatumumab (available as TRM-I), Apolizumab (available as HuIDlO, Remitogen™) and Volociximab (available as M200).

Cancer immunotherapy also includes a cytokine-based therapy. The cytokine-based cancer therapy utilizes one or more cytokines that modulate a subject's immune response. Non- limiting examples of cytokines useful in cancer treatment include interferon-a (IFN-a), interleukin-2 (IL-2), Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12).

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques),

microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P.

Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

Example 1 - Construction of VHH fragments targeting the N-terminal domain of nucleolin

The nucleolin-binding 10-amino acid sequence from F3 peptide (PQRRSARLSA) (SEQ

ID NO: 7) (Porkka et al. 2002. Proceedings of the National Academy of Sciences of the United States of America, 99(1 1), pp.7444-7449), which binds to the N-terminal domain of nucleolin (Christian et al. 2003. The Journal of Cell Biology, 163(4), pp.871-878), was grafted onto CDR1 or CDR3 of an anti-human TNF-a VHH, by polymerase chain reaction (PCR), as represented in Figure 1. In addition, a variant of this sequence, flanked by the linker SGGGS (SEQ ID NO: 3) at both ends, was also grafted onto each CDR. This type of sequence promotes loop flexibility and can therefore improve the binding of the protein towards its target (Fellouse et al. 2004. Proceedings of the National Academy of Sciences of the United States of America, 101(34), pp.12467-12472; Birtalan et al. 2010. Molecular BioSystems, 6, pp.1186-1 194). Primers (Table 1) were used to amplify the 10-amino acid sequence (with or without the linker) with primers adding sequence to the framework region flanking the CDR being replaced. PCR reactions were carried out with Phusion DNA Polymerase (Thermo Scientific, USA), under the conditions described in Table 2. PCR products were visualized on an agarose gel and recovered with the NzyGelPure kit (Nzytech, Portugal). A second PCR was carried out to overlap the two obtained sequences ( i.e., the first sequence being from the beginning of the VHH to the end of the grafted CDR(1 or 3) and the second sequence being from the beginning of the grafted CDR(1 or 3) to the end of the VHH). PCR conditions were the same as for the first PCR.

However primers not added until 10 cycles had passed. Upon recovery, the DNA was digested with Hindlll and Bglll (Thermo Scientific, USA) and inserted onto a pT7 vector with a peptide leader sequence, which exports the protein to the periplasm, and a histidine tag, used for purification. T4 DNA Ligase (Thermo Scientific, USA) was used for the ligation reaction, at 22°C for 2 h, followed by enzyme inactivation at 70°C for 5 min. The mixture was then used to transform JM109 bacteria by electroporation. A schematic of the cloning reaction is shown in Figure 1.

Table 1. Primers used for grafting of nucleolin-binding sequence in VHH CDRl and CDR3 and cloning in pFuse vector. Forward primers are represented with F and reverse primers with R.

5'

aNCL-CDRl-L R CGCGCTCAGACGCGCGCTACGACGCTGCGGGCTGCCGCCGCCGCTAGAG

GCGGCACAGCT (SEQ ID NO: 22)

5'

aNCL-CDR3 F TATTACTGTGCAGCGCCGCAGCGTCGTAGCGCGCGTCTGAGCGCGTGGGG

TCAGGGCACC (SEQ ID NO: 23)

5 ' CGCGCTCAGACGCGCGCTACGACGCTGCGGCGCTGCACAGTAATA aNCL-CDR3 R

(SEQ ID NO: 24)

5'

aNCL-CDR3-L F TATTACTGTGCAGCGCCGCAGCGTCGTAGCGCGCGTCTGAGCGCGTGGGG

TCAGGGCACC (SEQ ID NO: 25)

5 ' CGCGCTCAGACGCGCGCTACGACGCTGCGGCGCTGCACAGTAATA aNCL-CDR3-R R

(SEQ ID NO: 26)

5'

VHH Overlap F AAGCTTATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTT

CGCTACCGTGGCCCAGGCGGCCCA (SEQ ID NO: 27)

5' AATGGAAGATCTTTATTAGCTCGCGTAATCAGGCACGTCGTAG (SEQ ID

VHH Overlap R

NO: 28)

VHH-Fc F 5' AAACCATGGAACAGGTGCAGCTGCAG (SEQ ID NO: 29)

VHH-Fc R 5' ATAAGATCTGCTACTAACGGTCACCC (SEQ ID NO: 30)

Table 2. PCR conditions used for grafting of nucleolin-binding sequence onto VHH

DNA from positive clones (Figure 2), as determined by sequencing, was recovered with the Zyppy Plasmid Miniprep Kit (Zymo Research, USA) and BL21 (DE3) E. coli cells were transformed for VHH expression. Bacteria were grown in SB medium until reaching an optical density between 0.7 and 0.9, at 600 nm, after which protein expression was induced with 1 mM isopropyl β-D-l -thiogalactopyranoside (IPTG, Thermo Scientific, USA), at 16°C, for 16 h. VHH was purified by affinity chromatography with nickel columns and further desalted with PD-10 columns (GE Healthcare, UK).

The parental VHH nucleic acid and amino acid sequence are shown in SEQ ID NO: 9- 10, respectively. The nucleic acid and amino acid sequences of the novel anti-nucleolin VHH constructs are shown in SEQ ID NO: 1 1-14 and 15-18, respectively. The amino acid sequences of the CDRs are shown in Table 3.

Table 3. CDRs of the parental and novel anti-nucleolin VHH constructs

Example 2 - Binding of anti-nucleolin VHH fragments to human nucleolin and human TNF- a proteins

Binding of the generated anti-nucleolin VHH fragments to human nucleolin and to human TNF-a proteins was assessed by enzyme-linked immunosorbent assay (ELISA). Plates were first coated with 100 ng of human nucleolin or 200 ng of human TNF-a in carbonate buffer (50 mM sodium carbonate, pH 9.6), at 4°C, overnight. Nonspecific binding sites were then blocked with 3% (w/v) bovine serum albumin (BSA) in phosphate buffer saline (PBS, 137 mM NaCl, 2.7 mM KC1, 10 mM Na2HP04, 1.8 mM KH2P04, pH 7.4) for 1 h, at 37°C, washed once with PBS and further incubated with 100 pmol of each VHH construct (diluted in 1%, w/v, BSA in PBS), for 1 h at 37°C. After five washing steps, 100 μΐ of anti-HA-peroxidase antibody (clone 3F10 from Roche, Switzerland, diluted at 1 : 1000 in 1% BSA in PBS) was added and the plate was incubated for 1 h at 37°C. Following five additional washing steps, 100 μΐ of 0.06% (v/v) of H2O2 in 0.4 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) solution was added to the wells. Absorbance (405 nm/495 nm) was measured after 5-15 min on a microplate reader (Model 680, BioRad, USA).

All of the anti-nucleolin VHH fragments bound nucleolin, regardless the CDR grafted, with aNCL-CDR3 and aNCL-CDR3-L showing the highest degree of binding. The nucleolin- binding VHHs with F3 grafted onto CDR1 or CDR3 showed a 2- and 3-fold increased nucleolin binding, respectively, relative to the parental VHH, without the F3 peptide-derived sequence. This demonstrates a role of the F3 peptide-derived sequence in the binding of the anti-nucleolin VHH constructs to nucleolin, regardless the CDR grafted (Figure 3A). The anti-nucleolin VHHs presented a limited level of binding to TNF-a, which was approximately 40% lower than that observed for the parental VHH (Figure 3B).

Example 3 - Binding of anti-nucleolin VHH fragments to nucleolin-overexpressing cells

Human MDA-MB-435S cancer cells, human MDA-MB 231 cells, and mouse 4T1 breast cancer cells (ATCC, USA) were maintained in RPMI-1640 (Lonza, Switzerland), supplemented with 10%) (v/v) heat-inactivated fetal bovine serum (FBS, HyClone, USA), 2 mM of L- glutamine (Lonza, Switzerland) and 1% (v/v) Pen/Strep/Fungizone solution (HyClone, USA), at 37°C in a humidified atmosphere of 5% CO2.

To evaluate cell binding of the anti-nucleolin VHH fragments, one hundred thousand nucleolin-overexpressing cells previously treated with dissociation buffer were incubated with 10, 100 or 1000 nM VHH protein at 4°C for 45 min. After washing, a second incubation with anti-HA-FITC antibody (Y- 11 sc-805, Santa Cruz Biotechnology, USA) was performed at room temperature for 30 min. Cells were again washed, fixed and analyzed by flow cytometry (Guava easyCyte 5HT, Merck Millipore, USA), using the InCyte software module (Merck Millipore, USA). In a competitive inhibition assay, cells were pre-incubated with 75 μΜ of F3 peptide (custom synthesized by Genecust, Luxemburg) or 1 μΜ of infliximab (Janssen Biologies BV, The Netherlands), at 4°C for 30 min, followed by incubation with 1000 nM VHH fragments at 4°C for 45 min.

Grafting of the nucleolin-binding F3 peptide-derived sequence onto either CDR1 or CDR3 of the VHH led to a concentration-dependent binding of the protein to nucleolin- overexpressing cancer cells (Figures 4A-4C). In the case of MDA-MB-435S cells, a significant increase in binding, at 100 nM, of aNCL-CDR3 (p<0.01) or aNCL-CDR3-L (pO.001) relative to aNCL-CDRl and aNCL-CDRl-L or to the parental VHH (pO.001), was observed. This trend was confirmed at the 1000 nM protein concentration (p<0.001). At this protein concentration, a significant difference between the binding of a-NCL-CDRl VHH or a-NCL- CDR1-L VHH and the parental VHH (p<0.001) was also observed. At the highest concentration tested, CDR1- and CDR3-grafted VHHs bound approximately 40% and 80% of cells, respectively, whereas the parental VHH bound to less than 10% of the cells (Figure 4A). The binding profile of anti-nucleolin VHH constructs to the 4T1 breast cancer cell line, relative to the parental VHH (p<0.01), was similar to binding profile for MDA-MB-435S (Figure 4B). The difference between peptide grafting either onto CDR1 or CDR3 was not so pronounced and a decrease of up to four- fold of anti-nucleolin VHH was observed relative to MDA-MB-435S cells, depending on the protein concentration. In the MDA-MB-231 cell line, a similar concentration-dependent binding was observed for both anti-nucleolin VHHs and parental VHH, with 20-30% of the cells being targeted by the incubated nanobodies at the highest concentration tested (Figure 4C). For all tested cell lines, the presence of SGGGS sequences flanking the F3 peptide-derived sequence did not alter significantly the anti-nucleolin VHHs binding.

Pre-incubation of each of the cell lines tested with the F3 peptide resulted in a reduction of the anti-nucleolin VHH binding of at least 50% (Figures 4D-4F). The binding reduction of aNCL-CDR3 VHH and aNCL-CDR3-L VHH with MDA-MD-435S cells was greater than the reduction observed with pre-incubation with infliximab (Figure 4D). With the MDA-MB-231 cells, competitive inhibition with infliximab resulted in a reduction of binding for all anti- nucleolin VHHs, in similar levels to the ones observed when competitive inhibition was performed with the F3 peptide (Figure 4E). In contrast, binding of anti-nucleolin VHHs to 4T1 cells was not altered in the presence of infliximab (Figure 4F). These results suggest the involvement of cell surface nucleolin in the binding of the novel anti-nucleolin VHHs with the three cell lines tested. While in the 4T1 cells, binding was exclusively mediated by nucleolin, in the other cells lines (especially in MDA-MB-231) the results presented herein suggest TNF-a is also involved. In line with the results from Example 2, these assays also suggested that nucleolin binding was enabled by grafting of the F3 peptide-derived sequence onto either CDRl or CDR3.

Example 4 - Cytotoxicity of anti-nucleolin VHH against nucleolin-overexpressing cells

To evaluate the cytotoxicity of the anti-nucleolin VHH fragments, different cell densities of nucleolin-overexpressing cancer cell lines were seeded in 96-well plates (5000 MDA-MB- 435S or MDA-MB-231 cells or 3000 4T1 cells, per well). After 24 h, cell culture medium was exchanged, and cells were incubated with serial dilutions of VHH proteins, for a total of 72 h. Cell viability was then evaluated using the MTT assay (Mosmann 1983. Journal of

Immunological Methods, 65(1-2), pp.55-63), as following:

ODuntreated cells— ODtreated cells

% cell viability = :— xlOO

ODuntreated cells

All anti-nucleolin VHH fragments have cytotoxic effects in the micromolar range against nucleolin-overexpressing cancer cells, in a concentration-dependent manner (Figure 5A-5C). In the case of MDA-MB-435S cells, differences relative to the parental VHH are evident at 4 μΜ, with cell viability reduced to 60% (pO.001 for aNCL-CDRl ; p<0.01 for aNCL-CDRl-L) for the CDRl -grafted VHHs or 30% (p<0.001) for the CDR3 -grafted VHHs (corresponding to 1.5- or 2.5-fold decrease in cell viability, respectively). At 8 μΜ, differences in cell viability between anti-nucleolin VHH fragments were no longer visible, resulting in less than 20% of viable cells and reaching a 1.5-fold decrease in cell viability relative to the parental VHH (pO.001).

Parental VHH reduced cell viability to 45% (Figure 5A). This effect was expected, as the parental VHH is an anti-human TNFa VHH. These results suggest a bispecific effect of the anti- nucleolin VHHs. The extent of the decrease in cell viability achieved with aNCL-CDR3 VHH and aNCL- CDR3-L VHH in 4T1 cancer cells, was similar to that seen in MDA-MB-435S cells at all protein concentrations tested (at 4 μΜ, p<0.05 and p<0.01 for aNCL-CDR3 and aNCL-CDR3- L, respectively, and at 8 μΜ, p<0.001 for all anti-nucleolin VHHs, relative to the parental VHH). These results suggest that grafting the F3 peptide onto CDR3 improved the cytotoxicity (e.g., resulted in decreased of cell viability) of anti-nucleolin VHHs (Figure 5B).

Improved cytotoxicity against MDA-MB-231 cancer cells was only evident for the aNCL- CDR3 VHH and aNCL-CDR3-L VHH, as compared with aNCL-CDRl VHH, aNCL-CDRl-L VHH or the parental VHH (p<0.05, compared to CDR1 -grafted VHHs at 4 μΜ and p<0.01 or p<0.001, respectively, compared to aNCL-CDRl VHH; p<0.001 compared to aNCL-CDRl -L VHH; p<0.01 or pO.001 , respectively, compared to parental VHH at 8 μΜ). CDR1- and CDR3 -grafted anti-nucleolin VHHs showed a decrease of cell viability that was 60% and 40% (at 4 μΜ) or 55% and 20% (at 8 μΜ) lower, respectively, than that observed against MDA-MB- 435 S cancer cells. These results are consistent with the reduced binding levels observed with MDA-MB-231 cells relative to MDA-MB-435S cells. Parental VHH also reduced cell viability (down to 60%), suggesting a bispecific effect of the anti-nucleolin VHHs in these cells as in MDA-MB-435S cells (Figure 5C).

Example 5 - Construction of an anti-nucleolin VHH-Fc antibody

One of the VHH fragments presenting with the highest binding and cytotoxicity, aNCL-

CDR3, was cloned into a pFuse vector for construction of a fusion protein (aNCL- VHH-Fc) (Figures 6A-6B). This vector incorporates an IL2 signal sequence that causes secretion of the protein to the extracellular medium. The parental VHH was also cloned into this vector to generate the parental VHH-Fc antibody. DNA of the parental and aNCL-CDR3 VHH was digested from the pT7 vector using Ncol and Bglll (Thermo Scientific, USA). Ligation of the digested fragments in the pFuse vector and subsequent transformation of JM109 bacteria were performed as described in Example 1. HEK293T cells were then transfected, by the calcium phosphate method, with a positive clone of each sequence for protein expression (Graham & Van der Eb 1973. Virology, 52, pp.456-467; Jordan et al. 1996. Nucleic Acids Research, 24(4), pp.596-601). HEK293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Lonza, Switzerland), supplemented with 10% (v/v) heat-inactivated FBS (HyClone, USA), 2 mM of L-glutamine (Lonza, Switzerland) and 1% (v/v) Penicillin/Streptomycin/Fungiezone solution (HyClone, USA). Cell culture medium was exchanged for FBS-free fresh medium 6 h after transfection. After 48 h, cell medium was collected and the protein was purified by affinity chromatography using protein A column (Pierce, USA). Desalting was carried out with PD-10 desalting columns (GE Healthcare, UK), equilibrated in 20 mM HEPES, 100 mM NaCl, 5% (v/v) glycerol, pH 8.0. The nucleic acid sequence of the anti-nucleolin VHH-Fc antibody is shown in SEQ ID NO: 31. The amino acid sequence of the anti-nucleolin VHH-Fc antibody is shown in SEQ ID NO : 32.

Example 6 - Cytotoxicity of anti-nucleolin VHH-Fc against nucleolin-overexpressing cells

Cytotoxicity of the VHH-Fc antibodies against nucleolin-overexpressing cells was evaluated as described in Example 4, but using a different range of protein concentrations. The developed anti-nucleolin VHH-Fc antibody presented cytotoxic effects in the nanomolar range (Figures 7A-7C). Similar to what was observed for the anti-nucleolin VHH fragments, the decrease of cell viability observed upon incubation with the anti-nucleolin VHH-Fc antibody was more pronounced in MDA-MB-435S and 4T1 cell lines (less than 25% viable cells at the highest concentration tested) when compared with MDA-MB-231 (approximately 50% viable cells at the highest concentration tested). At the highest concentration tested, the anti-nucleolin VHH-Fc antibody led to a 1.7- or a 1.5-fold decrease in viability of MDA-MB-435S or MDA- MB-231 cells, respectively, relative to the parental VHH-Fc antibody. In accordance with what was observed in the cytotoxicity assays with the parental VHH, the parental VHH-Fc antibody did not alter the viability of 4T1 cells but decreased the viability of MDA-MB-435S and MDA- MB-231 cells to 53% and 64%, respectively.

Example 7 - ADCC of anti-nucleolin VHH-Fc against nucleolin-expressing cancer cells

The ADCC potential of the anti-nucleolin VHH-Fc antibody was tested against MDA- MB-435S (target) cancer cells, with the xCelligence Real-Time Cell Analyzer (RTCA; ACEA Biosciences, USA). PBMCs, used as effector cells, were isolated from buffy coat harvested from four healthy donors by a Ficoll-Paque PLUS density gradient (GE Healthcare, UK). Following culture in RPMI-1640 (supplemented as described in Example 3) at 37°C in a humidified atmosphere of 5% CO2, PBMCs were stored at -80°C in freezing medium (10% v/v dimethyl sulfoxide in FBS) until use. When needed, PBMCs were thawed, resuspended in culture medium and incubated overnight at 37°C in a humidified atmosphere of 5% CO2. To assess the ADCC effect, 7500 MDA-MB-435S adherent cancer cells were incubated on a RTCA 96-well plate for 24 h and then incubated with PBMCs at a final 5: 1 or 10:1 effector/target cell ratio and 25 nM of anti-nucleolin VHH-Fc. Cancer cells incubated only with effector cells or antibody were included as additional controls. Since ADCC is an Fc-dependent mechanism, MDA-MB-435S cells were also incubated with the VHH counterpart of the VHH-Fc antibodies. As the VHH-Fc antibodies are dimeric, the VHH fragments were added in a concentration of 50 nM.

Cell index was measured every 15 min for 96 h and the resulting curves were plotted and normalized to 1.0, matching the beginning of the incubation of PBMC with the different tested proteins and the cancer cells. Data were analyzed with RTCA Software Package and cancer cell death resulting from incubation with the fusion protein and PBMCs was calculated from the area under the curve (AUC) values, between approximately 24 h to 72 h, as following:

AUC(protein + PBMC) - AUC(protein) - AUC(PBMC)

death = -——

AUC (untreated cells)

The anti-nucleolin VHH-Fc antibody enabled higher cell death than the parental VHH-Fc (Figure 8A). This effect was partly dependent on the presence of the Fc region, as the anti- nucleolin VHH protein did not trigger the same level of cancer cell death in the presence of PBMCs (Figure 8B). These results confirmed that the anti-nucleolin VHH-Fc antibody was able to trigger ADCC. This was a nucleolin-specific effect, as there were no differences in cell death between parental VHH and parental VHH-Fc, in the presence of PBMCs (Figure 8C). The ADCC effect of the anti-nucleolin VHH-Fc was observed using PBMCs harvested from four donors (Table 4), showing different levels of ADCC activity. Upon PBMC incubation, and depending on the donor, the anti-nucleolin VHH-Fc protein enabled a 1.6- to 2.2-fold increase in cell death, relative to the parental VHH-Fc and a 1.3- to 2.1 -fold increase when compared to the anti-nucleolin VHH counterpart. Therefore, regardless of the PBMC origin, an Fc-dependent, nucleolin-specific effect has been shown, demonstrating an ADCC effect of the anti-nucleolin VHH-Fc antibody.

Table 4. Table 4 summarizes the cytotoxicity of anti-nucleolin and parental VHH-Fc or anti- nucleolin VHH constructs in the presence of PBMCs harvested from four different donors (values indicate percentage of cell death). Differences in the ADCC capacity among the proteins tested, were evaluated with repeated measures ANOVA, followed by Tukey's Multiple 5 Comparison Test.

Sequences of Nucleolin Constructs

SEQID NO: 4

10 GRTFSDHSGYTYTIG

SEQID NO: 33

CGCATCTATTGGAGCTCTGGTAACACCTATTACGCAGATAGTGTTAAAGGC

The amino acid sequence coded for by SEQ ID NO: 33 is SEQ ID NO: 5

SEQID NO: 5

15 Rl YWSSG NTYYADSVKG

SEQID NO: 6

RDGI PTSRSVESYNY SEQID NO: 7

PQRRSARLSA

20 SEQ ID NO: 8

SGGGSPQRRSARLSASGGGS SEQ ID NO: 9 - Parental VHH DNA sequence

ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGTGCAGCTGCAGGA ATCTGGCGGTGGCCTGGTTCAGCCGGGTGGCAGTCTGCGCCTGAGCTGTGCCGCCTCTGGTCGTACCTTTAGCGA TCATTCTGGTTATACCTACACGATTGGCTGGTTTCGTCAGGCGCCGGGCAAAGAACGTGAATTCGTGGCCCGCATC TATTGGAGCTCTGGTAACACCTATTACGCAGATAGTGTTAAAGGCCGTTTCGCCATTAGCCGCGATATCGCAAAAA ATACCGTGGATCTGACGATGAACAATCTGGAACCGGAAGATACCGCCGTTTATTACTGTGCAGCGCGTGATGGCA TTCCGACGTCTCGCAGTGTGGAAAGCTATAACTACTGGGGTCAGGGCACCCAGGTGACCGTTAGTAGCGGCCAG GCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAG SEQ ID NO: 10 - Parental VHH Amino acid sequence

M KKTAI AI AVALAG FATVAQAAQVQLQESG G G LVQP GG SLR LSCAASG RTFS D HS GYTYTI GW F RQAPG K E R E FVAR I YWSSG NTYYADSVKG R FAIS R D I AKNTVD LTM N N LE P E DTAVYYCAAR DG I PTS RSVESYNYWG QGTQVTVSSG QAG QH H H H H H G AYPYDVP DYAS Stop

SEQ ID NO: 11 - aNCL-CDRl DNA sequence

ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCCCAGGTGCA GCTGCAGGAATCTGGCGGTGGCCTGGTTCAGCCGGGTGGCAGTCTGCGCCTGAGCTGTGCCGCCTCTCCGCAGC GTCGTAGCGCGCGTCTGAGCGCGTGGTTTCGTCAGGCGCCGGGCAAAGAACGTGAATTCGTGGCCCGCATCTATT GGAGCTCTGGTAACACCTATTACGCAGATAGTGTTAAAGGCCGTTTCGCCATTAGCCGCGATATCGCAAAAAATA CCGTGGATCTGACGATGAACAATCTGGAACCGGAAGATACCGCCGTTTATTACTGTGCAGCGCGTGATGGCATTC CGACGTCTCGCAGTGTGGAAAGCTATAACTACTGGGGTCAGGGCACCCAGGTGACCGTTAGTAGCGGCCAGGCC GGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAG SEQ ID NO: 12 - aNCL-CDR3 DNA sequence

ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGTGCAGCTGCAGGA ATCTGGCGGTGGCCTGGTTCAGCCGGGTGGCAGTCTGCGCCTGAGCTGTGCCGCCTCTGGTCGTACCTTTAGCGA TCATTCTGGTTATACCTACACGATTGGCTGGTTTCGTCAGGCGCCGGGCAAAGAACGTGAATTCGTGGCCCGCATC TATTGGAGCTCTGGTAACACCTATTACGCAGATAGTGTTAAAGGCCGTTTCGCCATTAGCCGCGATATCGCAAAAA ATACCGTGGATCTGACGATGAACAATCTGGAACCGGAAGATACCGCCGTTTATTACTGTGCAGCGCCGCAGCGTC GTAGCGCGCGTCTGAGCGCGTGGGGTCAGGGCACCCAGGTGACCGTTAGTAGCGGCCAGGCCGGCCAGCACCA TCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAG

SEQ ID NO: 13 - aNCL-CDRl-L DNA sequence

ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCCCAGGTGCA GCTGCAGGAATCTGGCGGTGGCCTGGTTCAGCCGGGTGGCAGTCTGCGCCTGAGCTGTGCCGCCTCTAGCGGCG GCGGCAGCCCGCAGCGTCGTAGCGCGCGTCTGAGCGCGAGCGGCGGCGGCAGCTGGTTTCGTCAGGCGCCGGG CAAAGAACGTGAATTCGTGGCCCGCATCTATTGGAGCTCTGGTAACACCTATTACGCAGATAGTGTTAAAGGCCG TTTCGCCATTAGCCGCGATATCGCAAAAAATACCGTGGATCTGACGATGAACAATCTGGAACCGGAAGATACCGC CGTTTATTACTGTGCAGCGCGTGATGGCATTCCGACGTCTCGCAGTGTGGAAAGCTATAACTACTGGGGTCAGGG CACCCAGGTGACCGTTAGTAGCGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGT TCCGGACTACGCTTCTTAG

SEQ ID NO: 14 - QLNCL-CDR3-L DNA sequence

ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGTGCAGCTGCAGGA ATCTGGCGGTGGCCTGGTTCAGCCGGGTGGCAGTCTGCGCCTGAGCTGTGCCGCCTCTGGTCGTACCTTTAGCGA TCATTCTGGTTATACCTACACGATTGGCTGGTTTCGTCAGGCGCCGGGCAAAGAACGTGAATTCGTGGCCCGCATC TATTGGAGCTCTGGTAACACCTATTACGCAGATAGTGTTAAAGGCCGTTTCGCCATTAGCCGCGATATCGCAAAAA ATACCGTGGATCTGACGATGAACAATCTGGAACCGGAAGATACCGCCGTTTATTACTGTGCAGCGAGCGGCGGC GGCAGCCCGCAGCGTCGTAGCGCGCGTCTGAGCGCGAGCGGCGGCGGCAGCTGGGGTCAGGGCACCCAGGTGA CCGTTAGTAGCGGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTCCGGACTACG CTTCTTAG

SEQ ID NO: 15 - aNCL-CDRl Amino acid sequence

M KKTAI AI AVALAG FATVAQAAQVQLQESG G G LVQP GG SLR LSCAASPQR RSAR L SAW F RQAPG K E RE FVAR I YWSSG NTYYADSVKG R FAI SR D IAKNTVD LTM N N L E P E DTAVYYCAAR DG I PTS RSVE SYNYWG QGTQVTVSSG QAGQH H H H H H GAYPYD V P D YAS

SEQ ID NO: 16 - ctNCL-CDR3 Amino acid sequence

M KKTAI AI AVALAG FATVAQAAQVQLQESG G G LVQP GG SLR LSCAASG RTFS D HS GYTYTI GW F RQAPG K E R E FVAR I YWSSG NTYYADSVKG R FAIS R D I AKNTVD LTM N N LE P E DTAVYYCAAP QR RSAR LSAWGQGTQVTVSSGQAGQH H H H H H GAYPYD V P D Y A S Stop

SEQ ID NO: 17 - aNCL-CDRl-L Amino acid sequence

M KKTAI AI AVALAG FATVAQAAQVQLQESG G G LVQP GG SLR LSCAASSG G GS PQ R RSAR LSASG G GSWF RQAPG KE R E FVAR I YWSSG NTYYADSV KG R FAIS RD I AK N TVD LTM N N LE P E DTAVYYCAAR D G I PTS RSV ESYN YWG QGTQVTVSSG QAGQH H H H H H GAYPYDVP DYAS SEQ ID NO: 18 - QLNCL-CDR3-L Amino acid sequence M KKTAI AI AVALAG FATVAQAAQVQLQESG G G LVQP GG SLR LSCAASG RTFS D HS GYTYTI GW F RQAPG K E R E FVAR I YWSSG NTYYADSVKG R FAIS R D I AKNTVD LTM N N LE P E DTAVYYCAA SG G GSPQR RSAR LSASG G GSWG QGTQVTVSSG QAG QH H H H H H GAYPYDVP DYAS Stop

SEQ ID NO: 31 - ctNCL-VHH-Fc DNA sequence

ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGATATCGGCCATGGAAC AGGTGCAGCTGCAGGAATCTGGCGGTGGCCTGGTTCAGCCGGGTGGCAGTCTGCGCCTGAGCTGTGCCGCCTCT GGTCGTACCTTTAGCGATCATTCTGGTTATACCTACACGATTGGCTGGTTTCGTCAGGCGCCGGGCAAAGAACGT GAATTCGTGGCCCGCATCTATTGGAGCTCTGGTAACACCTATTACGCAGATAGTGTTAAAGGCCGTTTCGCCATTA GCCGCGATATCGCAAAAAATACCGTGGATCTGACGATGAACAATCTGGAACCGGAAGATACCGCCGTTTATTACT GTGCAGCGAGCGGCGGCGGCCCGCAGCGTCGTAGCGCGCGTCTGAGCGCGTGGGGTCAGGGCACCCAGGTGAC CGTTAGTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCA AGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACC TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAG GTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTAAATGA

SEQ ID NO: 32 - aNCL-VHH-Fc Amino acid sequence

M YR M QLLSC I ALSLALVTNS I SAM EQVQLQESG G G LVQP G GS LR LSCAASG RTFS D H SGYTYT I GW F RQAP G KE R E FVAR I YWSSG NTYYADSVKG RFAI S R D I AKNTVD L TM N N LE P E DTAVYYCAAPQR RSAR LSAWG QGTQVTVSS D KTHTCP PCPAP E LLG G PSV F LF P P KP KDTLM I SRTP EVTCVVVDVS H E D P EVK F NWYVDG VEVH N AKTKP R E EQYN STY RVVSVLTVLH QDWL N G K EYKC KVS N KALPAP I E KTI S KAKG QP R E PQ VYTL PPS R E E MTK NQVS LTC LVKG FYPSD I AV EW ES N G QP E N NY KTTP PVLDSD G S F F LYS KLTVD KS RWQQG N VFSCSV M H EALH N HYTQKS LSLS PG K Stop

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination.

Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

What Is Claimed Is:

1. An antibody or antigen binding fragment thereof that (i) specifically binds the N- terminal domain of nucleolin; and (ii) promotes antibody dependent cellular cytotoxicity (ADCC) towards a cell expressing nucleolin.

2. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment comprises an F3 peptide.

3. The antibody or antigen binding fragment thereof of claim 2, wherein the F3 peptide comprises 8-31 contiguous amino acids of F3.

4. The antibody or antigen binding fragment thereof of claims 2 or 3, wherein the F3 peptide comprises residues 5-14 of F3. 5. The antibody or antigen binding fragment thereof of claims 1-4, wherein the antibody or antigen binding fragment comprises a variable domain comprising a CDRl , a CDR2, and a CDR3.

6. The antibody or antigen binding fragment thereof of claim 5, wherein one or more of CDRl , CDR2, and CDR3 comprises the F3 peptide.

7. The antibody or antigen binding fragment thereof of claim 6, wherein one or both of CDRl or CDR3 comprising the F3 peptide. 8. The antibody or antigen binding fragment thereof of claim 7, wherein the variable domain comprises a CDRl, a CDR2, and a CDR3 comprising the F3 peptide.

9. The antibody or antigen binding fragment thereof of claim 7, wherein the variable domain comprises a CDRl, a CDR2, and the F3 peptide substituted for CDR3.

10. The antibody or antigen binding fragment thereof of any claims 1 -9, wherein the N- terminal domain of nucleolin comprises amino acids 1-283 of nucleolin.

1 1. The antibody or antigen binding fragment thereof of any of claims 1-10, wherein the antibody or antigen binding fragment specifically binds amino acids 1-283 of the N-terminal domain of nucleolin.

12. The antibody or antigen binding fragment thereof of any of claims 1-10, wherein the antibody or antigen binding fragment specifically binds amino acids 43-51 of the N-terminal domain of nucleolin.

13. The antibody or antigen binding fragment thereof of any of claims 1-10, wherein the antibody or antigen binding fragment specifically binds amino acids 221-233 of the N-terminal domain of nucleolin.

14. The antibody or antigen binding fragment thereof of any of claims 1-13, wherein the antibody or antigen binding fragment is a human or humanized monoclonal antibody.

15. The antibody or antigen binding fragment thereof of any of claims 1-14, wherein the antibody is a full length antibody.

16. The antibody or antigen binding fragment thereof of claim 15, wherein the antibody comprises an IgGl Fc domain.

17. The antigen binding fragment of any of claims 1-14, wherein the antigen binding fragment is a Fab, Fab', F(ab')2, Fv, a VHH, or a scFv.

18. The antigen binding fragment thereof of any of claims 1-14, wherein the antigen binding fragment comprises a scFv-Fc.

19. The antigen binding fragment thereof of any of claims 1-14, wherein the antigen binding fragment comprises a VHH-Fc.

20. The antigen binding fragment thereof of claims 18 or 19, wherein the Fc domain is an IgGl Fc domain.

21. The antibody or antigen binding fragment thereof of any of claims 1 -20, wherein the Fc domain comprises one or more modifications to increase ADCC. 22. The antibody or antigen binding fragment thereof of claim 21, wherein the one or more modifications comprises one or more of L234Y, S239D, T256A, K290A, K290Y, Y296W, S298A, A330F, A330L, I332E, E333A, K334A, K334V, A339T, E356K, K392D, D339K and K409D. 23. The antibody or antigen binding fragment thereof of claim 21, wherein the one or more modifications comprises the level of Fc-bound carbohydrate structure, including, but not limited to, alterations at the level of fructose, galactose, mannose, bisecting sugars and/or sialic acid.

24. The antibody or antigen binding fragment thereof of any claims 1 -23, wherein the antibody is a bispecific antibody, and wherein the antibody specifically binds a molecule present on the surface of immune cells.

25. The antibody or antigen binding fragment thereof of claim 24, wherein the molecule present on the surface of immune cells comprises MHC class I or MHC class II proteins, T cell receptors, B cell receptors, CD28, ICOS, TLT2, CD27, CD 137, OX40, HVEM, DR3, NKG2D, TIM- 1, TIM-2, DNAM-1, CRTAM, CTLA-4, PD-1 , PD-Ll, PD-L2, CXCR4, CD3, B7-1, B7-2, BTLA, CD 160, LAG-3, TIM-3, TIGIT, LAIR-1, CAR, CD40, GITR, BAFF-R, TACI, BCMA, CD72, CD22, CD96, 2B4, NTB-A, CRACC, Siglec-3.7.9, KLRG1, NKR-P1A, ILT2,

KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, CD94-NKG2A, CEACAM1, Plexin- Al, Plexin-A4, CD300b, CD300e, TREMl, TREM2, TREM3, ILT7, ILT3,4, TLT- 1, CD200R, TAM family, CD300a, CD300f, DC-SIGN, Kit, Allergin- 1, Pir-B, MAFA, or Gp49B l .

26. An isolated nucleic acid or a set of nucleic acids, which collectively encode the antibody of any one of claims 1-25.

27. A vector or vector set, comprising the nucleic acid or the set of nucleic acids of claim 26.

28. A host cell or host cell set, comprising the vector or vector set of claim 27.

29. The cell or cell set of claim 28, wherein the cell(s) is a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

30. A pharmaceutical composition comprising:

(a) the antibody or antibody fragment of any one of claims 1-25, the nucleic acid or the set of nucleic acids of claim 24, or the vector or vector set of claim 27; and

(b) a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 30, which is for treating a disease associated with cell-surface localized nucleolin.

32. The pharmaceutical composition of claim 30, for use as a medicament for treating cancer.

33. A method of treating a disease associated with cell-surface localized nucleolin, wherein the method comprises administering a therapeutically effective amount of the antibody or antibody fragment of claims 1-25 to a subject in need thereof.

34. The method of claim 33, wherein the disease is cancer.

35. The method of claim 34, wherein the cancer is a solid tumor forming cancer.

36. The method of any of claims 33-35, wherein the subject is administered a treatment for cancer.

37. The method of claim 36, wherein the treatment for cancer is a chemotherapy, a radiation therapy, or an immunotherapy.

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