Anti-pd-l1 Nanobody And Use Thereof

  • Published: Jun 20, 2019
  • Earliest Priority: Aug 04 2016
  • Family: 11
  • Cited Works: 3
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AU 2017305366 B2
(12) STANDARD PATENT (11) Application No. AU 2017305366 B2 
(19) AUSTRALIAN PATENT OFFICE 
(54) Title 
Anti-PD-L1 nanobody and use thereof 
(51) International Patent Classification(s) 
C07K 16/28 (2006.01) A61P 35/00 (2006.01) 
A61K 39/395 (2006.01) C12N 15/13 (2006.01) 
A61K 51/10 (2006.01) C12N 15/85 (2006.01) 
(21) Application No: 2017305366 (22) Date of Filing: 2017.08.03 
(87) WIPO No: W018/024237 
(30) Priority Data 
(31) Number (32) Date (33) Country 
201610634596.X 2016.08.04 CN 
(43) Publication Date: 2018.02.08 
(44) Accepted Journal Date: 2019.06.20 
(71) Applicant(s) 
Innovent Biologics (Suzhou) Co., Ltd 
(72) Inventor(s) 
Shen, Xiaoning;Miao, Xiaoniu;Liu, Xiaolin 
(74) Agent / Attorney 
Griffith Hack, GPO Box 1285, MELBOURNE, VIC, 3001, AU 
(56) Related Art 
WO 2016007235 Al 
WO 2016111645 Al 
SERGE MUYLDERMANS, "Nanobodies: Natural Single-Domain Antibodies", 
ANNUAL REVIEW OF BIOCHEMISTRY, (2013-06-02), vol. 82, no. 1, doi:10.1146/ 
annurev-biochem-063011-092449, ISSN 0066-4154, pages 775 - 797 
AHMAD SHAMAILA MUNIR ET AL, "PD-L1-specific T cells", CANCER 
IMMUNOLOGY, IMMUNOTHERAPY, SPRINGER, BERLIN/HEIDELBERG, vol. 65, 
no. 7, doi:10.1007/S00262-015-1783-4, ISSN 0340-7004, (2016-01-02), pages 797 
804,(2016-01-02) 
J. M. KIM ET AL, "Immune escape to PD-L1/PD-1 blockade: seven steps to 
success (or failure)", ANNALS OF ONCOLOGY., NL, (2016-05-20), vol. 27, no. 8, 
doi:10.1093/annonc/mdw217, ISSN 0923-7534, pages 1492 - 1504

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(54) Title: ANTI-PD-L NANOBODY AND USE THEREOF 
- (54)& 1:tPDLtlW fi 
(57) Abstract: Disclosed is a nanobody against the human programmed death factor PD-L1. The antibody has the function of blocking 
the binding of PD-L Ito the receptor PD-1. Disclosed are the nanobody and the gene sequence encoding the nanobody, the corresponding 
expression vector and the host cell capable of expressing the nanobody, and the method for producing the nanobody. At the same time, 
4 also disclosed is the sequence of the humanized PD-L nanobody. The humanized nanobody still has the function of blocking the 
binding of PD-Li to PD-1, and has a relatively high affinity and a relatively good specificity.  
(57) U:-$ 3t TA f t 3E FfPN T PD-L Y, r½MtPD-L 1 t'j # u PD-1

Anti-PD-Li Nanobody and Use Thereof 
Technical field 
The present disclosure relates to the field of biomedical or 
biopharmaceutical technology, and more specifically to the nanobodies against 
PD-Li, the coding sequences and the uses thereof.  
Background 
Programmed death 1 ligand 1 (PD-L), also known as CD274, is a member of 
the B7 family and is a ligand for PD-1. PD-Li is a type I transmembrane protein 
with a total of 290 amino acids, including one IgV-like region, one IgC-like 
region, one transmembrane hydrophobic region, and one intracellular region 
composed of 30 amino acids.  
Different from other B7 family molecules, PD-Li has an effect of negative 
regulation on immune response. The study found that PD-Li is mainly expressed 
in activated T cells, B cells, macrophages, dendritic cells and the like. In 
addition to lymphocytes, PD-Li is also expressed in the endothelial cells of 
other tissues such as thymus, heart, and placenta, as well as in the 
non-lymphoid system such as melanoma, liver cancer, gastric cancer, renal cell 
carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, 
head and neck cancer, etc. PD-Li has an extensive effect on the regulation 
of autoreactive T, B cells and immune tolerance, and it plays a role in 
peripheral T and B cell responses. The high expression of PD-Li on tumor cells 
correlates with the poor prognosis of cancer patients.  
Programmed death-i (PD-1) factor, which binds to PD-Li and is also known 
as CD279, is a member of the CD28 family. It contains two tyrosine residues 
in the cytoplasmic region. One residue near to the N-terminus is located in 
the immunoreceptor tyrosine-based inhibitory motif (ITIM), and the other near 
to the C-terminal is located in the immunoreceptor tyrosine-based switch motif 
(ITSM). PD-i is mainly expressed on the surface of activated T lymphocytes, 
B lymphocytes, and macrophages. Normally, PD-i can inhibit the function of 
T lymphocytes, promote the function ofTreg, thereby inhibiting the autoimmune 
response and preventing from the occurrence of autoimmune diseases. However, 
in the development of tumors, the binding of PD-Li expressed by tumor cells 
and PD- can promote the immune escape of tumors through the inhibitory effect 
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10290327_1(GHMatters)7P108776.AU

oflymphocytes. The binding ofPD-L1 to PD-i can lead to a variety ofbiological 
changes, causing immune regulation, such as inhibiting the proliferation and 
activation oflymphocytes, inhibiting the differentiation ofCD4+ T cells into 
Thi and Thi7 cells, and inhibiting the release of inflammatory cytokines, etc.  
The successful application of monoclonal antibodies in cancer detection 
and bio-targeted therapy has led to a revolution in the treatment of cancer.  
However, the molecular weight of the conventional monoclonal antibody (150 
kD) is too large to allow the antibody to penetrate the tissue, resulting in 
a lower effective concentration in the tumor region and insufficient 
therapeutic effect. The immunogenicity of traditional antibodies is high 
while the modified antibody can hardly achieve the intrinsic affinity. In 
addition, a number of facts, such as the long development cycle, high 
production costs, and lacking of stability of fully humanized traditional 
antibodies, limit their application and popularity in clinical practice.  
Nanobodies are the smallest antibody molecules so far, and their molecular 
weight is 1/10 that of ordinary antibodies. In addition to the antigen 
reactivity of monoclonal antibodies, nanobodies also possess unique 
functional properties such as low molecular weight, high stability, good 
solubility, easy expression, weak immunogenicity, strong penetration, and 
strong targeting, simple humanization, and low preparation cost, etc. It 
almost perfectly overcomes the shortcomings of traditional antibody, such as 
long development cycle, low stability, stringent conditions for storage, etc.  
However, there is still a lack of satisfactory nanobodies against PD-L1 
in the field. Therefore, it is an urgent need in the art to develop new specific 
nanobodies that are effective against PD-L1.  
Summary of Disclosure 
The present disclosure relates to a class of specific nanobodies that are 
effective against PD-L1.  
W In a first aspect, the present disclosure provides a VHH chain of an 
anti-PD-L1 nanobody, comprising complementary determining region (CDR) 1 as 
set forth by SEQ ID NO.: 5, CDR2 as set forth by SEQ ID NO.: 6 and CDR3 as 
set forth by SEQ ID NO.: 7.  
In a preferred embodiment, said CDR1, CDR2 and CDR3 are separated by frame 
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regions FRI, FR2, FR3, and FR4 of the VHH chain.  
Also disclosed is a VHH chain of anti-PD-L nanobodies, said VHH chain 
comprises a frame region (FR) and the complementary determining region (CDR) 
of the first aspect, and said frame region 
(a) is consisting of FRI as set forth by SEQ ID NO.:1, FR2 as set forth 
by SEQ ID NO.: 2, FR3 as set forth by SEQ ID NO.: 3, and FR4 as set forth by 
SEQ ID NO.: 4; or 
(b) is consisting of FRI as set forth by SEQ ID NO.:10, FR2 as set forth 
by SEQ ID NO.: 11, FR3 as set forth by SEQ ID NO.: 12, and FR4 as set forth 
by SEQ ID NO.: 13.  
In another preferred embodiment, the VHH chain of said anti-PD-Li 
nanobodies is as set forth by SEQ ID NO.: 8 or 14.  
A second aspect of the present disclosure provides an anti-PD-Lnanobody, 
comprising an amino acid sequence of SEQ ID NO.: 8 or SEQ ID NO.: 14.  
The third aspect of the present disclosure provides a polynucleotide 
encoding the VHH chain of an anti-PD-Li nanobody according to the first aspect 
of the present disclosure, or the anti-PD-Li nanobody according to the second 
aspect of the present disclosure.  
In another preferred embodiment, said polynucleotide has a nucleotide 
sequence of SEQ ID NO.: 9 or 15.  
In another preferred embodiment, said polynucleotide includes DNA or RNA.  
A fourth aspect of the present disclosure provides an expression vector 
comprising the polynucleotide according to the third aspect of the present 
disclosure.  
A fifth aspect of the present disclosure provides a host cell comprising 
the polynucleotide according to the third aspect of the present disclosure, 
optionally wherein the polynucleotide is integrated within the host cell 
genome, or the expression vector according to the fourth aspect of the present 
disclosure.  
W In another preferred embodiment, said host cell includes prokaryocyte or 
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eukaryocyte.  
In another preferred embodiment, said host cell is selected from the group 
consisting of E.coli. and yeast cells.  
A sixth aspect of the present disclosure provides a method for producing 
an anti-PD-Li nanobody, comprising: 
(a) culturing said host cell according to the fifth aspect of the present 
disclosure under conditions suitable for producing the anti-PD-L nanobody, 
thereby obtaining a culture comprising said anti-PD-L nanobody; and 
(b) isolating or recovering said anti-PD-Li nanobodies from said culture.  
In another preferred embodiment, said anti-PD-Li nanobody has an amino 
acid sequence of SEQ ID NO.: 8 or 14.  
A seventh aspect provides an anti-PD-Li nanobody when produced according 
to the method of the sixth aspect.  
An eighth aspect of the present disclosure provides an immunoconjugate, 
comprising: 
(a) the VHH chain of an anti-PD-Li nanobody according to the first aspect 
of the present disclosure, or said anti-PD-Li nanobody according to the second 
or seventh aspect of the present disclosure; and 
(b) a conjugating part selected from the group consisting of a detectable 
marker, drug, toxin, cytokine, radionuclide, and enzyme.  
In another preferred embodiment, said conjugating part is a drug or toxin.  
In another preferred embodiment, said conjugating part is a detectable 
marker.  
In another preferred embodiment, said conjugate is selected from the group 
consisting of fluorescent or luminescent markers, radiomarkers, MRI (magnetic 
resonance imaging) or CT (computed tomography) contrast agents, or enzymes, 
radionuclides, biotoxins, cytokines (eg, IL-2, etc.), antibodies, antibody 
Fe fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral 
particles, liposomes, nanomagnetic particles, prodrug activating enzymes (eg, 
W DT-diaphorase (DTD) or biphenyls hydrolase-like protein (BPHL), 
chemotherapeutic agents (eg, cisplatin) or any form of nanoparticles, etc.  
that produce detectable products.  
In another preferred embodiment, said immunoconjugate contains 
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multivalent (such as bivalent) VHH chains of the anti-PD-Li nanobodies 
according to the second aspect of the present disclosure, or the anti-PD-Li 
nanobodies according to the third aspect of the present disclosure.  
In another preferred embodiment, said multivalent refers that the amino 
acid sequence of the immunoconjugate contains several repeated VHH chains of 
the anti-PD-Li nanobodies according to the second aspect of the present 
disclosure, or the anti-PD-Li nanobodies according to the third aspect of the 
present disclosure.  
A ninth aspect provides a pharmaceutical composition comprising the VHH 
chain of an anti-PD-Li nanobody according to the first aspect, the anti-PD-Li 
nanobody according to the second or seventh aspect, or the immunoconjugate 
according to the eighth aspect, and a pharmaceutically acceptable carrier.  
A tenth aspect of the disclosure provides use of the VHH chain of an 
anti-PD-Li nanobody according to the first aspect, the anti-PD-Li nanobody 
according to the second or seventh aspect of the present disclosure, the 
polynucleotide according to the third aspect, the expression vector according 
to the fourth aspect, or the host cell according to the fifth aspect in the 
manufacture of an agent for detecting PD-L molecule.  
An eleventh aspect provides use of the VHH chain of an anti-PD-L nanobody 
according to the first aspect, the anti-PD-Li nanobody according to the second 
or seventh aspect, the polynucleotide according to the third aspect, the 
expression vector according to the fourth aspect, or the host cell according 
to the fifth aspect in the manufacture of a medicament for treating cancer.  
A twelfth aspect provides a method for treating cancer, comprising 
administering to a subject in need the VHH chain of an anti-PD-L nanobody 
according to the first aspect, the anti-PD-Li nanobody according to the second 
or seventh aspect, the immunoconjugate according to the eighth aspect, or the 
pharmaceutical composition according to the ninth aspect.  
In another preferred embodiment, said detecting comprises detection 
W conducted by flow cytometry or cell immunofluorescence.  
In one embodiment, the pharmaceutical composition comprises: 
11376334_1 (GHMatters) P108776.AU 24 May 19

(i) the complementary determining region (CDR) of VHH chain of the 
anti-PD-Li nanobodies according to the first aspect of the present disclosure, 
the VHH chain of the anti-PD-Li nanobodies according to the second aspect of 
the present disclosure, the anti-PD-Li nanobodies according to the third 
aspect of the present disclosure, or the immunoconjugate according to eighth 
aspect of the present disclosure; and 
(ii) a pharmaceutically acceptable carrier.  
In another preferred embodiment, said pharmaceutical composition is in 
a form of injection.  
In another preferred embodiment, said pharmaceutical composition is used 
for preparing a medicament for treating cancers, and said cancer is selected 
from the group consisting of gastric cancer, liver cancer, leukemia, renal 
tumor, lung cancer, small intestinal cancer, bone cancer, prostate cancer, 
colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical 
cancer, lymphoma, adrenal tumor and bladder tumor.  
The eleventh aspect of the present disclosure provides one or more use 
of the anti-PD-Li nanobodies according to the present disclosure: 
(i) for detecting human PD-L molecule; 
(ii) for flow cytometry assay; 
(iii) for cell immunofluorescence detection; 
(iv) for treating cancer; 
(v) for diagnosing cancer.  
In another preferred embodiment, said use is non-diagnostic and 
non-therapeutic.  
Also disclosed is arecombinant protein, and said recombinant protein has: 
(i) the sequence of variable region of heavy chain VHH according to the 
present disclosure or the sequence of nanobodies according to the present 
disclosure; and 
(ii) an optional tag sequence assisting expression and/or purification.  
W In another preferred embodiment, said tag sequence includes 6His tag or 
HA tag.  
In another preferred embodiment, said recombinant protein specifically 
binds to the PD-L protein.  
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Also disclosed is use of the VHH chain of the anti-PD-L nanobodies 
according to the present disclosure, the anti-PD-Li nanobodies according to 
the present disclosure, or the immunoconjugate according to the present 
disclosure for preparing a medicament, agent, detecting plate or kit; 
wherein, said agent, detecting plate or kit is used for detecting PD-L 
protein in the testing sample; 
wherein, said medicament is used for treating or preventing cancers 
expressing PD-Li (i.e. PD-Li positive).  
In another preferred embodiment, said cancer comprises gastric cancer, 
lymphoma, liver cancer, leukemia, renal tumor, lung cancer, small intestinal 
cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon 
cancer, prostate cancer, or adrenal tumors.  
Also disclosed is a method for detecting PD-Li protein in a sample, and 
said method comprises the steps of: 
(1) contacting the sample with the nanobodies according to the present 
disclosure; 
(2) detecting the antigen - antibody complex, wherein the detected complex 
indicated the presence of PD-L protein.  
Also disclosed is a method for treating a disease, said method comprising 
administering the nanobodies according to the present disclosure or the 
immunoconjugate according to the present disclosure to a subject in need.  
In another preferred embodiment, said subject includes mammals, such as 
human.  
Also disclosed is a frame region (FR) of a VHH chain of an anti- PD-L 
nanobody, and said frame region (FR) of the VHH chain is composed of FRI as 
set forth by SEQ ID NO. : 1, FR2 as set forth by SEQ ID NO. : 2, FR3 as set forth 
by SEQ ID NO.: 3, and FR4 as set forth by SEQ ID NO.: 4.  
It is to be understood that within the scope of the present disclosure, 
the above-described technical features of the present disclosure and the 
W technical features specifically described in the following (e.g., examples) 
may be combined with each other to form a new or preferred technical solution, 
which will not be repeated herein due to the limited space.  
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Brief Description of the Figures 
Figure 1 shows the SDS-PAGE result of the purified antigen protein and 
nanobody, in which A is the nucleic acid molecule for reference, B is the 
purified hPD-L1(ECD)-Fc protein, and Cis the purified hPD-L1(ECD)-Fc protein 
after the Fe tag protein being removed by TEV enzyme, D is the purified PD-L 
Nb-Fc protein, and E is the biotinylated PD-1-Fc protein. All of the above 
proteins were expressed by HEK293F cells.  
Figure 2 shows the detection result for the library capacity of the 
constructed library. The constructed library was coated onto a plate after 
being serially diluted. The figure shows 1/5 of the clones with gradient 
-7a
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dilution of 104 fold, 10' fold, and 10fold, and the number of clones was counted 
to determine the size of the library.  
Figure 3 is a detection result for the insertion rate of the constructed 
nanobody library. The DNA bands in the gel pores from left to right 
respectively correspond to DNA molecule marker for the first lane, and PCR 
products of detected insertion fragment for the other lanes. The PCR product 
lane is about 500bp. The insertion rate as detected is up to 95.8%.  
Figure 4 shows the screening and enrichment process of PD-L nanobodies.  
There is no enrichment after the first round of panning. It is 4 times enriched 
after the second round of panning and 210 times enriched after the third round 
of panning.  
Figure 5 is the illustration of purified PD-L1 nanobodies (corresponding 
to the nanobody of the amino acid of SEQ ID NO. : 8) expressed by E. col. It 
is an SDS-PAGE electrophoretogram of PD-L1 nanobody upon the resin gel 
affinity chromatography purification by nickel column. The results turned out 
that the purity of PD-L1 reaches over 90% after the purification.  
Figure 6 shows the blocking effects of PD-L1 nanobodies tested by FACS.  
It is conducted by the co-reaction of HEK293F cells instantly expressing human 
full-length PD-L1 protein, various groups of nanobodies and biotinylated 
hPD-1-Fc protein.  
Figure 7 illustrates the humanized PD-L1 nanobodies by eukaryotic 
expression upon purification. Four kinds of humanized PD-L1 nanobodies are 
expressed by HEK293F cells, wherein A is the protein molecule as a standard, 
B is the humanized PD-L1 Nb protein coded by the amino acid sequence of SEQ 
ID NO. : 10. The expressed nanobody has an Fe-tag protein and the protein purity 
reaches over 90%.  
Figure 8 shows the blocking effects of humanized PD-L1 nanobodies tested 
by FACS. It is conducted by the co-reaction of EBC-1 cells naturally expressing 
PD-L1 protein, humanized nanobodies and biotinylated hPD-1-F protein. It 
shows that the binding rate of PD-1-Fe-biotin and EBC-1 cells in blank group 
and negative control group is over 90%, while after the PD-L1 nanobodies and 
humanized nanobodies are added, the binding rate of PD-1-Fc-biotin and EBC-1 
cells is only less than 10%. This demonstrates the interaction between PD-1 
and PD-L1 can be significantly blocked by the added nanobodies.  
Figure 9 shows the testing results of the affinity of PD-L1 nanobodies.  
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The affinity of PD-Li nanobodies is tested by BiaCore T200. It shows that the 
affinity before humanization is 2.34X10- M and the affinity after 
humanization is 2.26X10, M. Humanization does not affect the affinity of the 
nanobodies.  
Figure 10 shows the specificity results of PD-Li nanobodies tested by 
ELISA. It could be seen that PD-L nanobodies before and after humanization 
only interact with human and Cercopithecidae PD-Li instead of iroidea or 
other member of PD-Li family. Both of the two nanobody strains have good 
specificity.  
Figure 11 shows the inhibition effects of nanobodies on the interaction 
between PD-i and PD-Li by MOA method, wherein the nanobodies before 
humanization have stronger activity than that of the antibodies in the 
positive control group while the nanobodies after humanization have 
comparable activity to that of the antibodies in the positive control group.  
Figure 12 shows the nanobodies and the humanized nanobodies can 
effectively activate T cells and have comparable effect of activation to that 
of the antibodies in the positive control group.  
Figure 13 shows the administration manner of the nanobodies of the 
invention in the study on tumor inhibition activity.  
Figure 14 shows the tumor volume is better inhibited than that in the 
control group in the mice inoculated with humanized Nb-Fe, and no significant 
increase (in tumor size) is observed, suggesting that the humanized Nb-Fc 
has significant tumor inhibiting effect.  
Figure 15 shows the humanized Nb-Fc of the invention has better solubility 
than that of the control antibody.  
Figure 16 shows there is no significant change in purity of the humanized 
Nb-Fc.  
Figure 17 shows that there is no significant change of binding between 
the humanized Nb-Fc and CHO-PDLI cells.  
Detailed description 
Upon extensive and intensive studies, the inventors have successfully 
obtained a class of anti-PD-Li nanobodies after numerous screening. The 
experimental results show that the anti-PD-Li nanobodies of the invention can 
effectively block the interaction between PD-Li and PD-1. Surprisingly, the 
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humanized anti-PD-Li nanobodies of the invention can even more effectively 
block the binding between PD-Li and PD-1. The BiaCore T200 analysis shows that 
the humanized anti-PD-Li nanobodies have high affinity, superior stability 
and significant tumor inhibitory effect. Based on this discovery, the 
invention is completed.  
In particular, the human PD-Li protein as antigen was used to immunize 
a camel, thereby obtaining a gene library of nanobodies with high quality.  
The PD-Li protein molecules were conjugated onto an ESLIA board and exhibited 
correct spatial structure of PD-Li protein. The antigens in such configuration 
were used to screen the gene library of nanobodies by phage exhibition 
technology (phage exhibition of a gene library of camel heavy chain antibody) 
thereby obtaining genes of nanobodies with PD-Li specificity. Then the genes 
were transferred into E. coli thereby obtaining the stains which can be 
effectively expressed in E.coli with high specificity.  
As used herein, the terms "nanobodies of the invention", "anti-PD-Li 
nanobodies of the invention", and "PD-Li nanobodies of the present invention" 
are exchangeable and refer to nanobodies that specifically recognize and bind 
to PD-Li (including human PD-Li). The more preferable nanobody is one 
comprising a VHH chain of amino acid sequence as set forth by SEQ ID NO.:8 
or 14.  
As used herein, the term "antibody" or "immunoglobulin" is a 
heterotetrameric glycosaminoglycan protein of about 150,000 Dalton with the 
same structural features, consisting of two identical light (L) chains and 
two identical heavy (H) chains. Each light chain is linked to the heavy chain 
through a covalent disulfide bond, and the number of disulfide bonds between 
the heavy chains of different immunoglobulin isoforms is different. Each heavy 
and light chainalso has intra-chain disulfide bonds whichare regular spaced.  
Each heavy chain has a variable region (VH) at one end followed by a plurality 
of constant regions. Each light chain has a variable region (VL) at one end 
and a constant region at the other end; the constant region of the light chain 
is opposite to the first constant region of the heavy chain, and the variable 
region of the light chain is opposite to the variable region of the heavy chain.  
Special amino acid residues form an interface between the variable regions 
of the light and heavy chains.  
As used herein, the terms "single domain antibody (VHH)" and "nanobodies" 
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have the same meaning referring to a variable region of a heavy chain of an 
antibody, and construct a single domain antibody (VHH) consisting of only one 
heavy chain variable region. It is the smallest antigen-binding fragment with 
complete function. Generally, the antibodies with a natural deficiency of the 
light chain and the heavy chain constant region 1 (CH1) are first obtained, 
the variable regions of the heavy chain of the antibody are therefore cloned 
to construct a single domain antibody (VHH) consisting of only one heavy chain 
variable region.  
As used herein, the term "variable" refers that certain portions of the 
variable region in the nanobodies vary in sequences, which forms the binding 
and specificity of various specific antibodies to their particular antigen.  
However, variability is not uniformly distributed throughout the nanobody 
variable region. It is concentrated in three segments called 
complementarity-determining regions (CDRs) or hypervariable regions in the 
variable regions of the light and heavy chain. The more conserved part of the 
variable region is called the framework region (FR). The variable regions of 
the natural heavy and light chains each contain four FR regions, which are 
substantially in a -folded configuration, joined by three CDRs which form 
a linking loop, and in some cases can form a partially -folded structure.  
The CDRs in each chain are closely adjacent to the others by the FR regions 
and form an antigen-binding site of the nanobody with the CDRs of the other 
chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669.  
(1991)). The constant regions are not directly involved in the binding of the 
nanobody to the antigen, but they exhibit different effects or functions, for 
example, involve in antibody-dependent cytotoxicity of the antibodies.  
As known by those skilled in the art, immunoconjugates and fusion 
expression products include: conjugates formed by binding drugs, toxins, 
cytokines, radionuclides, enzymes, and other diagnostic or therapeutic 
molecules to the nanobodies or fragments thereof of the present invention.  
The invention also includes a cell surface marker or an antigen that binds 
to said anti-PD-L1 protein nanobody or the fragment thereof.  
As used herein, the term "heavy chain variable region" and V" can be 
used interchangeably.  
As used herein, the terms "variable region" and "complementary 
determining region (CDR)" can be used interchangeably.  
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In another preferred embodiment, the heavy chain variable region of said 
nanobody comprises 3 complementary determining regions: CDR1, CDR2, and CDR3.  
In another preferred embodiment, the heavy chain of said nanobody 
comprises the above said heavy chain variable region and a heavy chain constant 
region.  
According to the present invention, the terms "nanobody of the invention", 
"protein of the invention", and "polypeptide of the invention" are used 
interchangeably and all refer to a polypeptide, such as a protein or 
polypeptide having a heavy chain variable region, that specifically binds to 
PD-L1 protein. They may or may not contain a starting methionine.  
The invention also provides other proteins or fusion expression products 
having the nanobodies of the invention. Specifically, the present invention 
includes any protein or protein conjugate and fusion expression product (i. e.  
immunoconjugate and fusion expression product) having a heavy chain 
containing a variable region, as long as the variable region are identical 
or at least 90% identical, preferably at least 95% identical to the heavy chain 
of the nanobody of the present invention.  
In general, the antigen-binding properties of a nanobody can be described 
by three specific regions located in the variable region of the heavy chain, 
referred as variable regions (CDRs), and the segment is divided into four frame 
regions (FRs). The amino acid sequences of four FRs are relatively 
conservative and do not directly participate in binding reactions. These CDRs 
form a loop structure in which the 0 -sheets formed by the FRs therebetween 
are spatially close to each other, and the CDRs on the heavy chain and the 
CDRs on the corresponding light chain constitute the antigen-binding site of 
the nanobody. The amino acid sequences of the same type of nanobodies can be 
compared to determine which amino acids constitute the FR or CDR regions.  
The variable regions of the heavy chains of the nanobodies of the invention 
become a particular interest because at least a part of them is involved in 
binding antigens. Thus, the present invention includes those molecules having 
a nanobody heavy chain variable region with a CDR, provided that their CDRs 
are 90% or more (preferably 95% or more, the most preferably 98% or more) 
identical to the CDRs identified herein.  
The present invention includes not only intact nanobodies but also 
fragment(s) of immunologically active nanobody or fusion protein(s) formed 
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from nanobodies with other sequences. Therefore, the present invention also 
includes fragments, derivatives and analogs of the nanobodies.  
As used herein, the terms "fragment," "derivative," and "analog" refer 
to a polypeptide that substantially retains the same biological function or 
activity of a nanobody of the invention. Polypeptide fragments, derivatives 
or analogs of the invention may be (i) polypeptides having one or more 
conservative or non-conservative amino acid residues (preferably 
non-conservative amino acid residues) substituted. Such substituted amino 
acid residues may or may not be encoded by the genetic code; or (ii) a 
polypeptide having a substituent group in one or more amino acid residues; 
or (iii) a polypeptide formed by fusing a mature polypeptide and another 
compound (such as a compound that increases the half-life of the polypeptide, 
for example, polyethylene glycol) ; or (iv) a polypeptide formed by fusing an 
additional amino acid sequence to the polypeptide sequence (e.g., a leader 
or secretory sequence or a sequence used to purify this polypeptide or a 
proprotein sequence, or a fusion protein formed with a 6 His tag). According 
to the teachings herein, these fragments, derivatives, and analogs are within 
the scope of one of ordinary skill in the art.  
The nanobody of the present invention refers to a polypeptide including 
the above CDR regions having PD-L protein binding activity. The term also 
encompasses variant forms of polypeptides comprising the above CDR regions 
that have the same function as the nanobodies of the invention. These 
variations include, but are not limited to, deletion insertions and/or 
substitutions of one or several (usually 1-50, preferably 1-30, more 
preferably 1-20, optimally 1-10) amino acids, and addition of one or several 
(generally less than 20, preferably less than 10, and more preferably less 
than 5) amino acids at C-terminus and/or N-terminus. For example, in the art, 
the substitution of amino acids with analogical or similar properties usually 
does not alter the function of the protein. For another example, addition of 
one or several amino acids at the C-terminus and/or N-terminus usually does 
not change the function of the protein. The term also includes active fragments 
and active derivatives of the nanobodies of the invention.  
The variant forms of the polypeptide include: homologous sequences, 
conservative variants, allelic variants, natural mutants, induced mutants, 
proteins encoded by DNAs capable of hybridizing with DNA encoding the nanobody 
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of the present invention under high or low stringent conditions, and 
polypeptides or proteins obtained using antiserum against the nanobodies of 
the invention.  
The invention also provides other polypeptides, such as a fusion protein 
comprising nanobodies or fragments thereof. In addition to almost full-length 
polypeptides, the present invention also includes fragments of the nanobodies 
of the invention. Typically, the fragment has at least about 50 contiguous 
amino acids of the nanobody of the invention, preferably at least about 50 
contiguous amino acids, more preferably at least about 80 contiguous amino 
acids, and most preferably at least about 100 contiguous amino acids.  
In the present invention, "a conservative variant of a nanobody of the 
present invention" refers to the polypeptides in which there are up to 10, 
preferably up to 8, more preferably up to 5, and most preferably up to 3 amino 
acids substituted by amino acids having analogical or similar properties, 
compared to the amino acid sequence of the nanobody of the present invention.  
These conservative variant polypeptides are preferably produced according to 
the amino acid substitutions in Table 1.  
Table 1 
Original residue Representative substitution Preferable substitution 
Ala (A) Val; Leu; Ile Val 
Arg (R) Lys; Glin; Asa Lys 
Asn (N) Gln; His; Lys; Arg Gln 
Asp (D) Glu Glu 
Cys (C) Ser Ser 
Gln (Q) Asn Asn 
Glu (E) Asp Asp 
Gly (G) Pro; Ala Ala 
His (H) Asn; Gln; Lys; Arg Arg 
Ile (I) Len; Val; Met; Ala; Phe Leu 
Leu (L) Ile; Val; Met; Ala; Phe Ile 
Lys (K) Arg; Gln; Asn Arg 
Met (M) Leu; Phe; Ile Leu 
Phe (F) Leu; Val; Ile; Ala; Tyr Leu 
Pro (P) Ala Ala 
Ser (S) Thr Thr 
Thr (T) Ser Ser 
Trp (W) Tyr; Phe Tyr 
Tyr (Y) Trp; Phe; Thr; Ser Phe 
Val (V) Ile; Leu; Met; Phe; Ala Leu 
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The present invention also provides a polynucleotide molecule encoding 
the above nanobody or fragment or fusion protein thereof. Polynucleotides of 
the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic 
DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can 
be a coding strand or a non-coding strand.  
Polynucleotides encoding the mature polypeptides of the invention include: 
coding sequences only encoding mature polypeptide; coding sequences for the 
mature polypeptide and various additional coding sequences; coding sequences 
(and optional additional coding sequences) and non-coding sequences for the 
mature polypeptide.  
The term "polynucleotide encoding a polypeptide" may include a 
polynucleotide that encodes the polypeptide, and may also include a 
polynucleotide that includes additional coding and/or non-coding sequences.  
The invention also relates to polynucleotides that hybridize to the 
sequences described above and that have at least 50%, preferably at least 70%, 
and more preferably at least 80% identity between the two sequences. The 
present invention specifically relates to polynucleotides that can be 
hybridized to the polynucleotides of the present invention under stringent 
conditions. In the present invention, "stringent conditions" refers to: (1) 
hybridization and elution at lower ionic strength and higher temperature, such 
as 0.2 x SSC, 0.1% SDS, 600 C; or (2) additional denaturants during 
hybridization, such as 50% (v/v) formamide, 0.1% fetal bovine serum / 0.1% 
Ficoll, 420 C, etc.; or (3) hybridization occurs only under the identity 
between the two sequences at least over 90%, preferably over 95%. Also, 
polypeptides encoded by hybridizable polynucleotides have the same biological 
functions and activities as mature polypeptides.  
The full-length nucleotide sequence of the nanobody of the present 
invention or a fragment thereof can generally be obtained by a PCR 
amplification method, a recombination method, or an artificial synthesis 
method. One possible method is to synthesize related sequences using synthetic 
methods, especially when the fragment length is short. In general, a long 
sequence of fragments can be obtained by first synthesizing a plurality of 
small fragments and then connecting them. In addition, the coding sequence 
of the heavy chain and the expression tag (eg, 6His) can be fused together 
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to form a fusion protein.  
Once the concerned sequences have been obtained, the concerned sequences 
can be obtained in large scale using recombinant methods. Usually, sequences 
can be obtained by cloning it into a vector, transferring it into cells, and 
then isolating the sequences from the proliferated host cells by conventional 
methods. Bio-molecules (nucleic acids, proteins, etc.) to which the present 
invention relates include bio-molecules that exist in isolated form.  
At present, DNA sequences encoding the protein of the present invention 
(or a fragment thereof, or a derivative thereof) can be obtained completely 
by chemical synthesis. The DNA sequence then can be introduced into various 
existing DNA molecules (or e.g. vectors) and cells known in the art. In 
addition, mutations can also be introduced into the protein sequences of the 
invention by chemical synthesis.  
The invention also relates to vectors comprising the above-mentioned 
suitable DNA sequences and suitable promoters or control sequences. These 
vectors can be used to transform an appropriate host cell so that it can express 
the protein.  
The host cell can be a prokaryotic cell, such as a bacterial cell; or a 
lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such 
as a mammalian cell. Representative examples are: Escherichia col, 
Streptomyces, bacterial cells such as Salonella typhimurium, fungal cells 
such as yeast, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS7, 
293 cells, and the like.  
The transformation of the host cell with the recombinant DNA can be 
performed using conventional techniques well known to those skilled in the 
art. When the host is a prokaryotic organism such as E. col, competent cells 
capable of absorbing DNA can be harvested after the exponential growth phase 
and treated with the CaC 2 method. The procedures used are well known in the 
art. Another method is to use MgCl2. If necessary, conversion can also be 
performed by electroporation. When the host is eukaryotic, the following DNA 
transfection methods can be used: calcium phosphate coprecipitation, 
conventional mechanical methods such as microinjection, electroporation, 
liposome packaging, and the like.  
The obtained transformants can be cultured in a conventional manner to 
express the polypeptide encoded by the gene of the present invention.  
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Depending on the host cells used, the medium used in the culture may be selected 
from various conventional media. The culture is performed under conditions 
suitable for the host cells growth. After the host cells are grown to an 
appropriate cell density, the selected promoter is induced by a suitable 
method (such as temperature shift or chemical induction) and the cells are 
incubated for a further period of time.  
The recombinant polypeptide in the above method may be expressed 
intracellularly, or on the cell membrane, or secreted extracellularly. If 
necessary, the recombinant protein can be isolated and purified by various 
separation methods by utilizing its physical, chemical and other 
characteristics. These methods are well-known to those skilled in the art.  
Examples of these methods include, but are not limited to: conventional 
renaturation treatment, treatment with a protein precipitation agent (salting 
out method), centrifugation, osmotic disruption, super treatment, 
ultracentrifugation, molecular sieve chromatography (gel filtration), 
adsorption layer analysis, ion exchange chromatography, high performance 
liquid chromatography (HPLC), and various other liquid chromatography 
techniques and the combinations thereof.  
The nanobodies of the invention may be used alone or in combination or 
conjugated with a detectable marker (for diagnostic purposes), a therapeutic 
agent, a PK (protein kinase) modification moiety, or a combination thereof.  
Detectable markers for diagnostic purposes include, but are not limited 
to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic 
resonance imaging) or CT (computed tomography) contrast agents, or enzymes 
capable of producing detectable products.  
Therapeutic agents that can be binded or conjugated to the nanobodies of 
the present invention include, but are not limited to: 1. Radionuclides; 2.  
Biological poisons; 3. Cytokines such as IL-2, etc.; 4. Gold 
nanoparticles/nanorods; 5. Viruses Particles; 6. Liposome; 7. Nano magnetic 
particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) 
or biphenyl hydrolase-like protein (BPHL)) ; 10. Chemotherapeutic agents (for 
example, cisplatin) or any form of nanoparticles, etc.  
Pharmaceutical composition 
The invention also provides a composition. Preferably, said composition 
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is a pharmaceutical composition comprising the above nanobody or active 
fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.  
In general, these materials can be formulated in non-toxic, inert, and 
pharmaceutically acceptable aqueous carrier media wherein the pH is generally 
about 5-8, preferably about 6-8, although the pH can be varied with the nature 
of the formulation material and the condition to be treated. The formulated 
pharmaceutical compositions can be administered by conventional routes 
including, but not limited to, intratumoral, intraperitoneal, intravenous, 
or topical administration.  
The pharmaceutical composition of the present invention can be directly 
used to bind PD-L1 protein molecules and thus can be used to treat tumors.  
In addition, other therapeutic agents can also be used at the same time.  
The pharmaceutical composition of the present invention contains a safe 
and effective amount (for example, 0.001-99 wt%, preferably 0.01-90 wt%, and 
more preferably 0.1-80 wt%) of the above-mentioned nanobodies of the present 
invention (or their conjugates) and pharmaceutically acceptable carriers or 
excipients. Such carriers include, but are not limited to: saline, buffer, 
dextrose, water, glycerol, ethanol, and the combinations thereof. The drug 
formulation should be suitable for the mode of administration. The 
pharmaceutical composition of the present invention may be prepared in the 
form of an injection, for example, by a conventional method using 
physiological saline or an aqueous solution containing glucose and other 
adjuvant. Pharmaceutical compositions such as injections and solutions are 
preferably made under aseptic conditions. The amount of active ingredient 
administered is a therapeutically effective amount, for example, about 10 
micrograms/kilogram body weight to about 50 milligrams/kilogram body weight 
per day. In addition, the polypeptides of the invention can also be used with 
other therapeutic agents.  
When a pharmaceutical composition is used, a safe and effective amount 
of the immune-conjugate is administered to the mammal, wherein the safe and 
effective amount is usually at least about 10 micrograms/kilogram body weight, 
and in most cases, no more than about 50 mg/kilogram body weight, preferably 
the dose is about 10 micrograms/kilogram body weight to about 10 
milligrams/kilogram body weight. Of course, factors such as the route of 
administration and the patient's health status should be considered to define 
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the specific doses, all of which are within the skills of skilled physicians.  
Nanobodies with markers 
In a preferred embodiment of the invention, the nanobodies carry 
detectable markers. More preferably, the marker is selected from the group 
consisting of isotopes, colloidal gold markers, colored markers, and 
fluorescent markers.  
Colloidal gold markers can be performed using methods known to those 
skilled in the art. In a preferred embodiment of the invention, the anti-PD-Li 
nanobodies are marked with colloidal gold to obtain colloidal gold-markered 
nanobodies.  
The anti-PD-Li nanobodies of the present invention have very good 
specificity and high potency.  
Detection method 
The invention also relates to a method of detecting PD-L protein. The 
method steps are basically as follows: obtaining a sample of cells and/or 
tissue; dissolving the sample in a medium; and detecting the level of PD-L1 
protein in the dissolved sample.  
According to the detection method of the present invention, the sample 
used is not particularly limited, and a representative example is a sample 
containing cells which is present in a cell preservation solution.  
Kits 
The present invention also provides a kit containing a nanobody (or a 
fragment thereof) or a detection board of the present invention. In a preferred 
embodiment of the present invention, the kit further includes a container, 
an instruction, a buffer, and the like.  
The present invention also provides a detection kit for detecting the 
level ofPD-Li, and said kit comprises nanobodies that recognize PD-Li protein, 
a lysis medium for dissolving a sample, a general reagent and a buffer needed 
for the detection, such as various buffer, detection markers, detection 
substrates, etc. The test kit can be an in vitro diagnostic device.  
Application 
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As described above, the nanobodies ofthe present invention have extensive 
biological application value and clinical application value. Said 
applications involve various fields such as diagnosis and treatment of 
diseases related to PD-LI, basic medical research, and biological research.  
One preferred application is for clinical diagnosis and targeted treatment 
of PD-Li.  
The main advantages of the present invention include: 
(a) the nanobodies of the invention are anti-PD-Li proteins with high 
specificity for humans and a correct spatial structure; 
(b) the nanobodies of the invention have a strong affinity; and 
(c) the nanobodies of the invention are simple to produce.  
The present invention is further described in combination with specific 
embodiments. It should be understood that these examples are only for 
illustrating the present invention and are not intended to limit the scope 
of the present invention. The experimental methods that do not specify the 
specific conditions in the following examples are generally performed 
according to conventional conditions such as those described in Sambrook et 
al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor 
Laboratory Press, 1989), or according to the conditions recommended by the 
manufacturer. Unless otherwise indicated, percentages and parts are 
percentages by weight and parts by weight.  
Example 1: Expression and purification of human PD-Li protein 
(1) The human PD-Li nucleotide sequence was integrated into pCDNA3.I(-) 
vector (commercially available from Invitrogen) and the sequence of the 
extracellular domain was sub-cloned into pFUSE-IgG1 vector (commercially 
available from Invitrogen), wherein a TEV cleavage site was introduced at 
C-terminal of hPD-LI(ECD) to facilitate the preparation of a hPD-Ll(ECD) with 
Fe-tag.  
(2) An Omega plasmid maxi kit was used to extract the constructed 
pFMSE-lgGl-hPD-Ll(ECD) plasmid.  
(3) HEK293F cells were cultured to an OD of 2.OXO0cells/mL.  
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(4) The plasmid and the transfection agent PEI were mixed (1:3) well and 
placed for 20 min, and then the product was added into HEK293F cells culture 
for further incubation in a shaker under 6% CO2 at 37V for 5-6 days.  
(5) The supernatant of the cells was collected and subjected to binding 
with Protein A beads at R.T. for 1 hour.  
(6) After the beads were washed by PBS (pH 7.0), 0.1 M of Glycine (pH3.0) 
was used to elute the proteins.  
(7) The eluted proteins were ultrafiltrated into PBS and sampled for an 
SDS-PAGE test after yield measurement (the test results are shown in Figure 
1B). The rest of the proteins were stored in a fridge at -80C.  
(8) The expressed hPD-L1(ECD)-Fc protein was cleavaged by using 0.1 mg 
TEV enzyme per 1mg hPD-L1(ECD)-Fc protein at 4C for 16 hours. The protein 
solution was loaded onto a Ni column and a Protein A column subsequentially 
and the flow-through was collected and sampled to an SDS-PAGE test (the test 
results are shown in Figure IC).  
Example 2: The construction of PD-Li nanobody library 
(1) 1 mg of hPD-L1 (ECD)-Fc antigen was mixed with Freund's adjuvant in 
equal volumes to immunize a Xinjiang bactrian camel once a week for a total 
of 7 times to stimulate B cells to express antigen-specific nanobodies; 
(2) After the 7 immunizations were completed, 100 mL of camel peripheral 
blood lymphocytes were sampled and total RNA was extracted.  
(3) cDNA was synthesized and VHH was amplified using nested PCR; 
(4) 20 u g pMECS phage display vector (purchased from Biovector) and 10 
4g VHH were digested with restriction endonucleases PstI and NotI and the 
two fragments were ligated together; 
(5) The ligated product was electronically transfected into competent TG1 
cells, and the PD-L1 nanobody library was constructed and the capacity thereof 
was determined. The capacity was 1.3X109 CFM (the results are shown in Figure 
2).  
At the same time, 24 clones were picked randomly for PCR detection of 
colony. The results showed that the insertion rate of the constructed library 
was 100%. Figure 3 shows the PCR results of colony.  
Example 3: Screening and verification of PD-Li nanobodies 
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Screening of nanobodies 
(1) 10 u g hPD-L1 (ECD) antigens dissolved in 100 mM NaHCO, (pH 8.2) was 
coupled to the NUNC ELISA board and left overnight at 4 °C; 
(2) 100 P L of 0.1% BSA was added on the next day and blocked at room 
temperature for 2 h.  
(3) After 2 h, 100 i L of phages (2 X 10" CFM of phage display gene library 
with nanobodies of immunized camel) was added and reacted at room temperature 
for 1 h; 
(4) 0.05% PBS + Tween-20 were used for washing for 5 times to wash away 
non-specific phages; 
(5) The phages specifically binded to PD-L1 were dissociated by 100 mM 
triethanolamine, and E. coliTG1 cells in logarithmic phase were infected and 
incubated at 37 C for 1 h. The phages were generated and purified for the 
next round of screening. The screening process was repeated for 3 rounds. The 
enrichment results are shown in Figure 4.  
Screening specific single positive clones by using phage-based 
enzyme-linked immunosorbent assay (ELISA): 
(1) From the cell culture dishes containing bacteriophages obtained by 
above 2-3 rounds of screening, 96 single colonies were picked and inoculated 
in TB medium containing 100 u g/mL ampicillin (2.3 liter KHPO4 in 1 liter TB 
medium. 12.52 g K2HPO 4, 12 g peptone, 24 g yeast extract, 4 mL glycerol). After 
the cells grew to logarithmic phase, IPTG was added to a final concentration 
of 1 mM and cultured overnight at 28C.  
(2) Crude nanobodies were obtained by osmotic method, and the nanobodies 
were transferred to an antigen-coated ELISA board and allowed to place at room 
temperature for 1 hour.  
(3) Unbound nanobodies were washed away with PBST and anti-mouse anti-HA 
nanobodies (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.).  
The product was placed at room temperature for 1 hour.  
(4) Unbound nanobodies ware washed away with PBST, and goat anti-mouse 
alkaline phosphatase-labeled nanobodies were added. The product was placed 
at room temperature for 1 hour.  
(5) Unbound nanobodies were washed with PBST, and alkaline phosphatase 
staining solution was added. The absorbance was read at 405 nm on an ELISA 
instrument.  
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(6) When D value of the sample well was over 3 times of the GD value of 
the control well (Ratio +/->3), it is confirmed as a positive clone well.  
(7) The bacteria in the positive clone wells were shaken in an LB liquid 
containing 1009 g/mL for plasmid extraction and sequencing.  
Example 4: Expression of nanobodies in E.coli host and purification: 
(1) The plasmids obtained from the previously sequenced clones were 
electro-transformed into E coli WK6 and then coated onto LA+Glucose (a 
culture plate containing ampicillin and glucose) for incubation overnight at 
37°C.  
(2) A single colony was picked, inoculated into 5 mL LB culture medium 
which contains ampicillin, and cultured in a shaker overnight at 370C; 
(3) 1 mL overnight-cultured strains were inoculated into 330 mL TB medium 
and culture in a shaker at 37 TC to an D value of 0.6-1. IPTG was added and 
the product was cultured in a shaker at 28 TC overnight.  
(4) The product was subjected to centrifugation and the strains were 
collected.  
(5) Using the osmosis method to obtain the crude nanobody extract.  
(6) Nanobodies with a purity of over 90% were prepared by Ni ion column 
affinity chromatography. The purification results are shown in Figure 5.  
Example 5: The blocking effects of nanobodies tested by flow cytometry 
(1) hPD-1-Fc-Biotin proteins were prepared (The preparation method for 
hPD-1-Fc was identical with Example 1. The SDS-PAGE test results are shown 
in Figure 1E). The biotinylation of the proteins were conducted according to 
the biotin reagent instructions.  
(2) 1X10' of HEK293F cells transiently expressing human PD-L1 full-length 
protein were taken from each sample and resuspended in 0.5% BSA-PBS buffer, 
and 10 i g of the above-mentioned purified PD-L1 nanobodies were added. hIgG1 
was set as a negative control and PBS was for blank group. 5 9g of 
hPD-1-Fc-biotins were added into all the samples for each and subjected to 
incubation at 4 TC for 20 min.  
(3) The cells were washed twice with PBS, and SA-PE (purchased from 
eBioscience) was added. The product was incubated at 4 for 20 minutes. A 
flow cytometry (BD FACS Calibur) was used for determine the cells after they 
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were washed twice with PBS. The determination results were shown in Figure 
6.  
Humanization of PD-Li nanobodies 
(1) Firstly, the PD-Li nanobody sequence of SEQ ID NO. : 8 was used as a 
template to search for homologous structures in the structural database. A 
total of 1306 structures were found, wherein 34 structures were taken (E value 
= 0.0, and sequence identity 70%); 
(2) These 34 structures were subjected to structural comparison. Based 
on the resolution of the crystal structure and the constructed evolutionary 
tree, 9 proteins including 3dwt were finally selected for multi-template 
homology modeling based on the PD-Li nanobody sequence of SEQ ID NO.: 8.  
Finally, 10 structures were obtained. The structures with lowest molpdf were 
selected according to the ranking of the scoring function from top to bottom 
and then left for the further process.  
(3) For those best structures obtained from modeling, the solvent 
accessibility of the residues calculated by ProtSA server (i.e. the ratio of 
the solvent contactable surface of the residues between folding and folding 
state) was used as a cut-off value. The residues with a value over 40% were 
taken as the residues exposed to the solvent.  
(4) An alignment was conducted between the best structures obtained from 
modeling and DP-47 sequence and the corresponding residues exposed to the 
solvent were substituted. A humanized PD-L1 nanobody of the amino acid 
sequence as set forth by SEQ ID NO.: 14 was ultimately determined. The 
sequences of the nanobodies before and after humanization were shown in Table 
2: 
Table 2 
nanobody domain SEQ ID NO.: 
Before humanization After humanization 
FR1 1 10 
CDR1 5 5 
FR2 2 11 
CDR2 6 6 
FR3 3 12 
CDR3 7 7 
FR4 4 13 
Full amino acid sequence 8 14 
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Full nucleotide sequence 9 15 
The comparison of the identity between the nanobody framework region and 
the DP-47 framework region before and after humanization is shown in Table 
3 below: 
Table 3 
nanobody The identity with DP-47 
domain Before After 
humanization humanization 
FR1 80% 92% 
FR2 66.67% 80% 
FR3 76.32% 89.47% 
FR4 90.91% 100% 
Example 6: The activity of anti-PD-L1 nanobodies determined by using MOA 
method 
In this experiment, two commercially available anti-PD-L1 nanobodies 
(Atezolizumab, ATE and Durvalumab, DUR) was taken as positive control 
nanobodies, and the cell lines (Promega) were detected using MOA. The 
activation of the NFAT signal was reflected by determing the fluorescent 
reporter gene, thereby detecting the inhibitory effects of the nanobodies 
(sequence shown in Example 5) on PD-1/PD-L1 binding. The steps were shown as 
follows: 
(1) CHOKI-PDL1 cells were plated one day before the activity assay: 
CHOK1-PDLl was passaged 1-2 days before. The culture supernatant was discarded 
and the resultant was washed with PBS. Appropriate amounts of trypsin were 
added to digest at 37C/5%CO2 for 3-5 min. Culture medium at 4 times the volume 
of trypsin was added, and the cells were transferred to a 50 ml centrifuge 
tube and subjected to cells counting. Cells with required volume were 
centrifuged for 10 min at 230 g. The medium was added and the cells were 
resuspended to 4 x 10" cells/mL. The cells were added to a white 96-well cell 
culture plate at 100 i I/well. PBS was added to the side wells at 200 u 1/well.  
Cells were incubated in a 37'C/5% CO0 incubator overnight.  
(2) Treatment of Jurkat-PD1 cells: Cells were passaged two days prior to 
the activity assay. After counting, cells of required volume were centrifuged 
for 5 min at 170 g. The cells were resuspended in assay buffer to 1.25 x 10° 
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cells/ml.  
(3) The samples and Jurkat-PD1 cells were added to the assay plate: the 
supernatant of CHOK1-PDL1 cells (95 P 1/well) was discarded. 40 91 of sample 
(purify nanobodies obtained from hybridoma supernatant or serially diluted 
hybridoma supernatant) positive controls, and negative controls were added.  
v 1 of Jurkat-PD1 cells were added and the resultant was incubated in a 
37C/5% C02incubator for 6 hours.  
(4) Assay: The Bio-GloTM buffer was thawed in advance, and Bio-GloTM 
substrate was added and mixed well. After 6 hours, Bio-GloTM Reagent was added 
at 80 91/well and placed at room temperature for 5-10 minutes for reading.  
The results of the experiments are shown in Table 4 and Figure 11. Under 
various concentrations, the nanobodies of the present invention before 
humanization were generally more active than the positive control, and the 
activity of the nanobodies of the invention after humanization was comparable 
to that of the positive control. Therefore, both Nb-Fc and humanized Nb-Fc 
nanobodies can effectively block PD1/PD-L1 interactions.  
Table 4 
Concentration Nb-Fc humanized ATE DUR IgG1 Cell (nm) Nb-Fc 
100.000 89222 89341 94006 94061 15659.5 23860 
33.333 95361 92060 97992 102218 
11.111 95122 92012 96453 97936 
3.704 97307 74598 96932 102944 
1.235 96119 68937 70803 89708 
0.412 95249 43734 29286 53071 
0.137 85291 23035 19240 23210 
0.046 60302 19080 17246 20308 
0.015 36885 19016 18355 17757 
0.005 26336 17079 16927 18243 
Example 7: Expression of humanized PD-Li nanobodies in eukaryocyte HEK293 
and purification 
(1) The PD-Li Nb sequences before and after humanization were synthesized 
to the pFUSE-IgG1 vector (purchased from Invivogen), and the pFUSE-IgG1-Nb 
plasmid (humanized) was extracted using Omega plasmid maxi kit.  
(2) HEK293F cells were cultured to an OD of 2.0X0'cells/mL; 
(3) The plasmid and the transfection reagent PEI (1:3) were mixed and 
allowed to stand for 20 min, then added to HEK293F cells, and cultured in a 
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6% C02shaker at 370 C for 5-6 days; 
(4) The cell supernatants were collected and subjected to the binding with 
Protein A beads at room temperature for 1 hour; 
(5) After washing the beads with phosphate buffer (pH 7.0), the proteins 
were eluted with 0.IM Glycine pH 3.0; 
(6) The eluted proteins were ultrafiltrated into PBS, and the yield was 
measured. Then the samples were analyzed by SDS-PAGE (the results are shown 
in Figure ID and Figure 7). The remaining proteins were stored in a 
refrigerator at -80° C. It can be seen from Figure 7 that the purity of 
humanized nanobodies reaches more than 90%.  
Example 8: Blocking effects of the humanized PD-L1 nanobodies determined 
by flow cytometry 
The method is identical with Example 5: 
(1) 2Xl105 human lung cancer cell lines (EBC-1) naturally expressing PD-L1 
in each sample were resuspended in 0.5% BSA-PBS buffer and 10 p g of purified 
humanized PD-Li nanobodies were added. hIgGi was set as the negative control 
group and PBS as the blank group. 5P g hPD-1-Fc-biotin was added into each 
sample, and the products were incubated at 4 0 C for 20min; 
(2) The cells were washed with PBS twice, and SA-PE from eBioscience was 
added. The resultants were incubated for 20 minutes at 4 C, and the cells 
were washed with PBS twice and loaded for tests. The results are shown in Figure 
8: it could be seen from the blank and the negative control, the binding rate 
of PD-1-Fc-biotin to EBC-1 cells was above 90%. While after the addition of 
PD-Li nanobodies and humanized nanobodies, the binding rate of PD-1-Fc-biotin 
to EBC-1 cells was less than 10 %. This indicates that the added nanobodies 
can significantly block the interaction of PD- with PD-Li.  
Example 9: Determination on the affinity of the nanobodies 
BiaCore T200 was used for detection. (1) Immobilization: The immobile 
phase antigens were immobilized on the surface of a CM-5 sensor chip using 
a carboxy-amino reaction; 
(2) Binding: The ananobodies were diluted with HBS buffer to an 
appropriate concentration (five concentration gradients) to observe the 
antigen-nanobody binding process; 
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(3) Chip regeneration: When performing the next nanobody measurement, 10 
mM Glycine was used for regeneration.  
(4) Analysis of the experimental results. The results of the assay are 
shown in Figure 9. The affinity of nanobodies before humanization was 
2. 34X 10- M, and the affinity of humanized nanobodies was 2.26X10-9 M.  
Humanization does not change the affinity of the nanobody.  
Example 10: The specificity of purified nanobodies by ELISA 
(1)The nanobodies before and after humanization were biotinylated by 
conventional methods; 
(2) The antigen proteins PD-Li (human), PD-Li (rat), PD-Li (monkey), PD-L2 
(human), B7H4 (human), B7H3 (human) were coated: 0. 5 9 g per well ( 5 P g/mL, 
100 UL), IgGi was coated as a control, left overnight at 40 C; 
(3) The products were washed by PBST 3 times, and 200 i L, 1% BSA was added 
to block in RT for 2 hours; 
(4) Each biotinylated nanobodies were diluted to 10 P g/mL, and 100 u L 
of each was incubated in each well and allowed to react at RT for 1 hour.  
(5) The unbound nanobodies were washed with PBST, 100 P L of 
streptavidin-HRP (1:1000 dilution) was added, and the resultant was let stand 
for 1 hour at room temperature.  
(6) The color development solution was added and the absorbance at the 
wavelength of 450 nm was read on ELISA. The specificity of the nanobodies was 
determined based on the absorbance values. The results are shown in Figure 
10. Both of the nanobodies before and after humanization interacted with human 
and monkey-derived PD-Li but not with the mouse-derived PD-L. The two 
nanobodies had good species specificity. Neither of the nanobodies before or 
after humanization interacted with PD-Li family members and had good family 
specificity.  
Example 11: Tests on mixed lymphocytes 
In this experiment, nanobodies were incubated with mature DC cells and 
CD4+ T cells derived from different donors and cultured in vitro. The relative 
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expression levels of IL2 and IFN-y in the system were detected to reflect 
the activation of T cells by different nanobodies. The steps are as follows: 
(1) PBMC isolation: 50 ml fresh blood from the donors was taken, and 2.5 
times ofPBS was added. The product was gently added into FiColl (Thermo) (12.5 
ml, 4 tubes), centrifuged at 400g for 30min, and stopped at 0 deceleration.  
The middle white band was aspirated into PBS (Gibco) and washed twice with 
PBS.  
(2) DC cell isolation: The isolated PBMC cells were taken and 5 ml of T 
cell culture medium was added. The cells were subject to adherent culture at 
37 ° C under 6% C0 for 2 hrs. The suspending cells were taken to separate CD4+ 
cells. 3 ml of DC was added to the remaining cells. After 2 days of culture, 
3 ml of DC medium was added for further culture to the fifth day. Then, rTNFa 
(R&D Systems) (1000 U/ml), IL-lb (R&D Systems) (5 ng/ml), IL-6 (R&D Systems 
(10 ng/ml) and1 I M PGE2 (Tocris) were cultured for 2 days as the DC cells 
for mixed lymphocyte reaction (MLR).  
(3) Isolation of CD4+ cells: PBMCs were incubated for 2 hr and the 
suspended cells were drawed into 15 ml centrifuge tubes, centrifuged at 200 
g for 10 min, resuspended in 500 91 of serum, 100 P1 of AB serum, and 100 
1 1of purified nanobodies, incubated for 20 min at 4° C, and washed once with 
the separation solution. 500 i 1 of Bead Buffer was added for incubation for 
min. The Bead was removed by magnetic field, washed once with T cell culture 
medium, resuspended with 8 ml culture medium, and incubated at 37 C under 
6% C 2. (The procedures were conducted according to the instruction of 'Human 
CD4+ T Cell Enrichment Kit' (19052, Stemcell)).  
(4)MLR experiment: The matured DC cells were mixed with CD4+ cells at a 
volume of 200 I1 per well, 10, 000 DC cells, and 100,000 CD4+ cells. nanobodies 
were added, DCs, T cells, and MLR were used as negative controls, and DC+T 
ceIls+anti-CD3/CD28 magnet beads were used as the positive control. The beads 
were subjected to mixed culture for 5 days, and a cisbio kit (Human IL2 Kit 
1000 Test, Human IFN gamma 1000 test) was used to detect IL2 and IFN-gamma 
concentration.  
The experimental results are shown in Figure 12. The nanobodies of the 
present invention (sequence shown in Example 5) stimulated the donor to 
produce more cytokines than the positive control. After humanization, the 
produced cytokines were comparable to the positive control. Therefore, both 
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10290327_1 (GHMatters) P108776.AU

of the nanobodies of the present invention and the humanized nanobodies can 
effectively activate T cells, and the activation effect is similar to that 
of the antibody of positive control group.  
Example 12 The study on tumor-inhibiting activities of anti-PD-Li 
nanobodies.  
In this study, human PD-Li expressing MC38 cells (MC38-PDLI) (Nanjing 
Galaxy) were used to determine the anti-tumor effects of humanized Nb-Fe in 
PD-Li transgenic mice. Firstly, MC38-PDL1 tumor-bearing mice model was 
established by subcutaneous inoculation. After tumor formation, different 
nanobodies (sequences are shown in Example 5) and different doses of treatment 
were administered, and the tumor volumes and body weight changes in each group 
of mice were monitored during administration. Dosing frequency was 2 
times/week, monitoring frequency was 2 times/week, and continuous monitoring 
last for 5 weeks. The dosage and methods were shown in Table 5 and Figure 13.  
Table 5 
Group Testing dosage Administration concentration Administration 
subjects volume route 
h-IgG IgG control 20 mg/kg 10 ml/kg 2.0 mg/ml Intraperitoneal 
injection 
humanized humanized 10 mg/kg 10 ml/kg 1.0 mg/ml Intraperitoneal 
Nb-Fc Nb-Fc injection 
The steps are shown as follows: 
1) The preparation of MC38/PD-L1 cell suspension: MC38 cells were 
dispensed with PBS (X) to a cell density of 1X10' cells/ml to prepare the 
MC38 cell suspension; 
2) Inoculation: 25 C57B1/6 background PD-Li mice were shaved at the right 
side of the back, and MC38/PD-LI cells were subcutaneously injected with 1 
x 10 cells/0.1 ml/body. After 6 days of tumor cell inoculation, the tumor 
volumes of each mouse were examined, and 25 mice with a tumor volume ranging 
from 87.4 mm" to 228.4 mm' were selected and grouped by average tumor volume.  
3) Dosing: See Figure 13.  
4) Test: The weight before and after each administration and the body 
weight and tumor volumes were measured. Weights were measured using an 
electronic balance, 2 times per week.  
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5) Measurement ofthe tumor volumes: The maximum length (L) and the maximum 
width (W) of the tumors were measured using a vernier caliper. The tumor 
volumes were calculated according to the following formula: V = LXW/2.  
The results of the experiments are shown in Figure 14. The mice vaccinated 
with humanized Nb-Fe had a very good control of tumor volumes comparing to 
the control group and showed no significant increase, indicating humanized 
Nb-Fc has a significant tumor inhibiting effect.  
Example 13. The solubility of the nanobodies detected by PEGprecipitation 
method 
In this experiment, the solubility of the nanobodies was reflected by PEG 
precipitation method through detecting the dissolution of alternative 
nanobodies (sequence shown in Example 5) in different concentrations of PEG.  
Proceeds are shown as follows: 
1) The nanobody sample was concentrated to 5 mg/ml.  
2) The samples were added into a 96-well cell culture plate with 40 i I 
nanobody samples per well to a final concentration of 1 mg/ml. 26.7 u 1, 40 
P 1, 46.7 p 1, 53.3 P 1, 60 v 1, 66.7 P 1, 73.3 u 1, 80 P1, 86.7 u 1, 93.3 
i1, 100 il1, and 106.7 PI1 of 30% PEG was added into columns 1 to 12, 
respectively, and the IBI301 Buffer was added to a total volume of 200 P91.  
The PEG concentration gradients were 4%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 
14%, 15%, and 16%, respectively.  
3) The product was place at room temperature for 1 hr and D500 nm was 
measured.  
The results of the study are shown in Table 6 and Figure 15. Humanized 
Nb-Fc appeared turbid at 8% (w/v) PEG, while the control antibody 
(post-marketing agent, Humira) appeared turbid at 6% (w/v) PEG. This indicates 
that the humanized Nb-Fe of the present invention has superior solubility than 
the control nanobody.  
Table 6 
OD500 nm 4% 6% 7% 8% 9% 10% 
humanized 0.0352 0.0356 0.0354 0.0602 0.0411 0.1792 Nb-Fc 
Humira 0.0365 0.0367 0.0387 0.2144 0.3627 0.6293 
OD500 nm 11% 12% 13% 14% 15% 16% 
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humanized 0.4778 0.5807 0.8418 0.8687 0.928 1.0514 
Nb-Fc 
Humira 0.8409 0.7891 0.9194 0.9589 0.9086 0.9444 
Example 14. Test on accelerated stability 
In this experiment, the long-term thermal stability of the nanobodies was 
evaluated by detecting changes in the purity and biological activity of the 
nanobodies (sequence shown in Example 5) after leaving at 40 C for 30 days.  
The purity of the desired nanobodies after 0, 14 and 30 days of storage at 
40° C was determined by using SEC. As shown in Table 7 and Figure 16, the purity 
of humanized Nb-Fe did not change significantly. In this experiment, the 
combination of accelerated stability test sample and CHO-PDL1 cells was 
detected by FACS method. The steps are as follows: 
1) Cell preparation: CH-PDL1 cells were counted and diluted to 2 x 10' 
cells/ml, then the cells were added into a U-bottom 96-well plate at 100 
91/well, and 50 P1 of cells was added to the wells in the first column; 
2) Detection steps: nanobodies were added to the first well to the final 
concentration of 200 nM, and mixed. 50 iy1 was pipetted into the second well, 
and so forth. The negative control is IgG Control. The product was subjected 
to ice bath for 20 minutes. PBS was added at 100 p 1/well. The resultant was 
centrifuged at 400 g for 5 min to remove the supernatant and the cells were 
washed with PBS once. The diluted (1:100) goat anti-human IgG-PE (eBioscience) 
was added at 100 p1/well. The resultant was subjected to ice bath for 20 min, 
centrifuged at 400 g for 5 min to remove the supernatant, washed once with 
PBS at 100 i1/well, resuspended with 100 p I1 PBS and detected by FACS.  
As shown in Figure 17, the binding of humanized Nb-Fe and CHO-PDL1 cells 
did not change significantly. The results show that humanized Nb-Fc has good 
thermal stability.  
Table 7 
SEC (%) Day 0 Day 14 Day 30 
humanized 
Nb-Fc 99.55 99.68 99.71 
All references mentioned in the present invention are incorporated herein 
by reference, as each of them is individually cited herein by reference.  
Further, it should be understood that, after reading the above contents, the 
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10290327_1 (GHMatters) P108776.AU

skilled person can make various modifications or amendments to the present 
invention. All these equivalents also fall into the scope defined by the 
pending claims of the subject application.  
It is to be understood that if any prior art publication is referred to 
herein, such reference does not constitute an admission that the publication 
forms a part of the common general knowledge in the art in Australia or any 
other country.  
In the claims which follow and in the preceding description of the 
invention, except where the context requires otherwise due to express language 
or necessary implication, the word "comprise" or variations such as 
"comprises" or "comprising" is used in an inclusive sense, i. e. to specify 
the presence of the stated features but not to preclude the presence or 
addition of further features in various embodiments of the invention.  
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11376334_1 (GHMatters) P108776.AU 24 May 19

Claims 
1. A VHH chain of an anti-PD-Li nanobody, comprising complementary 
determining region (CDR) 1 as set forth by SEQ ID NO.: 5, CDR2 as set forth 
by SEQ ID NO.: 6 and CDR3 as set forth by SEQ ID NO.: 7.  
2. The VHH chain of an anti-PD-Li nanobody of claim 1, further comprising: 
a. frame region (FR) 1 as set forth by SEQ ID NO.:1, FR2 as set forth 
by SEQ ID NO.: 2, FR3 as set forth by SEQ ID NO.: 3, and FR4 as set forth by 
SEQ ID NO.: 4; or 
b. FR1 as set forth by SEQ ID NO. :10, FR2 as set forth by SEQ ID NO.: 11, 
FR3 as set forth by SEQ ID NO.: 12, and FR4 as set forth by SEQ ID NO.: 13.  
3. An anti-PD-Li nanobody, comprising an amino acid sequence of SEQ ID 
NO.: 8 or SEQ ID NO.: 14.  
4. A polynucleotide encoding the VHH chain of an anti-PD-Li nanobody of 
claim 1 or claim 2, or the anti-PD-Li nanobody of claim 3.  
5. The polynucleotide of claim 4, comprising a nucleotide sequence of SEQ 
ID NO.: 9 or SEQ ID NO.: 15.  
6. An expression vector comprising the polynucleotide ofclaim4 or claim 5.  
7. A host cell comprising the polynucleotide of claim 4 or claim 5, 
optionally wherein the polynucleotide is integrated within the host cell 
genome, or the expression vector of claim 6.  
8. A method for producing an anti-PD-L nanobody, comprising: 
a. culturing said host cell of claim 7 under conditions suitable for 
producing the anti-PD-L nanobody, thereby obtaining a culture comprising 
said anti-PD-Li nanobody; and 
b. isolating or recovering said anti-PD-Li nanobody from said culture.  
9. An anti-PD-Li nanobody when produced according to the method of claim 8.  
10. An immunoconjugate comprising: 
a. the VHH chain of an anti-PD-Li nanobody of claim 1 or claim 2, or 
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11376334_1 (GHMatters) P108776.AU 24 May 19

the anti-PD-L nanobody of claim 3 or claim 9; and 
b. a conjugating part selected from a group consisting of a detectable 
marker, drug, toxin, cytokine, radionuclide, and enzyme.  
11. A pharmaceutical composition comprising the VHH chain of an anti-PD-Li 
nanobody of claim 1 or claim 2, the anti-PD-Li nanobody of claim 3 or claim 9, 
or the immunoconjugate ofclaim 10, and a pharmaceutically acceptable carrier.  
12. Use of the VHH chain of an anti-PD-Li nanobody of claim 1 or claim 2, the 
anti-PD-L nanobody of claim 3 or claim 9, the polynucleotide of claim 4 or 
claim 5, the expression vector of claim 6, or the host cell of claim 7 in the 
manufacture of an agent for detecting PD-L.  
13. Use of the VHH chain of an anti-PD-Li nanobody of claim 1 or claim 2, the 
anti-PD-L nanobody of claim 3 or claim 9, the polynucleotide of claim 4 or 
claim 5, the expression vector of claim 6, or the host cell of claim 7 in the 
manufacture of a medicament for treating cancer.  
14. A method for treating cancer, comprising administering to a subject in 
need the VHH chain of an anti-PD-L nanobody of claim 1 or claim 2, the 
anti-PD-L nanobody of claim 3 or claim 9, the immunoconjugate of claim 10, 
or the pharmaceutical composition of claim 11.  
-35
11376334_1 (GHMatters) P108776.AU 24 May 19

                         ÐòÁбí
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<120>  ¿¹PD-L1ÄÉÃ׿¹Ìå¼°ÆäÓ¦ÓÃ
<130>  P2017-1215
<150>  CN201610634596.X
<151>  2016-08-04
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caggtgcagc tgcaggagtc tgggggaggc tcggtacagg ctggagggtc 
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<223>  ÈËÔ´»¯VHH
<400>  15
caggtgcagc tgcaggagag cggcggcggc ctggtgcagc ccggcggcag 
cctgaggctg       60
agctgcgccg ccagcgccta caccatcagc aggaacagca tgggctggtt 
caggcaggcc      120
cccggcaagg gcctggaggg cgtggccgcc atcgagagcg acggcagcac 
cagctacagc      180
gacagcgtga agggcaggtt caccatcagc ctggacaaca gcaagaacac 
cctgtacctg      240
gagatgaaca gcctgagggc cgaggacacc gccgtgtact actgcgccgc 
ccccaaggtg      300
ggcctgggcc ccaggaccgc cctgggccac ctggccttca tgaccctgcc 
cgccctgaac      360
tactggggcc agggcaccct ggtgaccgtg agcagc                                
396

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