Anti-CCR8 antibody and Anti-CCR8 / CTLA-4 bispecific antibody, and use thereof

Abstract

Provided are an anti-CCR8 antibody and an anti-CCR8 / CTLA-4 bispecific antibody, and the use thereof. Further provided are a polynucleotide, recombinant vector, host cell, chimeric antigen receptor, antibody conjugate, pharmaceutical composition and kit related to the anti-CCR8 antibody and anti-CCR8 / CTLA-4 bispecific antibody, and the related use and method. The provided antibodies can specifically target and eliminate regulatory T lymphocytes with an immunosuppressive activity infiltrating tumor tissue, and can be used in anti-tumor immunotherapy.

Description

Anti-ccr8 antibodies, anti-ccr8 / ctla-4 bispecific antibodies, and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202510030754X filed on January 8, 2025, to the China National Intellectual Property Office, and claims priority to Chinese Patent Application No. 2025107921532 filed on June 13, 2025, to the China National Intellectual Property Office, the entire contents of which are incorporated by reference in their entirety for all purposes. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, in particular to an anti-CCR8 antibody and an anti-CCR8 / CTLA-4 bispecific antibody and uses thereof. BACKGROUND

[0004] The first immunotherapeutic antibody drug approved by FDA in 2011, Ipilimumab (trade name YERVOY), opened a new era of tumor immunotherapy. Immune checkpoints (IC), such as CTLA-4 or PD-1 / PD-L1, are important immunosuppressive molecules expressed by T cells or other cells. Immune checkpoint inhibitors (ICIs) that inhibit such molecules have achieved unprecedented success in the treatment of various cancers. Compared with PD-1 inhibitors, the incidence of immune-related adverse events (irAEs) of CTLA-4 inhibitors is high. Due to side effects, the dose of CTLA-4 inhibitors in clinical application is severely limited, resulting in relatively low efficacy, but long-term benefits have been observed in patients treated with anti-CTLA-4 monoclonal antibodies. A landmark analysis involving 1861 patients treated with Ipilimumab showed that the survival of some patients was prolonged from around the third year to 10 years, indicating the enduring value of anti-CTLA-4 antibodies as tumor immunotherapy agents. CTLA-4 targets the immune response upstream of the antigen presentation stage, while PD-1 targets terminal effector T lymphocytes, so theoretically, without considering toxicity, CTLA-4 targets will be more effective, so the development of low-toxicity anti-CTLA-4 antibodies has great application potential.

[0005] CCR8 belongs to G protein-coupled receptors, is a chemokine receptor highly expressed in tumor-infiltrating Tregs, and is expressed in lower amounts in Treg cells in the thymus, spleen and peripheral blood, and in Th2 cells. The known ligand of CCR8 is chemokine CCL1, which is up-regulated at the site of inflammation. CCL1 can recruit Foxp3 + CCR8 + Tregs cells, the surface CCR8 expression is up-regulated, and the expression of STAT3-dependent Foxp3, CD39, IL-10 and the like is induced, thereby enhancing the immunosuppressive activity of these tumor-infiltrating Tregs cells. Compared with Tregs in normal tissues, CCR8 is significantly up-regulated in tumor-infiltrating Tregs, indicating that CCR8 is a promising specific therapeutic target on Tregs in the tumor microenvironment (TME), and targeting CCR8 will not cause serious systemic autoimmune diseases. Therefore, antibodies targeting CCR8 can improve the immune environment of TME by eliminating Tregs cells in the tumor microenvironment and blocking the biological function of CCR8, and have a relatively specific anti-tumor immunotherapy potential.

[0006] It should be noted that the methods described in this section are not necessarily the methods that have been previously conceived or adopted. Unless otherwise indicated, nothing in this section should be construed as a recognition that any method described in this section is an art that has been previously conceived or adopted. Similarly, unless otherwise indicated, the problems mentioned in this section should not be considered to have been recognized in any prior art. SUMMARY

[0007] To solve the above problems, the present application provides a bispecific antibody or antigen binding fragment thereof which can specifically bind to CTLA-4 and CCR8 at the same time. Compared with ipilimumab, the bispecific antibody provided by the present application has higher efficacy, lower side effects, and lower treatment dosage, and is expected to become a new cornerstone drug for tumor immunotherapy.

[0008] According to an embodiment of the present application, the present application provides an antibody or antigen binding fragment thereof binding to CCR8, characterized in that the amino acid sequences of the complementarity determining regions (CDRs) defined according to the IMGT system are as follows: the amino acid sequence of heavy chain CDR1 is X1X2X3X4X5X6X7X8; the amino acid sequence of heavy chain CDR2 is X9X 10 X 11 X 22 X 23 X 24 X 25 X 26 X 27 X 28 ; the amino acid sequence of heavy chain CDR3 is X19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 the amino acid sequence of the heavy chain CDR1 is X 33 X 34 X 35 X 36 the amino acid sequence of the heavy chain CDR2 is X 37 X 38 X 39 X 40 X 41 X 41 X 42 the amino acid sequence of the heavy chain CDR3 is X 43 X 44 X 45 X 46 X 47 X 47 X 48 X 41 X 49 X 41 X 50 X 41 X 51 X 41 X 52 X 41 X 53 X 41 X 54 ; wherein: X1is G or A; X2is F or A; X3is T or A; X4is F or A; X5is N or A; X6is G, S or A; X7is Y or A; X8is G, S, V, L, I, T, Y, D, K or A; X9is I or A; X 10 is H, K, G, D, L, S, W, A or R; X 11 is S, Q, Y, N, G, L, M, W, A or T; X 12 is K or A; X 13 is S or A; X 14 is N or A; X 15 is N or A; X 16 is Y or A; X 17 is G, S or A; X 18 is T or A; X 19 is V or A; X 20 is R or A; X 21 is H or A; X 22 is A, E, Y, P, Q, T, I, V, N, M, F or K; X 23 is R, H, G, L, S, W, D, A, V, E, T, Q, N, M, I, P, F, Y or K; X 24 is V or A; X25 For A, K, H, G, L, S, W, D, or R; X 26 For E or A; X 27 For A, V, L, I, M, W, S, D, K, E, P, Q, H, T, R, N, F, Y, or G; X 28 For A, N, Q, M, W, S, D, K, L, G, F, R, H, I, T, E, V, P, or Y; X 29 For G, S, or A; X 30 For M or A; X 31 For A, G, L, M, W, S, K, E, R, P, Q, H, N, T, Y, V, F, I, or D; X 32 For Y or A; X 33 For K or A; X 34 For S or A; X 35 For V or A; X 36 For S or A; X 37 For T or A; X 38 For S or A; X 39 For G or A; X 40 For Y or A; X 41 For T or A; X 42 For Y or A; X 43 For L or A; X 44 For G, S, or A; X 45 For S or A; X 46 For Q or A; X 47 For H or A; X 48 For S or A; X 49 For A, K, H, G, L, M, W, S, D, E, V, I, Q, F, P, T, N, Y, or R; X 50 For E or A; X 51 For A, G, V, I, M, W, S, D, K, T, P, Q, N, H, E, Y, F, R, or L; X 52 For F, G, L, S, D, K, Q, W, A, or P; X 53 For A, P, G, L, S, D, K, Q, W, or F; and X 54 It can be T or A.

[0009] According to one embodiment of this application, this application provides a bispecific antibody or its antigen-binding fragment, characterized in that it comprises a first antigen-binding domain specifically binding to CTLA-4 and a second antigen-binding domain specifically binding to CCR8, wherein the first antigen-binding domain comprises a complementarity-determining region (CDR) as defined by the IMGT system, and the amino acid sequence of the complementarity-determining region is as follows: the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, GAF, and SEQ ID NO:6, respectively; and the second antigen-binding domain is selected from at least one of the following: (1) the second antigen-binding domain comprises a complementarity-determining region (CDR) as defined by the IMGT system, and the amino acid sequence of the complementarity-determining region is as follows: the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:6, and SEQ ID NO:15, respectively. ID NO:14, LAS, SEQ ID NO:16; or (2) the second antigen-binding domain contains the antibody or its antigen-binding fragment as described in this application.

[0010] According to one embodiment of this application, a polynucleotide is also provided, said polynucleotide encoding the antibody or antigen-binding fragment thereof described in this application and / or the bispecific antibody or antigen-binding fragment thereof described in this application.

[0011] According to one embodiment of this application, a recombinant vector is also provided, the recombinant vector comprising the polynucleotide described in this application.

[0012] According to one embodiment of this application, a host cell is also provided, the host cell comprising the polynucleotides described in this application and / or the recombinant vectors described in this application.

[0013] According to one embodiment of this application, a chimeric antigen receptor is also provided, the chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof described in this application or the bispecific antibody or antigen-binding fragment thereof described in this application.

[0014] According to one embodiment of this application, an antibody conjugate is also provided, the antibody conjugate comprising the antibody or antigen-binding fragment thereof described in this application or the bispecific antibody or antigen-binding fragment thereof described in this application, and a payload operatively linked to the antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof.

[0015] According to an embodiment of the present application, there is also provided a pharmaceutical combination comprising: an antibody or antigen-binding fragment thereof as described herein, or a bispecific antibody or antigen-binding fragment thereof as described herein; and a PD-1 antagonist.

[0016] According to an embodiment of the present application, there is also provided a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody or antigen-binding fragment thereof as described herein, a chimeric antigen receptor as described herein, an antibody conjugate as described herein, and / or a pharmaceutical combination as described herein; and a pharmaceutically or physiologically acceptable carrier.

[0017] According to an embodiment of the present application, there is also provided a kit comprising: an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody or antigen- binding fragment thereof as described herein, a polynucleotide as described herein, a recombinant vector as described herein, a host cell as described herein, a chimeric antigen receptor as described herein, an antibody conjugate as disclosed herein, a pharmaceutical combination as described herein, and / or a pharmaceutical composition as described herein.

[0018] According to an embodiment of the present application, there is also provided use of an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody or antigen-binding fragment as described herein, a polynucleotide as described herein, a recombinant vector as disclosed herein, a host cell as described herein, a chimeric antigen receptor as described herein, a pharmaceutical combination as described herein, a pharmaceutical composition as described herein, and / or a kit as described herein, in the preparation of a medicament or agent for detecting, preventing, alleviating or treating a disease.

[0019] According to an embodiment of the present application, there is also provided a method of detecting, preventing, alleviating or treating a disease, characterized in that the method comprises administering to a subject in need thereof an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody or antigen-binding fragment described herein, a polynucleotide as described herein, a recombinant vector as described

[0020] It is to be understood that the details set forth in this section are not intended to limit or otherwise define the key or critical features of the embodiments of the present application. Other features of the present application will become readily apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0021] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments and together with the description serve to explain exemplary implementations of the application. The illustrated embodiments are exemplary only and not limiting of the scope of the appended claims. In all the drawings: like reference numerals refer to like parts throughout the various figures.

[0022] FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D are ELISA binding activity graphs of 13F3H6-hz heavy chain alanine mutations and core sites in Example 3.

[0023] FIG. 2 is a schematic diagram of anti-CCR8 / CTLA-4 bispecific antibodies with “knobs-into-holes” heterodimeric structure prepared in Example 5.

[0024] FIG. 3 is a SEC-HPLC result graph of SGT003 heterodimer prepared in Example 5.

[0025] FIG. 4 is a result graph of binding activity of anti-CCR8 / CTLA-4 bispecific antibodies to hCTLA-4 antigen determined by ELISA in Example 6.

[0026] FIG. 5 is a result graph of binding activity of anti-CCR8 / CTLA-4 bispecific antibody to HEK293T cells expressing hCCR8 determined by flow cytometry in Example 6.

[0027] FIG. 6 is a result graph of binding activity of anti-CCR8 / CTLA-4 bispecific antibody to HEK293T cells expressing hCCR8 and hCTLA-4 determined by flow cytometry in Example 6.

[0028] FIG. 7A, 7B are result graphs of stability detection of anti-CCR8 / CTLA-4 bispecific antibodies in human serum, PBS determined by ELISA in Example 6.

[0029] FIG. 8A is a result graph of affinity detection of anti-CCR8 / CTLA-4 bispecific antibodies to hCTLA-4 determined by Biacore in Example 6.

[0030] FIG. 8B is a result graph of affinity detection of control antibody YERVOY to hCTLA-4 determined by Biacore in Example 6.

[0031] FIG. 9 is a result graph of binding activity detection of anti-CCR8 / CTLA-4 bispecific antibodies to hCCR8, hCTLA-4, and other irrelevant antigens determined by ELISA in Example 6.

[0032] Figures 10A and 10B are graphs showing results of flow cytometry assays to determine the ability of anti-CCR8 / CTLA-4 bispecific antibodies to block binding of CTLA-4 ligands B7-1 and B7-2 to cell surface hCTLA-4 in Example 7.

[0033] Figure 11 is a graph showing results of flow cytometry assays to determine the ability of anti-CCR8 / CTLA-4 bispecific antibodies to block binding of human CCL1 to cell surface human CCR8 in Example 7.

[0034] Figure 12 is a graph showing results of assays to determine the ability of anti-CCR8 / CTLA-4 bispecific antibodies to inhibit CCL1 -induced cell chemotaxis in Example 7.

[0035] Figure 13A is a graph showing results of assays to determine the ability of anti-CCR8 / CTLA-4 bispecific antibodies to co-stimulate IL-2 release from human PBMC cells with Staphylococcus aureus enterotoxin B (SEB) in Example 8.

[0036] Figure 13B is a graph showing results of assays to determine the ability of anti-CCR8 / CTLA -4 bispecific antibodies to co-stimulate TNF-a release from human PBMC cells with SEB in Example 8.

[0037] Figure 13C is a graph showing results of assays to determine the ability of anti-CCR8 / CTLA4 bispecific antibodies to co-stimulate IL-6 release from human PBMC cells with SEB in Example 8.

[0038] Figure 13D is a graph showing results of assays to determine the ability of anti-CCR8 / CTLA 4 bispecific antibodies to co-stimulate IL-23 release from human PBMC cells with SEB in Example 8.

[0039] Figure 14 is a graph showing statistical results of assays to determine the ability of anti-CCR8 / CTLA-4 bispecific antibody to induce Treg cell depletion in Example 9.

[0040] Figure 15 is a graph showing time-concentration curves of anti-CCR8 / CTLA-4 bispecific antibodies in mice in Example 10.

[0041] Figure 16 is a graph showing changes in tumor volume in a mouse MCA205 xenograft model in Example 11.

[0042] Figure 17 is a graph showing changes in tumor volume in a mouse MC38 xenograft model in Example 11.

[0043] Figure 18 is a graph showing changes in tumor volume in a mouse MB49 xenograft model in Example 11.

[0044] Figure 19 is a graph showing changes in tumor volume in a mouse EO771 xenograft model in Example 11. DETAILED DESCRIPTION

[0045] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, percent, relative quantity, price, cost, time, temperature, thickness, and so forth, used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. Each numerical parameter should be considered in light of the number of significant figures used in that respective number and by applying ordinary rounding techniques, or as understood by one of ordinary skill in the art to be at the parity of the value of the numerated value. Numerical parameters should be considered in the context of the other numerical parameters in the specification and the desired properties set forth in the specification, as would be understood by one of ordinary skill in the art.

[0046] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range falling within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0047] Unless otherwise indicated, the terms or expressions used herein are to be understood in the context of the entire document and as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0048] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meaning of similar expressions can be extrapolated in a like manner.

[0049] As used herein, the expressions "comprising", "including", "containing", mean that additional elements are not excluded.

[0050] As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.

[0051] As used herein, the terms "polypeptide" and "peptide" are used interchangeably to refer to a polymer of amino acids of any length. Thus, polypeptides, oligopeptides, proteins, antibodies, and enzymes are included within the definition of polypeptides.

[0052] As used herein, "identity" refers to the degree of similarity between a pair of sequences (nucleotide or amino acid). Identity is determined by dividing the number of identical residues by the total number of residues and multiplying the quotient by 100 to obtain a percentage. Gaps are not counted when evaluating identity. Thus, two copies of a perfectly identical sequence have 100% identity, but a sequence with deletions, additions, or substitutions can have a lower degree of identity. Those skilled in the art know that there are computer programs that can be used to determine the identity of sequences, such as those that employ algorithms such as BLAST. BLAST nucleotide searches are performed using the NBLAST program, and BLAST protein searches are performed using the BLASTP program, with the default parameters for each program.

[0053] As used herein, the expression "D130K" means that the amino acid at position 130 is substituted from D (aspartic acid) to K (lysine), and the expression "A50S" means that the amino acid at position 50 is substituted from A (alanine) to S (serine). In some embodiments, the positions are numbered with reference to the amino acid sequence of the variable region set forth in SEQ ID NO: 17 or SEQ ID NO: 19. Similar expressions can be interpreted accordingly.

[0054] As used herein, "antibody" refers to a specific immunoglobulin directed against an antigenic site. Antibodies can be produced according to methods well known in the art. Forms of antibodies include polyclonal or monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies (e.g., bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, fully human antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule that comprises an antigen-binding site so long as the antibody exhibits the desired biological binding activity.

[0055] As used herein, "antigen-binding fragment" refers to one or more portions of an antibody that retain binding specificity for an antigen of interest. Antigen-binding fragments include, but are not limited to, VHHs, CDR-containing fragments, and the like.

[0056] "Specifically binds" as described herein refers to a non-covalent interaction between an antibody or antigen-binding fragment thereof and an antigen. The strength or affinity of the interaction can be expressed in terms of the equilibrium dissociation constant (KD or Kd) of the antigen and the corresponding antibody: the smaller the KD value, the stronger the binding between the epitope and the antibody. The equilibrium dissociation constant (KD) is calculated as the ratio of Koff / kon, where the "on rate constant (Kon)" and the "off rate constant (Koff)" can be determined by calculating the concentration and the actual association and dissociation rates (see Nature 361 : 186-87 (1993)).

[0057] The precise amino acid sequence boundaries of a given complementarity determining region (CDR) or framework region (FR) can be readily determined using any of a number of numbering schemes well known in the art, including: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.” Dev Comp Immunol. 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol. 2001 Jun 8;309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0058] The boundaries of a given CDR or FR can differ depending on the scheme used to define the CDRs. For example, the Kabat scheme is based on sequence alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes are based on the most common antibody region sequence lengths, with insertions provided by the insertion letter (e.g., "30a") and deletions occurring in some antibodies. Both schemes place certain insertions and deletions (indels) in different positions, resulting in different numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on the scheme used by Oxford Molecular's AbM antibody modeling software.

[0059] Thus, unless otherwise specified, it should be understood that "CDRs" of a given antibody or region thereof, such as a variable region thereof, encompass CDRs as defined by any of the above schemes or other known schemes. For example, where a particular CDR (e.g., CDR3) is specified to contain a given amino acid sequence, it is understood that such CDR can also have the sequence of the corresponding CDR (e.g., CDR3) as defined by any of the above schemes or other known schemes. Likewise, unless otherwise specified, it should be understood that FRs of a given antibody or region thereof, such as a variable region thereof, encompass FRs as defined by any of the above schemes or other known schemes. Unless specifically indicated otherwise, the numbering scheme used to define the boundaries of CDRs and FRs herein employs the IMGT scheme.

[0060] As used herein, "exogenous" or "heterologous" are used interchangeably to refer to a source that is different from the inherent (original) organism, e.g., an organism from another species. As used herein, "heterologous gene" or "exogenous gene" refers to a gene that does not naturally occur in the host organism, which is introduced into the host organism by gene transfer.

[0061] As used herein, "vector" refers to a self-replicating DNA molecule that transfers foreign genes of interest into a host organism, and is often in the form of a circular double-stranded DNA molecule. Typical vectors include plasmids, viruses, phages, cosmids, and minichromosomes. Among them, plasmids are the most common vector form, which refers to a circular double-stranded DNA that can accept foreign nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0062] As used herein, "expression vector" and "recombinant vector" are used interchangeably to refer to a vector containing a foreign gene, which also contains regulatory elements for expression in a given host organism. The expression vector, when introduced into an appropriate host organism, can enable the expression of the inserted gene of interest.

[0063] "Transformation" as used herein refers to the transfer of an exogenous gene to a host organism, such as a host cell, resulting in genetically stable inheritance. The transformed gene can be maintained in the host organism in the form of a plasmid or can be integrated into the host organism genome. A host organism containing a transformed gene is referred to as a "transgenic" or "recombinant" or "transformed" organism or an "engineered" organism. Transformation of a host organism with an expression vector can be performed using routine techniques well known to those skilled in the art. When the host is a prokaryote, competent cells that are capable of DNA uptake can be made competent by harvesting during the exponential phase, treating with CaCl2, and using steps well known in the art. If desired, microinjection, electroporation, or liposome packaging can also be used. These are well known techniques in the art and will not be described in detail herein.

[0064] "Chimeric antigen receptor" or "CAR" as used herein refers to a set of polypeptides, in some non-limiting embodiments two polypeptides; when in an immune effector cell, it provides the cell with specificity for a target cell, e.g., for a cancer cell, and provides intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen binding domain comprises a bispecific antibody provided herein.

[0065] "Alleviate" and "treat" and synonyms thereof as used herein refer to an improvement in a disease, disorder, and / or condition. "Alleviate" and "treat" can be an improvement in at least one measurable physical parameter including, but not necessarily limited to, a parameter that can be discerned by a patient. "Alleviate" and "treat" can also mean to suppress the development of a disease, disorder, and / or condition, either physically (e.g., stabilize identifiable symptoms), physiologically (e.g., stabilize a physical parameter), or both. "Alleviate" and "treat" can also mean to slow down or reverse the development of a disease, disorder, and / or condition.

[0066] "Prevent" and synonyms thereof as used herein refer to delaying the onset or reducing the risk of acquiring a particular disease, disorder, and / or condition or symptoms associated with these diseases, disorders, and / or conditions.

[0067] In order to make the above objects, features and advantages of the present application more clear, a detailed description of the preferred embodiments of the present application is given below.

[0068] Anti-CCR8 antibodies or antigen-binding fragments thereof

[0069] According to one embodiment of this application, an antibody or antigen-binding fragment thereof that binds to CCR8 is provided, characterized in that the amino acid sequence of its complementarity-determining region (CDR) as defined by the IMGT system is as follows: the amino acid sequence of heavy chain CDR1 is X1X2X3X4X5X6X7X8; the amino acid sequence of heavy chain CDR2 is X9X... 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 The amino acid sequence of the heavy chain CDR3 is X. 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 The amino acid sequence of the light chain CDR1 is X. 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 The amino acid sequence of the light chain CDR2 is X. 43 X 44 X 45 The amino acid sequence of the light chain CDR3 is X. 46 X 47 X 48 X 49 X 50 X 51 X 52 X 53 X 54 Where: X1 is G or A; X2 is F or A; X3 is T or A; X4 is F or A; X5 is N or A; X6 is G, S or A; X7 is Y or A; X8 is G, S, V, L, I, T, Y, D, K or A; X9 is I or A; X 10 For H, K, G, D, L, S, W, A, or R; X 11 S, Q, Y, N, G, L, M, W, A, or T; X 12 For K or A; X 13is S or A; X 14 is N or A; X 15 is N or A; X 16 is Y or A; X 17 is G, S or A; X 18 is T or A; X 19 is V or A; X 20 is R or A; X 21 is H or A; X 22 is A, E, Y, P, Q, T, I, V, N, M, F or K; X 23 is R, H, G, L, S, W, D, A, V, E, T, Q, N, M, I, P, F, Y or K; X 24 is V or A; X 25 is A, K, H, G, L, S, W, D or R; X 26 is E or A; X 27 is A, V, L, I, M, W, S, D, K, E, P, Q, H, T, R, N, F, Y or G; X 28 is A, N, Q, M, W, S, D, K, L, G, F, R, H, I, T, E, V, P or Y; X 29 is G, S or A; X 30 is M or A; X 31 is A, G, L, M, W, S, K, E, R, P, Q, H, N, T, Y, V, F, I or D; X 32 is Y or A; X 33 is K or A; X 34 is S or A; X 35 is V or A; X 36 is S or A; X 37 is T or A; X 38 is S or A; X 39 is G or A; X 40 is Y or A; X 41 is T or A; X 42 is Y or A; X 43 is L or A; X 44 is G, S or A; X 45 is S or A; X 46 is Q or A; X 47 is H or A; X 48 is S or A; X 49 is A, K, H, G, L, M, W, S, D, E, V, I, Q, F, P, T, N, Y or R; X 50 is E or A; X 51 is A, G, V, I, M, W, S, D, K, T, P, Q, N, H, E, Y, F, R or L; X 52F, G, L, S, D, K, Q, W, A, or P; X 53 F, G, L, S, D, K, Q, W, A, or P; X 54 T or A.

[0070] In some embodiments, the amino acid sequence of the complementarity determining region thereof is selected from at least one of the following groups:

[0071] (1) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 29, LAS, and SEQ ID NO: 16, respectively;

[0072] (2) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 30, LAS, and SEQ ID NO: 16, respectively;

[0073] (3) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 31, LAS, and SEQ ID NO: 16, respectively;

[0074] (4) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 32, LAS, and SEQ ID NO: 16, respectively;

[0075] (5) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 33, LAS, and SEQ ID NO: 16, respectively;

[0076] (6) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 34, LAS, SEQ ID NO: 16, respectively;

[0077] (7) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 35, LAS, SEQ ID NO: 16, respectively;

[0078] (8) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 36, LAS, SEQ ID NO: 16, respectively;

[0079] (9) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,

[0080] (10) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2

[0081] (11) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, AAS, SEQ ID NO: 16, respectively;

[0082] (12) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LGS, SEQ ID NO: 16, respectively;

[0083] (13) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LSS, SEQ ID NO: 16, respectively;

[0084] (14) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAA, SEQ ID NO: 16, respectively;

[0085] (15) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR 2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO : 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 43, respectively;

[0086] (16) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID N0: 44, respectively;

[0087] (17) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ I D NO: 45, respectively;

[0088] (18) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 46, respectively;

[0089] (19) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 47, respectively;

[0090] (20) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 48, respectively;

[0091] (21) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 49, respectively;

[0092] (22) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 50, respectively;

[0093] (23) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR 2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO : 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS,SEQ ID NO: 51, respectively;

[0094] (24) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 52, respectively;

[0095] (25) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 53, respectively;

[0096] (26) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 54, respectively;

[0097] (27) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 55, respectively;

[0098] (28) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 56, respectively;

[0099] (29) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO : 14, LAS, SEQ ID NO: 57, respectively;

[0100] (30) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 58, respectively;

[0101] (31) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 59, respectively;

[0102] (32) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 60, respectively;

[0103] (33) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 61, respectively;

[0104] (34) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 62, respectively;

[0105] (35) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 63, respectively;

[0106] (36) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 64, respectively;

[0107] (37) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 65, respectively;

[0108] (38) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 66, respectively;

[0109] (39) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 67, respectively;

[0110] (40) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 68, respectively;

[0111] (41) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 69, respectively;

[0112] (42) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 70, respectively;

[0113] (43) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 71, respectively;

[0114] (44) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CD 1, the light chain CDR2, the light chain CDR3 are SEQ ID NO:

[0115] (45) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,

[0116] (46) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2

[0117] (47) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 13, SEQ ID NO: 14, LAS, SEQ ID NO: 75, respectively;

[0118] (48) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 76, respectively;

[0119] (49) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 77, respectively;

[0120] (50) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 78, respectively;

[0121] (51) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 79, respectively;

[0122] (52) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 80, respectively;

[0123] (53) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID N0: 14, LAS, SEQ ID NO: 81, respectively;

[0124] (54) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 82, respectively;

[0125] (55) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 83, respectively;

[0126] (56) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 84, respectively;

[0127] (57) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 85, respectively;

[0128] (58) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 86, respectively;

[0129] (59) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 87, respectively;

[0130] (60) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 88, respectively;

[0131] (61) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 89, respectively;

[0132] (62) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 90, respectively;

[0133] (63) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 91, respectively;

[0134] (64) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 92, respectively;

[0135] (65) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 93, respectively;

[0136] (66) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 94, respectively;

[0137] (67) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 95, respectively;

[0138] (68) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 96, respectively;

[0139] (69) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 97, respectively;

[0140] (70) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 98, respectively;

[0141] (71) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDF 1, the light chain CDR2, the light chain CDR3 are SEQ ID NO : 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID N0: 14, LAS, SEQ ID NO: 99, respectively;

[0142] (72) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 100, respectively;

[0143] (73) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 101, respectively;

[0144] (74) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 102, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0145] (75) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 103, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0146] (76) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 104, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0147] (77) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 105, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0148] (78) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 106, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0149] (79) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 107, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0150] (80) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 108, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0151] (81) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 109, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0152] (82) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 110, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0153] (83) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 111, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0154] (84) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 112, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0155] (85) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 113, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0156] (86) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 114, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0157] (87) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 115, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0158] (88) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 116, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0159] (89) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 117, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0160] (90) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 118, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0161] (91) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 119, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0162] (92) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 120, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0163] (93) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 121, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0164] (94) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,

[0165] (95) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2

[0166] (96) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 124, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0167] (97) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 125, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0168] (98) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 126, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0169] (99) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 127, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0170] (100) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 128, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0171] (101) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 129, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0172] (102) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 130, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0173] (103) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 131, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0174] (104) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 132, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0175] (105) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 133, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0176] (106) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,

[0177] (107) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2

[0178] (108) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 136, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0179] (109) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 137, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0180] (110) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 138, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0181] (111) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 139, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0182] (112) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 140, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0183] (113) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 141, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0184] (114) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 142, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0185] (115) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 143, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0186] (116) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 144, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0187] (117) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 145, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0188] (118) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 146, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0189] (119) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 147, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0190] (120) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 148, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0191] (121) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 149, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0192] (122) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 150, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0193] (123) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 151, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0194] (124) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 152, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0195] (125) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 153, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0196] (126) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 154, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0197] (127) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 155, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0198] (128) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 156, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0199] (129) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 157, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0200] (130) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 158, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0201] (131) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12,

[0202] (132) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 160, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0203] (133) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 161, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0204] (134) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 162, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0205] (135) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 163, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0206] (136) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 164, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0207] (137) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 165, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0208] (138) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 166, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0209] (139) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 167, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0210] (140) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 168, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0211] (141) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 169, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0212] (142) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 170, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0213] (143) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 171, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0214] (144) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 172, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0215] (145) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 173, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0216] (146) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 174, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0217] (147) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 175, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0218] (148) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 176, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0219] (149) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 177, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0220] (150) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:178, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0221] (151) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:179, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0222] (152) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:180, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0223] (153) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:181, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0224] (154) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:182, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0225] (155) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:183, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0226] (156) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 184, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0227] (157) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 185, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0228] (158) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 186, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0229] (159) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 187, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0230] (160) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 188, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0231] (161) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 189, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0232] (162) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 190, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0233] (163) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 191, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0234] (164) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 192, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0235] (165) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 193, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0236] (166) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 194, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0237] (167) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 195, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0238] (168) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 196, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0239] (169) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 197, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0240] (170) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 198, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0241] (171) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 199, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0242] (172) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 200, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0243] (173) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 201, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0244] (174) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:202, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0245] (175) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:203, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0246] (176) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:204, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0247] (177) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:205, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0248] (178) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:206, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0249] (179) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:207, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0250] (180) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:208, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0251] (181) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:209, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0252] (182) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:210, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0253] (183) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:211, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0254] (184) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:212, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0255] (185) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:213, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0256] (186) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:214, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0257] (187) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:215, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0258] (188) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:216, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0259] (189) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:217, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0260] (190) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:218, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0261] (191) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:219, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0262] (192) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:220, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0263] (193) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:221, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0264] (194) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:222, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0265] (195) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:223, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0266] (196) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:224, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0267] (197) The amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:225, SEQ ID NO:14, LAS, and SEQ ID NO:16, respectively.

[0268] (198) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 226, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0269] (199) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 227, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0270] (200) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 228, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0271] (201) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 229, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0272] (202) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 230, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0273] (203) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 231, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0274] (204) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 232, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0275] (205) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 233, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0276] (206) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 234, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0277] (207) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 235, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0278] (208) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 236, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0279] (209) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 237, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0280] (210) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 238, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0281] (211) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 239, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0282] (212) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 240, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0283] (213) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 241, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0284] (214) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 242, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0285] (215) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 243, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0286] (216)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 244, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively.

[0287] In some preferred embodiments, the amino acid sequences of the complementarity determining regions (CDRs) defined according to the IMGT system are as follows: the amino acid sequence of the heavy chain CDR1 is GX2X3FNX6YA, wherein X2 is F or A, X3 is T or A, X6 is S or A; the amino acid sequence of the heavy chain CDR2 is SEQ ID NO: 12; the amino acid sequence of the heavy chain CDR3 is VRHKKVREGYAMX 31 Y, X 31 is R, P, Q, H, N, T, V or I; the amino acid sequence of the light chain CDR1 is SEQ ID NO: 14; the amino acid sequence of the light chain CDR2 is LAS; and the amino acid sequence of the light chain CDR3 is SEQ ID NO: 16.

[0288] In some preferred embodiments, the amino acid sequences of the complementarity determining regions are selected from at least one of the following groups:

[0289] (1) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 103, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0290] (2) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 104, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0291] (3) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 108, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0292] (4) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 234, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0293] (5) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 235, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0294] (6) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 236, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0295] (7) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 237, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0296] (8) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 238, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0297] (9) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 239, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0298] (10) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 241, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively;

[0299] (11) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 243, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively.

[0300] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 17; and

[0301] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 19.

[0302] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 17, wherein the mutated site comprises G45A, F46A, T47A, F48A, N49A, A50G, A50S, Y51A, A52G, A52S, A52V, A52L, A52I, A52T, A52Y, A52D, A52K, I70A, R71H, R71K, R71G, R71D, R71L, R71S, R71W, R71A, T72S, T72Q, T72Y, T72N, T72G, T72L, T72M, T72W, T72A, K73A, S74A, N75A, N76A, Y77A, A78G, A78S, T79A, V118A, R119A, H120A, K121A, K121E, K121Y, K121P, K121Q, K121T, K121I, K121V, K121N, K121M, K121F, K122R, K122H, K122G, K122L, K122S, K122W, K122D, K122A, K122V, K122E, K122T, K122Q, K122N, K122M, K122I, K122P, K122F, K122Y, V123A, R124A, R124K, R124H, R124G, R124L, R124S, R124W, R124D, E125A, G126A, G126V, G126L, G126I, G126M, G126W, G126S, G126D, G126K, G126E, G126P, G126Q, G126H, G126T, G126R, G126N, G126F, G126Y, Y127A, Y127N, Y127Q, Y127M, Y127W, Y127S, Y127D, Y127K, Y127L, Y127G, Y127F, Y127R, Y127H, Y127I, Y127T, Y127E, Y127V, Y127P, A128G, A128S, M129A, D130A, D130G, D130L, D130M, D130W, D130S, D130K, D130E, D130R, D130P, D130Q, D130H, D130N, D130T, D130Y, D130V, D130F, D130I, or Y131A.

[0303] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 17, wherein the mutated site comprises F46A, T47A, A50S, D130R, D130P, D130Q, D130H, D130N, D130T, D130V, or D130I.

[0304] In some preferred embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 17, wherein the mutated site comprises F46A, T47A, A50S, D130R, D130P, D130Q, D130H, D130N, D130T, D130V, or D130I.

[0305] In some preferred embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 17, wherein the mutated site comprises F46A, T47A, A50S, D130R, D130P, D130Q, D130H, D130N, D130T, D130V, or D130I.

[0306] Bispecific antibody or antigen-binding fragment thereof

[0307] According to an embodiment of the present application, the present application provides a bispecific antibody or an antigen-binding fragment thereof, characterized in that it comprises a first antigen-binding domain specifically binding to CTLA-4 and a second antigen-binding domain specifically binding to CCR8, the first antigen-binding domain comprising complementarity determining regions (CDRs) defined according to the IMGT system, the amino acid sequences of which are as follows: the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, GAF, SEQ ID NO: 6, respectively; and the second antigen-binding domain is selected from at least one of the following groups: (1) the second antigen-binding domain comprises complementarity determining regions (CDRs) defined according to the IMGT system, the amino acid sequences of which are as follows: the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; or (2) the second antigen-binding domain comprises an antibody or an antigen-binding fragment thereof described in the present application.

[0308] In some embodiments, the first antigen-binding domain comprises a first heavy chain variable region and a first light chain variable region. In some embodiments, the first heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 7, or an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the first light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 9, or an amino acid sequence that is at least 80%, at least 81%, at last 82%, at least 83%, at least 84%, at least 85%, at last 86%, at least 87%, at least 88%, at least 89%, at last 90%, at least 91%, at least 92%, at least 93%, at last 94%, at least 95%, at least 96%, at least 97%, at last 98%, or at least 99% identical to the amino acid sequence as set forth in

[0309] In some embodiments, the second antigen binding domain comprises a second heavy chain variable region and a second light chain variable region. In some embodiments, the second heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the second light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence as set forth in SEQ ID NO: 19.

[0310] In some embodiments, the second heavy chain variable region comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 17, wherein the mutated site comprises G45A, F46A, T47A, F48A, N49A, A50G, A50S, Y51A, A52G, A52S, A52V, A52L, A52I, A52T, A52Y, A52D, A52K, I70A, R71H, R71K, R71G, R71D, R71L, R71S, R71W, R71A, T72S, T72Q, T72Y, T72N, T72G, T72L, T72M, T72W, T72A, K73A, S74A, N75A, N76A, Y77A, A78G, A78S, T79A, V118A, R119A, H120A, K121A, K121E, K121Y, K121P, K121Q, K121T, K121I, K121V, K121N, K121M, K121F, K122R, K122H, K122G, K122L, K122S, K122W, K122D, K122A, K122V, K122E, K122T, K122Q, K122N, K122M, K122I, K122P, K122F, K122Y, V123A, R124A, R124K, R124H, R124G, R124L, R124S, R124W, R124D, E125A, G126A, G126V, G126L, G126I, G126M, G126W, G126S, G126D, G126K, G126E, G126P, G126Q, G126H, G126T, G126R, G126N, G126F, G126Y, Y127A, Y127N, Y127Q, Y127M, Y127W, Y127S, Y127D, Y127K, Y127L, Y127G, Y127F, Y127R, Y127H, Y127I, Y127T, Y127E, Y127V, Y127P, A128G, A128S, M129A, D130A, D130G, D130L, D130M, D130W, D130S, D130K, D130E, D130R, D130P, D130Q, D130H, D130N, D130T, D130Y, D130V, D130F, D130I, or Y131A.

[0311] In some preferred embodiments, the second heavy chain variable region comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 17, wherein the mutated site comprises F46A, T47A, A50S, D130R, D130P, D130Q, D130H, D130N, D130T, D130V, or D130I.

[0312] In some embodiments, the second light chain variable region comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 19, wherein the mutated site comprises K27A, S28A, V29A, S30A, T31A, S32A, G33A, Y34A, T35A, Y36A, L54A, A55G, A55S, S56A, Q94A, H95A, S96A, R97A, R97K, R97H, R97G, R97L, R97M, R97W, R97S, R97D, R97E, R97V, R97I, R97Q, R97F, R97P, R97T, R97N, R97Y, E98A, L99A, L99G, L99V, L99I, L99M, L99W, L99S, L99D, L99K, L99T, L99P, L99Q, L99N, L99H, L99E, L99Y, L99F, L99R, P100F, P100G, P100L, P100S, P100D, P100K, P100Q, P100W, P100A, F101A, F101P, F101G, F101L, F101S, F101D, F101K, F101Q, F101W, or T102A.

[0313] In some preferred embodiments, the first antigen binding domain comprises a first heavy chain variable region set forth in SEQ ID NO: 7 and a first light chain variable region set forth in SEQ ID NO: 9.

[0314] In some preferred embodiments, the second antigen binding domain comprises a second heavy chain variable region set forth in SEQ ID NO: 17 and a second light chain variable region set forth in SEQ ID NO: 19.

[0315] In some embodiments, the bispecific antibody or antigen binding fragment thereof further comprises a constant region. Suitable constant region sequences are known in the art, and any suitable constant region sequence can be selected by one of skill in the art for use in the bispecific antibodies provided herein.

[0316] In some embodiments, the bispecific antibody or antigen binding fragment thereof further comprises a first constant region and a second constant region. In some preferred embodiments, the first and second constant regions are human IgG constant regions. In some more preferred embodiments, the human IgG constant regions are human IgG Fc regions.

[0317] In some more preferred embodiments, the first constant region comprises a first heavy chain constant region comprising a T366S, L368A, and Y407V mutation according to EU numbering. In some more preferred embodiments, the second heavy chain constant region comprises a second heavy chain constant region comprising a T366W mutation according to EU numbering. In some more preferred embodiments, the first heavy chain constant region is operably linked to the second heavy chain constant region.

[0318] In some more preferred embodiments, the first heavy chain constant region comprises a Y349C mutation according to EU numbering. In some more preferred embodiments, the second heavy chain constant region comprises a S354C mutation according to EU numbering. In some more preferred embodiments, the first heavy chain constant region is operably linked to the second heavy chain constant region by a disulfide bond.

[0319] In some more preferred embodiments, the sequence of the first heavy chain constant region is set forth in SEQ ID NO: 21. In some more preferred embodiments, the nucleic acid sequence encoding the first heavy chain constant region is set forth in SEQ ID NO: 22. In some more preferred embodiments, the sequence of the first light chain constant region is set forth in SEQ ID NO: 23. In some more preferred embodiments, the nucleic acid sequence encoding the first light chain constant region is set forth in SEQ ID NO: 24.

[0320] In some more preferred embodiments, the sequence of the second heavy chain constant region is set forth in SEQ ID NO: 25. In some more preferred embodiments, the nucleic acid sequence encoding the second heavy chain constant region is set forth in SEQ ID NO: 26. In some more preferred embodiments, the sequence of the second light chain constant region is set forth in SEQ ID NO: 27. In some more preferred embodiments, the nucleic acid sequence encoding the second light chain constant region is set forth in SEQ ID NO: 28.

[0321] Polynucleotides, recombinant vectors, host cells

[0322] According to an embodiment of the present application, there is also provided a polynucleotide encoding the antibody or antigen-binding fragment thereof described herein and / or the bispecific antibody or antigen-binding fragment thereof described herein. Due to the degeneracy of the code, one of skill in the art can appreciate that there are a number of nucleic acid sequences that can encode the bispecific antibody or antigen-binding fragment thereof provided herein, which are not limited herein. One of skill in the art can make appropriate codon optimization and select an appropriate nucleic acid sequence for expressing the bispecific antibody or antigen-binding fragment thereof provided herein, depending on the expression use or host.

[0323] According to an embodiment of the present application, there is also provided a recombinant vector comprising the polynucleotide described herein. Recombinant vectors suitable for expressing the bispecific antibody or antigen-binding fragment thereof described herein are well known in the art and are not limited herein.

[0324] In some embodiments, the recombinant vector comprises a cloning vector, an expression vector. Both viral and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically having an expression cassette for expressing a protein or RNA), and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet. 15:345, 1997, the contents of which are hereby incorporated by reference herein). Viral-derived vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors, herpesviral vectors.

[0325] In some embodiments, the recombinant vector further comprises a promoter. The promoter can be any promoter sequence suitable, i.e., a nucleic acid sequence that is recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the antibody. The promoter can be any nucleic acid sequence that serves as a transcriptional control element for a gene in the chosen host cell, including mutated, truncated, and hybrid promoters, and can be derived from a gene that encodes a protein or polypeptide that is homologous or heterologous to the host cell, either extracellularly or intracellularly.

[0326] The recombinant vector of the present application can be constructed using methods well known in the art. For example, appropriate restriction sites can be added to the ends of the polynucleotide of the present application according to the restriction sites contained in the backbone vector used, and then the polynucleotide can be inserted into the backbone vector.

[0327] According to an embodiment of the present application, there is also provided a host cell comprising the polynucleotide of the present application and / or the recombinant vector of the present application. The host cell can be selected according to the type of the expression vector. The polynucleotide and / or the expression vector can be delivered into the host cell using any suitable means known in the art, without being limited thereto.

[0328] In some embodiments, the bispecific antibodies or antigen-binding fragments thereof of the present application are expressed and produced using mammalian host cells. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines containing exogenous expression vectors. These include any normal non-immortal or normal or abnormal immortal animal or human cell. For example, a number of suitable host cell lines have been developed that are capable of secreting intact immunoglobulins, including CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells, and hybridomas. Exemplary host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells (e.g., HEK293, HEK293T, HEK293F), monkey kidney (COS) cells (e.g., COS-1, COS-7), baby hamster kidney (BHK) cells (e.g., BHK-21), African green monkey kidney cells (e.g., BSC-1), HeLa cells, human hepatocellular carcinoma cells (e.g., Hep G2), myeloma cells (e.g., NS0, 653, SP2 / 0), lymphoma cells, oocytes, and cells from transgenic animals (e.g., mammary epithelial cells) or any derivative, immortalized, or transformed cells thereof.

[0329] Chimeric antigen receptors, antibody conjugates

[0330] According to an embodiment of the present application, there is also provided a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof of the present application or the bispecific antibody or antigen-binding fragment thereof of the present application. In some embodiments, the chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0331] In some embodiments, the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment thereof and / or the bispecific antibody or antigen-binding fragment thereof provided herein. The antibody or antigen-binding fragment thereof, the bispecific antibody or antigen-binding fragment thereof provided herein can be conjugated to other moieties to construct a chimeric antigen receptor using methods known in the art.

[0332] According to an embodiment of the present application, there is also provided an antibody conjugate comprising an antibody or antigen-binding fragment thereof described herein or a bispecific antibody or antigen-binding fragment thereof described herein, and a payload operably linked to the antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof. In some embodiments, the payload comprises a cytotoxin, an immunomodulator, a cell reprogramming drug, or a radioisotope. Suitable radioisotopes, immunomodulators, cell reprogramming drugs, and cytotoxins, among others, are known in the art, and a skilled person can select a suitable type as needed and couple the bispecific antibody provided herein to a cytotoxin or radioisotope using methods known in the art.

[0333] In some non-limiting embodiments, the antibody conjugate comprises a bispecific antibody provided herein and a cytotoxin to form an antibody-drug conjugate (ADC). In some embodiments, examples of cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine, lomustine, 1-methyl- l-nitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin, cis-dichlorodiamine platinum, cisplatin, carboplatin, zorubicin, doxorubicin, detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, dactinomycin, bleomycin, calicheamicin, plicamycin, procarbazine, vinblastine, vincristine, paclitaxel, ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, teniposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane, interferons, and combinations thereof.

[0334] In some non-limiting embodiments, the antibody conjugate comprises a bispecific antibody provided herein, and a radioisotope to form a Radionuclide Drug Conjugates (RDC). Examples of radioisotopes useful in the present application include, but are not limited to, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, 99mTc, 123I, 18F, and 68Ga.

[0335] In addition to the above disclosed chimeric antigen receptors, antibody conjugates, bispecific antibodies or antigen-binding fragments thereof disclosed herein can also be conjugated to other factors either chemically or by genetic engineering. These factors provide the effect of targeting the antibody to a desired functional site or improve or provide other properties to the antibody, which will not be described here.

[0336] Pharmaceutical combination, pharmaceutical composition, kit

[0337] According to an embodiment of the present application, a pharmaceutical combination is also provided, comprising: an antibody or antigen-binding fragment thereof as described herein or a bispecific antibody or antigen-binding fragment thereof as described herein; and a PD-1 antagonist.

[0338] In some embodiments, the PD-1 antagonist comprises an antibody or antigen-binding fragment that binds to PD-1.

[0339] According to an embodiment of the present application, a pharmaceutical composition is also provided, comprising an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody or antigen-binding fragment thereof as described herein, a chimeric antigen receptor as described herein, an antibody conjugate as described herein, and / or a pharmaceutical combination as described herein.

[0340] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable carrier. The carrier can be any compatible physiologically acceptable nontoxic substance suitable for delivering the polypeptide, polynucleotide or recombinant vector provided herein into a mammal, e.g., a human.

[0341] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable excipient. In some embodiments, the excipient comprises at least one of a solubilizing agent, a disintegrant, a wetting agent, a stabilizer, a thickening agent, a diluent, a buffer, a flavoring agent.

[0342] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable agent. In some embodiments, the agent comprises at least one of a solubilizing agent, a disintegrant, a wet agent, a stabilizer, a thickening agent, a diluent, a buffer, a flavouring agent.

[0343] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable diluent. In some embodiments, the diluent comprises at least one of a solubilizing agent, a disintegrant, a wetter, a stabilizer, a thickening agent, a diluent, a buffer, a flavor agent.

[0344] In some non-limiting embodiments, the carrier and / or adjuvant for the pharmaceutical composition used in the present application can comprise, for example, a liquid, gel or solid carrier, an aqueous vehicle, a non-aqueous vehicle, an antimicrobial agent, an isotonic agent, a buffering agent, an antioxidant, a suspending agent, a dispersing agent, a chelating agent, a diluent, an adjuvant, an excipient or a non-toxic auxiliary substance, other components known in the art or various combinations thereof.

[0345] According to an embodiment of the present application, a kit is also provided, which comprises the antibody or antigen binding fragment thereof described in the present application, the bispecific antibody or antigen binding fragment thereof described in the present application, the polynucleotide described in the present application, the recombinant vector described in the present application, the host cell described in the present application, the chimeric antigen receptor described in the present application, the antibody conjugate described in the present application, the pharmaceutical combination described in the present application, and / or the pharmaceutical composition described in the present application.

[0346] Uses and methods

[0347] According to an embodiment of the present application, the use of the antibody or antigen binding fragment thereof described in the present application, the bispecific antibody or antigen binding fragment thereof described in the present application, the polynucleotide described in the present application, the recombinant vector described in the present application, the host cell described in the present application, the chimeric antigen receptor described in the present application, the antibody conjugate described in the present application, the pharmaceutical combination described in the present application, the pharmaceutical composition described in the present application, and / or the kit described in the present application in the preparation of a medicament or reagent for detecting, preventing, alleviating or treating a disease is also provided.

[0348] According to an embodiment of the present application, a method for detecting, preventing, alleviating or treating a disease is also provided, which is characterized in that the method comprises administering the antibody or antigen binding fragment thereof described in the present application, the bispecific antibody or antigen-binding fragment thereof described in the present application, the polynucleotide described in the present application, a recombinant vector described in the present application, a host cell described in the present application, a chimeric antigen receptor described in the present application, an antibody conjugate described in the present application, a pharmaceutical combination described in the present application, a pharmaceutical composition described in the present application, and / or a kit described in the present application to a subject in need thereof.

[0349] In some embodiments, the disease comprises a disease associated with CTLA-4 and / or CCR8. In some embodiments, the disease comprises a cancer or a tumor. In some embodiments, the disease comprises a cancer or a tumor expressing CTLA-4 and / or CCR8. In some preferred embodiments, the cancer or tumor comprises fibrosarcoma, colon cancer, bladder cancer, breast cancer.

[0350] In the uses described herein, the dosage of the bispecific antibody provided herein can depend on several factors, including the severity and responsiveness of the symptoms, the route of administration, the timing and the period of treatment (from a few days to several months to several years), and the time until improvement of the symptoms. The dosage regimen can be adjusted to provide a therapeutic response according to the specific situation of the patient by a person skilled in the art. For example, a single dose can be administered, several separate doses can be administered over a predetermined period of time, or the dose can be reduced or increased as indicated by the therapeutic situation. The size of the dose is determined by the specific therapeutic effect to be achieved. The dose value can also vary depending on the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the treating clinician.

[0351] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by the combination are all considered part of the disclosure of the present application.

[0352] Exemplary embodiments of the present application will be described below with reference to the accompanying drawings, which include various details of the embodiments of the present application to assist in understanding. It should be understood that these are considered only exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined only by the claims. Therefore, those of ordinary skill in the art should recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of the present application. Also, descriptions of well-known functions and structures are omitted in the following description for clarity and conciseness.

[0353] Embodiments

[0354] Unless otherwise specified, the techniques or conditions in the present embodiments are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are all conventional products that can be obtained by commercial purchase.

[0355] Example 1: Preparation of the antibody Ipilimumab against CTLA-4

[0356] The preparation method of the approved anti-CTLA-4 monoclonal antibody Ipilimumab (trade name YERVOY, Bristol-Myers Squibb, drug product identification code 8391005) is referred to the Chinese published patent CN1371416A, which is briefly described as follows:

[0357] The amino acid sequence of the Ipilimumab heavy chain variable region (Ipilimumab-VH) is shown in SEQ ID NO:7, and its encoding nucleic acid sequence is shown in SEQ ID NO:8; the amino acid sequence of the Ipilimumab light chain variable region (Ipilimumab-Vκ) is shown in SEQ ID NO:9, and its encoding nucleic acid sequence is shown in SEQ ID NO:10. The nucleic acid sequences encoding the heavy and light chain variable regions were synthesized by Genscript Biotech Inc.

[0358] The heavy and light chain cDNAs of Ipilimumab were cloned into commercially available vectors (purchased from NovoPro, catalog number: V010115) using homologous recombination to obtain recombinant heavy and light chain expression plasmids of the antibody Ipilimumab. KPM (purchased from Zhuhai Kairui, catalog number: K03125), the heavy chain recombinant plasmid, and the light chain recombinant plasmid were mixed at a volume ratio of 100:1:1 (the concentration of all recombinant plasmids was adjusted to 1 mg / mL) to prepare solution A. KPM and TA-293 (purchased from Zhuhai Kairui, catalog number: K20001) were mixed at a volume ratio of 10:1 to prepare solution B. Solution B was transferred to solution A and incubated at room temperature for 10 minutes to prepare the transfection complex. After incubation, the transfection complex was added to HEK293 cell culture medium and cultured at 37°C and 5% CO2. Twenty-four hours after transfection, 0.6% KE-293 (purchased from Zhuhai Kairui, catalog number: K30001) was added to increase product expression. The cells were cultured at 37℃ and 5% CO2 for another 5 days. After culture, the cell supernatant was collected by high-speed centrifugation and eluted with Elution Buffer using a Protein A affinity chromatography column (medium: Protein At Beads 4FF, purchased from Tiandi Renhe, catalog number: SA023100) to collect the target antibody. The elution buffer was then changed to PBS for subsequent experiments. The sequence of the obtained Ipilimumab antibody is shown in Table 1 below.

[0359] Table 1. Ipilimumab antibody-related sequences

[0360] Example 2: Preparation of anti-CCR8 antibody 13F3H6-hz

[0361] The preparation method of the anti-CCR8 monoclonal antibody 13F3H6-hz is described in accordance with the published patent WO2024008110A1, and is briefly described below:

[0362] The amino acid sequence of the 13F3H6-hz heavy chain variable region (13F3H6-hz-VH) is shown in SEQ ID NO:17, and its encoding nucleic acid sequence is shown in SEQ ID NO:18; the amino acid sequence of the 13F3H6-hz light chain variable region (13F3H6-hz-Vκ) is shown in SEQ ID NO:19, and its encoding nucleic acid sequence is shown in SEQ ID NO:20. The nucleic acid sequences encoding the heavy chain and light chain variable regions were synthesized by Genscript Biotech Inc.

[0363] The heavy and light chain cDNAs of 13F3H6-hz were cloned into commercially available vectors (NovoPro, catalog number: V010115) using homologous recombination to obtain the heavy and light chain expression plasmids of the 13F3H6-hz monoclonal antibody. KPM (purchased from Zhuhai Kairui, catalog number: K03125), the heavy chain recombinant plasmid, and the light chain recombinant plasmid were mixed at a volume ratio of 100:1:1 (the concentration of all recombinant plasmids was adjusted to 1 mg / mL) to prepare solution A. KPM and TA-293 (purchased from Zhuhai Kairui, catalog number: K20001) were mixed at a volume ratio of 10:1 to prepare solution B. Solution B was transferred to solution A and incubated at room temperature for 10 minutes to prepare the transfection complex. After incubation, the transfection complex was added to HEK293 cell culture medium and cultured at 37°C and 5% CO2. Twenty-four hours after transfection, 0.6% KE-293 (purchased from Zhuhai Kairui, catalog number: K30001) was added to increase product expression. The cells were cultured at 37℃ and 5% CO2 for another 5 days. After culture, the cell supernatant was collected by high-speed centrifugation and eluted with Elution Buffer using a Protein A affinity chromatography column (protein at beads 4FF, purchased from Tiandi Renhe, catalog number: SA023100) to collect the target antibody. The elution buffer was then replaced with PBS for subsequent experiments. The sequence of the obtained 13F3H6-hz antibody is shown in Table 2 below.

[0364] Table 2. 13F3H6-hz antibody-related sequences

[0365] Example 3: 13F3H6-hz heavy light chain alanine mutation and core site

[0366] The six CDR regions of the heavy and light chains of the 13F3H6-hz antibody obtained in Example 2 were subjected to site-directed mutagenesis using plasmids, and a large number of mutants were obtained. Their sequence information is shown in Tables 3-4.

[0367] The binding activity of the obtained mutants to hCCR8 was detected by ELISA method, and the detection method is briefly described as follows: the recombinant hCCR8-mFc (purchased from Kayka Biological, Recombinant Human CCR8 Protein is expressed from HEK293 with mFc at the C-Terminus, It contains Met1-Lys35, item number: CCR-HM308) was coated on a 96-well plate at a concentration of 2 μg / mL, 50 μL / well, and incubated at 2-8°C overnight. The initial concentration of the antibody was 10 μg / mL, and it was diluted by 5 times to set 8 dilution degrees. The secondary antibody was mouse anti-human IgG Fc / horseradish peroxidase labeled (Kingsway Biological, item number: A01854).

[0368] The results of the change of the binding activity of the mutants are shown in Tables 3 and 4, and FIGS. 1A, 1B, 1C and 1D, and the results show that all the mutants have good binding activity to hCCR8.

[0369] It is also found that the affinity of the antibody after mutation of multiple sites is changed compared with the antibody before mutation. Among them: LCDR3 and HCDR3 are crucial to the binding activity, and the binding activity of most antibodies after mutation is decreased; the binding activity of most antibodies after mutation of the LCDR1 region is slightly reduced; the binding activity after mutation of the LCDR2 region is greatly reduced; the binding activity of a few sites in the HCDR1 region is improved after mutation; the binding activity of most antibodies in the HCDR2 region is not significantly changed. According to the binding activity of the mutants, we limit the core sites of the HCDR region to G45, F46, A46, T47, A47, F48, A48, N49, A49, A50, S50, Y51, A51, A52, S52, I70, A70, R71, T72, Y77, A77, A78, G78, S78, T79, A79, V118, A118, K121, E121, Y121, P121, V121, N121, F121, K122, R124, G126, Y127, F127, D130, E130, R130, P130, Q130, H130, N130, T130, V130, I130, Y131, A131; the core sites of the LCDR region are V29, Y36, S56, Q94, R97, M97, Q97, F97, L99, F99, P100, F101, T102, A102.

[0370] Meanwhile, based on the binding activity of the mutants, we also found that the binding activity of the antibody was significantly improved after mutation at a few sites, including F46A, T47A, A50S, D130R, D130P, D130Q, D130H, D130N, D130T, D130V, and D130I.

[0371] Table 3: Summary of 13F3H6-Hz-VL site-directed mutagenesis and EC50 values Note: ↑ indicates increased binding activity of the mutant antibody; + / - indicates a decrease in binding activity of the mutant antibody by no more than 2 times; ↓ indicates a decrease in binding activity of the mutant antibody by no more than 2-5 times; ↓↓ indicates a decrease in binding activity of the mutant antibody by no more than 5-10 times; ↓↓↓ indicates a decrease in binding activity of the mutant antibody by more than 10 times.

[0372] Table 4: Summary of Site-Local Mutations and EC Values ​​of 13F3H6-Hz-VH Note: ↑ indicates increased binding activity of the mutant antibody; + / - indicates a decrease in binding activity of the mutant antibody by no more than 2 times; ↓ indicates a decrease in binding activity of the mutant antibody by no more than 2-5 times; ↓↓ indicates a decrease in binding activity of the mutant antibody by no more than 5-10 times; ↓↓↓ indicates a decrease in binding activity of the mutant antibody by more than 10 times; an EC50 value of / indicates that the antibody does not bind to the antigen and the EC50 cannot be fitted.

[0373] Example 4: Preparation of antigen and high-expression tool cell line

[0374] The amino acid sequence of h.CCR8 protein (Human CC chemokine receptor type 8, NCBI GenBank: NM_005201), including the full-length h.CCR8 protein and its N-terminal ECD, was obtained and expressed and purified by fusion with the Fc protein fragment of poly-His or mouse / human IgG. Virus was prepared using a lentiviral packaging system and used to infect HEK293T and BaF3 cells. Stable cell lines of HEK293T-hCCR8 (HEK293T cells highly expressing hCCR8) and BaF3-hCCR8 (BaF3 cells highly expressing hCCR8) were obtained through screening with puromycin (purchased from InvivoGen, catalog number: anti-pr-1).

[0375] h. The amino acid sequence of h.CTLA-4 (Human Cytotoxic T-lymphocyte protein 4, NCBI GenBank: NM_001037631), including the full length or extracellular segment of h.CTLA-4 protein, was fused with poly-His or Fc protein fragment of mouse / human IgG for expression and purification. The h.CTLA-4 plasmid was transfected into CHO and HEK293T-hCCR8 cells respectively using Lonza 4D-Nucleofector cell nucleus transfection system, and CHO-hCTLA4 (CHO cells with high expression of hCTLA4) and HEK293T-hCCR8 / hCTLA4 (HEK293T cells with high expression of hCCR8 and hCTLA4) cell strains were obtained by screening with hygromycin (purchased from Invitrogen, item number 10687010).

[0376] The amino acid sequence of CD80 / B7-1 (NCBI GenBank: NM_005191), including the full length or extracellular segment of CD80 protein, was fused with poly-His or Fc protein fragment of mouse / human IgG to express and purify.

[0377] The amino acid sequence of CD86 / B7-2 (NCBI GenBank: NM_175862), including the full length or extracellular segment of CD86 protein, was fused with poly-His or Fc protein fragment of mouse / human IgG, to express and purify.

[0378] The amino acid sequence of h.CCL1 protein (Human C-C motif chemokine 1, NCBI GenBank: NM_002981), including the full length of CCL1 protein and the Lys 24-Lys 96 segment without signal peptide, was fused with poly-His or Fc protein fragment of mouse / human IgG to perform expression and purification.

[0379] Example 5: Construction, expression and purification of anti-CCR8 / CTLA-4 bispecific antibody

[0380] The structural format of the bispecific antibody in the present application is the KIH (Knobs-into-Holes) IgG structural mode, the Fc modification of which is disclosed in patent US7695936B2, which introduces a "hole" at the interface of the first Fc polypeptide and a corresponding "knob" at the interface of the second Fc polypeptide, so that the protrusion can be positioned in the recess to facilitate the formation of Fc heterodimer and prevent the formation of homodimer form, the structure is shown in Figure 2. The "knobs-into-holes" mode replaces the threonine residue at position 366 (T366S) with a serine residue, replaces the leucine residue at position 368 (L368A) with an alanine residue, and replaces the tyrosine residue at position 407 (Y407V) with a valine residue in the CH3 domain of the first subunit of the Fc domain, and replaces the threonine residue at position 366 (T366W) with a tryptophan residue in the CH3 domain of the second subunit of the Fc domain.

[0381] In addition, in the CH3 domain of the first subunit of the Fc, the tyrosine residue at position 349 (Y349C) is also replaced with a cysteine residue, and in the CH3 domain of the second subunit of the Fc, the tyrosine residue at position 354 (S354C) is also replaced with a cysteine residue. The introduction of these two cysteine residues leads to the formation of a disulfide bond between the two subunits of the CH3 domain of the Fc, further stabilizing the heterodimer.

[0382] The sequence of the first heavy chain constant region connected to the first antigen binding domain of anti-CTLA-4 is shown in SEQ ID NO: 21 (the encoding nucleic acid sequence is shown in SEQ ID NO: 22), and the sequence of the first light chain constant region is shown in SEQ ID NO: 23 (the encoding nucleic acid sequence is shown in SEQ ID NO: 24); the sequence of the second heavy chain constant region connected to the second antigen binding domain of anti-CCR8 is shown in SEQ ID NO: 25 (the encoding nucleic acid sequence is shown in SEQ ID NO: 26), and the sequence of the second light chain constant region is shown in SEQ ID NO: 27 (the encoding nucleic acid sequence is shown in SEQ ID NO: 28).

[0383] After the monoclonal antibodies were expressed and purified, they were replaced into PBS (pH 7.4), and the two monoclonal antibodies were adjusted to similar concentrations, mixed according to the molar concentration n(knob):n(hole)=1:1, replaced into PBS+10mM EDTA+0.05M 2-MEA (pH 7.4), incubated at 37°C for 3 hours, then replaced into PBS+0.2mM 2-MEA (pH 7.4), and placed in a refrigerator at 2-8°C for 24 hours. The purity of the bispecific antibody was verified by SEC-HPLC, and the obtained antibody was named SGT003. The SEC-HPLC results are shown in FIG. 3, and the overlay from left to right is 13F3H6-hz-knob, SGT003, Ipilimumab-hole. The retention time of SGT003 after treatment with a reducing agent was single, and the purity was above 98%.

[0384] Example 6: Evaluation of the in vitro activity of the anti-CCR8 / CTLA-4 bispecific antibody

[0385] 1. Detection of the binding activity of SGT003 to hCTLA-4 antigen by ELISA

[0386] The binding activity of the bispecific antibody (hereinafter referred to as bispecific antibody or SGT003) prepared in Example 5 to hCTLA-4 antigen was analyzed by ELISA. Recombinant hCTLA-4 (purchased from ACRO, Recombinant Human CTLA-4 protein is expressed from HEK293 with His Tag at the C-Terminus, It contains Ala 37-Phe 162, item number: CT4-H5229) was coated on a 96-well plate at a concentration of 2 μg / mL, 50 μL / well, and incubated at 2-8°C overnight. The initial concentration of the antibody was 50 μg / mL, and it was diluted by 4 times to set 12 dilution levels. The secondary antibody was goat anti-human IgG / horseradish peroxidase labeled (Beijing Zhongsu Jinqiao Biotechnology Co., Ltd., item number: ZB-2304).

[0387] The results are shown in FIG. 4. The binding activity EC 50 value of SGT003 antibody to hCTLA-4 was about 0.06 μg / mL, which was determined by ELISA.

[0388] 2. Detection of the binding activity of SGT003 to HEK293T-hCCR8 by FACS

[0389] The binding activity of SGT003 and tool cells HEK293T-hCCR8 cells expressing CCR8 was analyzed by FACS method. The HEK293T-hCCR8 cells were trypsinized, the cells were collected by centrifugation, washed with pre-cooled PBS three times, and the cells were resuspended with 1% BSA (1xPBS, pH 7.4). 1x10 5 cells were plated in each well of a 96-well deep-well plate, and the antibody was added in gradient dilution and mixed well. The initial concentration of the antibody was 20 μg / mL, and 4-fold gradient dilution was performed to set 8 dilution degrees. Incubate at 4°C for 1 hour, wash with pre-cooled PBS three times, add 1 μL of APC-labeled goat anti-human IgG per well and mix well (Beijing Solabio Technology Co., Ltd., Catalog No: K0001G-APC), incubate at 4°C for 0.5 hours, resuspend after washing with pre-cooled PBS three times, and detect on a flow cytometer (BD, Model: Accuri C6 plus).

[0390] The results are shown in Figure 5. The binding activity EC 50 value of SGT003 and hCCR8 was about 0.64 μg / mL, determined by FACS method.

[0391] 3. Detection of the binding activity of SGT003 and hCCR8 and hCTLA-4 double transgenic HEK293T (HEK293T-hCCR8 / hCTLA-4) cells by FACS

[0392] The hCCR8 and hCTLA-4 double transgenic HEK293T-hCCR8 / hCTLA-4 cells were trypsinized, the cells were collected by centrifugation, washed with pre-cooled PBS three times, and the cells were resuspended with 1% BSA (1xPBS, pH 7.4). 1x10 5 cells were plated in each well of a 96-well deep-well plate, and 1 μL of APC-labeled goat anti-human IgG per well was added and mixed well (Beijing Solabio Technology Co., Ltd., Catalog No: K0001G -APC). The initial concentration of the antibody was 50 μg / mL, and 4-fold gradient dilution was performed to set 8 dilution

[0393] The results are shown in Figure 6. The binding activity EC 50 value of SGT003 and double transgenic cell strain was about 0.039 μg / mL, determined by FACS method.

[0394] 4. Evaluation of the stability of SGT003 in human serum and PBS at 37°C

[0395] SGT003 was diluted to 100 μg / mL with sterile PBS and human serum respectively, the volume was 200 μL / branch, and it was placed in a 37℃ incubator for 0 days, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days respectively. After 14 days, the binding of the samples in serum and PBS to hCTLA-4 antigen was analyzed by ELISA method.

[0396] The results are shown in Figures 7A and 7B. SGT003 has good antigen binding stability in human serum and PBS at 37℃.

[0397] 5. Biacore assay of the affinity of SGT003 to hCTLA-4

[0398] The sample chamber and flow cell temperature of Biacore 1K were set to 25℃, and the data collection frequency was 10 Hz. Protein A chips (purchased from Cytiva, item number: 29127556) were used to capture SGT003 and YERVOY respectively, with an antibody concentration of 0.5 μg / mL, a flow rate of 10 μL / min, and a capture time of 60 s (SGT003 Relative response was about 186.5, and YERVOY Relative response was about 88). The analyte was hCTLA-4 (purchased from ACRO, item number: CT4-H5229) diluted with 1x HBS-EP + pH 7.4 (10x HBS-EP + purchased from Cytiva, item number: BR100669) buffer, with an initial concentration of 100 nM, 2-fold dilution, and a final concentration of 0.78125 nM, a total of 8 concentrations, and an analyte flow rate of 30 μL / min, binding for 180 s, dissociation for 300 s, and regeneration conditions of glycine (pH about 1.5, purchased from Cytiva, item number: BR100354) with a flow rate of 30 μL / min and regeneration for 30 s. Using Biacore Insight Evaluation Software based on the principle of SPR, the affinity detection data was analyzed using 1:1 binding mode and Fit Local Kinetics mode.

[0399] The results are shown in Figures 8A and 8B and Table 5. The affinity of SGT003 to hCTLA-4 was 7.91E-10, and the affinity of YERVOY to hCTLA-4 was 1.08E-9, which was at a similar level.

[0400] Table 5. Biacore assay of the affinity of antibodies to hCTLA-4

[0401] 6. Evaluation of the specificity of SGT003

[0402] The specificity of SGT003 double antibody and irrelevant antigens was detected by ELISA. Irrelevant antigens (31 types in total, specific antigen information is as follows: huIL-2 (NM_000586), huIL-4 (NM_000589), huIL-5 (NM_000879), huIL-8 (NM_000584), huIL-10 (NM_000572), huIL-17A (NM_002190), huILLRB1 (NM_001081637), huILLR) were coated on 96-well ELISA plates. B2(NM_001080978), huLILRB3(XM_006726314), huLILRB4(NM_001278426), huLILRB5(NM_001081442), h uROR1(NM_001083592), humROR2(NM_013845.5), huPD-1(NM_005018), mPD1(NM_008798), huTREM2(NM_0 01271821), mTREM2 (NM_001272078), huHER2 (NM_001005862), mHER2 (NM_001003817), hCD206 (NM_0024 38), mCD206(NM_008625), huTSLP(NM_033035), mFSHR(NM_000145), huGP73(NM_016548), huFABP5(NM_0 The antigens 01444), huTK1 (NM_003258), huCLEC7A (NM_022570), huAPOE (NM_000041), huGPNMB (NM_001005340), huZnT8 (NM_001172811), and huCKMB (HAEA01008583) were prepared using the same method as in Example 3. The antigen concentration was 5 μg / mL, 50 μL / well, and the coating was incubated overnight at 2–8°C. The next day, after blocking the ELISA plate, SGT003 secondary antibody was added to each well at a concentration of 10 μg / mL, 50 μL / well, and the plate was incubated at 37°C for 1 hour. The secondary antibody was goat anti-human IgG / horseradish enzyme labeled (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: ZB-2304).

[0403] As shown in Figure 9, SGT003 showed no non-specific binding to any irrelevant antigens except for its specific binding to the target antigen.

[0404] Example 7: Evaluation of the in vitro blocking bioactivity of SGT003

[0405] 1. SGT003 blocks the binding of CTLA-4 ligand to CTLA-4 on the cell surface.

[0406] SGT003 and the isotype control antibody (Isotype, the anti-CTLA-4 monoclonal antibody YERVOY prepared in Example 1) were prepared into antibody dilutions with an initial concentration of 1200 μg / mL, serially diluted 3-fold (8 gradients in total), and added to 96-well plates (purchased from Corning, catalog number 3894), 50 μL per well. Biotinylated B7-1 (purchased from ACRO, catalog number B71-H82E9) and B7-2 (purchased from ACRO, catalog number CD6-H82F5) ligand proteins were prepared at a concentration of 2 μg / mL, 50 μL per well, and added to 96-well plates. The ligand proteins were mixed with equal volumes of the serially diluted antibodies and incubated at room temperature for 30 min. Recombinant CHO cells expressing h.CTLA-4 (CHO-hCTLA-4) were collected, centrifuged, resuspended in pre-chilled PBS buffer, and the cell density was adjusted to 1 × 10⁶ cells / well. 6 Cells / mL, 100 μL per well, was added to the corresponding well of a 96-well plate and incubated at 4°C for 1 h. After incubation, the cells were centrifuged at 3000 rpm, and the cell pellet was collected. 200 μL of pre-chilled PBS buffer was added to each well, and the cells were washed once by centrifugation at 3000 rpm. The cell pellet was then collected again. 100 μL of pre-pre-chilled Streptavidin Protein-FITC (purchased from ACRO, catalog number STN-NF113) dilution was added to the cell pellet, and the plate was incubated at 4°C for 1 h. After incubation, 100 μL of pre-chilled PBS buffer was added to each well, and the cells were washed once at 3000 rpm. After resuspending the cells in 200 μL of pre-chilled PBS buffer, the plate was analyzed by flow cytometry, and the fluorescence signal in the FL1-A channel was collected.

[0407] The results are shown in Figures 10A and 10B and Table 6. SGT003 can inhibit the binding of B7-1 and B7-2 to CTLA-4 on the cell surface, and exhibits a significant dose-response effect.

[0408] Table 6. SGT003's ability to block the binding of B7-1, B7-2 to cell surface hCTLA-4

[0409] 2. SGT003 blocks the binding of human CCL1 to human CCR8 on the cell surface.

[0410] SGT003 and isotype control antibody (Isotype, anti-CCR8 monoclonal antibody 13F3H6-hz prepared in Example 2) were prepared into antibody dilutions with an initial concentration of 400 μg / mL, serially diluted 5× in 10 steps. 50 μL of each solution was added to a 96-well plate (purchased from Corning, catalog number 3894). hCCL1 ligand protein (purchased from ACRO, catalog number CC1-H5253) was prepared at a concentration of 80 ng / mL, 50 μL of which was added to each well of the 96-well plate. The ligand protein was mixed with an equal volume of the serially diluted antibody and incubated at room temperature for 30 min. Ba / F3-hCCR8 cells were harvested, centrifuged, and the cell pellet was resuspended in pre-chilled PBS buffer. The cell density was adjusted to 2×10⁶ cells / well. 6 Cells / mL, 100 μL per well, was added to the corresponding position in a 96-well plate and incubated at 4°C for 1 h. After incubation, the cells were centrifuged at 3000 rpm, and the cell pellet was collected. 200 μL of pre-chilled PBS buffer was added to each well, and the cells were washed once by centrifugation at 3000 rpm. The cell pellet was then collected. 100 μL of pre-prepared pre-chilled PE anti-mouse IgG2a antibody (purchased from Biolegend, catalog number 407107) dilution was added to the cell pellet, and the plate was incubated at 4°C for 1 h. After incubation, 100 μL of PBS buffer was added to each well, and the cells were washed once at 3000 rpm. After resuspending the cells in 200 μL of pre-chilled PBS buffer, the plate was analyzed by flow cytometry, and the fluorescence signal in the FL2-A channel was collected.

[0411] As shown in Figure 11, SGT003 can block the binding of hCCL1 to CCR8 on the cell surface, and exhibits a significant dose-response effect, inhibiting the activity of IC50. 50 The value is approximately 0.68 μg / mL.

[0412] 3. SGT003 inhibits hCCL1-induced cell chemotaxis.

[0413] SGT003 was prepared into an initial concentration of 400 μg / mL, with five 2× serial dilutions. 150 μL of each dilution was added to each well of a 96-well plate (purchased from Corning, catalog number 3799). Ba / F3-hCCR8 cells were collected, and the cell pellet was collected by centrifugation. The cell density was adjusted to 6.4 × 10⁻⁶ cells / well using RPMI 1640 complete medium. 5cells / mL, 150 μL / well, and the cells were added to a 96-well plate and incubated with gradient-diluted antibodies at room temperature for 30 min. After the incubation, 250 μL of the suspension was transferred to a Transwell (purchased from Millipore, catalog number PTSP24H48) chamber, which was placed in a 24-well plate (purchased from Corning, catalog number 3524) under the chamber, and 500 μL / well of a prepared human CCL1 ligand protein at a final concentration of 10 ng / mL was added to the 24-well plate. The cell plate was incubated in a 37°C CO2incubator for 24 h. After the incubation, the chamber was discarded, and 500 μL / well of Cell-ATP Viability Detection Reagent (purchased from MCE, catalog number HY-K0302) was added to the 24-well plate. After incubation with shaking at room temperature, the chemiluminescence signal was detected by a microplate reader.

[0414] The results are shown in Table 7 and FIG. 12. SGT003 can block the hCCL1-induced chemotactic migration of Ba / F3-hCCR8 cells and exhibit a clear dose effect.

[0415] Table 7. Inhibition of hCCL1-induced chemotaxis of Ba / F3-hCCR8 cells by SGT003

[0416] Example 8: Co-stimulatory activation activity of SGT003 and Staphylococcus aureus enterotoxin (SEB) on human PBMC cells

[0417] SGT003 and the positive control antibody (anti-CTLA-4 monoclonal antibody YERVOY prepared in Example 1) were prepared into dilutions at an initial concentration of 800 μg / mL, 2-fold gradient dilution of 3 concentrations, and then 3-fold gradient dilution of 5 concentrations, for a total of 8 concentration gradients. The dilutions were added to a 96-well plate (purchased from Corning, catalog number 3799) at 50 μL per well. SEB (purchased from Tox Tech, catalog number BT202) was prepared into a dilution at a concentration of 40 ng / mL, and 50 μL / well was added to the 96-well plate. Human peripheral blood mononuclear cells (PBMCs) (purchased from Shanghai Yaju Biotechnology Co., Ltd., catalog number CZPB010M) were recovered, and the cell density was adjusted to 5×10 5cells / mL, 100 μL per well, mixed with drugs evenly, and blank control group (PBMC only) and SEB single control group (PBMC + SEB) were set up, the volume of the medium in the control group was supplemented, and the cell plate was placed in a 37°C CO2 incubator, and detected after incubation for about 24 h and 72 h. All drugs, antigen dilution and cell density adjustment used RPMI 1640 complete medium containing 10% FBS (purchased from Thermo Fisher, item number 10099141C) (purchased from Thermo Fisher, item number A1049101). After incubation, the cell supernatant was taken, diluted, and the human IL-2 ELISA detection kit (purchased from Beijing Dakewo Biotechnology Co., Ltd., item number 1110203), human TNF-α ELISA detection kit (purchased from Beijing Dakewo Biotechnology Co., Ltd., item number 1117203), human IL-6 ELISA detection kit (purchased from Beijing Dakewo Biotechnology Co., Ltd., item number 1110603) and human IL-23 ELISA detection kit (purchased from Beijing Dakewo Biotechnology Co., Ltd., item number 1112302) were used to detect cytokines according to the detection instruction, and the OD 450nm was read, and the dose-effect curve was drawn.

[0418] The results are shown in Figures 13A-13D. SGT003 and YERVOY can both activate immune cells to release IL-2, and the activation levels are comparable. At the same time, SGT003 and YERVOY do not activate human PBMC cells to release inflammatory factors IL-6 and IL-23 under SEB co-stimulation conditions.

[0419] At the same time, it has been reported that the increase in the level of TNF-α release is one of the side effects of YERVOY treatment, which can cause inflammation-related adverse reactions (such as colitis, hepatitis, etc.). The results of this example found that the release level of TNF-α inflammatory factor in the SGT003 group was lower than that in the YERVOY group, suggesting that SGT003 has better safety than YERVOY.

[0420] Example 9: SGT003 induces Tregs cell elimination

[0421] Prepare SGT003 and YERVOY antibody dilution buffers, starting at a concentration of 800 μg / mL. Perform three 2× serial dilutions, followed by five 3× serial dilutions, for a total of eight dilutions. Add 50 μL to each well of a 96-well plate (purchased from Corning, catalog number 3799). Prepare SEB (purchased from Tox Tech, catalog number BT202) dilution buffer at a concentration of 40 ng / mL, adding 50 μL / well to each 96-well plate. Resuscitate human PBMC cells (purchased from Shanghai Yayu Biotechnology Co., Ltd., catalog number CZPB010M) and adjust the cell density to 5 × 10⁶ cells / well. 5 100 μL of the cell culture medium was seeded into each well of a 96-well plate and mixed thoroughly with the drug. A blank control group (PBMC only) and a SEB-only control group (PBMC+SEB) were also set up. The culture medium volume in the control groups was replenished to a certain level. The cell culture plates were placed in a 37°C CO2 incubator and incubated for approximately 72 hours. The cell pellet was then collected by centrifugation. Surface marker antibodies, such as anti-hCD3 antibody (purchased from Invitrogen, catalog number 317308), anti-hCD4 antibody (purchased from Invitrogen, catalog number 35-0047-42), and anti-hCD8a antibody (purchased from Invitrogen, catalog number 62-0086-42), were diluted 1:100 with PBS to obtain the flow cytometry antibody dilution. 100 μL of the above flow cytometry antibody dilution was added to each well of the cells and mixed thoroughly. The cells were incubated at 4°C in the dark for 30 minutes. Collect the cell pellet by centrifugation. Prepare fixation and permeabilization buffer according to the reagent instructions. Resuspend the cells in the buffer at 200 μL / well and incubate at 4°C in the dark for 1 h. Collect the cell pellet by centrifugation and add 1:100 diluted human Foxp3. + Antibodies (purchased from Invitrogen, catalog number 17-4776-42) were incubated at 4°C in the dark for 30 min. Cell pellets were collected by centrifugation, resuspended in PBS, and the positivity rate of each antibody's corresponding fluorescence channel was detected by flow cytometry. All data were processed using FlowJo software. All drug and antigen dilutions and cell density adjustments were performed using RPMI 1640 complete medium (purchased from Thermo Fisher, catalog number A1049101) containing 10% FBS (purchased from Thermo Fisher, catalog number 10099141C).

[0422] As shown in Figure 14, flow cytometry analysis revealed that the SGT003 antibody significantly reduced the proportion of Treg cells in peripheral blood, and its effect was significantly better than that of YERVOY (P<0.01).

[0423] Example 10: Preliminary pharmacokinetic study of SGT003 in mice

[0424] To investigate the metabolism of the bispecific antibody of the present application in mice, 3 female C57BL6 mice were selected, and tail vein injection was used for administration, and the administration dose was 10 mg / kg. The serum antibody concentration was detected by blood sampling at different time points, and the blood sampling points were 30 min, 2 h, 4 h, 8 h, 1 d, 4 d, 7 d and 14 d after administration.

[0425] The serum samples at the blood sampling points for pharmacokinetics were detected for the concentrations of total antibodies and active antibodies by ELISA method. Goat anti-human Fc antibody (1:2000, Sigma, I8885) and CTLA-4 protein (1 μg / mL, ACRO, CT4-H52H9) were diluted with PBS respectively, coated in 100 μL / well at 4°C overnight. The next day, the coating solution in the well plate was discarded, 3% skimmed milk powder (diluted with PBS) was added at 300 μL / well, and 37°C was blocked for 2 h. The standard antibody with known concentration and mouse serum were added to the well plate respectively at 100 μL / well, and 37°C was incubated for 1 h. The well plate was washed for 3 times, the secondary antibody was goat anti-human IgG / horseradish peroxidase labeled (Beijing Zhongshanjinqiao Biotechnology Co., Ltd., ZB-2304), 100 μL / well, 37°C incubated for 30 min. The well plate was washed for 4 times, TMB was added for color development for 10 min, and finally the stop solution was added. The absorbance at 450 nm was measured on the enzyme label instrument. The sample concentration was fitted with the standard curve of OD color development value, and the sample concentration was calculated by substituting the standard curve. The pharmacokinetic parameters of 3 mice were calculated.

[0426] The pharmacokinetic parameters calculated by non-compartment model are the most commonly used in the art, which are summarized as follows:

[0427] “Lambda_z” is the first-order elimination rate constant k; “t 1 / 2” is the terminal half-life, also known as the elimination half-life; “T max” is the time at which the maximum plasma concentration is observed; “C max” is the maximum plasma concentration observed; “AUC” is the “area under the curve” of plasma concentration over time; “AUClast” is the area under the curve from zero time to the final blood sampling time; “AUCINF” is the area under the curve from zero time to infinite time; “Vz” is the apparent distribution volume; “Cl” is the plasma clearance rate; “MRTlast” is the mean residence time from zero time to the final blood sampling time.

[0428] The results are shown in Table 8 and FIG. 15. The pharmacokinetic determination results of SGT003 antibody show that SGT003 shows a relatively standard antibody pharmacokinetic characteristic, and the average half-life (t 1 / 2 )>5.4 days.

[0429] Table 8 Pharmacokinetic study of SGT003 in mice

[0430] Example 11: Pharmacodynamic study of SGT003 in tumor-bearing mice

[0431] In this example, the anti-CTLA-4 monoclonal antibody (YERVOY) prepared in Example 1, the anti-CCR8 monoclonal antibody (13F3H6-hz) prepared in Example 2, and the commercially available anti-PD-1 monoclonal antibody (trade name RMP1-14, manufacturer BioXcell, item number BE0146) were used as controls or in combination.

[0432] 1. Pharmacodynamic evaluation of SGT003 in tumor-bearing MCA205 cell mouse xenograft model

[0433] C57BL / 6hCTLA-4 / hCCR8 double transgenic mice were purchased from the supplier Beijing Boao S&T Pharmaceutical Co., Ltd. The animals were all SPF (specific pathogen free) level, and the mice were about 5-6 weeks old when they arrived at the animal house.

[0434] MCA205 cells (2x10 6 were inoculated subcutaneously in the right flank of C57BL / 6hCTLA-4 / hCCR8 mice. After 14 days of inoculation, the tumor volume was about 265mm 3 . Animals with large deviations in body weight, too large or too small tumors were removed, and the mice were randomly divided into 9 groups according to tumor volume, with 8 mice in each group.

[0435] The samples were injected intraperitoneally immediately after grouping (D14 after inoculation, D0 of administration), once every 3 days, a total of 6 times, with 0.1 mL / 10 g per injection according to body weight. Among them:

[0436] 1) The dosages of the YERVOY group, the PD-1 monoclonal antibody group, and the CCR8 monoclonal antibody group were all 4 mg / kg;

[0437] 2) The dosages of the PD-1 monoclonal antibody and SGT003 double antibody combination group were 4 mg / kg and 2 mg / kg, respectively;

[0438] 3) The dosages of the YERVOY and CCR8 monoclonal antibody combination group were both 4 mg / kg;

[0439] 4) The dosages of the SGT003 double antibody group were 4 mg / kg, 2 mg / kg, and 1 mg / kg, respectively;

[0440] 5) The negative control group was injected with the same volume of solvent.

[0441] The tumor was measured using a vernier caliper, with the long diameter recorded as L 长 and the short diameter recorded as L 短 . The tumor volume was measured once every 3 days, and the data were recorded for a total of 18 days (D32). The animal tumor grew to 2000mm3 Then, humane execution will be carried out.

[0442] Data was recorded using Excel software: average values ​​were calculated using AVG; SEM values ​​were calculated using STDEV / SQRT (number of animals per group); and graphs were generated using analysis software.

[0443] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2 .

[0444] Tumor inhibition rate (TGI) (%) = 1 - T / C (%), where T / C is the relative tumor proliferation rate. The relative tumor volume (RTV) is calculated based on the measurement results using the formula: RTV = Vt / V0. Where V0 is the tumor volume measured at the time of administration (d0), and Vt is the tumor volume at each measurement. The relative tumor proliferation rate (T / C) (%) is calculated using the formula: T / C = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group).

[0445] The results are shown in Table 9 and Figure 16. SGT003 showed stronger inhibitory effects on MCA205 xenografts than equal doses of CCR8 monoclonal antibody, Yervoy monoclonal antibody, and PD-1 monoclonal antibody, with significant differences (P < 0.01). This suggests that SGT003 alone can produce good antitumor activity.

[0446] Meanwhile, the results showed that SGT003 had a stronger inhibitory effect on MCA205 xenograft tumors than the combination of Yervoy monoclonal antibody and CCR8 monoclonal antibody, confirming that the CTLA-4 / CCR8 bispecific antibody SGT003 could produce superior antitumor activity compared with the combination of CTLA-4 monoclonal antibody and CCR8 monoclonal antibody alone.

[0447] Furthermore, the results showed that when SGT003 was used in combination with PD-1 monoclonal antibody, the inhibitory effect on transplanted tumors was further enhanced (tumor inhibition rate reached 96.25%), confirming that SGT003 and PD-1 monoclonal antibody have a good synergistic effect and are an effective drug combination regimen.

[0448] Table 9. Inhibitory effect of test products on MCA205 xenografts in C57BL / 6hCTLA-4 / hCCR8 mice

[0449] 2. Efficacy evaluation of SGT003 in a mouse xenograft model bearing MC38 cells.

[0450] C57BL / 6hCTLA4 / hCCR8 double transgenic mice were purchased from the supplier, Beijing Bao'sai Technology Co., Ltd. Medical Science and Technology Co., Ltd. The animals were all SPF (specific pathogen free) level, and the mice were about 5-6 weeks old when they arrived at the animal house.

[0451] MC38 cells (2 x 10 6 were inoculated subcutaneously in the right flank of C57BL / 6hCTLA4 / hCCR8 mice. After 15 days of inoculation, the tumor volume was about 180 mm 3 . Animals with excessive weight, tumor size or small tumor size were removed, and the mice were randomly divided into 5 groups according to the tumor volume, with 6 mice in each group.

[0452] The samples were injected intraperitoneally immediately after grouping (D15 after inoculation, D0 after administration), once every 3 days, a total of 6 times, and each mouse was injected with 0.1 mL / 10 g according to the body weight. Among them:

[0453] 1) The positive control group was injected with YERVOY at a dose of 4 mg / kg;

[0454] 2) The test product group was injected with SGT003 at a dose of 4 mg / kg, 2 mg / kg and 1 mg / kg, respectively;

[0455] 3) The negative control group was injected with the same volume of solvent.

[0456] The tumor length was measured using a vernier caliper and recorded as L 长 , and the short diameter was recorded as L 短 . The tumor volume was measured every 3 days, and the data was recorded for a total of 27 days (D42). If the tumor volume of the animals in the negative control group reached 2000 mm 3 , they were humanely sacrificed.

[0457] The data was recorded using Excel software: the average value was calculated using AVG; the SEM value was calculated using STDEV / SQRT (number of animals in each group); and the analysis software was used for plotting.

[0458] The formula for calculating the tumor volume (V) is: V = 1 / 2 x L 长 x L 短 2 .

[0459] TGI (%) = 1 - T / C (%), wherein T / C is the relative tumor growth rate. The relative tumor volume (RTV) was calculated according to the measured results, and the calculation formula is: RTV = Vt / V0. Wherein V0 is the tumor volume measured at the time of caging (i.e. d0), and Vt is the tumor volume at each measurement. The calculation formula of the relative tumor growth rate T / C (%) is: T / C = TRTV / CRTV x 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group).

[0460] The results are shown in Table 10 and Figure 17, and SGT003 test product has good tumor inhibition effect on MC38 transplanted tumors at 4 mg / kg, 2 mg / kg and 1 mg / kg doses.

[0461] It is also found that the tumor inhibition effect of SGT003 test product at 2 mg / kg dose group is equivalent to that of YERVOY test product at 4 mg / kg dose group, indicating that SGT003 has good effect at low dose.

[0462] Table 10. Tumor inhibition effect of test product on MC38 transplanted tumors in tumor-bearing C57BL / 6h.CTLA4 / hCCR8 mice

[0463] 3. Pharmacodynamic evaluation of SGT003 in tumor-bearing MB49 cell mouse transplanted tumor model

[0464] C57BL / 6h.CTLA4 / hCCR8 mice were purchased from Beijing Baoao Sida Tech Co., Ltd. hCTLA- 4 / hCCR8 The double transgenic mice were all SPF (specific pathogen free) level, and the mice were about 5-6 weeks old when they arrived at the animal house.

[0465] MB49 cells (2 x 10 6 were inoculated subcutaneously in the right flank of C57BL / 6h.CTLA4 / hCCR8 mice, and after 8 days of inoculation, the tumor volume was about 187 mm 3 The animals with large weight deviation, too large or too small tumors were removed, and the mice were randomly divided into 10 groups according to the tumor volume, 8 mice in each group.

[0466] The samples were injected intraperitoneally immediately after grouping (D8 after inoculation, D0 after administration), and the administration was once every 3 days, a total of 6 times, and each mouse was injected 0.1 mL / 10 g according to the body weight. Among them:

[0467] 1) The administration amount of YERVOY group, PD-1 monoclonal antibody group and CCR8 monoclonal antibody group was 4 mg / kg;

[0468] 2) PD-1 mAb combined with SGT003 bispecific antibody group, the dosages are both 4 mg / kg;

[0469] 3) YERVOY combined with CCR8 mAb group, the dosages are both 4 mg / kg;

[0470] 4) SGT003 bispecific antibody group, the dosages are 8 mg / kg, 4 mg / kg and 2 mg / kg respectively;

[0471] 5) Negative control group, the same volume of solvent is injected.

[0472] The long diameter of tumor is measured as L 长 and the short diameter as L 短 The tumor volume is measured every 3 days, and the data is recorded for 24 days (D32). When the tumor volume of the animal reaches 2000 mm 3 , the animal is humanely sacrificed.

[0473] The data is recorded by using Excel software: the average value is calculated by AVG; the SEM value is calculated by STDEV / SQRT (the number of animals in each group); and the analysis software is used for plotting.

[0474] The formula for calculating the tumor volume (V) is: V = 1 / 2 x L 长 x L 短 2 .

[0475] The tumor inhibition rate TGI (%) = 1-T / C (%), wherein T / C is the relative tumor proliferation rate. According to the measured results, the relative tumor volume (RTV) is calculated, and the formula is: RTV = Vt / V0. Wherein V0 is the tumor volume measured at the time of caging (i.e. d0), and Vt is the tumor volume at each measurement. The formula for calculating the relative tumor proliferation rate T / C (%) is: T / C = TRTV / CRTV x 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group).

[0476] The results are shown in Table 11 and Figure 18. SGT003 has a stronger inhibitory effect on MB49 transplanted tumors than the same dose of CCR8 mAb, YERVOY mAb and PD-1 mAb, and the difference is significant (P<0.01), indicating that SGT003 alone can produce better anti-tumor activity.

[0477] The results also show that SGT003 has a stronger inhibitory effect on MB49 transplanted tumors than YERVOY mAb + CCR8 mAb combination, confirming that CTLA-4 / CCR8 bispecific antibody SGT003 can produce more excellent anti-tumor activity compared with simple combination of CTLA-4 mAb and CCR8 mAb.

[0478] Table 11. Anti-tumor effect of test products on MB49 transplanted tumor in C57BL / 6hCTLA4 / hCCR8 tumor-bearing mice

[0479] 4: Pharmacodynamic evaluation of SGT003 in a mouse transplanted tumor model with EO771 tumor-bearing cells

[0480] C57BL / 6hCTLA4 / hCCR8 double transgenic mice were purchased from the supplier, Beijing Baoao S&T Pharmaceutical Co., Ltd. The animals were all SPF (specific pathogen free) level, and were about 5-6 weeks old when they arrived at the animal house.

[0481] EO771 cells (2 x 10 6 were inoculated subcutaneously in the right flank of C57BL / 6hCTLA4 / hCCR8 mice. After 8 days, when the average tumor volume was close to 90mm 3 , animals with excessive weight, tumor size or small tumor size were removed, and the mice were randomly divided into 2 groups according to tumor volume, 4 mice in each group.

[0482] The samples were injected intraperitoneally immediately after grouping (D8 after inoculation, D0 after administration), once every 3 days, a total of 6 times, 0.1 mL / 10 g per injection according to body weight. Among them:

[0483] 1) The test drug group was injected with SGT003, and the dose was 10 mg / kg;

[0484] 2) The negative control group was injected with the same volume of solvent.

[0485] The tumor length was measured using a vernier caliper, denoted as L 长 , and the short diameter was denoted as L 短 . The tumor volume was measured every 3 days, and the data was recorded, a total of 27 days (D35). When the tumor volume of the animals in each group reached 2000mm 3 , the animals were humanely sacrificed.

[0486] The data was recorded using Excel statistical software: the average value was calculated by AVG; the SEM value was calculated by STDEV / SQRT (number of animals in each group); and the analysis software was used for plotting.

[0487] The formula for calculating the tumor volume (V) is: V = 1 / 2 x L 长 x L 短 2 .

[0488] TGI(%) = 1 - T / C(%), wherein T / C is the relative tumor growth rate. The relative tumor volume (RTV) was calculated according to the measured results, and the calculation formula was: RTV = Vt / V0. Wherein V0 is the tumor volume measured at the time of caging (i.e. d0), and Vt is the tumor volume at each measurement. The calculation formula of the relative tumor growth rate T / C (%) is: T / C = TRTV / CRTV x 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group)

[0489] The results are shown in Table 12 and Figure 19, which show that SGT003 has a strong tumor inhibition effect on EO771 transplanted tumors.

[0490] Table 12. Tumor inhibition effect of the test product on EO771 transplanted tumors of tumor-bearing C57BL / 6hCTLA4 / hCCR8 mice

[0491] It should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application can have various changes and variations. Although the specific embodiments have been described, there can be or currently cannot be foreseeable alternatives, modifications, changes, improvements and substantial equivalents of the above-described embodiments for the applicant or other skilled in the art. Therefore, the appended claims submitted and possibly modified are intended to cover all such alternatives, modifications, changes, improvements and substantial equivalents. It is important that as the technology evolves, many of the elements described herein can be replaced by equivalent elements that appear after the present application.

Claims

1. An antibody or antigen-binding fragment thereof that binds CCR8, characterized in that, The amino acid sequences of the complementarity determining regions (CDRs) defined according to the IMGT system are as follows: The amino acid sequence of heavy chain CDR1 is X1X2X3X4X5X6X7X8; The amino acid sequence of heavy chain CDR2 is X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 ; The amino acid sequence of the heavy chain CDR3 is X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 X 32 ; The amino acid sequence of the heavy chain CDR1 is X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 ; The amino acid sequence of the heavy chain CDR2 is X 43 X 44 X 45 ; and The amino acid sequence of the heavy chain CDR3 is X 46 X 47 X 48 X 49 X 50 X 51 X 52 X 53 X 54 ;​ wherein: X1is G or A; X2is F or A; X3is T or A; X4is F or A; X5is N or A; X6is G, S or A; X7is Y or A; X8is G, S, V, L, I, T, Y, D, K or A; X9is I or A; X 10 is H, K, G, D, L, S, W, A or R; X 11 is S, Q, Y, N, G, L, M, W, A or T; X 12 is K or A; X 13 S or A; X 14 is N or A; X 15 is N or A; X 16 Y or A; X 17 is G, S or A; X 18 is T or A; X 19 V or A; X 20 R or A; X 21 is H or A; X 22 is A, E, Y, P, Q, T, I, V, N, M, F, or K; X 23 R, H, G, L, S, W, D, A, V, E, T, Q, N, M, I, P, F, Y or K; X 24 is V or A; X 25 is A, K, H, G, L, S, W, D or R; X 26 E or A; X 27 is A, V, L, I, M, W, S, D, K, E, P, Q, H, T, R, N, F, Y or G; X 28 is A, N, Q, M, W, S, D, K, L, G, F, R, H, I, T, E, V, P or Y; X 29 is G, S or A; X 30 M or A; X 31 is A, G, L, M, W, S, K, E, R, P, Q, H, N, T, Y, V, F, I, or D; X 32 is Y or A; X 33 is K or A; X 34 S or A; X 35 V or A; X 36 S or A; X 37 is T or A; X 38 S or A; X 39 is G or A; X 40 Y or A; X 41 is T or A; X 42 Y or A; X 43 L or A; X 44 is G, S or A; X 45 S or A; X 46 Q or A; X 47 is H or A; X 48 S or A; X 49 is A, K, H, G, L, M, W, S, D, E, V, I, Q, F, P, T, N, Y or R; X 50 E or A; X 51 is A, G, V, I, M, W, S, D, K, T, P, Q, N, H, E, Y, F, R or L; X 52 is F, G, L, S, D, K, Q, W, A or P; X 53 is A, P, G, L, S, D, K, Q, W or F; and X 54 is T or A.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequences of the complementarity determining regions are selected from at least one of the following groups: (1) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 29, LAS, SEQ ID NO: 16, respectively; (2) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID N0: 13, SEQ ID NO: 30, LAS, SEQ ID NO: 16, respectively; (3) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy CDR3, light chain CDR1, light chain CDR2, light chain CCR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO : 13, SEQ ID NO: 31, LAS, SEQ ID NO: 16, respectively; (4) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy Chain CDR3, light chain CDR1, light chain CDR2, light chain CDDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO 13, SEQ ID NO: 32, LAS, SEQ ID NO: 16, respectively; (5) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy CHDR3, light chain CDR1, light chain CDR2, light chain CDRS are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 33, LAS, SEQ ID NO: 16, respectively; (6) the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy CD3, light chain CDR1, light chain CDR2, light chain CDRS is SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 1 3, SEQ ID NO: 34, LAS, SEQ ID NO: 16, respectively; (7) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 35, LAS, SEQ ID NO: 16 respectively; (8) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (9) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (10) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (11) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (12) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (13) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (14) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (15) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 43, respectively; (16) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (17) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (18) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (19) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQID NO: 47, respectively; (20) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (21) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR23, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ IDNO: 49, respectively; (22) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (23) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 51, respectively; (24) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (25) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (26) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (27) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQID NO: 55, respectively; (28) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (29) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 14, LAS, SEQ ID NO: 57, respectively; (30) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ IDNO: 58, respectively; (31) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 59, respectively; (32) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (33) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (34) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (35) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQID NO: 63, respectively; (36) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (37) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR 2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: (38) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (39) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 67, respectively; (40) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (41) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (42) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (43) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQID NO: 71, respectively; (44) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2; the heavy chain CDR3, the light chain CDR1, the light chain CDR1, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ IDNO: 72, respectively; (45) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2; (46) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2. (47) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 75, respectively; (48) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (49) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (50) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (51) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQID NO: 79, respectively; (52) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (53) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR 2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: (54) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ IDNO: 82, respectively. (55) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 83, respectively; (56) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (57) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (58) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (59) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQID NO: 87, respectively; (60) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2thegroup of CDR3, the light chain CDR1, the light chain CDR2, the group of CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 88, respectively; (61) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 group of CDR3, the light chain CDR1, the light chain CDR2, group of CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (62) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2group of CDR3, the light chain CDR1, the light chain CDR2, CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 90, respectively. (63) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 91, respectively; (64) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (65) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (66) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (67) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQID NO: 95, respectively; (68) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (69) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ IDNO: 97, respectively; (70) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (71) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 99, respectively; (72) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (73) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (74) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 102, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, LAS, SEQ ID NO: 16, respectively; (75) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 103, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:13, LAS, SEQ ID NO: 16, respectively; (76) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (77) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR23, the light chain CDR3 are SEQ ID NO: 105, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:12, LAS, SEQ ID NO: 16, respectively; (78) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR22, the light chain CDR3 are SEQ ID NO: 106, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:11, LAS, SEQ ID NO: 16, respectively; (79) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 107, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (80) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (81) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (82) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (83) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (84) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (85) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (86) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (87) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 115, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (88) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (89) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (90) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (91) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (92) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (93) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (94) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (95) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 123, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (96) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (97) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (98) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (99) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (100) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (101) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (102) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (103) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 131, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (104) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (105) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (106) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (107) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (108) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (109) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (110) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (111) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 139, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (112) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (113) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (114) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (115) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (116) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (117) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (118) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (119)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 147, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (120)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (121)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (122)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (123)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 151, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the (125)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (126)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (127) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 155, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (128) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (129) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (130) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (131) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (132) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (133) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (134) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (135)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 163, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (136)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (137)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (138)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (139)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (140)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (141)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (142)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (143) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 171, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (144) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (145) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (146) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (147) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (148) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (149) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (150) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (151) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 179, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (152) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (153) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (154) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (155) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (156) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (157) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (158) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (159)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 187, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (160)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (161)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (162)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (163)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 191, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the (165)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (166)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (167) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 195, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (168) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (169) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (170) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (171) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (172) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (173) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (174) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (175)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 203, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (176)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (177)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (178)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (179)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (180)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (181)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (182)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (183)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 211, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (184)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (185)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (186)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (187)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR3, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 215, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the (189)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (190)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (191) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 219, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (192) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (193) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (194) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (195) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (196) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (198) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 ​ (199) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 227, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (200) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (201) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (202) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (203) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (204) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (205) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (206) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (207) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 235, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (208) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (209) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (210) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (211) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (212) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2、 (213) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (214) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (215)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 243, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (216)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, 3.The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequences of the complementarity determining regions (CDRs) defined according to the IMGT system are as follows: the amino acid sequence of the heavy chain CDR1 is GX 2X 3FNX 6YA, wherein X 2 is F or A, X 3 is T or A, and X 6 is S or A; the amino acid sequence of the heavy chain CDR2 is SEQ ID NO: 12; The amino acid sequence of the heavy chain CDR3 is VRHKKVREGYAMX 31 Y, X 31 is R, P, Q, H, N, T, V, or I; the amino acid sequence of the light chain CDR1 is SEQ ID NO: 14; the amino acid sequence of the light chain CDR2 is LAS; and the amino acid sequence of the light chain CDR3 is SEQ ID NO:

16. 4.The antibody or antigen-binding fragment thereof of claim 3, wherein the amino acid sequences of the complementarity determining regions are selected from at least one of the following groups: (1)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2; (2)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2; (3)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2. (4)the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2. ​ (5) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 235, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; (6) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, (7) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2,the heavy chain CDR3, the light chain CDR1, the light chain CDR2, (8) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2the heavy chain CDR3, the light chain CDR1, the light chain CDR2,the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, (9) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 the heavy chain CDR3, the light chain CDR1, the light chain CDR 2, the light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: (10) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 (11) the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein: the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 17; and the light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247 The heavy chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

18.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein: the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 17, the mutated site comprising G45A, F46A, T47A, F48A, N49A, A50G, A50S, Y51A, A52G, A52S, A52V, A52L, A52I, A52T, A52Y, A52D, A52K, I70A, R71H, R71K, R71G, R71D, R71L, R71S, R71W, R71A, T72S, T72Q, T72Y, T72N, T72G, T72L, T72M, T72W, T72A, K73A, S74A, N75A, N76A, Y77A, A78G, A78S, T79A, V118A, R119A, H120A, K121A, K121E, K121Y, K121P, K121Q, K121T, K121I, K121V, K121N, K121M, K121F, K122R, K122H, K122G, K122L, K122S, K122W, K122D, K122A, K122V, K122E, K122T, K122Q, K122N, K122M, K122I, K122P, K122F, K122Y, V123A, R124A, R124K, R124H, R124G, R124L, R124S, R124W, R124D, E125A, G126A, G126V, G126L, G126I, G126M, G126W, G126S, G126D, G126K, G126E, G126P, G126Q, G126H, G126T, G126R, G126N, G126F, G126Y, Y127A, Y127N, Y127Q, Y127M, Y127W, Y127S, Y127D, Y127K, Y127L, Y127G, Y127F, Y127R, Y127H, Y127I, Y127T, Y127E, Y127V, Y127P, A128G, A128S, M129A, D130A, D130G, D130L, D130M, D130W, D130S, D130K, D130E, D130R, D130P, D130Q, D130H, D130N, D130T, D130Y, D130V, D130F, D130I, or Y131A; and the light chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence obtained by mutating at least one site on the sequence set forth in SEQ ID NO: 19, the mutated site comprising K27A, S28A, V29A, S30A, T31A, S32A, G33A, Y34A, T35A, Y36A, L54A, A55G, A55S, S56A, Q94A, H95A, S96A, R97A, R97K, R97H, R97G, R97L, R97M, R97W, R97S, R97D, R97E, R97V, R97I, R97Q, R97F, R97P, R97T, R97N, R97Y, E98A, L99A, L99G, L99V, L99I, L99M, L99W, L99S, L99D, L99K, L99T, L99P, L99Q, L99N, L99H, L99E, L99Y, L99F, L99R, P100F, P100G, P100L, P100S, P100D, P100K, P100Q, P100W, P100A, F101A, F101P, F101G, F101L, F101S, F101D, F101K, F101Q, F101W, or T102A.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein: the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence obtained by mutating the sequence set forth in SEQ ID NO: 17 at at least one site, the mutated site comprising F46A, T47A, A50S, D130R, D130P, D130Q, D130H, D130N, D130T, D130V, or D130I; and the light chain variable region of the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:

19.

8. A bispecific antibody or antigen-binding fragment thereof, characterized in that, which comprises a first antigen binding domain that specifically binds to CTLA-4 and a second antigen binding domain that specifically binds to CCR8, wherein: the first antigen binding domain comprises complementarity determining regions (CDRs) defined according to the IMGT system, the amino acid sequences of which are as follows: heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, GAF, SEQ ID NO: 6, respectively; and the second antigen binding domain is selected from at least one of the following group: (1) the second antigen binding domain comprises complementarity determining regions (CDRs) defined according to the IMGT system, the amino acid sequences of which are as follows: the amino acid sequences of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3 are SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, LAS, SEQ ID NO: 16, respectively; or (2) the second antigen binding domain comprises the antibody or antigen binding fragment thereof of any one of claims 1-7.

9. The bispecific antibody or antigen binding fragment thereof of claim 8, wherein: the first antigen binding domain comprises a first heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 7, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 7; and a first light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 9, or an amino acid sequence that is at least 80%, 85%, 90%,95%, 98%, or 99% identical to the amino acid sequence as set forth in the second antigen binding domain comprises a second heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80%, 85%, 90%, or 95%, 98%, or 99% identical to the amino acid sequence as set forth SEQ ID NO: 17; and a second light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence that is at least 80%, 85%, 90%,or 95%, 98%, or 99% identical to the amino acid sequence as set forth 10. The bispecific antibody or antigen binding fragment thereof of claim 8 or 9, wherein: the first antigen binding domain comprises a first heavy chain variable region as set forth in SEQ ID NO: 7 and a first light chain variable region as set forth in SEQ ID NO: 9; and the second antigen binding domain comprises a second heavy chain variable region as set forth in SEQ ID NO: 17 and a second light chain variable region as set forth in SEQ ID NO:

19.

11. The bispecific antibody or antigen binding fragment thereof of any one of claims 8-10, further comprising a first constant region and a second constant region, the first and second constant regions being human IgG constant regions.

12. The bispecific antibody or antigen binding fragment thereof of claim 11, the human IgG constant regions being human IgG Fc regions.

13. The bispecific antibody or antigen-binding fragment thereof of claim 12, wherein: the first constant region comprises a first heavy chain constant region comprising T366S, L368A, and Y407V mutations according to EU numbering; and the second heavy chain constant region comprises a second heavy chain constant region comprising a T366W mutation according to EU numbering.

14. The bispecific antibody or antigen-binding fragment thereof of claim 13, wherein the first heavy chain constant region is operably linked to the second heavy chain constant region.

15. The bispecific antibody or antigen-binding fragment thereof of claim 12, wherein: the first heavy chain constant region comprises a Y349C mutation according to EU numbering; and the second heavy chain constant region comprises a S354C mutation according to EU numbering.

16. The bispecific antibody or antigen-binding fragment thereof of claim 15, wherein the first heavy chain constant region is operably linked to the second heavy chain constant region by a disulfide bond.

17. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-16. the polynucleotide encodes the antibody or antigen-binding fragment thereof of any one of claims 1-7 and / or the bispecific antibody or antigen-binding fragment thereof of any one of claims 8-16.

18. A recombinant vector, characterized in that, the recombinant vector comprises the polynucleotide of claim 17.

19. A host cell, characterized in that, the host cell comprises the polynucleotide of claim 17 and / or the recombinant vector of claim 18.

20. A chimeric antigen receptor, characterized in that, the chimeric antigen receptor comprises the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the bispecific antibody or antigen-binding fragment thereof of any one of claims 8-16.

21. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the bi specific antibody or antigen-binding fragment thereof of any one of claims 8-16, and a payload operably linked to the antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof.

22. A pharmaceutical combination comprising, The pharmaceutical combination comprises: the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the bispecific anti body or antigen-binding fragment thereof of any one of claims 8-16; and a PD-1 antagonist.

23. The pharmaceutical combination of claim 22, wherein the PD-1 antagonist comprises an antibody or antigen-binding fragment that binds PD-1.

24. A pharmaceutical composition comprising, The pharmaceutical composition comprises: the antibody or antigen-binding fragment thereof of any one of claims 1-7, the bispecific antibody or antigen-binding fragment thereof of any one of claims 8-16, the chimeric antigen receptor of claim 20, the antibody conjugate of claim 21, and / or the pharmaceutical combination of claim 22; and a pharmaceutically or physiologically acceptable carrier.

25. A kit comprising, The kit comprises the antibody or antigen-binding fragment thereof of any one of claims 1-7, the bispecific antibody or antigen-binding fragment thereof of any one of claims 8-16, the polynucleotide of claim 17, the recombinant vector of claim 18, the host cell of claim 19, the chimeric antigen receptor of claim 20, the antibody conjugate of claim 21, the pharmaceutical combination of claim 22 or 23, and / or the pharmaceutical composition of claim 24.

26. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7, the bispecifc antibody or antigen-binding fragment thereof of any one of claims 8-16, the polynucieotide of claim 17, the recombinant vector of claim 18, the host cell o claim 19, the chimeric antigen receptor of claim 20, the antibody conjugate o claim 21, the pharmaceutical combination of claim 22 or 23, the pharmaceutical composition of claim 24, and / or the kit of claim 25 in the manufacture of a medicament or agent for detecting, preventing, alleviating or treating a disease.

27. The use of claim 26, wherein the disease comprises a cancer or a tumor.

28. The use of claim 27, wherein the cancer or tumor comprises fibrosarcoma, colon cancer, bladder cancer, breast cancer.

29. A method of detecting, preventing, alleviating or treating a disease, characterized in that, The method comprises administering to a subject in need thereof the antibody or antigen-binding fragment thereof of any one of claims 1-7, the bispeefic antibody or antigen-binding fragment thereof of any one of claims 8-16, the polynueieotide of claim 17, the recombinant vector of claim 18, the host ceil of claim 19, the chimeric antigen receptor of claim 20, the antibody conjugates of claim 21, the pharmaceutical combination of claim 22 or 23, the pharmaceutical composition of ciaim 24, and / or the kit of claim 25.

30. The method of claim 29, wherein the disease comprises a cancer or a tumor.

31. The method of claim 30, wherein the cancer or tumor comprises fibrosarcoma, colon cancer, bladder cancer, breast caneer.

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