Anti-gitr antibodies and uses thereof

Abstract

The present application describes anti-GITR antibodies and antigen-binding fragments thereof. The present application also describes nucleic acids encoding these antibodies, compositions comprising these antibodies, methods of producing these antibodies, and methods of using these antibodies for treating or preventing diseases, such as cancer.

Description

Technical Field

[0001] This invention relates to antibodies or antigen-binding fragments thereof capable of specifically binding to the GITR protein (preferably human GITR protein), and the use of such agents. In some embodiments, this application relates to mouse and humanized monoclonal antibodies against GITR, and the use of these antibodies. These antibodies or antigen-binding fragments thereof can be used for diagnosis and treatment of diseases associated with GITR activity and / or expression.

[0002] References to sequence lists submitted electronically

[0003] This application includes a sequence list, which was submitted electronically via EFS-Web as an ASCII sequence list with the filename "688096.129 sequence list", created on December 28, 2019, and is 82kb in size. The sequence list submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0004] Cancer immunotherapy utilizes the body's own immune system to better identify, manage, and even reverse disease phenotypes by stimulating, amplifying, or supplementing the immune system. One of the most commonly used classes of immunotherapies involves targeting immune checkpoints such as programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T-cell immunoglobulin and mucin-containing domain protein-3 (TIM-3), lymphocyte activation gene 3 (LAG-3), and T-cell activation V domain Ig inhibitor (VISTA), which are key regulators of the immune response. The discovery of antibodies as immune checkpoint inhibitors has been one of the most successful methods in cancer drug discovery. Following the approval of ipilimumab, the first CTLA4-targeting checkpoint inhibitor, in 2011, immune checkpoint inhibitors are now available as first-line treatments for a variety of cancers, such as metastatic melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), breast cancer, urothelial carcinoma, and liver cancer.

[0005] Despite revolutionary successes in immune checkpoint blockade, a certain percentage of patients still do not benefit or show any positive response. Therefore, the development of alternative treatment options remains a major focus for further enhancing anti-tumor immunity. Among these, glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR) has attracted significant attention as an attractive immunotherapy target.

[0006] GITR belongs to the tumor necrosis factor receptor superfamily (TNFRSF). It is a cytokine receptor that binds to tumor necrosis factor (TNF) via its extracellular cysteine-rich domain. TNFRSF is involved in various cellular processes, from apoptosis and inflammation to other signal transduction pathways in proliferation, survival, and differentiation. GITR is identified as a member of TNFRSF and protects T cells from apoptosis upon exposure to glucocorticoids (GCs), which are potent inducers of T cell death and are commonly used as immunosuppressants and anti-inflammatory agents. GITR is located at CD25... + CD4 + GITR and GITRL are constitutively and exclusively expressed at high levels on regulatory T cells. Their ligand, GITRL, is primarily expressed by activated antigen-presenting cells (APCs). Expression of GITR and GITRL has also been observed on epidermal keratinocytes, osteoclast precursors, and epithelial cells. This suggests that the functions of GITR and GITRL may extend beyond regulating immune responses and involve mediating leukocyte adhesion and migration. The anticancer therapeutic potential of GITR-modulated function is attributed to its role in effector T cells (T cells). eff Co-stimulatory effects in tumors and regulatory T(T) on tumor invasiveness reg Cell suppression or depletion. Its role has been elucidated using preclinical tumor models of mouse GITR monoclonal antibodies (i.e., DTA-1), including increasing CD4+. + CD8 + T and CD4 + IL9 + (Th9) cell infiltration; decreased CD4 count + FoxP3 + T reg The level; and overall enhancement of T eff With T reg ratio.

[0007] Preclinical combinations of anti-GITR with other therapies, such as anti-PD-1 and anti-CTLA4, also potentially overcome T-cell depletion and enhance CD8. + T eff Promising results were observed in cell proliferation and invasion. Immunomodulation targeting GITR offers a promising opportunity to expand anticancer treatment options and could be considered in combination therapy with other immunomodulatory antibodies. Summary of the Invention

[0008] In one general aspect, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), preferably human GITR.

[0009] An isolated monoclonal antibody or its antigen-binding fragment is provided, comprising heavy chain complementarity-determining regions 1 (HCDR1), 1, 1, and 1; light chain complementarity-determining regions 1 (LCDR1), 1, 1, and 1; and LCDR3, having the following polypeptide sequence:

[0010] a. These are SEQ ID NO: 36, 37, 38, 39, 40, and 41, respectively;

[0011] b. These are SEQ ID NOs: 42, 43, 44, 45, 46, and 47, respectively;

[0012] c. These are SEQ ID NOs: 48, 49, 50, 54, 55, and 56, respectively;

[0013] d. These are SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively;

[0014] e. These are SEQ ID NOs: 57, 58, 59, 60, 61, and 62, respectively;

[0015] f. These are SEQ ID NOs: 63, 64, 65, 66, 67, and 68, respectively;

[0016] g. These are SEQ ID NO: 69, 70, 71, 72, 73, and 74, respectively;

[0017] h. are SEQ ID NO: 75, 76, 77, 78, 79, and 80, respectively;

[0018] i. These are SEQ ID NOs: 81, 82, 83, 84, 85, and 86, respectively;

[0019] j. are SEQ ID NO: 87, 88, 89, 93, 94, and 95 respectively;

[0020] k. are SEQ ID NO: 87, 88, 89, 96, 97, and 98 respectively;

[0021] l. These are SEQ ID NO: 90, 91, 92, 93, 94, and 95, respectively;

[0022] m. are SEQ ID NO: 90, 91, 92, 96, 97, and 98 respectively;

[0023] n. are SEQ ID NO:99, 100, 101, 102, 103, and 104 respectively;

[0024] o. are SEQ ID NO: 105, 106, 107, 108, 109, and 110 respectively;

[0025] p. are SEQ ID NO: 111, 112, 113, 114, 115, and 116 respectively;

[0026] q. are SEQ ID NO: 117, 118, 119, 120, 121, and 122 respectively;

[0027] r. are SEQ ID NO: 123, 124, 125, 126, 127, and 128 respectively;

[0028] s. are SEQ ID NO: 123, 124, 125, 129, 130, and 131, respectively;

[0029] t. are respectively SEQ ID NO: 123, 124, 125, 132, 133, and 134; or

[0030] u. are SEQ ID NO: 135, 136, 137, 138, 139, and 140 respectively;

[0031] Or variants of these isolated monoclonal antibodies or their antigen-binding fragments, which contain up to about three amino acid substitutions (e.g., one, two, or three amino acid substitutions) in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3.

[0032] The antibody or its antigen-binding fragment specifically binds to GITR, preferably human GITR.

[0033] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region or a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, 3, 5, 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 26, 28, 30, or 34, and the light chain variable region having a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 20, 21, 23, 25, 27, 29, 31, 32, 33, or 35.

[0034] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment comprises:

[0035] a. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:1 and the light chain variable region having the polypeptide sequence of SEQ ID NO:2;

[0036] b. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:3 and the light chain variable region having the polypeptide sequence of SEQ ID NO:4;

[0037] c. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:5 and the light chain variable region having the polypeptide sequence of SEQ ID NO:7;

[0038] d. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:6 and the light chain variable region having the polypeptide sequence of SEQ ID NO:7;

[0039] e. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:8 and the light chain variable region having the polypeptide sequence of SEQ ID NO:9;

[0040] f. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:10 and the light chain variable region having the polypeptide sequence of SEQ ID NO:11;

[0041] g. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:12 and the light chain variable region having the polypeptide sequence of SEQ ID NO:13;

[0042] h. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:14 and the light chain variable region having the polypeptide sequence of SEQ ID NO:15;

[0043] i. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:16 and the light chain variable region having the polypeptide sequence of SEQ ID NO:17;

[0044] j. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:18 and the light chain variable region having the polypeptide sequence of SEQ ID NO:20;

[0045] k. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:18 and the light chain variable region having the polypeptide sequence of SEQ ID NO:21;

[0046] l. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:19 and the light chain variable region having the polypeptide sequence of SEQ ID NO:20;

[0047] m. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:19 and the light chain variable region having the polypeptide sequence of SEQ ID NO:21;

[0048] n. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:22 and the light chain variable region having the polypeptide sequence of SEQ ID NO:23;

[0049] o. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:24 and the light chain variable region having the polypeptide sequence of SEQ ID NO:25;

[0050] p. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:26 and the light chain variable region having the polypeptide sequence of SEQ ID NO:27;

[0051] q. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:28 and the light chain variable region having the polypeptide sequence of SEQ ID NO:29;

[0052] r. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30 and the light chain variable region having the polypeptide sequence of SEQ ID NO:31;

[0053] s. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30 and the light chain variable region having the polypeptide sequence of SEQ ID NO:32;

[0054] t. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30, and the light chain variable region having the polypeptide sequence of SEQ ID NO:33; or

[0055] u. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:34 and the light chain variable region having the polypeptide sequence of SEQ ID NO:35.

[0056] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment is chimeric. In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment is chimeric, and the isolated monoclonal antibody or its antigen-binding fragment contains a human IgG1 constant region or a variant thereof. Variants of the human IgG1 constant region, for example, contain at least one amino acid modification selected from K214R, D356E, L358M, and ΔK447.

[0057] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment is human or humanized.

[0058] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment is humanized, and the isolated monoclonal antibody or its antigen-binding fragment comprises:

[0059] a. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:143 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0060] b. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:143 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0061] c. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:144 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0062] d. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:144 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0063] e. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:145 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0064] f. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:145 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0065] g. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:148 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0066] h. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:149 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0067] i. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:150 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0068] j. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:152, and the light chain variable region having the polypeptide sequence of SEQ ID NO:154; or

[0069] k. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:153 and the light chain variable region having the polypeptide sequence of SEQ ID NO:154.

[0070] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment binds to GITR and is able to induce effector-mediated tumor cell lysis.

[0071] Also provided are isolated nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0072] Vectors comprising isolated nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention are also provided.

[0073] Host cells comprising vectors containing isolated nucleic acids encoding the monoclonal antibody of the present invention or an antigen-binding fragment thereof are also provided.

[0074] In some embodiments, a pharmaceutical composition is provided comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0075] A method of treating a subject with cancer in need is also provided, comprising administering the pharmaceutical composition of the invention to the subject. The cancer can be any liquid or solid tumor, for example, selected from, but not limited to, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0076] Methods for generating the monoclonal antibody or antigen-binding fragment thereof of the present invention are also provided. These methods include culturing a cell containing a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for generating the monoclonal antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof from the cell or culture.

[0077] Methods for generating pharmaceutical compositions comprising the monoclonal antibody or its antigen-binding fragment of the present invention are also provided. These methods include combining the monoclonal antibody or its antigen-binding fragment with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0078] Methods for determining the GITR level of a subject are also provided. These methods include: (a) obtaining a sample from the subject; (b) contacting the sample with the isolated monoclonal antibody or its antigen-binding fragment of the present invention; and (c) determining the GITR level of the subject. The sample may be, for example, a tissue sample or a blood sample. The tissue sample may be, for example, a cancer tissue sample. Attached Figure Description

[0079] The foregoing summary and the following detailed description of preferred embodiments of this application will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that this application is not limited to the precise embodiments shown in the drawings.

[0080] Figure 1A-1K The results show the antigen-specific humoral response. The results present antiserum titers against the following assays using ELISA: allotype control ( Figure 1A ); pre-serum ( Figure 1B ); PBS Figure 1C ); mouse AD94 ( Figure 1D ); mouse AD95 ( Figure 1E ); mouse AD96 ( Figure 1F ); mouse AD97 ( Figure 1G ); mouse AD98 ( Figure 1H ); mouse AD99 ( Figure 1I ); mouse AD100 ( Figure 1J ); and mouse AD101 ( Figure 1K ).

[0081] Figures 2A-2D The figure shows the FACS results for the anti-GITR mouse antibody.

[0082] Figure 3 The figure shows the results of the dose-response assay of the functional reporter gene of the anti-GITR mouse antibody.

[0083] Figures 4A-4C The figure shown illustrates the FACS results using both human GITR / Cho-K1 and cyno-GITR / Cho-K1 cell lines. Figure 4A and 4B The diagram showing the combination of FACS for the GITR of the human is displayed. Figure 4C The diagram shows the FACS integration of cyno-GITR.

[0084] Figures 5A-5C The figure shown illustrates the FACS binding assay results of the humanized anti-GITR antibody. Figure 5A :45F1F3; Figure 5B : 270C5C10; and Figure 5C :225H7D12).

[0085] Figures 6A-6B The figure shows the results of a functional bioassay of the humanized anti-GITR antibody reporter gene. Detailed Implementation

[0086] Various publications, articles, and patents are cited or described in the background and throughout this specification; each of these references is incorporated herein by reference in its entirety. Discussions of documents, bills, materials, devices, articles, etc., included in this specification are for the purpose of providing background to the invention. Such discussion is not an admission that any or all of these matters constitute part of the prior art with respect to any disclosed or claimed invention.

[0087] 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 to which this invention pertains. In other respects, certain terms used herein have the meanings set forth in the specification.

[0088] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a / an” and “the” as used herein and in the appended claims include plural indicators.

[0089] Unless otherwise stated, any numerical value (such as concentrations or concentration ranges as described herein) should be understood to be modified by the term “about” in all cases. Therefore, numerical values ​​generally include ±10% of the listed values. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions of values ​​within such ranges, unless the context clearly indicates otherwise.

[0090] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Such equivalents are intended to be covered within the scope of this invention.

[0091] As used herein, the terms “comprises,” “comprising,” “includes,” “has,” “having,” “contains,” or any other variation thereof will be understood to mean including the stated integers or groups of integers, but not excluding any other integers or groups of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed, or elements inherent to such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, “or” means inclusive or rather than exclusive or. For example, any of the following satisfies condition A or B: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0092] As used herein, the connecting term “and / or” between multiple listed elements should be understood to encompass both individual and combined options. For example, when two elements are connected by “and / or”, the first option refers to the applicability of the first element in the absence of the second element. The second option refers to the applicability of the second element in the absence of the first element. The third option refers to the applicability of the first and second elements together. Any one of these options should be understood to fall within this meaning and thus satisfy the requirement of the term “and / or” as used herein. The simultaneous applicability of more than one of these options should also be understood to fall within this meaning and thus satisfy the requirement of the term “and / or”.

[0093] As used herein, “subject” means any animal, preferably a mammal, and most preferably a human. The term “mammal” as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and more preferably humans.

[0094] It should also be understood that the terms “about,” “approximately,” “usually,” “substantially,” and similar terms used herein, when referring to the size or feature of a component of a preferred invention, indicate that the described size / feature is not a strict boundary or parameter and does not exclude minor variations that are functionally identical or similar to it, as would be understood by one of ordinary skill in the art. At a minimum, such indicators including numerical parameters will include variations that do not change the least significant figure using mathematical and industrial principles accepted in the art (e.g., rounding, measurement errors or other systematic errors, manufacturing tolerances, etc.).

[0095] In the context of two or more nucleic acid or polypeptide sequences (e.g., anti-GITR antibodies and polynucleotides encoding these antibodies, GITR polypeptides and GITR polynucleotides encoding these polypeptides), the term "identity" or "identity percentage" refers to the fact that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned to the maximum correspondence, as measured by visual inspection using one of the following sequence comparison algorithms.

[0096] For sequence comparisons, typically one sequence is used as a reference sequence, and the test sequence is compared to this reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. Based on the specified program parameters, the sequence comparison algorithm then calculates the percentage of sequence identity between one or more test sequences and the reference sequence.

[0097] The optimal alignment of sequences for comparison can be performed using, for example: Smith and Waterman’s local homology algorithm, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch’s homology alignment algorithm, J. Mol. Biol. 48:443 (1970); Pearson and Lipman’s similarity search method, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); or visual inspection (generally see Current Protocols in Molecular Biology, FMAusubel et al., editors, Current Protocols, Greene Publishing Associates, Inc. and John). A joint venture of Wiley & Sons, Inc. (1995 Supplement) (Ausubel)

[0098] Examples of suitable algorithms for determining sequence identity percentages and sequence similarity are the BLAST and BLAST2.0 algorithms, described by Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-3402, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) in the query sequence by identifying short words of length W that match or satisfy a positive threshold score T when aligned with words of the same length in a database sequence. T is called the neighborhood word score threshold (Altschul et al., ibid.). These initial neighborhood word hits act as seeds for a search to find longer HSPs containing them. These word hits are then extended in both directions along each sequence until the cumulative alignment score can be increased.

[0099] For nucleotide sequences, a cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped if: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score tends to zero or falls below zero due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and a comparison of two strands as default values. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix as default values ​​(see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0100] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of the test nucleic acid with a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0101] A further indication that two nucleic acid sequences or peptides are substantially identical is the immune cross-reaction between a peptide encoded by the first nucleic acid and a peptide encoded by the second nucleic acid, as described below. Therefore, for example, if the difference between the two peptides lies only in conserved substitutions, the two peptides are generally substantially identical. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0102] As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated from, produced separately from, or purified from other biological components of the organism in which it is naturally present, namely other chromosomal and extrachromosomal DNA and RNA, as well as proteins. Therefore, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified using standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a composition and are considered isolated when the composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also includes nucleic acids, peptides, and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0103] As used herein, the term "polynucleotide" is synonymous with "nucleic acid molecule," "nucleotide," or "nucleic acid," and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, and DNA of mixtures of single-stranded and double-stranded regions; single-stranded and double-stranded RNA, and RNA of mixtures of single-stranded and double-stranded regions; and hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. Additionally, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA, or simultaneously comprising RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and unusual bases such as inosine. DNA and RNA can be modified in a variety of ways; therefore, "polynucleotides" include polynucleotides in chemically, enzymatically, or metabolically modified forms commonly found in nature, as well as chemical forms of DNA and RNA specific to viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains, often called oligonucleotides.

[0104] As used in this article, the term "vector" is a replicon in which another nucleic acid segment can be efficiently inserted to cause replication or expression of that segment.

[0105] As used herein, the term "host cell" refers to a cell containing the nucleic acid molecules of the present invention. A "host cell" can be any type of cell, such as a primary cell, a cell in a culture, or a cell derived from a cell line. In one embodiment, a "host cell" is a cell transfected or transduced with the nucleic acid molecules of the present invention. In another embodiment, a "host cell" is a progeny or potential progeny of such transfected or transduced cells. The progeny of a cell may be the same as or may differ from the parent cell due to, for example, mutations or environmental influences that may occur in the offspring or when the nucleic acid molecules are integrated into the host cell genome.

[0106] As used herein, the term "expression" refers to the biosynthesis of a gene product. This term encompasses the transcription of a gene into RNA. It also encompasses the translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed polypeptide can reside in the cytoplasm of the host cell, enter the extracellular environment (such as the growth medium of a cell culture), or anchor to the cell membrane.

[0107] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule composed of amino acids and can be considered a protein by those skilled in the art. The conventional single-letter or three-letter codes for amino acid residues are used herein. The terms “peptide,” “polypeptide,” and “protein,” used interchangeably herein, refer to an amino acid polymer of any length. This polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acid components. These terms also cover naturally modified or intervened amino acid polymers; for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. These definitions also include, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. For example, the peptides, polypeptides, and proteins of the present invention may contain one or more amino acid substitutions.

[0108] The peptide sequences described herein are written according to usual convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although amino acid isomers are known, the L-form of the amino acid is represented unless otherwise explicitly indicated.

[0109] The term "amino acid modification" at a specified location, such as an amino acid modification in the Fc region, refers to the substitution or deletion of a specified residue, or the insertion of at least one amino acid residue adjacent to that specified residue. An insertion "adjacent" to a specified residue means insertion within one or two residues therein. Insertion can be at the N-terminus or C-terminus of the specified residue. Substitution is the preferred amino acid modification described herein.

[0110] As used herein, the term "amino acid substitution" refers to the replacement of one amino acid residue in a polypeptide sequence with another amino acid residue. "Conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue with a side chain having similar chemical characteristics. Families of amino acid residues with similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, phenylalanine replacing tyrosine is a conservative substitution. Typically, conserved substitutions in the sequences of the peptides, soluble proteins, and / or antibodies disclosed herein do not eliminate the binding of the amino acid sequence to the target binding site. Methods for identifying conserved substitutions of amino acids that do not eliminate binding are well known in the art.

[0111] As used herein, the term "variant" relating to an antibody or antigen-binding fragment thereof ("reference antibody") having a polypeptide with a specific sequence characteristic refers to a different antibody having a polypeptide that, relative to the reference binding portion, comprises one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and / or additions. Anti-GITR antibodies or antigen-binding fragment variants thereof at least retain specific binding to GITR. In some embodiments, anti-GITR antibodies or antigen-binding fragment variants thereof may originate from one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to the amino acid sequence of the reference antibody. In some embodiments, anti-GITR antibodies or antigen-binding fragment variants thereof may comprise at least three (3) amino acid substitutions.

[0112] Antibody

[0113] This invention generally relates to isolated antibodies against glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), nucleic acids encoding these antibodies and expression vectors, recombinant cells containing these vectors, and compositions comprising these antibodies. Methods for preparing these antibodies and methods for treating diseases including cancer using these antibodies are also provided. The antibodies of this invention possess one or more desired functional properties, including but not limited to high affinity binding to GITR, high specificity for GITR, and the ability to inhibit tumor growth in desired subjects and animal models when administered alone or in combination with other anticancer therapies.

[0114] In general, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to GITR.

[0115] As used herein, the term "antibody" is used broadly and includes immunoglobulins or antibody molecules, including human antibodies, humanized antibodies, complex antibodies, chimeric antibodies, and monoclonal or polyclonal antibody fragments. Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. Antibody structures are well known. Immunoglobulins can be classified into five main classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of their heavy chain constant domain. IgA and IgG can be further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Accordingly, the antibodies of the present invention can belong to any of the five main classes or their corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. The light chains of antibodies in vertebrate species can be classified into one of two distinct types based on the amino acid sequence of their constant domains, namely κ and λ. Accordingly, the antibodies of the present invention can contain either a κ or λ light chain constant domain. According to specific embodiments, the antibodies of the present invention comprise constant regions of the heavy and / or light chains derived from rat or human antibodies. In addition to the constant heavy and light chain domains, the antibody also contains an antigen-binding region composed of variable light and heavy chain regions, each of which contains three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains may alternatively be referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains may alternatively be referred to as HCDR1, HCDR2, and HCDR3.

[0116] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to GITR is substantially free of antibodies that do not bind to GITR). Additionally, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0117] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies (i.e., the individual antibodies constituting the group are identical except for a small amount of potentially naturally occurring mutations). The monoclonal antibodies of this invention can be prepared using hybridoma methods, phage display technology, single-lymphocyte gene cloning technology, or recombinant DNA methods. For example, monoclonal antibodies can be produced from hybridomas comprising B cells obtained from transgenic nonhuman animals (such as transgenic mice or rats) having a genome containing human heavy chain transgenes and light chain transgenes.

[0118] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as a biantibody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stable Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stable biantibody (ds biantibody), single-chain antibody molecule (scFv), single-domain antibody (sdAb), scFv dimer (bivalent biantibody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, humped single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen that the parent antibody or the parent antibody fragment binds to. According to a particular embodiment, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the heavy chain. According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab')2.

[0119] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that comprises a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region, or comprises only the heavy chain variable region.

[0120] As used herein, the term "human antibody" means an antibody produced by a human being or an antibody having an amino acid sequence corresponding to that of a human-produced antibody, prepared using any technique known in the art. This definition of human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide.

[0121] As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology with human antibodies in order to retain the antigen-binding properties of the antibody, but whose antigenicity is reduced in the human body.

[0122] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable regions of both the light and heavy chains typically correspond to the variable regions of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions correspond to the sequence of antibodies derived from another mammalian species (e.g., human) to avoid triggering an immune response in that species.

[0123] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In embodiments, the first and second epitopes are located on the same antigen (e.g., the same protein (or a subunit of a multimeric protein)). In embodiments, the first and second epitopes overlap or substantially overlap. In embodiments, the first and second epitopes do not overlap or substantially do not overlap. In embodiments, the first and second epitopes are located on different antigens (e.g., different proteins (or different subunits of a multimeric protein)). In embodiments, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In embodiments, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0124] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In embodiments, the first and second epitopes are on the same antigen (e.g., the same protein (or a subunit of a multimeric protein)). In embodiments, the first and second epitopes overlap or substantially overlap. In embodiments, the first and second epitopes are on different antigens (e.g., different proteins (or different subunits of a multimeric protein)). In embodiments, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to the second epitope. In embodiments, a bispecific antibody comprises a hapten or a fragment thereof having binding specificity to the first epitope, and a hapten or a fragment thereof having binding specificity to the second epitope. In this embodiment, the bispecific antibody comprises an scFv or a fragment thereof that binds specifically to a first epitope, and an scFv or a fragment thereof that binds specifically to a second epitope. In this embodiment, the first epitope is located on a GITR, and the second epitope is located on an immune checkpoint molecule and / or other tumor-associated immunosuppressive factors or surface antigens.

[0125] As used herein, the term "GITR" refers to the glucocorticoid-induced tumor necrosis factor receptor-associated protein. GITR belongs to the tumor necrosis factor receptor superfamily (TNFRSF) and is a cytokine receptor that binds to tumor necrosis factor (TNF) via an extracellular cysteine-rich domain. TNFRSF is involved in various cellular processes, from apoptosis and inflammation to other signal transduction pathways in proliferation, survival, and differentiation. GITR is identified as a member of TNFRSF and protects T cells from apoptosis upon exposure to glucocorticoids (GCs), which are potent inducers of T cell death and are commonly used as immunosuppressants and anti-inflammatory agents. GITR is located at CD25 + CD4 + GITR and GITRL are constitutively and exclusively expressed at high levels on regulatory T cells. Their ligand, GITRL, is primarily expressed by activated antigen-presenting cells (APCs). Expression of GITR and GITRL has also been observed on epidermal keratinocytes, osteoclast precursors, and epithelial cells. This suggests that the functions of GITR and GITRL may extend beyond regulating immune responses and may also involve mediating leukocyte adhesion and migration. The anticancer therapeutic potential of GITR-modulated function is attributed to its role in effector T cells (T cells). eff Co-stimulatory effects in tumors and regulatory T(T) on tumor invasiveness regGITR can inhibit or deplete cells. Therefore, GITR is a tumor-associated / tumor-specific antigen, and anti-GITR monoclonal antibodies (mAbs) may be potential anticancer therapies. Furthermore, GITR can be used to specifically target cancer cells with therapeutic molecules. Exemplary amino acid sequences of human GITR are represented by GenBank accession numbers NP_004186.1 (isotype 1), NP_683699.1 (isotype 2), and / or NP_683700.1 (isotype 3).

[0126] As used in this article, an antibody that "specifically binds to GITR" refers to an antibody with a concentration of 1×10⁻⁶. -7 M or less, preferably 1×10 -8 M or less, more preferably 5×10 -9 M or below, 1×10 -9 M or below, 5×10 -10 M or less, or 1×10 -10 M or below K D Antibodies that bind to GITR (preferably human GITR). The term "K" D "" refers to the dissociation constant, which is derived from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as molar concentration (M). In view of this disclosure, the Kd of an antibody can be determined using methods in the art. D Value. For example, the K value of an antibody. D It can be determined by using surface plasmon resonance, for example, by using a biosensor system (e.g., (System), or by using biofilm layer interference techniques (such as the Octet RED96 system).

[0127] antibody K D The smaller the value, the higher the affinity of the antibody for binding to the target antigen.

[0128] According to a particular aspect, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof, comprising heavy chain complementarity-determining regions 1 (HCDR1), 1, 1, and 1, and light chain complementarity-determining regions 1 (LCDR1), 1, 1, and 1, having the following polypeptide sequence:

[0129] a. These are SEQ ID NO: 36, 37, 38, 39, 40, and 41, respectively;

[0130] b. These are SEQ ID NOs: 42, 43, 44, 45, 46, and 47, respectively;

[0131] c. These are SEQ ID NOs: 48, 49, 50, 54, 55, and 56, respectively;

[0132] d. These are SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively;

[0133] e. These are SEQ ID NOs: 57, 58, 59, 60, 61, and 62, respectively;

[0134] f. These are SEQ ID NOs: 63, 64, 65, 66, 67, and 68, respectively;

[0135] g. These are SEQ ID NO: 69, 70, 71, 72, 73, and 74, respectively;

[0136] h. are SEQ ID NO: 75, 76, 77, 78, 79, and 80, respectively;

[0137] i. These are SEQ ID NOs: 81, 82, 83, 84, 85, and 86, respectively;

[0138] j. are SEQ ID NO: 87, 88, 89, 93, 94, and 95 respectively;

[0139] k. are SEQ ID NO: 87, 88, 89, 96, 97, and 98 respectively;

[0140] l. These are SEQ ID NO: 90, 91, 92, 93, 94, and 95, respectively;

[0141] m. are SEQ ID NO: 90, 91, 92, 96, 97, and 98 respectively;

[0142] n. are SEQ ID NO:99, 100, 101, 102, 103, and 104 respectively;

[0143] o. are SEQ ID NO: 105, 106, 107, 108, 109, and 110 respectively;

[0144] p. are SEQ ID NO: 111, 112, 113, 114, 115, and 116 respectively;

[0145] q. are SEQ ID NO: 117, 118, 119, 120, 121, and 122 respectively;

[0146] r. are SEQ ID NO: 123, 124, 125, 126, 127, and 128 respectively;

[0147] s. are SEQ ID NO: 123, 124, 125, 129, 130, and 131, respectively;

[0148] t. are respectively SEQ ID NO: 123, 124, 125, 132, 133, and 134; or

[0149] u. are SEQ ID NO: 135, 136, 137, 138, 139, and 140 respectively;

[0150] Or a variant of the isolated monoclonal antibody or its antigen-binding fragment, which contains up to about three (e.g., one, two, or three) amino acid substitutions in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3.

[0151] The antibody or its antigen-binding fragment specifically binds to GITR, preferably human GITR.

[0152] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment comprises heavy chain complementarity-determining regions 1 (HCDR1), 1, ...

[0153] a. These are SEQ ID NO: 36, 37, 38, 39, 40, and 41, respectively;

[0154] b. These are SEQ ID NOs: 42, 43, 44, 45, 46, and 47, respectively;

[0155] c. These are SEQ ID NOs: 48, 49, 50, 54, 55, and 56, respectively;

[0156] d. These are SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively;

[0157] e. These are SEQ ID NOs: 57, 58, 59, 60, 61, and 62, respectively;

[0158] f. These are SEQ ID NOs: 63, 64, 65, 66, 67, and 68, respectively;

[0159] g. These are SEQ ID NO: 69, 70, 71, 72, 73, and 74, respectively;

[0160] h. are SEQ ID NO: 75, 76, 77, 78, 79, and 80, respectively;

[0161] i. These are SEQ ID NOs: 81, 82, 83, 84, 85, and 86, respectively;

[0162] j. are SEQ ID NO: 87, 88, 89, 93, 94, and 95 respectively;

[0163] k. are SEQ ID NO: 87, 88, 89, 96, 97, and 98 respectively;

[0164] l. These are SEQ ID NO: 90, 91, 92, 93, 94, and 95, respectively;

[0165] m. are SEQ ID NO: 90, 91, 92, 96, 97, and 98 respectively;

[0166] n. are SEQ ID NO:99, 100, 101, 102, 103, and 104 respectively;

[0167] o. are SEQ ID NO: 105, 106, 107, 108, 109, and 110 respectively;

[0168] p. are SEQ ID NO: 111, 112, 113, 114, 115, and 116 respectively;

[0169] q. are SEQ ID NO: 117, 118, 119, 120, 121, and 122 respectively;

[0170] r. are SEQ ID NO: 123, 124, 125, 126, 127, and 128 respectively;

[0171] s. are SEQ ID NO: 123, 124, 125, 129, 130, and 131, respectively;

[0172] t. are respectively SEQ ID NO: 123, 124, 125, 132, 133, and 134; or

[0173] u. are SEQ ID NO: 135, 136, 137, 138, 139, and 140 respectively;

[0174] The antibody or its antigen-binding fragment specifically binds to GITR, preferably human GITR.

[0175] According to another specific aspect, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof, comprising a heavy chain variable region or a light chain variable region, the heavy chain variable region having at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to one of SEQ ID NO: 1, 3, 5, 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 26, 28, 30, or 34, and the light chain variable region having 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to one of SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 20, 21, 23, 25, 27, 29, 31, 32, 33, or 35 ... According to a preferred embodiment, the isolated monoclonal antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:1, 3, 5, 6, 8, 10, 12, 14, 16, 18, 19, 22, 24, 26, 28, 30, or 34, and the light chain variable region has a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:2, 4, 7, 9, 11, 13, 15, 17, 20, 21, 23, 25, 27, 29, 31, 32, 33, or 35 ...

[0176] According to another particular aspect, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof of the present invention, comprising:

[0177] a. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:1 and the light chain variable region having the polypeptide sequence of SEQ ID NO:2;

[0178] b. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:3 and the light chain variable region having the polypeptide sequence of SEQ ID NO:4;

[0179] c. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:5 and the light chain variable region having the polypeptide sequence of SEQ ID NO:7;

[0180] d. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:6 and the light chain variable region having the polypeptide sequence of SEQ ID NO:7;

[0181] e. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:8 and the light chain variable region having the polypeptide sequence of SEQ ID NO:9;

[0182] f. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:10 and the light chain variable region having the polypeptide sequence of SEQ ID NO:11;

[0183] g. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:12 and the light chain variable region having the polypeptide sequence of SEQ ID NO:13;

[0184] h. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:14 and the light chain variable region having the polypeptide sequence of SEQ ID NO:15;

[0185] i. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:16 and the light chain variable region having the polypeptide sequence of SEQ ID NO:17;

[0186] j. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:18 and the light chain variable region having the polypeptide sequence of SEQ ID NO:20;

[0187] k. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:18 and the light chain variable region having the polypeptide sequence of SEQ ID NO:21;

[0188] l. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:19 and the light chain variable region having the polypeptide sequence of SEQ ID NO:20;

[0189] m. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:19 and the light chain variable region having the polypeptide sequence of SEQ ID NO:21;

[0190] n. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:22 and the light chain variable region having the polypeptide sequence of SEQ ID NO:23;

[0191] o. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:24 and the light chain variable region having the polypeptide sequence of SEQ ID NO:25;

[0192] p. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:26 and the light chain variable region having the polypeptide sequence of SEQ ID NO:27;

[0193] q. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:28 and the light chain variable region having the polypeptide sequence of SEQ ID NO:29;

[0194] r. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30 and the light chain variable region having the polypeptide sequence of SEQ ID NO:31;

[0195] s. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30 and the light chain variable region having the polypeptide sequence of SEQ ID NO:32;

[0196] t. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30, and the light chain variable region having the polypeptide sequence of SEQ ID NO:33; or

[0197] u. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:34 and the light chain variable region having the polypeptide sequence of SEQ ID NO:35.

[0198] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO:36, 37, 38, 39, 40, and 41, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:1, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:2. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1; and a light chain variable region having the polypeptide sequence of SEQ ID NO:2.

[0199] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO:42, 43, 44, 45, 46, and 47, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:3, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:4. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3; and a light chain variable region having the polypeptide sequence of SEQ ID NO:4.

[0200] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO:48, 49, 50, 54, 55, and 56, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:5, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:7. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5; and a light chain variable region having the polypeptide sequence of SEQ ID NO:7.

[0201] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO:51, 52, 53, 54, 55, and 56, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:6, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:7. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:6; and a light chain variable region having the polypeptide sequence of SEQ ID NO:7.

[0202] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 57, 58, 59, 60, 61, and 62, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 8, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 9. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:8; and a light chain variable region having the polypeptide sequence of SEQ ID NO:9.

[0203] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 63, 64, 65, 66, 67, and 68, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 10, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 11. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:10; and a light chain variable region having the polypeptide sequence of SEQ ID NO:11.

[0204] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO:69, 70, 71, 72, 73, and 74, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:12, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:13. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:12; and a light chain variable region having the polypeptide sequence of SEQ ID NO:13.

[0205] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO:75, 76, 77, 78, 79, and 80, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:14, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:15. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:14; and a light chain variable region having the polypeptide sequence of SEQ ID NO:15.

[0206] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 81, 82, 83, 84, 85, and 86, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 16, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 17. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:16; and a light chain variable region having the polypeptide sequence of SEQ ID NO:17.

[0207] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 87, 88, 89, 93, 94, and 95, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 18, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 20. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:18; and a light chain variable region having the polypeptide sequence of SEQ ID NO:20.

[0208] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 87, 88, 89, 96, 97, and 98, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 18, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 21. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:18; and a light chain variable region having the polypeptide sequence of SEQ ID NO:21.

[0209] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 90, 91, 92, 93, 94, and 95, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 19, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 20. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:19; and a light chain variable region having the polypeptide sequence of SEQ ID NO:20.

[0210] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 90, 91, 92, 96, 97, and 98, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 19, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 21. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:19; and a light chain variable region having the polypeptide sequence of SEQ ID NO:21.

[0211] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 99, 100, 101, 102, 103, and 104, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 22, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 23. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:22; and a light chain variable region having the polypeptide sequence of SEQ ID NO:23.

[0212] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 105, 106, 107, 108, 109, and 110, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 24, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 25. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:24; and a light chain variable region having the polypeptide sequence of SEQ ID NO:25.

[0213] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 111, 112, 113, 114, 115, and 116, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 26, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 27. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:26; and a light chain variable region having the polypeptide sequence of SEQ ID NO:27.

[0214] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 117, 118, 119, 120, 121, and 122, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 28, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 29. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:28; and a light chain variable region having the polypeptide sequence of SEQ ID NO:29.

[0215] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 123, 124, 125, 126, 127, and 128, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 30, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 31. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:30; and a light chain variable region having the polypeptide sequence of SEQ ID NO:31.

[0216] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 123, 124, 125, 129, 130, and 131, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 30, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 32. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:30; and a light chain variable region having the polypeptide sequence of SEQ ID NO:32.

[0217] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 123, 124, 125, 132, 133, and 134, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 30, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 33. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:30; and a light chain variable region having the polypeptide sequence of SEQ ID NO:33.

[0218] In one embodiment, the present invention relates to an isolated monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 135, 136, 137, 138, 139, and 140, respectively. In another embodiment, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 34, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 35. Preferably, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:34; and a light chain variable region having the polypeptide sequence of SEQ ID NO:35.

[0219] According to another specific aspect, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof, wherein the antibody or antigen-binding fragment is chimeric. In some embodiments, the isolated monoclonal antibody or antigen-binding fragment is chimeric, and the isolated monoclonal antibody or antigen-binding fragment comprises a human IgG1 constant region or a variant thereof. The human IgG1 constant region comprises a human IgG1 heavy chain constant region and a light chain κ constant region. Variants of the human IgG1 heavy chain constant region, for example, comprise at least one amino acid modification selected from K214R, D356E, L358M, and ΔK447. In some embodiments, the constant region of the human IgG1 heavy chain comprises the polypeptide sequence of SEQ ID NO:141, and the constant region of the human IgG1 light chain comprises the polypeptide sequence of SEQ ID NO:142. In some embodiments, the constant region of the variant human IgG1 heavy chain comprises the polypeptide sequence of SEQ ID NO:155, and the constant region of the human IgG1 light chain comprises the polypeptide sequence of SEQ ID NO:142. In some embodiments, the constant region of the human IgG1 heavy chain variant comprises the polypeptide sequence of SEQ ID NO:156, and the constant region of the human IgG1 light chain comprises the polypeptide sequence of SEQ ID NO:142. For example, the variable regions of clones 45F1F3, 225H7D12, 270C5C10, 163H12G7, 172H7B9, and 223F12C4 described below are fused to the human IgG1 constant region having modifications of K214R, D356E, L358M, and ΔK447. As another example, the variable regions of clones 204C10G12, 225A8D9, 274C7H2, 384H3H11, 134D7B3, 177C6B11, 215F2A5, and 223H11H1 described below are fused to the human IgG1 constant region having a modification of K214R.

[0220] In some embodiments, the anti-GITR monoclonal antibody or its antigen-binding fragment is a chimeric monoclonal antibody or its antigen-binding fragment. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a murine animal such as a mouse) and a human constant region. In another instance, the chimeric antibody is a “class-switching” antibody, wherein the class or subclass has been changed relative to the class or subclass of the parent antibody.

[0221] According to another particular aspect, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof, wherein the antibody or antigen-binding fragment is human or humanized.

[0222] In some embodiments, the chimeric antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies contain one or more variable domains, wherein, for example, the HVR (or a portion thereof) of the CDR is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies may also optionally contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), for example, to restore or enhance antibody specificity or affinity.

[0223] Humanized antibodies and their preparation methods are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (Describes SDR(a-CDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (Describes “Surface Rework”); Dall'Acqua et al., Methods 36:43-60 (2005) (Describes “FR Reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (Describes the “guided selection” approach to FR reorganization).

[0224] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best fit” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions derived from the common sequence of human antibodies from specific light chain or heavy chain variable region subgroups (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)). ); human mature (somatic mutation) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and frame regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0225] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment is humanized, and the isolated monoclonal antibody or its antigen-binding fragment comprises:

[0226] a. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:143 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0227] b. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:143 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0228] c. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:144 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0229] d. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:144 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0230] e. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:145 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0231] f. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:145 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0232] g. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:148 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0233] h. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:149 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0234] i. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:150 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0235] j. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:152, and the light chain variable region having the polypeptide sequence of SEQ ID NO:154; or

[0236] k. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:153 and the light chain variable region having the polypeptide sequence of SEQ ID NO:154.

[0237] In one embodiment, the present invention relates to an isolated humanized monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO: 36, 37, 38, 39, 40, and 41, respectively. In another embodiment, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 143, 144, or 145, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 146 or 147. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:143, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:146. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:143, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:147. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:144, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:146. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:144, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:147.Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:145, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:146. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:145, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:147.

[0238] In one embodiment, the present invention relates to an isolated humanized monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO:42, 43, 44, 45, 46, and 47, respectively. In another embodiment, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:148, 149, or 150, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:151. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:148, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:151. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:149, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:151. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:150, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:151.

[0239] In one embodiment, the present invention relates to an isolated humanized monoclonal antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NO:48, 49, 50, 54, 55, and 56, respectively. In another embodiment, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:152 or 153, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO:154. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:152, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:154. Preferably, the isolated humanized monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:153, and the light chain variable region has a polypeptide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:154.

[0240] Antibody CDRs are defined by those skilled in the art using various methods / systems. These systems and / or definitions have been developed and refined over many years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is widely used. The Chothia definition is based on the location of structural loop regions. The IMGT system is based on sequence variability and location within variable domain structures. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. An exemplary system is Kabat.

[0241] In another general aspect, the present invention relates to isolated nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. Those skilled in the art will understand that the coding sequence of a protein can be altered without changing the amino acid sequence of the protein (e.g., substitution, deletion, insertion, etc.). Accordingly, those skilled in the art will understand that the nucleic acid sequence encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0242] In another general aspect, the present invention relates to a vector comprising isolated nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof of the present invention. In view of this disclosure, any vector known to those skilled in the art, such as plasmids, granules, phage vectors, or viral vectors, may be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements for establishing the conventional function of the expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive, inducible, or repressive promoter. Many expression vectors capable of delivering nucleic acids to cells are known in the art and may be used herein to generate antibodies or antigen-binding fragments thereof in cells. Recombinant expression vectors according to embodiments of the present invention may be generated using conventional cloning techniques or artificial gene synthesis. Such techniques are well known to those skilled in the art in view of this disclosure.

[0243] In another general aspect, the present invention relates to host cells comprising isolated nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. In view of this disclosure, any host cell known to those skilled in the art can be used for recombinant expression of the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is *E. coli* TG1 or BL21 cells (for expressing, for example, scFv or Fab antibodies), CHO-DG44 or CHO-K1 cells, or HEK293 cells (for expressing, for example, full-length IgG antibodies). According to a particular embodiment, the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, wherein it is stably integrated into the host cell genome, thereby enabling efficient expression of the recombinant nucleic acid.

[0244] In another general aspect, the present invention relates to a method for producing the monoclonal antibody or antigen-binding fragment thereof of the present invention, the method comprising culturing a cell containing a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for producing the monoclonal antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cell or culture (e.g., from a supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested from the cell and purified according to conventional techniques known in the art as described herein.

[0245] Pharmaceutical Composition

[0246] In another general aspect, the present invention relates to pharmaceutical compositions comprising isolated monoclonal antibodies of the present invention or antigen-binding fragments thereof, and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutical composition" means a product comprising the antibodies of the present invention and a pharmaceutically acceptable carrier. The antibodies of the present invention and compositions comprising such antibodies can also be used to prepare medicaments for the therapeutic applications mentioned herein.

[0247] As used herein, the term "carrier" means any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicles, microspheres, liposome encapsulations, or other materials well known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of a carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceuticalally acceptable carrier" means a non-toxic material that does not interfere with the effectiveness or bioactivity of the compositions according to the invention. According to specific embodiments, in view of this disclosure, any pharmaceutically acceptable carrier suitable for use in antibody pharmaceutical compositions may be used in this invention.

[0248] Formulations of the active pharmaceutical ingredient and a pharmaceutically acceptable carrier are known in the art, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions). Non-limiting examples of other components include buffers, diluents, solvents, tension modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used to formulate the pharmaceutical compositions of the present invention.

[0249] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation containing water. Liquid formulations may comprise solutions, suspensions, emulsions, microemulsions, gels, etc.

[0250] In one embodiment, the pharmaceutical composition may be formulated as an injectable agent that can be administered, for example, via an injection device (e.g., a syringe or infusion pump). For example, the injectable agent may be delivered subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0251] In another embodiment, the pharmaceutical composition is a solid dosage form, such as a freeze-dried or spray-dried composition, which can be used as is, or a solvent and / or diluent can be added to it prior to use by a physician or patient. Solid dosage forms may include tablets (such as compressed tablets and / or coated tablets) and capsules (e.g., hard or soft gelatin capsules). For example, the pharmaceutical composition may also be in the form of capsules, sugar-coated pills, powders, granules, lozenges, or powders for reconstitution.

[0252] The dosage form can be immediately released, in which case the dosage form may contain a water-soluble or dispersible carrier, or the dosage form can be delayed-release, sustained-release, or modified-release, in which case the dosage form may contain a water-insoluble polymer that modifies the dissolution rate of the dosage form in the gastrointestinal tract or under the skin.

[0253] In other embodiments, the pharmaceutical composition may be delivered intranasally, intrabuccally, or sublingually.

[0254] In another embodiment of the invention, the pharmaceutical composition comprises a buffer solution. In another embodiment of the invention, the pharmaceutical composition comprises a preservative. In another embodiment of the invention, the pharmaceutical composition comprises an isotonic agent. In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer.

[0255] In further embodiments of the invention, the pharmaceutical composition comprises one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecule or ion consisting of a water-soluble (hydrophilic) portion and a lipophilic (lipophilic) portion. Surfactants can be, for example, selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants.

[0256] In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as EDTA and / or benzoamidine hydrochloride (HCl).

[0257] In another general aspect, the present invention relates to a method for producing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof of the present invention, the method comprising combining the monoclonal antibody or an antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0258] How to use

[0259] In another general aspect, the present invention relates to a method for targeting GITRs on the surface of cancer cells in a subject, the method comprising administering to the subject an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GITRs, or a pharmaceutical composition of the present invention. The binding of the monoclonal antibody or its antigen-binding fragment to GITRs can mediate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cell cytotoxicity (ADCC), or other effects leading to the death of target cancer cells. The monoclonal antibody or its antigen-binding fragment may, for example, be used to recruit conjugated pharmaceuticals, and / or may form bispecific antibodies with another monoclonal antibody to mediate the death of target cancer cells.

[0260] The functional activity of antibodies binding to GITR and their antigen-binding fragments can be characterized by methods known in the art and as described herein. Methods for characterizing antibodies binding to GITR and their antigen-binding fragments include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, and OctetRed assays; and binding assays by FACS to detect the binding of antibodies to GITR on cancer cells or cells recombinantly expressing GITR. According to specific embodiments, methods for characterizing antibodies binding to GITR and their antigen-binding fragments include those described below.

[0261] In another general aspect, the present invention relates to a method of treating a subject with cancer, the method comprising administering to the subject an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GITR, or a pharmaceutical composition of the present invention. Cancer may be selected, for example, but not limited to, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0262] According to embodiments of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of an anti-GITR antibody or an antigen-binding fragment thereof. As used herein, the term "therapeutically effective amount" refers to the amount of an active ingredient or component that elicits a desired biological or pharmaceutical response in a subject. The therapeutically effective amount may be determined empirically and in a conventional manner for the purposes described.

[0263] As used in this article with respect to anti-GITR antibodies or their antigen-binding fragments, the therapeutically effective amount means the amount of anti-GITR antibody or its antigen-binding fragment that modulates the immune response in subjects in need.

[0264] According to a specific embodiment, a therapeutically effective amount refers to an amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of a disease, disorder, or condition to be treated, or symptoms associated with such disease, disorder, or condition; (ii) shortening the duration of a disease, disorder, or condition to be treated, or symptoms associated with such disease, disorder, or condition; (iii) preventing the progression of a disease, disorder, or condition to be treated, or symptoms associated with such disease, disorder, or condition; (iv) causing the remission of a disease, disorder, or condition to be treated, or symptoms associated with such disease, disorder, or condition; (v) preventing the development or onset of a disease, disorder, or condition to be treated, or symptoms associated with such disease, disorder, or condition; (vi) (vii) to prevent the recurrence of a disease, disorder, or condition to be treated or symptoms associated with such disease, disorder, or condition; (viii) to reduce hospitalization of subjects with a disease, disorder, or condition to be treated or symptoms associated with such disease, disorder, or condition; (viii) to shorten the length of hospitalization of subjects with a disease, disorder, or condition to be treated or symptoms associated with such disease, disorder, or condition; (ix) to increase the survival rate of subjects with a disease, disorder, or condition to be treated or symptoms associated with such disease, disorder, or condition; (xi) to suppress or reduce the disease, disorder, or condition to be treated or symptoms associated with such disease, disorder, or condition in subjects; and / or (xii) to enhance or improve the preventive or therapeutic effects of another therapy.

[0265] The effective therapeutic dose or dosage can vary depending on various factors, such as the disease, disorder, or condition to be treated, the route of administration, the target site, the subject's physiological state (including, for example, age, weight, and health), whether the subject is human or animal, other medications administered, and whether the treatment is preventative or therapeutic. Therapeutic doses can be titrated to optimize safety and efficacy.

[0266] According to specific embodiments, the compositions described herein are formulated to be administered to a subject via the intended route. For example, the compositions described herein may be formulated to be administered intravenously, subcutaneously, or intramuscularly.

[0267] As used herein, the terms "treat," "treating," and "treatment" are all intended to refer to improving or reversing at least one measurable physical parameter associated with cancer, which may not necessarily be identifiable in the subject but can be identified in the subject. The terms "treat," "treating," and "treatment" can also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refers to reducing or preventing the development or onset of one or more symptoms associated with a disease, disorder, or condition (such as a tumor or more preferably cancer), or shortening the duration of one or more symptoms associated with a disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refers to preventing the recurrence of a disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refers to increasing the survival rate of a subject suffering from a disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refers to eliminating the disease, disorder, or condition of a subject.

[0268] According to specific embodiments, compositions for treating cancer are provided. For cancer therapy, the compositions can be used in combination with another treatment, including but not limited to chemotherapy, anti-CD20 mAb, anti-CD47 mAb, anti-LAG-3 mAb, anti-CD73 mAb, anti-CTLA-4 mAb, anti-PD-L1 mAb, anti-PD-1 mAb, PD-1 / PD-L1 therapy, other immuno-oncology drugs, anti-angiogenic agents, radiotherapy, antibody-drug conjugates (ADCs), targeted therapy, or other anticancer drugs. Anti-GITR antibodies can be used to construct bispecific antibodies with chaperone mAbs targeting immune checkpoint molecules and / or other tumor surface antigens to treat cancers / tumors that simultaneously express GITR and specific tumor-associated antigens.

[0269] As used herein, the term “combination” refers to the use of more than one therapy in the context of administering two or more therapies to a subject. The use of the term “combination” does not limit the order in which the therapies are administered to the subject. For example, a first therapy (e.g., the combination described herein) may be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to administering a second therapy to the subject, concurrently with administering the second therapy to the subject, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to administering the second therapy to the subject).

[0270] In another general aspect, the present invention relates to methods for determining the GITR level of a subject. These methods include: (a) obtaining a sample from the subject; (b) contacting the sample with a monoclonal antibody of the present invention or an antigen-binding fragment thereof; and (c) determining the GITR level of the subject.

[0271] As used herein, “sample” means a biological sample isolated from a subject and may include, but is not limited to, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy (e.g., cancerous tissue), lymph, ascites, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluid. “Blood sample” means whole blood or any part thereof, including blood cells, serum, and plasma.

[0272] In some embodiments, the GITR level of a subject can be determined using an assay selected from, but not limited to, Western blot, ELISA, and / or immunohistochemistry (IHC). Relative protein levels can be determined using Western blot analysis and immunohistochemistry (IHC), and absolute protein levels can be determined using ELISA. When determining the relative level of GITR, the level can be determined between at least two samples (e.g., between samples from the same subject at different time points, between samples from different tissues of the same subject, and / or between samples from different subjects). Alternatively, when determining the absolute level of GITR, for example by ELISA, the absolute level of GITR in a sample can be determined by generating a standard for the ELISA assay prior to testing the sample. Those skilled in the art will understand which analytical techniques are used to determine the level of GITR in a subject's sample using the antibody or its antigen-binding fragment of the present invention.

[0273] Methods for determining GITR levels in subject samples can facilitate the diagnosis of abnormal (elevated, decreased, or insufficient) GITR levels and lead to appropriate treatment decisions. Such diseases can include, for example, cancer. Furthermore, by monitoring a subject's GITR levels, the risk of developing a disease, as indicated above, can be determined based on knowledge of GITR levels during and / or the progression of a specific disease.

[0274] Example

[0275] The present invention provides the following non-limiting embodiments.

[0276] Example 1 is an isolated monoclonal antibody or its antigen-binding fragment, comprising heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, and light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequence:

[0277] a. These are SEQ ID NO: 36, 37, 38, 39, 40, and 41, respectively;

[0278] b. These are SEQ ID NOs: 42, 43, 44, 45, 46, and 47, respectively;

[0279] c. These are SEQ ID NOs: 48, 49, 50, 54, 55, and 56, respectively;

[0280] d. These are SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively;

[0281] e. These are SEQ ID NOs: 57, 58, 59, 60, 61, and 62, respectively;

[0282] f. These are SEQ ID NOs: 63, 64, 65, 66, 67, and 68, respectively;

[0283] g. These are SEQ ID NO: 69, 70, 71, 72, 73, and 74, respectively;

[0284] h. are SEQ ID NO: 75, 76, 77, 78, 79, and 80, respectively;

[0285] i. These are SEQ ID NOs: 81, 82, 83, 84, 85, and 86, respectively;

[0286] j. are SEQ ID NO: 87, 88, 89, 93, 94, and 95 respectively;

[0287] k. are SEQ ID NO: 87, 88, 89, 96, 97, and 98 respectively;

[0288] l. These are SEQ ID NO: 90, 91, 92, 93, 94, and 95, respectively;

[0289] m. are SEQ ID NO: 90, 91, 92, 96, 97, and 98 respectively;

[0290] n. are SEQ ID NO:99, 100, 101, 102, 103, and 104 respectively;

[0291] o. are SEQ ID NO: 105, 106, 107, 108, 109, and 110 respectively;

[0292] p. are SEQ ID NO: 111, 112, 113, 114, 115, and 116 respectively;

[0293] q. are SEQ ID NO: 117, 118, 119, 120, 121, and 122 respectively;

[0294] r. are SEQ ID NO: 123, 124, 125, 126, 127, and 128 respectively;

[0295] s. are SEQ ID NO: 123, 124, 125, 129, 130, and 131, respectively;

[0296] t. are respectively SEQ ID NO: 123, 124, 125, 132, 133, and 134; or

[0297] u. are SEQ ID NO: 135, 136, 137, 138, 139, and 140 respectively;

[0298] Or a variant of the isolated monoclonal antibody or its antigen-binding fragment, which contains up to about three (e.g., one, two, or three) amino acid substitutions in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3.

[0299] The antibody or its antigen-binding fragment specifically binds to glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), preferably human GITR.

[0300] Example 2 is a monoclonal antibody or its antigen-binding fragment isolated as described in Example 1, wherein the isolated monoclonal antibody or its antigen-binding fragment comprises heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, and has the following polypeptide sequence:

[0301] a. These are SEQ ID NO: 36, 37, 38, 39, 40, and 41, respectively;

[0302] b. These are SEQ ID NOs: 42, 43, 44, 45, 46, and 47, respectively;

[0303] c. These are SEQ ID NOs: 48, 49, 50, 54, 55, and 56, respectively;

[0304] d. These are SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively;

[0305] e. These are SEQ ID NOs: 57, 58, 59, 60, 61, and 62, respectively;

[0306] f. These are SEQ ID NOs: 63, 64, 65, 66, 67, and 68, respectively;

[0307] g. These are SEQ ID NO: 69, 70, 71, 72, 73, and 74, respectively;

[0308] h. are SEQ ID NO: 75, 76, 77, 78, 79, and 80, respectively;

[0309] i. These are SEQ ID NOs: 81, 82, 83, 84, 85, and 86, respectively;

[0310] j. are SEQ ID NO: 87, 88, 89, 93, 94, and 95 respectively;

[0311] k. are SEQ ID NO: 87, 88, 89, 96, 97, and 98 respectively;

[0312] l. These are SEQ ID NO: 90, 91, 92, 93, 94, and 95, respectively;

[0313] m. are SEQ ID NO: 90, 91, 92, 96, 97, and 98 respectively;

[0314] n. are SEQ ID NO:99, 100, 101, 102, 103, and 104 respectively;

[0315] o. are SEQ ID NO: 105, 106, 107, 108, 109, and 110 respectively;

[0316] p. are SEQ ID NO: 111, 112, 113, 114, 115, and 116 respectively;

[0317] q. are SEQ ID NO: 117, 118, 119, 120, 121, and 122 respectively;

[0318] r. are SEQ ID NO: 123, 124, 125, 126, 127, and 128 respectively;

[0319] s. are SEQ ID NO: 123, 124, 125, 129, 130, and 131, respectively;

[0320] t. are respectively SEQ ID NO: 123, 124, 125, 132, 133, and 134; or

[0321] u. are SEQ ID NO: 135, 136, 137, 138, 139, and 140 respectively;

[0322] The antibody or its antigen-binding fragment specifically binds to glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR), preferably human GITR.

[0323] Example 3 is an isolated monoclonal antibody or its antigen-binding fragment as described in Example 1 or 2, comprising a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, 3, 5, 6, 8, 10, 12, 14, 16, 22, 24, 26, 28, 30, or 34, and the light chain variable region has a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, 4, 7, 9, 11, 13, 15, 17, 23, 25, 27, 29, 31, 32, 33, or 35.

[0324] Example 4 is an isolated monoclonal antibody or its antigen-binding fragment as described in any one of Examples 1 to 3, comprising:

[0325] a. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:1 and the light chain variable region having the polypeptide sequence of SEQ ID NO:2;

[0326] b. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:3 and the light chain variable region having the polypeptide sequence of SEQ ID NO:4;

[0327] c. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:5 and the light chain variable region having the polypeptide sequence of SEQ ID NO:7;

[0328] d. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:6 and the light chain variable region having the polypeptide sequence of SEQ ID NO:7;

[0329] e. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:8 and the light chain variable region having the polypeptide sequence of SEQ ID NO:9;

[0330] f. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:10 and the light chain variable region having the polypeptide sequence of SEQ ID NO:11;

[0331] g. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:12 and the light chain variable region having the polypeptide sequence of SEQ ID NO:13;

[0332] h. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:14 and the light chain variable region having the polypeptide sequence of SEQ ID NO:15;

[0333] i. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:16 and the light chain variable region having the polypeptide sequence of SEQ ID NO:17;

[0334] j. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:22 and the light chain variable region having the polypeptide sequence of SEQ ID NO:23;

[0335] k. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:24 and the light chain variable region having the polypeptide sequence of SEQ ID NO:25;

[0336] l. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:26 and the light chain variable region having the polypeptide sequence of SEQ ID NO:27;

[0337] m. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:28 and the light chain variable region having the polypeptide sequence of SEQ ID NO:29;

[0338] n. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30 and the light chain variable region having the polypeptide sequence of SEQ ID NO:31;

[0339] o. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30 and the light chain variable region having the polypeptide sequence of SEQ ID NO:32;

[0340] p. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:30 and the light chain variable region having the polypeptide sequence of SEQ ID NO:33; or

[0341] q. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:34 and the light chain variable region having the polypeptide sequence of SEQ ID NO:35.

[0342] Example 5 is an isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of Examples 1 to 4, wherein the antibody or antigen-binding fragment thereof is chimeric.

[0343] Example 6 is an isolated monoclonal antibody or its antigen-binding fragment as described in Example 5, wherein the monoclonal antibody or its antigen-binding fragment contains the human IgG1 constant region.

[0344] Example 7 is an isolated monoclonal antibody or its antigen-binding fragment as described in Example 6, wherein the constant region of these individuals' IgG1 contains at least one amino acid modification selected from K214R, D356E, L358M, and ΔK447.

[0345] Example 8 is an isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of Examples 5 to 7, wherein the antibody or antigen-binding fragment thereof is human or humanized.

[0346] Example 9 is a monoclonal antibody or its antigen-binding fragment isolated as described in Example 8, comprising:

[0347] a. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:143 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0348] b. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:143 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0349] c. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:144 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0350] d. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:144 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0351] e. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:145 and the light chain variable region having the polypeptide sequence of SEQ ID NO:146;

[0352] f. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:145 and the light chain variable region having the polypeptide sequence of SEQ ID NO:147;

[0353] g. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:148 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0354] h. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:149 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0355] i. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:150 and the light chain variable region having the polypeptide sequence of SEQ ID NO:151;

[0356] j. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:152, and the light chain variable region having the polypeptide sequence of SEQ ID NO:154; or

[0357] k. The heavy chain variable region having the polypeptide sequence of SEQ ID NO:153 and the light chain variable region having the polypeptide sequence of SEQ ID NO:154.

[0358] Example 10 is an isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of Examples 1 to 9, wherein the monoclonal antibody or antigen-binding fragment thereof is capable of binding to GITR and inducing effector-mediated tumor cell lysis.

[0359] Example 11 is an isolated nucleic acid that encodes a monoclonal antibody or its antigen-binding fragment as described in any one of Examples 1 to 10.

[0360] Example 12 is a vector containing the isolated nucleic acid as described in Example 11.

[0361] Example 13 is a host cell that contains the vector as described in Example 12.

[0362] Example 14 is a pharmaceutical composition comprising an isolated monoclonal antibody or an antigen-binding fragment thereof as described in any one of Examples 1 to 10, and a pharmaceutically acceptable carrier.

[0363] Example 15 is a method for treating cancer in a subject in need, the method comprising administering to the subject a pharmaceutical composition as described in Example 14.

[0364] Example 16 is a method for targeting GITR on the surface of cancer cells in a subject of need, the method comprising administering to the subject a pharmaceutical composition as described in Example 14.

[0365] Example 17 is a method for producing a monoclonal antibody or an antigen-binding fragment thereof as described in any one of Examples 1 to 10, the method comprising culturing a cell containing a nucleic acid encoding the monoclonal antibody or an antigen-binding fragment thereof under conditions for producing the monoclonal antibody or an antigen-binding fragment thereof, and recovering the monoclonal antibody or an antigen-binding fragment thereof from the cell or culture.

[0366] Example 18 is a method for producing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of Examples 1 to 10, the method comprising combining the monoclonal antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0367] Example 19 is a method for determining the GITR level of a subject, the method comprising:

[0368] a. Obtain samples from the subject;

[0369] b. Contact the sample with the isolated monoclonal antibody or its antigen-binding fragment as described in any one of Examples 1 to 10; and

[0370] c. Determine the subject's GITR level.

[0371] Example 20 is the method as described in Example 19, wherein the sample is a tissue sample or a blood sample.

[0372] Example 21 is the method as described in Example 20, wherein the tissue sample is a cancer tissue sample.

[0373] Example

[0374] The following examples are intended purely as illustrations of the invention and should not be construed as limiting the invention in any way. The following examples and detailed descriptions are provided by way of illustration rather than limitation.

[0375] Example 1: Production of anti-GITR monoclonal antibody (mAb)

[0376] immunity

[0377] In accordance with current animal welfare regulations, Balb / c and C57 mice were immunized with human GITR-Fc protein (GenScript; catalog number Z03440). For immunization, the antigen was administered either in PBS solution or formulated as an emulsion with CFA (complete Freund's adjuvant; primary immunization) or IFA (incomplete Freund's adjuvant; booster immunization). In multi-arm immunization, the antigen was administered to the abdominal skin of mice via intraperitoneal injection or subcutaneous injection in the back using a gene gun. Each animal received four doses (the first dose was 50 μg / mL, followed by three doses of 25 μg / mL each). Seven days after each time point during the immunization program, 20 μL of blood samples were collected from the animals. Figure 1A-1K Using immobilized GITR-Fc protein as a control, antiserum titers were monitored in an ELISA-based assay until fusion criteria were met. Specific recognition of GITR-Fc was validated by an ELISA (results) of its binding to biotin-GITR ligand-Fc.

[0378] Selection of hybridomas that secrete anti-GITR antibodies

[0379] Three days after the final immunization, spleen cells from selected mice were extracted and fused with sp2 / 0 cells under sterile conditions according to a standard hybridoma generation protocol. The fused cells were cultured for 6 days in DMEM medium (supplemented with 10% FBS) containing 1X HAT (hypoxanthine-aminopterin-thymidine). The binding capacity of the supernatant contents to human GITR-Fc was analyzed by ELISA, while reverse selection against human IgG1 was performed. Only single positive clones targeting human GITR-Fc were selected, with a cutoff value of OD. 450>1. The ability of selected clones to bind to a stable Cho-K1 cell line overexpressing GITR was analyzed by FACS. Positive parental clones were subcloned by limiting dilution and cultured in DMEM medium (supplemented with 10% FBS) containing 1X HT (hypoxanthine-thymidine). 100 μL of cell suspension was aliquoted to maintain 1–3 cells / well. After 1 week of cell culture, a new round of screening using ELISA and FACS was performed until positive single clones were obtained. Each unique clone was selected to generate 0.5 mg of purified antibody for further characterization. The antibody isotype was tested (Clonotyping System-HRP, Southern Biotech; Birmingham, AL) and purified using protein-A magnetic beads (GenScript, catalog number L00695), eluted with 0.5 M sodium citrate solution (pH 3.5), and neutralized with 0.5 M Tris-HCl (pH 9.0). The storage buffer was replaced with PBS to determine the concentration using nanodrop.

[0380] Example 2: In vitro characterization of anti-GITR mouse antibodies

[0381] GITR analyzed by FACS + / Cho-K1 cell binding

[0382] A total of 38 clones were sent to small-scale production, producing mAb antibody products, including 204C10G12, 384H3H11, 289H7D4, 197E9G3, 45F1F3, 31D7D10, 136E1F3, 225H7H7, 100E12D5, 7E1A7, 263H6G11, 358E3C9, 222H11G10, 206H9G11, 225A8D9, 223F12C4, and 225H7D. 12, 11A4E11, 223H11H1, 153F5C6, 377C9H2, 320E12H10, 209E10F11, 201A3A11, 274C7H2, 287F1E11, 275A4D7, 172H7B9, 163H12G7, 270C5C10, 78D7D8, 114A5D8, 114A5D8, 84F3H4, 366B6H9, 85D2H9, and 366G5C11.

[0383] To verify the cell surface antigen binding of the antibody product, approximately 1 × 10⁻⁶ cells were harvested. 5 Cho-K1 cells expressing human GITR were used in a reaction mixture, and these cells were compared with 10... -3 Up to 10 2Incubate mM of anti-GITR mAb in a 3-fold serially diluted solution, then detect the results using 1 μg / mL of goat anti-mouse IgG (H+L) secondary antibody labeled with the fluorophore (iFluor 647). Results are presented in... Figures 2A-2D China. EC 50 The values ​​are summarized in Table 1. A total of 34 mAbs are displayed for EC. 50 EC of less than 100 ng / ml, with 30 mAbs 50 Less than 10 ng / ml.

[0384] Table 1. FACS binding data of anti-GITR mouse monoclonal antibody

[0385]

[0386] Functional reporter gene assay of anti-GITR mouse antibody

[0387] Functional screening of anti-GITR mouse antibodies was performed using the reporter gene-based anti-GITR agonist assay kit developed by Promega Corp. (catalog number CS184009; Promega; Madison, WI). This kit contains an effector cell line expressing GITR and a promoter-regulated luciferase reporter gene. The agonist effect of the anti-GITR antibody stimulates effector cells to elicit a pro-inflammatory response, thereby activating certain transcription factors that bind to upstream response elements of the luciferase reporter gene, resulting in the production of luciferase. Addition of a substrate will produce a detectable luminescent signal.

[0388] In short, GITR-expressing effector cell lines were thawed and counted, and their viability was tested. Cells were transferred to 96-well plates at 50,000 cells per well. Antibody samples (i.e., anti-GITR human antibody (GenScript, B50011812)) as a positive control, or mouse IgG as a negative control, were prepared in a series of dilutions and added to the effector cells, and incubated at 37°C, 5% CO2 for 6 hours. Then, luciferase substrate solution was added to the mixture, and the entire plate was incubated in the dark at room temperature for 10 minutes. Finally, the plate was read in luminescence mode in a PheraStar (BMGLabtech, PheraStarPlus FSX). If the antibody sample had anti-GITR agonist activity, the luminescence signal showed an S-shaped curve with increasing antibody concentration. Figure 3 ).

[0389] EC 50 The values ​​are shown in Table 2. Given the high EC value of 134D7B3... 50However, it did not show good activity. 172H7B9, 45F1F3, and 163H12G7 exhibited the desired EC activity. 50 And a reasonable span.

[0390] Table 2. GITR functional reporter gene assay of anti-GITR mouse monoclonal antibody

[0391]

[0392] Example 3: Antibody CDR sequence alignment

[0393] A total of 14 mouse antibodies were sequenced. CDR3 alignment results are summarized in Table 3, CDR sequences are summarized in Table 4, and the variable region sequences are shown below. Sequencing of three clones revealed more than one heavy or light chain.

[0394] Table 3. Alignment of CDR3 sequences of individual antibodies

[0395]

[0396]

[0397] Table 4: CDR sequences of mAb clones

[0398]

[0399]

[0400]

[0401] Example 4: Chimeric Antibody Production and Analysis

[0402] Construction of chimeric antibodies for characterization

[0403] Will Figure 3All candidate antibodies tested, along with 204C10G12, 206H9G11, 225A8D9, 274C7H2, 384H3H11, 177C6B11, and 215F2A5, were selected for the synthesis and sequencing of chimeric antibodies targeting the constant region of human IgG1. The variable region coding sequences of these 14 mAbs were optimized for human codon-biased expression using the GenScript online tool. Variable DNA fragments of 45F1F3, 163H12G7, 172H7B9, 223F12C4, 225H7D12, 270C5C10, 204C10G12, 225A8D9, 274C7H2, 206H9G11, 134D7B3, 177C6B11, 215F2A5, and 384H3H11 were synthesized and fused to the human IgG1 heavy chain domain (CH1-hinge-CH2-CH3) and the light chain κ constant region (CL) for transient expression in a chimeric form. The variable DNA fragments 45F1F3, 225H7D12, 270C5C10, 163H12G7, 172H7B9, and 223F12C4 were fused into the human IgG1 constant region modified with K214R, D356E, L358M, and ΔK447. The variable DNA fragments 204C10G12, 225A8D9, 274C7H2, 384H3H11, 134D7B3, 177C6B11, 215F2A5, and 223H11H1 were fused into the human IgG1 constant region modified with K214R. Heavy and light chain expression constructs were cloned into single pTT5-based plasmids containing a synthetic signal peptide for secretory expression. Cloning 206H9G11 failed to express and showed non-clonal characteristics upon sequencing.

[0404] Chimeric antibodies were expressed in HEK293-6E cells (National Research Council) transfected with antibody heavy / light chain plasmids using PEImax 40,000 (Polysciences, Inc.; Warrington, PA). Expression / secretion was enhanced 24 hours later with tryptone N-1 supplementation. After 5 days of shaking culture at 37°C and 5% CO2, the supernatant was collected, and the antibody contents were purified using the aforementioned protein-A beads. The chimeric antibody product was stored in PBS for analysis.

[0405] Chimeric antibody FACS binding analysis

[0406] The binding pattern of the chimeric antibody to GITR expressed on Cho-K1 cells was plotted using 3-fold serial dilutions of the antibody starting at a concentration of 300 nM. The antibody-GITR binding curves were generated using geometric mean. The raw data were plotted with four parameters using GraphPadPrism v6.02 software, and ECGs were analyzed using a best-fit program. 50 ( Figures 4A-4C The results are summarized in Table 5.

[0407] The cross-species reactivity of all candidate antibodies was tested on FACS using the cynomolgus monkey GITR / Cho-K1 cell line (cyno GITR / Cho-K1). Non-cynomolgus monkey conjugates and weak cynomolgus monkey conjugates were removed for further testing. In human and cynomolgus monkey FACS analyses, antibodies with the desired EC were selected. 50 45F1F3, 225H7D12, and 270C5C10 were selected as lead antibody candidates. Compared to the baseline antibody used as a positive control, both 45F1F3 and 225H7D12 showed superior EC50. 50 The 270C5C10 has a slightly higher EC. 50 Furthermore, in epitope combination experiments, 270C5C10 showed different epitope bindings with 45F1F3 and 225H7D12. Therefore, all three clones were selected for further development.

[0408] Table 5. Chimeric antibody binding on GITR / Cho-K1 cells in FACS analysis

[0409]

[0410] Chimeric anti-GITR antibody functional reporter gene assay

[0411] As described in Example 2, EC from three candidate chimeric antibodies derived from GITR functional reporter assays 50 The values ​​are summarized in Table 6. Clones 45F1F3 show the lowest EC50. 50 The value was 0.01446 μg / ml.

[0412] Table 6. Functional reporter gene assay for chimeric antibody GITR

[0413]

[0414] Example 5. Humanized Antibody Production and Analysis

[0415] Humanization design of candidate antibodies

[0416] Based on the antibody variable domain sequence, the CDR, HV loop, and FR were analyzed, and homology modeling was performed to obtain the modeled structure of the mouse antibody. The solvent-accessible surface area of ​​the framework residues was calculated. Based on the results, buried framework residues (i.e., solvent-accessible surface area <15%) were identified. A human receptor VH and VL with the same top sequence as the mouse control were selected. The CDR of the mouse antibody was directly transplanted onto the human receptor framework to obtain the transplanted antibody sequence without any reversion mutations, where some amino acids were modified back to the mouse framework sequence. For candidate antibody 45F1F3, four VH sequences and two VL sequences with reversion mutations were synthesized. For candidate antibody 225H7D12, four VH sequences and three VL sequences with reversion mutations were synthesized. For candidate antibody 270C5C10, five VH sequences and two VL sequences with reversion mutations were synthesized. Post-translational modifications and chemical degradations, including deamidation, isomerization oxidation, and glycosylation, in the transplanted sequences were analyzed through exploitability assessment. PTM hotspots, such as N-glycosylation sites, aberrant proline residues, deamidation sites, isomerization sites, oxidation sites, and unpaired cysteine ​​residues, were identified, which may affect the binding activity and manufacturability of transplanted antibodies.

[0417] DNA sequences encoding the humanized light and heavy chain variable regions were synthesized. Antibody characteristics were compared to select the best candidate antibody. For each chimeric antibody, low EC50 values ​​were determined based on FACS binding assays with GITR / Cho-K1. 50 and the minimum number of reversion mutations ( Figures 5A-5C ), and select a humanized antibody as the final lead antibody. EC 50 The values ​​are summarized in Table 7. All three humanized antibodies of chimeric antibody 45F1F3 showed low EC50 values. 50 Both humanized antibodies to the chimeric antibody 270C5C10 exhibited low EC50. 50 Two humanized antibodies to the chimeric antibody 225H7D12 showed lower EC50. 50 Besides VH1.4+VL1.2, EC 50 The concentration was 0.1810 μg / mL, while it was 0.1122 μg / mL before humanization.

[0418] Table 7. FACS binding to EC of humanized antibodies 50

[0419]

[0420] Cell-based reporter gene assay for humanized antibodies

[0421] Functional screening of anti-GITR antibodies was performed using a reporter gene-based anti-GITR agonist assay developed by GenScript. This assay included an effector cell line expressing GITR and a promoter-regulated luciferase reporter gene. Effector cells were stimulated by the anti-GITR antibody agonist to elicit a pro-inflammatory response, thereby activating an NFkB transcription factor that binds to an upstream response element of the luciferase reporter gene, resulting in the production of luciferase. Substrate addition produced a luminescent signal, which was recorded by a luminometer. The functional bioassay results for all humanized lead candidate antibodies, their corresponding chimeric antibodies, and the positive control anti-GITR antibody (Genscript, anti-GITRAb BA20190125CFT02) are shown in [the table / image / incomplete]. Figure 6A In B, all 11 candidate antibodies tested showed greater potency than the baseline antibody. Candidate antibody 270C5C10 VL1+VH1.4 showed the lowest EC50. 50 (0.03552 μg / mL), its potency is higher than EC. 50 It is more than 5 times larger than the baseline antibody of 0.1781 μg / mL (Table 8).

[0422] Table 8. Assay of functional reporter gene for humanized antibody GITR

[0423]

[0424] Those skilled in the art will understand that changes can be made to the above embodiments without departing from the broad inventive concept of the invention. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined in this specification.

[0425] sequence list

[0426] SEQ ID NO:1 mAb clone 45F1F3-H heavy chain variable region

[0427]

[0428] SEQ ID NO:2 mAb clone 45F1F3-L light chain variable region

[0429]

[0430] SEQ ID NO:3 mAb clone 225H7D12-H heavy chain variable region

[0431]

[0432] SEQ ID NO:4 mAb clone 225H7D12-L light chain variable region

[0433]

[0434] SEQ ID NO:5 mAb clone 270C5C10-Ha heavy chain variable region a(VHa)

[0435]

[0436] SEQ ID NO:6 mAb clone 270C5C10-Hb heavy chain variable region b (VHb)

[0437]

[0438] SEQ ID NO:7 mAb clone 270C5C10-L light chain variable region (VL)

[0439]

[0440] SEQ ID NO: 8 mAb clone 134D7B3-H heavy chain variable region (VH)

[0441]

[0442] SEQ ID NO: 9 mAb clone 134D7B3-L light chain variable region (VL)

[0443]

[0444] SEQ ID NO: 10 mAb clone 177C6B11-H heavy chain variable region (VH)

[0445]

[0446] SEQ ID NO: 11 mAb clone 177C6B11-L light chain variable region (VL)

[0447]

[0448] SEQ ID NO: 12 mAb clone 215F2A5-H heavy chain variable region (VH)

[0449]

[0450] SEQ ID NO: 13 mAb clone 215F2A5-L light chain variable region (VL)

[0451]

[0452] SEQ ID NO: 14 mAb clone 223H11H1-H heavy chain variable region (VH)

[0453]

[0454] SEQ ID NO: 15 mAb clone 223H11H1-L light chain variable region (VL)

[0455]

[0456] SEQ ID NO: 16 mAb clone 204C10G12-H heavy chain variable region (VH)

[0457]

[0458] SEQ ID NO: 17 mAb clone 204C10G12-L light chain variable region (VL)

[0459]

[0460] SEQ ID NO: 18 mAb clone 206H9G11-Ha heavy chain variable region 1 (VH1)

[0461]

[0462] SEQ ID NO: 19 mAb clone 206H9G11-Hb heavy chain variable region 2 (VH2)

[0463]

[0464] SEQ ID NO: 20 mAb clone 206H9G11-La light chain variable region 1 (VL1)

[0465]

[0466] SEQ ID NO: 21 mAb clone 206H9G11-Lb light chain variable region 2 (VL2)

[0467]

[0468] SEQ ID NO: 22 mAb clone 225A8D9-H heavy chain variable region (VH)

[0469]

[0470] SEQ ID NO: 23 mAb clone 225A8D9-L light chain variable region (VL)

[0471]

[0472] SEQ ID NO: 24 mAb clone 274C7H2-H heavy chain variable region (VH)

[0473]

[0474] SEQ ID NO: 25 mAb clone 274C7H2-L light chain variable region (VL)

[0475]

[0476] SEQ ID NO: 26 mAb clone 384H3H11-H heavy chain variable region (VH)

[0477]

[0478] SEQ ID NO: 27 mAb clone 384H3H11-L light chain variable region (VL)

[0479]

[0480] SEQ ID NO: 28 mAb clone 163H12G7-H heavy chain variable region (VH)

[0481]

[0482] SEQ ID NO: 29 mAb clone 163H12G7-L light chain variable region (VL)

[0483]

[0484] SEQ ID NO: 30 mAb clone 172H7B9-H heavy chain variable region (VH)

[0485]

[0486] SEQ ID NO: 31 mAb clone 172H7B9-La light chain variable region a(VLa)

[0487]

[0488] SEQ ID NO: 32 mAb clone 172H7B9-Lb light chain variable region b (VLb)

[0489]

[0490] SEQ ID NO: 33 mAb clone 172H7B9-Lc light chain variable region c(VLc)

[0491]

[0492] SEQ ID NO: 34 mAb clone 223F12C4-H heavy chain variable region (VH)

[0493]

[0494] SEQ ID NO: 35 mAb clone 223F12C4-L light chain variable region (VL)

[0495]

[0496] SEQ ID NO: 141 human IgG1 CH

[0497]

[0498] SEQ ID NO: 155 Human IgG1 CH variant 1

[0499]

[0500] SEQ ID NO: 156 human IgG1 CH variant 2

[0501]

[0502] SEQ ID NO: 142 human IgG1 CL

[0503]

[0504] SEQ ID NO: 143 Humanized mAb 45F1F3 VH1.1

[0505]

[0506] SEQ ID NO:144 Humanized mAb 45F1F3 VH1.3

[0507]

[0508] SEQ ID NO:145 Humanized mAb 45F1F3 VH1.4

[0509]

[0510] SEQ ID NO:146 Humanized mAb 45F1F3 VL1

[0511]

[0512] SEQ ID NO:147 Humanized mAb 45F1F3 VL1.1

[0513]

[0514] SEQ ID NO:148 Humanized mAb 225H7D12 VH1.1

[0515]

[0516] SEQ ID NO:149 Humanized mAb 225H7D12 VH1.3

[0517]

[0518] SEQ ID NO:150 Humanized mAb 225H7D12 VH1.4

[0519]

[0520] SEQ ID NO:151 Humanized mAb 225H7D12 VL1.2

[0521]

[0522] SEQ ID NO:152 Humanized mAb 270C5C10 VH1.2

[0523]

[0524] SEQ ID NO: 153 Humanized mAb 270C5C10 VH1.4

[0525]

[0526] SEQ ID NO: 154 Humanized mAb 270C5C10 VL1

[0527] sequence list <110> Nanjing GenScript Biotech Co., Ltd. <120> Anti-GITR antibodies and their uses <130> 688096.129 <150> PCT / CN2020 / 070101 <151> 2020-01-02 <160> 156 <170> PatentIn version 3.5 <210> 1 <211> 118 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 variable heavy chain sequence <400> 1 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ile Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Phe Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Thr Tyr Tyr Cys 85 90 95 Thr Arg Ser Gly His Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 2 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 variable light chain sequence <400> 2 Asp Val Val Met Thr Gln Ala Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Phe Asp Arg 20 25 30 Asp Gly Lys Thr Tyr Leu Ser Trp Leu Leu Glu Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Asn Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Met Lys 100 105 110 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 225H7D12 Variable Heavy Chain Sequence <400> 3 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Thr Thr Gly Tyr Arg Phe Thr Gly Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Val Ser Asn Asn Asn Glu Lys Phe 50 55 60 Arg Asp Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Ile Gln Leu Ser Ser Leu Thr Thr Asp Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Lys Ala Ser Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 4 <211> 108 <212> PRT <213> Artificial sequence <220> <223> 225H7D12 variable light chain sequence <400> 4 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Phe 20 25 30 Tyr Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Ile Ser Asn Leu Ala Ser Gly Val Pro Thr Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 270C5C10 Heavy Chain Variable Region Sequence <400> 5 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30 Tyr Met Asn Trp Ala Lys Gln Ser Gln Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Gly Ser Arg Tyr Lys Glu Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Pro Gly Ser Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 6 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 270C5C10 Hb Variable Heavy Chain Sequence <400> 6 Glu Val Gln Leu Gln Gln Ser Gly Pro Val Gln Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asn Pro Asn Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 7 <211> 112 <212> PRT <213> artificial sequence <220> <223> 270C5C10 sequence sequence <400> 7 Asp Val Val Leu Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Arg Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Gln Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 8 <211> 121 <212> PRT <213> Artificial sequence <220> <223> 134D7B3 variable heavy chain sequence <400> 8 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Asn Asn Tyr 20 25 30 Val Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Tyr Pro Gly Asn Gly Gly Ser Asp Tyr Asn Glu Lys Phe 50 55 60 Lys Val Lys Ala Lys Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Phe Tyr Tyr Phe Gly Ser Ser Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 9 <211> 107 <212> PRT <213> Artificial sequence <220> <223> 134D7B3 variable light chain sequence <400> 9 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Thr Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 123 <212> PRT <213> Artificial sequence <220> <223> 177C6B11 variable heavy chain sequence <400> 10 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Phe 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro His Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Pro Ser Ser Ala Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Asp Tyr Asp Gly Val Ala Trp Leu Ala Phe 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 11 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 177C6B11 Variable Light Chain Sequence <400> 11 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 12 <211> 115 <212> PRT <213> Artificial sequence <220> <223> 215F2A5 variable heavy chain sequence <400> 12 Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Leu Ser Asp His 20 25 30 Tyr Met Thr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Ala Asp Ile Asn Pro Tyr Ser Gly Thr Val Thr Phe Asn Gln Arg Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Phe 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr 100 105 110 Val Ser Ser 115 <210> 13 <211> 112 <212> PRT <213> Synthetic Sequence <220> <223> 215F2A5 Variable Light Chain Sequence <400> 13 Gly Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Asp Gln Thr Ile Val Tyr Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 14 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> 223H11H1 Variable Heavy Chain Sequence <400> 14 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala His 20 25 30 Tyr Met Asn Trp Val Lys Lys Ser Gln Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asp Asn Gly Ile Ile Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val Phe 65 70 75 80 Leu Glu Phe Arg Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Glu Ala Leu Asp Tyr Trp Gly Gln Gly Ser Ser Val Thr 100 105 110 Val Ser Ser 115 <210> 15 <211> 112 <212> PRT <213> artificial sequence <220> <223> 223H11H1 sequence <400> 15 Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Thr Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser 20 25 30 Asn Gly Lys Val Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asp Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Gly 85 90 95 Ser His Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 16 <211> 118 <212> PRT <213> Artificial Sequence <220> [[ID=**18**]]<223> 204C10G12 Variable Heavy Chain Sequence <400> 16 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Ser Asp Ser Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Asn Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Asp Phe Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 17 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 204C10G12 Variable Light Chain Sequence <400> 17 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro<                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     &em​​​​​​​​​​​​​​<210> 18 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 Ha variable heavy chain sequence <400> 18 Glu Val Gln Leu Gln Gln Ser Gly Pro Ala Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Tyr Met His Trp Met Lys Gln Ser Pro Glu Lys Ser Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Thr Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Thr Arg Arg Asp Tyr Tyr Ala Met Asp Tyr Trp Asp Gln Gly Thr Ser 100 105 110 Val Ile Val Ser Ser 115 <210> 19 <211> 125 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 Hb variable heavy chain sequence <400> 19 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Phe 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile Ala Pro Ile Thr Thr Val Val Ala Thr Asn Tyr Phe 100 105 110 Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 20 <211> 107 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 La variable light chain sequence <400> 20 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Ile Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Phe Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Thr 100 105 <210> 21 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 206H9G11 Light Chain Variable Region Sequence <400> 21 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser[[ID=ll]] 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 22 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 225A8D9 Variable Heavy Chain Sequence <400> 22 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asn Ile Asn Tyr Asp Gly Lys Phe 50 55 60 Lys Gly Glu Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Ala Trp Leu Gly Lys Thr Tyr Val Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 23 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> 225A8D9 Variable Light Chain Sequence <400> 23 Glu Ile Val Val Thr Gln Ser Pro Ala Ile Thr Ala Ala Ser Leu Gly 1 5 10 15 Gln Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Glu Ile Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Asn Phe Pro Phe Thr Phe 85 90 95 Gly Ser Gly Thr Lys Val Glu Ile Arg 100 105 <210> 24 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 274C7H2 Variable Heavy Chain Sequence <400> 24 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Tyr Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Pro Ser Tyr Asp Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 25 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> 274C7H2 Variable Light Chain Sequence <400> 25 Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe[[ID=z36]] 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 His Phe Ile Phe Gly Ser Gly Thr Lys Val Thr Val Leu 100 105 <210> 26 <211> 116 <212> PRT <213> Artificial sequence <220> <223> 384H3H11 variable heavy chain sequence <400> 26 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ile Gly Leu Arg Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> 27 <211> 113 <212> PRT <213> Artificial sequence <220> <223> 384H3H11 variable light chain sequence <400> 27 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 28 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 163H12G7 variable heavy chain sequence <400> 28 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Ser Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Thr Tyr 20 25 30 Thr Val His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Ala Asp Arg Ser Ser Thr Thr Ala Tyr 65 7O 75 80 Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95<00O1885>Ala Arg Trp Ser Tyr Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 29 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 163H12G7 Variable Light Chain Sequence <400> 29 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Ser Ile Gly 1 5 10 15 [[ID=!8]]Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Leu Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Tyr Gln Ala 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 30 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> 172H7B9 Variable Heavy Chain Sequence <400> 30 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Ser Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Pro Ser 20 25 30 Gly Met Gly Val Thr Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Val Lys Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Gly Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Arg Gly Ser Tyr Ser Asp Tyr Gly Gly Trp Ser Phe Asp 100 105 110 Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 31 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 172H7B9 Lambda Variable Light Chain Sequence <400> 31 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 32 <211> 113 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 Lb variable light chain sequence <400> 32 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 33 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 172H7B9 Lc Variable Light Chain Sequence <400> 33 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 34 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> 223F12C4 Variable Heavy Chain Sequence <400> 34 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile His Pro Ser Asp Gly Asp Ile Asp His Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Leu Cys Tyr Ile Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 100 105 110 Ser Ser <210> 35 <211> 107 <212> PRT <213> Artificial sequence <220> <223> 223F12C4 variable light chain sequence <400> 35 Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Ile Ala Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ala Lys Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln Gly Gln Asp Tyr Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 36 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 HCDR1 <400> 36 Ile Asp Tyr Ala Trp Asn 1 5 <210> 37 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 HCDR2 <400> 37 Tyr Ile Ser Asn Ser Gly Tyr Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 38 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 HCDR3 <400> 38 Ser Gly His Tyr Asp Leu Phe Asp Tyr 1 5 <210> 39 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 LCDR1 <400> 39 Lys Ser Ser Gln Ser Leu Phe Asp Arg Asp Gly Lys Thr Tyr Leu Ser 1 5 10 15 <210> 40 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 LCDR2 <400> 40 Leu Val Ser Asn Leu Asp Ser 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 LCDR3 <400> 41 Trp Gln Gly Thr His Phe Pro Tyr Thr 1 5 <210> 42 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 225H7D12 HCDR1 <400> 42 Gly Tyr Trp Ile Glu 1 5 <210> 43 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 225H7D12 HCDR2 <400> 43 Glu Ile Leu Pro Gly Ser Gly Val Ser Asn Asn Asn Glu Lys Phe Arg 1 5 10 15 Asp <210> 44 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 225H7D12 HCDR3 <400> 44 Lys Ala Ser Tyr Tyr Thr Met Asp Tyr 1 5 <210> 45 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 225H7D12 LCDR1 <400> 45 Thr Ala Ser Ser Ser Val Ser Ser Phe Tyr Phe His 1 5 10 <210> 46 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 225H7D12 LCDR2 <400> 46 Ser Ile Ser Asn Leu Ala Ser 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 225H7D12 LCDR3 <400> 47 His Gln Tyr His Arg Ser Pro Arg Thr 1 5 <210> 48 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 Ha HCDR1 <400> 48 Asp Asn Tyr Met Asn 1 5 <210> 49 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 Ha HCDR2 <400> 49 Asp Ile Asn Pro Asn Asn Gly Gly Ser Arg Tyr Lys Glu Lys Phe Lys 1 5 10 15 Asp <210> 50 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 Ha HCDR3 <400> 50 Pro Gly Ser Tyr Gly Phe Ala Tyr 1 5 <210> 51 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 Hb HCDR1 <400> 51 Asp Tyr Tyr Met Asn 1 5 <210> 52 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 Hb HCDR2 <400> 52 Ile Ile Asn Pro Asn Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 53 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 Hb HCDR3 <400> 53 Arg Asp Tyr Asp Trp Phe Ala Tyr 1 5 <210> 54 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 LCDR1 <400> 54 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Arg Thr Tyr Leu Asn 1 5 10 15 <210> 55 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 LCDR2 <400> 55 Leu Val Ser Gln Leu Asp Ser 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 270C5C10 LCDR3 <400> 56 Trp Gln Gly Thr His Phe Pro Trp Thr 1 5 <210> 57 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 134D7B3 HCDR1 <400> 57 Asn Tyr Val Ile Glu 1 5 <210> 58 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 134D7B3 HCDR2 <400> 58 Val Ile Tyr Pro Gly Asn Gly Gly Ser Asp Tyr Asn Glu Lys Phe Lys 1 5 10 15 Val <210> 59 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 134D7B3 HCDR3 <400> 59 Phe Tyr Tyr Phe Gly Ser Ser Tyr Ala Met Asp Tyr 1 5 10 <210> 60 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 134D7B3 LCDR1 <400> 60 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 61 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 134D7B3 LCDR2 <400> 61 Tyr Thr Ser Arg Leu His Ser 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 134D7B3 LCDR3 <400> 62 Gln Gln Gly Asn Thr Leu Pro Phe Thr 1 5 <210> 63 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 177C6B11 HCDR1 <400> 63 Ser Phe Trp Met His 1 5 <210> 64 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 177C6B11 HCDR2 <400> 64 Arg Ile Asp Pro His Thr Gly Gly Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Thr <210> 65 <211> 14 <212> PRT <213> Artificial sequence <220> <223> 177C6B11 HCDR3 <400> 65 Gly Gly Tyr Tyr Asp Tyr Asp Gly Val Ala Trp Leu Ala Phe 1 5 10 <210> 66 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 177C6B11 LCDR1 <400> 66 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 67 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 177C6B11 LCDR2 <400> 67 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 177C6B11 LCDR3 <400> 68 Gln Gln Tyr Tyr Ser Tyr Pro Trp Thr 1 5 <210> 69 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 215F2A5 HCDR1 <400> 69 Asp His Tyr Met Thr 1 5 <210> 70 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 215F2A5 HCDR2 <400> 70 Asp Ile Asn Pro Tyr Ser Gly Thr Val Thr Phe Asn Gln Arg Phe Lys 1 5 10 15 Asp <210> 71 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 215F2A5 HCDR3 <400> 71 Arg Glu Ala Met Asp Tyr 1 5 <210> 72 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 215F2A5 LCDR1 <400> 72 Arg Ser Asp Gln Thr Ile Val Tyr Ser Asn Gly Asn Thr Tyr Leu Gln 1 5 10 15 <210> 73 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 215F2A5 LCDR2 <400> 73 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 74 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 215F2A5 LCDR3 <400> 74 Phe Gln Gly Ser His Val Pro Tyr Thr 1 5 <210> 75 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 223H11H1 HCDR1 <400> 75 Ala His Tyr Met Asn 1 5 <210> 76 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 223H11H1 HCDR2 <400> 76 Asp Ile Asn Pro Asp Asn Gly Ile Ile Arg Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 77 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 223H11H1 HCDR3 <400> 77 Arg Glu Ala Leu Asp Tyr 1 5 <210> 78 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 223H11H1 LCDR1 <400> 78 Arg Ser Ser Gln Ser Ile Val Tyr Ser Asn Gly Lys Val Tyr Leu Asn 1 5 10 15 <210> 79 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 223H11H1 LCDR2 <400> 79 Lys Val Ser Asp Arg Phe Ser 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 223H11H1 LCDR3 <400> 80 Leu Gln Gly Ser His Ile Pro Trp Thr 1 5 <210> 81 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 204C10G12 HCDR1 <400> 81 Ser Tyr Trp Ile Thr 1 5 <210> 82 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 204C10G12 HCDR2 <400> 82 Asp Ile Tyr Pro Gly Ser Asp Ser Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asn <210> 83 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 204C10G12 HCDR3 <400> 83 Gly Asp Asp Phe Trp Tyr Phe Asp Val 1 5 <210> 84 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 204C10G12 LCDR1 <400> 84 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 85 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 204C10G12 LCDR2 <400> 85 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 86 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 204C10G12 LCDR3 <400> 86 Phe Gln Gly Ser His Val Pro Leu Thr 1 5 <210> 87 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 H1 HCDR1 <400> 87 Asp Tyr Tyr Met His 1 5 <210> 88 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 H1 HCDR2 <400> 88 Glu Ile Asn Pro Ser Thr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Ala <210> 89 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 H1 HCDR3 <400> 89 Arg Asp Tyr Tyr Ala Met Asp Tyr 1 5 <210> 90 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 H2 HCDR1 <400> 90 Thr Phe Gly Met Gly Val Gly 1 5 <210> 91 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 H2 HCDR2 <400> 91 His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 92 <211> 15 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 H2 HCDR3 <400> 92 Ile Ala Pro Ile Thr Thr Val Val Ala Thr Asn Tyr Phe Asp Val 1 5 10 15 <210> 93 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 L1 LCDR1 <400> 93 Lys Ala Ser Gln Asp Val Arg Ile Ala Val Ala 1 5 10 <210> 94 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 L1 LCDR2 <400> 94 Ser Ala Ser Tyr Arg Phe Thr 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 L1 LCDR3 <400> 95 Gln Gln His Tyr Ser Phe Pro Leu Thr 1 5 <210> 96 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 L2 LCDR1 <400> 96 Arg Ala Ser Gln Ser Ile Gly Thr Ser Ile His 1 5 10 <210> 97 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 L2 LCDR2 <400> 97 Tyr Ala Ser Glu Ser Ile Ser 1 5 <210> 98 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 206H9G11 L2 LCDR3 <400> 98 Gln Gln Ser Asn Ser Trp Leu Thr 1 5 <210> 99 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 225A8D9 HCDR1 <400> 99 Ser Ser Trp Met Asn 1 5 <210> 100 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 225A8D9 HCDR2 <400> 100 Arg Ile Tyr Pro Gly Asp Gly Asn Ile Asn Tyr Asp Gly Lys Phe Lys 1 5 10 15 Gly <210> 101 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 225A8D9 HCDR3 <400> 101 Ser Ala Trp Leu Gly Lys Thr Tyr Val Met Asp Tyr 1 5 10 <210> 102 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 225A8D9 LCDR1 <400> 102 Ser Ala Ser Ser Ser Val Thr Tyr Met Asn 1 5 10 <210> 103 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 225A8D9 LCDR2 <400> 103 Glu Ile Ser Lys Leu Ala Ser 1 5 <210> 104 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 225A8D9 LCDR3 <400> 104 Gln Gln Trp Asn Phe Pro Phe Thr 1 5 <210> 105 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 274C7H2 HCDR1 <400> 105 Ser Tyr Trp Met His 1 5 <210> 106 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 274C7H2 HCDR2 <400> 106 Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 107 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 274C7H2 HCDR3 <400> 107 Asn Pro Ser Tyr Asp Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 108 <211> 14 <212> PRT <213> Artificial sequence <220> <223> 274C7H2 LCDR1 <400> 108 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 109 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 274C7H2 LCDR2 <400> 109 Gly Thr Asn Asn Arg Ala Pro 1 5 <210> 110 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 274C7H2 LCDR3 <400> 110 Ala Leu Trp Tyr Ser Asn His Phe Ile 1 5 <210> 111 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 383H3H11 HCDR1 <400> 111 Ser Tyr Gly Val His 1 5 <210> 112 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 383H3H11 HCDR2 <400> 112 Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser 1 5 10 15 <210> 113 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 383H3H11 HCDR3 <400> 113 Ile Gly Leu Arg Ser Phe Ala Tyr 1 5 <210> 114 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 383H3H11 LCDR1 <400> 114 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 115 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 383H3H11 LCDR2 <400> 115 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 116 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 383H3H11 LCDR3 <400> 116 Gln Asn Asp Tyr Ser Tyr Pro Phe Thr 1 5 <210> 117 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 163H12G7 HCDR1 <400> 117 Thr Tyr Thr Val His 1 5 <210> 118 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 163H12G7 HCDR2 <400> 118 Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 119 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 163H12G7 HCDR3 <400> 119 Trp Ser Tyr Trp Tyr Phe Asp Val 1 5 <210> 120 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 163H12G7 LCDR1 <400> 120 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 121 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 163H12G7 LCDR2 <400> 121 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 122 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 163H12G7 LCDR3 <400> 122 Tyr Gln Ala Thr His Phe Pro Leu Thr 1 5 <210> 123 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 HCDR1 <400> 123 Pro Ser Gly Met Gly Val Thr 1 5 <210> 124 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 HCDR2 <400> 124 His Ile Tyr Trp Asp Asp Val Lys Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 125 <211> 14 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 HCDR3 <400> 125 Arg Gly Ser Tyr Ser Asp Tyr Gly Gly Trp Ser Phe Asp Val 1 5 10 <210> 126 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 La LCDR1 <400> 126 Lys Ser Ser Gln Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 127 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 La LCDR2 <400> 127 Gly Ala Ser Thr Arg Glu Ser 1 5 <210> 128 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 La LCDR3 <400> 128 Gln Asn Asp His Ser Tyr Pro Pro Thr 1 5 <210> 129 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 Lb LCDR1 <400> 129 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 130 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 Lb LCDR2 <400> 130 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 131 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 Lb LCDR3 <400> 131 Gln Asn Asp Tyr Ser Tyr Pro Leu Thr 1 5 <210> 132 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 Lc LCDR1 <400> 132 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 133 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 Lc LCDR2 <400> 133 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 134 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 172H7B9 Lc LCDR3 <400> 134 Gln Asn Asp His Ser Tyr Pro Pro Thr 1 5 <210> 135 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 223F12C4 HCDR1 <400> 135 Ser Tyr Trp Ile His 1 5 <210> 136 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 223F12C4 HCDR2 <400> 136 Arg Ile His Pro Ser Asp Gly Asp Ile Asp His Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 137 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 223F12C4 HCDR3 <400> 137 Tyr Ile Met Asp Tyr 1 5 <210> 138 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 223F12C4 LCDR1 <400> 138 His Ala Ser Gln Asn Ile Asn Val Trp Leu Ser 1 5 10 <210> 139 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 223F12C4 LCDR2 <400> 139 Lys Ala Ala Lys Leu Gln Thr 1 5 <210> 140 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 223F12C4 LCDR3 <400> 140 His Gln Gly Gln Asp Tyr Pro Leu Thr 1 5 <210> 141 <211> 330 <212> PRT <213> Artificial sequence <220> <223> Human IgG1 CH <400> 141 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 142 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 CL <400> 142 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15[[ID=4​ 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 143 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 45F1F3 VH1.1 <400> 143 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ile Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Phe Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Gly His Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 144 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 45F1F3 VH1.3 <400> 144 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ile Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Gly His Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 145 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 45F1F3 VH1.4 <400> 145 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ile Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Phe Ser Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Gly His Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 146 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 45F1F3 L1 <400> 146 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Phe Asp Arg 20 25 30 Asp Gly Lys Thr Tyr Leu Ser Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Asn Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 147 <211> 112 <212> PRT <213> Artificial sequence <220> <223> 45F1F3 L1.1 <400> 147 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Phe Asp Arg 20 25 30 Asp Gly Lys Thr Tyr Leu Ser Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Asn Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 148 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 225H7D12 VH1.1 <400> 148 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Thr Gly Tyr Arg Phe Thr Gly Tyr 20 25 30 Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Val Ser Asn Asn Asn Glu Lys Phe 50 55 60 Arg Asp Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Lys Ala Ser Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 149 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> 225H7D12 VH1.3 <400> 149 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Gly Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Val Ser Asn Asn Asn Glu Lys Phe 50 55 60 Arg Asp Arg Ala Thr Met Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Lys Ala Ser Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 150 <211> 118 <212> PRT <213> artificial sequence <220> <223> 225H7D12 VH1.4 <400> 150 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Thr Gly Tyr Arg Phe Thr Gly Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Val Ser Asn Asn Asn Glu Lys Phe 50 55 60 Arg Asp Arg Ala Thr Phe Thr Ala Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Lys Ala Ser Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 151 <211> 108 <212> PRT <213> artificial sequence <220> <223> 225H7D12 L1.2 <400> 151 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Phe 20 25 30 Tyr Phe His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Ile Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 152 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 270C5C10 VHa1.2 <400> 152 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Gly Ser Arg Tyr Lys Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Pro Gly Ser Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 153 <211> 117 <212> PRT <213> artificial sequence <220> <223> 270C5C10 VHa1.4 <400> 153 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30 Tyr Met Asn Trp Ala Arg Gln Ser Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Gly Ser Arg Tyr Lys Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Pro Gly Ser Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 154 <211> 112 <212> PRT <213> artificial sequence <220> <223> 270C5C10 VL1 <400> 154 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Arg Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Gln Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 155 <211> 329 <212> PRT <213> Artificial sequence <220> <223> Human IgG1 CH variant 1 <400> 155 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 156 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 CH Variant 2 <400> 156 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 \nGly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330

Claims

1. An isolated monoclonal antibody or its antigen-binding fragment, comprising heavy chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3 and light chain complementarity-determining regions 1 (LCDR1), LCDR2 and LCDR3, wherein, The polypeptide sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 36, 37, 38, 39, 40 and 41, respectively; The antibody or its antigen-binding fragment specifically binds to glucocorticoid-induced tumor necrosis factor receptor-associated protein (GITR).

2. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 1, wherein the antibody or its antigen-binding fragment specifically binds to human GITR.

3. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 1, comprising a heavy chain variable region or a light chain variable region, wherein the polypeptide sequence of the heavy chain variable region has at least 95% identity with SEQ ID NO: 1, and the polypeptide sequence of the light chain variable region has at least 95% identity with SEQ ID NO:

2.

4. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 1, comprising a heavy chain variable region or a light chain variable region, wherein the polypeptide sequence of the heavy chain variable region has at least 96% identity with SEQ ID NO: 1, and the polypeptide sequence of the light chain variable region has at least 96% identity with SEQ ID NO:

2.

5. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 1, comprising a heavy chain variable region or a light chain variable region, wherein the polypeptide sequence of the heavy chain variable region has at least 97% identity with SEQ ID NO: 1, and the polypeptide sequence of the light chain variable region has at least 97% identity with SEQ ID NO:

2.

6. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 1, comprising a heavy chain variable region or a light chain variable region, wherein the polypeptide sequence of the heavy chain variable region has at least 98% identity with SEQ ID NO: 1, and the polypeptide sequence of the light chain variable region has at least 98% identity with SEQ ID NO:

2.

7. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 1, comprising a heavy chain variable region or a light chain variable region, wherein the polypeptide sequence of the heavy chain variable region has at least 99% identity with SEQ ID NO: 1, and the polypeptide sequence of the light chain variable region has at least 99% identity with SEQ ID NO:

2.

8. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 3, wherein the polypeptide sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the polypeptide sequence of the light chain variable region is as shown in SEQ ID NO:

2.

9. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 1, wherein the antibody or its antigen-binding fragment is chimeric.

10. The isolated monoclonal antibody or antigen-binding fragment thereof as claimed in claim 9, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a human IgG1 constant region.

11. The isolated monoclonal antibody or antigen-binding fragment thereof as claimed in claim 10, wherein the constant region of these individuals' IgG1 contains at least one amino acid modification selected from K214R, D356E, L358M, and ΔK447.

12. The isolated monoclonal antibody or its antigen-binding fragment as described in claim 9, wherein the antibody or its antigen-binding fragment is humanized.

13. The isolated humanized monoclonal antibody or its antigen-binding fragment as described in claim 12, wherein: a. The polypeptide sequence of the heavy chain variable region is shown in SEQ ID NO: 145, and the polypeptide sequence of the light chain variable region is shown in SEQ ID NO: 147; b. The polypeptide sequence of the heavy chain variable region is shown in SEQ ID NO: 143, and the polypeptide sequence of the light chain variable region is shown in SEQ ID NO: 146; c. The polypeptide sequence of the heavy chain variable region is shown in SEQ ID NO: 143, and the polypeptide sequence of the light chain variable region is shown in SEQ ID NO: 147; d. The polypeptide sequence of the heavy chain variable region is shown in SEQ ID NO: 144, and the polypeptide sequence of the light chain variable region is shown in SEQ ID NO: 146; e. The polypeptide sequence of the heavy chain variable region is shown in SEQ ID NO: 144, and the polypeptide sequence of the light chain variable region is shown in SEQ ID NO: 147; f. The polypeptide sequence of the heavy chain variable region is shown in SEQ ID NO: 145, and the polypeptide sequence of the light chain variable region is shown in SEQ ID NO:

146.

14. The isolated monoclonal antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 13, wherein the monoclonal antibody or antigen-binding fragment thereof is capable of binding to GITR and inducing effector-mediated tumor cell lysis.

15. An isolated nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 14.

16. A vector comprising the isolated nucleic acid as described in claim 15.

17. A host cell comprising the vector as described in claim 16.

18. A pharmaceutical composition comprising an isolated monoclonal antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 14, and a pharmaceutically acceptable carrier.

19. The use of the pharmaceutical composition of claim 18 in the preparation of a medicament for treating cancer in a subject in need, wherein the cancer is selected from lung cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, and non-Hodgkin's lymphoma.

20. A method for producing a monoclonal antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 14, the method comprising culturing a cell containing a nucleic acid encoding the monoclonal antibody or an antigen-binding fragment thereof under conditions for producing the monoclonal antibody or an antigen-binding fragment thereof, and recovering the antibody or an antigen-binding fragment thereof from the cell or culture.

21. A method for producing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 14, the method comprising combining the monoclonal antibody or the antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

22. Use of the isolated monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 14 in the preparation of a diagnostic agent or medicament for determining the GITR level of a subject, wherein determining the GITR level of the subject comprises: a. Obtain samples from the subject; b. Contacting the sample with the isolated monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 14; and c. Determine the subject's GITR level. The subject has cancer or is at risk of developing cancer, and the cancer is selected from lung cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, and non-Hodgkin's lymphoma.

23. The use as described in claim 22, wherein the sample is a tissue sample or a blood sample.

24. The use as described in claim 23, wherein the tissue sample is a cancer tissue sample.

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