Personalized Cancer Immunotherapy

Abstract

The present invention relates to a method for obtaining an antibody or an antigen-binding fragment thereof that specifically binds to a tumor sample and is directed against the tumor sample, the method comprising: administering autologous dendritic cells to an individual; obtaining immune cells and a tumor sample from the individual; constructing an antibody library of the immune cells; and screening the antibody library to obtain an antibody or a fragment thereof that specifically binds to the tumor sample and is directed against the tumor sample. The present invention also relates to a method for engineering immune cells, an antibody or an antigen-binding fragment thereof that specifically binds to a tumor sample and is directed against the tumor sample, and uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of cancer immunotherapy. Specifically, the present invention relates to methods for obtaining antibodies for personalized cancer immunotherapy. Background Art

[0002] Precision medicine and immunotherapy are currently two of the hottest areas in cancer research. Immunotherapy aims to stimulate (or restore) a patient's own immune system to fight cancer. One type of personalized immunotherapy that has been approved for patient use is chimeric antigen receptor (CAR)-T cell therapy, a novel immuno-oncology treatment modality.

[0003] There is a need for technologies that can achieve more effective personalized cancer immunotherapy against cancer. Summary of the Invention

[0004] The present invention provides a method for obtaining an antibody or an antigen-binding fragment thereof that specifically binds to and is directed against a tumor in an individual suffering from a tumor, comprising:

[0005] obtaining a tumor sample from the individual;

[0006] administering dendritic cells to the individual to elicit an anti-tumor immune response;

[0007] collecting immune cells of the individual;

[0008] constructing an antibody library of the immune cells; and

[0009] screening the antibody library to obtain the antibody or fragment thereof that specifically binds to and is directed against the tumor.

[0010] In one embodiment, the individual undergoes surgical resection or concurrent chemotherapy and radiotherapy.

[0011] In one embodiment, the individual undergoes surgical resection or concurrent chemotherapy and radiotherapy before administering dendritic cells.

[0012] In one embodiment, the tumor sample is obtained before administering dendritic cells.

[0013] In one embodiment, the dendritic cells are autologous or allogeneic; preferably autologous. In another embodiment of the present invention, autologous dendritic cells are collected from the individual before or after the individual undergoes radiotherapy, chemotherapy, or surgery. In yet another embodiment, autologous dendritic cells are derived from monocytes collected from the individual before or after the individual receives radiotherapy, chemotherapy, or surgery.

[0014] In one embodiment of the present invention, dendritic cells are administered to an individual more than once. In one embodiment, dendritic cells are administered more than twice at regular time intervals. In one embodiment, dendritic cells are administered twice. In another embodiment, the time interval is once a week. In one embodiment, dendritic cells are administered to the individual twice a week.

[0015] In one embodiment, the method comprises:

[0016] obtaining a tumor sample from an individual;

[0017] administering dendritic cells to the individual twice a week to elicit an anti-tumor immune response after the individual undergoes surgical resection or concurrent chemotherapy and radiotherapy;

[0018] collecting immune cells of the individual;

[0019] constructing an antibody library of the immune cells; and

[0020] screening the antibody library to obtain an antibody or a fragment thereof that specifically binds to the tumor sample and is directed against the tumor sample.

[0021] In one embodiment of the present invention, dendritic cells are administered to an individual at a dose of from 10 4 cells / kg body weight to 10 8 cells / kg body weight. In another embodiment, dendritic cells are administered to an individual at a dose of from 10 4 cells / kg body weight to 10 6 cells / kg body weight.

[0022] In one embodiment, the immune cells described herein are derived from peripheral blood mononuclear cells (PBMCs).

[0023] In one embodiment of the present invention, the immune cells described herein are B cells that are a major part of the construction of a combinatorial antibody library.

[0024] In one embodiment of the present invention, the antibody library is constructed by phage display.

[0025] In one embodiment of the present invention, the method further comprises establishing a primary tumor cell line based on the tumor sample and screening the antibody library with the primary tumor cell line.

[0026] In one embodiment of the present invention, the antibody library is screened by cell-based screening.

[0027] In one embodiment of the present invention, the antibody or a fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or an scFv antibody or a fragment thereof.

[0028] In one embodiment of the present invention, the antibody is an anti-carbonic anhydrase IX (CAIX) antibody.

[0029] In one embodiment of the present invention, the tumor is squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric / stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney / renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, melanoma, multiple myeloma, B-cell lymphoma, brain cancer, head and neck cancer or their related metastatic cancers.

[0030] The present invention also provides a method for engineering an immune cell that specifically binds to a tumor sample and is directed against the tumor sample, comprising:

[0031] Obtaining an antibody or an antigen-binding fragment thereof that specifically binds to a tumor sample and is directed against the tumor sample according to the method mentioned above;

[0032] Providing an immune cell; and

[0033] Expressing the antibody or an antigen-binding fragment thereof on the surface of the immune cell.

[0034] In one embodiment of the present invention, the immune cell is a chimeric antibody immune cell.

[0035] The present invention also provides an antibody or an antigen-binding fragment thereof that specifically binds to a tumor sample and is directed against the tumor sample, which comprises complementarity-determining regions (CDRs) of the heavy-chain variable region and complementarity-determining regions of the light-chain variable region, wherein the complementarity-determining regions of the heavy-chain variable region comprise CDRH1, CDRH2 and CDRH3 regions, and the complementarity-determining regions of the light-chain variable region comprise CDRL1, CDRL2 and CDRL3 regions, and wherein:

[0036] The CDRH1 region comprises the amino acid sequence of SEQ ID NO:1 (SYAMQ); the CDRH2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 (GMSDDGSWTDYGAAVKG) and SEQ ID NO:3 (GVSDDGSWTGYGAAVQG); the CDRH3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4 (GAGTGYCDNRSFGCASTIDA) and SEQ ID NO:5 (GAGTGYCNNRGFGCASTIDA); and

[0037] The CDRL1 region contains the amino acid sequence of SEQ ID NO: 6 (SGSSGSYG); the CDRL2 region contains an amino acid sequence selected from the group consisting of SEQ ID NO: 7 (HNDKRPS) and SEQ ID NO: 8 (YNDKRPS); and the CDRL3 region contains the amino acid sequence of SEQ ID NO: 9 (GSADRSGAGI).

[0038] In one embodiment of the present invention, the antibody or its antigen-binding fragment is a mammalian antibody.

[0039] In one embodiment of the present invention,

[0040] The CDRH1 region contains the amino acid sequence of SEQ ID NO: 1; the CDRH2 region contains the amino acid sequence of SEQ ID NO: 2; the CDRH3 region contains the amino acid sequence of SEQ ID NO: 4; and

[0041] The CDRL1 region contains the amino acid sequence of SEQ ID NO: 6; the CDRL2 region contains the amino acid sequence of SEQ ID NO: 7; and the CDRL3 region contains the amino acid sequence of SEQ ID NO: 9.

[0042] In one embodiment of the present invention,

[0043] The CDRH1 region contains the amino acid sequence of SEQ ID NO: 1; the CDRH2 region contains the amino acid sequence of SEQ ID NO: 3; the CDRH3 region contains the amino acid sequence of SEQ ID NO: 5; and

[0044] The CDRL1 region contains the amino acid sequence of SEQ ID NO: 6; the CDRL2 region contains the amino acid sequence of SEQ ID NO: 8; and the CDRL3 region contains the amino acid sequence of SEQ ID NO: 9.

[0045] In one embodiment of the present invention, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 (AVTLDESGGGLHTPGGGLSLVCRASGFTFSSYAMQWVRQAPGKGLEWVAGMSDDGSWTDYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKGAGTGYCDNRSFGCASTIDAWGHGTEVIVSS); and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYHNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVL).

[0046] In one embodiment of the present invention, the antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (AVTLDESGGGLQTPGGGLSLVCRASGFTMTSYAMQWVRQAPGKGLEWVAGVSDDGSWTGYGAAVQGRATISRDNGQSTVRLLLNNLRAEDTATYYCVKGAGTGYCNNRGFGCASTIDAWGHGTEVIVSS); and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVL).

[0047] In one embodiment of the present invention, the antibody or its antigen-binding fragment comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:14 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYHNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLHTPGGGLSLVCRASGFTFSSYAMQWVRQAPGKGLEWVAGMSDDGSWTDYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKGAGTGYCDNRSFGCASTIDAWGHGTEVIVSS) and SEQ ID NO:15 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRPSDIPS RFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGGLSLVCRASGFTMTSYAMQWVRQAPGKGLEWVAGVSDDGSWTGYGAAVQGRATISRDNGQSTVRLLLNNLRAEDTATYYCVKGAGTGYCNNRGFGCASTIDAWGHGTEVIVSS).

[0048] In one embodiment of the present invention, the antibody or its antigen-binding fragment is expressed on the surface of immune cells.

[0049] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned antibody or its antigen-binding fragment and, optionally, a pharmaceutically acceptable carrier or excipient.

[0050] The present invention provides the use of the above-mentioned pharmaceutical composition for the manufacture of a medicament for treating tumors in an individual in need thereof.

[0051] In one embodiment of the present invention, the medicament is administered intravenously, intraperitoneally, intraarterially, intrathecally, intravesically or intratumorally.

[0052] The present invention provides a method for treating tumors in an individual in need thereof, which comprises administering the above-mentioned pharmaceutical composition.

[0053] In one embodiment of the present invention, the administration is carried out intravenously, intraperitoneally, intraarterially, intrathecally, intravesically or intratumorally.

[0054] Brief Description of the Drawings

[0055] Figure 1 Shows the results of cell-based ELISA of K562-CAIX.

[0056] Figures 2A to 2C Shows the results of cell surface staining. 2A: Unstained control antibody (anti-CAIX); scFv L6. 2B: Long 1B, 3A, 2F, 3C antibodies. 2C: Short 1C, 1F, 1G, 2B antibodies.

[0057] Figure 3 Shows a schematic diagram of a method for obtaining an antibody or an antigen-binding fragment thereof that specifically binds to a tumor sample and is directed against the tumor sample.

[0058] Figure 4 Shows the results of cell-based ELISA of cells of KYL. Detailed Description

[0059] To facilitate understanding of the technology of the present invention, several terms and phrases are defined below. Other definitions are set forth throughout the description.

[0060] In addition, as used herein, unless the context clearly dictates otherwise, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or". Unless the context clearly dictates otherwise, the term "based on" is not exclusive and allows for additional factors not described. Also, throughout the specification, the meanings of "a / an" and "the" include plural references. The meaning of "in" includes "in" and "on".

[0061] As used herein, the term "specifically binds" means that an antibody does not cross-react to a significant extent with other epitopes.

[0062] As used herein, the term "epitope" refers to the site on an antigen to which an antibody binds.

[0063] As used herein, the term "antibody" refers to single-chain, double-chain, and multi-chain proteins and polypeptides belonging to the classes of polyclonal, monoclonal, chimeric, and humanized antibodies; it also includes synthetic and genetically engineered variants of such antibodies. "Antibody fragment" includes Fab, Fab', F(ab')2, and Fv fragments, as well as any portion of an antibody having specificity for one or more desired target epitopes.

[0064] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. In other words, a monoclonal antibody consists of homogeneous antibodies produced from the growth of a single cell line (e.g., a hybridoma cell, a eukaryotic host cell transfected with a DNA molecule encoding a homogeneous antibody, or a prokaryotic host cell transfected with a DNA molecule encoding a homogeneous antibody). These antibodies are directed against a single epitope and thus have a high degree of specificity.

[0065] As used herein, the term "humanized antibody" refers to a recombinant protein in which the antibody CDRs from one species (e.g., mouse or chicken) are transferred from the heavy chain variable region and the light chain variable region of the antibody of that species into the human heavy chain variable domain and the light chain variable domain (framework regions). The constant domains of the antibody molecule are derived from the constant domains of the human antibody. In some cases, specific residues in the framework regions of the humanized antibody (especially those that contact or are close to the CDR sequences) may be modified, e.g., they may be replaced by the corresponding residues from a source mouse, rodent, cynomolgus monkey, or other antibody. Humanized antibodies can be achieved by a variety of methods, including (a) transplanting only the non-human CDRs onto the human framework and constant regions, with or without retaining key framework residues, or (b) transplanting the entire non-human variable domain by replacing surface residues, but "masking" it with a human-like portion. Such methods suitable for practicing the present invention include those disclosed in Padlan, Mol. Immunol., 31(3):169-217(1994).

[0066] As used herein, the term "chimeric antibody" refers to a recombinant protein containing the variable domains of both the antibody heavy chain and the antibody light chain, which includes the complementarity-determining regions (CDRs) of an antibody from one species (preferably a rodent antibody or a chicken antibody, more preferably a mouse antibody), whereas the constant domains of the antibody molecule are derived from those of a human antibody.

[0067] As used herein, the term "complementarity-determining region" (CDR) refers to the non-contiguous antigen-combining sites found within the variable regions of the heavy and light chain polypeptides. The CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616(1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); by Chothia et al., J. Mol. Biol. 196:901-917(1987); and MacCallum et al., J. Mol. Biol. 262:732-745(1996), where the definitions include overlapping or subsets of amino acid residues when compared to each other.

[0068] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that when two peptide sequences have at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned using default gap weights, such as by the programs GAP or BESTFIT. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or similarity can be adjusted upward to correct for the nature of the conservative substitutions. The manner of making such an adjustment is well known to those skilled in the art. Examples of amino acid groups having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0069] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, an individual or a patient and can be used in diagnostic or monitoring assays. The definition encompasses blood and other liquid samples of biological origin; solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny.

[0070] As used herein, the term "treatment / treating" and its like terms encompass any treatment of a disease in a mammal (especially a human), and include: (a) preventing a disease from occurring in an individual who may be predisposed to the disease but has not been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing the disease to regress.

[0071] As used interchangeably herein, the terms "individual", "subject", "host", and "patient" refer to a mammal, including but not limited to rats (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, sheep, suids, goats), etc.

[0072] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an antibody that is sufficient to effect the treatment of a disease in a mammal or other individual when administered for treating the disease.

[0073] The present invention provides a method for obtaining an antibody or an antigen-binding fragment thereof that specifically binds to a tumor and is directed against the tumor in an individual having a tumor, comprising:

[0074] obtaining a tumor sample from the individual;

[0075] administering dendritic cells to the individual to elicit an anti-tumor immune response;

[0076] collecting immune cells of the individual;

[0077] constructing an antibody library of the immune cells; and

[0078] screening the antibody library to obtain the antibody or a fragment thereof that specifically binds to the tumor and is directed against the tumor.

[0079] In one embodiment, a tumor sample is obtained to establish a specific primary tumor cell line. In one embodiment, the immune cells described herein are derived from peripheral blood mononuclear cells (PBMCs).

[0080] It is believed (but not intended to be limited by any theory) that dendritic cells are antigen-presenting cells of the mammalian immune system that present antigens on the cell surface to T cells of the immune system. Administering autologous dendritic cells prior to collecting immune cells enhances the specificity and effectiveness of the screening and facilitates obtaining a powerful antibody that specifically binds to the tumor sample and is directed against the tumor sample. Autologous dendritic cells can be derived from monocytes of the individual or provided by proliferation. Preferably, the dendritic cells are derived from PBMCs of the individual; more preferably, directly from peripheral blood.

[0081] In a preferred embodiment, the individual is a mammal. Exemplary mammals include humans, pigs, sheep, goats, horses, mice, dogs, cats, cows, etc.

[0082] Clinically, cancer patients typically undergo radiotherapy, chemotherapy, or surgery. To collect effective dendritic cells, dendritic cells are preferably collected from an individual before the individual undergoes radiotherapy, chemotherapy, or surgery. The duration between radiotherapy, chemotherapy, or surgery and collection can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, preferably 7 days.

[0083] The administration of dendritic cells can be carried out more than once; preferably, dendritic cells are administered to the individual twice at regular time intervals. The interval between administrations can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, preferably 7 days.

[0084] In another aspect, dendritic cells are administered to the individual in an amount sufficient to demonstrate antigen presentation. Preferably, dendritic cells are administered to the individual at a dose of 10 4 cells / kg body weight to 10 6 cells / kg body weight.

[0085] In the case where the individual undergoes radiotherapy, chemotherapy, or surgery, dendritic cells are preferably administered to the individual after the individual undergoes radiotherapy, chemotherapy, or surgery.

[0086] The collected immune cells can be from lymph or blood, preferably from peripheral blood. Preferably, the immune cells are collected 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, preferably 7 days, after the administration of dendritic cells.

[0087] Examples of immune cells according to the present invention include but are not limited to lymphocytes, neutrophils, monocytes, and macrophages. Preferably, the immune cells are lymphocytes. Examples of lymphocytes include but are not limited to T cells, B cells, and NK cells. Preferably, the immune cells are B cells.

[0088] The manner of constructing the antibody library of immune cells can be altered. Examples of construction include but are not limited to phage display, in vitro display, yeast display, mammalian display, and bacterial display; preferably, the antibody library is constructed by phage display.

[0089] The tumor sample can be provided directly by the individual or processed. Preferably, the method further comprises establishing a primary tumor cell line from the tumor sample and screening the antibody library with the primary tumor cell line. In addition, preferably, the antibody library is screened by cell-based screening.

[0090] The antibody or its fragment can be a monoclonal antibody, chimeric antibody, humanized antibody, human antibody, or scFv antibody or its fragment, preferably an scFv antibody.

[0091] In one embodiment of the present invention, anti-carbonic anhydrase IX (CAIX) antibodies are screened according to the present invention.

[0092] Tumors that can be treated with the antibody or its pharmaceutical composition include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (epithelial squamous cell carcinoma); lung cancers including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; peritoneal cancer; hepatocellular carcinoma; gastric cancer (gastric / stomach cancer) including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer (liver cancer); bladder cancer; urethral cancer; hepatoma; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer (kidney / renal cancer); prostate cancer; vulvar cancer; thyroid cancer; hepatic carcinoma; anal cancer; penile cancer; melanoma; multiple myeloma and B-cell lymphoma, brain cancer, and head and neck cancer, and related metastatic cancers.

[0093] The present invention also provides a method for engineering an immunocyte that specifically binds to a tumor sample and is directed against the tumor sample, which comprises:

[0094] Obtaining an antibody or its antigen-binding fragment that specifically binds to a tumor sample and is directed against the tumor sample according to the method mentioned above;

[0095] Providing an immunocyte; and

[0096] Expressing the antibody or its antigen-binding fragment on the immunocyte.

[0097] Preferably, the immunocyte is a chimeric antibody immunocyte. In one embodiment of the present invention, the chimeric antibody comprises an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular T cell signaling domain. The antibody or its antigen-binding fragment serves as the antigen recognition domain.

[0098] The present invention also provides an antibody or its antigen-binding fragment that specifically binds to a tumor sample and is directed against the tumor sample, which comprises complementarity-determining regions (CDRs) of the heavy chain variable region and complementarity-determining regions of the light chain variable region, wherein the complementarity-determining regions of the heavy chain variable region comprise CDRH1, CDRH2, and CDRH3 regions, and the complementarity-determining regions of the light chain variable region comprise CDRL1, CDRL2, and CDRL3 regions, and wherein:

[0099] The CDRH1 region contains the amino acid sequence of SEQ ID NO:1 (SYAMQ); the CDRH2 region contains an amino acid sequence selected from the group consisting of SEQ ID NO:2 (GMSDDGSWTDYGAAVKG) and SEQ ID NO:3 (GVSDDGSWTGYGAAVQG); the CDRH3 region contains an amino acid sequence selected from the group consisting of SEQ ID NO:4 (GAGTGYCDNRSFGCASTIDA) and SEQ ID NO:5 (GAGTGYCNNRGFGCASTIDA); and

[0100] the CDRL1 region contains the amino acid sequence of SEQ ID NO:6 (SGSSGSYG); the CDRL2 region contains an amino acid sequence selected from the group consisting of SEQ ID NO:7 (HNDKRPS) and SEQ ID NO:8 (YNDKRPS); the CDRL3 region contains the amino acid sequence of SEQ ID NO:9 (GSADRSGAGI).

[0101] Specifically, the antibody or its antigen-binding fragment is anti-CAIX L3A, wherein:

[0102] the CDRH1 region contains the amino acid sequence of SEQ ID NO:1; the CDRH2 region contains the amino acid sequence of SEQ ID NO:2; the CDRH3 region contains the amino acid sequence of SEQ ID NO:4; and

[0103] the CDRL1 region contains the amino acid sequence of SEQ ID NO:6; the CDRL2 region contains the amino acid sequence of SEQ ID NO:7; the CDRL3 region contains the amino acid sequence of SEQ ID NO:9.

[0104] Specifically, the antibody or its antigen-binding fragment is anti-CAIX S1C, wherein:

[0105] the CDRH1 region contains the amino acid sequence of SEQ ID NO:1; the CDRH2 region contains the amino acid sequence of SEQ ID NO:3; the CDRH3 region contains the amino acid sequence of SEQ ID NO:5; and

[0106] the CDRL1 region contains the amino acid sequence of SEQ ID NO:6; the CDRL2 region contains the amino acid sequence of SEQ ID NO:8; the CDRL3 region contains the amino acid sequence of SEQ ID NO:9.

[0107] Preferably, anti-CAIX L3A comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10 (AVTLDESGGGLHTPGGGLSLVCRASGFTFSSYAMQWVRQAPGKGLEWVAGMSDDGSWTDYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKGAGTGYCDNRSFGCASTIDAWGHGTEVIVSS); and a light chain variable region comprising the amino acid sequence of SEQ ID NO:11 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYHNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVL).

[0108] Preferably, anti-CAIX S1C comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:12 (AVTLDESGGGLQTPGGGLSLVCRASGFTMTSYAMQWVRQAPGKGLEWVAGVSDDGSWTGYGAAVQGRATISRDNGQSTVRLLLNNLRAEDTATYYCVKGAGTGYCNNRGFGCASTIDAWGHGTEVIVSS); and a light chain variable region comprising the amino acid sequence of SEQ ID NO:13 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVL).

[0109] More preferably, anti-CAIX L3A comprises the amino acid sequence of SEQ ID NO:14 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYHNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLHTPGGGLSLVCRASGFTFSSYAMQWVRQAPGKGLEWVAGMSDDGSWTDYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKGAGTGYCDNRSFGCASTIDAWGHGTEVIVSS).

[0110] More preferably, the anti-CAIX S1C comprises the amino acid sequence of SEQ ID NO:15 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRPSDIPSRFSGSKS GSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQT PGGGLSLVCRASGFTMTSYAMQWVRQAPGKGLEWVAGVSDDGSWTGYGAAVQGRATISRDNGQSTVRLLLNNLRAEDTATYYCVKGAGTGYCNNRGFGCASTIDAWGHGTEVIVSS).

[0111] In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment is conjugated to a therapeutic agent. In a preferred embodiment of the present invention, the antibody or its antigen-binding fragment is expressed on the cell surface. More preferably, the cell is an immune cell, and even more preferably, a NK-cell.

[0112] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody or its antigen-binding fragment mentioned above and, optionally, a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of the present invention is formulated with suitable carriers, excipients, and other agents that provide improved delivery, transfer, tolerance, and the like. A variety of suitable formulations can be found in all pharmacists' formularies known: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic), DNA conjugates, anhydrous absorbent pastes, water-in-oil and oil-in-water emulsions, emulsion carbopol (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbopol.

[0113] The antibody dose administered to a patient can vary depending on the patient's age and body size, the target disease, the condition, the route of administration, and similar factors. The preferred dose is usually calculated based on body weight or body surface area. When the antibody of the present invention is used to treat tumors in adult patients, it is advantageous to administer the antibody of the present invention intravenously. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dose and duration of antibody administration can be determined empirically; for example, patient progression can be monitored by periodic evaluations and the dose adjusted accordingly. In addition, methods well known in the art can be used to adjust the dose between species.

[0114] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Introduction methods include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraarterial, intrathecal, intravesical or intratumoral, epidural and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as oral mucosa, rectal mucosa, and intestinal mucosa, etc.) and can be administered together with other biological active agents. Administration can be systemic or local. Preferably, the drug is administered intravenously, intraperitoneally, intraarterially, intrathecally, intravesically or intratumorally.

[0115] The present invention provides the use of the pharmaceutical composition mentioned above for manufacturing a drug for treating tumors of an individual in need.

[0116] The present invention provides a method for treating tumors of an individual in need, which comprises administering the pharmaceutical composition mentioned above.

[0117] In one embodiment of the present invention, the administration is carried out intravenously, intraperitoneally, intraarterially, intrathecally, intravesically or intratumorally.

[0118] The following examples are provided to assist those skilled in the art in practicing the present invention.

[0119] Examples

[0120] Example 1: Expression, purification and binding analysis of anti-CAIX Ab

[0121] K562 cells were obtained for negative selection; meanwhile, CAIX-overexpressing K562 cells were obtained for positive selection. An antibody phage library was established according to chickens immunized with CAIX Ag.

[0122] An antibody library was constructed based on a previously reported method (Andris-Widhopf J, Rader C, Steinberger P, et al. Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods 2000;242:159-81). Briefly, spleens collected from chickens after final immunization were immediately placed in Trizol (Gibco BRL., USA) for homogenization. 10 μg of RNA was reverse-transcribed into first-strand cDNA using the SuperScript RT kit (Invitrogen, USA). After amplification using chicken-specific primers, the PCR products of the heavy and light chain variable (VH and VL) regions were subjected to a second round of PCR to form full-length scFv fragments with short or long linkers, which were further digested with SfiI and cloned into the pComb3X vector. Recombinant phage DNA was transformed into Escherichia coli strain ER2738 by electroporation (MicroPulser from BioRad). The production of recombinant phage was initiated by adding wild-type VCS-M13 helper phage, followed by precipitation with 4% polyethylene glycol 8000 and 3% NaCl (w / v), and finally resuspension in 1× phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). Subsequently, the recombinant phage in the scFv antibody library was blocked with PBS containing 1% BSA at 4 °C (50 μ l) for 10 12 to 10 13 plaque-forming units (pfu) for 1 hour, and then incubated with 10 6 K562 cells (100 μ l) at room temperature twice for 30 minutes (negative selection). After collecting the unbound phage supernatant, the supernatant was incubated with CAIX-overexpressing K562 cells at 4 °C for 1 hour (positive selection). Subsequently, the cells were washed 5 - 8 times with PBS. The bound phage was eluted with 0.2 M glycine-HCl (pH 2.2), neutralized with 1 M Tris-HCl buffer, and then used to infect Escherichia coli strain ER2738. The amplified phage was precipitated and recovered as described above for the next round of selection.

[0123] Libraries with short and long linkers were constructed as shown in Table 1. The elution titers after each screening are shown (Table 1). After three rounds of screening, CAIX-binding phage variants were highly enriched. These results indicate that non-specific binding phages were removed during the screening process and pure lines with specific binding affinity were enriched. The sequences were confirmed to belong to the chicken immunoglobulin germline genes.

[0124] Table 1

[0125]

[0126] To examine its binding reactivity, 50 μ μl of the candidate phage was incubated with K562 or CAIX-overexpressing K562 cells in ELISA plate wells at room temperature with shaking at 150 rpm for 2 hours. After washing 3 times with PBS, it was incubated with HRP-conjugated anti-M13 antibody (1:5,000) at room temperature for 2 hours to detect the bound scFv. After washing 6 times with PBS, tetramethylbenzidine (TMB) substrate solution (Sigma, USA) was added to each well for color development. The reaction was stopped with 2N H2SO4 and the optical density was measured at 450 nm to 540 nm using an ELISA plate reader (BioTek Synergy HT). The results of cell-based ELISA are shown in Figure 1 . The candidate scFvs had higher binding activity to CAIX-overexpressing K562 cells compared to K562 cells.

[0127] The binding activity of the candidate phage clones was detected using an anti-HA antibody. Flow cytometry analysis was performed on 1×10 5 K562 or K562-CAIX cells and 20 μl of the candidate phage clones. The results of cell surface staining are shown in Figures 2A to 2C . All scFvs except anti-CAIX L1B scFv could specifically bind to CAIX-overexpressing cells.

[0128] Among the antibodies, anti-CAIX L3A (long 3A) and S1C (short 1C) were obtained.

[0129] Example 2: Anti-Thyroid Cancer Ab in In Vivo Analysis

[0130] A schematic diagram of the method for obtaining antibodies or antigen-binding fragments thereof that specifically bind to tumor samples and are directed against tumor samples is shown in Figure 3 .

[0131] Dendritic cells were collected from patients with thyroid cancer. After the patients underwent surgical resection or concurrent chemotherapy and radiotherapy, the patients received two injections (in week 1 and week 2) of 1×10 4 to 1×10 6 cells / kg body weight of autologous DC cells into the axillary lymph nodes (ALNs). Surgical resection samples were used to establish tumor cell lines, and peripheral blood collected in week 3 was used to establish an Ab phage library.

[0132] Thyroid cancer cells (KYL cells) were established from a patient and cultured in RPMI 1640 medium (HyUNK, SH30027) supplemented with 1.5% PLUS TM Human Plate Lysate (CPMPASS, CPS-PLS5-BAG) and 2% penicillin-streptomycin (Life technologies, 15140) at 37°C in humidified air with 5% CO2.

[0133] The cell-based screening protocol was as mentioned in Example 1.

[0134] Phage titers were performed according to Example 1, and the results are shown in Table 2.

[0135] Table 2:

[0136] Screening 1 Amplification 1 Screening 2 Amplification 2 Screening 3 Long linker 1.3*10^2 4*10^11 1.5*10^5 7.0*10^11 3.7*10^5 Short linker 1.2*10^2 2*10^10 1.1*10^5 1.0*10^12 1.22*10^6

[0137] The protocols for phage amplification and cell-based ELISA were the same as those in Example 1. The results are shown in Figure 4 . Clones D and F had high binding activity to thyroid cancer cells.

[0138] Although the invention has been described in connection with the specific embodiments set forth above, many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art. All such alternatives, modifications, and variations are considered to fall within the scope of the invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to carbonic anhydrase IX (CAIX), comprising complementarity-determining regions (CDRs) of the heavy-chain variable region and complementarity-determining regions of the light-chain variable region, wherein the complementarity-determining regions of the heavy-chain variable region comprise CDRH1, CDRH2, and CDRH3 regions, and the complementarity-determining regions of the light-chain variable region comprise CDRL1, CDRL2, and CDRL3 regions, and wherein: The amino acid sequence of the CDRH1 region is SEQ ID NO: 1 (SYAMQ); the amino acid sequence of the CDRH2 region is SEQ ID NO: 2 (GMSDDGSWTDYGAAVKG); the amino acid sequence of the CDRH3 region is SEQ ID NO: 4 (GAGTGYCDNRSFGCASTIDA); and the amino acid sequence of the CDRL1 region is SEQ ID NO: 6 (SGSSGSYG); the amino acid sequence of the CDRL2 region is SEQ ID NO: 7 (HNDKRPS); the amino acid sequence of the CDRL3 region is SEQ ID NO: 9 (GSADRSGAGI); or the amino acid sequence of the CDRH1 region is SEQ ID NO: 1; the amino acid sequence of the CDRH2 region is SEQ ID NO: 3 (GVSDDGSWTGYGAAVQG); the amino acid sequence of the CDRH3 region is SEQ ID NO: 5 (GAGTGYCNNRGFGCASTIDA); and the amino acid sequence of the CDRL1 region is SEQ ID NO: 6; the amino acid sequence of the CDRL2 region is SEQ ID NO: 8 (YNDKRPS); the amino acid sequence of the CDRL3 region is SEQ ID NO:

9.

2. The antibody or antigen-binding fragment thereof according to claim 1, which comprises a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 10 (AVTLDESGGGLHTPGGGLSLVCRASGFTFSSYAMQWVRQAPGKGLEWVAGMSDDGSWTDYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKGAGTGYCDNRSFGCASTIDAWGHGTEVIVSS); and a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 11 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYHNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVL); or a heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 12 (AVTLDESGGGLQTPGGGLSLVCRASGFTMTSYAMQWVRQAPGKGLEWVAGVSDDGSWTGYGAAVQGRATISRDNGQSTVRLLLNNLRAEDTATYYCVKGAGTGYCNNRGFGCASTIDAWGHGTEVIVSS); and a light-chain variable region comprising the amino acid sequence of SEQ ID NO: 13 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVL).

3. The antibody or antigen-binding fragment thereof according to claim 1, which comprises any amino acid sequence selected from the following: SEQ ID NO:14 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYHNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVLGQSSRSSGGGGSSGGGGSAVTLDESGGGLHTPGGGLSLVCRASGFTFSSYAMQWVRQAPGKGLEWVAGMSDDGSWTDYGAAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKGAGTGYCDNRSFGCASTIDAWGHGTEVIVSS) or SEQ ID NO:15 (ALTQPSSVSANLGETVEITCSGSSGSYGWYQQKSPGSAPVTVIYYNDKRPSDIPSRFSGSKSGSTGTLTITGVQAEDEAVYYCGSADRSGAGIFGAGTTLTVLGQSSRSSAVTLDESGGGLQTPGGGLSLVCRASGFTMTSYAMQWVRQAPGKGLEWVAGVSDDGSWTGYGAAVQGRATISRDNGQSTVRLLLNNLRAEDTATYYCVKGAGTGYCNNRGFGCASTIDAWGHGTEVIVSS).

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or an scFv antibody.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a humanized antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a human antibody.

8. The antibody or antigen-binding fragment thereof according to claim 1, which is expressed on the surface of immune cells.

9. Use of a pharmaceutical composition for the manufacture of a medicament for treating thyroid tumors in an individual in need thereof; wherein the pharmaceutical composition comprises a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and optionally a pharmaceutically acceptable carrier or excipient.

10. The use according to claim 9, wherein the drug is administered intravenously, intraperitoneally, intraarterially, intrathecally, intravesically or intratumorally.

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