Anti-ctla4 antibodies and uses thereof

Abstract

This refers to anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4 or CTLA-4) antibodies, antigen-binding fragments, and their uses.

Description

1 / 85 “ANTI-CTLA4 ANTIBODIES AND THEIR USES” TECHNICAL FIELD

[0001] This disclosure relates to cytotoxic T lymphocyte-associated protein 4 (CTLA4 or CTLA-4) antibodies and their uses. BACKGROUND

[0002] Cancer is currently one of the diseases with the highest human mortality rate. According to statistical data from the World Health Organization, in 2012, the global number of cancer cases and deaths reached 14 million and 8.2 million, respectively. In China, there are 3.07 million newly diagnosed cancer cases and 2.2 million deaths.

[0003] The recent clinical and commercial success of anticancer antibodies has created great interest in antibody-based therapeutic products. There is a need to develop anticancer antibodies for use in various antibody-based therapeutic products to treat cancers. SUMMARY

[0004] This disclosure relates to anti-CTLA4 antibodies, antigen-binding fragments thereof, and their uses.

[0005] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA4) comprising:

[0006] a variable heavy chain (VH) region comprising complementarity determination regions Petition 870200036579, dated 03 / 19 / 2020, p. 14 / 139 2 / 85 (CDRs) 1, 2 and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence;and a variable light chain (VL) region comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, wherein the selected VL CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs 1 and 3 amino acid sequences are one of the following: (1) the amino acid sequences of VH CDRs 1, 2 and 3 selected are established in SEQ ID NOs: 1, 2, 3, respectively, and the amino acid sequences of VL Selected CDRs 1, 2, and 3 are established in SEQID. Nos: 4, 5, 6, respectively; (2) the amino acid sequences of VH CDRs 1, 2 and 3 selected are established in SEQ ID NOs: 7, 8, 9, respectively, and the amino acid sequences of VL Selected CDRs 1, 2, and 3 are established in SEQ ID. Nos: 10, 11, 12, respectively; Petition 870200036579, dated 03 / 19 / 2020, p. 15 / 139 3 / 85 (3) the amino acid sequences of selected VH CDRs 1, 2 and 3 are established in SEQ ID NOs: 45, 46, 47, respectively, and the amino acid sequences of selected VL CDRs 1, 2, and 3 are established in SEQ. ID Nos: 48, 49, 50, respectively; (4) the amino acid sequences of selected VH CDRs 1, 2 and 3 are established in SEQ ID NOs: 51, 52, 53, respectively, and the amino acid sequences of selected VL CDRs 1, 2, and 3 are established in SEQ ID Nos: 54, 55, 56, respectively.

[0007] In some embodiments, VH comprises CDRs 1,2, with the amino acid sequences set in SEQID NOs: 1, 2 and 3, respectively, and VL comprises CDRs 1,2, with the amino acid sequences established in SEQID. Nos: 4, 5 and 6, respectively.

[0008] In some embodiments, VH comprises CDRs 1,2, with the amino acid sequences set in SEQID NOs: 7, 8 and 9, respectively, and VL comprises CDRs 1,2, with the amino acid sequences established in SEQID. Nos: 10, 11 and 12, respectively.

[0009] In some embodiments, the antibody or antigen-binding fragment binds specifically to human CTLA4. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0010] In one aspect, the revelation refers to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising: Petition 870200036579, dated 03 / 19 / 2020, page 16 / 139 4 / 85 (1) an immunoglobulin heavy chain or a fragment thereof comprising a variable heavy chain (VH) region comprising complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences set out in SEQ ID NOs: 1, 2 and 3, respectively, and wherein the VH, when paired with a variable light chain (VL) region comprising the amino acid sequence set out in SEQ ID NO: 18, 19, 20, or 70, if linked to CTLA4; (2) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2 and comprising the amino acid sequences set out in SEQ ID NOs: 4, 5 and 6, respectively, and wherein VL, when paired with a VL comprising the amino acid sequence set out in SEQ ID NO: 13, 14, 15, 16, 17 or 69, binds to CTLA4; (3) an immunoglobulin heavy chain or a fragment thereof comprising a variable heavy chain (VH) region comprising CDRs 1, 2 and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8 and 9, respectively, and wherein VH, when paired with a variable light chain (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 25, 26, 27, 28 or 72, binds to CTLA4; or (4) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2 and comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11 and 12, respectively, and wherein VL, when paired with a VL comprising the amino acid sequence Petition 870200036579, dated 03 / 19 / 2020, p. 17 / 139 5 / 85 established in SEQ ID NO: 21, 22, 23, 24 or 71, is linked to CTLA4; (5) an immunoglobulin heavy chain or a fragment thereof comprising a variable heavy chain (VH) region comprising CDRs 1, 2 and 3 comprising the amino acid sequences set out in SEQ ID NOs: 45, 46 and 47, respectively, and wherein VH, when paired with a variable light chain (VL) region comprising the amino acid sequence set out in SEQ ID NO: 74, binds to CTLA4; (6) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2 and comprising the amino acid sequences set out in SEQ ID NOs: 48, 49 and 50, respectively, and wherein VL, when paired with a VL comprising the amino acid sequence set out in SEQ ID NO: 73, binds to CTLA4; (7) an immunoglobulin heavy chain or a fragment thereof comprising a variable heavy chain (VH) region comprising CDRs 1, 2 and 3 comprising the amino acid sequences set out in SEQ ID NOs: 51, 52 and 53, respectively, and wherein VH, when paired with a variable light chain (VL) region comprising the amino acid sequence set out in SEQ ID NO: 76, binds to CTLA4; (8) an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2 and comprising the amino acid sequences set out in SEQ ID NOs: 54, 55 and 56, respectively, and wherein VL, when paired with a Petition 870200036579, dated 03 / 19 / 2020, p. 18 / 139 6 / 85 VL, comprising the amino acid sequence established in SEQ ID NO: 75, binds to CTLA4.

[0011] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2 and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2 and 3, respectively.

[0012] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2 and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively.

[0013] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2 and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8 and 9, respectively.

[0014] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2 and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11 and 12, respectively.

[0015] In some modalities, VH when paired with VL specifically binds to human CTLA4. In some modalities, VL when paired with VH specifically binds to human CTLA4. Petition 870200036579, dated 03 / 19 / 2020, p. 19 / 139 7 / 85

[0016] In some embodiments, the immunoglobulin heavy chain or fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.

[0017] In some embodiments, the nucleic acid encodes a single-stranded variable fragment (scFv). In some embodiments, the nucleic acid is cDNA.

[0018] In another aspect, the disclosure also provides a vector comprising one or more of the nucleic acids as described herein. In some embodiments, the vector encodes the VL region and the VH region which together bind to a CTLA4.

[0019] In one aspect, the disclosure relates to a pair of vectors, wherein each vector comprises one of the nucleic acids as described herein, wherein, together, the pair of vectors encodes the VL region and the VH region which, together, bind to CTLA4.

[0020] In one aspect, the disclosure provides a cell comprising the vector as described herein or the pair of vectors as described herein. In some embodiments, the cell is a CHO cell.

[0021] In another aspect, the disclosure refers to a cell comprising one or more of the nucleic acids as described in this document.

[0022] In one aspect, the revelation provides a cell comprising two of the nucleic acids as described herein. In some embodiments, the two nucleic acids together encode the VL region and the VH region. Petition 870200036579, dated 03 / 19 / 2020, page 20 / 139 8 / 85 which, together, connect to CTLA4.

[0023] In one aspect, the disclosure also provides a method of producing an antibody or an antigen-binding fragment thereof. The method includes the steps of culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; and collecting the antibody or antigen-binding fragment produced by the cell.

[0024] In one aspect, the revelation refers to an antibody or antigen-binding fragment thereof that binds to CTLA4 comprising a variable heavy chain (VH) region comprising an amino acid sequence that is at least 90% identical to a selected VH sequence and a variable light chain (VL) region comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 13, 14,15, 16, 17 or 69 and the selected VL sequence is SEQ ID NO: 18, 19, 20 or 70; (2) the selected VH sequence is SEQ ID NO: 21, 22, 23, or 71 and the selected VL sequence is SEQ ID NO: 25, 26, 27, 28, or 72; (3) the selected VH sequence is SEQ ID NO: 73, and the selected VL sequence is SEQ ID NO:74; (4) the selected VH sequence is SEQ ID NO: 75, and the selected VL sequence is SEQ ID NO:76.

[0025] In some embodiments, VH comprises the sequence of SEQ ID NO: 13 and VL comprises the sequence of Petition 870200036579, dated 03 / 19 / 2020, p. 21 / 139 9 / 85 SEQ ID NO: 19. In some embodiments, VH comprises the sequence with SEQ ID NO: 14 and VL comprises the sequence with SEQ ID NO: 19.

[0026] In some embodiments, VH comprises the sequence with SEQ ID NO: 21 and VL comprises the sequence with SEQ ID NO: 25. In some embodiments, VH comprises the sequence with SEQ ID NO: 22 and VL comprises the sequence with SEQ ID NO: 25.

[0027] In some embodiments, the antibody or antigen-binding fragment binds specifically to human CTLA4. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0028] In one aspect, the disclosure also provides an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof covalently or non-covalently linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colcicine, doxorubicin, daunorubicin, dihydroxyanthracine, maytansinoids such as DM-1 and DM-4, ​​dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogues).

[0029] In one respect, revelation provides a method of Petition 870200036579, dated 03 / 19 / 2020, p. 22 / 139 10 / 85 treatment of an individual who has cancer. The method includes the steps of administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment as described herein, or the antibody-drug conjugate as described herein, to the individual.

[0030] In some forms, the cancer is melanoma. In some forms, the cancer is unresectable melanoma or metastatic melanoma.

[0031] In some forms, the cancer is non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic, hormone-refractory prostate cancer.

[0032] In one aspect, the disclosure also provides a method of decreasing the rate of tumor growth. The method includes the steps of bringing a tumor cell into contact with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described in this document.

[0033] In another aspect, the disclosure relates to a method of exterminating a tumor cell, wherein the method includes the steps of bringing a tumor cell into contact with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein or the antibody-drug conjugate as described herein.

[0034] In one aspect, the disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein. Petition 870200036579, dated 03 / 19 / 2020, page 23 / 139 11 / 85 document and a pharmaceutically acceptable carrier.

[0035] In another aspect, the disclosure relates to a pharmaceutical composition comprising the antibody-drug conjugate as described herein and a pharmaceutically acceptable carrier.

[0036] As used in this document, the term cancer refers to cells that have the capacity for autonomous growth. Examples of such cells include cells that have an abnormal state or condition characterized by the growth of rapidly proliferating cells. The term means that it includes cancerous growths, for example, tumors; oncogenic processes, metastatic tissues and cells transformed into malignant tissues or organs, regardless of histopathological type or stage of invasiveness. It also includes malignancies of various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung carcinoma and small bowel cancer.Naturally occurring cancer includes any cancer that is not experimentally induced by the implantation of cancer cells into an individual and includes, for example, cancer that arises spontaneously, cancer caused by a patient's exposure to a carcinogen (or carcinogens), cancer resulting from the insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, for example, viral infections. The term. Petition 870200036579, dated 03 / 19 / 2020, page 24 / 139 12 / 85 Carcinoma is recognized in the art and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous or sarcomatous tissues. An adenocarcinoma refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term sarcoma is recognized in the art and refers to malignant tumors of mesenchymal origin. The term hematopoietic neoplastic disorders includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder may arise from myeloid, lymphoid, or erythroid lineages or precursor cells thereof.

[0037] As used in this document, the term antibody refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determinant region (CDR) (e.g., any of the three CDRs of an immunoglobulin light chain or any of the three CDRs of an immunoglobulin heavy chain) and has the ability to bind specifically to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody may contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bispecific antibodies, single-chain antibodies. Petition 870200036579, dated 03 / 19 / 2020, p. 25 / 139 13 / 85 simple, diabodies, linear antibodies and multispecific antibodies formed from antibody fragments.

[0038] As used in this document, the term “antigen-binding fragment” refers to a portion of a full-length antibody wherein the antibody portion has the ability to bind specifically to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of a light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0039] As used in this document, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a bovine) that contains a non-rearranged or rearranged human immunoglobulin locus (e.g., human immunoglobulin heavy or light chain locus).

[0040] As used in this document, the term “chimeric antibody” refers to an antibody that contains Petition 870200036579, dated 03 / 19 / 2020, page 26 / 139 14 / 85 a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human antibody and a mouse antibody). A non-limiting example of a chimeric antibody is an antibody that contains the variable domain sequences (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human antibody (e.g., mouse) and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

[0041] As used in this document, the term “humanized antibody” refers to a non-human antibody that contains minimal sequence derived from a non-human immunoglobulin (e.g., mouse) and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable region (CDR) residues of the recipient antibody are replaced by hypervariable region (CDR) residues of a non-human antibody (e.g., a donor antibody), for example, a mouse, rat, or rabbit antibody, that has the desired specificity, affinity, and capability. In some embodiments, the Fv frame residues of the human immunoglobulin are replaced by corresponding non-human immunoglobulin (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody.These modifications can be made to further refine antibody performance. In some cases... Petition 870200036579, dated 03 / 19 / 2020, page 27 / 139 In 15 / 85 embodiments, the humanized antibody contains substantially all of at least one, and typically two, variable domains, wherein all or substantially all hypervariable loops (CDRs) correspond to those of a non-human immunoglobulin (e.g., mouse) and all or substantially all frame regions are those of a human immunoglobulin. The humanized antibody may also contain at least a portion of an immunoglobulin constant (Fc) region, typically that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0042] As used in this document, the term “single-chain antibody” refers to a simple polypeptide containing at least two variable immunoglobulin domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that has the ability to bind specifically to an antigen. Non-limiting examples of single-chain antibodies are described in this document.

[0043] As used in this document, the term “multimeric antibody” refers to an antibody that contains four or more (e.g., six, eight, or ten) variable immunoglobulin domains. In some embodiments, the multimeric antibody has the ability to crosslink a target molecule (e.g., CTLA4) to at least one second target molecule (e.g., PD1) on the surface of a mammalian cell (e.g., a human T cell). Petition 870200036579, dated 03 / 19 / 2020, page 28 / 139 16 / 85

[0044] As used in this document, the terms “individual” and “patient” are used interchangeably throughout the descriptive report and describe an animal, human or non-human, to which treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients may be adult humans or young humans (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. These include, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

[0045] As used in this document, when referring to an antibody, the expressions “specifically binds” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., CTLA4) preferentially to other molecules because the interaction is dependent on the presence of a specific structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a specific target antibody. Petition 870200036579, dated 03 / 19 / 2020, p. 29 / 139 17 / 85 For example, an antibody that binds to a CTLA4 molecule may be referred to as a CTLA4-specific antibody or an anti-CTLA4 antibody.

[0046] As used in this document, the terms polypeptide, peptide, and “protein” are used interchangeably to refer to amino acid polymers of any length of at least two amino acids.

[0047] As used herein, the terms polynucleotide, nucleic acid molecule and nucleic acid sequence are used interchangeably herein to refer to nucleotide polymers of any length of at least two nucleotides and include, without limitation, DNA, RNA, DNA / RNA hybrids and modifications thereof.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to whom this invention pertains. The methods and materials described herein are for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein in their entirety by reference. In case of conflict, the present descriptive report, including the definitions, shall control.

[0049] Other features and advantages of the invention will be Petition 870200036579, dated 03 / 19 / 2020, page 30 / 139 18 / 85 evident from the detailed description and figures that follow and the claims. DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 is a flowchart showing the first part of an exemplary protocol for producing anti-CTLA4 antibodies.

[0051] FIG. 2 is a flowchart showing the second part of an exemplary protocol for producing anti-CTLA4 antibodies.

[0052] FIG. 3 is a set of flow cytometry graphs showing anti-CTLA4 antibodies blocking the binding between CTLA4 and CD80.

[0053] FIG. 4 is a set of flow cytometry graphs showing anti-CTLA4 antibodies blocking the binding between CTLA4 and CD86.

[0054] FIG. 5 is a set of graphs showing the flow cytometry results of cross-reactivity of anti-CTLA4 antibodies against monkey CTLA4 (rmCTLA4), mouse CTLA4 (mCTLA4) and chimeric human-mouse CTLA4 (chiCTLA4).

[0055] FIG. 6 is a graph showing the body weight over time of humanized mice with B-hCTLA-4 with MC-38 tumor treated with anti-CTLA4 antibodies 13A4 and 4G12.

[0056] FIG. 7 is a graph showing the change in percentage of body weight over time in humanized mice with B-hCTLA-4 with MC-38 tumor treated with anti-CTLA4 antibodies 13A4 and 4G12.

[0057] FIG. 8 is a graph showing tumor size over time in B-hCTLA-4 humanized mice. Petition 870200036579, dated 03 / 19 / 2020, page 31 / 139 19 / 85 with MC-38 tumor treated with anti-CTLA4 13A4 and 4G12 antibodies.

[0058] FIG. 9 is a graph showing body weight over time in B-hCTLA-4 humanized mice with MC-38 tumor treated with Yervoy and 13A4.

[0059] FIG. 10 is a graph showing the percentage change in body weight over time in B-hCTLA-4 humanized mice with MC-38 tumor treated with Yervoy and 13A4.

[0060] FIG. 11 is a graph showing tumor size over time in BhCTLA-4 humanized mice with MC-38 tumor treated with Yervoy and 13A4.

[0061] FIG. 12 is a graph showing the body weight over time of humanized mice with B-hCTLA-4 with MC-38 tumor treated with Yervoy, anti-hCTLA4 antibodies 4G12-H1K1-IgG1 and 4G12-H2K1-IgG1.

[0062] FIG. 13 is a graph showing the percentage change in body weight over time in B-hCTLA-4 humanized mice with MC-38 tumor treated with Yervoy, anti-hCTLA4 antibodies 4G12-H1K1-IgG1 and 4G12-H2K1-IgG1.

[0063] FIG. 14 is a graph showing tumor size over time in BhCTLA-4 humanized mice with MC-38 tumor treated with Yervoy, antihCTLA4 antibodies 4G12-H1K1-IgG1 and 4G12-H2K1-IgG1.

[0064] FIG. 15 is a graph showing body weight over time of humanized mice with B-hCTLA-4 with MC-38 tumor treated with Yervoy, humanized anti-hCTLA4 antibodies 13A4-H1K2-IgG1, 13A4-H2K2-IgG1, 13A4-H1K2-IgG4 and 13A4-H1K2-IgG1-N297A.

[0065] FIG. 16 is a graph showing the change of Petition 870200036579, dated 03 / 19 / 2020, p. 32 / 139 20 / 85 percent body weight over time of B-hCTLA-4 humanized mice with MC-38 tumor treated with Yervoy, humanized anti-hCTLA4 antibodies 13A4-H1K2-IgG1, 13A4H2K2-IgG1, 13A4-H1K2-IgG4 and 13A4-H1K2-IgG1-N297A.

[0066] FIG. 17 is a graph showing tumor size over time in BhCTLA-4 humanized mice with MC-38 tumor treated with Yervoy, humanized antihCTLA4 antibodies 13A4-H1K2-IgG1, 13A4-H2K2-IgG1, 13A4H1K2-IgG4 and 13A4-H1K2-IgG1-N297A.

[0067] FIG. 18 is a set of flow cytometry graphs showing anti-CTLA4 antibodies blocking the binding between CTLA4 and human CD86.

[0068] FIG. 19 is a set of graphs showing the flow cytometry results of cross-reactivity of anti-CTLA4 antibodies against monkey, mouse and chimeric human-mouse CTLA4.

[0069] FIG. 20 is a graph showing the body weight over time of humanized mice with B-hCTLA-4 with MC-38 tumor treated with CT4-04-13A4, CT4-20-6D2 and CT420-7E12.

[0070] FIG. 21 is a graph showing the percentage change in body weight over time in humanized mice with B-hCTLA-4 with MC-38 tumor treated with CT404-13A4, CT4-20-6D2 and CT4-20-7E12.

[0071] FIG. 22 is a graph showing tumor size over time in BhCTLA-4 humanized mice with MC-38 tumor treated with CT4-04-13A4, CT4-20-6D2 and CT4-20-7E12.

[0072] FIG. 23 lists CDR sequences of anti-CTLA4 antibodies 13A4, 4G12, 6D2, 7E12 and humanized antibodies. Petition 870200036579, dated 03 / 19 / 2020, p. 33 / 139 21 / 85 of them as defined by Kabat's numbering system.

[0073] FIG. 24 lists CDR sequences of anti-CTLA4 antibodies 13A4, 4G12, 6D2, 7E12 and humanized antibodies thereof as defined by Chothia numbering.

[0074] FIG. 25 lists amino acid sequences of variable heavy chain regions and variable light chain regions of humanized anti-CTLA4 antibodies.

[0075] FIG. 26 lists amino acid sequences of human CTLA4, mouse CTLA4, monkey CTLA4 and chimeric CTLA4.

[0076] FIG. 27 shows the amino acid sequence of the variable heavy chain and light chain regions of several mouse anti-hCTLA4 antibodies. DETAILED DESCRIPTION

[0077] The present disclosure provides examples of antibodies, antigen-binding fragment thereof, that bind to cytotoxic T lymphocyte-associated protein 4 (CTLA4, CTLA4, also known as CD152).

[0078] CTLA4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits an inhibitory signal to T cells. It is an immune checkpoint and acts as a switch when bound to CD80 or CD86, downregulating immune responses.

[0079] This disclosure also provides humanized anti-CTLA4 antibody sequences and methods for producing and using the antibodies. CTLA4 and Cancer

[0080] The immune system can differentiate between normal cells in the body and those seen as foreign, which Petition 870200036579, dated 03 / 19 / 2020, page 34 / 139 The 22 / 85 mechanism allows the immune system to attack foreign cells while leaving normal cells alone. This mechanism sometimes involves proteins called immune checkpoints. Immune checkpoints are molecules in the immune system that either increase a signal (co-stimulatory molecules) or decrease a signal.

[0081] Checkpoint inhibitors can prevent the immune system from attacking normal tissue and thus prevent autoimmune diseases. Many tumor cells also express checkpoint inhibitors. These tumor cells evade immune surveillance by co-opting specific immune checkpoint pathways, particularly in T cells that are specific for tumor antigens (Creelan, Benjamin C. “Update on immune checkpoint inhibitors in lung cancer”. Cancer Control 21.1 (2014): 80-89). Because many immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies against the ligands and / or their receptors.

[0082] The immune checkpoint pathway involves an elaborate series of cellular interactions that prevent excessive effector activity by T cells under normal conditions. Part of this pathway is a cell surface receptor, called cytotoxic T lymphocyte antigen 4 (CTLA4, CD152). CTLA4 is a member of the immunoglobulin superfamily that is expressed exclusively on T cells. CTLA4 acts to inhibit T cell activation and is reported to inhibit helper T cell activity and enhance T cell immunosuppressive activity. Petition 870200036579, dated 03 / 19 / 2020, page 35 / 139 23 / 85 regulatory. So, CTLA-4 acts as a “switch-off” and deactivates the immune response. Once a cytotoxic T cell becomes active, it expresses CTLA-4 on its cell surface, which then competes with the costimulatory molecule CD28 for their mutually shared ligands, B7-1 (CD80) or B7-2 (CD86) on the APC. This “yin-yang” balance keeps cytotoxic activity in checkpoint while allowing T cell function to proceed in a self-limiting manner (Creelan, Benjamin C. Update on immune checkpoint inhibitors in lung cancer. Cancer Control 21.1 (2014): 80-89).

[0083] Many cancer cells can stimulate abnormal CTLA-4 expression on T cells, and these CTLA-4 abnormal T cells exhibit an anergic phenotype. Cancer cells can then co-opt the CTLA-4 pathway to escape patrolling T cells. The introduction of monoclonal antibodies that inhibit CTLA-4 can achieve consistent and durable antitumor responses in various cancers, such as melanoma. These anti-CTLA4 antibodies (e.g., tremelimumab and ipilimumab (Yervoy)) bind to CTLA4, blocking signaling inhibitors, which allows cytotoxic T lymphocytes to destroy cancer cells. Therefore, anti-CTLA4 antibodies can be used to treat cancers.

[0084] The present disclosure provides several anti-CTLA4 antibodies, antigen-binding fragments thereof, and methods of using these anti-CTLA4 antibodies and antigen-binding fragments to treat cancers. Antibodies and Antigen-binding fragments

[0085] The present disclosure provides anti-CTLA4 antibodies. Petition 870200036579, dated 03 / 19 / 2020, p. 36 / 139 24 / 85 and antigen-binding fragments thereof. In general, antibodies (also called immunoglobulins) consist of up to two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure may be a four-chain immunoglobulin antibody comprising two heavy chains and two light chains. The heavy chain of the antibody may be of any isotype including IgM, IgG, IgE, IgA or IgD or subisotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a kappa light chain or a lambda light chain. An antibody may comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, each containing a variable domain (or variable region, VH) and multiple constant domains (or constant regions), link together via disulfide bonds in their constant domains to form the antibody "stem".The light chains, each containing a variable domain (or variable region, VL) and a constant domain (or constant region), each link to a heavy chain via a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is linked. The variable regions of both light and heavy chains contain three hypervariable regions sandwiched between more conserved frame regions (FR).

[0086] These hypervariable regions, known as complementary determination regions (CDRs), form loops that comprise the antigen-binding surface principle of the antibody. The four framework regions adopt Petition 870200036579, dated 03 / 19 / 2020, page 37 / 139 25 / 85 largely a confirmation of beta-lamina and the CDRs form loops that connect and, in some cases, form part of the beta-lamina structure. The CDRs in each chain are held in proximity by the frame regions and, with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0087] Methods for identifying the CDR regions of an antibody by analyzing the antibody's amino acid sequence are well known, and a number of definitions of CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described in, for example, Martin, Protein sequence and structure analysis of antibody variable domains, Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains, Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2):269-94 (Jan. 1998); Chothia et al., Nature 342(6252):877-83 (Dec.of 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); wherein each is incorporated into this document by reference in its entirety.

[0088] CDRs are important for recognizing an antigen epitope. As used in this document, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen-binding domain of Petition 870200036579, dated 03 / 19 / 2020, pp. 38 / 139 26 / 85 an antibody. The minimum size of an epitope can be about three, four, five, six, or seven amino acids, but these amino acids do not need to be in a consecutive linear sequence of the primary antigen structure, as the epitope can depend on a three-dimensional configuration of the antigen based on the secondary or tertiary structure of the antigen.

[0089] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant region, particularly in their hinge and domains. Upper CH2. The sequences and differences of the subclasses of IgG subclasses are known in the art and are described, for example, in Vidarsson, Gestur, Gillian Dekkers, and Theo Rispens. IgG subclasses and allotypes: from structure to effector functions. Frontiers in Immunology 5 (2014); Irani, Vashti, et al. Molecular properties of human IgG subclasses and their implications for the design of therapeutic monoclonal antibodies against infectious diseases. Molecular Immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The subclasses of human IgG: molecular analysis of structure, function and regulation. Elsevier, 2016; wherein each is incorporated into this document by reference in its entirety.

[0090] The antibody may also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid). The antibodies disclosed in this document also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific, and chimeric antibodies that Petition 870200036579, dated 03 / 19 / 2020, pp. 39 / 139 27 / 85 include an immunoglobulin-binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" is a portion of an antibody that retains specific binding activity of the intact antibody, that is, any portion of an antibody that is capable of specifically binding to an epitope on the intact antibody target molecule. This includes, for example, Fab, Fab', F(ab')2 and variants of these fragments. Thus, in some embodiments, an antibody or an antigen-binding fragment thereof may be, for example, an scFv, an Fv, an Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multispecific antibody formed of antibody fragments, and any polypeptide that includes a binding domain that is an antibody-binding domain, or is homologous to it.Non-limiting examples of antigen-binding domains include, for example, the heavy chain and / or light chain CDRs of an intact antibody, the variable regions of the heavy and / or light chain of an intact antibody, the full-length heavy or light chain of an intact antibody, or an individual heavy chain or light chain CDR of an intact antibody.

[0091] In some embodiments, the antigen-binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor consists of fusions of single-strand variable fragments (scFv) as described in this document, fused to the transmembrane and endodomain of CD3zeta. In some embodiments, the antigen receptor Petition 870200036579, dated 03 / 19 / 2020, p. 40 / 139 The chimeric 28 / 85 also comprises intracellular signaling domains of several co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD2841BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the development additionally provides cells (e.g., T cells) that express the chimeric antigen receptors as described in this document.

[0092] In some embodiments, the scFV has a variable heavy chain domain and a variable light chain domain. Anti-CTLA4 Antibodies and Antigen-Binding Fragments

[0093] The discovery provides antibodies and antigen-binding fragments thereof that specifically bind to CTLA4. The antibodies and antigen-binding fragments described herein are capable of binding to CTLA4 and can inhibit the inhibitory pathway of CTLA4, thereby enhancing the immune response. The discovery provides mouse anti-CTLA4 antibodies CT4-04-13A4 (13A4), CT4-03-4G12 (4G12), CT4-20-6D2 (6D2), and CT4-20-7E12 (7E12), and humanized antibodies thereof.

[0094] CDR sequences for antibodies derived from 13A4 and 13A4 (e.g., humanized antibodies) include CDRs of the variable heavy chain domain, SEQ ID NOs: 1-3, and CDRs of the variable light chain domain, SEQ ID NOs: 4-6, as defined by Kabat numbering. CDRs can also be defined by the Chothia system. Under Chothia numbering, CDR sequences of the variable heavy chain domain are established at SEQ ID NOs: 29-31 and the Petition 870200036579, dated 03 / 19 / 2020, page 41 / 139 29 / 85 CDR sequences of the variable light chain domain are established in SEQ ID Nos: 32-34.

[0095] Similarly, CDR sequences for antibodies derived from 4G12 and 4G12 include CDRs of the heavy chain variable domain, SEQ ID NOs: 7-9 and CDRs of the light chain variable domain, SEQ ID NOs: 10-12, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are established at SEQ ID NOs: 35-37 and the CDRs of the light chain variable domain are established at SEQ ID NOs: 3840.

[0096] The CDR sequences for antibodies derived from 6D2 and 6D2 include CDRs of the variable domain of the heavy chain, SEQ ID NOs: 45-47 and CDRs of the variable domain of the light chain, SEQ ID NOs: 48-50, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the variable domain of the heavy chain are established in SEQ ID NOs: 57-59 and the CDRs of the variable domain of the light chain are established in SEQ ID NOs: 60-62.

[0097] The CDR sequences for antibodies derived from 7E12 and 7E12 include CDRs of the variable domain of the heavy chain, SEQ ID NOs: 51-53 and CDRs of the variable domain of the light chain, SEQ ID NOs: 54-56, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the variable domain of the heavy chain are established in SEQ ID NOs: 63-65 and the CDRs of the variable domain of the light chain are established in SEQ ID NOs: 66-68.

[0098] The amino acid sequence for the variable heavy chain region and variable light chain region of humanized antibodies is also provided. As there are different Petition 870200036579, dated 03 / 19 / 2020, page 42 / 139 30 / 85 ways to humanize the mouse antibody (e.g., the sequence can be replaced with different amino acids), the heavy chain and light chain of an antibody can have more than one version of humanized sequences. The amino acid sequences for the variable region of the humanized 13A4 antibody heavy chain are established in SEQ ID NO: 13-17. The amino acid sequences for the variable region of the humanized 13A4 antibody light chain are established in SEQ ID NO: 18-20. Any of these variable region sequences for the heavy chain (SEQ ID NO: 13-17) can be paired with any of these variable region sequences for the light chain (SEQ ID NO: 18-20).

[0099] Similarly, the amino acid sequences for the humanized 4G12 antibody heavy chain variable region are established in SEQ ID NO: 21-24. The amino acid sequences for the humanized 4G12 antibody light chain variable region are established in SEQ ID NO: 25-28. Any of these heavy chain variable region sequences (SEQ ID NO: 21-24) may be paired with any of these light chain variable region sequences (SEQ ID NO: 25-28).

[0100] As shown in FIG. 25, percentage of humanization means the percentage identity of the variable region sequence of heavy chain or light chain compared to human antibody sequences in the International Immunogenetics Information System (IMGT) database. The maximum point means that the variable region sequence of heavy chain or light chain is closer to a specific species than to other species. For example, the maximum point for being human Petition 870200036579, dated 03 / 19 / 2020, page 43 / 139 31 / 85 means that the sequence is closer to human than to other species. The maximum point for human and Macaca fascicularis means that the sequence has the same percentage identity as the human sequence and the Macaca fascicularis sequence, and these percentage identities are the highest compared to sequences from other species. In some embodiments, the percentage of humanization is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. A detailed description regarding how to determine the percentage of humanization and how to determine the maximum points is known in the art and is described, for example, in Jones, Tim D., et al. The INNs and outs of antibody nonproprietary names. MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety.

[0101] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two or three variable region heavy chain CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 29-31, SEQ ID NOs: 35-37, SEQ ID NOs: 45-47, SEQ ID NOs: 51-53, SEQ ID NOs: 57-59 and SEQ ID NOs: 63-65; and / or one, two, or three variable region CDRs of light chain selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs 10-12, SEQ ID NOs: 32-34, SEQ ID NOs 38-40, SEQ ID NOs 48-50, SEQ ID NOs 54-56, SEQ ID NOs 60-62, and SEQ ID NOs 66-68.

[0102] In some embodiments, antibodies may have a variable heavy chain (VH) region comprising Petition 870200036579, dated 03 / 19 / 2020, p. 44 / 139 32 / 85 complementarity determination regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence, and a variable light chain (VL) region comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85% %, 90% or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%,90% or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. The selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs 1, 2, and 3 amino acid sequences are shown in FIG. 23 (Kabat CDR) and FIG. 24 (Chothia CDR).

[0103] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable heavy chain domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one, or two insertions, Petition 870200036579, dated 03 / 19 / 2020, p. 45 / 139 33 / 85 amino acid deletions or substitutions; SEQ ID NO: 3 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0104] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable heavy chain domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0105] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable heavy chain domain containing one, two, or three of the CDRs of SEQ ID NO: 29 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 31 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0106] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable heavy chain domain containing one, two, or three of the CDRs with SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 37 with zero, one, or two amino acid insertions, deletions, or substitutions. Petition 870200036579, dated 03 / 19 / 2020, p. 46 / 139 34 / 85

[0107] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable heavy chain domain containing one, two, or three of the CDRs of SEQ ID NO: 45 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 46 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 47 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0108] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable heavy chain domain containing one, two, or three of the CDRs of SEQ ID NO: 51 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 52 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 53 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0109] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable heavy chain domain containing one, two, or three of the CDRs of SEQ ID NO: 57 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 58 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 59 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0110] In some embodiments, the antibody or antigen-binding fragment described in this document may contain a variable heavy chain domain. Petition 870200036579, dated 03 / 19 / 2020, p. 47 / 139 35 / 85 containing one, two, or three of the CDRs of SEQ ID NO: 63 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 64 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 65 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0111] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable light chain domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0112] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable light chain domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0113] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable light chain domain containing one, two, or three of the CDRs of SEQ ID NO: 32 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 33 with zero, one, or two insertions, Petition 870200036579, dated 03 / 19 / 2020, p. 48 / 139 36 / 85 amino acid deletions or substitutions; SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0114] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable light chain domain containing one, two, or three of the CDRs of SEQ ID NO: 38 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 40 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0115] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable light chain domain containing one, two, or three of the CDRs of SEQ ID NO: 48 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 49 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 50 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0116] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable light chain domain containing one, two, or three of the CDRs of SEQ ID NO: 54 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 55 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 56 with zero, one, or two amino acid insertions, deletions, or substitutions. Petition 870200036579, dated 03 / 19 / 2020, p. 49 / 139 37 / 85

[0117] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable light chain domain containing one, two, or three of the CDRs of SEQ ID NO: 60 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 61 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 62 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0118] In some embodiments, the antibody or antigen-binding fragment described herein may contain a variable light chain domain containing one, two, or three of the CDRs of SEQ ID NO: 66 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 67 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 68 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0119] Insertions, deletions, and replacements can be within the CDR sequence or at one or both terminal ends of the CDR sequence.

[0120] The disclosure also provides antibodies or antigen-binding fragments thereof that bind to CTLA4. The antibodies or antigen-binding fragments thereof contain a variable heavy chain (VH) region comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH sequence and a variable light chain (VL) region comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH sequence Petition 870200036579, dated 03 / 19 / 2020, p. 50 / 139 38 / 85 selected VL sequence. In some modes, the selected VH sequence is SEQ ID NO: 13, 14, 15, 16, 17, or 69, and the selected VL sequence is SEQ ID NO: 18, 19, 20, or 70. In some modes, the selected VH sequence is SEQ ID NO: 21, 22, 23, 24, or 71, and the selected VL sequence is SEQ ID NO: 25, 26, 27, 28, or 72. In some modes, the selected VH sequence is SEQ ID NO: 73, and the selected VL sequence is SEQ ID NO: 74. In some modes, the selected VH sequence is SEQ ID NO: 75, and the selected VL sequence is SEQ ID NO: 76.

[0121] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of a first and second amino acid or nucleic acid sequence for optimal alignment, and non-homologous sequences may be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence and, in some embodiments, is at least 90%, 95%, or 100%. The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position.The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length. Petition 870200036579, dated 03 / 19 / 2020, p. 51 / 139 39 / 85 of each gap, which needs to be introduced for ideal alignment of the two sequences. For the purposes of this disclosure, the comparison between sequences and determination of percent identity between two sequences can be achieved using a Blossum 62 scoring matrix with a gap penalty of 12, an extended gap penalty of 4, and a frameshift gap penalty of 5.

[0122] The revelation also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or light chain comprises CDRs as shown in FIG. 23 or FIG. 24 or have sequences as shown in FIG. 25 or FIG. 27. When the polypeptides are paired with the corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to CTLA4.

[0123] Anti-CTLA4 antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific antibodies or antibody fragments (e.g., bispecific). The additional antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly produced antibodies (i.e., intrabody), and antigen-binding fragments thereof. Antibodies or antigen-binding fragments Petition 870200036579, dated 03 / 19 / 2020, page 52 / 139 40 / 85 The antigens themselves can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or antigen-binding fragment is an IgG antibody or antigen-binding fragment thereof.

[0124] Antibody fragments are suitable for use in the methods provided as long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to CTLA-4 will retain an ability to bind to CTLA4. An Fv fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of a variable heavy chain domain and a light chain domain in close association, which may be covalent in nature, for example, in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen-binding specificity to the antibody.However, even a simple variable domain (or half of a Fv comprising only three antigen-specific CDRs) can have the ability to recognize and bind to antigen, although typically at a lower affinity than the entire binding site.

[0125] Single-chain Fv or scFv antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present on a single polypeptide chain. Generally, the Fv polypeptide additionally comprises a binder of Petition 870200036579, dated 03 / 19 / 2020, page 53 / 139 41 / 85 polypeptide between the VH and VL domains, which enables scFv to form the desired structure for antigen binding.

[0126] The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments that are generally covalently linked near their carboxy termini by means of hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0127] Diabodies are small antibody fragments with two antigen-binding sites, whose fragments comprise a VH connected to a VL on the same polypeptide chain (VH and VL). Through the use of a binder that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and create two antigen-binding sites.

[0128] Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0129] The antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.

[0130] Antibody multimerization can be achieved through natural antibody aggregation or through Petition 870200036579, dated 03 / 19 / 2020, page 54 / 139 42 / 85 chemical or recombinant linkage techniques known in the art. For example, some percentages of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0131] Alternatively, antibody homodimers can be formed using chemical linking techniques known in the art. For example, heterofunctional crosslinking agents including, but not limited to, SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthioacetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94: 7509-7514, 1997). Antibody homodimers can be converted into Fab'2 homodimers via pepsin digestion. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0132] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be produced by genetically engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from the recombinant cell culture. For example, the interface may contain at least a portion of the CH3 domain of a constant antibody domain. In this method, one or more small amino acid side chains of the interface of the first antibody molecule are replaced by Petition 870200036579, dated 03 / 19 / 2020, p. 55 / 139 43 / 85 larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain (or side chains) are created at the interface of the second antibody molecule by replacing large amino acid side chains with smaller chains (e.g., alanine or threonine). This provides a mechanism for increasing heterodimer productivity into other undesirable end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated by reference in its entirety.

[0133] Bispecific antibodies include crosslinked or “heteroconjugated” antibodies. For example, one of the antibodies in the heteroconjugate may be coupled to avidin and the other to biotin. Heteroconjugated antibodies may also be produced using any convenient crosslinking methods. Suitable crosslinking agents and crosslinking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0134] Methods for generating bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229:81, 1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the diothiol complexing agent sodium arsenite to stabilize the vicinal diothiols and prevent the Petition 870200036579, dated 03 / 19 / 2020, page 56 / 139 44 / 85 intermolecular disulfide formation. The generated Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab' TNB derivatives is then reconverted into Fab' thiol via reduction with mercaptoethylamine and is mixed with an equimolar amount of another Fab' TNB derivative to form the bispecific antibody.

[0135] Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in an individual or in solution). Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin). Conjugation of a stabilizing molecule may increase the half-life or extend the biological activity of an antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human). Antibody Characteristics

[0136] Antibodies or antigen-binding fragments of the same described in this document can block the binding between CTAL4 and CD80 and / or the binding between CTLA4 and CD86. By blocking the binding between CTAL4 and CD80 and / or the binding between CTLA4 and CD86, anti-CTLA4 antibodies interrupt the inhibitory pathway of CTLA4 and upregulate the immune response.

[0137] In some implantations, the antibody (or Petition 870200036579, dated 03 / 19 / 2020, p. 57 / 139 45 / 85 antigen-binding fragments of the same) specifically binds to CTLA4 (e.g., human CTLA4, monkey CTLA4, mouse CTLA4, and / or chimeric CTLA4) with a dissociation constant (Kd) less than 1 x 10⁻⁶ M, less than 1 x 10⁻⁷ M, less than 1 x 10⁻⁸ M, less than 1 x 10⁻⁹ M, or less than 1 x 10⁻¹⁰ M. In some embodiments, Kd is less than 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.

[0138] In some disciplines, Kd is greater than 1 x 10-7M, greater than 1 x 10-8M, greater than 1 x 10-9M, greater than 1 x 1010M, greater than 1 x 10-11M or greater than 1 x 10-12M.

[0139] General techniques for measuring the affinity of an antibody for an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human CTLA4 (SEQ ID NO: 41), monkey CTLA4 (e.g., rhesus monkey CTLA4, SEQ ID NO: 43), chimeric CTLA4 (SEQ ID NO: 44), and / or mouse CTLA4 (SEQ ID NO: 42). In some embodiments, the antibody does not bind to human CTLA4 (SEQ ID NO: 41), monkey CTLA4 (e.g., rhesus monkey CTLA4, SEQ ID NO: 43), chimeric CTLA4 (SEQ ID NO: 44), and / or mouse CTLA4 (SEQ ID NO: 42).

[0140] In some embodiments, the antibody has a tumor growth inhibition percentage (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some modalities, the antibody has a percentage of tumor growth inhibition that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% Petition 870200036579, dated 03 / 19 / 2020, p. 58 / 139 46 / 85 can be determined, for example, in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after treatment begins or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after treatment begins. As used in this document, the percentage of tumor growth inhibition (TGI%) is calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100

[0141] Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero. Methods of Producing Anti-CTLA4 Antibodies

[0142] An isolated fragment of human CTLA4 can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be created in animals through multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than once (e.g., two, three, or four times).

[0143] The full-length polypeptide or protein may be used or, alternatively, antigenic peptide fragments thereof may be used as immunogens. The Petition 870200036579, dated 03 / 19 / 2020, page 59 / 139 47 / 85 An antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the CTLA4 amino acid sequence and encompasses an epitope of the protein such that an antibody grown against the peptide forms a specific immune complex with the protein. As described above, the full-length human CTLA4 sequence is known in the art (SEQ ID NO: 41).

[0144] An immunogen is typically used to prepare antibodies by immunizing a suitable individual (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). A suitable immunogenic preparation may contain, for example, a recombinantly expressed polypeptide or a chemically synthesized one (e.g., a fragment of human CTLA4). The preparation may additionally include an adjuvant, such as Freund's complete or incomplete adjuvant or a similar immunostimulatory agent.

[0145] Polyclonal antibodies can be prepared as described above by immunizing a suitable individual with a CTLA4 polypeptide or an antigenic peptide thereof (e.g., part of CTLA4) as an immunogen. The antibody titer in the immunized individual can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized CTLA4 polypeptide or peptide. If desired, antibody molecules can be isolated from mammals (e.g., from blood) and further purified by well-known techniques such as protein A chromatography of protein G to obtain the IgG fraction. At an appropriate time after immunization, for example, when Petition 870200036579, dated 03 / 19 / 2020, pp. 60 / 139 48 / 85 If specific antibody titers are highest, antibody-producing cells can be obtained from the individual and used to prepare monoclonal antibodies using standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96, 1985), or triome techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells that produce a monoclonal antibody are detected by screening hybridoma culture supernatants for antibodies that bind to a polypeptide or epitope of interest, for example, using a standard ELISA assay.

[0146] Variants of the antibodies or antigen-binding fragments described herein may be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequences that constitute the antibody's antigen-binding site or an antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein, for example, CTLA4. Any combination of Petition 870200036579, dated 03 / 19 / 2020, page 61 / 139 49 / 85 deletions, insertions and / or combinations can be made to arrive at an antibody or antigen-binding fragment that has increased binding affinity for the target. Amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications in the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that a different sugar is bound by enzymes present in a cell) or introducing new glycosylation sites.

[0147] The antibodies disclosed herein may be derived from any animal species, including mammals.Non-limiting examples of native antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically modified to produce human antibodies.

[0148] Human and humanized antibodies include antibodies that have variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or specific mutagenesis). Petition 870200036579, dated 03 / 19 / 2020, page 62 / 139 50 / 85 of the site in vitro or by somatic mutation in vivo), for example, in CDRs.

[0149] A humanized antibody typically has a human frame (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequences introduced into it from a non-human source. These non-human amino acid residues are often called import residues, which are typically taken from a variable import domain. Humanization can essentially be accomplished, for example, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); each of which is incorporated by reference in its entirety herein.Consequently, humanized antibodies are chimeric antibodies in which substantially less than one intact human V domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are replaced by residues from analogous sites in human antibodies.

[0150] The choice of human VH and VL domains to be used in the production of humanized antibodies is very important to reduce immunogenicity. According to the so-called best-fit method, the sequence of the V domain of a mouse antibody is screened against all Petition 870200036579, dated 03 / 19 / 2020, p. 63 / 139 51 / 85 the library of known human domain sequences. The human sequence that is closest to that of the mouse is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).

[0151] It is more important that antibodies be humanized with high specificity and affinity retention for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analyzing parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the probable role of residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen.Thus, RF residues can be selected and combined from receptor and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (or target antigens), is achieved.

[0152] Commonly, amino acid sequence variants of human, humanized, or chimeric anti-CTLA4 antibody will contain an amino acid sequence that has at least Petition 870200036579, dated 03 / 19 / 2020, p. 64 / 139 52 / 85 minus 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with a sequence present in the light or heavy chain of the original antibody.

[0153] Identity or homology with respect to an original sequence is normally the percentage of amino acid residues present in the candidate sequence that are identical to a sequence present in the human, humanized, or chimeric anti-CTLA4 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percentage sequence identity and not considering any conservative substitutions as part of the sequence identity. None of the N-terminal, C-terminal, or internal extensions, deletions, or insertions in the antibody sequence should be interpreted as affecting sequence identity or homology.

[0154] Further modifications to anti-CTLA4 antibodies or antigen-binding fragments can be made. For example, a cysteine ​​residue (or residues) can be introduced into the Fc region, thereby allowing the formation of an interchain disulfide bond in that region. The homodimeric antibody then generated can have any half-life increased in vitro and / or in vivo. Homodimeric antibodies with increased half-life in vitro and / or in vivo can also be prepared using heterobifunctional crosslinkers as described, for example, in Wolff et al. (Cancer Res. 53: 2560-2565, 1993). Alternatively, an antibody can be genetically modified which has double Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0155] In some embodiments, a covalent modification Petition 870200036579, dated 03 / 19 / 2020, p. 65 / 139 53 / 85 can be performed on the anti-CTLA4 antibody or its antigen-binding fragment. These covalent modifications can be made through chemical or enzymatic synthesis or through enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting the targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is capable of reacting with selected side chains or N- or C-terminal residues. Recombinant Vectors

[0156] The present disclosure also provides recombinant vectors (e.g., expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), host cells that are introduced into the recombinant vectors (i.e., so that the host cells contain the polynucleotide and / or a vector comprising the polynucleotide), and the production of recombinant antibody polypeptides or fragments thereof by means of recombinant techniques.

[0157] As used in this document, a “vector” is any construct capable of applying one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An “expression vector” is capable of applying and expressing one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, Petition 870200036579, dated 03 / 19 / 2020, page 66 / 139 54 / 85 The polynucleotide of interest is positioned for expression in the vector by being operatively linked with regulatory elements such as a promoter, enhancer, and / or poly-A tail, either within the vector or in the host cell genome at or near the flanking of the integration site of the polynucleotide of interest so that the polynucleotide of interest is translated in the host cell introduced with the expression vector.

[0158] A vector can be introduced into the host cell by means of methods known in the art, for example, electroporation, chemical transfection (e.g., DEAEdextran), transformation, transfection and infection and / or transduction (e.g., with recombinant virus). Then, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors and DNA or RNA expression vectors associated with cationic condensation agents.

[0159] In some implantations, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective) replication-competent virus or may use a defective virus for replication. In the latter case, viral propagation will generally occur in complementation virus packaging cells. Suitable systems are disclosed, for example, in FisherHoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Petition 870200036579, dated 03 / 19 / 2020, page 67 / 139 55 / 85 Flexner et al., 1989, Ann. NY Acad Sci 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; US Patents 4,603,112, 4,769,330 and 5,017,487; document WO 89 / 01973; Patent No. US 4,777,127; document GB 2,200,651; document EP 0,345,242; document WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Academic. Sci. USA, 91:215-219; KassEisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. The techniques for incorporating DNA into such expression systems are well known to those of common skill in the technique. DNA can also be denuded, as described, for example, in Plmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating the DNA in biodegradable microspheres that are efficiently transported into cells.

[0160] For expression, the DNA insert comprising an antibody-coding or polypeptide-coding polynucleotide disclosed in this document may be operatively ligated to a suitable promoter (e.g., a heterologous promoter), such as the phage PL lambda promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and retroviral LTR promoters, to name a few. Other suitable promoters are known to those skilled in the art. The expression constructs may additionally contain transcription initiation and termination sites and, in the transcript region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may Petition 870200036579, dated 03 / 19 / 2020, pp. 68 / 139 56 / 85 include a translation that starts at the beginning and a stop codon (UAA, μgA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0161] As indicated, expression vectors may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. The culture media and conditions suitable for the host cells described herein are known in the art.

[0162] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, and pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5, available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL, available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0163] Suitable non-limiting bacterial promoters for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, and the lambda PR promoters. Petition 870200036579, dated 03 / 19 / 2020, pp. 69 / 139 57 / 85 and PL and the trp promoter. Non-limiting eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, retroviral LTR promoters such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0164] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as factor alpha, alcohol oxidase and PGH can be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY and Grant et al., Methods Enzymol., 153: 516-544 (1997).

[0165] The introduction of the construct into the host cell can be effected by means of calcium phosphate transfection. DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).

[0166] The transcription of DNA encoding an antibody of the present revelation by means of higher eukaryotes can be enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting DNA elements, typically about 10 to 300 bp, that act to increase the transcriptional activator of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the origin of replication at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the Petition 870200036579, dated 03 / 19 / 2020, pp. 70 / 139 58 / 85 polyome enhancer on the late side of the origin of replication and adenovirus enhancers.

[0167] For secretion of the translated protein into the endoplasmic reticulum lumen, the periplasmic space, or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or they can be heterologous signals.

[0168] The polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST fusion) or with a histidine tag, and may include not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide to enhance stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide chemical fractions can be added to the polypeptide to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. The addition of peptide chemical fractions to polypeptides to generate secretion or excretion, to enhance stability, and to facilitate purification, among other things, are familiar and routine procedures in the techniques. Treatment Methods

[0169] The antibodies or antibody or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes. In one aspect, the disclosure provides methods for treating cancer in a Petition 870200036579, dated 03 / 19 / 2020, page 71 / 139 59 / 85 individual, methods of reducing the rate of increase in tumor volume in an individual over time, methods of reducing the risk of developing metastasis, or methods of reducing the risk of developing additional metastasis in an individual. In some modalities, treatment may stop, slow, delay, or inhibit the progression of a cancer. In some modalities, treatment may result in a reduction in the number, severity, and / or duration of one or more cancer symptoms in an individual.

[0170] In one aspect, the disclosure provides methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to an individual in need thereof (for example, an individual who has or is identified or diagnosed as having cancer), for example, breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy.In some forms, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), metastatic bladder cancer, or hormone-refractory prostate cancer.

[0171] In some embodiments, the compositions and methods disclosed in this document may be used for the Petition 870200036579, dated 03 / 19 / 2020, page 72 / 139 60 / 85 treatment of patients at risk of cancer. Cancer patients can be identified using several methods known in the art.

[0172] As used in this document, an “effective amount” means an amount or dosage sufficient to effect beneficial or desired results including stopping, slowing, delaying or inhibiting the progression of a disease, for example, cancer. An effective amount will vary depending on, for example, the age and body weight of an individual to whom the antibody, antigen-binding fragment, antibody-coding polynucleotide, vector comprising the polynucleotide and / or compositions thereof are to be administered, the severity of symptoms and the route of administration, and therefore administration can be determined on an individual basis.

[0173] An effective amount may be administered in one or more administrations. By way of example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to improve, stop, stabilize, reverse, inhibit, slow down, and / or delay the progression of a cancer in a patient or is an amount sufficient to improve, stop, stabilize, reverse, slow down, and / or delay the proliferation of a cell (e.g., a biopsied cell, any of the cancer cells as described herein, or a cell line (e.g., a cancer cell line)) in vitro. As understood in the art, an effective amount of an antibody or antigen-binding fragment may vary depending on, among other things, the patient's history. Petition 870200036579, dated 03 / 19 / 2020, page 73 / 139 61 / 85 as well as other factors such as the type (and / or dosage) of antibody used.

[0174] Effective amounts and schedules for administering the antibodies, antibody-coding polynucleotides and / or compositions disclosed herein can be determined empirically and making such determinations is within the skill of the art. Those skilled in the art will understand that the dosage to be administered will vary depending, for example, on the mammal that will receive the antibodies, antibody-coding polynucleotides and / or compositions disclosed herein, the route of administration, the specific type of antibodies, antibody-coding polynucleotides, antigen-binding fragments and / or compositions disclosed herein used, and other drugs that are administered to the mammal.Guidance on selecting appropriate doses for antibody or antigen-binding fragment can be found in the literature on therapeutic uses of antibodies and antigen-binding fragments, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, chapter 22 and pages 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pages 365-389.

[0175] A typical daily dose of an effective amount of an antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dosage may be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage may be greater than 10 mg / kg, 9 mg / kg, Petition 870200036579, dated 03 / 19 / 2020, page 74 / 139 62 / 85 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg or 0.01 mg / kg. In some embodiments, the dosage is approximately 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg or 0.1 mg / kg.

[0176] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent may be administered to the individual at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition).In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (for example, a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one agent). Petition 870200036579, dated 03 / 19 / 2020, page 75 / 139 63 / 85 additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, at least one additional therapeutic agent is administered in a sustained-release oral formulation.

[0177] In some embodiments, one or more additional therapeutic agents may be administered to the individual before or after administering at least one antibody, antigen-binding antibody fragment or pharmaceutical composition (for example, any of the antibodies, antigen-binding antibody fragments or pharmaceutical compositions described herein).In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (for example, any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the individual in such a way that there is an overlap in the bioactive period of one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (for example, any of the antibodies or antigen-binding fragments described herein) in the individual.

[0178] In some embodiments, the individual may be administered at least one antibody, antigen-binding antibody fragment or pharmaceutical composition (for example, any of the antibodies, antigen-binding antibody fragments or pharmaceutical compositions described herein) over an extended period of time (for example, over a period of at least 1 week, Petition 870200036579, dated 03 / 19 / 2020, page 76 / 139 64 / 85 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years or 5 years). A skilled medical professional can determine the length of the treatment period using any of the methods described in this document to diagnose or by monitoring the effectiveness of the treatment (e.g., observing at least one symptom of cancer).As described in this document, a skilled medical professional may also alter the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to the individual and may also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to the individual based on an assessment of treatment efficacy (e.g., using any of the methods described in this document and known in the art). Pharmaceutical Composition and Routes of Administration

[0179] Pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein are also provided herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein may be present in a pharmaceutical composition in any combination. Pharmaceutical compositions may be Petition 870200036579, dated 03 / 19 / 2020, page 77 / 139 65 / 85 formulated in any manner known in the art.

[0180] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may include a sterile diluent (e.g., water or sterile saline solution), a fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial or antifungal agents such as benzyl alcohol or methylparabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates and isotonic agents such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride) or any combination thereof.Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, for example, US Patent No. 4,522,811). The compositions can be formulated and encapsulated in ampoules, disposable syringes, or multiple dose vials. When necessary (as, for example, in injectable formulations), adequate fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant. The absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an absorption-delaying agent (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by means of implants and microencapsulated delivery systems, which may include... Petition 870200036579, dated 03 / 19 / 2020, pp. 78 / 139 66 / 85 biodegradable and biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0181] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein may be formulated for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) in the form of dosage units (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

[0182] The toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). An individual can, for example, determine the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population): the therapeutic index being the ratio of LD50:ED50. Agents exhibiting high therapeutic indices are preferred. When an agent exhibits an undesirable side effect, care should be taken to minimize potential harm (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0183] Data obtained from cell culture assays and animal studies can be used in the formulation of Petition 870200036579, dated 03 / 19 / 2020, pp. 79 / 139 67 / 85 an adequate dosage of any given agent for use in an individual (e.g., a human being). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in an individual (e.g., kills cancer cells) in an individual (e.g., a human individual identified as having cancer) or an individual identified as being at risk of developing the disease (e.g., an individual who previously developed cancer but is now cured), decreases the severity, frequency, and / or duration of one or more symptoms of a disease in an individual (e.g., a human being).The efficacy and dosage of any of the antibodies or antigen-binding fragments described in this document can be determined by a healthcare professional or veterinary professional using methods known in the art, as well as by observing one or more symptoms of disease in an individual (e.g., a human). Certain factors may influence the dosage and timing required to effectively treat an individual (e.g., the severity of the disease or disorder, previous treatments, the individual's general health and / or age, and the presence of other diseases).

[0184] Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of body weight (e.g., about 1 pg / kg to about 500 mg / kg; about 100 pg / kg to Petition 870200036579, dated 03 / 19 / 2020, pp. 80 / 139 68 / 85 approximately 500 mg / kg; approximately 100 mg / kg to approximately 50 mg / kg; approximately 10 mg / kg to approximately 5 mg / kg; approximately 10 mg / kg to approximately 0.5 mg / kg; or approximately 1 mg / kg to approximately 50 mg / kg). Although these doses cover a wide range, an individual with ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their potency and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered first, and the attending healthcare professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working in the development stage) may subsequently and gradually increase the dose until an adequate response is obtained.Furthermore, it is understood that the specific dose level for any particular individual will depend on a variety of factors including the activity of the specific compound employed, the individual's age, body weight, general health, gender and diet, the time of administration, the route of administration, the excretion rate and the half-life of the antibody or antibody fragment in vivo.

[0185] Pharmaceutical compositions may be included in a container, package or dispenser along with instructions for administration. EXAMPLES

[0186] The invention is further described in the following examples, which are not limited to the scope of the invention described in the claims. Example 1. Generation of Mouse Anti-hCTLA4 Antibodies Petition 870200036579, dated 03 / 19 / 2020, page 81 / 139 69 / 85

[0187] To generate mouse antibodies against human CTLA4 (hCTLA4; SEQ ID NO: 41), 6-8 week old female BALB / c mice were immunized with human CTLA4. Anti-hCTLA4 antibodies were collected using the methods described below (FIG. 1 and FIG. 2). Immunization of mice

[0188] Female BALB / c mice aged 6–8 weeks were immunized with human CTLA4 proteins with a his-tail at 20 pg / mouse at a concentration of 100 pg / ml. Human CTLA4 proteins with a his-tail were emulsified with adjuvant and injected at four positions on the backs of the mice. For the first subcutaneous (sc) injection, the diluted antigen was emulsified with Freund's Complete Adjuvant (CFA) in an equal volume. In subsequent subcutaneous injections, the protein was emulsified with Freund's Incomplete Adjuvant (IFA) in an equal volume. Three days after the third injection or booster immunization, blood (serum) was collected and analyzed for antibody titer using ELISA.

[0189] In another experiment, 6-8 week old female BALB / c mice were immunized by injecting the human CTLA4 expression plasmid into the mice. Plasmids encoding the antigen were injected into the anterior tibial muscle (intramuscular injection; IM injection) of the mice using gene guns at a concentration of 1000 pg / µL to 60 pg per mouse. At least four injections were performed with at least 14 days between each injection. Blood (serum) was collected days after the last immunization and the serum was tested for antibody titer by ELISA. Petition 870200036579, dated 03 / 19 / 2020, page 82 / 139 70 / 85

[0190] The procedures to enhance immunization were also performed at least fourteen days after the previous immunization (either by plasmid injection or by protein injection). CHO cells expressing CTLA4 antigen on their surface were injected intravenously into mice via the tail veins. The spleen was then collected four days after injection. Fusion of SP2 / 0 cells and splenic cells

[0191] Spleen tissues were ground. Spleen cells were first screened by CD3s Microspheres and Mouse IgM Microspheres and then fused with SP2 / 0 cells. The cells were then placed in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium. Primary screening for hybridoma

[0192] Primary screening of hybridoma supernatant in 96-well plates was performed using Fluorescence-Activated Cell Selection (FACS) according to standard procedures. Chinese hamster ovary (CHO) cells were added to 96-well plates (2 χ¹⁰⁴ cells per well) prior to screening. 50 pl of supernatant was used. The antibodies used in experiments were (1) Fluorescein-conjugated AffiniPure F(ab)2 Fragment Anti-Mouse IgG, Fcy Specific and (2) Alexa Fluor® 647-conjugated Alexa Fluor® 647 Fragment Anti-Human IgG, Fcy Specific. Subcloning Petition 870200036579, dated 03 / 19 / 2020, page 83 / 139 71 / 85

[0193] Subcloning was performed using ClonePix2. In summary, the positive wells identified during primary screening were transferred to semi-solid medium, and IgG-positive clones were identified and tested. The anti-mouse Fc IgG antibody FITC was used. Ascitic fluid antibodies

[0194] 1 x 106 positive hybridoma cells were injected intraperitoneally into B-NDG® mice (Beijing Biocytogen, Beijing, China). Monoclonal antibodies were produced by culturing hybridoma cells in the mouse peritoneal cavity. The hybridoma cells multiplied and produced ascitic fluid in the abdomens of the mice. The fluid contained a high concentration of antibody which could be harvested for later use. Antibody purification

[0195] Antibodies in ascites fluid were purified using GE AKTA protein chromatography (GE Healthcare, Chicago, Illinois, United States). CT4-0413A4 (13A4), CT4-03-4G12 (4G12), CT4-20-6D2 (6D2), and CT4-20-7E12 (7E12) were, among the mouse antibodies, produced using the methods described above. The amino acid sequences of the variable heavy chain and light chain regions of these antibodies are provided in FIG. 27. Example 2. Humanization of mouse antibodies

[0196] The starting point for humanization was mouse antibodies (e.g., 13A4 and 4G12). The amino acid sequences for the variable heavy chain region and the variable light chain region of these antibodies Petition 870200036579, dated 03 / 19 / 2020, page 84 / 139 72 / 85 mice were determined. Five humanized heavy chain variable region variants (SEQ ID NOs: 13-17) and three humanized light chain variable region variants (SEQ ID NOs: 18-20) for 13A4 were constructed containing different permutations of substitutions (FIG. 25). The heavy chain and light chain amino acid sequences CDR1, CDR2, and CDR3 for humanized 13A4 are shown in SEQ ID NO: 1-6 (Kabat numbering) or SEQ ID NO: 29-34 (Chothia numbering).

[0197] Four humanized heavy chain variable region variants (SEQ ID NOs: 21-24) and four humanized light chain variable region variants (SEQ ID NOs: 25-28) for 4G12 were constructed containing different permutations of substitutions (FIG. 25). The CDR1, CDR2, and CDR3 heavy chain and light chain amino acid sequences for humanized 4G12 are shown in SEQ ID NO: 7-12 (Kabat numbering) or SEQ ID NO: 35-40 (numbering of Chothia).

[0198] These humanized antibodies were generated using standard procedures and facilitated with the use of BioLuminate 1.0 (Schrodinger, Shanghai, China). Example 3. In vitro test of mouse anti-hCTLA4 antibodies: blocking the binding of CD80 and CD86 to CTLA4.

[0199] Blocking assays were performed to determine whether anti-CTLA4 antibodies can block the binding between CTLA4 and CD80 and the binding between CTLA4 and CD86.

[0200] Anti-CTLA4 antibodies were collected from mouse ascites fluid and purified by chromatography. 25 μl of CHO cells transiently transfected with human CTLA4 were added to each well. Petition 870200036579, dated 03 / 19 / 2020, page 85 / 139 73 / 85 in a plate. The purified antibodies were titrated to final concentrations of 50, 5, 0.5, 0.05, 0.005 μg / ml. The titrated antibodies were added to each well at 25 μl per well at 4 °C and incubated for 30 minutes.

[0201] Biotin-hCD80 or Biotin-hCD86 was titrated to 0.4 μg / ml. 50 μl of the ligand solution was added to each well, making the final concentration of Biotin-hCD80 or Biotin-hCD86 0.2 μg / ml. Cells with Biotin-hCD80 or Biotin-hCD86 were incubated at 4 °C for 15 minutes.

[0202] After washing with phosphate-buffered saline (PBS), 50 μl of fluorescein isothiocyanate antibody conjugate of Fc IgG anti-mouse (IgG FcFITC) and streptavidin-phycoerythrin (streptavidin-PE) were added at a 1:100 dilution to each well at 4 °C and incubated for 15 minutes, followed by washing with PBS. Signals for FITC and PE were determined by flow cytometry.

[0203] As shown in FIG. 3, when the concentration of mouse anti-hCTLA4 antibody (CT4-04-13A4 and CT403-4G12) increased, the signal for PE decreased, suggesting that the binding between human CTLA4 and Biotin-hCD80 was blocked by CT4-04-13A4 and CT4-03-4G12 antibodies.

[0204] Similarly, in FIG. 4, when the concentration of anti-hCTLA4 antibody (CT4-04-13A4 and CT4-03-4G12) increased, the signal for PE decreased, suggesting that the binding between human CTLA4 and Biotin-hCD86 was blocked by CT4-04-13A4 and CT4-03-4G12 antibodies. Example 4. Cross-reactivity of chimeric anti-hCTLA antibodies against monkey, mouse, and chimeric human-mouse CTLA4. Petition 870200036579, dated 03 / 19 / 2020, p. 86 / 139 74 / 85

[0205] CHO cells were transfected with rhesus monkey CTLA4 (rmCTLA4, SEQ ID NO: 43), mouse CTLA4 (mCTLA4, SEQ ID NO: 42), and chimeric CTLA4 (mouse and human) (chiCTLA4, SEQ ID NO: 44).

[0206] 25 μl of CHO cells were added to each well. 25 μl of purified chimeric anti-hCTLA antibodies (1 μg / ml) (CT4-03-4G12-mHvKv-IgG1 and CT4-04-13A4-mHvKv-IgG1) were added to each well and incubated at 4 °C for 30 minutes. CT4-03-4G12-mHvKv-IgG1 and CT4-04-13A4-mHvKv-IgG1 are chimeric anti-hCTLA antibodies. CT4-03-4G12-mHvKv-IgG1 has the mouse 4G12 antibody's variable heavy chain domain and variable light chain domain, and the human IgG1 antibody's constant domains (CL, CH1, CH2, CH3). Similarly, CT4-04-13A4-mHvKv-IgG1 te, variable heavy chain domain and variable light chain domain of the mouse 13A4 antibody and constant domains of the human IgG1 antibody (CL, CH1, CH2, CH3).

[0207] After washing twice with PBS (1200 rpm, 5 min), 50 μl of fluorescein isothiocyanate antibody-dependent IgG Fc antibody (IgG Fc-FITC) were added at a 1:100 dilution to each well and incubated at 4 °C for 30 minutes, followed by washing with PBS. Signals for FITC were determined by flow cytometry.

[0208] As shown in FIG. 5, CT4-04-13A4-mHvKvIgG1 did not cross-react with mouse CTLA4 and had strong cross-reactivity with rmCTLA4 and chimeric CTLA4. Similarly, CT4-03-4G12-mHvKv-IgG1 did not cross-react with mouse CTLA4 and had strong cross-reactivity with rmCTLA4 and chimeric CTLA4. In FIG. 5, NC means control. Petition 870200036579, dated 03 / 19 / 2020, page 87 / 139 75 / 85 negative and PC means negative control. Example 5. In vivo test of anti-hCTLA4 antibodies in mice.

[0209] In order to test anti-hCTLA4 antibodies in vivo and to predict the effects of these antibodies in the human body, a humanized CTLA-4 model was generated. The humanized mouse CTLA4 model was genetically engineered to express a chimeric CTLA4 protein (SEQ ID NO: 44) in which a portion of the extracellular region of the mouse CTLA4 protein was replaced by the extracellular region of human CTLA4. The amino acid residues at positions 41-143 of SEQ ID NO: 44 are derived from human CTLA4. The humanized mouse model (humanized mice with BhCTLA-4) may provide a novel tool for testing new therapeutic treatments in a clinical setting by significantly reducing the difference between clinical outcome in humans and in common mice expressing mouse CTLA4.

[0210] Anti-hCTLA4 antibodies were tested to demonstrate their effect on tumor growth in vivo in a colon carcinoma model. MC-38 cancer tumor cells (colon adenocarcinoma cell) were injected subcutaneously into B-hCTLA-4 humanized mice. When the tumors in the mice reached a volume of 150 ± 50 mm3, the mice were randomly placed into different groups based on tumor volume. The mice were then intravenously injected with PBS and anti-hCTLA4 antibodies. The antibody was given every three days for a total of 15 days (6 injections in total). The injected volume was calculated based on the mouse weight. Petition 870200036579, dated 03 / 19 / 2020, pp. 88 / 139 76 / 85 at 10 μl / g. The long geometric axis and short geometric axis length of the tumor were measured twice weekly, and the tumor volume was calculated as 0.5 χ² (long geometric axis) χ² (short geometric axis). The weight of the mice was also measured before injection, when the mice were placed in different groups (before the first antibody injection), twice weekly during the antibody injection period, and before euthanasia.

[0211] The percentage of tumor growth inhibition (TGI%) was calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero.

[0212] The T-test was performed for statistical analysis. A TGI% greater than 60% indicates significant suppression of tumor growth. P < 0.05 is a threshold indicating a significant difference. In vivo results for anti-hCTLA4 antibodies from 13A4 and 4G12 mice.

[0213] The weight of the mice was monitored throughout the treatment period. The weight of the mice in all different groups increased (FIG. 6 and FIG. 7). No significant difference in weight was observed between the control group and the anti-hCTLA4 treatment groups. The results showed that the anti-hCTLA4 antibodies were well tolerated and are not toxic to the mice.

[0214] Tumor size, however, showed a significant difference in groups treated with 13A4 and 4G12 antibodies. Petition 870200036579, dated 03 / 19 / 2020, page 89 / 139 77 / 85 (FIG. 8). As shown in FIG. 8, 13A4 and 4G12 inhibited tumor growth in a concentration-dependent manner. Interestingly, 4G12 at a dose of 0.3 mg / kg had better results compared to 4G12 at a dose of 1 mg / kg, suggesting that a relatively low dose (e.g., less than 0.5 mg / kg or from 0.1 mg / kg to 0.5 mg / kg) of 4G12 may achieve the best results.

[0215] The TGI% on day 22 for each treatment group was also calculated as shown in the table below. Table 1 Group Antibodies TGI% G2 13A4 (3 mg / kg) 103.00 % G3 13A4 (1 mg / kg) 82.90 % G4 13A4 (0.3 mg / kg) 69.00 % G5 4G12 (3 mg / kg) 45.20 % G6 4G12 (1 mg / kg) 10.10 % G7 4G12 (0.3 mg / kg) 73.60% In vivo results for 13A4 and Yervoy

[0216] To compare the efficacy of CTLA4 antibodies, Yervoy was used in the same experiments with 13A4. The weight of mice in different groups increased, all of them, during the treatment period (FIG. 9 and FIG. 10). No toxic effects were observed.

[0217] Treatment with 13A4 and Yervoy resulted in a significant decrease in tumor size (FIG. 11). Notably, treatment with 13A4 resulted in at least comparable effects compared to Yervoy. The TGI% on day 28 for both treatment groups is shown below. Table 2 Petition 870200036579, dated 03 / 19 / 2020, pp. 90 / 139 78 / 85 TGI Antibodies Group% G3 Yervoy 104.60% G5 13A4 104.70% Example 6. In vivo results for humanized anti-hCTLA4 antibodies.

[0218] Humanized antibodies were generated by the methods described in Example 2. To confirm the therapeutic effects of humanized antibodies, six humanized anti-hCTLA4 antibodies (4G12-HlKl-IgGl; 4G12H2Kl-IgGl; 13A4-HlK2-IgGl; 13A4-H2K2-IgGl; 13A4-HlK2-IgG4; 13A4-HlK2-IgGl-N297A) were tested in B-hCTLA-4 humanized mice to demonstrate their effects on tumor growth in vivo (FIGS. 12-17). Furthermore, to reduce glycan heterogeneity, the Fc region of the humanized antibody 13A4-HlK2-IgGl was further genetically modified to replace Asparagine at position 297 with Alanine (FIG. 17). The variable regions of the light and heavy chains of the antibodies are shown in the table below. Table 3 Humanized Antibodies Variable Region of Heavy Chain Variable Region of Light Chain Type 4G12-H1K1- IgGl SEQ ID NO: 21 SEQ ID NO: 25 IgGl 4G12-H2K1- IgGl SEQ ID NO: 22 SEQ ID NO: 25 IgGl Petition 870200036579, dated 03 / 19 / 2020, pp. 91 / 139 79 / 85 Humanized Antibodies Variable Region of Heavy Chain Variable Region of Light Chain Type 13A4-H1K2- IgGl SEQ ID NO: 13 SEQ ID NO: 19 IgGl 13A4-H2K2- IgGl SEQ ID NO: 14 SEQ ID NO: 19 IgGl 13A4-H1K2- IgG4 SEQ ID NO: 13 SEQ ID NO: 19 IgG4 13A4-H1K2- IgGl-N297A SEQ ID NO: 13 SEQ ID NO: 19 IgGl with N297A mutation in the Fc region.

[0219] Similar procedures as described in Example 5 were used. MC-38 cancer tumor cells (colon adenocarcinoma cell) were injected subcutaneously into B-hCTLA-4 humanized mice. When the tumors in the mice reached a volume of 150 ± 50 mm3, the mice were randomly placed into different groups based on tumor volume. The mice were then injected intravenously with PBS and anti-CTLA4 antibodies. The antibody was given twice weekly (days 1 and 4 of each week) for 3 weeks (6 injections in total). The dosage was calculated based on mouse weight at 10 mg / kg. The long geometric axis and short geometric axis length of the tumor were measured twice weekly, and the tumor volume was calculated as 0.5 * (long geometric axis) χ² (short geometric axis). The weight of the mice was also measured before and during injection. Petition 870200036579, dated 03 / 19 / 2020, pp. 92 / 139 80 / 85 time point when the mice were placed into different groups (before the first antibody injection). Weight was also measured twice a week during the antibody injection period and at the right time point before euthanasia.

[0220] The percentage of tumor growth inhibition (TGI%) was calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]*100. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day zero. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day zero.

[0221] The T-test was performed for statistical analysis. A TGI% greater than 60% indicates significant suppression of tumor growth. P < 0.05 was considered to indicate a significant difference. In vivo results for 4G12-H1K1-IgG1 and 4G12-H2K1-IgG1

[0222] Mice were divided into 4 groups: 1) In 1) In G1, human IgG was used as the control; 2) In G4, Yervoy was administered to mice for comparison purposes; 3) In G10, 4G12-H1K1-IgG1 was administered to mice; and 4) In G11, 4G12-H2K1-IgG1 was administered to mice.

[0223] The weight of the mice in the four groups was monitored throughout the treatment period (FIG. 12 and FIG. 13). The mice in each group were generally healthy and the results showed that the anti-CTLA4 antibodies were well tolerated and were not toxic to the mice.

[0224] The tumor size, however, was significantly smaller in groups treated with Yervoy, Petition 870200036579, dated 03 / 19 / 2020, pp. 93 / 139 81 / 85 4G12-HlKl-IgGl and 4G12-H2Kl-IgGl (FIG. 14). As shown in FIG. 14, 4G12-HlKl-IgGl (P=0.002) and 4G12-H2Kl-IgGl (P=0.002) inhibited tumor growth compared to the control group and had better results compared to Yervoy (P=0.007). The TGI% on day 21 for each treatment group is shown below. Table 4 Antibody Group TGI% G4 Yervoy 82.20 % G10 4G12-HlKl-IgGl 90.50 % Gll 4G12-H2Kl-IgGl 90.50 % In vivo results for 13A4-H1K2-IgG1, 13A4-H2K2-IgG1, 13A4-H1K2-IgG4 and 13A4-H1K2-IgG1~N297A

[0225] The mice were placed into 6 groups: 1) In G1, human IgG was used as the control group; 2) In G4, Yervoy was administered to the mice for comparison purposes; 3) In G6, 13A4-HlK2-IgGl was administered to the mice; 4) In G7, 13A4-H2K2-IgGl was administered to the mice; 5) In G8, 13A4-HlK2-IgG4 was administered to the mice; and 6) In G9, 13A4-HlK2-IgGl-N297A was administered to the mice.

[0226] The weight of the mice in the six groups was monitored throughout the treatment period (FIG. 15 and FIG. 16). The results showed that the mice in each group were healthy and the anti-CTLA4 antibodies were not toxic to the mice.

[0227] Tumor sizes, however, were different in each group. As shown in FIG. 17, all anti-CTLA4 antibodies can inhibit tumor growth in Petition 870200036579, dated 03 / 19 / 2020, pp. 94 / 139 82 / 85 comparison with the control group. In particular, 13A4HlK2-IgGl and 13A4-H2K2-IgGl had better results compared to Yervoy.

[0228] The TGI% on day 21 for each treatment group was shown below. Table 5 Group Antibodies TGI% G4 Yervoy 82.20 % G6 13A4-HlK2-IgGl 99.00 % G7 13A4-H2K2-IgGl 9 6.60 % G8 13A4-HlK2-IgG4 56.10 % G9 13A4-HlK2-IgGl-N297A 47.30% Example 7. In vitro test of mouse anti-hCTLA4 antibodies: CT4-20-6D2 (6D2) and CT4-20-7E12 (7E12)

[0229] Anti-CTLA4 antibodies were collected from mouse ascites fluid and purified by chromatography. 25 μL of CHO cells transiently transfected with human CTLA4 were added to each well in a plate. The purified antibodies were titrated to final concentrations of 50, 5, 0.5, 0.05, 0.005 pg / ml. The titrated antibodies were added to each well at 25 μL per well at 4 °C and incubated for 30 minutes. Biotin-hCD86 was titrated to 0.4 pg / ml. 50 μL of the ligand solution were added to each well, making the final concentration of Biotin-hCD86 0.2 pg / ml. Cells with Biotin-hCD86 were incubated at 4 °C for 15 minutes. After being washed with phosphate-buffered saline (PBS), 50 μL of fluorescein isothiocyanate conjugate of anti-mouse Fc IgG antibody (Fc-FITC IgG) and streptavidin-phycoerythrin Petition 870200036579, dated 03 / 19 / 2020, pp. 95 / 139 83 / 85 (streptavidin-PE) were added at a 1:100 dilution to each well at 4 °C and incubated for 15 minutes, followed by washing with PBS. Signals for FITC and PE were determined by flow cytometry.

[0230] As shown in FIG. 18, when the concentration of anti-hCTLA4 antibody (CT4-20-6D2 and CT4-207E12) increased, the signal for PE decreased, suggesting that the binding between human CTLA4 and Biotin-hCD86 was blocked by CT4-20-6D2 and CT4-20-7E12 antibodies. Example 8. Cross-reactivity of anti-hCTLA antibodies against monkey, mouse, and chimeric human-mouse CTLA4.

[0231] CHO cells were transfected with rhesus monkey CTLA4 (rmCTLA4, SEQ ID NO: 43), mouse CTLA4 (mCTLA4, SEQ ID NO: 42), and chimeric CTLA4 (mouse and human) (chiCTLA4, SEQ ID NO: 44).

[0232] 25 μl of CHO cells were added to each well. 25 μl of purified mouse anti-hCTLA antibodies (1 μg / ml) (CT4-20-6D2 and CT4-20-7E12) were added to each well and incubated at 4 °C for 30 minutes.

[0233] After washing twice with PBS (1200 rpm, 5 min), 50 μl of fluorescein isothiocyanate antibody-dependent IgG Fc antibody (IgG Fc-FITC) were added at a 1:100 dilution to each well and incubated at 4 °C for 30 minutes, followed by washing with PBS. Signals for FITC were determined by flow cytometry.

[0234] As shown in FIG. 19, CT4-20-6D2 and CT4-207E12 did not cross-react with mouse CTLA4, they had Petition 870200036579, dated 03 / 19 / 2020, pp. 96 / 139 84 / 85 weak cross-reactivity with chimeric CTLA4 and relatively strong cross-reactivity with rmCTLA4. Example 9. In vivo test of anti-hCTLA4 antibodies from mice.

[0235] Anti-hCTLA4 antibodies were tested to demonstrate their effect on tumor growth in vivo in a colon carcinoma model. MC-38 cancer tumor cells (colon adenocarcinoma cells) were injected subcutaneously into B-hCTLA-4 humanized mice. When the tumors in the mice reached a volume of 150 ± 50 mm3, the mice were randomly placed into different groups based on tumor volume. The mice were then intravenously injected with PBS and anti-hCTLA4 antibodies. The antibody was given twice a week (6 injections in total). In the control group, saline solution was administered to the mice. The injected volume was calculated based on the mouse weight at 10 µl / g. The long geometric axis and short geometric axis lengths of the tumor were measured twice weekly, and the tumor volume was calculated as 0.5 χ² (long geometric axis) χ² (short geometric axis).The weight of the mice was also measured before injection, when the mice were placed into different groups (before the first antibody injection), twice a week during the antibody injection period, and before euthanasia.

[0236] The T-test was performed for statistical analysis. A TGI% greater than 60% indicates significant suppression of tumor growth. P < 0.05 was considered to indicate a significant difference.

[0237] The weight of the mice was monitored throughout Petition 870200036579, dated 03 / 19 / 2020, pp. 97 / 139 The 85 / 85 treatment period saw an increase in the weight of mice in all different groups (FIG. 20 and FIG. 21). No significant difference in weight was observed between the control group and the anti-hCTLA4 treatment groups. The results showed that anti-hCTLA4 antibodies were not toxic to mice.

[0238] Tumor size, however, showed a significant difference in groups treated with antibodies 13A4, 6D2 and 7E12 (FIG. 22). As shown in FIG. 22, 13A4, 6D2 and 7E12 all inhibited tumor growth in mice.

[0239] The TGI% on day 21 for each treatment group is shown below. Table 6 TGI Antibodies Group% G2 13A4 109.70% G3 6D2 69.80% G4 7E12 65.60% OTHER MODALITIES

[0240] It should be understood that although the invention has been described in combination with the detailed description thereof, the preceding description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims. Petition 870200036579, dated 03 / 19 / 2020, pp. 98-139

Claims

1 / 3 CLAIMS 1. Antibody or antigen-binding fragment thereof that binds to cytotoxic T lymphocyte-associated protein 4 (CTLA4) characterized by comprising: a variable heavy chain (VH) region comprising complementarity-determining regions (CDRs) 1, 2 and 3; and a variable light chain (VL) region comprising CDRs 1, 2 and 3, wherein (1) the amino acid sequences of VH CDRs 1, 2 and 3 are established in SEQ ID NOs: 1, 2, 3, respectively, as defined by Kabat numbering, and the amino acid sequences of VL CDRs 1, 2 and 3 are established in SEQ ID NOs: 4, 5, 6, respectively, as defined by Kabat numbering; or the amino acid sequences of VH CDRs 1, 2, and 3 are established in SEQ ID NOs: 29, 30, 31, respectively, as defined by Chothia numbering; and the amino acid sequences of VL CDRs 1, 2, and 3 are established in SEQ ID NOs: 32, 33, 34, respectively.as defined by the Chothia numbering; or (2) the amino acid sequences of VH CDRs 1, 2 and 3 are established in SEQ ID NOs: 7, 8, 9, respectively, as defined by the Kabat numbering; and the amino acid sequences of VL CDRs 1, 2 and 3 are established in SEQ ID NOs: 10, 11, 12, respectively, as defined by the Kabat numbering; or; the amino acid sequences of VH CDRs 1, 2 and 3 are established in SEQ ID NOs: 35, 36, 37, respectively, as defined by the Chothia numbering; and the amino acid sequences of VL CDRs 1, 2 and 3 are established in SEQ ID NOs: 38, 39, 40, respectively, as defined by the Chothia numbering.

2. Antibody or antigen-binding fragment thereof, according to claim 1, as per Petition 870260069106, dated 13 / 07 / 2026, page 14 / 16 2 / 3, characterized in that VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2 and 3, respectively, and VL comprises CDRs 1, 2,3 with the amino acid sequences established in SEQ ID NOs: 4, 5 and 6, respectively.

3. Antibody or antigen-binding fragment thereof, according to claim 1, characterized in that VH comprises CDRs 1, 2, 3 with the amino acid sequences established in SEQ ID NOs: 7, 8 and 9, respectively, and VL comprises CDRs 1, 2, 3 with the amino acid sequences established in SEQ ID NOs: 10, 11 and 12, respectively.

4. Antibody or antigen-binding fragment thereof, according to claim 1, characterized in that VH sequence is SEQ ID NO: 13 or 14 and VL sequence is SEQ ID NO: 19; (2) the VH sequence comprising the amino acid sequence established in SEQ ID NO: 69, and the VL sequence comprising the amino acid sequence established in SEQ ID NO: 70; (3) the VH sequence is SEQ ID NO: 21 or 22 and the VL sequence is SEQ ID NO: 25; or (4) the VH sequence comprising the amino acid sequence set forth in SEQ ID NO: 71, and the VL sequence comprising the amino acid sequence set forth in SEQ ID NO:

72.

5. Antibody or antigen-binding fragment thereof, according to any one of claims 1-4, the antibody or antigen-binding fragment being characterized as a humanized antibody or antigen-binding fragment thereof, or a single-chain variable fragment (scFV). Petition 870260069106, dated 13 / 07 / 2026, p. 15 / 16 3 / 3 6. Antibody-drug conjugate characterized as comprising the antibody or antigen-binding fragment thereof as defined in any one of claims 1-5, covalently linked to a therapeutic agent. 7.Antibody-drug conjugate according to claim 6, characterized in that the therapeutic agent is a cytotoxic or cytostatic agent.

8. Use of an antibody or antigen-binding fragment thereof, according to any one of claims 1-5, characterized in that it is for the manufacture of a medicament for the prevention or treatment of cancer.

9. Use of an antibody or antigen-binding fragment thereof, according to claim 8, characterized in that the cancer is breast cancer, carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer or hematological malignancy. 10.Use of an antibody or antigen-binding fragment thereof, according to claim 8, characterized in that the cancer is liver cancer or small cell lung cancer. Petition 870260069106, dated 07 / 13 / 2026, p. 16 / 16.

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