Monoclonal antibody against the IgV domain of B7-H3 and its use

Abstract

The present invention relates to monoclonal antibodies that bind to the IgV domain of human B7-H3 and to methods of using these antibodies for the treatment of cancer and infectious diseases and for imaging of B7-H3-positive cells.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 768,128, filed on November 16, 2018, the content of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. A copy of the ASCII format sequence listing created on November 15, 2019, has a file name of SequenceListing.txt and a size of 19,181 bytes.

Background Art

[0003] Throughout this specification, various documents are referenced by numbers in parentheses. The bibliographic details of these references are shown at the end of the specification. The contents of these documents are incorporated herein by reference in their entirety to more fully explain the technology related to the present invention.

[0004] B7 - H3 is a type I transmembrane protein and belongs to the B7 family. B7 - H3 is overexpressed in human malignant tumors (1, 2) including prostate cancer (3), liver cancer (4), melanoma (5), leukemia (6), breast cancer (7), ovarian cancer (8), pancreatic cancer (9), colorectal cancer (10), lung cancer (11), bladder cancer (12), renal cancer (13), brain cancer (14), and osteosarcoma (15). Some studies have shown a correlation between high expression of B7 - H3 and poor prognosis (1, 2). These studies suggest that B7 - H3 is an immune checkpoint used by human cancer cells to inhibit the function of immune cells (1, 2). The extracellular region of B7 - H3 is composed of IgV - IgC - IgV - IgC or IgV - IgC. Previous studies have shown that the FG loop of the IgV domain of B7 - H3 plays an important role in T - cell suppression by B7 - H3 (16).

[0005] Cancer and infectious diseases are serious public health problems in the United States and other countries. The present invention relates to antibodies that can be used for the treatment of these diseases.

Disclosure of the Invention

[0006] The present invention relates to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the IgV domain of human B7-H3.

[0007] In some embodiments, the antibody comprises a heavy chain having (a) complementarity-determining region (CDR) 1 comprising GYTFTSYWIT (SEQ ID NO: 1), CDR2 comprising DIYPGSGSTNYNEKFKS (SEQ ID NO: 2), and / or CDR3 comprising ARGGTRFSPFAY (SEQ ID NO: 3); and / or (b) a light chain having CDR1 comprising RSSQSIVHSNGNTYLE (SEQ ID NO: 4), CDR2 comprising KVSNRFS (SEQ ID NO: 5), and / or CDR3 comprising FQGSHVPWT (SEQ ID NO: 6). In another embodiment, the antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence shown in SEQ ID NO: 14 and / or a light chain variable domain (VL) comprising the amino acid sequence shown in SEQ ID NO: 16, and these sequences do not include a leader sequence.

[0008] In some embodiments, the antibody comprises a heavy chain having (a) CDR1 comprising GYTFTSYWMH (SEQ ID NO: 7), CDR2 comprising MIHPNSGSTNYNEKFKS (SEQ ID NO: 8), and / or CDR3 comprising YYYGSSYAMDY (SEQ ID NO: 9); and / or (b) a light chain having CDR1 comprising SASSSVSYMH (SEQ ID NO: 10), CDR2 comprising STSNLAS (SEQ ID NO: 11), and / or CDR3 comprising QQRSSYPYT (SEQ ID NO: 12). In another embodiment, the antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 18 and / or a VL comprising the amino acid sequence shown in SEQ ID NO: 20, and these sequences do not include a leader sequence.

[0009] In some embodiments, the antibody comprises a heavy chain having (a) CDR1 comprising GYTFTSYWMH (SEQ ID NO: 7); CDR2 comprising MIHPNSGSTNYNEKFKS (SEQ ID NO: 8), and / or CDR3 comprising YYGTNV (SEQ ID NO: 15); and / or (b) a light chain having CDR1 comprising KSVSTSGYSYMH (SEQ ID NO: 13), CDR2 comprising LVSNLES (SEQ ID NO: 14), and / or CDR3 comprising QHIREAYT (SEQ ID NO: 16). In another embodiment, the antibody comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 23 and / or a VL comprising the amino acid sequence set forth in SEQ ID NO: 25, wherein these sequences do not include a leader sequence.

[0010] In some embodiments, the antibody competes with the above antibody or fragment thereof with respect to binding to the IgV domain of human B7-H3.

[0011] In one embodiment, the antibody is a humanized antibody or a chimeric antibody.

[0012] The present invention also relates to the use of the antibody or fragment thereof for the treatment of cancer and infectious diseases and for the imaging of B7-H3 positive cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

Figure 1

Figure 2

Figure 3

Best Mode for Carrying Out the Invention

[0014] The present invention relates to I) a) GYTFTSYWIT (SEQ ID NO: 1) (Complementary Determining Region (CDR), CDR1), DIYPGSGSTNYNEKFKS (SEQ ID NO: 2) (CDR2), or ARGGTRFSPFAY (SEQ ID NO: 3) (CDR3) a heavy chain comprising one or more of; and b) RSSQSIVHSNGNTYLE (SEQ ID NO: 4) (CDR1), KVSNRFS (SEQ ID NO: 5) (CDR2), or FQGSHVPWT (SEQ ID NO: 6) (CDR3) a light chain comprising one or more of; II) a) GYTFTSYWMH (SEQ ID NO: 7) (CDR1), MIHPNSGSTNYNEKFKS (SEQ ID NO: 8) (CDR2), or YYYGSSYAMDY (SEQ ID NO: 9) (CDR3) a heavy chain comprising one or more of; and b) SASSSVSYMH (SEQ ID NO: 10) (CDR1), STSNLAS (SEQ ID NO: 11) (CDR2), or QQRSSYPYT (SEQ ID NO: 12) (CDR3) a light chain comprising one or more of; or, III) An antibody or fragment thereof that competes with the antibody or fragment of I) or II) with respect to binding to the IgV domain of human B7-H3 relates to a monoclonal antibody or an antigen-binding fragment thereof that binds to the IgV domain of human B7-H3.

[0015] The fragment that binds to the B7-H3 IgV domain can be, for example, Fab, F(ab)2 or scFv.

[0016] In some embodiments, the antibody or fragment is GYTFTSYWIT (SEQ ID NO:1) (CDR1), DIYPGSGSTNYNEKFKS (SEQ ID NO:2) (CDR2) and ARGGTRFSPFAY (SEQ ID NO:3) (CDR3) containing heavy chain; and RSSQSIVHSNGNTYLE (SEQ ID NO:4) (CDR1), KVSNRFS (SEQ ID NO:5) (CDR2) and FQGSHVPWT (SEQ ID NO:6) (CDR3) containing light chain, comprising.

[0017] In some embodiments, the antibody or fragment is a) GYTFTSYWMH (SEQ ID NO:7) (CDR1), MIHPNSGSTNYNEKFKS (SEQ ID NO:8) (CDR2), and YYYGSSYAMDY (SEQ ID NO:9) (CDR3) containing heavy chain; and b) SASSSVSYMH (SEQ ID NO:10) (CDR1), STSNLAS (SEQ ID NO:11) (CDR2), and QQRSSYPYT (SEQ ID NO:12) (CDR3) containing light chain, comprising.

[0018] In one aspect, the framework regions of the light and heavy chains are human framework regions or have 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to human framework regions. In one aspect, the anti-B7-H3 antibody or its B7-H3 binding fragment comprises (i) a VH framework comprising the framework sequence of human germline GHV1-46*01, IGHV1-46*02, IGHV1-46*03, IGHD1-1*01, IGHD1-26*01, IGHD2-8*01, IGHD3-10*01, IGHD3-22*01, IGHD4-23*01, IGHJ3*01, IGHJ4*01, IGHJ4*03, IGHJ6*01 or IGHJ6*02; and / or (ii) a VL framework comprising the framework sequence of human germline IGKV2-18*01, IGKV2D-29*02, IGKV2-29*03, IGKV3-11*01, IGKV3-15*01, IGKV3-20*02, IGKJ1*01, IGKJ2*01, IGKJ4*01 or IGKJ4*02.

[0019] In one aspect, the antibody or fragment has a human heavy chain constant domain, such as a human Fc region. In some aspects, the human heavy chain constant domain is derived from human IgG such as IgG1, IgG2, IgG3 or IgG4. In some aspects, the antibody or fragment comprises a human κ light chain constant domain.

[0020] In certain aspects, the antibody or fragment is a blocking antibody or fragment, or an inhibitory antibody or fragment, that antagonizes the biological activity of B7-H3.

[0021] The invention also relates to bispecific antibodies containing the antigen-binding domain of the anti-B7-H3 antibodies described herein, and to the antigen-binding domain of monoclonal antibodies that bind to the CD3 component of the T cell receptor (TCR) complex of T cells.

[0022] The invention also relates to GYTFTSYWIT (SEQ ID NO: 1) (heavy chain CDR1), DIYPGSGSTNYNEKFKS (SEQ ID NO: 2) (heavy chain CDR2), ARGGTRFSPFAY (SEQ ID NO: 3) (heavy chain CDR3), RSSQSIVHSNGNTYLE (SEQ ID NO: 4) (light chain CDR1), KVSNRFS (SEQ ID NO: 5) (light chain CDR2), and FQGSHVPWT (SEQ ID NO: 6) (light chain CDR3) An isolated nucleic acid or cDNA encoding a complementarity determining region of an anti-B7-H3 antibody comprising one or more of the above.

[0023] Furthermore, the present invention relates to GYTFTSYWMH (SEQ ID NO: 7) (heavy chain CDR1), MIHPNSGSTNYNEKFKS (SEQ ID NO: 8) (heavy chain CDR2), YYYGSSYAMDY (SEQ ID NO: 9) (heavy chain CDR3), SASSSVSYMH (SEQ ID NO: 10) (light chain CDR1), STSNLAS (SEQ ID NO: 11) (light chain CDR2), and QQRSSYPYT (SEQ ID NO: 12) (light chain CDR3) An isolated nucleic acid or cDNA encoding a complementarity determining region of an anti-B7-H3 antibody comprising one or more of the above.

[0024] Furthermore, the present invention relates to GYTFTSYWMH (SEQ ID NO: 7) (heavy chain CDR1), KSVSTSGYSYMH (SEQ ID NO: 13) (light chain CDR1), MIHPNSGSTNYNEKFKS (SEQ ID NO: 8) (heavy chain CDR2), LVSNLES (SEQ ID NO: 14) (light chain CDR2), YYGTNV (SEQ ID NO: 15) (heavy chain CDR3), and QHIREAYT (SEQ ID NO: 16) (light chain CDR3) An isolated nucleic acid or cDNA encoding a complementarity determining region of an anti-B7-H3 antibody comprising one or more of the above.

[0025] In any of the above-described embodiments, the nucleic acid may be isolated. As used herein, an "isolated" or "purified" nucleic acid is a nucleic acid that is (1) separated from genomic DNA or intracellular RNA and / or (2) not naturally occurring.

[0026] The present invention also relates to a vector or host cell comprising an isolated nucleic acid (e.g., cDNA).

[0027] The present invention also relates to a method for producing an anti-B7-H3 IgV domain antibody or a B7-H3 IgV domain-binding fragment thereof, comprising culturing a host cell under conditions such that the host cell described herein produces the anti-B7-H3 IgV domain antibody or a B7-H3 IgV domain-binding fragment thereof.

[0028] Furthermore, the present invention relates to a pharmaceutical composition comprising an antibody or a fragment thereof described herein and a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include, but are not limited to, Additive Solution - 3 (AS-3), physiological saline, phosphate-buffered saline, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann's balanced saline solution, and heparinized sodium citrate dextrose solution.

[0029] Compositions or pharmaceutical compositions comprising an antibody or an antibody fragment described herein preferably contain a stabilizer to prevent loss of protein activity or structural integrity due to denaturation, oxidation, or aggregation during storage and transportation. The composition or pharmaceutical composition can contain one or more of a combination of salts, surfactants, pH, and tonicity agents such as sugars that can contribute to aggregation inhibition. When the composition or pharmaceutical composition of the present invention is an injection, it is preferably at a nearly neutral pH. It is also advantageous to minimize the amount of surfactant to avoid the formation of air bubbles in the formulation that are harmful in injection. In some embodiments, the composition or pharmaceutical composition is a liquid in which the bioactive antibody is stable at a high concentration and is suitable for administration by inhalation or parenterally.

[0030] The pharmaceutically acceptable carrier to be used can depend on the route of administration. The pharmaceutical composition can be formulated for administration by methods known in the art. In some embodiments, the composition or pharmaceutical composition is suitable for intravenous, intramuscular, intraperitoneal, intradermal and / or subcutaneous injection. In some embodiments, the composition or pharmaceutical composition is liquid and the risk of foam formation and anaphylaxis is minimized. In some embodiments, the composition or pharmaceutical composition is isotonic. In some embodiments, the pH of the composition or pharmaceutical composition is between 6.8 and 7.4.

[0031] The present invention also relates to a method of enhancing the immune function of a subject, comprising administering to the subject an effective amount of an antibody or fragment thereof described herein that is effective in enhancing the immune function. The subject may be suffering from, for example, cancer or an infectious disease.

[0032] The present invention further relates to a method for treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or fragment thereof described herein for treating cancer. The cancer can be, for example, prostate cancer, liver cancer, melanoma, leukemia, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, osteosarcoma, adrenal cancer, anal canal cancer, basal cell and squamous cell skin cancer, cholangiocarcinoma, bladder cancer, bone cancer, brain and spinal cord tumors, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing family tumors, eye cancer (intraocular melanoma), gallbladder cancer, gastrointestinal neuroendocrine (carcinoid) tumors, gastrointestinal stromal tumors (GIST), gestational trophoblast disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumors, malignant mesothelioma, melanoma skin cancer, Merkel cell skin cancer, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, central nervous system (CNS) neoplasms, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor (NET), penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, Wilms tumor, squamous cell carcinoma, environmentally induced cancer, cancer combinations, metastatic lesions of cancer, myeloid tumors, acute myeloid leukemia (AML), AML with recurrent genetic abnormalities, AML with myelodysplastic changes, therapy-related AML, ambiguous lineage acute leukemia, myeloproliferative tumors, essential thrombocythemia, polycythemia vera, myelofibrosis (MF), primary myelofibrosis, systemic mastocytosis, myelodysplastic syndrome (MDS), myeloproliferative / myelodysplastic syndrome, chronic myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, myelodysplastic syndrome (MDS), refractory anemia with ring sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts (type 1), refractory anemia with excess blasts (type 2), MDS with isolated del(5q), unclassifiable MDS, myeloproliferative / myelodysplastic syndrome, chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, juvenile myelomonocytic leukemia, unclassifiable myeloproliferative / myelodysplastic syndrome, lymphoid neoplasms, precursor lymphoid neoplasms, B-cell lymphoblastic leukemia, B-cell lymphoblastic lymphoma, T-cell lymphoblastic leukemia, T-cell lymphoblastic lymphoma, mature B-cell neoplasms,Diffuse large B-cell lymphoma, primary central nervous system lymphoma, primary mediastinal B-cell lymphoma, Burkitt lymphoma / leukemia, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, mantle cell lymphoma, marginal zone lymphoma, post-transplant lymphoproliferative disorder, HIV-related lymphoma, primary effusion lymphoma, intravascular large B-cell lymphoma, primary cutaneous B-cell lymphoma, hairy cell leukemia, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma or solitary plasmacytoma (solitary bone and extramedullary) may also be present. The cancer may be, for example, at an early, intermediate, advanced stage, locally advanced or metastatic, recurrent or refractory to other treatments, or for which standard treatments have not been established. As used herein, "treatment" of cancer means reducing the number of cancer cells or metastatic cells in an organ or tissue, slowing tumor growth, killing tumor cells, metastatic tumor cells or potentially metastatic tumor cells, preventing or reducing the spread of cancerous cells from the original site in the body to another site, arresting the progression of metastatic cancer, preventing recurrence of metastasis, slowing or reducing the progression of the disease, and / or increasing survival rate.

[0033] The present invention further relates to a method for treating an infectious disease in a subject, comprising administering to the subject an effective amount of an antibody or fragment thereof described herein for treating the infectious disease. The infection may be caused by, for example, a virus, viroid, bacterium, prion, nematode, arthropod, fungus or protozoan. As used herein, "treatment" of an infectious disease means reducing the signs or symptoms of the infectious disease and / or reducing the number of infectious pathogens in the subject.

[0034] The present invention also contemplates the use of the sequences of the antibodies or fragments described herein (e.g., the sequences of the six CDRs or VH and VL) in the manufacture of chimeric antigen receptors that can be used in CAR-T technology.

[0035] The antibodies and fragments thereof, compositions, and bispecific binding molecules of the present invention may be used in the treatment methods described herein, may be for use in the treatment described herein, and / or may be for use in the manufacture of a medicament for performing the treatment described herein. It is understood. The present invention also relates to kits and articles of manufacture comprising the antibodies and antigen-binding portions thereof, compositions, and bispecific binding molecules described herein.

[0036] The anti-B7-H3 antibody or fragment thereof described herein may further have a detectable marker such as a fluorescent label or a radioactive label bound thereto. The present invention also provides a method for detecting B7-H3 positive cells in a subject, comprising administering to the subject an effective amount of an antibody or fragment (e.g., labeled with a detectable marker) capable of detectably binding to B7-H3 positive cells, and then detecting the antibody or fragment, thereby detecting B7-H3 positive cells. The label can be detected, for example, by imaging. B7-H3 positive cells can be, for example, cancer cells.

[0037] Furthermore, the anti-B7-H3 antibody or fragment thereof described herein can be part of an antibody-drug conjugate (ADC). The ADC composition according to the present invention may comprise, for example, the antibody or fragment thereof of the present invention in combination with a cytotoxic agent and a linker. Cytotoxic agents that may be used include alkylating agents, bifunctional alkylating agents, monofunctional alkylating agents, anthracyclines, cytoskeleton disrupting agents, taxanes, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotide analogs, nucleotide precursor analogs, peptide antibiotics, platinum-based drugs, retinoids, vinca alkaloids and their derivatives, actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vemurafenib, vinblastine, vincristine or vindesine, but are not limited thereto. The ADC according to the present invention may comprise the antibody or fragment thereof of the present invention (e.g., covalently) bound to a small molecule or a drug.

[0038] As used herein, the term "antibody" refers to an intact antibody, i.e., an antibody having a complete Fc and Fv region. The variable domains of the mature light and heavy chains are composed, from the N-terminal to the C-terminal direction, of the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid assignment to each domain follows the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) or Chothia et al., Nature 342:878-883 (1989). "Fragment" refers to a portion of an antibody or a linked portion of an antibody, such as, but not limited to, Fab, F(ab)2 or single-chain Fv (scFv), which is the antigen-binding portion rather than the whole antibody and competes with the intact antibody for specific binding. In this case, the antigen is the IgV domain of human B7-H3. Such fragments can be produced, for example, by cleavage of an intact antibody or by recombination. See Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). The antigen-binding fragment may be produced by DNA recombination techniques, enzymatic or chemical cleavage of an intact antibody, or molecular biology techniques.

[0039] In some embodiments, the fragment is a polypeptide comprising at least a portion of an antibody sufficient to specifically bind to the IgV domain of human B7-H3, including Fab, Fab’, F(ab’)2, Fd, Fv, complementarity determining region (CDR) fragments, single-chain antibody (scFv), a light chain variable domain (VL) and a heavy chain variable domain (VH) linked via a peptide linker, or a diabody. As used herein, an Fd fragment means a fragment of an antibody consisting of VH and CH1 domains; an Fv fragment consists of the V1 and VH domains of a single chain of an antibody; a dAb fragment (Ward et al., Nature 341:544-546 (1989), incorporated herein by reference in its entirety) consists of a VH domain. In some embodiments, the amino acid length of the fragment is at least 5, 6, 8, or 10. In another embodiment, the amino acid length of the fragment is at least 14, at least 20, at least 50, or at least 70, 80, 90, 100, 150, or 200.

[0040] In some embodiments, the scFv described herein includes a framework sequence of a variable domain having the same sequence as a human variable domain FR1, FR2, FR3, and / or FR4. In some embodiments, the scFv includes a linker peptide having a length of 5 to 30 amino acids. In some embodiments, the scFv includes a linker peptide comprising one or more of glycine, serine, and threonine. In some embodiments, the amino acid length of the linker of the scFv is 10 to 25. In one embodiment, the peptide linker comprises glycine, serine, and / or threonine. See, for example, Bird et al., Science, 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988).

[0041] As used herein, the term "monoclonal antibody" refers to an antibody in a substantially homogeneous antibody population, i.e., the individual antibodies that make up the population are identical except for possible minor mutants, e.g., naturally occurring mutants. Thus, the modifier "monoclonal" indicates the property of not being a mixture of different antibodies. In one aspect, such monoclonal antibodies typically include antibodies that contain a polypeptide sequence that binds to the IgV domain of human B7-H3. In contrast to polyclonal antibodies, which include antibodies against different determinants (epitopes), monoclonal antibodies typically bind to a single determinant of an antigen. In addition to their specificity, monoclonal antibodies are typically advantageous in that they are not contaminated with other immunoglobulins. Thus, once their sequence has been determined, monoclonal antibodies can be produced by non-hybridoma techniques, e.g., by appropriate recombinant means.

[0042] In some aspects described herein, the antibody or fragment is isolated. As used herein, the term "isolated antibody" or "isolated antigen-binding fragment" refers to an antibody or fragment that has one or more of the following characteristics due to its origin or derivation: (1) does not contain components that are present in its natural state, (2) does not contain proteins of the same species, (3) is expressed by cells of a different species, (4) does not occur naturally without human intervention.

[0043] In certain embodiments, the antibody is humanized. A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal non-human immunoglobulin sequences. In some embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the recipient's hypervariable regions (HVRs) (or CDRs) have been replaced by the residues of the HVRs of a non-human species such as a mouse, rat, rabbit, or non-human primate (donor antibody) that have the desired specificity, affinity, and / or potency. In embodiments, the antibody has one, two, three, four, five, or all six of the CDRs (CDR1-3 of the heavy and light chains) of the murine antibodies described herein. In certain embodiments, the residues of the framework (FR) of the murine mAb are replaced by the residues of the framework (FR) of the corresponding human immunoglobulin variable domain. These may be modified in the practice of the invention to further refine the performance of the antibody. Additionally, in certain embodiments, the humanized antibody may contain residues that are not present in the recipient antibody or donor antibody. In some embodiments, the humanized antibody does not contain residues that are not present in the recipient antibody or donor antibody. Generally, a humanized antibody substantially comprises at least one, and usually two, variable domains, wherein all, or in certain embodiments substantially all, of the hypervariable loops in the variable domains correspond to those of a non-human immunoglobulin, and all, or in certain embodiments substantially all, of the FRs are those of a human immunoglobulin sequence. A humanized antibody may also, in some cases, include at least a portion of the constant region (Fc) of the immunoglobulin, usually the Fc of a human immunoglobulin.See, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994) and U.S. Pat. Nos. 6,982,321 and 7,087,409; these references and the specification are incorporated herein by reference in their entirety. In embodiments where the humanized antibody contains residues not present in the recipient or donor antibody, the Fc region of the antibody may be modified as described in WO 99 / 58572; the contents of said specification are incorporated herein by reference in their entirety.

[0044] Techniques for humanizing monoclonal antibodies are well known and are described, for example, in U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089 and 6,180,370; the entire disclosures of which are incorporated herein by reference. Many "humanized" antibodies containing the antigen-binding site of a non-human immunoglobulin are known, e.g., antibodies having murine or modified murine V regions and their complementarity-determining regions (CDRs) fused to human constant domains. See, for example, Winter et al., Nature 349: 293-299 (1991); Lobuglio et al., Proc. Nat. Acad. Sci. USA 86: 4220-4224 (1989); Shaw et al., J. Immunol. 138: 4534-4538 (1987); Brown et al., Cancer Res. 47: 3577-3583 (1987). Other references describe murine hypervariable regions or CDRs transplanted into human framework regions (FRs) prior to fusion with appropriate human antibody constant domains. See, for example, Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988); Jones et al., Nature 321: 522-525 (1986). Another reference describes murine CDRs having a recombinant engineered murine framework region - European Patent Application Publication No. 0519596 (the entire disclosure of which is incorporated). These "humanized" molecules are designed to minimize the undesirable immune responses that limit the duration and effectiveness of treatment in human recipients by murine anti-human antibodies. The constant region of the antibody can be modified to be immunologically inert (e.g., not cause complement lysis). See, for example, International Publication No. 99 / 58572; UK Patent Application No. 9809951.8.Other methods of humanizing antibodies are described in Daugherty et al., Nucl. Acids Res. 19: 2471-2476 (1991), U.S. Pat. Nos. 6,180,377, 6,054,297, 5,997,867, 5,866,692, 6,210,671 and 6,350,861; and International Publication No. 01 / 27160 (incorporated herein by reference in its entirety).

[0045] In some embodiments, the antibodies or fragments herein can be produced recombinantly; for example, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., mouse) into which a human immunoglobulin gene has been introduced, and the like.

[0046] The term "K d " is intended herein to refer to the dissociation constant of an antibody-antigen interaction. The affinity constant is the reciprocal of the dissociation constant. One method for determining the K d or binding affinity of an antibody for the IgV domain of B7-H3 is to measure the binding affinity of a monovalent Fab fragment of the antibody. To obtain a monovalent Fab fragment, an antibody (e.g., IgG) can be cleaved with papain or expressed recombinantly. The affinity of fragments of anti-human B7-H3 IgV domain antibodies can be determined, for example, by surface plasmon resonance (BIAcore3000® surface plasmon resonance (SPR) system, BIAcore Inc., Piscataway N.J.). The CM5 chip can be activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen can be diluted in 10 mM sodium acetate at pH 4.0 and injected into the activated chip at a concentration of 0.005 mg / mL. Using variable flow times across individual chip channels, two antigen densities can be achieved: 100-200 response units (RU) for detailed kinetic studies and 500-600 RU for screening assays. Purified and serially diluted (estimated K dInject the Fab (0.1 to 10 times that amount) at 100 mL / min for 1 minute, allowing a dissociation time of up to 2 hours. The concentration of the Fab protein is determined by ELISA and / or SDS-PAGE electrophoresis using a Fab of known concentration (determined by amino acid analysis). The association rate (k on ) and the dissociation rate (k off ) can be obtained simultaneously by fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110, the content of which is incorporated herein by reference in its entirety) using a BIA evaluation program. The value of the equilibrium dissociation constant (K d ) is calculated as k off / k on . This procedure is suitable for determining the binding affinity of an antibody or fragment for an antigen. Other methods known in the art can also be used (e.g., ELISA).

[0047] An antibody or polypeptide that "specifically binds" to an epitope is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule "specifically binds" or "preferentially binds" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer time and / or with a higher affinity than with another cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with a higher affinity, binding strength, more readily, and / or for a longer time than it binds to other substances. For example, an antibody that specifically or preferentially binds to the human B7-H3 IgV domain is an antibody that binds to this epitope with a higher affinity, binding strength, more readily, and / or for a longer time than it binds to other epitopes of B7-H3 or non-B7-H3 epitopes. From this definition, it is also understood that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily have to be exclusive binding (although it can include exclusive binding). In some embodiments, when K d is 1 mM or less, preferably 100 nM or less, the antibody is said to specifically bind to the antigen.

[0048] Antibodies (immunoglobulins) can be assigned to different classes according to the amino acid sequence of the constant domain of the heavy chain. The antibody or fragment can be, for example, any of an IgG, IgD, IgE, IgA or IgM antibody or a fragment thereof. In some embodiments, the antibody is an immunoglobulin G. In some embodiments, the antibody fragment is a fragment of immunoglobulin G. In some embodiments, the antibody is IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4. In some embodiments, the antibody comprises the sequence of human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3 or human IgG4. Combinations of these antibody subtypes can also be used. One consideration in selecting the type of antibody to use is the desired serum half-life of the antibody. For example, the serum half-life of IgG is typically 23 days, IgA is 6 days, IgM is 5 days, IgD is 3 days and IgE is 2 days. (Abbas AK, Lichtman AH, Pober JS. Cellular and Molecular Immunology, 4th edition, W.B. Saunders Co., Philadelphia, 2000, which is incorporated herein by reference in its entirety.)

[0049] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be called "VH". The variable domain of the light chain may be called "VL". These domains are generally the most variable parts of the antibody and contain the antigen-binding site. The term "variable" refers to the fact that certain parts of the variable domains have sequences that vary greatly between antibodies and contribute to the binding and specificity of the antibody for a particular antigen. However, this variation is not evenly distributed throughout the entire variable domain of the antibody. It is concentrated in three segments called hypervariable regions (HVRs) (or CDRs) of the variable domains of the light and heavy chains. The parts of the variable domain that are highly conserved are called framework regions (FRs). The native variable domains of the heavy and light chains each contain four FR regions, mainly having a β-sheet structure, connected by three CDRs that form loops and in some cases form part of the β-sheet structure. The CDRs of each chain that are positioned close to each other by the FR regions contribute to the formation of the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains do not directly participate in binding to the antigen but exhibit various effector functions such as involvement in antibody-dependent cell cytotoxicity.

[0050] The "light chain" of a vertebrate antibody (immunoglobulin) has one of two distinct types called κ and λ, based on the amino acid sequence of the constant domain.

[0051] "Framework" or "FR" residues are residues of the variable domain other than the HVR residues as defined herein.

[0052] As used herein, the terms "hypervariable region", "HVR", or "CDR" refer to regions of the variable domain of an antibody that are hypervariable and / or form defined loops. In general, an antibody contains six CDRs, three (H1, H2, H3) in VH and three (L1, L2, L3) in VL. In native antibodies, H3 and L3 among the six CDRs show the greatest diversity, and in particular, H3 is thought to play a unique role in conferring fine specificity to the antibody. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). In fact, natural camel antibodies consisting only of heavy chains are functional and stable even without light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). There are several ways to describe CDRs, and they are included herein. Kabat's complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is incorporated herein by reference in its entirety). The heavy and light chains each have CDR1, 2, and 3. Chothia's CDRs are based on the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM CDRs are a compromise between Kabat's CDRs and Chothia's structural loops and use Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on the analysis of complex crystal structures.CDR may include "extended CDR" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) of VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3) of VH. The residues of the variable domain are numbered by the method of Kabat et al. within the definitions described above.

[0053] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The boundaries of the Fc region of an immunoglobulin heavy chain may vary, but in the case of the Fc region of a human IgG heavy chain, it is usually defined from the amino acid residue of Cys226 or Pro230 to the C-terminus. The C-terminal lysine of the Fc region can be removed, for example, during the production or purification of an antibody, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody as used herein may or may not have a lysine at the C-terminus.

[0054] The subject may be a mammal, preferably a human.

[0055] In some embodiments, the amino acid sequence of human B7-H3 is the amino acid sequence shown below (GenBank: CAE47548.1, SEQ ID NO: 17), with the underlined part being the IgV domain.

[0056]

Chemical formula

[0057] As used herein, "and / or" meaning, for example, alternative A and / or alternative B means (i) alternative A, (ii) alternative B, and (iii) either alternative A or alternative B.

[0058] All combinations of the various elements described herein, including all subsets, are within the scope of the invention unless otherwise indicated herein or clearly inconsistent with the context.

[0059] The present invention will be better understood from the following examples. However, the present invention is as described in the claims, and those skilled in the art will readily understand that the following methods and their results are merely illustrative of the present invention.

Example

[0060] Production of mAbs against the IgV domains of human B7-H3 and mouse B7-H3 The IgV domain is a functional domain of B7-H3. As described in reference (17), a human B7-H3 IgV-Ig fusion protein was produced by fusing the B7-H3 IgV coding region (E35 - A139) to the human IgG1Fc tag of plasmid pMT / BiP. The fusion protein was expressed in the S2 system and purified. Mice were immunized with the B7-H3 IgV-Ig fusion protein, and hybridomas were obtained from spleen cells fused to NSO myeloma cells by standard methods.

[0061] Examination of the characteristics of mAbs 8B12 and 12B4 Monoclonal antibodies (mAbs) 8B12 and 12B4 were produced. 8B12 is an IgG3 having a κ light chain, and 12B4 is an IgG2b having a κ light chain.

[0062] The binding affinities (K d ) for human B7-H3 and mouse B7-H3 determined by surface plasmon resonance were 0.30 nM and 4.01 nM for mAb 8B12, and 8.23 nM and 15.38 nM for mAb 12B4, respectively (Table 1). Both 8B12 and 12B4 strongly bound to the endogenous B7-H3 expressed by human cancer cell lines PC3 and MDA-MB231 (Figure 1).

[0063] Since dendritic cells and activated T cells express B7-H3 (Figure 2), the antagonistic activities of 8B12 and 12B4 were determined in the mixed lymphocyte reaction. Mature dendritic cells differentiated from monocytes isolated from PBMC of a certain donor were incubated for 4 days in the presence of 8B12, 12B4, or control mouse IgG together with purified T cells obtained from PBMC of another donor. 8B12 and 12B4 were able to regulate cytokine production of T cells (Figures 3A and 3B).

[0064]

Table 1

[0065] Sequences of mAbs 8B12, 12B4, and 24D12 The sequence of 8B12 was determined and found to have unique VH and VL sequences. The polypeptide sequence and coding sequence of 8B12 are shown below.

[0066]

Chem.

[0067] The polypeptide sequence and coding sequence of 12B4 are shown below.

[0068]

Chem.

[0069] The polypeptide sequence and coding sequence of 24D12 are shown below.

[0070]

Chem.

[0071]

Chem.

[0072] References 1. Picarda, E., Ohaegbulam, K.C. & Zang, X. Molecular Pathways: Targeting B7-H3 (CD276) for Human Cancer Immunotherapy. Clin Cancer Res 22, 3425-3431 (2016). 2. Zang, X. & Allison, J.P. The B7 family and cancer therapy: costimulation and coinhibition. Clin Cancer Res 13, 5271-5279 (2007). 3. Zang, X., et al. B7-H3 and B7x are highly expressed in human prostate cancer and associated with disease spread and poor outcome. Proc Natl Acad Sci U S A 104, 19458-19463 (2007). 4. Sun, T.W., et al. B7-H3 is expressed in human hepatocellular carcinoma and is associated with tumor aggressiveness and postoperative recurrence. Cancer Immunol Immunother 61, 2171-2182 (2012). 5. Wang, J., et al. B7-H3 associated with tumor progression and epigenetic regulatory activity in cutaneous melanoma. J Invest Dermatol 133, 2050-2058 (2013). 6. Hu, Y., et al. Expression of costimulatory molecule B7-H3 and its prognostic implications in human acute leukemia. Hematology 20, 187-195 (2015). 7. Sun, J., et al. B7-H3 expression in breast cancer and upregulation of VEGF through gene silence. Onco Targets Ther 7, 1979-1986 (2014). 8. Zang, X., et al. Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas. Mod Pathol 23, 1104-1112 (2010). 9. Chen, Y., et al. The coexpression and clinical significance of costimulatory molecules B7-H1, B7-H3, and B7-H4 in human pancreatic cancer. Onco Targets Ther 7, 1465-1472 (2014). 10. Ingebrigtsen, V.A., et al. B7-H3 expression in colorectal cancer: associations with clinicopathological parameters and patient outcome. BMC Cancer 14, 602 (2014). 11. Sun, Y., et al. B7-H3 and B7-H4 expression in non-small-cell lung cancer. Lung Cancer 53, 143-151 (2006). 12. Xylinas, E., et al. Association of T-cell co-regulatory protein expression with clinical outcomes following radical cystectomy for urothelial carcinoma of the bladder. Eur J Surg Oncol 40, 121-127 (2014). 13. Qin, X., et al. B7-H3 is a new cancer-specific endothelial marker in clear cell renal cell carcinoma. Onco Targets Ther 6, 1667-1673 (2013). 14. Baral, A., Ye, H.X., Jiang, P.C., Yao, Y. & Mao, Y. B7-H3 and B7-H1 expression in cerebral spinal fluid and tumor tissue correlates with the malignancy grade of glioma patients. Oncol Lett 8, 1195-1201 (2014). 15. Wang, L., et al. B7-H3 is overexpressed in patients suffering osteosarcoma and associated with tumor aggressiveness and metastasis. PLoS One 8, e70689 (2013). 16. Vigdorovich, V., et al. Structure and T cell inhibition properties of B7 family member, B7-H3. Structure 21, 707-717 (2013). 17. Zhao, R., et al. HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function. Proc Natl Acad Sci U S A 110, 9879-9884 (2013).

Claims

1. An antibody that specifically binds to the IgV domain of the B7-H3 protein, wherein the antibody is an antibody comprising a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 19 and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 21, an antibody comprising a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region of the amino acid sequence shown in SEQ ID NO: 25, or an antibody comprising a heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:

29.

2. The antibody according to claim 1, wherein the antibody is a chimeric antibody or a humanized antibody.

3. The antibody according to claim 2, wherein the antibody is a humanized antibody.

4. The antibody according to claim 1, further comprising an antigen-binding fragment of a monoclonal antibody that binds to the CD3 component of the T cell receptor (TCR) complex of T cells.

5. The antibody according to claim 1, further comprising a detectable marker.

6. The antibody according to claim 1, further comprising a cytotoxic agent bound to the antibody.

7. The antibody according to claim 1 for use in the manufacture of a medicament.

8. A composition comprising an isolated nucleic acid containing a nucleotide sequence encoding the antibody according to claim 1.

9. A composition comprising a host cell or a vector containing an isolated nucleic acid containing a nucleotide sequence encoding the antibody according to claim 1.

10. A pharmaceutical composition for treating a subject suffering from a disease, comprising a therapeutically effective amount of the antibody according to claim 1, wherein the disease is a disease from which the subject benefits by enhancing the immune function of the subject.

11. The pharmaceutical composition according to claim 10, wherein the disease is cancer.

12. wherein the cancer is prostate cancer, liver cancer, melanoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, bladder cancer, brain cancer, osteosarcoma, adrenal cancer, anal canal cancer, basal cell and squamous cell skin cancer, cholangiocarcinoma, bone cancer, spinal cord tumor, cervical cancer, endometrial cancer, esophageal cancer, Ewing family tumor, eye cancer (intraocular melanoma), gallbladder cancer, gastrointestinal neuroendocrine (carcinoid) tumor, gastrointestinal stromal tumor (GIST), gestational trophoblast disease, Kaposi sarcoma, renal cancer, laryngeal and hypopharyngeal cancer, pulmonary carcinoid tumor, malignant mesothelioma, Merkel cell skin cancer, nasal and paranasal cavity cancer, nasopharyngeal cancer, neuroblastoma, central nervous system (CNS) neoplasm, oral and oropharyngeal cancer, pancreatic neuroendocrine tumor (NET), penile cancer, pituitary tumor, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms tumor, squamous cell carcinoma, environmentally induced cancer, cancer combination, cancer metastatic lesion, myeloid tumor, acute myeloid leukemia (AML), AML with recurrent genetic abnormalities, AML with myelodysplastic changes, therapy-related AML, ambiguous lineage acute leukemia, myeloproliferative tumor, essential thrombocythemia, polycythemia vera, myelofibrosis (MF), primary myelofibrosis, systemic mastocytosis, myelodysplastic syndrome (MDS), myeloproliferative / myelodysplastic syndrome, chronic myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, refractory anemia with ring sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts (type 1), refractory anemia with excess blasts (type 2), MDS with isolated deletion (5q), unclassifiable MDS, chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, juvenile myelomonocytic leukemia, unclassifiable myeloproliferative / myelodysplastic syndrome, lymphoid neoplasm, precursor lymphoid neoplasm, B-cell lymphoblastic leukemia, B-cell lymphoblastic lymphoma, T-cell lymphoblastic leukemia, T-cell lymphoblastic lymphoma, mature B-cell neoplasm, diffuse large B-cell lymphoma, primary central nervous system lymphoma, primary mediastinal B-cell lymphoma, Burkitt lymphoma / leukemia, follicular lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, mantle cell lymphoma, marginal zone lymphoma, post-transplant lymphoproliferative disorder, HIV-related lymphoma, primary effusion lymphoma,The pharmaceutical composition according to claim 11, which is selected from the group consisting of intravascular large B-cell lymphoma, primary cutaneous B-cell lymphoma, hairy cell leukemia, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma or solitary plasmacytoma (isolated bone and extramedullary).

13. The pharmaceutical composition according to claim 10, wherein the disease is an infection.

14. The pharmaceutical composition according to claim 13, wherein the infection is caused by a virus, viroid, bacterium, prion, nematode, arthropod, fungus or protozoan.

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