Anti-B7-H3 antibody and its use

Abstract

Anti-B7-H3 (B7 homolog 3) antibodies, their antigen-binding fragments, ADCs, and their uses are provided.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to PCT / CN2023 / 113363, PCT / CN2023 / 113350, PCT / CN2023 / 113348, and PCT / CN2024 / 105244, filed on 16 August 2023. The entirety of the aforementioned applications is incorporated herein by reference.

[0002] Technical field This disclosure relates to anti-B7-H3 (B7 homolog 3) antibodies and methods of using them. [Background technology]

[0003] Cancer is currently one of the leading causes of death in humans. According to World Health Organization statistics, in 2012, there were 14 million new cancer cases and 8.2 million cancer deaths worldwide. In China, there were 3.07 million new cancer cases and 2.2 million deaths.

[0004] Recent clinical and commercial successes of anti-cancer antibodies have generated considerable interest in antibody-based therapies. There is a need for the development of antibodies for use in various antibody-based therapies to treat cancer or autoimmune diseases. [Overview of the project]

[0005] This disclosure relates to anti-B7-H3 antibodies, their antigen-binding fragments, antibody-drug conjugates, and the use thereof.

[0006] In one embodiment, the disclosure relates to a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein in some embodiments, the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR1, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR2, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VH CDR3, and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein in some embodiments, the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR1, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a selected VL CDR2, and the VL CDR3 region comprises a selected VL With respect to the light chain variable region, which includes an amino acid sequence that is at least 80% identical to the amino acid sequence of CDR3, and an antibody or antigen-binding fragment thereof that binds to B7 homolog 3 (B7-H3), in some embodiments, the amino acid sequences of the selected VH CDR1, 2, and 3, and the amino acid sequences of the selected VL CDR1, 2, and 3 are one of the following.(1) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 4-6, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1-3, respectively. (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 7-9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1-3, respectively. (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 10-12, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1-3, respectively. (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 13-15, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs. 1-3, respectively. (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs. 16-18, respectively, and the selected VL The amino acid sequences of CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively, and (6) the amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively.

[0007] In some embodiments, according to the Kabat numbering system, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 4 to 6, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively. In some embodiments, according to the Kabat numbering system, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 7 to 9, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively. In some embodiments, according to the Kabat numbering system, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively. In some embodiments, according to the Chothia numbering system, VH includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 13 to 15, respectively, and VL includes CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively. In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 16-18, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1-3, respectively. In some embodiments, according to the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 19-21, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1-3, respectively. In some embodiments, the antibody or antigen-binding fragment specifically binds to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3. In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or its antigen-binding fragment (e.g., a human IgG1 antibody or its fragment). In some embodiments, the antibody or antigen-binding fragment is a single-strand variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

[0008] In one embodiment, the disclosure relates to nucleic acids comprising polynucleotides that encode a polypeptide including the following: (1) An immunoglobulin heavy chain or fragment thereof comprising heavy chain variable regions (VH) comprising complementarity-determining regions (CDR) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 4 to 6, respectively, wherein in some embodiments, the VH binds to B7-H3 when paired with a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO. 22. (2) An immunoglobulin light chain or fragment thereof comprising a light chain variable region (VL) comprising complementarity-determining regions (CDRs) 1, 2, and 3, respectively, which comprise the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein in some embodiments, the VL binds to B7-H3 when paired with a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 23. (3) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 7 to 9, respectively, wherein in some embodiments, the VH binds to B7-H3 when pairing with VL comprising the amino acid sequence shown in SEQ ID NO. 22. (4) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR 1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein in some embodiments, the VL binds to B7-H3 when pairing with a VH comprising the amino acid sequence shown in SEQ ID NO: 24. (5) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 10 to 12, respectively, wherein in some embodiments, the VH binds to B7-H3 when pairing with VL comprising the amino acid sequence shown in SEQ ID NO. 23. (6) An immunoglobulin light chain or fragment thereof comprising a VL comprising CDR 1, 2, and 3, each comprising the amino acid sequences shown in SEQ ID NOs: 1 to 3, wherein in some embodiments, the VL binds to B7-H3 when pairing with a VH comprising the amino acid sequence shown in SEQ ID NO: 25. (7) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 13 to 15, respectively, wherein in some embodiments, the VH binds to B7-H3 when pairing with VL comprising the amino acid sequence shown in SEQ ID NO. 22. (8) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 16 to 18, respectively, wherein in some embodiments, the VH binds to B7-H3 when pairing with VL comprising the amino acid sequence shown in SEQ ID NO. 22, or (9) An immunoglobulin heavy chain or fragment thereof comprising VH comprising CDR 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NOs. 19 to 21, respectively, wherein in some embodiments, the VH binds to B7-H3 when pairing with VL comprising the amino acid sequence shown in SEQ ID NO. 22.

[0009] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin heavy chain or fragment thereof containing VH, and the VH comprising CDRs 1, 2, and 3 comprising amino acid sequences shown in SEQ ID NOs. 4, 5, and 6, or SEQ ID NOs. 13, 14, and 15, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin heavy chain or fragment thereof containing VH, and the VH comprising CDRs 1, 2, and 3 comprising amino acid sequences shown in SEQ ID NOs. 7, 8, and 9, or SEQ ID NOs. 16, 17, and 18, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin heavy chain or fragment thereof containing VH, and the VH comprising CDRs 1, 2, and 3 comprising amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, or SEQ ID NOs. 19, 20, and 21, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide, the polypeptide comprising an immunoglobulin light chain or fragment thereof containing a VL, the VL comprising CDRs 1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3. In some embodiments, the VH specifically binds to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3 when paired with the VL, or the VL specifically binds to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3 when paired with the VH. In some embodiments, the immunoglobulin heavy chain or fragment thereof is a human or humanized immunoglobulin heavy chain or fragment thereof (e.g., a human IgG1 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human or humanized immunoglobulin light chain or fragment thereof (e.g., a human IgG1 light chain or fragment thereof). In some embodiments, the nucleic acid encodes a single-stranded variable fragment (scFv), a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid is cDNA.

[0010] In one embodiment, the disclosure relates to a vector comprising one or more nucleic acids described herein. In one embodiment, the disclosure relates to a vector comprising two nucleic acids described herein, wherein in some embodiments, the vectors together encode a VH region and a VL region that bind to B7-H3. In one embodiment, the disclosure relates to a pair of vectors, wherein in some embodiments, each vector comprises one of the nucleic acids described herein, and in some embodiments, the pair of vectors together encode a VH region and a VL region that bind to B7-H3.

[0011] In one embodiment, the disclosure relates to a cell comprising a vector or a pair of vectors described herein. In some embodiments, the cell is a CHO cell. In one embodiment, the disclosure relates to a cell comprising one or more nucleic acids described herein. In one embodiment, the disclosure relates to a cell comprising two nucleic acids described herein. In some embodiments, the two nucleic acids both encode a VH region and a VL region that bind together to B7-H3.

[0012] In one embodiment, the present disclosure relates to a method for producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing cells described herein under conditions sufficient to enable the cells to produce the antibody or the antigen-binding fragment, and (b) recovering the antibody or the antigen-binding fragment produced by the cells.

[0013] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to B7-H3, comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80% identical to a selected VL sequence, wherein in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 23 and the selected VL sequence is SEQ ID NO: 22, (2) the selected VH sequence is SEQ ID NO: 24 and the selected VL sequence is SEQ ID NO: 22, and (3) the selected VH sequence is SEQ ID NO: 25 and the selected VL sequence is SEQ ID NO: 22. In some embodiments, the VH sequence comprises the sequence of SEQ ID NO: 23 and the VL sequence comprises the sequence of SEQ ID NO: 22. In some embodiments, the VH sequence comprises the sequence of SEQ ID NO: 24 and the VL sequence comprises the sequence of SEQ ID NO: 22. In some embodiments, VH includes the sequence of SEQ ID NO: 25, and VL includes the sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigen-binding fragment specifically binds to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3. In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or its antigen-binding fragment (e.g., a human IgG1 antibody or its fragment). In some embodiments, the antibody or antigen-binding fragment is a single-strand variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

[0014] In one embodiment, this disclosure relates to an antibody or an antigen-binding fragment that cross-competes with an antibody or an antigen-binding fragment described herein.

[0015] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that binds to B7-H3, comprising a heavy chain variable region (VH) containing the same VH CDR1, VH CDR2, and VH CDR3 as those of a selected VH sequence, and a light chain variable region (VL) containing the same VL CDR1, VL CDR2, and VL CDR3 as those of a selected VL sequence, wherein in some embodiments, the selected VH sequence and the selected VL sequence are one of the following: (1) the selected VH sequence is SEQ ID NO: 23 and the selected VL sequence is SEQ ID NO: 22, (2) the selected VH sequence is SEQ ID NO: 24 and the selected VL sequence is SEQ ID NO: 22, and (3) the selected VH sequence is SEQ ID NO: 25 and the selected VL sequence is SEQ ID NO: 22.

[0016] In one embodiment, this disclosure relates to an antibody-drug conjugate comprising an antibody described herein or an antigen-binding fragment thereof covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor. In some embodiments, the therapeutic agent is an MMAE or MMAF.

[0017] In some embodiments, the therapeutic agent is selected from the following: [ka]

[0018] In some embodiments, the therapeutic agent is linked to an antibody or its antigen-binding fragment via a linker. In some embodiments, the linker has the following structure: [ka]

[0019] In some embodiments, the antibody-drug conjugate has the following structure: [ka] In some embodiments, n=1 to 8, and in some embodiments, "Ab" represents an antibody or its antigen-binding fragment.

[0020] In some embodiments, the drug-antibody ratio (DAR) is approximately 4 or 8.

[0021] In one embodiment, the present disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein. In some embodiments, the subject has liver cancer, pancreatic cancer, prostate cancer, osteosarcoma, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bladder cancer, kidney cancer, brain tumor, head and neck cancer, or melanoma. In some embodiments, the subject has cancer cells expressing B7-H3. In some embodiments, the methods described herein further include administering a therapeutically effective dose of anti-OX40 antibody, anti-PD-1 antibody, anti-PDL1 antibody, anti-PDL2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, and / or anti-CD40 antibody to a target.

[0022] In one embodiment, the present disclosure relates to a method for reducing the rate of tumor growth, the method comprising contacting tumor cells with a composition comprising an effective amount of an antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, as described herein.

[0023] In one embodiment, the present disclosure relates to a method for killing tumor cells, the method comprising contacting the tumor cells with a composition comprising an effective amount of an antibody or an antigen-binding fragment thereof, or an antibody-drug conjugate, as described herein.

[0024] In one embodiment, this disclosure relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, a pharmaceutically acceptable carrier, as described herein. In one embodiment, this disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate, a pharmaceutically acceptable carrier, as described herein.

[0025] In one embodiment, this disclosure relates to a chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof as described herein.

[0026] As used herein, the term “cancer” means cells capable of autonomous proliferation. Examples of such cells include cells in an abnormal state or condition characterized by rapid proliferation. The term means cancerous growth, e.g., tumors, oncogenic processes, metastatic tissues, and malignant transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. Malignancies of various organ systems, e.g., head and neck, respiratory, cardiovascular, renal, reproductive, hematological, nervous, liver, gastrointestinal, and endocrine systems, as well as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, gliomas, and small intestine cancers. “Spontaneously occurring” cancers include any cancer that is not experimentally induced by transplanting cancer cells into a subject, e.g., spontaneously occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers resulting from transgenic oncogene insertions or tumor suppressor gene knockouts, and cancers caused by infections, e.g., viral infections. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine glandular tissue. This term also includes carcinosarcoma, which is a malignant tumor composed of cancerous and sarcomatoid tissue. "Adenocarcinoma" refers to a carcinoma of glandular origin, or a carcinoma in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases involving hematopoietic hyperplasia / neoplastic cells. Hematopoietic neoplastic diseases may originate from the bone marrow, lymphoid system, or erythrocyte lineage, or their progenitor cells. Hematological cancers are cancers that begin in blood-forming tissues such as bone marrow, or in cells of the immune system. Examples of hematological cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0027] As used herein, the term “antibody” means any antigen-binding molecule that contains at least one (e.g., 1, 2, 3, 4, 5, or 6) complementarity-determining regions (CDRs) (e.g., any of three CDRs derived from immunoglobulin light chains or any of three CDRs derived from immunoglobulin heavy chains) and is capable of specifically binding 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, antibodies may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0028] As used herein, the term “antigen-binding fragment” means a portion of a full-length antibody, the portion of which is specifically capable of binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain in the heavy chain or a variable domain in the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0029] As used herein, the term “human antibody” means an antibody encoded by endogenous nucleic acids of human origin (e.g., rearranged human immunoglobulin heavy or light chain loci). In some embodiments, human antibodies are recovered from humans or produced in human cell culture media (e.g., in human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacterial or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., cattle) containing unarranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0030] As used herein, the term “chimeric antibody” means an antibody containing sequences present in at least two different species (e.g., an antibody derived from two different mammalian species, such as a human and a mouse antibody). Non-limiting examples of chimeric antibodies include antibodies containing a variable domain sequence (e.g., all or part of the light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody, as well as a constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0031] As used herein, the term “humanized antibody” means a non-human antibody that contains minimal sequences derived from non-human (e.g., mouse) immunoglobulin and sequences derived from human immunoglobulin. In non-limiting examples, a humanized antibody is a human antibody (recipient antibody) in which residues in the hypervariable (e.g., CDR) region of the recipient antibody are replaced by residues in the hypervariable (e.g., CDR) region of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody having the desired specificity, affinity, and capability. In some embodiments, Fv framework residues of human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications can further refine the performance of the antibody. In some embodiments, the humanized antibody contains substantially all, at least one, and typically two, variable domains, with all or substantially all of the hypervariable loop (CDR) corresponding to the hypervariable loop of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework region being a human immunoglobulin sequence. The humanized antibody may also contain an immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. The humanized antibody can be produced using molecular biological methods well known in the art. Non-limiting examples of methods for producing the humanized antibody are described herein.

[0032] As used herein, the term “single-chain antibody” means a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of mammalian immunoglobulin heavy or light chains) that are specifically capable of binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0033] Where used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and refer to an animal, human, or non-human being to be treated according to the methods of the present invention. Veterinary and non-veterinary uses are conceived by this disclosure. 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. Examples include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, pigs (e.g., pigs, miniature pigs), equids, canids, felines, bovines, and other domestic, livestock, and zoo animals.

[0034] As used herein, when referring to an antibody, the terms "specifically bind" and "specifically bind" mean that the interaction depends on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the target molecule (e.g., B7-H3). In other words, the antibody interacts with its target molecule, preferably more than other molecules, in order for the reagent to recognize and bind to a molecule containing a specific structure, rather than the entire molecule. An antibody that specifically binds to a target molecule may also be called a target-specific antibody. For example, an antibody that specifically binds to the B7-H3 molecule may be called a B7-H3-specific antibody or an anti-B7-H3 antibody.

[0035] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and mean polymers of at least two amino acids of any length.

[0036] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and mean, but are not limited to, polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Methods and materials for use in the present invention are described herein, but other suitable methods and materials well known in the art may also be used. Materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0038] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0039] [Figure 1] List the Kabat CDR sequences of anti-B7-H3 antibodies. [Figure 2] The Chothia CDR sequences of anti-B7-H3 antibodies are listed below. [Figure 3] The sequences of the heavy chain variable region and light chain variable region of the anti-B7-H3 antibody, as well as the amino acid sequences discussed in this disclosure, are listed below. [Figure 4-1] The amino acid sequences discussed in this disclosure are listed below. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 5] This paper shows the mean tumor volume in different groups of B-NDG mice transplanted with patient-derived lung tumor tissue and treated with PBS (G1), ISO-CPT2 (G2), 21A9-CPT2 (G3), and 20H8-CPT2 (G4), respectively. PBS was used as a negative control. ISO-CPT2 was used as an isotype control. [Figure 6] This shows the mean tumor volume in different groups of B-NDG mice that were injected with tumor fragments derived from colorectal cancer patients and treated with PBS or ADC. [Figure 7] This shows the mean tumor volume in various groups of B-NDG mice injected with tumor fragments derived from breast cancer patients and treated with PBS or ADC. [Modes for carrying out the invention]

[0040] The B7 family consists of structurally similar cell surface protein ligands that bind to receptors on lymphocytes that regulate the immune response. While the activation of T and B lymphocytes is initiated by the binding of antigen-specific T cell receptors or B cell receptors on the cell surface, additional signals transmitted simultaneously by B7 ligands determine the final immune response. These "costimulatory" or "co-inhibitory" signals are transmitted by B7 ligands via CD28 family receptors on lymphocytes. Interactions between B7 family molecules and costimulatory receptors enhance the immune response, while interactions with co-inhibitory receptors attenuate it. Molecules belonging to the B7 family include, for example, B7.1 (CD80), B7.2 (CD86), the inducible costimulatory ligand (ICOS-L), programmed DES-1 ligand (PD-L1), programmed DES-2 ligand (PD-L2), B7-H3 (CD276), and B7-H4. These are all transmembrane or glycosylphosphatidylinositol (GPI)-bound proteins, characterized by extracellular IgV and IgC domains associated with the variable and constant regions of immunoglobulins. The IgV and IgC domains of B7 family molecules are each encoded by a single exon, with additional exons encoding the leader sequence, transmembrane region, and cytoplasmic domain, respectively. B7-H3 (CD276) is unique in that its major human form contains two extracellular tandem IgV-IgC domains.

[0041] Members of the B7 family are expressed in antigen-presenting cells such as dendritic cells, macrophages, and B cells. Unlike B7.1, which is expressed only in lymphocytes, CD276 is also expressed in various normal tissues, but at low levels. CD276 expression is regulated by various cytokines. For example, IFN-γ upregulates CD276 expression, while IL4 downregulates its expression. When members of the B7 family bind to different receptors, either co-stimulatory or co-inhibitory effects can occur. For example, the binding of B7 and CD28 promotes co-stimulation, while the binding of B7 and CTLA4 promotes co-inhibition. Early studies found that CD276 promotes positive regulation of T cells, but subsequent studies have shown that CD276 exerts co-inhibitory effects in T cell immunity. Thus, CD276 has a dual immune action: both co-stimulatory and co-inhibitory.

[0042] In normal tissues (non-tumor tissues), B7-H3 (CD276) primarily plays an inhibitory role in adaptive immunity, suppressing T cell activation and proliferation. In malignant tissues, B7-H3 is an immune checkpoint molecule that inhibits specific immune responses to tumor antigens. B7-H3 also has tumor-promoting functions distinct from immunological ones, such as promoting migration, invasion, angiogenesis, chemotherapy resistance, epithelial-mesenchymal transition, and effects on tumor cell metabolism. Therefore, anti-B7-H3 (CD276) antibodies may have potential utility as a cancer treatment.

[0043] This disclosure provides examples of antibodies, antigen-binding fragments thereof, and ADCs that bind to B7-H3.

[0044] B7-H3 B7 homolog 3 (also known as B7-H3, CD276, differentiation cluster 276, B7RP-2, or 4Ig-B7-H3) is a type I transmembrane protein encoded by mouse chromosome 9 and human chromosome 15. Its extracellular domain in mice (2Ig-B7-H3 isoform) consists of a pair of immunoglobulin variable (IgV) and immunoglobulin constant (IgC) domains. In humans (4Ig-B7-H3 isoform), however, two identical pairs exist as a result of exon duplication. The intracellular tail of B7-H3 is short, and no known signaling motifs have been identified. B7-H3 was first discovered in humans and later identified in mice, but it is widely expressed across species. A soluble form, produced by cleavage by matrix metalloproteinases (MMPs) on the surface of activated T cells, monocytes, or dendritic cells, or by alternative splicing of introns, is also detectable in human serum. Soluble CD276 activates the NF-κB signaling pathway, promoting the invasion and metastasis of pancreatic cancer cells. Furthermore, levels of soluble CD276 in the pleural fluid of non-small cell lung cancer patients are significantly higher than those of healthy individuals. Therefore, CD276 may be useful as a diagnostic and prognostic marker for related tumors.

[0045] B7-H3 is expressed in many tissues and cell types. At the mRNA level, it is widely found in non-lymphoid organs such as the liver, heart, and prostate, as well as lymphoid organs such as the spleen and thymus. Despite widespread mRNA expression, protein expression is restricted in the steady state, suggesting the existence of an important post-transcriptional regulatory mechanism. B7-H3 is constitutively present in non-immune quiescent fibroblasts, endothelial cells (ECs), osteoblasts, and amniotic fluid stem cells. Furthermore, B7-H3 expression is induced on immune cells, particularly antigen-presenting cells. In particular, co-culture with regulatory T cells (Tregs), and in vitro stimulation with IFN-γ, lipopolysaccharide (LPS), or anti-CD40 all induce B7-H3 expression on dendritic cells (DCs). Monocytes upregulate B7-H3 after LPS stimulation, and monocyte-derived dendritic cells upregulate it after cytokine-induced differentiation. Furthermore, B7-H3 is also detected in natural killer (NK) cells, B cells, and some T cell subsets after PMA / ionomycin stimulation.

[0046] The B7-H3 pathway plays a dual role in regulating the innate immune response. One study revealed that B7-H3 expressed on the cell surface of neuroblastoma cells protects them from NK cell-mediated lysis. Another group argues that B7-H3 co-stimulates innate immunity by enhancing the release of pro-inflammatory cytokines from LPS-stimulated monocytes / macrophages in a manner dependent on both Toll-like receptor (TLR) 4 and TLR2.

[0047] B7-H3 plays a crucial role in T cell-mediated adaptive immunity, but the nature of its signaling remains controversial. The co-stimulatory role of B7-H3 to human T cells was first reported in vitro. Mouse studies showing that B7-H3 exacerbates experimental autoimmune encephalomyelitis (EAE), arthritis, bacterial meningitis, and chronic graft rejection supported this claim. However, subsequent studies have largely shown that B7-H3 functions as a co-inhibitor of T cells. B7-H3 inhibits the activation, proliferation, and effector cytokine production (IFN-γ and IL-2) of polyclonal or alloreactive CD4+ and CD8+ T cells in mouse and human. This negative regulation of T cells is associated with decreased activity of NFAT, NF-κB, and AP-1 transcription factors. Independent studies using protein inhibition or gene knockout mice have reported that B7-H3 improves graft-versus-host disease, extends cardiac graft survival, reduces airway hyperresponsiveness, and delays the onset of EAE, particularly by downregulating the Th1 response. These findings further confirm the co-inhibitory properties of B7-H3.

[0048] The receptor(s) for B7-H3 (possibly multiple) have not yet been identified. However, the crystalline structure of mouse B7-H3 reveals that its receptor binding on T cells involves a specific segment (FG loop) that connects the F and G chains of the immunoglobulin variable domain of B7-H3. Furthermore, B7-H3 crystallizes as a glycosylated monomer but also undergoes abnormal dimerization in vitro. Taken together, the nature of the receptor, differences in the cellular environment, and various disease models certainly explain the discrepancies in the function of the B7-H3 pathway, which modulates both innate and adaptive immunity during homeostasis and inflammation.

[0049] Beyond the immune system, the B7-H3 pathway has a non-immunological role in promoting osteoblast differentiation and osteomineralization in mice, ensuring normal bone formation. In fact, B7-H3 knockout mice had lower bone mineral density and were more susceptible to fractures compared to wild-type mice. Furthermore, like other immune checkpoints in the B7-CD28 pathway, B7-H3 is also expressed in human cancers and participates in tumorigenesis through the regulation of both immune-related and non-immune-related pathways.

[0050] For detailed descriptions of CD276 and its function, see, for example, Picarda, E. et al., “Molecular pathways: targeting B7-H3 (CD276) for human cancer immunotherapy.” Clinical Cancer Research 22.14(2016):3425-3431, Collins, M. et al., “The B7 family of immune-regulatory ligands.” Genome Biology 6.6(2005):1-7, Castellanos, JR et al., “B7-H3 role in the immune landscape of cancer.” American Journal of Clinical and Experimental Immunology 6.4(2017):66, and Yang, S. et al., “B7-H3, a checkpoint molecule, as a target for cancer immunotherapy.” International Journal of Biological Sciences 16.11(2020):1767, the entirety of each of these works is incorporated herein by reference.

[0051] The present invention provides anti-B7-H3 antibodies, their antigen-binding fragments, ADCs, and methods for inhibiting tumor growth and treating various diseases, including cancer, using these anti-B7-H3 antibodies, their antigen-binding fragments, or ADCs.

[0052] Anti-B7-H3 antibody The present invention provides antibodies that specifically bind to B7-H3 (e.g., human B7-H3) and antigen-binding fragments thereof. In some embodiments, the antibodies and antigen-binding fragments described herein bind to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3. In some embodiments, these antibodies bind to cells expressing B7-H3 (B7-H3-positive cells). In some embodiments, these antibodies do not bind to cells that do not express B7-H3 (B7-H3-negative cells). In some embodiments, these antibodies can be taken up into target cells (e.g., cancer cells expressing B7-H3) via endocytosis. In some embodiments, these antibodies can bind to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3 with high affinity. In some embodiments, these antibodies can initiate complement-dependent cell-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0053] This disclosure provides, for example, anti-B7-H3 antibodies 19A1, 20H8, and 21A9, including modified antibodies therefrom, such as chimeric antibodies, humanized antibodies, and human antibodies.

[0054] CDR sequences for 19A1 and antibodies derived from 19A1 (e.g., human antibodies) include the CDRs of the heavy chain variable domain, SEQ ID NOs: 4-6, and the CDRs of the light chain variable domain, SEQ ID NOs: 1-3, as defined by Kabat. CDRs can also be defined by the Chothia system. Under the Chothia definition, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NOs: 13-15, and the CDR sequences of the light chain variable domain are shown in SEQ ID NOs: 1-3.

[0055] CDR sequences for 20H8 and antibodies derived from 20H8 (e.g., human antibodies) include the heavy chain variable domain CDRs, SEQ ID NOs. 7-9, and the light chain variable domain CDRs, SEQ ID NOs. 1-3, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in SEQ ID NOs. 16-18, and the light chain variable domain CDR sequences are shown in SEQ ID NOs. 1-3.

[0056] CDR sequences of 21A9 and antibodies derived from 21A9 (e.g., human antibodies) include the heavy chain variable domain CDRs, sequence numbers 10-12, and the light chain variable domain CDRs, sequence numbers 1-3, as defined by Kabat. Under Chothia's definition, the heavy chain variable domain CDR sequences are shown in sequence numbers 19-21, and the light chain variable domain CDR sequences are shown in sequence numbers 1-3.

[0057] The amino acid sequence of the heavy chain variable region of the 19A1 antibody is shown in SEQ ID NO: 23. The amino acid sequence of the light chain variable region of the 19A1 antibody is shown in SEQ ID NO: 22.

[0058] The amino acid sequence of the heavy chain variable region of the 20H8 antibody is shown in SEQ ID NO: 24. The amino acid sequence of the light chain variable region of the 20H8 antibody is shown in SEQ ID NO: 22.

[0059] The amino acid sequence of the heavy chain variable region of the 21A9 antibody is shown in SEQ ID NO: 25. The amino acid sequence of the light chain variable region of the 21A9 antibody is shown in SEQ ID NO: 22.

[0060] The amino acid sequences for the heavy chain variable region and light chain variable region of the modified antibody are also provided. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, 24, or 25. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 22. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, which together bind to B7-H3.

[0061] In some embodiments, the variable region is entirely human, for example, derived from human heavy chain immunoglobulin loci (e.g., a combination of human IGHV, IGHD, and IGHJ genes) and / or human kappa chain immunoglobulin loci (e.g., a combination of human IGKV and IGKJ genes).

[0062] Furthermore, in some embodiments, the antibodies or antigen-binding fragments described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 4-6, SEQ ID NOs: 7-9, SEQ ID NOs: 10-12, SEQ ID NOs: 13-15, SEQ ID NOs: 16-18, and SEQ ID NOs: 19-21, and / or one, two, or three light chain variable region CDRs selected from the group consisting of SEQ ID NOs: 1-3.

[0063] In some embodiments, the antibody may have a heavy chain variable region (VH) containing complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region contains or comprises 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 contains or comprises 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 contains or comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR1; the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR2; and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of the selected VL CDR3. The amino acid sequences of the selected VH CDR1, 2, and 3 and the selected VL CDR1, 2, and 3 are shown in Figure 1 (CDR under Kabat's definition) and Figure 2 (CDR under Chothia's definition).

[0064] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, which have 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0065] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9

[0066] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three heavy chain variable domains of the CDRs of SEQ ID NO: 10 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 11 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 12 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0067] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15

[0068] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18

[0069] In some embodiments, the antibody or antigen-binding fragments described herein may contain heavy chain variable domains containing one, two, or three CDRs of SEQ ID NO: 19 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, SEQ ID NO: 20 having 0, 1, or 2 amino acid insertions, deletions, or substitutions, or SEQ ID NO: 21 having 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0070] In some embodiments, the antibody or antigen-binding fragments described herein may contain one, two, or three light chain variable domains of the CDRs of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3

[0071] Insertions, deletions, and substitutions may be present within the CDR sequence or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on a Kabat definition scheme. In some embodiments, the CDR is determined based on a Chothia definition scheme. In some embodiments, the CDR is determined based on a combination of the Kabat and Chothia definition schemes. In some embodiments, the CDR is determined based on the IMGT definition. In some embodiments, the CDR is determined based on the contact definition.

[0072] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to B7-H3. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) 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 light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 23, and the selected VL sequence is SEQ ID NO: 22. In some embodiments, the selected VH sequence is SEQ ID NO: 24, and the selected VL sequence is SEQ ID NO: 22. In some embodiments, the selected VH sequence is SEQ ID NO: 25, and the selected VL sequence is SEQ ID NO: 22.

[0073] To measure the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences to ensure optimal alignment for comparison, and non-homologous sequences may be ignored). Subsequently, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of 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 that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparison and measurement of the percentage of identity between two sequences can be performed using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0074] This disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides containing an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain contains a CDR as shown in Figure 1 or Figure 2, or has a sequence as shown in Figure 3. When a polypeptide forms a pair with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to B7-H3 (e.g., human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3).

[0075] In some embodiments, the antibody or its antigen-binding fragment may have three VH CDRs identical to those of any VH sequence CDR described herein. In some embodiments, the antibody or its antigen-binding fragment may have three VL CDRs identical to those of any VL sequence CDR described herein.

[0076] Anti-B7-H3 antibodies and antigen-binding fragments may also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein include polyclonal, monoclonal, multimer, multispecific (e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments may 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 its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0077] Antibody fragments are suitable for use in the provided manner, insofar as they retain the desired affinity and specificity of the full-length antibody. Therefore, an antibody fragment that binds to B7-H3 retains its ability to bind to B7-H3. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are closely associated, which can essentially be covalent, for example, in scFv. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Together, the six CDRs, or subsets thereof, confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind to an antigen, though usually with lower affinity than the entire binding site. A single-stranded Fv or (scFv) antibody fragment contains the VH and VL domains (or regions) of the antibody, and these domains are located within a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form a desirable structure for antigen binding.

[0078] This disclosure also provides an antibody or an antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., “Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein.” Journal of Virology 70.3(1996):1863-1872, which is incorporated herein by reference in its entirety. In one embodiment, this disclosure also provides an antibody or an antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment described herein. Epitope-binding assays are well known in the art and are described, for example, in Estep et al., “High throughput solution-based measurement of antibody-antigen affinity and epitope binning.” MAbs. Vol.5. No.2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0079] Antibody and antigen-binding fragments This disclosure provides various antibodies and their antigen-binding fragments derived from the anti-B7-H3 antibody described herein. Generally, an antibody (also called an immunoglobulin) consists of two classes of polypeptide chains: a light chain and a heavy chain. An unspecified example of an antibody in this disclosure may be an intact four-immunoglobulin chain antibody containing two heavy chains and two light chains. The heavy chain of the antibody may be any isotype including IgM, IgG, IgE, IgA, or IgD, or a subisotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain may be a κ light chain or a λ light chain. The antibody may contain two identical copies of the light chain and two identical copies of the heavy chain, each containing one variable domain (or variable region, V HThe heavy chain, which contains a variable domain (or variable region), binds to each other via disulfide bonds within its constant domains, forming the "stem" of the antibody. Each of these contains one variable domain (or variable region, V L Each light chain containing a constant domain (or constant region) is bonded 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 bonded. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0080] The hypervariable regions, known as complementarity-determining regions (CDRs), form loops containing the antigen-binding surface of the antibody. The four framework regions are largely adapted to the β-sheet structure, and the CDRs form loops connecting the β-sheet structure, and in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity to the framework regions and, together with the CDRs of other chains, contribute to the formation of the antigen-binding region.

[0081] Methods for identifying the CDR region of an antibody by analyzing its amino acid sequence are well-known, and several definitions of CDRs are commonly used. Kabat's definition is based on sequence variability, while Chothia's definition is based on the location of the structural loop region. These methods and definitions are, for example, found in 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, T. 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 This is described in et al., Nature 342(6252):877-83 (Dec. 1989), and Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), the entirety of each of these works is incorporated herein by reference.

[0082] CDRs are important for recognizing the epitopes of antigens. As used herein, an “epitope” is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, as epitopes can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structures of the antigen.

[0083] 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 regions, particularly the hinge and upper CH2 domain. The sequences and differences of IgG subclasses are well known in the art, for example, in Vidarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2 (2015):171-182; and Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference.

[0084] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies containing an immunoglobulin-binding domain fused to another polypeptide. The terms “antigen-binding domain” or “antigen-binding fragment” refer to any portion of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of an antibody that is specifically capable of binding to an epitope on the target molecule of an intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or its antigen-binding fragment may be any polypeptide containing, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and a binding domain that is an antibody-binding domain, or a binding domain homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0085] Antibody fragments suitable for use in the methods described herein are also provided. Fab fragments contain variable and constant domains of the light chain, as well as a variable domain and a first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments generally contain a pair of Fab fragments, commonly linked near the carboxyl terminus by a hinge cysteine ​​between them. Other chemical linkages of antibody fragments are well known in the art.

[0086] A diabody is a small antibody fragment containing two antigen-binding sites, and this fragment contains VH (VH and VL) attached to VL within the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same chain, the domains can be paired with complementary domains on another chain, thereby generating two antigen-binding sites.

[0087] Linear antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0088] The antibodies and antibody fragments of this disclosure can be modified within the Fc region to provide a desired effector function or serum half-life. In some embodiments, the antibody or its antigen-binding fragment may include one, two, or three heavy chain variable region CDRs selected from Figures 1-2. In some embodiments, the antibody or its antigen-binding fragment may include one, two, or three light chain variable region CDRs selected from Figures 1-2.

[0089] Antibody multimerization can be achieved by the natural aggregation of antibodies or by chemical or recombinant conjugation techniques known in the art. For example, a certain proportion of purified antibody preparations (e.g., one purified IgG molecule) naturally form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0090] Alternatively, antibody homodimers can be formed by chemical bonding techniques well known in the art. For example, antibody polymers can be formed using heterobifunctional crosslinking agents, including but not limited to SMCC (4-(maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl) and SATA (S-acetylthioacetate N-succinimidyl). Exemplary procedures for forming antibody homodimers are described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by pepsin digestion. Another method for forming antibody homodimers is by using the autoaffinity T15 peptide, as described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0091] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be produced by recombining the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the larger amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers. This method is described, for example, in WO96 / 27011, which is incorporated in its entirety by reference.

[0092] Antibodies or their antigen-binding fragments can also take various forms. Many different forms of antigen-binding constructs are known in the art, for example, as described in Suurs, et al. "A review of bispecific antibodies and antibody constructs in oncology and clinical challenges," Pharmacology & Therapeutics (2019), which is incorporated in its entirety by reference.

[0093] In some embodiments, the antibody or its antigen-binding fragment is BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. In some embodiments, the antibody or its antigen-binding fragment is VHH-scAb, VHH-Fab, Dual scFab, F(ab')2, Diabody, crossMab, DAF(2in1), DAF(4in1), DutaMab, DT-IgG, Knob-in-hole common light chain, Knob-in-hole assembly, Charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, Orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH These include IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Mini-antibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, or scFv1-PEG-scFv2.

[0094] In some embodiments, the antibody or its antigen-binding fragment may be a TrioMab. In a TrioMab, the two heavy chains originate from different species, where different sequences restrict the heavy-light chain pairing.

[0095] In some embodiments, the antibody or its antigen-binding fragment has two distinct heavy chains and one common light chain. Heterodimerization of the heavy chains can be performed based on the knob-into-hole method or other heavy chain pairing techniques.

[0096] In several embodiments, the CrossMAb technology can be used to generate bispecific antibodies. The CrossMAb technology can be used to perform the correct light chain association in bispecific heterodimer IgG antibodies, enabling the generation of various bispecific antibody formats, including bi(1+1), tri(2+1), and tetra(2+2) valent bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab) based antibodies. These formats can be derived from any existing antibody pair using domain crossover without requiring the identification of a common light chain, post-translational processing / ex vitro chemical assembly, or the introduction of a series of mutations to force the correct light chain association. This method is incorporated in its entirety by reference from Klein et al., “The use of CrossMAb technology for the generation of bi- and multispecific antibodies.” MAbs. Vol. 8. No. 6. Taylor & Francis, 2016. In several embodiments, the CH1 domain in the heavy chain and the CL domain in the light chain are exchanged.

[0097] The antibody or its antigen-binding fragment can be a duobody. The Fab exchange mechanism naturally present in IgG4 antibodies is mimicked in a controlled manner in IgG1 antibodies, a mechanism called controlled Fab exchange. This format can ensure specific pairing between the heavy and light chains.

[0098] In the bivariable domain antibody (DVD-Ig), additional VH and variable light chain (VL) domains are added to each N-terminus for bispecific targeting. This format is similar to IgG-scFv, but the added binding domains bind individually to the corresponding N-terminus of each heavy chain, instead of the scFv.

[0099] In scFv-IgG, two scFv molecules are linked to the C-terminus of the heavy chain (CH3). The scFv-IgG format has two distinct divalent binding sites and is therefore also called tetravalent. There is no problem with heavy-light chain pairing in scFv-IgG.

[0100] In some embodiments, the antibody or its antigen-binding fragment may have an IgG-IgG format. Two intact IgG antibodies are conjugated by chemically linking the C-terminuses of their heavy chains.

[0101] Antibodies or their antigen-binding fragments may also have a Fab-scFv-Fc format. In the Fab-scFv-Fc format, a light chain, a heavy chain, and a third chain containing the Fc region and scFv are assembled. This ensures efficient production and purification.

[0102] In some embodiments, the antibody or its antigen-binding fragment may be a TF. Three Fab fragments are linked by disulfide crosslinks. Two fragments target tumor-associated antigens (TAAs), and one fragment targets a hapten. The TF format does not have an Fc region.

[0103] ADAPTIR has two scFvs bound to both sides of a certain Fc region. It discards the intact IgG that serves as the basis for its construct, but preserves the Fc region, extending its half-life and facilitating purification.

[0104] Bispecific T cell-inducing antibodies ("BiTE") consist of two scFv molecules, VLA VHA and VHB VLB, on a single peptide chain. They possess only a binding domain and lack an Fc region.

[0105] In BiTE-Fc, the Fc region is fused to the BiTE construct. The addition of the Fc region increases the half-life, thereby extending the effective concentration and avoiding continuous intravenous administration (IV).

[0106] Biaffinity retargeting (DART) involves two peptide chains linking opposite fragments (i.e., VLA to VHB and VLB to VHA), and a sulfur bond fusing them together at the C-terminus. In DART, the sulfur bond can improve stability compared to BiTE.

[0107] In DART-Fc, the Fc region is bound to DART. This can be generated by assembling three chains (two via disulfide bonds, similar to DART). One chain contains half of the Fc region, which dimerizes with the third chain, expressing only the Fc region. The addition of the Fc region extends the half-life, resulting in a longer effective concentration and avoiding consecutive IV injections.

[0108] In tetravalent DART, four peptide chains are assembled. Essentially, two DART molecules are created by half of the Fc region and then dimerized. This format has divalent bonds to both targets, and therefore, it is a tetravalent molecule.

[0109] A tandem diabody (TandAb) contains two diabodies. Each diabody consists of a VHA and a VLB fragment, and another VHA and a VLB fragment, associated by covalent bonds. The two diabodies are linked by a peptide chain. This can improve stability compared to a diabody composed of two scFvs. It has two divalent bonding sites.

[0110] The scFv-scFv-toxin comprises a toxin and two scFvs containing a stabilizing linker. This can be used for the specific delivery of the payload.

[0111] In the module scFv-scFv-scFv, one scFv for TAA is tagged with a short, recognizable peptide and assembled into a bsAb consisting of two scFvs, one for CD3 and the other for the recognizable peptide.

[0112] In ImmTAC, a stable and soluble T cell receptor fuses to a CD3-recognizing scFv. By using a TCR, ImmTAC is suitable for targeted proteins, such as intracellular proteins.

[0113] Triple-specific nanobodies consist of two single variable domains (nanobodies) and other modules for extending the half-life. The half-life can be increased by adding additional modules.

[0114] In the triple-specific killer cell engager (TriKE), two scFv cells are linked via a polypeptide linker incorporating human IL-15. Adding a linker that binds to IL-15 increases NK cell survival and proliferation.

[0115] Any antibody or antigen-binding fragment described herein can be conjugated with a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin such as human serum albumin). Conjugation with a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment or extend its biological activity, either in vitro (e.g., in tissue culture medium or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0116] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of the single-stranded variable fragments (scFv) described herein, fused to the CD3-ζ transmembrane and endodomains. In some embodiments, the chimeric antigen receptor also includes intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor includes multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, for increased potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) expressing the chimeric antigen receptor described herein.

[0117] In some embodiments, the scFv has one heavy chain variable domain and one light chain variable domain. In some embodiments, the scFv has two heavy chain variable domains and two light chain variable domains. In some embodiments, the scFv has two antigen-binding regions (antigen-binding regions: A and B), and the two antigen-binding regions can bind to their respective target antigens with different affinities.

[0118] In some embodiments, a bispecific antibody targeting B7-H3 and an additional antigen (e.g., OX40, CD28, CD3, 4-1BB, CD314, CD47, PD-1, CTLA4, CD40, or PD-L1) can be generated using the antibody or antigen-binding fragment sequence described herein (e.g., CDR or VH / VL sequence). In some embodiments, the antibody has a common light chain.

[0119] In some embodiments, the antibodies, antigen-binding fragments thereof, or ADCs described herein can bind to one or more antigens or one or more epitopes. In some embodiments, the antibodies or antigen-binding fragments thereof described herein (e.g., 20H8 and 21A9) can bind to the same epitope or substantially identical epitopes.

[0120] In some embodiments, the antibody or its antigen-binding fragment has a heavy chain constant region which is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26.

[0121] In some embodiments, the bispecific antibodies or their antigen-binding fragments described herein have a common light chain.

[0122] The anti-B7-H3 antibodies (e.g., 19A1, 20H8, and 21A9) in this disclosure are from RenLite. (登録商標)These are human antibodies produced in mice. Because these antibodies have the same fully humanized common light chain, the anti-B7-H3 antibodies were generated to have two identical or different heavy chain variable regions targeting B7-H3 (e.g., any one of the B7-H3 targeting VH regions described herein) and two identical common light chain variable regions. Exemplary antibodies obtained are shown in Figures 1-3. In some embodiments, the anti-B7-H3 antigen-binding domain includes the CDR of the anti-B7-H3 antibody as shown in Figures 1 and 2. In some embodiments, the anti-B7-H3 antigen-binding domain includes the VH and VL of the anti-B7-H3 antibody as shown in Figure 3.

[0123] In several embodiments, all of these B7-H3 antibodies showed good endocytosis rates in cancer cell lines.

[0124] Antibody-drug conjugate (ADC) In some embodiments, the antibodies or antigen-binding fragments described herein can be optionally conjugated with a therapeutic agent using a linker to form an antibody-drug conjugate. The antibody-drug conjugate, comprising the antibody or its antigen-binding fragment, can be covalently or noncovalently bound to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cell proliferation inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, camptothecin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, meitansinoids (such as DM-1 and DM-4), zione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide, as well as its analogues). In some embodiments, the therapeutic agent is MMAE or MMAF.

[0125] Definitions of specific functional groups and chemical terms are described in detail below. For the purposes of the present invention, chemical elements are identified based on the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition, inside the cover, and specific functional groups are generally defined as described herein. Furthermore, general principles of organic chemistry, as well as specific functional groups and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0126] All ranges recited herein are inclusive, unless expressly stated to the contrary. When a range of values is recited, it is intended to include every value and subrange within that range. For example, "C 1-6 " is intended to include C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 .

[0127] The compounds of this disclosure or any formula describing and explaining the compounds may have one or more chiral (asymmetric) centers. The compounds or any formula describing and explaining the compounds of this disclosure may have one or more chiral (asymmetric) centers. The present invention encompasses all stereoisomers of the compounds herein or any formula describing and explaining the compounds of the present invention. All asymmetric centers present in a compound or any formula describing and explaining the compounds of the present invention may independently have (R) or (S) configurations. When the bond to the chiral carbon is depicted as a straight line in the structural formula, or when the compound name is written without a (R) or (S) chiral designation for the chiral carbon, it is understood that both the (R) and (S) configurations of each chiral carbon, and therefore each enantiomer or diastereomer and mixtures thereof, are encompassed in the formula or name.

[0128] This disclosure includes all possible enantiomers and diastereomers, as well as mixtures of two or more stereoisomers, e.g., mixtures of enantiomers and / or diastereomers in any proportion. Thus, enantiomers are subject to this disclosure in the form of enantiomerically pure forms, both levorotatory and dextrorotatory anticellar forms, in racemic forms, and in mixtures of two enantiomers in any proportion. In the case of cis / trans isomerism, this disclosure includes both cis and trans forms, as well as mixtures of these forms in any proportion. Preparation of individual stereoisomers can be carried out, as necessary, by separation of mixtures by conventional methods such as chromatography or crystallization, by the use of stereochemically homogeneous starting materials for synthesis, or by stereoselective synthesis. Optionally, derivatization can also be performed before separation of stereoisomers. Separation of mixtures of stereoisomers can be carried out as an intermediate step in the synthesis of the compound, or on the final racemic product. Absolute stereochemistry can be determined, if necessary, by X-ray crystallography of crystalline products or intermediates derivatized with reagents containing stereocenters of known configurations. Alternatively, absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopy.

[0129] Unless otherwise specified, the structures described herein include compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the atomic mass or mass number that is dominant in nature. These compounds are referred to as “isotope variants.” This disclosure is intended to encompass all pharmaceutically acceptable isotopic variants of the compounds of the present invention, or any formulations that describe and explain the compounds of the present invention. Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen (e.g., 2 H (i.e., D) and 3 H), carbon isotopes (e.g., 11 C, 13 C, and 14 C) Isotopes of chlorine (for example, 36 Cl), fluorine isotopes (for example, 18 F) Iodine isotopes (for example, 123 I, 125 I) Nitrogen isotopes (for example, 13 N, 15 N), oxygen isotopes (for example, 15 O, 17 O, and 18 O), phosphorus isotopes (for example, 32 P), and sulfur isotopes (e.g., 35 Examples include, but are not limited to, S). Specific isotopic variants of compounds, or any formulas that describe and explain the compounds of this disclosure, for example, incorporating radioisotopes, may be useful for studying the tissue distribution of drugs and / or substrates. In particular, hydrogen is replaced with deuterium ( 2 Compounds having the described structures, differing only in that they can be replaced with heavier isotopes, such as by substituting H or D, may be useful in several situations because they may offer certain therapeutic benefits, such as improved metabolic stability, extended half-life in the body, or reduced dose requirements. The compounds of this disclosure, or any isotopic variants of the compounds of this disclosure, can generally be prepared by techniques known to those skilled in the art, or by processes similar to those described in the accompanying examples and synthesis, using appropriate isotope-labeled reagents instead of previously used unlabeled reagents.

[0130] The compounds provided herein are described by reference to both general formulas and specific compounds. Furthermore, all compounds of this disclosure may exist in numerous different forms or derivatives within the scope of this disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and active metabolites.

[0131] As used herein, the term “pharmaceutically acceptable salt” includes, unless otherwise specified, salts that retain the biological efficacy of the free acid / base form of a particular compound and are not biologically or otherwise undesirable. Pharmaceutically acceptable salts may include salts formed with inorganic bases or acids and organic bases or acids. Where a compound of the Disclosure contains one or more acidic or basic groups, the Disclosure also includes the corresponding pharmaceutically acceptable salts thereof. Thus, compounds of the Invention containing acidic groups such as carboxyl groups can exist in the form of salts and can be used in accordance with the Invention, for example, as alkali metal salts, alkaline earth metal salts, aluminum salts, or ammonium salts. More non-limiting examples of these salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, or salts with ammonia or organic amines such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine, or amino acids. These salts can be readily obtained, for example, by reacting a compound having an acidic group with a suitable base, such as lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide. Other base salts of the compounds of this disclosure include, but are not limited to, copper(I), copper(II), iron(II), iron(III), manganese(II), and zinc salts. Compounds of this disclosure containing one or more basic groups, such as protonable groups, can exist in the form of salts and can be used according to the present invention in the form of addition salts with inorganic or organic acids.Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, embonic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, and other acids known to those skilled in the art. The salts formed include, among others, hydrochlorides, chlorides, hydrobroms, bromides, iodides, sulfates, phosphates, methanesulfons (mesylates), tosylates, carbonates, bicarbonates, formates, acetates, sulfoacetates, triflates, oxalates, malons, maleates, succinates, tartrates, malates, emponates, mandelates, fumarates, lactates, citrates, glutarates, stearates, aspartates, and glutamates. The stoichiometry of the salts formed from the compounds of this disclosure may further be an integer multiple of 1 or a non-integer multiple.

[0132] The compounds of this disclosure containing a basic nitrogen-containing group include, for example, methyl, ethyl, isopropyl, and C2-butyl chloride, bromide, and iodide. 1-4 Alkyl halides, such as dimethyl, diethyl, and diamyl sulfates, are diC 1-4 Alkyl sulfates, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides, etc. 10-18 Alkyl halides, and aryl C compounds such as benzyl chloride and phenethyl bromide. 1-4 Quaternization can be performed using reagents such as alkyl halides.

[0133] Where the compounds of this disclosure contain both acidic and basic groups in their molecules, this disclosure also includes internal salts or betaines (amphoteric ions) in addition to the salt forms described above. Each salt can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in a solvent or dispersant, or by anion exchange or cation exchange with other salts. This disclosure also includes all salts of the compounds of this disclosure that are not suitable for direct use in pharmaceuticals due to their poor physiological compatibility, but can be used, for example, as intermediates in chemical reactions or in the preparation of pharmaceutically acceptable salts. For a more appropriate review of salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002).

[0134] Any formula describing and explaining a compound or a compound of the disclosure and its pharmaceutically acceptable salts may exist in both non-solvated and solvated forms. As used herein, the term “solvate” refers to a molecular complex comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules. For example, the term “hydrate” is used when the solvent is water.

[0135] The pharmaceutically acceptable solvates provided in this disclosure may include those in which the crystallization solvent is isotope-substituted, such as D2O, d6-acetone, and d6-DMSO.

[0136] Linker (binding compound) In some embodiments, the therapeutic agent is conjugated via a linker (or binding compound). As used herein, the terms “linker” or “binding compound” refer to a compound that can form a ligand-drug conjugate by conjugating a ligand (e.g., an antibody or its antigen-binding fragment as described herein) and a therapeutic agent (e.g., any of the therapeutic agents as described herein) with the groups of the ligand compound and the therapeutic agent compound, respectively, by a coupling reaction.

[0137] In some embodiments, the linker described herein is a compound having the following formula: [ka] Equation (I), Alternatively, it may be a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof, where Q represents a junction that can be coupled to a ligand via a bond selected from the group consisting of carbonyl, thioether, amide, disulfide, and hydrazone bonds, and L represents a linker that can bind Q to the therapeutic agent.

[0138] In some embodiments, the joint portion (Q in equation (I)) has the following structure. [ka]

[0139] In some embodiments, the linker portion (L in equation (I)) has the following equation: [ka] Here, L1 is a polypeptide residue consisting of 3 to 8 amino acid residues, including at least one amino acid residue having a side chain carboxyl group, such as a glutamic acid residue or an aspartic acid residue, where "-COOH" indicates the carboxyl group of the C-terminal amino acid residue of the polypeptide residue. L2 is either absent or a monodentate, bidentate, or tridentate hydrophilic group attached to the side chain carboxyl group on the amino acid residue of polypeptide residue L1, and L2 is -NHC(R L2a )(R L2b )(R L2c ) has a structure, where R L2a , R L2b , and R L2c These are independently H, -(CH2O)(CH2CH2O) m (CH2) p C(O)OH, and -(CH2O)(CH2CH2O)m (CH2) p C(O)NHR L2d Each of the following groups is selected, R L2d is either H or C, which is optionally substituted with 1 to 6 hydroxyl groups. 1-6 It is an alkyl group, where each m is independently 0 to 10, preferably 0 to 4, for example 0, 1, 2, 3, or 4, and particularly preferably m is 0, and each p is independently 1 to 4, for example 1, 2, 3, or 4, and JPEG2026529918000008.jpg89 shows the N-terminal side of the polypeptide residue covalently bonded to junction Q.

[0140] In some embodiments, polypeptide residue L1 is NH -Glu-Val-Ala- COOH In some embodiments, the hydrophilic group L2 has the following structure. [ka] Here, "*" represents the site covalently bonded to polypeptide residue L1, for example. NH -Glu-Val-Ala- COOH This shows the side chain of the Glu residue.

[0141] In some embodiments, the linker described herein is a compound having the following structure. [ka] CPT-L

[0142] In some embodiments, the linker is a VC linker. Details of the linker used in ADCs can be found, for example, in Su, Z. et al. “Antibody-drug conjugates: Recent advances in linker chemistry.” Acta Pharmaceutica Sinica B (2021), which is incorporated in its entirety by reference.

[0143] Therapeutic drugs In some embodiments, therapeutic agents conjugated to antibodies or antigen-binding fragments described herein are described below.

[0144] In some embodiments, the therapeutic agent described herein is a cytotoxic agent. In some embodiments, the cytotoxic agent is a camptothecin compound, its analogue, or derivative. In some preferred embodiments, the camptothecin compound is a compound having the following structure. [ka] Here, X is selected from the group consisting of -CH2-, O, and S, and Y is selected from the group consisting of H, D, and F.

[0145] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[3',4':6,7]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione) (CPT-1). The structure of CPT-1 is shown below: [ka] CPT-1

[0146] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13(2H)-dione (CPT-2). The structure of CPT-2 is shown below: [ka] CPT-2

[0147] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[3',4':6,7]indolidino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione (CPT-3). The structure of CPT-3 is shown below: [ka] CPT-3

[0148] In some embodiments, the therapeutic agent is (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,9,12,15-tetrahydro-13H-pyrano[4,3,2-de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-10,13(2H)-dione (CPT-4). The structure of CPT-4 is shown below: [ka] CPT-4

[0149] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (also known in the art as a derivative of drastatin-10), or a derivative thereof. The auristatin can be, for example, an ester formed of auristatin E and a keto acid. For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. Exemplary auristatin synthesis and structure are described in U.S. Patent Publication No. 2003-0083263, International Patent Publication No. WO04 / 010957, International Patent Publication No. WO02 / 088172, and U.S. Patents No. 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, and 5,599. These are described in Nos. 902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, each of which is incorporated herein by reference for all purposes.

[0150] Auristatin has been shown to interfere with microtubule dynamics, as well as nuclear and cell division, and has been shown to possess anticancer activity. Auristatin can bind to tubulin and exert cytotoxic or cell proliferation inhibitory effects in cancer cells. Numerous well-known assays exist in the art that can be used to measure whether auristatin or the resulting antibody-drug conjugate exerts cell proliferation inhibitory or cytotoxic effects in desired cells.

[0151] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include thiotepa and cyclosphosphamide (CYTOXAN). TMAlkylating agents such as busulfan, improsulfan, and biposulfan, alkyl sulfonates, aziridines such as benzodopa, carbocone, metsuredopa, and uredopa, ethyleneimines and methylamelamamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamamine, chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, f Nitrogen mustards such as enesterine, prednimastine, trophosphamide, uracil mustard, carmastine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, nitrosoureas, acrasinomycin, actinomycin, anthramycin, azaserine, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophiline, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epi Antibiotics such as rubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; fludarabine, 6-mercaptopri Purine analogs such as thiamiprine and thioguanine, pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU, androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone, anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane, folic acid supplements such as folinic acid, acegraton, aldofamide glycoside, aminolevulinic acid, amsacrine,Best Love Sil, Bisanthren, Edatrexate, Defofamine, Demecoltin, Diadiquan, Elfomitin, Elptinium Acetate, Etoglucid, Gallium Nitrate, Hydroxyurea, Lentinan, Ronidamin, Mitoguazone, Mytoxantrone, Mopidamol, Nitracrine, Pentostatin, Fenamet, Pirarubicin, Podophyllic Acid, 2-Ethylhydrazide, Procarbazine, PSK 7. Lazoxane, schizophyllan, spirogermanium, tenuazonic acid, triadiquan, 2,2',2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitractol, pipobromane, gasitosine, arabinoside ("Ara-C"), cyclophosphamide, taxane, e.g., paclitaxel (TAXOL®, Bristol-Myers Examples include Squibb Oncology (Princeton, New Jersey), doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, platinum analogs such as cisplatin or carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoic acid, esperamicin, capecitabine, and any pharmaceutically acceptable salts, acids, or derivatives thereof. This definition also includes antihormone agents that modulate or inhibit hormonal activity in tumors, such as tamoxifen, raloxifene, aromatase inhibitor 4(5)imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston), as well as antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, and antiestrogens including any pharmaceutically acceptable salts, acids, or derivatives of the above. A detailed description of chemotherapeutic agents is available at:For example, it can be found in US20180193477A1, and the entire thing is incorporated by reference.

[0152] Linker - therapeutic compound In some embodiments, a linker (e.g., any of the linkers described herein) and a therapeutic agent (e.g., any of the therapeutic agents described herein) can be linked to form a "linker-therapeutic agent" compound.

[0153] In some embodiments, the linker therapeutic compound has the following structure. [ka]

[0154] In some embodiments, the linker therapeutic compound has the following structure. [ka]

[0155] In some embodiments, an antibody ("Ab"), for example, any of the antibodies or antigen-binding fragments described herein, can be linked to a linker therapeutic compound (for example, any of the linker therapeutic compounds described herein) to generate an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate has the following structure. [ka] Here, n = 1-8. In some embodiments, n = 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, or approximately 8. In some embodiments, n is approximately 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, or 7-8. In some embodiments, n is an integer multiple or a non-integer multiple of 1.

[0156] Antibody and ADC characteristics The present invention provides antibodies, antigen-binding fragments thereof, and ADCs thereof that specifically bind to B7-H3 (e.g., any of the human B7-H3 proteins or isoforms described herein). These antibodies may be agonists or antagonists. In some embodiments, the anti-B7-H3 antibodies or antigen-binding fragments described herein can bind to B7-H3 and inhibit the binding of B7-H3 to its receptor (which is little known). By inhibiting the binding of B7-H3 to its receptor, the anti-B7-H3 antibodies can inhibit B7-H3-related signaling pathways and thus treat cancer. In some embodiments, the anti-B7-H3 antibodies or antigen-binding fragments described herein can confer specificity to B7-H3-expressing cancer cells, resulting in the enrichment of the conjugated therapeutic agent in the ADC (e.g., any therapeutic agent described herein) near the cancer cells, thereby effectively killing the cancer cells. In some embodiments, the anti-B7-H3 antibodies or antigen-binding fragments can induce CDC or ADCC.

[0157] The affinity of an antibody to an antigen can be measured using common techniques, including, for example, ELISA, RIA, and surface plasmon resonance (SPR). The affinity can be derived from the quotient of the dynamic rate constant (KD = koff / on). In some embodiments, the antibody or its antigen-binding fragment described herein is 0.1s -1 Less than 0.01s-1 Less than 0.001s -1 Less than 0.0001s -1 Less than or 0.00001s -1 It can bind to B7-H3 (e.g., human B7-H3, monkey B7-H3, mouse B7-H3, canine B7-H3, and / or chimeric B7-H3) with a dissociation rate (koff) of less than 0.01s. In some embodiments, the dissociation rate (koff) is 0.01s. -1 The above is 0.001s -1 More than 0.0001s -1 More than 0.00001s -1 or greater than or equal to 0.000001s -1 That's all.

[0158] In some embodiments, the motor velocity (kon) is 1 × 10⁻⁶ 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms greater than, or 1 × 10⁻⁶ 6 It is greater than / Ms. In some embodiments, the motor velocity (kon) is 1 × 10 5 / Ms less than 1 × 10 6 Less than / Ms, or 1 × 10 7 It is less than / Ms.

[0159] The affinity can be estimated from the quotient of the velocity constant (KD = koff / kon). In some embodiments, KD is 1 × 10⁻¹⁰. -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, or 1 × 10 -10 It is less than M. In some embodiments, KD is 50nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or less than 1nM. In some embodiments, KD is 1 × 10⁻¹⁶ -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11M or 1 × 10 -12 It is greater than M.

[0160] In some embodiments, the antibodies or antigen-binding fragments described herein bind to human B7-H3(4Ig) (SEQ ID NO: 33), human B7-H3(2Ig) (SEQ ID NO: 34), mouse B7-H3 (SEQ ID NO: 35), monkey B7-H3 (SEQ ID NO: 36), and / or canine B7-H3 (e.g., SEQ ID NO: 37). In some embodiments, the antibodies do not bind to human B7-H3(4Ig), B7-H3(2Ig), mouse B7-H3, monkey B7-H3, and / or canine B7-H3.

[0161] Unlike MGC-018 analogues, in some embodiments, the antibodies or antigen-binding fragments described herein can specifically bind to B7-H3 but not to other B7-H3 family proteins, such as CD80, CD86, PD-L1, B7-H4, B7-H5, B7-H6, or B7-H7. In some embodiments, these B7-H3 family proteins are derived from humans, mice, monkeys, or dogs. The specificity of the antibodies or antigen-binding fragments described herein to B7-H3 rather than other B7-H3 family proteins is thought to be due to specific binding to a B7-H3 epitope not shared among other B7-H3 family proteins.

[0162] Thermal stability can also be determined. The Tm of the antibody or antigen-binding fragment described herein may be above 60°C, above 61°C, above 62°C, above 63°C, above 64°C, above 65°C, above 66°C, above 67°C, above 68°C, above 69°C, above 70°C, above 71°C, above 72°C, above 73°C, above 74°C, above 75°C, above 76°C, above 77°C, above 78°C, above 79°C, above 80°C, above 81°C, above 82°C, above 83°C, above 84°C, above 85°C, above 86°C, above 87°C, above 88°C, above 89°C, above 90°C, above 91°C, above 92°C, above 93°C, above 94°C, or above 95°C. Since IgG is described as a multi-domain protein, the melting curve may show two transitions, namely a first denaturation temperature Tm D1 and a second denaturation temperature Tm D2. The presence of these two peaks typically indicates denaturation of the Fc domain (Tm D1) and the Fab domain (Tm D2), respectively. When two peaks are present, Tm usually refers to Tm D2. Accordingly, in some embodiments, the Tm D1 of the antibody or antigen-binding fragment described herein is above 60°C, above 61°C, above 62°C, above 63°C, above 64°C, above 65°C, above 66°C, above 67°C, above 68°C, above 69°C, above 70°C, above 71°C, above 72°C, above 73°C, above 74°C, above 75°C, above 76°C, above 77°C, above 78°C, above 79°C, above 80°C, above 81°C, above 82°C, above 83°C, above 84°C, above 85°C, above 86°C, above 87°C, above 88°C, above 89°C, above 90°C, above 91°C, above 92°C, above 93°C, above 94°C, or above 95°C. In some embodiments, the Tm D2 of the antibody or antigen-binding fragment described herein is above 60°C, above 61°C, above 62°C, above 63°C, above 64°C, above 65°C, above 66°C, above 67°C, above 68°C, above 69°C, above 70°C, above 71°C, above 72°C, above 73°C, above 74°C, above 75°C, above 76°C, above 77°C, above 78°C, above 79°C, above 80°C, above 81°C, above 82°C, above 83°C, above 84°C, above 85°C, above 86°C, above 87°C, above 88°C, above 89°C, above 90°C, above 91°C, above 92°C, above 93°C, above 94°C, or above 95°C.In some embodiments, Tm, Tm D1, and Tm D2 are less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0163] In some embodiments, the antibody or antigen-binding fragment thereof described herein has a purity of more than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, for example, as measured by HPLC. In some embodiments, the purity is less than 30%, 40%, 50%, 60%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, for example, as measured by HPLC.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof described herein has a tumor growth inhibition percentage (TGI TVIt has a TGI%. In some embodiments, the antibody has a tumor growth inhibition rate of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be measured, for example, 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 the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition rate (TGI%) is calculated using the following formula: TGI(%)=[1-(T i -T0) / (V i -V0)] × 100% T i V is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. i V0 is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0165] In some embodiments, the antibody or antigen-binding fragment described herein is a B7-H3 antagonist. In some embodiments, the antibody or antigen-binding fragment described herein is a B7-H3 agonist.

[0166] In some embodiments, the antibodies or antigen-binding fragments described herein are non-toxic.

[0167] In some embodiments, antibodies or antigen-binding fragments can induce complement-dependent cell-mediated cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) and kill tumor cells.

[0168] In some embodiments, the antibody or antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the antibody or antigen-binding fragment can induce complement-dependent cell-mediated cytotoxicity (CDC). In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody.

[0169] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is Fab, Fab', F(ab')2, and Fv fragments. In some embodiments, the protein construct described herein has an Fc region without effector function. In some embodiments, Fc is human IgG4 Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (L234A and L235A mutations in EU numbering) or an LALA-PG mutation (L234A, L235A, and P329G mutations in EU numbering).

[0170] Other modifications can also be made to the Fc region. For example, introducing a cysteine ​​residue into the Fc region allows for the formation of interchain disulfide bonds in this region. The homodimeric fusion protein thus produced may have an extended half-life both in vitro and / or in vivo.

[0171] In some embodiments, IgG4 has the S228P mutation (EU number). The S228P mutation prevents IgG4 Fab arm replacement both in vivo and in vitro. In some embodiments, Fc has the SI mutation (S239D and I332E mutations in the EU numbering).

[0172] In some embodiments, we provide an Fc region having a sugar structure that lacks fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an Fc region composition may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the total amount of sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297 as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 refers to the asparagine residue at approximately position 297 of the Fc region (position 314 in the Eu numbering or Kabat numbering of the Fc region residue), however, due to minute sequence variations in the Fc region sequence, Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucose-modified mutants can have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region can be further modified, and asparagine at position 297 can be replaced with alanine (N297A).

[0173] In some embodiments, the bispecific antibodies described herein can conjugate to a therapeutic agent to form an antibody-drug conjugate (ADC). In some embodiments, the DAR of the ADCs described herein is approximately 7.5 to 8.5, 7.6 to 8.5, 7.7 to 8.5, 7.8 to 8.5, 7.9 to 8.5, 8.0 to 8.5, 8.1 to 8.5, 8.2 to 8.5, 8.3 to 8.5, 8.4 to 8.5, 7.5 to 8.4, and 7.6. ~approximately 8.4, approximately 7.7~approximately 8.4, approximately 7.8~approximately 8.4, approximately 7.9~approximately 8.4, approximately 8.0~approximately 8.4, approximately 8.1~approximately 8.4, approximately 8.2~approximately 8.4, approximately 8.3~approximately 8.4, approximately 7.5~approximately 8.3, approximately 7.6~approximately 8.3, approximately 7.7~approximately 8.3, approximately 7.8~approximately 8.3, approximately 7.9~approximately 8.3, approximately 8.0~approximately 8.3, approximately 8.1~approximately 8.3, approximately 8.2~approx. 8.3, approx. 7.5~approx. 8.2, approx. 7.6~approx. 8.2, approx. 7.7~approx. 8.2, approx. 7.8~approx. 8.2, approx. 7.9~approx. 8.2, approx. 8.0~approx. 8.2, approx. 8.1~approx. 8.2, approx. 7.5~approx. 8.1, approx. 7.6~approx. 8.1, approx. 7.7~approx. 8.1, approx. 7.8~approx. 8.1, approx. 7.9~approx. 8.1, approx. 8.0~approx. 8.1, approx. 7.5~approx. 8. 0, approximately 7.6-8.0, approximately 7.7-8.0, approximately 7.8-8.0, approximately 7.9-8.0, approximately 7.5-7.9, approximately 7.6-7.9, approximately 7.7-7.9, approximately 7.8-7.9, approximately 7.5-7.8, approximately 7.6-7.8, approximately 7.7-7.8, approximately 7.5-7.7, approximately 7.6-7.7, or approximately 7.5-7.6.

[0174] In some embodiments, an anti-B7-H3 antibody, its antigen-binding fragment, or an ADC as described herein can be internalized into target cells (e.g., cell lines expressing B7-H3 or primary cancer cells). In some embodiments, the target cells include any cell type described herein. In some embodiments, internalization is achieved by endocytosis. In some embodiments, the endocytosis rate is indicated by the percentage of cells in the total cell population that are signal-positive for the antibody. For example, the percentage of positive cells may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. In some embodiments, the percentage of cells positive for the antibody signal is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to an isotype control antibody (e.g., human IgG1).

[0175] In some embodiments, the anti-B7-H3 ADCs described herein can inhibit the proliferation of in vivo cancer cells (e.g., lung cancer, gastric cancer, or skin cancer) in xenograft mouse models at dose levels of less than 6 mg / kg, less than 5 mg / kg, less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg.

[0176] Method for producing anti-B7-H3 antibodies Isolated fragments of human B7-H3 can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injection) of the antigen peptide or protein. In some embodiments, the antigen peptide or protein is injected with at least one adjuvant. In some embodiments, the antigen peptide or protein can be conjugated with a drug that is immunogenic in the immunized species. Animals may be injected with the antigen peptide or protein two or more times (e.g., two, three, or four times).

[0177] A full-length polypeptide or protein may be used, or an antigenic peptide fragment thereof may be used as an immunogen. The antigenic peptide of the protein contains at least eight (e.g., at least 10, 15, 20, or 30) amino acid residues of the B7-H3 amino acid sequence and includes the protein epitope, such that an antibody produced against the peptide forms a specific immune complex with the protein. As described above, the full-length sequence of human B7-H3 is known in the art (SEQ ID NO: 33). In some embodiments, an Fc-tagged human B7-H3 protein is used as an immunogen.

[0178] Immunogens are typically used for antibody preparation by immunizing a suitable target (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). A suitable immunogenic preparation may contain, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., a fragment of human B7-H3). The preparation may further contain an adjuvant, such as a Freund complete or incomplete adjuvant, or a similar immunostimulant.

[0179] Polyclonal antibodies can be prepared as described above by immunizing a suitable target with a B7-H3 polypeptide or its antigenic peptide (e.g., a part of B7-H3, e.g., the extracellular region) as an immunogen. The antibody titer in the immunized target can be monitored over time using standard techniques such as enzyme-linked immunosorbent assay (ELISA) with the immobilized B7-H3 polypeptide or peptide. If desired, the antibody molecule can be isolated from a mammal (e.g., from blood) and further purified by well-known techniques such as protein A chromatography or protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, for example, when the titer of specific antibodies is at its maximum, antibody-producing cells can be obtained from the target organism 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., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (generally, see Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). For example, hybridoma cells that produce monoclonal antibodies can be detected by screening hybridoma culture supernatants for antibodies that bind to a target polypeptide or epitope using a standard ELISA assay.

[0180] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human antibody, a humanized antibody, or a chimeric antibody, or an antibody or antigen-binding fragment described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence that produces the antigen-binding site or antigen-binding domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments may exhibit increased affinity for a target, such as B7-H3. Any combination of deletions, insertions, and / or combinations can be realized in an antibody or antigen-binding fragment with increased binding affinity to a target. Antibodies or antigen-binding fragments can be modified, or novel post-translational modifications can be introduced, by changing the amino acids introduced into the antibody or antigen-binding fragment, such as changing the number of glycosylation sites (e.g., increasing or decreasing them), changing the type of glycosylation sites (e.g., altering the amino acid sequence so that different sugars are bound by enzymes present in the cell), or introducing novel glycosylation sites.

[0181] The antibodies disclosed herein may be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, 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.

[0182] Phage display (panning) can be used to optimize antibody sequences with desired binding affinity. In this technique, a gene encoding single-stranded Fv (including VH or VL) is inserted into the phage's coat protein gene, causing the phage to "display" scFv on its outside while containing the gene for the protein internally, thereby creating a correlation between genotype and phenotype. These display phages can then be screened against a target antigen to detect the interaction between the displayed antigen-binding site and the target antigen. Thus, the amplified protein library, obtained through a process called in vitro selection, can be screened and amplified to obtain antibody sequences with desired binding affinity.

[0183] Examples of human antibodies and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as) human germline immunoglobulin sequences. Examples of human antibodies include amino acid residues within the CDR that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo).

[0184] Humanized antibodies typically have a human framework (FR) into which non-human CDRs have been transplanted. Therefore, humanized antibodies have one or more amino acid sequences introduced into humans from a non-human source. These non-human amino acid residues are often called “import” residues, and are typically derived from “import” variable domains. Humanization can essentially be carried out by substituting, for example, a rodent CDR or CDR sequence with the corresponding sequence of a human antibody. These methods are described, for example, in Jones et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse." Nature 321.6069(1986):522; Riechmann et al. "Reshaping human antibodies for therapy." Nature 332.6162(1988):323; and Dall'Acqua et al. "Antibody humanization by framework shuffling." Methods 36.1(2005):43-60, each of which is incorporated herein by reference in its entirety. Therefore, a "humanized" antibody is a chimeric antibody in which a portion considerably smaller than the intact human V domain is substituted with a corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which several CDR residues and several FR residues are substituted with residues derived from similar sites within the human antibody.

[0185] The selection of human VH and VL domains for humanized antibody production is crucial for reducing immunogenicity. According to the so-called "best-fit" method, the V domain sequence of a mouse antibody is screened against an entire library of known human domain sequences. The human sequence that most closely matches the mouse sequence is then selected as the human FR domain for the humanized antibody (Sims et al. "A humanized CD18 antibody can block function without cell destruction." The Journal of Immunology 151.4(1993):2296-2308; Chothia, et al., "Canonical structures for the hypervariable regions of immunoglobulins." Journal of Molecular Biology 196.4(1987):901-917).

[0186] Furthermore, it is important to humanize antibodies while maintaining high specificity and affinity for antigens, as well as other desirable biological properties. To achieve this goal, humanized antibodies can be prepared by analytical processes of parental sequences and various conceptual humanization products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that can illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. By observing these displays, it is possible to analyze the roles that residues can play in the functionalization of candidate immunoglobulin sequences, i.e., the residues that affect the candidate immunoglobulin's ability to bind to its antigen. In this way, FR residues can be selected and combined from recipient and import sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0187] Typically, amino acid sequence variants of human antibodies, humanized antibodies, or chimeric anti-B7-H3 antibodies contain amino acid sequences that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences present in the light or heavy chain of the original antibody.

[0188] In some embodiments, mice having humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenMab) TM Antibodies are generated using mice. A heavy chain immunoglobulin locus is a region on a chromosome that contains the gene for the heavy chain of the antibody. Loci can include, for example, the human IGHV (variable) gene, the human IGHD (variability) gene, the human IGHJ (binding) gene, and the mouse heavy chain constant domain gene. A κ chain immunoglobulin locus is a region on a chromosome that contains the gene encoding the light chain (κ chain) of the antibody. Examples of κ chain immunoglobulin loci include the human IGKV (variable) gene, the human IGKJ (binding) gene, and the mouse light chain constant domain gene. A detailed description of RenMab mice can be found in PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety. Antibodies generated by mice include complete human VH, complete human VL, and the mouse constant region. In some embodiments, human VH and human VL are ligated to the human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4).

[0189] In some embodiments, mice having humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci (e.g., RenLite TMMice) are used to generate antibodies. A heavy chain immunoglobulin locus is a chromosomal region that contains the gene encoding the heavy chain of an antibody. The locus can comprise, for example, a human IGHV (variable) gene, a human IGHD (diversity) gene, a human IGHJ (joining) gene, and a mouse heavy chain constant domain gene. A kappa chain immunoglobulin locus is a chromosomal region that contains the gene encoding the common light chain. Examples of the kappa chain immunoglobulin locus include a human IGKV (variable) gene, a human IGKJ (joining) gene, and a mouse light chain constant domain gene. RenLite TM Detailed descriptions of RenLite mice can be found in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety.

[0190] Identity or homology to an original sequence generally refers to the percentage of amino acid residues in a candidate sequence that are identical to residues present in a human, humanized, or chimeric anti-OX40 antibody or fragment, after aligning the sequences, introducing gaps as necessary to achieve the maximum percent sequence identity, and not counting conservative substitutions as part of sequence identity.

[0191] An anti-B7-H3 antibody or antigen-binding fragment thereof can be subjected to further modifications. For example, cysteine residues can be introduced into the Fc region to allow the formation of interchain disulfide bonds within this region. The homodimeric antibody thus generated may have an increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life can also be prepared using heterobifunctional cross-linking agents, such as those described in Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies with dual Fc regions can be recombinantly produced (see, e.g., Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0192] In some embodiments, covalent modifications can be added to anti-B7-H3 antibodies or their antigen-binding fragments. These covalent modifications can be added by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that can react with selected side chains or N or C-terminal residues.

[0193] In some embodiments, to enhance production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further recombined, replacing the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is provided, for example, in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated in its entirety by reference.

[0194] In some embodiments, the methods described herein are designed to produce bispecific antibodies. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell cultures. For example, the interface may include at least a portion of the CH3 domain of the antibody constant domain. In the method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with small amino acid side chains (e.g., alanine or threonine), a compensated "cavity" of the same or similar size as the large side chains is formed at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products (such as homodimers). The method is described, for example, in WO 96 / 27011, which is incorporated herein by reference in its entirety.

[0195] Bispecific antibodies include, for example, crosslinked antibodies or "heteroconjugate" antibodies. For example, one antibody in a heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can be produced by any convenient crosslinking method. Suitable crosslinking agents and 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.

[0196] Techniques for producing bispecific antibodies from antibody fragments are also known in this field. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al. (Science 229:81, 1985) described a method for generating F(ab')2 fragments by cleaving intact antibodies by proteolysis. These fragments are reduced in the presence of the dithiol conjugate sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'TNB derivatives is then reduced with mercaptoethylamine to convert it back to a Fab'-thiol, which is then mixed with an equimolar amount of another Fab'TNB derivative to form a bispecific antibody.

[0197] In some embodiments, a knob-into-hole (KIH) technique may be used, which involves engineering the CH3 domain to produce a “knob” or “hole” in each heavy chain to promote heterodimerization. The KIH technique is, for example, Xu, Yiren et al., “Production of bispecific antibodies in 'knobs-into-holes' using a cell-free expression system,” MAbs, Vol. 7, No. 1, Taylor & Francis, 2015, which is incorporated in its entirety by reference. In some embodiments, one heavy chain has the T366W and / or S354C (knob) substitution (EU numbering), and the other heavy chain has the Y349C, T366S, L368A and / or Y407V (hole) substitution (EU numbering). In some embodiments, one heavy chain has one or more of the substitutions Y349C and T366W (EU numbering). The other heavy chain may have one or more substitutions from E356C, T366S, L368A, and Y407V (EU numbering). Furthermore, substitutions (-ppcpScp-->-ppcpPcp-) can be introduced into the hinge region of the two-substituted IgG.

[0198] Recombination vectors This disclosure also provides recombinant vectors (e.g., expression vectors) containing isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such host cells contain polynucleotides and / or the polynucleotide-containing vectors), and the production of recombinant antibody polypeptides or fragments thereof by recombinant technology.

[0199] As used herein, “vector” is any construct that, when introduced into a host cell, can deliver one or more polynucleotides of interest to the host cell. An “expression vector” can deliver and express one or more polynucleotides of interest as encoded polypeptides within the host cell into which the expression vector has been introduced. Thus, within the expression vector, the polynucleotides of interest are positioned for expression within the vector by being operably bound to regulatory elements such as promoters, enhancers, and / or poly-A tails, either within the vector or in the host cell genome, at, near, or adjacent to the integration site of the polynucleotides of interest, so that the polynucleotides of interest are translated within the host cell into which the expression vector has been introduced.

[0200] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (that can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0201] In some embodiments, polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein) are introduced using a viral expression system (e.g., a smallpox or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (deficient) replicable virus or a non-replicable virus. In the latter case, viral replication generally occurs only in complementary viral packaging cells. Suitable systems include, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321, Flexner et al., 1989, Ann. NYAcad Sci. 569:86-103, Flexner et al., 1990, Vaccine, 8:17-21, U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487, WO 89 / 01973, U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242, WO 91 / 02805, Berkner-Biotechniques, 6:616-627, 1988, Rosenfeld et al. Preferred systems are disclosed in al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler 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. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA may also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749 and Cohen, 1993, Science, 259:1691-1692.By coating DNA onto biodegradable beads that efficiently transport it to cells, the uptake of naked DNA can be increased.

[0202] For expression, a DNA insert containing a polynucleotide encoding an antibody or polypeptide, as disclosed herein, can be operably ligated to a suitable promoter (e.g., a heterologous promoter), such as, to name a few, the phage λPL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoter of a retroviral LTR. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is the cytomegalovirus (CMV) promoter. The expression construct may further contain sites for transcription start and end, and within the transcription region, a ribosome-binding site for translation. The coding portion of the mature transcript expressed by the construct may include a translation start at the beginning and a stop codon (UAA, UGA, or UAG) located approximately at the end of the polypeptide being translated.

[0203] As shown, the expression vector may contain at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, and tetracycline or ampicillin resistance genes for Escherichia coli and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are well known in the art.

[0204] Non-restrictive vectors for bacterial use include pQE70, pQE60, and pQE-9 from Qiagen, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restrictive eukaryotic cell vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene, and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0205] Suitable non-limiting bacterial promoters for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic cell promoters include the CMV pre-early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTR promoters such as those for Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0206] In the yeast Saccharomyces cerevisiae, several vectors containing constitutional or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used.

[0207] The construct can be introduced into host cells by 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), which are incorporated herein by reference.

[0208] Transcription of the antibody-encoding DNA in more eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are typically cis-acting elements of DNA, approximately 10–300 bp in length, that enhance the transcriptional activity of promoters in a given host cell type. Examples of enhancers include the SV40 enhancer, located behind the origin of replication at base pairs 100–270, the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the origin of replication, and the adenovirus enhancer.

[0209] Appropriate secretory signals can be incorporated into expressed polypeptides to induce the secretion of translated proteins into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment. These signals can be endogenous to the polypeptide, or they can be heterologous signals.

[0210] Polypeptides (e.g., antibodies) can be expressed in modified forms such as fusion proteins (e.g., GST fusions) or by histidine tagging, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of a polypeptide to improve stability and endurance in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides to induce secretion or excretion, improve stability, and facilitate purification is a well-known and common technique, particularly in the art.

[0211] This disclosure relates to nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with any of the nucleotide sequences described herein, and Further, we provide amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with any of the amino acid sequences described herein.

[0212] This disclosure relates to nucleic acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology to any of the nucleotide sequences described herein, and Further, we provide amino acid sequences having at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with any of the amino acid sequences described herein.

[0213] In some embodiments, the disclosure relates to a nucleotide sequence encoding any of the peptides described herein, or an amino acid sequence encoded by any of the nucleotide sequences described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0214] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence, or (ii) consists of an amino acid sequence, wherein the amino acid sequence is one of the sequences described herein.

[0215] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence, or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0216] The percentage of sequence homology (e.g., amino acid sequence homology or nucleic acid homology) can also be determined. Methods for determining the percentage of sequence homology are known in the art. In some embodiments, conserved amino acid residues having similar physicochemical properties (homology percentage), such as leucine and isoleucine, can be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties are defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0217] This disclosure provides one or more nucleic acids encoding any of the polypeptides described herein. In some embodiments, the nucleic acid (e.g., cDNA) comprises a polynucleotide encoding a heavy-chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding a light-chain polypeptide described herein. In some embodiments, the nucleic acid comprises a polynucleotide encoding an scFv polypeptide described herein.

[0218] In some embodiments, a vector may comprise both nucleic acids encoding the VL region and the VH region that commonly bind to B7-H3 described herein. In some embodiments, a pair of vectors is provided, wherein each vector comprises one of the nucleic acids described herein, and together encodes the VL region and the VH region that commonly bind to B7-H3.

[0219] Therapeutic Methods The antibody or antigen-binding fragment thereof of the present disclosure can be used for various therapeutic purposes.

[0220] The methods described herein include methods for treating cancer-associated conditions. Generally, the method comprises administering a therapeutically effective amount of the engineered antibody or antigen-binding fragment thereof described herein to a subject in need of or determined to be in need of such treatment.

[0221] As used herein, "treating" means ameliorating at least one symptom of a cancer-associated medical condition. Cancer generally causes death, and therefore treatment can increase life expectancy (e.g., at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years). For the treatment of cancer-associated conditions, administration of a therapeutically effective amount of an agent described herein results in a reduction in the number of cancer cells and / or alleviation of symptoms.

[0222] As used herein, the term “cancer” refers to an abnormal condition or situation characterized by cells having the ability to self-replicate, i.e., rapid cell proliferation. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of their histopathological type or stage of invasion. As used herein, the term “tumor” refers to cancer cells, e.g., cancer cell masses. Cancers that can be treated or diagnosed using the methods described herein include malignancies of various organ systems, such as malignancies affecting the lungs, mammary glands, thyroid gland, lymphatic system, gastrointestinal tract, and urogenital tract, and adenocarcinomas, including most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell lung cancer, small intestine cancer, and esophageal cancer. In some embodiments, the agents described herein are designed to treat or diagnose cancer in a subject. The term “cancer” is known in the art and refers to malignant tumors of epithelial or endocrine tissue, including respiratory cancers, gastrointestinal cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. In some embodiments, cancer is kidney cancer or melanoma. Exemplary cancers include cancers that form from tissues of the cervix, lungs, prostate, mammary glands, head and neck, colon, and ovaries. The term further includes carcinosarcoma, which includes, for example, malignant tumors consisting of cancerous and sarcomatous tissues. “Adenocarcinoma” refers to cancer originating from glandular tissue, or, here, cancer in which tumor cells form recognizable glandular structures. The term “sarcoma” is known in the art and refers to malignant tumors of mesenchymal origin. In some embodiments, cancer is chemotherapy-resistant cancer.

[0223] In one embodiment, the disclosure provides a method for treating cancer in a subject, a method for slowing the rate of increase of tumor volume in a subject over time, a method for reducing the risk of metastasis, or a method for reducing the risk of further metastasis in a subject. In some embodiments, the treatment can interrupt, slow, stop, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction of the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0224] In one aspect, the Disclosure provides a method comprising administering a therapeutically effective amount of an antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate disclosed herein to a subject in need, for example, a subject having or being identified or diagnosed with cancer, such cancer being, for example, liver cancer, pancreatic cancer, prostate cancer, osteosarcoma, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, non-small cell lung cancer (NSCLC), bladder cancer, kidney cancer, brain tumor, head and neck cancer, or melanoma.

[0225] In one embodiment, the Disclosure provides a method comprising administering a therapeutically effective amount of an antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed with, cancer), such cancer being, for example, lymphoma, non-small cell lung cancer (NSCLC), leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, pancreatic cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, or sarcoma. In some embodiments, leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia. In some embodiments, lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia. In some embodiments, sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0226] In some embodiments, anti-B7-H3 antibodies are designed to treat non-small cell lung cancer, cutaneous squamous cell carcinoma, pancreatic cancer, primary hepatocellular carcinoma, colorectal cancer, clear cell renal cell carcinoma, or breast cancer.

[0227] In some embodiments, the subject is human, and the B7-H3 antibody is designed to treat lung cancer, melanoma, gastric cancer, astroblastoma, glioblastoma, epidermal carcinoma, ductal carcinoma, and breast cancer. In some embodiments, the subject is dog, and the B7-H3 antibody is designed to treat canine osteosarcoma.

[0228] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients at risk of cancer can be identified by various methods well known in the art.

[0229] In some embodiments, the present disclosure relates to a method for treating an autoimmune disease or inflammation, the method comprising administering to a subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0230] In one embodiment, the present disclosure provides methods for treating, preventing, or reducing the risk of developing diseases associated with abnormal or undesirable immune responses, such as autoimmune diseases. These autoimmune diseases include alopecia areata, lupus (or systemic lupus erythematosus), ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behçet's disease, pernicious anemia, bullous pemphigoid, polyarteritis nodosa, myocarditis, relapsing polychondritis, celiac disease, polyglandular autoimmune syndrome, chronic fatigue syndrome (CFS), polymyalgia rheumatica, chronic inflammatory demyelinating polyradiculoneuritis, polymyositis / dermatomyositis, chronic inflammatory demyelinating polyneuropathy, primary agammaglobulinemia, and Churg-Strauss syndrome. Examples of conditions that can be treated include, but are not limited to, syndromes, primary biliary cholangitis, pemphigoid scarring, psoriasis, CREST syndrome, Raynaud's disease, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus erythematosus, rheumatoid arthritis, cryoglobulinemia, sarcoidosis, fibromyalgia, scleroderma (systemic sclerosis), Graves' disease, Sjögren's syndrome, Guillain-Barré syndrome, stiff-person syndrome, Hashimoto's thyroiditis, Takayasu's arteritis, idiopathic pulmonary fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes mellitus (type 1), vasculitis, lichen planus, and vitiligo. In some embodiments, inflammation can be treated using antibodies or antigen-binding fragments. In some embodiments, anti-B7-H3 antibodies are designed to treat microbial infections and other diseases or disorders such as allergic disorders.

[0231] In some embodiments, the antibody is designed to treat bone mineralization disorders, such as rickets, renal diseases (renal osteodystrophy, Fanconi syndrome), tumor-induced osteomalacia, hypophosphatasia, McCune-Albright syndrome, or osteogenesis imperfecta with mineralization disorders (osteogenesis imperfecta-like syndromes (SROIs)). In some embodiments, the bone mineralization disorder is osteoporosis.

[0232] In some embodiments, the present disclosure relates to a method for inhibiting an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein. In some embodiments, the subject has an autoimmune disease.

[0233] As used herein, “effective dose” means an amount or dosage sufficient to produce a beneficial or desired outcome, including interrupting, slowing, stopping, or inhibiting the progression of a disease, such as cancer. The effective dose varies depending on the age and weight of the person to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector containing the polynucleotide, and / or composition thereof is administered, the severity of the symptoms, and the route of administration, and therefore the dose can be determined on an individual basis.

[0234] An effective dose can be administered in one or more doses. For example, an effective dose of antibody or antigen-binding fragment is an amount sufficient to mitigate, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to mitigate, halt, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, an effective dose of antibody or antigen-binding fragment may vary depending on other factors, in particular, the patient's medical history, as well as the type (and / or dose) of antibody used.

[0235] The effective doses and schedules for administering the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein can be determined experimentally, and making such determinations is within the scope of the art. Those skilled in the art will understand that the dose to be administered will vary depending, for example, on the mammal receiving the antibodies, polynucleotides encoding the antibodies, and / or compositions disclosed herein, the route of administration, the specific type of antibody, the polynucleotide encoding the antibody, the antigen-binding fragment, and / or the compositions disclosed herein used, and other agents administered to the mammal. Guidelines for selecting an appropriate dose for an antibody or antigen-binding fragment can be found in literature on the therapeutic use of antibodies and antigen-binding fragments, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch.22 and pp.303-357, and Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp.365-389.

[0236] An effective daily dose of antibody is typically 0.01 mg / kg to 100 mg / kg (mg per kg of patient body weight). In some embodiments, the dose may be 100 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 25 mg / kg, 20 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, 0.3 mg / kg, or less than 0.1 mg / kg. In some embodiments, the dose may be 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 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, 0.3 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or greater than 0.01 mg / kg. In some embodiments, the dose is approximately 50 mg / kg, 40 mg / kg, 30 mg / kg, 25 mg / kg, 20 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, 0.9 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.

[0237] In any of the methods described herein, at least one antibody, its antigen-binding fragment, 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, can be administered to a target 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 (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one 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.

[0238] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., 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 (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that the biological activity periods of the one or more additional therapeutic agents and the biological activity periods of at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) overlap within the subject.

[0239] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) can be administered to a subject over a long period of time (e.g., over a period of at least one week, two weeks, three weeks, one month, two months, three months, four months, twelve months, one year, two years, three years, four years, or five years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein to diagnose or follow up on the effectiveness of the treatment (e.g., to observe at least one symptom of cancer). As described herein, skilled medical professionals may also change (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and may adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to a subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0240] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agents may include one or more inhibitors selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0241] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades an estrogen receptor.

[0242] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reolysin, Alimta, dicaine, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalidomide, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0243] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-6 antagonist (e.g., an IL-6 receptor), an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a therapy targeting CX3CL1, a therapy targeting CXCL9, a therapy targeting CXCL10, a therapy targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0244] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to a subject.

[0245] In some embodiments, additional therapeutic agents include anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, or anti-CD40 antibody.

[0246] Pharmaceutical composition and route of administration Pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein are also provided herein. Two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein may be present in any combination in the pharmaceutical composition. The pharmaceutical composition may be formulated in any form well known in the art.

[0247] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may include sterile diluents (e.g., sterile water or saline), non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antimicrobial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphoric acid), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposome suspensions are also available as pharmaceutically acceptable carriers. The composition preparations can be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. Where necessary (for example, in injectable formulations), adequate fluidity can be maintained by coatings such as lecithin or by the use of surfactants. The absorption of antibodies or their antigen-binding fragments can be prolonged by including absorption-delaying agents (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulation delivery systems, which include biodegradable and biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0248] Compositions containing one or more antibodies or antigen-binding fragments described herein can be formulated in unit dosage forms (i.e., physically distinct units containing a predetermined amount of the active compound to facilitate administration and ensure uniformity of dose) for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0249] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be supplied in unit dosage forms (i.e., doses for single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. Formulation depends on the chosen route of administration. For injection, antibodies can be formulated in aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain compounding agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, antibodies may be in lyophilized form for preparation with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0250] The toxicity and therapeutic effect of a composition can be determined by standard pharmaceutical procedures in cell culture media or experimental animals (e.g., monkeys). For example, the LD50 (lethal dose for 50% of the population) and ED50 (effective dose for 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs exhibiting a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize the potential harm (i.e., mitigate the undesirable side effects). Toxicity and therapeutic effect can be determined by other standard pharmaceutical procedures.

[0251] Data obtained from cell culture assays and animal studies can be used in the formulation of appropriate doses of any given agent for use in subjects (e.g., humans). A therapeutically effective dose of one or more antibodies (e.g., 1, 2, 3, or 4) or their antigen-binding fragments (e.g., any of the antibodies or antibody fragments described herein) is the amount that treats the disease (e.g., kills cancer cells) or reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human subject identified as having cancer) or a subject identified as being at risk of developing the disease (e.g., a subject who has previously had cancer but is now cured), or the amount that reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a healthcare professional or veterinary professional using methods well known in the art, in addition to observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors may influence the dose and timing required to effectively treat the subject (e.g., severity of the disease or disability, previous treatments, the subject's overall health and / or age, and the presence of other diseases).

[0252] Exemplary doses include the amount (milligrams or micrograms) of either the antibody or antigen-binding fragment described herein per kilogram of body weight of the subject (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, about 1 μg / kg to about 50 μg / kg, about 0.3 mg / kg to about 25 mg / kg, about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg). Although these doses cover a wide range, those skilled in the art will understand that the efficacy and effective dose of therapeutic agents containing antibodies and their antigen-binding fragments can be determined by methods well known in the art. Typically, a relatively low dose is administered first, and the dose can be subsequently and gradually increased by the healthcare professional or veterinary professional (in the case of therapeutic use), or the researcher (if still working in the development stage), until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject depends on various factors, including the activity of the specific compound used, the subject's age, weight, overall health, sex, and diet, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in the body.

[0253] The pharmaceutical composition may be included in a container, pack, or dispenser, along with instructions for administration. This disclosure also provides methods for producing antibodies or their antigen-binding fragments for various applications described herein. Examples

[0254] The present invention will be further illustrated by the following embodiments, which are not intended to limit the scope of the invention as described in the claims.

[0255] Example 1. Production of anti-B7-H3 antibody His-tagged human B7-H3 protein (hB7-H3(4Ig)-His, Beijing ACRO Biosystems Co. Ltd., catalog number: B7B-H52E7) and / or His-tagged mouse B7-H3 protein (mB7-H3-His, Beijing ACRO Biosystems Co. Ltd., catalog number: B73-M52H4) are emulsified with an adjuvant, and this is used to make RenLite TM Mice were immunized with Biocytogen (in situ, with a fully human heavy chain variable domain combined with a common light chain substitution). RenLite TM The mice are described, for example, in PCT / CN2021 / 097652, which is incorporated herein by reference in its entirety. Postorbital blood was collected as a negative control prior to immunization.

[0256] Antibody titers in serum were detected by fluorescence-activated cell sorting (FACS). If a desirable immune response was achieved, antigen-specific immune cells were isolated from immunized mice to further obtain anti-B7-H3 antibodies or the light and heavy chain variable region sequences of the anti-B7-H3 antibody. For example, plasma cells secreting antigen-specific monoclonal antibodies were screened and identified using single-cell techniques (e.g., using Beacon® Optofluidic System, Berkeley Lights Inc.). The nucleotide sequences encoding the antibody variable regions were then obtained using reverse transcription PCR and sequencing. The obtained sequences were cloned into vectors containing sequences encoding the human IgG1 constant region for antibody expression. The binding affinity of the expressed antibodies to B7-H3 was validated using FACS.

[0257] The exemplary antibodies obtained by this method included 19A1, 20H8, and 21A9. These antibodies contain the same light chain, and their Kabat-defined or Chothia-defined sequences of the VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3 are shown in Figures 1 and 2. The amino acid sequences of the VH and VL regions of these antibodies are shown in Figure 3.

[0258] Example 2. Interspecies binding of anti-B7-H3 antibody CHO-S-hB7-H3(4Ig) cells, CHO-S-hB7-H3(2Ig) cells, CHO-S-mB7-H3 cells, CHO-S-fasB7-H3 cells, or CHO-S-dB7-H3 cells, each 1 × 10⁻¹⁶ 5 Cells were transferred to a 96-well plate at a cell / well density. 30 μL of purified anti-B7-H3 antibody (1 μg / mL) was added to each well of the 96-well plate, and the plate was incubated at 4°C for 30 minutes. Next, after washing with PBS, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alex Flour® 647(RL1-H) (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170) at 4°C in the dark for 15 minutes, followed by flow cytometry analysis. Human IgG1 protein was used as the isotype control (ISO). The test results are shown in the table below. CHO-S-hB7-H3(4Ig) cells, CHO-S-hB7-H3(2Ig) cells, CHO-S-mB7-H3 cells, CHO-S-fasB7-H3 cells, and CHO-S-dB7-H3 cells were obtained by transfecting CHO-S cells with vectors expressing human B7-H3(4Ig) amino acid sequences (hB7-H3(4Ig), SEQ ID NO: 33), human B7-H3(2Ig) amino acid sequences (hB7-H3(2Ig), SEQ ID NO: 34), mouse B7-H3 amino acid sequence (mB7-H3, SEQ ID NO: 35), monkey B7-H3 amino acid sequence (fasB7-H3, SEQ ID NO: 36), and canine B7-H3 amino acid sequence (dB7-H3, SEQ ID NO: 37).

[0259] [Table 1]

[0260] The results show that all anti-B7-H3 antibodies can bind to human B7-H3, monkey B7-H3, and canine B7-H3. Furthermore, 19A1 and 20H8 can also bind to mouse B7-H3, but 21A9 cannot.

[0261] Example 3. Binding activity of anti-B7-H3 antibody against B7-H3 positive tumor cells. This experiment was conducted to test the binding activity of anti-B7-H3 antibodies to several different tumor cell lines. Functional genomic RNA sequencing technology was used to determine the mRNA expression levels of B7-H3 in these cell lines, which are shown in the table below.

[0262] [Table 2]

[0263] Human non-small cell lung cancer NCI-H358 cells (ATCC, catalog number: CRL-5807), human melanoma A375 cells (ATCC, catalog number: CRL-1619), human gastric cancer NCI-N87 cells (ATCC, catalog number: CRL-5822), human glioblastoma U87-MG cells (ATCC, catalog number: HTB-14), human epidermal carcinoma A431 cells (ATCC, catalog number: CRL-1555), or human ductal carcinoma BT474 cells (ATCC, catalog number: HTB-20) are placed in 5 × 10⁶ well plates. 4 Cells were seeded at a density of 10 cells / well. Sample anti-B7-H3 antibody (2.5 μg / mL) was added to a 96-well plate, and the plate was incubated at 4°C for 30 minutes. Next, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alexa Flour 647 (RL1-H) (Jackson ImmunoResearch Laboratories, Inc., catalog number: 109-606-170) in the dark at 4°C for 15 minutes, and flow cytometry analysis was performed. The results shown in the table below demonstrate that all anti-B7-H3 antibodies were able to bind to NCI-H358 cells, A375 cells, NCI-N87 cells, U87-MG cells, A431 cells, and BT474 cells.

[0264] [Table 3]

[0265] Example 4. Binding activity of anti-B7-H3 antibody against B7-H3 negative tumor cells. Place 5 × 10⁶ B7-H3 negative tumor cells, such as Jurkat cells (Cobioer Biosciences, catalog number: CBP60520), Daudi cells (Cobioer Biosciences, catalog number: CBP60262), or Raji cells (Cobioer Biosciences, catalog number: CBP60272) into a 96-well plate. 4 Cells were seeded at a density of 10 cells / well. Sample anti-B7-H3 antibodies (0.1 μg / mL, 1 μg / mL, 10 μg / mL, or 100 μg / mL) were added to 96-well plates, and the plates were incubated at 4°C for 30 minutes. Next, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alexa Flour 647 in the dark at 4°C for 15 minutes, and flow cytometry analysis was performed. The results showed that none of the anti-B7-H3 antibodies bound to Jurkat cells, Daudi cells, or Raji cells.

[0266] Example 5. Internalization of anti-B7-H3 antibody Anti-B7-H3 antibody (2.5 μg / mL) and the secondary antibody anti-hIgG-Fc-Alexa Flour 647 were added to NCI-H358, A375, NCI-N87, U87-MG, A431, or BT474 cells and incubated for 6 hours. After incubation, the cells were centrifuged and subsequently washed with FACS buffer. Next, the MFI was measured by flow cytometry. The endocytosis rate of the antibody was calculated by comparing the MFI values ​​between samples. Human IgG1 was used as an isotype control (ISO). The results are shown in the table below.

[0267] [Table 4]

[0268] According to the data, 19A1, 20H8, and 21A9 showed favorable endocytosis rates in NCI-H358 cells, A375 cells, U87-MG cells, A431 cells, and BT474 cells.

[0269] Example 6. Binding affinity of anti-B7-H3 antibody Anti-B7-H3 antibodies include hB7-H3(4Ig)-His, His-tagged human B7-H3 (hB7-H3(2Ig)-His, Beijing ACRO Biosystems Co. Ltd., catalog number: B73-M52H4), His-tagged monkey (cynomolgus macaque) B7-H3 protein (fasB7-H3-His, ACRO Biosystems Inc., catalog number: B73-C52Ha), His-tagged mouse B7-H3 protein (mB7-H3-His, ACRO Biosystems Inc., catalog number: B73-M52H4), or His-tagged canine B7-H3 protein (dB7-H3-His, ACRO Biosystems The binding affinity to Protein A (B73-C52H9) was validated using a Biacore™ (Biacore, Inc., Piscataway, New Jersey) 8K biosensor equipped with a pre-immobilized Protein A sensor chip.

[0270] Purified anti-B7-H3 antibody was diluted to 2 μg / mL and then injected into a Biacore™ 8K biosensor at a flow rate of 10 μL / min for approximately 100 seconds to achieve the desired protein density (e.g., approximately 50 response units (RU)). Next, His-tagged B7-H3 protein at a concentration of 200 nM was injected at a flow rate of 30 μL / min for 180 seconds. Dissociation was monitored for 400 seconds. After each titration of the final injection, the tip was regenerated by elution with glycine (pH 1.5) at a flow rate of 30 μL / min for 30 seconds. For isotype controls (ISO), antibodies targeting unrelated target proteins were used.

[0271] Using Biacore™ 8K Evaluation software 3.0, the entire dataset was fitted to a 1:1 Langmuir coupled model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) to simultaneously obtain the motor association velocity (kon) and dissociation velocity (koff). Affinity was estimated from the quotient of the motor velocity constant (KD = koff / kon).

[0272] The same method, with appropriately adjusted parameters (e.g., antibody concentration) as those skilled in the art would understand, was performed for each test antibody. The results for each test antibody are summarized in the table below.

[0273] [Table 5] (The "-" indicates that they are not connected.)

[0274] Enobrituzumab is a B7-H3 targeted, Fc-optimized, humanized monoclonal antibody developed by MacroGenics, Inc. The VH and VL sequences are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

[0275] MGC-018 is an antibody-drug conjugate (ADC) targeting B7-H3, consisting of a humanized anti-B7-H3 monoclonal antibody conjugated to seco-DUBA, a DNA alkylating prodrug, via a cleavable linker developed by Macrogenics. The heavy chain and light chain sequences of the humanized anti-B7-H3 monoclonal antibody are shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively. The heavy and light chains combine to form an MGC-018 analog (monoclonal antibody), which functions as a positive control.

[0276] The results show that 19A1, 20H8, and 21A9 all exhibited high affinity binding to human B7-H3 and monkey B7-H3. Furthermore, 19A1 can also bind to mouse B7-H3 and canine B7-H3.

[0277] Example 7. Binding activity of anti-B7-H3 antibody against B7-H3 family proteins. This experiment was conducted to test the binding activity of anti-B7-H3 antibodies to other B7-H3 family proteins, such as CD80, CD86, PD-L1, B7-H4, B7-H5, B7-H6, and B7-H7. Specifically, CHO-S-hB7-H3 cells, CHO-S-hCD80 cells, CHO-S-hCD86 cells, CHO-S-hPD-L1 cells, CHO-S-hB7-H4 cells, CHO-S-hB7-H5 cells, CHO-S-hB7-H6 cells, or CHO-S-hB7-H7 cells were bound to 5 × 10⁶ cells each. 4 The cells were transferred to a 96-well plate at a cell / well density. Sample anti-B7-H3 antibody (10 μg / mL) was added to the 96-well plate, and the plate was incubated at 4°C for 30 minutes. Next, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alexa Flour 647 (RL1-H) in the dark at 4°C for 15 minutes, and flow cytometry analysis was performed.

[0278] CHO-S-hCD80 cells, CHO-S-hCD86 cells, CHO-S-hPD-L1, CHO-S-hB7-H4 cells, CHO-S-hB7-H5 cells, CHO-S-hB7-H6 cells, and CHO-S-hB7-H7 cells contain human CD80 (hCD80, having the amino acid sequence described in SEQ ID NO: 40), human CD86 (hCD86, having the amino acid sequence described in SEQ ID NO: 41), and human PD-L1 (hPD-L1, having the amino acid sequence described in SEQ ID NO: 42). These were obtained by transfecting CHO-S cells with vectors expressing human B7-H4 (hB7-H4, having the amino acid sequence described in SEQ ID NO 27), human B7-H5 (hB7-H5, having the amino acid sequence described in SEQ ID NO 28), human B7-H6 (hB7-H6, having the amino acid sequence described in SEQ ID NO 29), and human B7-H7 (hB7-H7, having the amino acid sequence described in SEQ ID NO 30).

[0279] The results are summarized in the table below, demonstrating that none of 19A1, 20H8, and 21A9 can bind to human CD80, CD86, PD-L1, B7-H4, B7-H5, B7-H6, or B7-H7. No binding was observed in the negative control ISO, but the positive control MGC-018 analog showed clear binding to the above B7-H3 family proteins.

[0280] [Table 6]

[0281] Example 8. Epitope assay of anti-B7-H3 antibody To determine whether the two anti-B7-H3 antibodies targeted the same or overlapping epitopes, a biolayer interferometry (BLI) epitope binding assay was performed using the ForteBio Octet® system. A 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% polysorbate-20 (P20), pH 7.4), diluted from 10× HBS-EP+ buffer, was used as the running buffer throughout the experiment. Approximately 10 μg / mL hB7-H3(4Ig)-his was captured at 1000 rpm / min for 200 seconds, and 200 nM test antibody (analyte 1) for ligand binding was injected at 1000 rpm / min. To determine whether the binding of different antibodies interfered with each other, another comparison antibody (analyte 2) was injected under the same conditions. The binding time was 300 seconds for each antibody.

[0282] The binding values ​​for each antibody were obtained using Data Analysis HT 12.0. To quantify the interference of one antibody's binding to another, binding ratios were calculated and each pair of antibodies was compared. The binding ratio was defined by dividing the binding value of the second antibody (sample 2) by the binding value of the first antibody (sample 1). A threshold of 0.5 was used to determine whether the two antibodies could interfere with each other. For example, a binding ratio greater than 0.5 indicates that the second antibody can bind to B7-H3 even after the first antibody has bound. Therefore, the two antibodies may recognize different epitopes. The results are summarized in the table below, showing that 20H8 and 21A9 are likely to recognize the same epitope, while 19A1 is likely to recognize a different epitope. The results also show that the epitopes recognized by 19A1, 20H8, and 21A9 are different from those recognized by the positive control, the MGC-018 analog.

[0283] [Table 7]

[0284] Example 9. Antibody-drug conjugate The purified antibody was conjugated to CPT1, CPT2, CPT3, or CPT4 via a CPT-L linker. For the antibody-drug conjugate name, add CPTx (x=1, 2, 3, or 4) immediately after the antibody name. For example, if 19A1 is coupled to CPT1, it is named 19A1-CPT1. As another example, if 19A1 is coupled to CPT2, it is named 19A1-CPT2.

[0285] HIC-HPLC was used to detect the coupling between antibodies and drug molecules. Human IgG1 molecules were conjugated to CPT2 to form isotype-CPT2 (ISO-CPT2), which was used as an isotype control. The HIC-HPLC detection results showed that the drug-antibody ratio (DAR) of the ADC was approximately 8.

[0286] Example 10. Antitumor activity of ADC in a human patient-derived lung xenograft (PDX) model We tested the inhibitory effects of ADCs on tumor growth in a lung cancer model. Immunohistochemical (IHC) staining of patient-derived lung tumor tissue revealed that the histochemical score (H score) for B7-H3 expression levels in the lung PDX model was 98.49.

[0287] The tumors in the mouse were approximately 200-300 mm in size. 3 When the tumor volume reached a certain level, the mice were randomly divided into different groups based on tumor volume. The mice were then administered either PBS (as a negative control) or ADC intravenously (iv). ADC was administered once a week (a total of one dose). Details of the administration schedule, route of administration, and frequency are shown in the table below.

[0288] [Table 8]

[0289] The long and short axes of the tumor were measured, and the tumor volume was calculated using the following formula: 0.5×(long axis)×(short axis) 2 .

[0290] Tumor growth inhibition (TGI) is 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 0. 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 0. A t-test was performed for statistical analysis. A TGI greater than 60% indicates a clear suppression of tumor growth. P<0.05 is the threshold for showing a statistically significant difference.

[0291] The table below summarizes the results of this experiment, including tumor volume, mouse survival rate, and TGI (%) at the day of group assignment (day 0), 14 days after group assignment (day 14), and at the end of the experiment (day 21).

[0292] [Table 9]

[0293] The tumor sizes in the groups treated with 21A9-CPT2 and 20H8-CPT2 are shown in Figure 5. The results showed that both 21A9-CPT2 and 20H8-CPT2 exhibited excellent tumor-inhibiting effects.

[0294] Example 11. Antitumor activity in a human colorectal cancer PDX model The efficacy of ADC was tested in a xenograft (PDX) model derived from human colorectal cancer patients. B7H3 expression was assessed by immunohistochemistry (IHC) in patient-derived colorectal tumor tissue, with an H score of 28.05. Patient-derived tumor fragments (2 mm × 2 mm × 2 mm) were transplanted into the right flank of B-NDG mice. The tumor volume was approximately 200-300 mm². 3Upon reaching a certain tumor volume, the mice were randomly divided into different groups based on their tumor volume. The mice were then administered either PBS or ADC intravenously (iv). Details of the administration scheme are shown in the table below.

[0295] [Table 10]

[0296] The administration frequency was twice a week (a total of four doses). Tumor volume was measured twice a week, and the body weight of the mice was recorded.

[0297] As shown in Figure 6, both 21A9-CPT2 and 20H8-CPT2 demonstrated excellent tumor-inhibiting effects in a human colorectal cancer model.

[0298] Example 12. Antitumor activity in a human breast cancer PDX model The effects of ADC were tested in a xenograft model derived from human breast cancer patients. A patient-derived tumor fragment (2 mm × 2 mm × 2 mm) (B7H3 H score: 73.79) was transplanted into the right flank of B-NDG mice. The tumor volume of the mice was approximately 250 mm². 3 Upon reaching a certain tumor volume, the mice were randomly divided into different groups based on tumor volume. The mice were then injected with either PBS or ADC intravenously. Details of the administration scheme are shown in the table below. As shown in Figure 7, both 21A9-CPT2 and 20H8-CPT2 demonstrated excellent tumor inhibitory effects.

[0299] [Table 11]

[0300] Other Embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to B7 homolog 3 (B7-H3), A heavy chain variable region (VH) including complementarity-determining regions (CDRs) 1, 2, and 3, The VH CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR1, the VH CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR2, and the VH CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VH CDR3, and the heavy chain variable region, A light chain variable region (VL) including CDR1, 2, and 3, The VL CDR1 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR1, the VL CDR2 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR2, and the VL CDR3 region includes an amino acid sequence that is at least 80% identical to the amino acid sequence of the selected VL CDR3, and comprises a light chain variable region. The amino acid sequences of the selected VH CDR1, 2, and 3, and the amino acid sequences of the selected VL CDR1, 2, and 3 are as follows: (1) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 4 to 6, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (2) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 7 to 9, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (3) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 10 to 12, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (4) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 13 to 15, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. (5) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 16 to 18, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively, and (6) The amino acid sequences of the selected VH CDR1, 2, and 3 are shown in SEQ ID NOs: 19 to 21, respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are shown in SEQ ID NOs: 1 to 3, respectively. One of these is the antibody or its antigen-binding fragment.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 4 to 6, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 7 to 9, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with the Kabat numbering scheme, VH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 10 to 12, respectively, and VL comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 13 to 15, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 16 to 18, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, in accordance with the Chothia numbering scheme, VH comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 19 to 21, respectively, and VL comprises CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NOs. 1 to 3, respectively.

8. The antibody or antigen-binding fragment according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment specifically binds to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3.

9. The antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is a human or humanized antibody or an antigen-binding fragment thereof (for example, a human IgG1 antibody or a fragment thereof).

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

11. A nucleic acid comprising a polynucleotide encoding a polypeptide, The polypeptide is as follows: (1) An immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 4 to 6, and the VH, when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO. 22, binds to B7-H3. (2) An immunoglobulin light chain or fragment thereof containing VL, wherein VL comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs: 1 to 3, and VL, when paired with VH containing the amino acid sequence shown in SEQ ID NO: 23, binds to B7-H3. (3) An immunoglobulin heavy chain or fragment thereof containing VH, wherein VH comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 7 to 9, and VH, when paired with VL containing the amino acid sequence shown in SEQ ID NO. 22, binds to B7-H3. (4) An immunoglobulin light chain or fragment thereof containing VL, wherein VL comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 1 to 3, and VL, when paired with VH containing the amino acid sequence shown in SEQ ID NO. 24, binds to B7-H3. (5) An immunoglobulin heavy chain or fragment thereof containing VH, wherein VH comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 10 to 12, and VH, when paired with VL containing the amino acid sequence shown in SEQ ID NO. 22, binds to B7-H3. (6) An immunoglobulin light chain or fragment thereof comprising VL, wherein VL comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 1 to 3, and VL, when paired with VH containing the amino acid sequence shown in SEQ ID NO. 25, binds to B7-H3. (7) An immunoglobulin heavy chain or fragment thereof containing VH, wherein VH comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 13 to 15, and VH, when paired with VL containing the amino acid sequence shown in SEQ ID NO. 22, binds to B7-H3. (8) An immunoglobulin heavy chain or fragment thereof containing VH, wherein VH comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 16 to 18, and VH, when paired with VL containing the amino acid sequence shown in SEQ ID NO. 22, binds to B7-H3, or (9) An immunoglobulin heavy chain or fragment thereof containing VH, wherein VH comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 19 to 21, and VH, when paired with VL containing the amino acid sequence shown in SEQ ID NO. 22, binds to B7-H3. Nucleic acids, including

12. A nucleic acid comprising a polynucleotide encoding a polypeptide, The nucleic acid according to claim 11, wherein the polypeptide comprises an immunoglobulin heavy chain or fragment thereof containing VH, and the VH comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 4, 5, and 6, or SEQ ID NOs. 13, 14, and 15, respectively.

13. A nucleic acid comprising a polynucleotide encoding a polypeptide, The nucleic acid according to claim 11, wherein the polypeptide comprises an immunoglobulin heavy chain or fragment thereof containing VH, and the VH comprises CDRs 1, 2, and 3, each containing the amino acid sequence shown in SEQ ID NOs. 7, 8, and 9, or SEQ ID NOs. 16, 17, and 18, respectively.

14. A nucleic acid comprising a polynucleotide encoding a polypeptide, The nucleic acid according to claim 11, wherein the polypeptide comprises an immunoglobulin heavy chain or fragment thereof containing VH, and the VH comprises CDRs 1, 2, and 3 each containing the amino acid sequences shown in SEQ ID NOs. 10, 11, and 12, or SEQ ID NOs. 19, 20, and 21, respectively.

15. A nucleic acid comprising a polynucleotide encoding a polypeptide, The nucleic acid according to claim 11, wherein the polypeptide comprises an immunoglobulin light chain or fragment thereof containing a VL, and the VL comprises CDRs 1, 2, and 3, each containing the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3.

16. The nucleic acid according to any one of claims 11 to 15, wherein the VH specifically binds to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3 when paired with VL, or the VL specifically binds to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3 when paired with VH.

17. The nucleic acid according to any one of claims 11 to 16, wherein the immunoglobulin heavy chain or fragment thereof is a human or humanized immunoglobulin heavy chain or fragment thereof (for example, a human IgG1 heavy chain or fragment thereof), and the immunoglobulin light chain or fragment thereof is a human or humanized immunoglobulin light chain or fragment thereof (for example, a human IgG1 light chain or fragment thereof).

18. The nucleic acid according to any one of claims 11 to 17, wherein the nucleic acid encodes a single-stranded variable fragment (scFv), a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

19. The nucleic acid according to any one of claims 11 to 18, wherein the nucleic acid is cDNA.

20. A vector comprising one or more nucleic acids as described in any one of claims 11 to 19.

21. A vector comprising two nucleic acids according to any one of claims 11 to 19, encoding the VH region and the VL region that bind together to B7-H3.

22. A pair of vectors, Each vector comprises one nucleic acid as described in any one of claims 11 to 19, wherein the pair of vectors both encode the VH region and the VL region that bind together to B7-H3.

23. A cell comprising the vector according to claim 20 or 21, or the pair of vectors according to claim 22.

24. The cell according to claim 23, wherein the cell is a CHO cell.

25. A cell comprising one or more nucleic acids as described in any one of claims 11 to 19.

26. A cell comprising two nucleic acids according to any one of claims 11 to 19.

27. The cell according to claim 26, wherein the two nucleic acids both encode the VH region and the VL region that bind to B7-H3.

28. A method for producing an antibody or its antigen-binding fragment, (a) Culturing the cells according to any one of claims 23 to 27 under conditions sufficient for the cells to produce the antibody or the antigen-binding fragment, (b) recovering the antibody or antigen-binding fragment produced by the cells, The aforementioned method.

29. An antibody that binds to B7-H3 or an antigen-binding fragment thereof, It comprises a heavy chain variable region (VH) containing an amino acid sequence that is at least 80% identical to the selected VH sequence, and a light chain variable region (VL) containing an amino acid sequence that is at least 80% identical to the selected VL sequence, The selected VH sequence and the selected VL sequence are as follows: (1) The selected VH sequence is sequence number 23, and the selected VL sequence is sequence number 22. (2) The selected VH sequence is sequence number 24, and the selected VL sequence is sequence number 22, (3) The selected VH sequence is sequence number 25, and the selected VL sequence is sequence number 22. One of these is an antibody or its antigen-binding fragment.

30. The antibody or antigen-binding fragment thereof according to claim 29, wherein VH comprises the sequence of SEQ ID NO: 23 and VL comprises the sequence of SEQ ID NO:

22.

31. The antibody or antigen-binding fragment thereof according to claim 29, wherein VH comprises the sequence of SEQ ID NO: 24 and VL comprises the sequence of SEQ ID NO:

22.

32. The antibody or antigen-binding fragment thereof according to claim 29, wherein VH comprises the sequence of SEQ ID NO: 25 and VL comprises the sequence of SEQ ID NO:

22.

33. The antibody or antigen-binding fragment according to any one of claims 29 to 32, wherein the antibody or antigen-binding fragment specifically binds to human B7-H3, monkey B7-H3, mouse B7-H3, and / or canine B7-H3.

34. The antibody or antigen-binding fragment according to any one of claims 29 to 33, wherein the antibody or antigen-binding fragment is a human or humanized antibody or an antigen-binding fragment thereof (for example, a human IgG1 antibody or a fragment thereof).

35. The antibody or antigen-binding fragment according to any one of claims 29 to 34, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multispecific antibody (e.g., a bispecific antibody).

36. An antibody or an antigen-binding fragment that cross-competes with the antibody or antigen-binding fragment described in any one of claims 1 to 10 and 29 to 35.

37. An antibody that binds to B7-H3 or an antigen-binding fragment thereof, A heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3 which are identical to VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence, A light chain variable region (VL) including VL CDR1, VL CDR2, and VL CDR3 which are identical to VL CDR1, VL CDR2, and VL CDR3 of the selected VL sequence, The selected VH sequence and the selected VL sequence are as follows: (1) The selected VH sequence is sequence number 23, and the selected 22 sequence is sequence number 22. (2) The selected VH sequence is sequence number 24, and the selected VL sequence is sequence number 22, (3) The selected VH sequence is sequence number 25, and the selected VL sequence is sequence number 22. One of these is an antibody or its antigen-binding fragment.

38. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and 29 to 37, covalently bound to a therapeutic agent.

39. The antibody-drug conjugate according to claim 38, wherein the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor.

40. The antibody-drug conjugate according to claim 39, wherein the therapeutic agent is MMAE or MMAF.

41. The aforementioned therapeutic agent is as follows: 【Chemistry 1】 An antibody-drug conjugate according to claim 39, selected from the following.

42. The antibody-drug conjugate according to claim 41, wherein the therapeutic agent is linked to the antibody or its antigen-binding fragment via a linker.

43. The linker is as follows: 【Chemistry 2】 The antibody-drug conjugate according to claim 42, having the structure of [the specified structure].

44. The antibody-drug conjugate is as follows: 【Transformation 3】 (Here, n = 1 to 8, and "Ab" represents an antibody or its antigen-binding fragment.) An antibody-drug conjugate according to any one of claims 41 to 43, having the structure of the antibody-drug conjugate described in any one of claims 41 to 43.

45. The antibody-drug conjugate according to any one of claims 38 to 44, wherein the drug-antibody ratio (DAR) is approximately 4 or 8.

46. A treatment method for patients with cancer, The method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and 29 to 37, or an antibody-drug conjugate according to any one of claims 38 to 45.

47. The method according to claim 46, wherein the subject is liver cancer, pancreatic cancer, prostate cancer, osteosarcoma, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bladder cancer, kidney cancer, brain tumor, head and neck cancer, or melanoma.

48. The method according to claim 46 or 47, wherein the subject has cancer cells expressing B7-H3.

49. The method according to any one of claims 46 to 48, further comprising administering to the subject a therapeutically effective amount of anti-OX40 antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-PDL2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM-3 antibody, anti-4-1BB antibody, and / or anti-CD40 antibody.

50. A method for reducing the rate of tumor growth, The method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and 29 to 37, or an antibody-drug conjugate according to any one of claims 38 to 45.

51. A method for killing tumor cells, The method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and 29 to 37, or an antibody-drug conjugate according to any one of claims 38 to 45.

52. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and 29 to 37, and a pharmaceutically acceptable carrier.

53. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 38 to 45 and a pharmaceutically acceptable carrier.

54. A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10 and 29 to 37.

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