Anti-b7h3 antibodies and uses thereof

Abstract

This invention provides anti-B7H3 antibodies or antigen-binding fragments and their applications. The anti-B7H3 antibodies or antigen-binding fragments of this invention can specifically bind to B7H3 and have high affinity (both are around 1 nM). They can be used for the diagnosis and / or treatment of tumors (including cancer and benign tumors).

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to anti-B7H3 antibodies or antigen-binding fragments and their applications. Background Technology

[0002] B7H3 (B7 homolog 3 protein), also known as CD276, is a type I transmembrane glycoprotein belonging to the B7 ligand family.

[0003] Currently, two forms of B7H3 have been identified: 2Ig-B7H3 and 4Ig-B7H3. 2Ig-B7H3 is expressed in mouse and human cells and has an extracellular IgV-IgC structure; 4Ig-B7H3 is expressed only in human cells and consists of tandem repeats of IgV-IgC-IgV-IgC structures. The dominant form of B7H3 in humans is 4Ig-B7H3.

[0004] B7H3 expression levels are limited in normal tissues, but it is abnormally high in a variety of advanced human solid tumors, including but not limited to head and neck cancer, kidney cancer, prostate cancer, lung cancer, breast cancer, gastric cancer, and liver cancer. Its overexpression is generally associated with poorer patient prognosis and clinical outcomes. Therefore, B7H3 is considered a diagnostic biomarker for certain tumors and could serve as an effective target for the development of anti-tumor drugs. Summary of the Invention

[0005] This invention provides an anti-B7H3 antibody or antigen-binding fragment that can specifically bind to B7H3. In some embodiments, the anti-B7H3 antibody or antigen-binding fragment has high affinity.

[0006] This invention provides an antibody or antigen-binding fragment that specifically binds to B7H3 and comprises one or more amino acid sequences from (a) to (f):

[0007] (a) HCDR1, comprising an amino acid sequence as shown in any one of SEQ ID NO: 14, 6, 9, 12, 16, 18, 20, 22-24;

[0008] (b) HCDR2, comprising an amino acid sequence as shown in any one of SEQ ID NO: 15, 7, 10, 13, 17, 19, 21, 25;

[0009] (c) HCDR3, which contains the amino acid sequence shown in SEQ ID NO: 11 or 8;

[0010] (d) LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 1;

[0011] (e) LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 2;

[0012] (f) LCDR3, which contains the amino acid sequence shown in SEQ ID NO: 3.

[0013] In some embodiments, the antibody or antigen-binding fragment specifically binds to B7H3 and comprises:

[0014] (a) HCDR1, comprising an amino acid sequence as shown in any one of SEQ ID NO: 14, 6, 9, 12, 16, 18, 20, 22-24;

[0015] (b) HCDR2, comprising an amino acid sequence as shown in any one of SEQ ID NO: 15, 7, 10, 13, 17, 19, 21, 25;

[0016] (c) HCDR3, which contains the amino acid sequence shown in SEQ ID NO: 11 or 8.

[0017] In some embodiments, the antibody or antigen-binding fragment specifically binds to B7H3 and comprises:

[0018] (d) LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 1;

[0019] (e) LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 2;

[0020] (f) LCDR3, which contains the amino acid sequence shown in SEQ ID NO: 3.

[0021] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:14, HCDR2 as shown in SEQ ID NO:15, and HCDR3 as shown in SEQ ID NO:11; or,

[0022] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:8; or...

[0023] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11; or...

[0024] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:12, HCDR2 as shown in SEQ ID NO:13, and HCDR3 as shown in SEQ ID NO:11; or...

[0025] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:17, and HCDR3 as shown in SEQ ID NO:11; or...

[0026] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, and HCDR3 as shown in SEQ ID NO:11; or...

[0027] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:20, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:11; or...

[0028] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:22, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:11; or...

[0029] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:11; or...

[0030] The antibody or antigen-binding fragment includes HCDR1 as shown in SEQ ID NO:24, HCDR2 as shown in SEQ ID NO:25, and HCDR3 as shown in SEQ ID NO:8.

[0031] In some embodiments, the antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, and LCDR3 as shown in SEQ ID NO:3.

[0032] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:14, HCDR2 as shown in SEQ ID NO:15, and HCDR3 as shown in SEQ ID NO:11; or,

[0033] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:8; or...

[0034] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11; or...

[0035] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:12, HCDR2 as shown in SEQ ID NO:13, and HCDR3 as shown in SEQ ID NO:11; or...

[0036] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:17, and HCDR3 as shown in SEQ ID NO:11; or...

[0037] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, and HCDR3 as shown in SEQ ID NO:11; or...

[0038] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:20, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:11; or...

[0039] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:22, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:11; or...

[0040] The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:11; or...

[0041] The antibody or antigen-binding fragment includes HCDR1 as shown in SEQ ID NO:24, HCDR2 as shown in SEQ ID NO:25, and HCDR3 as shown in SEQ ID NO:8;

[0042] The antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, and LCDR3 as shown in SEQ ID NO:3.

[0043] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:14, HCDR2 as shown in SEQ ID NO:15, HCDR3 as shown in SEQ ID NO:11, LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, and LCDR3 as shown in SEQ ID NO:3.

[0044] Amino acid sequences of HCDR1-3 (AbM number)

[0045]

[0046] Amino acid sequences (AbM numbers) of LCDR1-3

[0047]

[0048] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO: 51 or 26-35, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 51 or 26-35, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 51 or 26-35; and / or

[0049] The variable region of the light chain of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 49, 50 or 4, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 49, 50 or 4, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 49, 50 or 4.

[0050] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in any one of SEQ ID NO:51 or 26-35.

[0051] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO:49, 50 or 4.

[0052] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO:51 or 26-35, and the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:49, 50 or 4.

[0053] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:29 or 51, and the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:49, 50, or 4.

[0054] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:29, and the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:49.

[0055] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:51, and the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:50.

[0056] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:29, and the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:4.

[0057] In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0058] In some embodiments, the heavy chain constant region is a constant region of IgG, IgA, IgE, IgM, or IgD. In some embodiments, the heavy chain constant region is a constant region of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain constant region is a constant region of IgG1.

[0059] In some embodiments, the light chain constant region is a κ or λ chain constant region. In some embodiments, the light chain constant region is a κ chain constant region.

[0060] In some embodiments, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 47, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 47.

[0061] In some embodiments, the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:46, or an amino acid sequence having at least 80% or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:46, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO:46.

[0062] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:36-45, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO:36-45, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in any one of SEQ ID NO:36-45.

[0063] In some embodiments, the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO:5.

[0064] In some embodiments, the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:36-45, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:5.

[0065] In some embodiments, the antibody or antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4.

[0066] In some embodiments, the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0067] In some implementations, the antibody or antigen-binding fragment is scFv, Fab, Fab', or F(ab)2.

[0068] In some implementations, the antibody or antigen-binding fragment is scFv.

[0069] In some embodiments, the structure of the scFv is NH2-heavy chain variable region (VH)-linker-light chain variable region (VL)-COOH or NH2-light chain variable region-linker-heavy chain variable region-COOH.

[0070] In some embodiments, the connector is a GS connector. In some embodiments, the GS connector is (GGGGS)n, where n is an integer. In some embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, the sequence of the GS connector is GGGGSGGGGSGGGGS (SEQ ID NO: 48). In some embodiments, the sequence of the GS connector is GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 52).

[0071] In some embodiments, the scFv has the structure NH2-heavy chain variable region-linker-light chain variable region-COOH. In some embodiments, the scFv has the structure NH2-heavy chain variable region-linker-light chain variable region-COOH, wherein the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 51 or 26-35, and the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 49, 50, or 4.

[0072] In some embodiments, the scFv comprises an amino acid sequence as shown in any one of SEQ ID NO: 53-55.

[0073] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody (including full-length monoclonal antibodies), a multispecific antibody, or an antigen-binding fragment (e.g., a bispecific antibody or antigen-binding fragment). In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

[0074] In some embodiments, the antibody has two identical heavy chains and two identical light chains. In some embodiments, the Fc regions pair to form disulfide bonds.

[0075] In some embodiments, the antibody or antigen-binding fragment is a separated antibody or antigen-binding fragment.

[0076] The variable region, constant region, and full-length amino acid sequence are as follows:

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Example scFv sequence:

[0083] QVQLVQSGAEVKKPGSSVKVSCKTSGGTFNENAISWVRQAPGQGLEWMGGIIPDIHIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGWGYWFDPWGQGTLVTVSSGG GGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPRTFGQGTKVEIK (SEQ ID NO: 53)

[0084] QVQLVQSGAEVKKPGSSVKVSCKTSGGTFNENAISWVRQAPGQGLEWMGGIIPDIHIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGWGYWFDPWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPRTFGQGTKVEIK (SEQ ID NO: 54)

[0085] QVQLVQSGAEVKKPGSSVKVSCKTSGGTFNENAISWVRQAPGQCLEWMGGIIPDIHIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGWGYWFDPWGQGTLVTVSSGG GGSGGGGSGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPRTFGCGTKVEIK (SEQ ID NO: 55)

[0086] The present invention also provides a polynucleotide encoding the antibody or antigen-binding fragment. In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 56, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 56; and / or the polynucleotide comprises the sequence shown in SEQ ID NO: 57, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 57.

[0087] In some embodiments, the polynucleotide is a separated polynucleotide.

[0088] The present invention also provides expression vectors comprising the polynucleotides. In some embodiments, the expression vector comprising the polynucleotides is a nucleic acid fragment, plasmid, bacteriophage, or virus. In some embodiments, the expression vector is an isolated expression vector.

[0089] An example nucleic acid sequence encoding VH:

[0090] CAAGTTCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCTCCTCCGTGAAGGTGTCCTGCAAGACTTCTGGCGGCACCTTTAACGAGAACGCCATCTCCTGGGTCCGACAGGCTCCTGGACAAGGCTTGGAATGGATGGGCGGCATCATCCCCGACATCCATATCGCCA ACTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACAAGTCTACCTCCACCGCCTACATGGAACTGTCCAGTCTGAGATCCGAGGACACCGCCGTGTACTACTGTGCTAGAGGCTGGGGCTATTGGTTCGACCTTGGGGACAGGGCACACTGGTCACAGTTTCAAGC (SEQ ID NO: 56)

[0091] An example nucleic acid sequence encoding VL:

[0092] GAAATCGTGATGACCCAGTCTCCTGCCACACTGTCCGTGTCTCCAGGCGAGAGAGCTACCCTGTCTTGCAGAGCCTCTCAGTCCGTGTCCTCTAACCTGGCCTGGTATCAGCAGAAGCCTGGACAGGCACCTCGGCTGTTGATCTACGGCGCTTCTACCA GAGCCACAGGCATCCCTGCCAGATTCTCCGGCCTCGGATCTGGCACCGAGTTTACCCTGACCATCTCCAGCCTGCAGAGCGAGGACTTTGCTGTGTACTATTGCCAGCAGTACAACAACTGGCCCCGGACCTTTGGCGGCGGAACAAAGGTGGAAATCAAG (SEQ ID NO: 57)

[0093] The present invention also provides host cells comprising the polynucleotide or expression vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, HEK293 cell (such as HEK293F cell), BHK cell, COS1 cell, COS7 cell, CV1 cell, or mouse L cell.

[0094] The present invention also provides pharmaceutical compositions comprising the antibody or antigen-binding fragment or biological material, and a pharmaceutically acceptable carrier.

[0095] The present invention also provides the use of the antibody or antigen-binding fragment, the biomaterial, or the pharmaceutical composition in the preparation of medicaments for diagnosing and / or treating diseases. In some embodiments, the present invention provides the use of the antibody or antigen-binding fragment, the biomaterial, or the pharmaceutical composition in the diagnosis and / or treatment of diseases. In some embodiments, the present invention provides a method for diagnosing and / or treating a disease, the method comprising administering an effective dose of the antibody or antigen-binding fragment, the biomaterial, or the pharmaceutical composition to a patient in need.

[0096] In some implementations, the disease is selected from tumors, respiratory diseases, skin and musculoskeletal diseases, genitourinary diseases, nervous system diseases, and digestive system diseases.

[0097] In some implementations, the tumor is a benign tumor or cancer.

[0098] In some implementations, the tumor is a hematologic malignancy or a solid tumor.

[0099] In some implementations, the disease includes tumors that are positive for B7H3 expression.

[0100] In some implementations, the tumor is selected from neuroblastoma, prostate cancer, breast cancer, ovarian cancer, melanoma, renal cell carcinoma, osteosarcoma, colorectal cancer, lung cancer such as non-small cell lung cancer, glioma such as glioblastoma, pancreatic cancer, head and neck cancer, leukemia, cervical cancer, kidney cancer, squamous cell carcinoma, gastric cancer, liver cancer, mesothelioma, anal cancer, skin cancer, vulvar cancer, fibroblastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignancy, sarcoma, brain cancer, central nervous system tumors, and brain metastases.

[0101] This invention also provides diagnostic methods and uses. In some embodiments, a method for detecting B7H3 expression in a sample is provided, which involves contacting the sample with the antibody or antigen-binding fragment, causing the antibody or antigen-binding fragment to bind to B7H3, and detecting the binding, i.e., the amount of B7H3 in the sample. In some embodiments, the use of the antibody or antigen-binding fragment in the preparation of a kit for diagnosing or prognosing tumors (including benign tumors and cancers) is provided. In some embodiments, a diagnostic or prognostic kit comprising the antibody or antigen-binding fragment is provided.

[0102] This invention provides anti-B7H3 antibodies or antigen-binding fragments and their applications. The anti-B7H3 antibodies or antigen-binding fragments of this invention can specifically bind to B7H3, exhibiting high specificity and high affinity, and can also be used for the diagnosis and prognosis of tumors (including benign tumors and cancers). Attached Figure Description

[0103] Figure 1 The binding curves of human-B7H3-his with different anti-B7H3 antibodies are shown.

[0104] Figure 2 The binding curves of mouse-B7H3-his with different anti-B7H3 antibodies are shown.

[0105] Figure 3a and 3b The binding curves of Calu-6 cells to different anti-B7H3 antibodies are shown.

[0106] Figure 4a and 4b The binding curves of MDA-MB-468 cells with different anti-B7H3 antibodies are shown. Detailed Implementation

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0108] definition

[0109] It should be noted that the term “a” entity refers to one or more of the same entity. For example, “an antibody” should be understood as one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably in this document.

[0110] As used herein, the terms “comprising” or “including” mean that antibodies, compositions, or methods include the listed elements, such as components or steps, but do not exclude others. “Substantially composed of” means that antibodies, compositions, or methods exclude other elements that fundamentally affect the characterization of the composition, but do not exclude elements that do not substantially affect the antibody, composition, or method. “Composed of” means excluding elements not specifically listed.

[0111] The term "polypeptide" is intended to encompass both the singular and plural forms of "polypeptide" and refers to a molecule formed by amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not imply a specific length of the product. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" can be used in place of any of the foregoing terms or interchangeably with any of the foregoing terms. The term "polypeptide" is also intended to refer to products modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced through recombinant technologies, but they do not necessarily have to be translated from a specified nucleic acid sequence; they can be produced in any manner, including chemical synthesis.

[0112] An amino acid is an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by nucleic acids directly or in its precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids.

[0113] As used herein, the twenty common amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty common amino acids (e.g., D-amino acids), non-natural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be components suitable for the peptides provided herein. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the peptide representation used herein, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxyl-terminal direction, consistent with standard usage and convention. Conventional (or natural) amino acids are L-amino acids, including alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0114] Minor variations in the amino acid sequence of antibody or immunoglobulin molecules are covered within this invention, provided that the amino acid sequence identity is maintained at at least 75%, such as at least 80%, 90%, 95%, and even 99%. In some embodiments, the variation is a conserved amino acid substitution. A conserved amino acid substitution is a substitution that occurs within the relevant amino acid family in its side chain. Genetically encoded amino acids are broadly classified into the following categories: (1) acidic amino acids, such as aspartate and glutamate; (2) basic amino acids, such as lysine, arginine, and histidine; (3) nonpolar amino acids, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, such as glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) serine and threonine from the aliphatic-hydroxy family; (ii) asparagine and glutamine from the amide family; (iii) alanine, valine, leucine, and isoleucine from the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine from the aromatic family. In some embodiments, the conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. For example, it is reasonable to predict that leucine can be replaced by isoleucine or valine alone, aspartate by glutamate, threonine by serine, or a structurally related amino acid can be replaced by another amino acid, without significantly affecting the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the binding site. Whether an amino acid change results in a functional peptide can be readily determined by measuring the specific activity of the polypeptide derivative. Fragments or analogues of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art.

[0115] In some embodiments, amino acid substitution has the following effects: (1) reducing sensitivity to proteolytic activity, (2) reducing sensitivity to oxidation, (3) altering the binding affinity for forming protein complexes, (4) altering the binding affinity, and (5) imparting or improving other physicochemical or functional properties to such analogs. Analogs may include various mutant proteins with sequences different from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conserved amino acid substitutions) may be made in the naturally occurring sequence (preferably in the polypeptide portion outside the domains forming intermolecular contacts). Conserved amino acid substitutions should not significantly alter the structural characteristics of the parental sequence (e.g., the substituted amino acid should not tend to disrupt the helical structure present in the parental sequence or disrupt other types of secondary structures characterizing the parental sequence).

[0116] The number of conserved amino acid substitutions in VL and VH is approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 8, approximately 9, approximately 10, approximately 11, approximately 13, approximately 14, or approximately 15, or any range between any two of these values ​​(including the end values) or any of these values. The number of conserved amino acid substitutions in the heavy chain constant region, the light chain constant region, the heavy chain, or the light chain is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, or about 45, or a range between any two of these values ​​(including the end values) or any of these values.

[0117] The term "isolated" as used in this invention with respect to cells, nucleic acids, peptides, antibodies, etc., such as "isolated" DNA, RNA, peptides, and antibodies, refers to molecules isolated from one or more other components such as DNA or RNA, respectively, in the cellular native environment. The term "isolated" as used in this invention also refers to nucleic acids or peptides that, when produced by recombinant DNA technology, are substantially free of cellular material, viral material, or cell culture medium, or chemical precursors or other chemicals used in chemical synthesis. Furthermore, "isolated nucleic acids" is intended to include nucleic acid fragments that are not present in their native state and will not be present in their native state. The term "isolated" is also used in this invention to refer to cells or peptides isolated from other cellular proteins or tissues. Isolated peptides are intended to include purified and recombinant peptides. Isolated peptides, antibodies, etc., are typically prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acids, peptides, antibodies, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including extreme values) between any two of these values, or any value therein.

[0118] When the term “encoding” is applied to polynucleotides, it refers to a polynucleotide called the “encoding” polypeptide that, in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce the polypeptide and / or fragments thereof.

[0119] The term “recombination” refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not exist naturally. Unrestricted embodiments can be created by combining polynucleotides or polypeptides that do not normally exist.

[0120] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology or identity can be determined by comparing comparable positions in each sequence. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous or identical at that position. The degree of homology between sequences is a function of the number of common matching or homologous positions. "At least 80% identity" means approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 90%, approximately 91%, approximately 92%, approximately 94%, approximately 95%, approximately 98%, approximately 99%, or a range between any two of these values ​​(including the end values) or any value within that range. "At least 90% identity" means approximately 90% identity, approximately 91% identity, approximately 92% identity, approximately 93% identity, approximately 95% identity, approximately 96% identity, approximately 97% identity, approximately 98% identity, approximately 99% identity, or a range (including the endpoints) between any two of these values ​​or any value therein.

[0121] Polynucleotides are specific sequences of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T), or, when the polynucleotide is RNA, thymine replaced by uracil (U). The "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.

[0122] The terms "polynucleotide," "polynucleotide," and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Examples of unrestricted polynucleotides include: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonucleases, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, expression vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modification is present, structural modifications to the nucleotides can be made before or after assembly of the polynucleotide. The sequence of the nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the polynucleotide of the present invention includes a double-stranded form and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0123] "Identity or sequence identity" between a nucleic acid or polynucleotide sequence (or polypeptide or antibody sequence) and another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that, when sequence alignment is performed, that percentage of bases (or amino acids) are identical in the two compared sequences. This alignment and identity percentage or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignment is preferably performed using default parameters. One alignment procedure is BLAST using default parameters, such as BLASTN and BLASTP, which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of identity and encode polypeptides with the same or similar biological activities.

[0124] "Antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody, any antigen-binding fragment, or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the complementarity-determining region (CDR), heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), framework region (FR), or any portion thereof of the heavy chain or light chain or its ligand-binding moiety, or at least a portion of the binding protein. CDR regions include the CDR regions of the light chain variable region (VL CDR1-3) and the CDR regions of the heavy chain variable region (VH CDR1-3). Antibodies or antigen-binding fragments can specifically recognize and bind to polypeptides or polypeptide complexes containing one or more (e.g., two) antigens. Antibodies or antigen-binding fragments that specifically recognize and bind to multiple (e.g., two) antigens can be referred to as multispecific (e.g., bispecific) antibodies or antigen-binding fragments.

[0125] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody. The antibody fragments of this invention may have a composition similar to F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc., in monospecific antibody fragments. Regardless of their structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0126] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin. In some respects, these regions are linked to a short linker peptide (or linker) of about 10 to about 25 amino acids. The linker / linker may be enriched with glycine to increase flexibility and with serine or threonine to increase solubility, and may link the N-terminus of the VH to the C-terminus of the VL, and vice versa. Although the constant region is removed and the linker is introduced, the protein retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example, as described in U.S. Patent 5,892,019. Suitable linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (e.g., Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448). Other linkers can also be used in this invention, such as those described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur. J. Immunol. 2001; 31:94-106, Hu et al, Cancer Res. 1996; 56:3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293:41-56, and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0127] The term "antibody" encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are categorized as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with further subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, which is IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and their assigned functional specificities are known. All immunoglobulin types are within the scope of this invention. In some embodiments, the immunoglobulin molecule is of the IgG type. Two heavy chains and two light chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues through a variable region surrounding the heavy chain.

[0128] The antibodies, antigen-binding fragments or derivatives of the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies (scFv), and epitope-binding fragments (e.g., Fab, Fab', and F(ab')2).

[0129] The terms "constant" and "variable" are used according to function. The light chain variable region (VL) and heavy chain variable region (VH) determine antigen recognition and specificity. The light chain constant region (CL) and heavy chain constant region (CH) confer important biological properties such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the constant regions are numbered as they move further away from the antibody's antigen-binding site or N-terminus. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0130] In naturally occurring antibodies, assuming the antibody presents its three-dimensional conformation in an aqueous environment, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous amino acid sequences that specifically bind to the antigen, forming the antigen-binding domain. The remaining amino acids in the antigen-binding domain, referred to as the "framework" region, exhibit less intermolecular variability. The framework region mostly adopts a β-sheet conformation, with the CDR forming a ring structure attached to it, or in some cases, forming part of a β-sheet structure. Thus, the framework region positions the CDR in the correct orientation by forming a scaffold through non-covalent interchain interactions. The antigen-binding domain with a CDR at a specific position forms a surface complementary to the epitope on the antigen, which facilitates non-covalent binding of the antibody to its antigenic epitope. For a given heavy or light chain variable region, those skilled in the art can identify the amino acids containing the CDRs and framework regions using known methods.

[0131] As used herein, the term "CDR" refers to the complementarity-determining region within the variable region of an antibody. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3 (or specifically, VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) for each variable region. The boundaries of the CDRs within the variable region of the same antibody may vary depending on the assignment system. Therefore, when referring to antibodies defined with a specific CDR sequence as defined in this invention, the scope of the antibody also includes antibodies whose variable region sequence contains the CDR sequence of this invention, but whose claimed CDR boundaries differ from the specific CDR boundaries defined in this invention due to the application of different schemes. CDRs as defined by Kabat and Chothia include overlaps or subsets of amino acid residues when compared with each other. Nevertheless, the application of either definition to refer to the CDR of an antibody or its variants is within the scope of this invention. The exact residue numbering containing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can typically determine which specific residues are contained in the CDR based on the amino acid sequence of the antibody's variable region.

[0132] As used herein, the term "frame" or "frame sequence" refers to the sequence remaining after subtracting the CDR from the variable region. Because the precise definition of the CDR sequence can be determined using different systems, the meaning of a frame sequence requires correspondingly different interpretations. Six CDRs (CDR1, CDR2, and CDR3 of the light chain and CDR1, CDR2, and CDR3 of the heavy chain) divide the frame region on both the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying a particular subregion as FR1, FR2, FR3, or FR4, the frame region may be referred to as a combination of FRs within the variable region of a single naturally occurring immunoglobulin chain, as used herein. As used herein, FR represents one of the four subregions, and FRs represent two or more of the four subregions constituting the frame region.

[0133] The frame region and CDR region of a humanized antibody do not necessarily correspond precisely to the parental sequence. For example, the donor antibody CDR or common frame can be mutated by substitution, insertion, and / or deletion of at least one amino acid residue, such that the CDR or frame residue at that site does not correspond to the donor antibody or common frame. Typically, at least 80%, at least 85%, more than 90%, or at least 95% of the humanized antibody residues will correspond to those residues in the parental FR and CDR sequences. As used herein, the term "common frame" refers to a frame region in a common immunoglobulin sequence. As used herein, the term "common immunoglobulin sequence" refers to a sequence formed by the most frequently occurring amino acid (or nucleotide) in a family of related immunoglobulin sequences. In an immunoglobulin family, each position in the common sequence is occupied by the amino acid that most frequently occurs at that position in that family. If two amino acids occur with equal frequency, either one may be included in the common sequence. In some embodiments, the frame region is a human receptor frame, such as a human immunoglobulin frame or a human common frame. A "human common frame" is a frame representing the most common amino acid residues in a series of human immunoglobulin VL or VH frame sequences. Generally, the selection of human immunoglobulin VL or VH sequences comes from a subgroup of variable domain sequences. Typically, these subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3.

[0134] Where a term used and / or accepted in this field has two or more definitions, the definition of the term used herein includes all of those meanings unless explicitly stated otherwise. A concrete example is the use of the term “complementarity-determining region” (“CDR”) to describe discontinuous antigen-binding sites found within the variable regions of heavy and light chain polypeptides.

[0135] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can apply this "Kabat numbering" system to any variable region sequence without relying on experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., US Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also be numbered using systems such as the Chothia numbering system, the AbM numbering system, and the Contact numbering system.

[0136] Kabat numbering is a standard used to number residues in antibodies, typically for identifying CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothia numbering system is similar to the Kabat system, but it takes into account the location of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The AbM numbering system uses a set of integrated computer programs mimicking antibody structures, created by the Oxford Molecular Group. See, for example, Martin et al., ProcNatl Acad Sci (USA), 86: 9268-9272 (1989); "AbM" TM , A Computer Program for ModelingVariable Regions of Antibodies (AbM TM "Computer programs for modeling antibody variable regions" Oxford, UK; Oxford Molecular, Ltd. The AbM numbering system uses known databases and an ab initio method to model the tertiary structure of antibodies from primary sequences, as described, for example, in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198 (1999). The Contact numbering system is based on the analysis of valid complex crystal structures; see, for example, MacCallum et al., J. Mol. Biol., 5: 732-45 (1996).

[0137] In some embodiments, the CDR of the antibodies of the present invention is defined according to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, and the Contact numbering system. In some embodiments, the CDR of the antibodies of the present invention is defined according to the AbM numbering system.

[0138] "Treatment" refers to therapeutic treatments and preventative or preventative measures aimed at preventing, mitigating, improving, or stopping adverse physiological changes or disorders, such as disease progression, including but not limited to the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, mitigation, reduction, or disappearance of the disease state (whether partial or complete), and prolongation of expected survival without treatment. Patients requiring treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those needing prevention of the condition or disorder, as well as those who can or are expected to benefit from the application of the antibody or pharmaceutical composition provided by this invention for detection, diagnostic procedures, and / or treatment.

[0139] The term "cancer" refers to or is intended to describe a physiological state in mammals characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoma. More specific examples of such cancers include neuroblastoma, prostate cancer, breast cancer, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, osteosarcoma, melanoma, lung cancer such as non-small cell lung cancer, glioma such as glioblastoma, pancreatic cancer, head and neck cancer, leukemia, cervical cancer, kidney cancer, squamous cell carcinoma, gastric cancer, liver cancer such as hepatocellular carcinoma, mesothelioma, anal cancer, skin cancer, vulvar cancer, fibroblastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignancy, sarcoma, brain cancer, central nervous system tumors, and brain metastases.

[0140] The terms “overexpression” or “overexpressed” are interchangeable in referring to a gene that is typically transcribed or translated at a detectably higher level in certain cells, such as cancer cells, compared to normal cells. Overexpression can be the overexpression of a protein or RNA (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability, and altered protein degradation), as well as localized overexpression (increased nuclear localization) and enhanced functional activity resulting from altered protein transport patterns, such as increased enzymatic hydrolysis of substrates. Overexpression can be 50%, 60%, 70%, 80%, 90%, or more compared to normal or control cells.

[0141] As used herein, the term "administer" means the delivery of a substance to achieve a therapeutic purpose. Administration can be parenteral, enteral, or local. Parenteral administration is typically by injection and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0142] As used herein, the term "effective dose" or "therapeutic effective dose" refers to an amount of a drug, such as an antibody, sufficient to reduce or improve the severity and / or duration of a condition (e.g., cancer) or one or more of its symptoms; prevent disease progression; induce disease remission; prevent recurrence, development, onset, or progression of one or more symptoms associated with the condition; detect the condition; or enhance or improve the preventive or therapeutic effect of another therapy (e.g., a prophylactic or therapeutic agent). For example, an effective dose of an antibody can inhibit tumor growth (e.g., inhibit an increase in tumor volume); reduce tumor growth (e.g., reduce tumor volume); reduce the number of cancer cells; and / or alleviate one or more symptoms associated with cancer to some extent. For example, an effective dose can improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of recurrence.

[0143] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody or derivative used, the patient's age and weight, general health condition, sex and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors will be determined by a healthcare professional, including those skilled in the art. The dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using pharmacological and pharmacokinetic principles well known in the art. In some embodiments, the effective dosage ranges from about 0.01 mg / kg to about 100 mg / kg, and may be, for example, once daily or up to once monthly. It should be noted that dosage values ​​may vary depending on the type and severity of the symptoms to be relieved. Furthermore, it should be understood that a specific dosing regimen for any particular patient may be adjusted at any time based on the patient's needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges set forth herein are merely illustrative and are not intended to limit the scope or practice of the required composition.

[0144] The terms "patient" and "subject" are used interchangeably and refer to any mammal requiring diagnosis, prognosis, or treatment, including but not limited to humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc. In some implementations, the patient is a human patient.

[0145] As used herein, the term "need" means that a patient has been identified as requiring a particular method or treatment. In some embodiments, this identification can be made through any diagnostic approach. The patient may require any of the methods and treatments described herein.

[0146] As used in this article, the term "administration" refers to the application of a substance to achieve a therapeutic purpose (e.g., treating a tumor).

[0147] The term "reagent" as used in this article refers to chemical compounds, mixtures of chemical compounds, biological macromolecules, or extracts derived from biological materials.

[0148] The term "medicine" or "drug" refers to a compound or composition that, when properly administered to a patient, can induce a desired therapeutic effect.

[0149] "Approximately" refers to a typical error range for the corresponding value that is readily known to those skilled in the art. In some embodiments, "approximately" as used herein refers to the described value and its range of ±10%, ±5%, or ±1%.

[0150] EC 50 "That is, the half-maximal concentration (EC50)." 50 () refers to the concentration that can cause 50% of the maximum effect.

[0151] The binding specificity of the antibody or antigen-binding fragment of the present invention can be detected by in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA), surface plasmon resonance, flow cytometry (FACS), or enzyme-linked immunosorbent assay (ELISA).

[0152] The term "pharmaceuticalally acceptable" refers to a substance approved by a government regulatory agency or listed in a recognized pharmacopoeia for use in animals, and especially in humans. Furthermore, "pharmaceuticalally acceptable excipients" generally refer to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0153] "Excipients" refer to diluents, adjuvants, excipients, or carriers that can be administered to patients together with the active ingredient. These drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline, glucose, and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid, chelating agents, and tonic agents such as dextran or dextrose are also foreseeable. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions will contain a clinically effective dose of antibody or antigen-binding fragments or fusion proteins, preferably in a purified form, along with an appropriate amount of excipients to provide a dosage form suitable for the patient. The formulation should be suitable for the dosing regimen. Parent formulations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0154] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be used, allowing the active ingredient to be mixed prior to administration.

[0155] The antibody or antigen-binding fragment or fusion protein of the present invention may be in neutral or salt form. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0156] Antibody preparation

[0157] Various methods for preparing antibodies are known in this field, such as hybridoma technology, recombinant DNA technology, transgenic mouse technology, and phage display library methods.

[0158] Antibodies can be prepared using conventional recombinant DNA techniques. Vectors and cell lines for antibody production can be selected, constructed, and cultured using techniques well-known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as *Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells*, DL Hacker, FM Wurm, in *Reference Module in Life Sciences*, 2017, the entire contents of which, including supplementary information, are incorporated herein by reference.

[0159] In some embodiments, antibody-encoding DNA can be designed and synthesized according to the antibody amino acid sequence described herein using conventional methods, placed into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in a culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody-encoding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters of long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as actin promoters. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX, pcDNA3.1 (+ / -), pcDNA3.1 / Zeo (+ / -), pcDNA3.1 / Hygro (+ / -), pcDNA3.1 / G418 (+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian host cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0160] In some implementations, the inserted gene fragment needs to contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, and hygromycin resistance genes to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the aforementioned genes. After culturing in a selective medium, successfully transfected cells grow in large numbers, producing the desired target protein. The resulting antibody can be separated or purified using conventional techniques, such as protein A-agarose gel chromatography, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0161] All content of the references cited in this article, including supplementary information, is incorporated into the full text through citation.

[0162] The technical solution of the present invention will be further illustrated below through specific embodiments. These specific embodiments do not represent a limitation on the scope of protection of the present invention, and any non-essential modifications or adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0163] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0164] Example 1: Screening of anti-B7H3 antibodies

[0165] scFv phage display technology was used to screen for antibodies specifically targeting B7H3. The general screening protocol was as follows: Magnetic beads were washed with PBS and divided into two portions. One portion was used to bind to the human-B7H3-his antigen (catalog number: B73-H52E2, Acro), while the other portion was used to block a phage library. The mixture was incubated overnight at 4°C. The next day, the antigen-magnetic bead complex was added to the blocked phage library and incubated. After incubation, the mixture was washed multiple times to remove non-specifically bound phages. 100 μL of trypsin was added to elute the phages specifically bound to the human-B7H3-his antigen from the magnetic beads. The phages were then infected with E. coli TG1 cells in the logarithmic growth phase and cultured overnight for amplification. On the third day, the amplified phages were purified and extracted. The above steps were repeated, and after multiple rounds of screening, positive phages specifically binding to human-B7H3-his were finally enriched.

[0166] After screening the scFv phage display library, plasmids were extracted, digested with enzymes, and ligated into the pET-22b vector (containing a Myc tag). The plasmids were then electroporated into BL21 competent cells. The electroporation products were transferred to resuscitation medium for recovery, and the recovered products were serially diluted, plated, and incubated overnight. The next day, single clones were picked from the plates with the titers determined and transferred to *E. coli* expression medium (Invitrogen). Amplification was performed at 37°C and 240 rpm for 6 hours (h), followed by overnight incubation at 28°C. After incubation, the supernatant was transferred by centrifugation. The supernatant contained the scFv antibody to be identified. This antibody contained a Myc tag, and the specific binding of the antigen and antibody could be determined by detecting this tag. The structure of scFv is VH-GS linker-VL (sequence composition shown in Table 1-1), and the sequence of the GS linker is GGGGSGGGGSGGGGS (SEQ ID NO: 48). For example, the full sequence of 1C1-scFv is shown in SEQ ID NO: 53.

[0167] Positive clones were identified by ELISA. Human-B7H3-his antigen was coated at a concentration of 2 μg / mL, 100 μL / well, onto a 96-well ELISA plate and incubated overnight at 4 °C. On the second day, wash three times with PBST, add bovine serum albumin diluted with 5% PBST solution for blocking (200 μL / well), and incubate at 37°C for 3 h; wash three times with PBST, take 60 μL of the above scFv expression supernatant and mix thoroughly with 60 μL of PBS, transfer 100 μL / well to ELISA plates, and incubate at 37°C for 1 h; wash five times with PBST to remove unbound antibodies, add anti-Myc-HRP (9B11) enzyme-labeled antibody, and incubate at 37°C for 1 h; wash eight times with PBST to remove unbound antibodies, add 100 μL of TMB chromogenic solution (Shanghai Sangon Biotech, catalog number E66100), and incubate at 37°C for 15 min; finally, add 50 μL of 0.1 M H2SO4 to stop the reaction, read the absorbance at 450 nm using a microplate reader, and send clones with high OD values ​​for sequencing analysis. After ELISA identification and sequencing analysis, the positive clones obtained finally included: 1A9-scFv, 1B1-scFv, 1B8-scFv, 1C1-scFv, 1D2-scFv, 1D4-scFv, 1F5-scFv, 1H6-scFv, 2C3-scFv, 2D3-scFv, and 2G10-scFv.

[0168] Table 1-1 Sequence composition of anti-B7H3 scFv

[0169]

[0170] Example 2: Preparation of anti-B7H3 antibody

[0171] The nucleotide sequences encoding the heavy and light chains of the anti-B7H3 antibody were cloned into recombinant expression vectors, respectively. All anti-B7H3 antibodies had identical light chain sequences, as shown in SEQ ID NO: 5, and the variable region sequence of the light chain was shown in SEQ ID NO: 4. The sequence composition of the anti-B7H3 antibody is shown in Tables 1-2, and the CDR was defined according to the AbM numbering system.

[0172] HEK293F cells (ATCC) were transiently transfected at a 1:1 ratio with an expression vector for both the light and heavy chains of the antibody using PEI reagent (catalog number: 24765-1, Polysciences). Prior to transfection, cell viability was above 95%. The plasmid-PEI complex was added to the cells during transfection. At 24 h and 72 h post-transfection, 10% (v / v) of OPM-CHO PFF05 (catalog number: F81279-001; OPMI) was added to the cells. The expression supernatant was collected after 5–7 days, centrifuged at high speed to remove impurities, and purified using a Protein A column. The column was washed with PBS, and the antibody protein was eluted with acidic elution buffer (pH 3.0–3.5). The pH was adjusted to neutral with 1M Tris-HCl. The eluted sample was concentrated using an ultrafiltration tube, replaced with PBS buffer, aseptically filtered, and aliquoted for storage.

[0173] Table 1-2 Sequence composition of anti-B7H3 antibody

[0174]

[0175] Example 3: ELISA detection of antibody-antigen binding

[0176] Human antigen B7H3-his (catalog number: B73-H52E2, Acro) was coated at a concentration of 2 μg / mL, 100 μL / well, onto a 96-well ELISA plate and incubated overnight at 4 °C. On the second day, after washing three times with PBST, 200 μL of bovine serum albumin diluted with 5% PBST solution was added for blocking, and the plates were incubated at 37°C for 3 hours. After washing three times with PBST, all positive antibodies were serially diluted 12 times (3-fold) starting at 33.3 nM, and 100 μL / well was transferred to each well of an ELISA plate, incubated at 37°C for 1 hour. After washing five times with PBST to remove unbound antibodies, anti-human kappa light chain-HRP (catalog number: A7164, Sigma) was added, and the plates were incubated at 37°C for 1 hour. After washing eight times with PBST to remove unbound antibodies, 100 μL of TMB chromogenic buffer (Shanghai Sangon Biotech, catalog number: E66100) was added, and the plates were incubated at 37°C for 15 minutes. Finally, 50 μL of 0.1 M H₂SO₄ was added to stop the incubation, and the absorbance was read at 450 nm using a microplate reader. A curve was plotted with OD value on the ordinate and antibody concentration on the abscissa to analyze the antigen-antibody binding. The results are as follows: Figure 1 As shown, all positive antibodies have strong binding ability, EC 50 The value is between 0.05nM and 0.1nM.

[0177] Example 4: ELISA detection of interspecies specificity of antibodies

[0178] The mouse antigen B7H3-his (catalog number: B73-M52H4, Acro) was coated at a concentration of 1 μg / mL, 100 μL / well, onto a 96-well ELISA plate and incubated overnight at 4 °C. On the second day, after washing three times with PBST, 200 μL of bovine serum albumin diluted with 5% PBST solution was added for blocking, and the plates were incubated at 37°C for 3 hours. After washing three times with PBST, all positive antibodies were serially diluted eight times (3-fold serially) starting at 33.3 nM, and 100 μL / well was transferred to each well of the ELISA plate, incubated at 37°C for 1 hour. After washing five times with PBST to remove unbound antibodies, anti-human kappa light chain-HRP (catalog number: A7164, Sigma) was added, and the plates were incubated at 37°C for 1 hour. After washing eight times with PBST to remove unbound antibodies, 100 μL of TMB chromogenic solution (Shanghai Sangon Biotech, catalog number: E66100) was added, and the plates were incubated at 37°C for 15 minutes. Finally, 50 μL of 0.1 M H₂SO₄ was added to stop the incubation, and the absorbance was read at 450 nm using a microplate reader. A curve was plotted with OD value on the ordinate and antibody concentration on the abscissa to analyze the antigen-antibody binding. The results are as follows: Figure 2 As shown.

[0179] Example 5: FACS detection of the binding of B7H3 positive cells to antibodies

[0180] Flow cytometry was used to detect the binding of anti-B7H3 antibody to human breast cancer cells MDA-MB-468 and human degenerative cancer cells Calu-6 (Shanghai Cell Bank, Chinese Academy of Sciences), which highly express B7H3. The experimental procedure was as follows: MDA-MB-468 and Calu-6 cells were collected, washed once with PBS, and resuspended in PBS. The cell density was adjusted to 3 million / mL, and 100 μL / well was seeded into 96-well V plates (catalog number: 3897, Costa). The antibody was pre-diluted to 20 μg / mL with PBS, and then 3-fold diluted 10 times, resulting in 11 concentration gradients. 50 μL / well was transferred to the cells in the 96-well V plates, mixed, and incubated at 4°C for 1 h. The cells were washed twice with PBS, and 100 μL (1:1000 dilution) of goat anti-human IgG-Fc PE fluorescent secondary antibody (catalog number: 12-4998-82, eBioscience) was added. Incubate at 4℃ for 1 hour, wash twice with PBS, and analyze using a CytoFLEX flow cytometer (model: AOO-1-1102, Beckman Coulter). Plot a curve with fluorescence value on the ordinate and antibody concentration on the abscissa to analyze the binding of the antibody to B7H3 positive cells (two different flow cytometers were used for the flow cytometry analysis, hence the difference in fluorescence intensity). The binding curve of the antibody to B7H3 positive Calu-6 cells is shown below. Figure 3a and Figure 3b As shown, the binding curve with MDA-MB-468 is as follows: Figure 4a and 4b As shown.

[0181] Example 6: SPR detection of anti-B7H3 antibody kinetics and affinity constant

[0182] The kinetic constants (including binding rate (Ka), dissociation rate (Kd), and binding-dissociation equilibrium constant (K) of the human-B7H3-his antigen with the aforementioned anti-B7H3 antibody were determined by surface plasmon resonance (SPR) on a Biacore T200 (GE Healthcare) instrument. DThe general experimental procedure was as follows: Following the manufacturer's instructions (GE Healthcare), detection was performed using a protein A chip. 5 μg / mL antibody dilution was passed through the experimental flow paths (Fc2, Fc4) at a flow rate of 10 µL / min for 15 seconds. The flow rate was then adjusted to 30 µL / min, and different concentrations of human-B7H3-his (catalog number: B73-H52E2, Acro) antigen dilutions (0 nM, 1.235 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM) were analyzed sequentially, simultaneously flowing through the experimental flow paths (Fc2, Fc4) and the reference flow paths (Fc1, Fc3). The binding time was 90 s, and the dissociation time was 300 s. Finally, the chip was regenerated by introducing glycine pH 1.5 buffer for 60 s before starting the next cycle. The kinetic binding constant was estimated by nonlinearly fitting the sensing data to a 1:1 binding model provided by the Biacore T200 evaluation software. The antibody affinity results are shown in the table below:

[0183] Table 2. Affinity constants of antibody binding to human-B7H3-His

[0184]

Claims

1. An antibody or antigen-binding fragment that specifically binds to B7H3 and comprises one or more amino acid sequences from (a) to (f): (a) HCDR1, comprising an amino acid sequence as shown in any one of SEQ ID NO: 14, 6, 9, 12, 16, 18, 20, 22-24; (b) HCDR2, comprising an amino acid sequence as shown in any one of SEQ ID NO: 15, 7, 10, 13, 17, 19, 21, 25; (c) HCDR3, which contains the amino acid sequence shown in SEQ ID NO: 11 or 8; (d) LCDR1, which contains the amino acid sequence shown in SEQ ID NO: 1; (e) LCDR2, which contains the amino acid sequence shown in SEQ ID NO: 2; (f) LCDR3, which contains the amino acid sequence shown in SEQ ID NO:

3.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises HCDR1 as shown in any one of SEQ ID NO: 14, 6, 9, 12, 16, 18, 20, 22-24, HCDR2 as shown in any one of SEQ ID NO: 15, 7, 10, 13, 17, 19, 21, 25, or HCDR3 as shown in SEQ ID NO: 11 or 8.

3. The antibody or antigen-binding fragment as described in claim 1 or 2, wherein the antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, and LCDR3 as shown in SEQ ID NO:

3.

4. The antibody or antigen-binding fragment according to any one of claims 1-3, wherein the antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:14, HCDR2 as shown in SEQ ID NO:15, and HCDR3 as shown in SEQ ID NO:11; or, The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:6, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:8; or... The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11; or... The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:12, HCDR2 as shown in SEQ ID NO:13, and HCDR3 as shown in SEQ ID NO:11; or... The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:17, and HCDR3 as shown in SEQ ID NO:11; or... The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, and HCDR3 as shown in SEQ ID NO:11; or... The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:20, HCDR2 as shown in SEQ ID NO:21, and HCDR3 as shown in SEQ ID NO:11; or... The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:22, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:11; or... The antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:7, and HCDR3 as shown in SEQ ID NO:11; or... The antibody or antigen-binding fragment includes HCDR1 as shown in SEQ ID NO:24, HCDR2 as shown in SEQ ID NO:25, and HCDR3 as shown in SEQ ID NO:8; The antibody or antigen-binding fragment comprises LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, and LCDR3 as shown in SEQ ID NO:

3.

5. The antibody or antigen-binding fragment according to any one of claims 1-4, wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO: 51 or 26-35, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 51 or 26-35, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NO: 51 or 26-35.

6. The antibody or antigen-binding fragment according to any one of claims 1-5, wherein the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 49, 50 or 4, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 49, 50 or 4, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO: 49, 50 or 4.

7. An antibody or antigen-binding fragment, wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in any one of SEQ ID NO: 51 or 26-35, and the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 49, 50 or 4.

8. The antibody or antigen-binding fragment according to any one of claims 1-7, wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 29 or 51, and the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 49, 50 or 4.

9. The antibody or antigen-binding fragment according to any one of claims 1-8, wherein the antibody or antigen-binding fragment is an IgG isotype; or, the antibody or antigen-binding fragment is an IgG1 or IgG4 isotype.

10. The antibody or antigen-binding fragment according to any one of claims 1-9, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, the heavy chain constant region comprising an amino acid sequence as shown in SEQ ID NO: 47, or an amino acid sequence having at least 80% or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 47, or an amino acid sequence having one or more conserved amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 47; and / or The light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:46, or an amino acid sequence having at least 80% or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:46, or an amino acid sequence having one or more conserved amino acid substitutions with the amino acid sequence shown in SEQ ID NO:

46.

11. The antibody or antigen-binding fragment according to any one of claims 1-8, wherein the antibody or antigen-binding fragment is scFv, Fab, Fab', or F(ab)2; or, The antibody or antigen-binding fragment is scFv, and the structure of the scFv is NH2-heavy chain variable region (VH)-linker-light chain variable region (VL)-COOH or NH2-light chain variable region-linker-heavy chain variable region-COOH, wherein the linker is (GGGGS)n, and n is 1, 2, 3, 4, 5 or 6; or the n is 3 or 4; or the scFv contains an amino acid sequence as shown in any one of SEQ ID NO: 53-55.

12. An antibody, wherein the heavy chain of the antibody comprises an amino acid sequence as shown in any one of SEQ ID NO:36-45, and the light chain of the antibody comprises an amino acid sequence as shown in SEQ ID NO:

5.

13. A biomaterial, comprising: 1) A polynucleotide encoding an antibody or antigen-binding fragment as described in any one of claims 1-11 or a portion or all of an antibody as described in claim 12; or, 2) An expression vector comprising a polynucleotide encoding a portion or all of an antibody or antigen-binding fragment as described in any one of claims 1-11 or an antibody as described in claim 12; or, 3) A cell comprising a portion or all of a polynucleotide encoding an antibody or antigen-binding fragment as claimed in any one of claims 1-11 or an antibody as claimed in claim 12.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of claims 1-11, a portion or all of an antibody as described in claim 12, or a biological material as described in claim 13, and a pharmaceutically acceptable carrier.

15. The use of the antibody or antigen-binding fragment of any one of claims 1-11, part or all of the antibody of claim 12, the biomaterial of claim 13, or the pharmaceutical composition of claim 14 in the preparation of a medicament for diagnosing and / or treating a disease.

16. The application as described in claim 15, wherein the disease is selected from tumors, respiratory diseases, skin and musculoskeletal diseases, genitourinary diseases, nervous system diseases, and digestive system diseases; or, the tumor is a benign tumor or cancer; or, the tumor is a hematologic malignancy or solid tumor; or, the tumor is a tumor that expresses B7H3 positively; or, the tumor is selected from neuroblastoma, prostate cancer, breast cancer, non-small cell lung cancer, ovarian cancer, renal cell carcinoma, osteosarcoma, melanoma, lung cancer such as non-small cell lung cancer, glioma such as glioblastoma, pancreatic cancer, head and neck cancer, leukemia, cervical cancer, kidney cancer, squamous cell carcinoma, gastric cancer, liver cancer such as hepatocellular carcinoma, mesothelioma, anal cancer, skin cancer, vulvar cancer, fibroblastic small round cell tumor, medulloblastoma, meningioma, peritoneal malignant tumor, sarcoma, brain cancer, central nervous system tumors, and brain metastases.

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