Anti-B7-H3 antibody and its use

Abstract

The present invention relates to novel antibodies and antigen-binding fragments thereof that specifically bind to B7-H3, and compositions comprising said antibodies or antigen-binding fragments thereof. The present invention further relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof, host cells comprising said nucleic acids, and related uses. Furthermore, the present invention relates to therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.

Description

[Technical Field]

[0001] This invention relates to a novel antibody that specifically binds to B7-H3 and its antigen-binding fragment, as well as a composition comprising the antibody or its antigen-binding fragment. Furthermore, this invention relates to nucleic acids encoding the antibody or its antigen-binding fragment, host cells containing the nucleic acid, and related uses. Moreover, this invention relates to the therapeutic and diagnostic uses of these antibodies or their antigen-binding fragments. [Background technology]

[0002] B7-H3 (also known as CD276) is a type I transmembrane glycoprotein that is very similar in structure to PD-L1, and both belong to the B7 / CD28 superfamily. B7-H3 is widely expressed at the transcriptional level (RNA) in lymphoid and non-lymphoid organs, but protein expression of B7-H3 is very limited, mainly expressed in activated dendritic cells, mononuclear cells, T lymphocytes, B lymphocytes, and Nk lymphocytes, with very low expression levels in other normal tissues. Recently, B7-H3 has been highly expressed in various solid tumors, such as lung cancer, gastric cancer, pancreatic cancer, prostate cancer, kidney cancer, ovarian cancer, endometrial cancer, colorectal cancer, liver cancer, and breast cancer, and its overexpression has been found to be closely associated with survival, prognosis, or tumor level. In addition to its high expression in tumors, B7-H3 may have a function similar to PD-L1-mediated T cell suppression signaling. It has been proposed that B7-H3 possesses co-stimulatory and co-inhibitory functions depending on tumor specificity, microenvironmental factors, and signal intensity. In addition to functioning as an immunomodulator, B7-H3 is associated with promoting cancer metastasis and angiogenesis.

[0003] Because B7-H3 expression is primarily limited to tumors, B7-H3 is a very important tumor-associated antigen and can be used as a potential target for broad-spectrum immunotherapy. [Overview of the project]

[0004] This invention provides an anti-B7-H3 antibody, its coding gene, and applications. The inventors obtain the anti-human B7-H3 antibody of the present invention, which has high affinity and specificity, by screening hybridomas, constructing chimeric antibodies, and humanizing them.

[0005] In one embodiment, the present invention provides a novel antibody or its antigen-binding fragment that binds to a B7-H3 molecule.

[0006] In some embodiments, the anti-B7-H3 antibody of the present invention is (i) It binds to human and cynomolgus monkey B7-H3 with high affinity, (ii) Effectively binds to B7-H3 on the cell surface, (iii) Effectively binds to soluble B7-H3, (iv) Effectively activate the ADCC effect, (v) To effectively suppress or reduce the growth and progression of tumors within the body, It possesses one or more of the above characteristics.

[0007] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH), wherein the VH is (i) Three complementarity-determining regions (CDRs) in the VH of any of the antibodies shown in Table B, or (ii) Three heavy chain complementarity determining regions (CDRs) of any antibody shown in Table A, or (iii) An amino acid sequence having one or more (preferably 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the VH sequence of any antibody shown in Table B, wherein the amino acid mutations do not occur in the CDR region, or (iv) A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH sequence of any of the antibodies shown in Table B, and comprising a sequence containing the corresponding CDR of the said sequence.

[0008] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment of the present invention comprises a light chain variable region (VL), wherein the VL is (i) Three complementarity-determining regions (CDRs) contained in the VL of any of the antibodies shown in Table B, or (ii) Three light chain complementarity determining regions (CDRs) of any antibody shown in Table A, or (iii) An amino acid sequence having one or more (preferably 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the VL sequence of any antibody shown in Table B, wherein the amino acid mutations do not occur in the CDR region, or (iv) A sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VL sequence of any of the antibodies shown in Table B, and comprising a sequence containing the corresponding CDR of the said sequence.

[0009] In some embodiments, the anti-B7-H3 antibody of the present invention or its antigen-binding fragment comprises a heavy chain variable region VH and / or a light chain variable region VL, where, (a) The VH is, (i) Three complementarity-determining regions (CDRs) in the VH of any of the antibodies shown in Table B, or (ii) Three heavy chain complementarity determining regions (CDRs) of any antibody shown in Table A, or (iii) An amino acid sequence having one or more (preferably 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the VH sequence of any antibody shown in Table B, wherein the amino acid mutations do not occur in the CDR region, or (iv) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the VH sequence of any of the antibodies shown in Table B and containing the corresponding CDRs of said sequence, and / or (b) said VL is (i) the three complementarity determining regions (CDRs) contained in the VL of any of the antibodies shown in Table B, or (ii) the three light chain complementarity determining regions (CDRs) of any of the antibodies shown in Table A, or (iii) an amino acid sequence having one or more (preferably 10 or less, more preferably 5 or less, 4 or less, 3 or less, 2 or less, 1 or less) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) with respect to the VL sequence of any of the antibodies shown in Table B, said amino acid mutations not occurring in the CDR regions, or (iv) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the VL sequence of any of the antibodies shown in Table B and containing the corresponding CDRs of said sequence,

[0010] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that binds to B7-H3, comprising the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region shown by any one of SEQ ID NOs: 16, 18, 20 and 22, and / or one of the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region shown by any one of SEQ ID NOs: 17, 19, 21 and 23, or variants of combinations of said CDR sequences.

[0011] In some other embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that binds to B7-H3, comprising three complementarity-determining regions HCDR of the heavy-chain variable region and three complementarity-determining regions LCDR of the light-chain variable region, wherein HCDR1 comprises or consists of the amino acid sequence represented by SEQ ID NO: 1 or 8, HCDR2 comprises or consists of the amino acid sequence represented by any one of SEQ ID NO: 2, 7, 9 and 14, HCDR3 comprises or consists of the amino acid sequence represented by SEQ ID NO: 3 or 10, LCDR1 comprises or consists of the amino acid sequence represented by any one of SEQ ID NO: 4, 11 and 15, LCDR2 comprises or consists of the amino acid sequence represented by SEQ ID NO: 5 or 12, and LCDR3 comprises or consists of the amino acid sequence represented by SEQ ID NO: 6 or 13.

[0012] In some embodiments, the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof that binds to a B7-H3 molecule, comprising a heavy-chain variable region VH and / or a light-chain variable region VL, wherein 1) the VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH represented by SEQ ID NO: 16, and the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL represented by SEQ ID NO: 17; 2) the VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH represented by SEQ ID NO: 18, and the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL represented by SEQ ID NO: 19; 3) the VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH represented by SEQ ID NO: 20, and the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL represented by SEQ ID NO: 21, or 4) the VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH represented by SEQ ID NO: 22, and the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL represented by SEQ ID NO: 23.

[0013] In some embodiments, the present invention provides an anti-B7-H3 antibody or its antigen-binding fragment, comprising a heavy chain variable region VH and / or a light chain variable region VL, wherein (i) The VH comprises HCDR1, HCDR2, and HCDR3, where HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1 or 8, HCDR2 comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 2, 7, 9, and 14, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or 10. and / or (ii) The VL includes LCDR1, LCDR2, and LCDR3, where LCDR1 includes or consists of the amino acid sequence shown in any one of SEQ ID NOs: 4, 11, and 15; LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NOs: 5 or 12; and LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NOs: 6 or 13.

[0014] In some embodiments, the present invention is 1) HCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 1, HCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 2, HCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 3, LCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 4, LCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 5, LCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 6, 2) HCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 1, HCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 7, HCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 3, LCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 4, LCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 5, LCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 6, 3) HCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 8, HCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 9, HCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 10, LCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 11, LCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 12, LCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 13, 4) HCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 8, HCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 14, HCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 10, LCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 15, LCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 12, LCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 13, This invention provides an anti-B7-H3 antibody or its antigen-binding fragment containing the antibody.

[0015] In some embodiments, the present invention provides an anti-B7-H3 antibody or its antigen-binding fragment, comprising a heavy chain variable region VH and / or a light chain variable region VL, wherein (a) The heavy chain variable region VH is, (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one of sequence numbers 16, 18, 20, and 22, and comprising or including the corresponding CDR sequence of said sequence, (ii) containing or consisting of the amino acid sequence shown in any one of sequence numbers 16, 18, 20, and 22, or (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) with respect to the amino acid sequence represented by any one of SEQ ID NOs. 16, 18, 20, and 22, preferably such amino acid mutations do not occur in the CDR region. and / or (b) The light chain variable region VL is (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one of sequence numbers 17, 19, 21, and 23, and comprising the corresponding CDR sequence of said sequence, (ii) containing or consisting of the amino acid sequence shown in any one of sequence numbers 17, 19, 21, and 23, or (iii) The amino acid sequence includes one or more (preferably 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the amino acid sequence represented by any one of SEQ ID NOs. 17, 19, 21, and 23, wherein the amino acid mutations do not occur in the CDR region.

[0016] In some embodiments, the present invention is 1) Heavy chain variable region VH containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16, and light chain variable region VL containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 17. 2) Heavy chain variable region VH containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 18, and light chain variable region VL containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 19. 3) Heavy chain variable region VH containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 20, and light chain variable region VL containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 21. 4) Heavy chain variable region VH containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 22, and light chain variable region VL containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 23. This invention provides an anti-B7-H3 antibody or its antigen-binding fragment containing the antibody.

[0017] In some embodiments, the present invention is 1) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 16, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 17, 2) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 19, 3) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 20, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 21, 4) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 22, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 23, This invention provides an antibody or its antigen-binding fragment that binds to B7-H3 containing the present antibody.

[0018] In some embodiments, the present invention provides an anti-B7-H3 antibody or its antigen-binding fragment comprising a heavy chain and / or a light chain, wherein (a) The heavy chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one of sequence numbers 24, 26, 28, and 30, and comprising or consisting of an amino acid sequence that includes the corresponding CDR sequence of said sequence, (ii) containing or consisting of the amino acid sequence shown in any one of sequence numbers 24, 26, 28, and 30, or (iii) The amino acid sequence having one or more (preferably 20 or fewer or 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the amino acid sequence represented by any one of SEQ ID NOs: 24, 26, 28, and 30, wherein the amino acid mutations do not occur in the CDR region of the heavy chain, and more preferably, the amino acid mutations do not occur in the variable region of the heavy chain. and / or (b) The light chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one of sequence numbers 25, 27, 29, and 31, and comprising or consisting of an amino acid sequence that includes the corresponding CDR sequence of said sequence, (ii) containing or consisting of the amino acid sequence shown in any one of sequence numbers 25, 27, 29, and 31, or (iii) The amino acid sequence comprises one or more (preferably 20 or fewer or 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) with respect to the amino acid sequence represented by any one of SEQ ID NOs. 25, 27, 29, and 31, wherein the amino acid mutations do not occur in the CDR region of the light chain, and more preferably, the amino acid mutations do not occur in the variable region of the light chain.

[0019] In some embodiments, the present invention is 1) A heavy chain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 24, and a light chain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 25. 2) A heavy chain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 26, and a light chain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 27. 3) A heavy chain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 28, and a light chain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 29. 4) A heavy chain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 30, and a light chain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 31. This invention provides an anti-B7-H3 antibody or its antigen-binding fragment containing the antibody.

[0020] In some embodiments, the present invention is 1) A heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 24, and a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 25, 2) A heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 27, 3) A heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 28, and a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 29, 4) A heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 30, and a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 31, This invention provides an antibody or its antigen-binding fragment that binds to B7-H3 containing the present antibody.

[0021] In some embodiments, the anti-B7-H3 antibody of the present invention is an antibody in the IgG1 form, an antibody in the IgG2 form, an antibody in the IgG3 form, or an antibody in the IgG4 form, and preferably the anti-B7-H3 antibody is an antibody in the IgG1 form.

[0022] In some embodiments, the anti-B7-H3 antibody is a monoclonal antibody.

[0023] In some embodiments, the anti-B7-H3 antibody is a chimeric antibody, and in preferred embodiments, the anti-B7-H3 antibody is a humanized antibody. The anti-B7-H3 antibody of the present invention also includes antibody fragments, which are preferably selected from Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain antibodies (such as scFv), single-domain antibodies, bispecific antibodies (dAb), or linear antibodies.

[0024] In some embodiments, the present invention provides isolated nucleic acids encoding an anti-B7-H3 antibody or an antigen-binding fragment thereof, a vector containing the nucleic acid, or a host cell containing the nucleic acid or the vector.

[0025] In some embodiments, the present invention provides a method for producing an anti-B7-H3 antibody or its antigen-binding fragment, comprising culturing host cells according to the present invention under conditions suitable for the expression of the nucleic acid of the antibody or its antigen-binding fragment according to the present invention. In another embodiment, the present invention provides an anti-B7-H3 antibody and its antigen-binding fragment produced by the method described above.

[0026] In some embodiments, the present invention provides immune complexes and pharmaceutical compositions comprising an anti-B7-H3 antibody or its antigen-binding fragment.

[0027] In some embodiments, the present invention further provides applications of anti-B7-H3 antibodies or their antigen-binding fragments, immune complexes, or pharmaceutical compositions in the preparation of drugs for preventing or / or treating B7-H3-related diseases or conditions (such as tumors).

[0028] In some embodiments, the present invention further provides methods for preventing and / or treating B7-H3-related diseases or conditions (such as tumors), the methods comprising administering an effective amount of an antibody or antigen-binding fragment thereof, an immune complex, or a pharmaceutical composition that conjugates to B7-H3 of the present invention to a target.

[0029] The present invention further relates to a method for detecting B7-H3 molecules in a sample, the method comprising (a) contacting an antibody or antigen-binding fragment described in the present invention with a sample, and (b) detecting a complex formed in the sample of the antibody or antigen-binding fragment and a B7-H3 molecule.

[0030] In another embodiment, the present invention further relates to a method for diagnosing tumors expressing the B7-H3 molecule in a subject, the method comprising (a) obtaining a sample of the subject, (b) contacting the sample with an antibody or antigen-binding fragment according to the present invention, and (c) detecting a complex formed in the sample of the antibody or antigen-binding fragment and the B7-H3 molecule. [Brief explanation of the drawing]

[0031] The following preferred embodiments of the invention, described in detail below, will be better understood when read in conjunction with the following drawings. For illustrative purposes, the drawings show the current preferred embodiments of the invention. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings. [Figure 1] This is the result of FACS detection of antibody binding to CHOS cells overexpressing human B7H3. [Figure 2] This is the result of detecting antibody ADCC activity. [Figure 3] These are the antitumor results of antibodies in the body. Here, Figure 3a shows the change in tumor volume in tumor-bearing mice, and Figures 3b and 3c show the change in body weight of tumor-bearing mice. [Modes for carrying out the invention]

[0032] I. Definition Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methodologies, forms, or reagents described herein, as these may be modified. Furthermore, the terms used in this invention are merely for the purpose of describing specific embodiments and are not intended to limit the scope of the invention; the scope of the invention is understood to be limited solely by the claims. Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0033] The following definitions are used to interpret this specification, and where appropriate, a singular term may include a plural form, and vice versa. It should be understood that the terms used herein are merely descriptive and not limiting to specific embodiments.

[0034] The term "approximately," when used with a number or figure, means covering a range of numbers or figures that is 5% smaller than the lower limit and 5% larger than the upper limit.

[0035] When the term "and / or" is used to connect two or more options, it should be understood to refer to any one of the options, or any two or more of the options.

[0036] The terms “contain” or “include” mean to include the aforementioned elements, integers, or steps, but not to exclude any other elements, integers, or steps. Where the terms “contain” or “include” are used herein, unless otherwise specified, they also include combinations of the aforementioned other elements, integers, or steps. For example, when it is referred to to “contain” an antibody variable region of a particular sequence, it is also intended to include the antibody variable region consisting of that particular sequence.

[0037] The term “antibody” is used herein in its broadest sense and includes, but is not limited to, multiple antibody structures, including monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (such as bispecific antibodies), single-chain antibodies, complete antibodies, or antibody fragments exhibiting desired antigen-binding activity. A complete antibody typically contains at least two full-length heavy chains and two full-length light chains, but in some cases it contains relatively fewer chains; for example, antibodies naturally occurring in camels may contain only heavy chains.

[0038] The term “antigen-binding fragment” (which may be used interchangeably with “antibody fragment” and “antigen-binding moiety” herein) refers to a molecule distinct from the complete antibody that contains a portion of the complete antibody and binds to an antigen bound to the complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bispecific antibodies (diabodies, dAb), linear antibodies, single-chain antibodies (such as scFv), single-domain antibodies, antigen-binding fragments of bivalent or bispecific antibodies, camelid antibodies, and other fragments exhibiting the desired ability to bind to an antigen (such as B7-H3).

[0039] "Affinity" or "binding affinity" refers to the affinity of intrinsic bonds, which reflects the interaction between members of a bond pair. The affinity of molecule X for its partner Y can usually be expressed by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by conventional methods known in this art. One specific method for measuring affinity is the ForteBio dynamical coupling assay described herein.

[0040] As used herein, the term “Fc region” defines the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the constant region. The term includes both native sequence Fc regions and mutant Fc regions. In some embodiments, the Fc region of a human IgG heavy chain is typically extended from Cys226 or Pro230 to the carbonyl end of the heavy chain. Meanwhile, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the amino acid residue numbers in the Fc region or constant region refer to Kabat et al., Sequences of Proteins of Immunological Interest, 5 th This follows the EU numbering system (also known as the EU Index) described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0041] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, within which each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (e.g., Kindt et al. Kuby Immunology, 6). th (See ed., WH Freeman and Co., page 91 (2007)). A single VH domain or VL domain can also confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening complementary libraries of VL domains or VH domains using the VH domain or VL domain of an antibody that binds to that antigen, see, for example, Portolano et al., J.Immunol. 150:880~887 (1993) and Clarkson et al., Nature 352:624~628 (1991).

[0042] A “complementarity-determining region,” “CDR region,” “CDR,” or “hypervariable region” (which can be used interchangeably with “HVR” in this specification) is a region within the antibody variable domain whose sequence is hypervariable and which forms a structurally determined ring (“hypervariable loop”) and / or contains antigen contact residues (“antigen contact sites”). CDRs primarily play a role in binding to antigen epitopes. Heavy chain and light chain CDRs are numbered sequentially from the N-terminus and are usually referred to as CDR1, CDR2, and CDR3. CDRs located within the heavy chain variable domain of an antibody are also called HCDR1, HCDR2, and HCDR3, and CDRs located within the light chain variable domain of an antibody are called LCDR1, LCDR2, and LCDR3. In the amino acid sequence of a given light chain variable region or heavy chain variable region, the CDR sequence can be determined by various methods known in the field. For example, the Kabat complementarity-determining region (CDR) is the most common, determined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, on the other hand, refers to the location of the structural ring (Chothia and Lesk, J. Mol. Biol. 196:901~917 (1987)). AbM CDRs are a trade-off between Kabat CDRs and Chothia structural rings and are used by Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of the complex crystalline structure obtained. Depending on the different CDR determination proposals, the residues of each HVR / CDR in these CDRs are as follows:

[0043] [Table 1] The CDR is a CDR sequence that is positioned at the following Kabat residue positions based on the Kabat numbering system.

[0044] These are positions 24-36 or 24-34 (LCDR1) in VL, positions 46-56 or 50-56 (LCDR2), and positions 89-97 or 89-96 (LCDR3), and positions 26-35 or 27-35B (HCDR1) in VH, positions 50-65 or 49-65 (HCDR2), and positions 93-102, 94-102, or 95-102 (HCDR3).

[0045] In one embodiment, the boundary of the HCDR1 antibody of the present invention is determined by the AbM rule, and the boundaries of HCDR2, HCDR3, and LCDR are determined by the Kabat rule, as shown in Table A below, for example.

[0046] The CDR may be determined by having the same Kabat numbering position as the sequence of reference CDRs (e.g., any one of the exemplary CDRs of the present invention).

[0047] Unless otherwise specified, in this invention, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbered positions based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0048] Unless otherwise specified, in the present invention, the terms "CDR" or "CDR sequence" include a CDR sequence determined by any one of the methods described above.

[0049] However, it should be recognized that there may be differences in the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems. That is, there are differences in the CDR sequences of the variable region of the same antibody as defined by different assignment systems. Therefore, when an antibody is limited by a specific CDR sequence as defined in the present invention, the range of the antibody may also include antibodies whose variable region sequence includes the specific CDR sequence, but whose CDR boundary differs from the specific CDR boundary defined in the present invention because a different approach (e.g., different rules or combinations of assignment systems) is used.

[0050] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, despite the differences in CDRs among antibodies, the number of amino acid positions directly involved in antigen binding in a CDR is limited. At least two of the Kabat, Chothia, AbM, Contact, and North methods can be used to determine the minimum overlapping region and provide a "minimal binding unit" for antigen binding. The minimal binding unit may be a subpart of the CDR. As is known to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody structure and protein folding. Accordingly, the present invention also considers variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimal binding unit may remain unchanged, while the remaining CDR residues, defined based on Kabat or Chothia, may be replaced with conserved amino acid residues.

[0051] The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which certain cytotoxic effector cells (e.g., NK cells, neutrophils, macrophages) specifically bind to antigen-carrying target cells by binding to secretory immunoglobulins at Fc receptors (FcRs) present in these cells, thereby killing the target cells with cytotoxins. ADCC-mediated cells are primarily NK cells expressing only FcγRIII, while mononuclear cells express FcγRI, FcγRII, and FcγRIII. To evaluate the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, or the ADCC activity of the target molecule can be evaluated in vivo, for example, in an animal model. The examples herein provide exemplary assays for evaluating ADCC activity.

[0052] The term "functional Fc region" refers to an Fc region that possesses "effector function" over a naturally occurring Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors, BCRs). Such effector functions are generally determined by the relationship between the Fc region and its binding domain (e.g., antibody-variable domain), which can be evaluated, for example, by several measurement methods disclosed in this invention.

[0053] As used in this invention, the term "therapeutic agent" includes any substance effective in preventing or treating tumors (such as cancer), and includes chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

[0054] As used herein, the term “immunomodulator” refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response may be a humoral response or a cellular response.

[0055] The term "effective dose" refers to the amount or dosage of the antibody or its fragments, complexes, or compositions of the present invention that, after administration to a patient, produces the desired effect in a patient requiring treatment or prevention. For therapeutic or preventive purposes, the "effective dose" can be divided into a "therapeutic effective dose" and a "preventive effective dose." The effective dose can be easily determined by a physician skilled in the art, taking into account various factors such as the mammalian species, volume, age, and health status, as well as the specific disease involved, the degree or severity of the disease, the individual patient's response, the specific antibody administered, the dosage form, the bioavailability characteristics of the administered formulation, the chosen dosage regimen, and the application of combination therapy.

[0056] In one embodiment, compared to a control, an effective amount of the B7-H3 antibody of the present invention suppresses measurable parameters (e.g., tumor growth rate, tumor volume, etc.) preferably by at least about 20%, and more preferably by at least about 40%.

[0057] The terms “host cell,” “host cell line,” and “host cell culture” may be used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including their offspring. Host cells include “transformed organisms” and “transformed cells,” including primary transformed cells and their offspring regardless of passage number. Offspring may not be exactly the same as the parent cells in terms of nucleic acid content and may contain mutations. This specification includes mutant offspring with the same function or biological activity screened or selected from primary transformed cells.

[0058] The term "chimeric antibody" refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species. For example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody.

[0059] The term "humanized antibody" refers to an antibody in which an antigen-binding site derived from another mammalian species, such as the mouse reproductive system, is attached to a human immunoglobulin sequence. Humanized antibodies are typically prepared using recombinant technology and are chimeric molecules that allow for additional framework region modifications within the human framework sequence. The antigen-binding site may include a fully variable domain fused to the constant region, or it may include only a complementarity-determining region transplanted into the appropriate framework sequence within the variable domain. In some embodiments, a humanized antibody contains at least one, typically two, of the nearly all variable domains, of which all or nearly all CDRs (e.g., six CDRs) correspond to portions derived from the non-human antibody, and all or nearly all FRs correspond to portions derived from the human antibody. A humanized antibody may also contain at least a portion of the antibody constant region derived from the human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to the antibody that has undergone humanization.

[0060] The term "immune complex" refers to an antibody that has been combined with one or more other substances (including, but not limited to, cytotoxic agents or labels).

[0061] As used herein, the term “labeling” refers to a compound or composition that is directly or indirectly compounded or fused to a reagent (e.g., a polynucleotide probe or antibody) and facilitates detection by the reagent to which it is compounded or fused. Labeling can either be detectable itself (e.g., radioisotope labeling or fluorescent labeling) or, when labeled by an enzyme catalyst, can catalyze the chemical modification of a detectable substrate compound or composition. The term is intended to include direct labeling of a probe or antibody by coupling (i.e., physical linking) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reaction with another directly labeled reagent. Examples of indirect labeling include the detection of a primary antibody by a fluorescently labeled secondary antibody, and the use of terminal labeling of a biotin-containing DNA probe detectable by a fluorescently labeled streptavidin protein.

[0062] The terms “individual” or “subject” include mammals. Mammals include, but are not limited to, livestock (e.g., cattle, goats, cats, dogs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0063] The term "isolated" antibody refers to an antibody that has been isolated from its natural environment. In some embodiments, the antibody is purified to a purity of 95% or more than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).

[0064] "Nucleic acid encoding an isolated anti-B7-H3 antibody or its antigen-binding fragment" means one or more nucleic acid molecules encoding the antibody heavy chain or light chain (or its antigen-binding fragment), and includes such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations within a host cell.

[0065] The sequence identity between sequences is calculated as follows.

[0066] To determine 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 sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison). In one preferred embodiment, the length of the reference sequence to be aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Next, 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.

[0067] Mathematical algorithms can be used to perform sequence comparisons between two sequences and calculate the percentage of identity. In one preferred embodiment, the percentage of identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444~453) algorithm (available at http: / / www.gcg.com), which is integrated into the GAP program of the GCG software package, using a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the NWSgapdna.CMP matrix, gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6, which is integrated into the GAP program of the GCG software package (available at http: / / www.gcg.com). A particularly preferred parameter set (and the one parameter set to be used unless otherwise specified) employs a Blossum 62 scoring matrix with a gap penalty of 12, a gap stretching penalty of 4, and a frameshift gap penalty of 5.

[0068] Alternatively, the percentage of identity between two amino acid or nucleotide sequences may be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11~17) integrated into the ALIGN program (version 2.0), utilizing the PAM120 weighted remainder table, gap length penalty 12, and gap penalty 4.

[0069] Additionally or selectively, by using the nucleic acid and protein sequences described herein as “query sequences” to perform searches on public databases, for example, sequences of other family members or related sequences can be identified.

[0070] The term "medicinal adjuvants" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete or incomplete)), excipients, vectors, and stabilizers that are administered together with the active substance.

[0071] The term "pharmaceutical composition" refers to a composition in which the active ingredient contained herein exists in a form that enables its biological activity, and which does not contain any other ingredient that is toxic to the subject to which the composition is administered.

[0072] As used herein, “treatment” means reducing, interrupting, delaying, relieving, stopping, decreasing, or reversing the progression or severity of any existing symptoms, symptoms, conditions, disease, or illness.

[0073] As used herein, “prevention” includes inhibiting the onset or progression of a disease, condition, or symptoms associated with a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a prevention program. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug before the onset of any signs or symptoms associated with cancer, particularly before cancer develops in subjects at risk of developing cancer.

[0074] The term “vector,” as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid ligated to it. The term includes vectors as self-replicating nucleic acid structures, and vectors linked to the genome of a host cell into which they are introduced. Some vectors can guide the expression of a nucleic acid manipulably ligated to them. Such vectors are referred to herein as “expression vectors.”

[0075] The term "subject / patient sample" refers to a collection of tissue or cell samples obtained from a patient or subject. The tissue or cell samples may be derived from solid tissue (such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples), blood or any blood component, body fluids (such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid), or cells derived from pregnancy or any stage of development in the subject. II. Antibodies Unless otherwise specified, the terms "B7-H3", "B7H3" and "CD276" can be used interchangeably herein. B7-H3 is a type I transmembrane glycoprotein belonging to the B7 / CD28 superfamily member, and has a sequence similar to the extracellular domain of PD-L1. B7-H3 has 316 amino acids including a putative signal peptide consisting of 28 amino acids, an extracellular region and transmembrane region consisting of 217 amino acids, and a cytoplasmic domain consisting of 45 amino acids, and has a molecular weight of about 45 kDa to 66 kDa. In the human body, due to exon duplication, the extracellular structure of B7-H3 may be an IgV-IgC-like domain (2Ig-B7-H3) or an IgV-IgC-IgV-IgC-like domain (4Ig-B7-H3). The sequence of cynomolgus monkey B7-H3 has about 90% homology with the corresponding human sequence.

[0076] As used herein, the terms "anti-B7-H3 antibody", "anti-B7-H3", "B7-H3 antibody" or "antibody against B7-H3" refer to an antibody whose antibody or antigen-binding fragment can bind to the B7-H3 protein with sufficient affinity. Said antibody can be used as a diagnostic agent and / or therapeutic agent targeting B7-H3.

[0077] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof of the present invention binds to B7-H3 (such as human or cynomolgus monkey B7-H3) with sufficient affinity, for example, binds to B7-H3 with the following equilibrium dissociation constant (K D ), and the K D ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (for example, 10 -7 M or less, for example, 10 -7 M to 10 -10 M). In some embodiments, B7-H3 is human or cynomolgus monkey B7-H3. In some embodiments, the antibody binding affinity is measured using a biosensing method by optical interference. For example, in the biosensing by optical interference, the antibody has a K -7 of about 1×10 D, about 5×10 -8 K below M D , about 1×10 -8 K below M D , about 5×10 -9 K below M D , about 1×10 -9 K below M D , about 1×10 -10 K below M D It then binds to human B7-H3.

[0078] In some embodiments, the antibody or antigen-binding fragment of the present invention binds to B7-H3 expressed on the surface of a cell.

[0079] In some embodiments, the antibody or antigen-binding fragment of the present invention can induce an ADCC effect. In some embodiments, the antibody or antigen-binding fragment of the present invention can suppress and / or reduce the growth and / or volume of tumors in the body.

[0080] In some embodiments, the antibody or antigen-binding fragment conjugating to B7-H3 of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), where the VH and VL comprise a combination selected from the six CDRs shown in Table A.

[0081] In one embodiment of the present invention, the amino acid mutation described herein includes amino acid substitution, insertion, or deletion. Preferably, the amino acid mutation described herein is an amino acid substitution, and preferably a conservative substitution.

[0082] In a preferred embodiment, the amino acid mutation described in the present invention occurs in a region outside the CDR (e.g., FR). More preferably, the amino acid mutation described in the present invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region.

[0083] In some embodiments, the substitutions are conservative substitutions. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same type, for example, one acidic amino acid being substituted with another acidic amino acid, one basic amino acid being substituted with another basic amino acid, or one neutral amino acid being substituted with another neutral amino acid. Exemplary substitutions are shown in the table below.

[0084] [Table 2] In one embodiment, the substitution occurs in the CDR region of the antibody. Generally, the resulting mutant has modifications (e.g., improvements) to certain biological properties (e.g., enhanced affinity) compared to the parent antibody, and / or retains some substantially preserved biological properties of the parent antibody. An exemplary substitution mutant is an affinity-mature antibody.

[0085] In some embodiments, antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites in antibodies can be easily achieved by modifying the amino acid sequence so that one or more glycosylation sites are produced or removed. If the antibody contains an Fc region, the sugars attached to it can be modified. In some applications, modifications to remove undesirable glycosylation sites are useful, such as removing a fucose module to improve antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, complement-dependent cell-mediated cytotoxicity (CDC) can be modified by galactosidation modification.

[0086] In one embodiment, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein in order to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions. For examples of Fc variants, see U.S. Patent No. 7,332,581, U.S. Patent No. 6,737,056, U.S. Patent No. 6,737,056, WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591~6604 (2001), U.S. Patent No. 6,194,551, WO 99 / 51642 and Idusogie et al. J. Immunol. 164:4178~4184 (2000), U.S. Patent No. 7,371,826, Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351.

[0087] In one embodiment, it may be necessary to produce antibodies modified by cysteine ​​engineering, for example, "thioMAb" antibodies in which one or more residues of the antibody are substituted with cysteine ​​residues. For example, cysteine-engineered antibodies can be produced as described in U.S. Patent No. 7,521,541.

[0088] In some embodiments, the antibodies provided herein can be further modified to include other known and readily available non-protein moieties in the art. Moieties suitable for the inducing action of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. III. Nucleic acids of the present invention, vectors containing the same, and host cells The present invention provides nucleic acids encoding any of the aforementioned anti-B7-H3 antibodies or antigen-binding fragments thereof. A vector comprising the nucleic acid is further provided. In one embodiment, the vector is an expression vector.

[0089] The present invention further provides host cells comprising the nucleic acid or the vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from Escherichia coli cells, mammalian cells (e.g., CHO cells or 293 cells) or other cells suitable for the production of antibodies or their antigen-binding fragments. In another embodiment, the host cell is prokaryotic. In one embodiment, the host cell is selected from Escherichia coli cells.

[0090] As will be apparent to those skilled in the art, due to the degenerative nature of the genetic code, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0091] These polynucleotide sequences can be generated using methods known in the field, either by solid-phase DNA synthesis from scratch or by PCR inducing a sequence encoding an antibody or antigen-binding fragment that binds to B7-H3.

[0092] In one embodiment, one or more vectors comprising the nucleic acid of the present invention are provided. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. The vector includes, but is not limited to, a virus, plasmid, cosmid, lambda phage, or yeast artificial chromosome (YAC).

[0093] In one embodiment, a host cell containing the vector is provided. Suitable host cells for cloning or expressing an antibody-encoding vector include prokaryotic or eukaryotic cells as described herein. For example, the antibody can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, 5,840,523, and Charlton, Methods in Molecular Biology, vol 248 (edited by BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, Expression of antibody fragments in Escherichia coli. After expression, the antibody can be isolated from a paste-like substance of bacterial cells in the soluble fraction and further purified.

[0094] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or their antigen-binding fragments. For example, eukaryotic microorganisms such as filamentous fungi and yeasts are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have already been "humanized" produce antibodies having a partially or completely human glycosylated form. See Gerngross, Nat. Biotech. 22:1409~1414 (2004) and Li et al., Nat. Biotech. 24:210~215 (2006). Host cells suitable for the expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells may be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of usable mammalian host cell lines include the SV40-transformed monkey kidney-derived CV1 line (COS-7) and the human fetal kidney line (293HEK or 293 cells, e.g., Graham et al., J. Gen Virol. 36:59 (1977)). Other usable mammalian host cell lines include Chinese hamster ovary (CHO) cells such as DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:216 (1980)) and myeloma cell lines such as Y0, NS0, and Sp2 / 0. Some mammalian host cell lines suitable for antibody production are briefly described, for example, in Yazaki & Wu, Methods in Molecular Biology, vol 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). IV. Production and Purification of Antibody Molecules of the Present Invention In one embodiment, the present invention provides a method for producing an anti-B7-H3 antibody or a fragment thereof (preferably an antigen-binding fragment), the method comprising culturing host cells under conditions suitable for the expression of a nucleic acid encoding the antibody or a fragment thereof (preferably an antigen-binding fragment), and optionally isolating the antibody or a fragment thereof. In one embodiment, the method further comprises isolating the anti-B7-H3 antibody or a fragment thereof from host cells. V. Multispecific antibodies In a further embodiment, the present invention provides a multispecific (including bispecific) antibody molecule that specifically binds to B7-H3. In one embodiment, in a multispecific antibody, the antibody of the present invention (or its antigen-binding fragment) forms a first binding specificity to B7-H3.

[0095] In one embodiment, binding specificity is provided by the antibody's "binding site" or "antigen-binding site" (the region in the antibody molecule that actually binds to the antigen). In a preferred embodiment, the antigen-binding site consists of a VH / VL pair comprising the antibody's light chain variable domain (VL) and heavy chain variable domain (VH). Thus, in one embodiment, a "multispecific" antibody is an antibody having at least two antigen-binding sites, where each of the at least two antigen-binding sites can bind to different epitopes of the same antigen or to different epitopes of different antigens.

[0096] For information on multispecific antibodies and their manufacturing, please refer to the descriptions in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792, and WO 2010 / 145793, for example. VI. Immune complexes In some embodiments, the present invention provides immune complexes generated by conjugating antibodies of the present invention to heterologous molecules. In some embodiments, the heterologous molecule is a therapeutic or diagnostic agent, such as a cytotoxic agent or a chemotherapeutic agent. The cytotoxic agent includes any agent that is harmful to cells. Examples of cytotoxic agents suitable for the generation of immune complexes are known in the art.

[0097] Linkers can be used to covalently bond various solids of a complex. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a “cleavable linker” that facilitates the release of polypeptides after delivery to the target site. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or linkers containing disulfides can be used (Chari et al., Cancer Research 52(1992)127~131;US5,208,020).

[0098] In some embodiments, the antibodies of the present invention can be conjugated with diagnostic agents or detectable reagents. Such complexes can be used as part of clinical testing methods (e.g., to confirm the efficacy of a specific therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or illness. Such diagnosis and detection can be achieved by coupling the antibodies with detectable reagents, which include, but are not limited to, a variety of enzymes such as horseradish peroxidase, prosthetic groups such as streptavidin / biotin and avidin / biotin, fluorescent substances, luminescent substances, radioactive substances, and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron-emitting imaging applications.

[0099] In some embodiments, the immune complex is used for the prevention or treatment of tumors. In some embodiments, the tumor is cancer. VII. Pharmaceutical Compositions, Pharmaceutical Formulations, and Combination Products The present invention further comprises compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an anti-B7-H3 antibody or its immune complex or a multispecific antibody, and compositions comprising a polynucleotide encoding an anti-B7-H3 antibody or its immune complex or a multispecific antibody. These compositions may also optionally contain appropriate medicinal adjuvants, such as pharmaceutically acceptable vectors known in the art and pharmaceutically acceptable excipients including buffers.

[0100] The pharmaceutical composition or formulation of the present invention may be further combined with one or more other active ingredients, the active ingredients being necessary for the treatment of a specific indication and preferably having complementary activities that do not adversely affect each other.

[0101] Accordingly, in one embodiment, the present invention also provides a combination product of drugs. In one embodiment, the combination product comprises the antibody, immune complex, or multispecific antibody of the present invention and a second therapeutic agent in the preparation of the same pharmaceutical composition or formulation. In another embodiment, the combination product comprises the antibody, immune complex, or multispecific antibody of the present invention and a second therapeutic agent, separately contained in different pharmaceutical compositions or formulations. The second therapeutic agent may be administered before, simultaneously with, or after administration of the antibody of the present invention (e.g., in the same formulation or a different formulation).

[0102] The pharmaceutical compositions, formulations, and combination products of the present invention can be provided as products for the treatment, prevention, and / or diagnosis of the diseases and / or conditions described herein. The products may include containers and labels or instructions for use. Suitable containers include, for example, bottles, syringes, and IV infusion bags. Containers can be manufactured from a variety of materials, such as glass or plastic. In one embodiment, the product may include (a) a first container containing the antibody or antibody fragment, immune complex, or multispecific antibody of the present invention, and optionally (b) a second container containing a second therapeutic agent. Furthermore, the product may also include other materials desirable from a commercial and user perspective, including pharmaceutically acceptable diluents such as buffers and sterile water for injection, needles, syringes, and injection pumps. VIII. Applications According to one aspect of the present invention, a method for preventing and / or treating a B7-H3-related disease or condition (such as cancer) is provided, comprising administering an effective amount of the anti-B7-H3 antibody or its antigen-binding fragment, immune complex, or pharmaceutical composition of the present invention to a target.

[0103] The subjects may be mammals such as primates, and preferably higher primates such as humans. In one embodiment, the subjects are suffering from or at risk of suffering from the diseases described herein. In one embodiment, the subjects are receiving or have already received other treatments such as chemotherapy and / or radiotherapy. In another embodiment, the present invention provides the application of anti-B7-H3 antibodies or their antigen-binding fragments, immune complexes or pharmaceutical compositions in the production or preparation of drugs for the prevention and / or treatment of B7-H3-related diseases or conditions referred to herein.

[0104] In some embodiments, the antibodies, antibody fragments, immune complexes, compositions, or products of the present invention can delay the onset of disease symptoms and / or symptoms associated with the disease.

[0105] The antibody of the present invention (and pharmaceutical compositions or immune complexes containing the same, and any additional therapeutic agents) may be administered by any suitable method, including parenteral administration, intrapulmonary administration, intranasal administration, and, in the case of local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. The method of administration is determined to some extent by whether the administration is short-term or long-term, and the administration may be by any suitable route, including injection, such as intravenous or subcutaneous injection. In this specification, however limited, various administration schedules include single doses or multiple doses at multiple times, bolus administration, pulse infusion, etc.

[0106] When the antibody of the present invention is used for the prevention or treatment of a disease, the appropriate dosage (when administered alone or in combination with one or more additional therapeutic agents) is determined by the type of disease being treated, the type of antibody, the severity and course of the disease, whether the administration of the antibody is for preventive or therapeutic purposes, the patient's previous treatment history, the patient's clinical history, the patient's response to the antibody, and the judgment of the attending physician. The antibody is administered appropriately to the patient in a single treatment or over a series of treatments.

[0107] In the methods of the present invention described above, the composition of the present invention, a multispecific antibody, or an immune complex may be administered instead of the antibody or antigen-binding moiety of the present invention. Alternatively, in these methods, the composition of the present invention, a multispecific antibody, or an immune complex may be further administered in addition to the antibody or antigen-binding moiety of the present invention. IX. Methods and compositions for diagnosis and detection In some embodiments, any anti-B7-H3 antibody or its antigen-binding fragment provided herein can be used to detect the presence of B7-H3 in a biological sample. In one embodiment, the results of the detection are used for the diagnosis or auxiliary diagnosis of a disease. When used herein, the term “detection” includes quantitative or qualitative detection, and exemplary detection methods include immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody molecule-compound magnetic beads, ELISA assay techniques, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid sample derived from an organism. In some embodiments, the biological sample includes cells or tissue. In some embodiments, the biological sample is derived from an overgrowth or cancerous lesion.

[0108] In one embodiment, the present invention provides a method and reagent kit for detecting B7-H3 in a biological sample. In one embodiment, B7-H3 is human B7-H3 or cynomolgus monkey B7-H3. In one embodiment, the method includes contacting a biological sample with an anti-B7-H3 antibody described herein under conditions that allow for the binding of the antibody to B7-H3, and detecting whether a complex has been formed between the anti-B7-H3 antibody and B7-H3. The method may be performed in vitro or in vivo. In some embodiments, the sample is derived from a cancer patient. The sample may be a tissue biopsy, tissue section, blood, plasma, or serum or other bodily fluid.

[0109] In some embodiments, the present invention provides a method for treating a B7-H3 related disease or condition (such as cancer or a tumor), comprising administering a therapeutically effective dose of an anti-B7-H3 antibody to a target. In another embodiment, the method further comprises administering one or more other therapies to a target.

[0110] In one embodiment, the anti-B7-H3 antibody is used to select a target suitable for treatment with the anti-B7-H3 antibody, for example, B7-H3 being a biomarker for selecting the target. In one embodiment, the antibody of the present invention can be used to diagnose tumors and, for example, to evaluate (e.g., monitor) the treatment or progression, diagnosis and / or staging of the disease described herein in the target. In one embodiment, the anti-B7-H3 antibody of the present application can be used to determine the presence or absence of cancer cells in various tissues (such as the ovaries, lungs, mammary glands, prostate, kidneys, pancreas, thyroid gland, and brain). The anti-B7-H3 antibody of the present application can be used to determine the presence or absence of cancer cells and B7-H3 levels released into the circulating blood from solid tumors, and the circulating B7-H3 antigen may be a complete B7-H3 molecule or a fragment thereof. The detection method is performed by a method such as FACS.

[0111] In one embodiment, a labeled anti-B7-H3 antibody is provided. The labeling includes, but is not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, high electron density labels, chemiluminescent labels, and radioactive labels), and moieties indirectly detectable by, for example, enzyme-catalyzed reactions or intermolecular interactions, such as enzymes or ligands. X. Exemplary anti-B7-H3 antibody of the present invention

[0112] [Table 3]

[0113] [Table 4]

[0114] [Table 5-1] [Table 5-2] [Table 5-3] Examples To aid in understanding the present invention, the following embodiments are described. These embodiments are not intended to limit the scope of the claims of the present invention in any way, and those skilled in the art can make various modifications based on the description in this specification.

[0115] Unless explicitly stated otherwise, the implementation of this invention will utilize the technical and routine methods of the art of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology.Descriptions of these methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Ed., 2001), Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed., 1989), Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience, Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985), Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992), Transcription and See also the translation (B. Hames & S. Higgins, Eds., 1984), Perbal, A Practical Guide to Molecular Cloning (1984), Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Current Protocols in Immunology QEColigan (AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and special publications such as Advances in Immunology. Example 1. Production of hybridoma cells immunized animal Following conventional methods, Bal b / c mice (Beijing Weitong Lihua) were immunized with recombinant human 4Ig-B7H3 protein (SEQ ID NO: 32) (SINO BIOLOGICAL, product number 11188-H08H). Recombinant human 4Ig-B7H3 protein (50 ug per mouse) was homogeneously mixed with an equal volume of TiterMax (Sigma, product number T2684-1ML) adjuvant, and administered subcutaneously every two weeks for a total of five immunizations.

[0116] Cell fusion After the serum titer met the requirements, the mouse spleen was removed according to conventional methods to prepare a B lymphocyte suspension, which was then mixed with SP2 / 0 myeloma cells (ATCC, CRL-1581) in a 1:2 to 1:1 ratio before electrofusion. The fused cells were transferred from the electrode dish to a 50 mL centrifuge tube, diluted with screening medium (the composition of the preparation is shown in Table 1), and a cell suspension (concentration: 1 × 10⁻⁶) was prepared. 4 cells / mL ~ 2 × 10 4 We obtained (100 μL of cells / mL). We added 100 μL of cell suspension per well to a 96-well plate. On day 5 after fusion, we replaced the screening medium with fresh medium. Depending on the cell growth stage, we screened positive clones by detection by flow cytometry (FACS) after culturing for 10 days (or longer).

[0117] [Table 6] High-throughput screening of hybridoma cells Hybridoma cells specifically expressing anti-B7H3 antibodies were screened using flow cytometry (FACS). Simply put, CHO cells expressing human B7H3 (CHO-huB7H3) were counted, and 1 × 10⁻⁶ cells were identified. 6Diluted to 100 μL / mL, then added 100 μL to each well of a U-bottom 96-well plate, centrifuged at 500 g for 5 min, and removed the cell medium. Subsequently, each culture supernatant from the hybridoma 96-well plate and the positive control antibody (MGA271) were added to a U-shaped plate containing CHO cells, respectively, to resuspend the cells at 100 μL per well, and incubated on ice for 30 min. Further centrifugation at 500 g for 5 min was performed to remove the supernatant, and the cells were washed once with PBS solution. Centrifugation at 500 g for 5 min was performed to remove the PBS solution. 100 μL of anti-mouse Fab FITC-labeled secondary antibody (diluted in PBS solution at a 1:500 ratio) was added to each well, and 100 μL of anti-human Fab FITC-labeled secondary antibody was added to the positive control antibody culture well. Incubated on ice under light-shielded conditions for 30 min, centrifuged at 500 g for 5 min to remove the supernatant, and the cells were washed once with PBS solution. Next, the cells were resuspended in 50 μL of PBS solution, detected by FACS, and positive clones were screened for identification.

[0118] The obtained positive clones were re-screened using CHO cells expressing cynomolgus monkey B7H3 (SEQ ID NO: 34) (CHO-cynoB7H3) in the same manner as described above, and a total of two hybridoma cell lines, 19A2 and 20G5, which bind to both human B7H3 and monkey B7H3, were obtained.

[0119] Using biofilm thin-layer interferometry (ForteBio), the affinity of the two resulting hybridoma cell lines for the antigen was measured, and the resulting KD values ​​are shown in Table 2.

[0120] [Table 7] Subcloning of positive hybridoma cells Based on the results of cell binding and affinity measurements, subcloning was performed on the aforementioned clone.

[0121] The specific steps were as follows: A basic medium was obtained by replacing HAT in the screening medium with HT (Gibco, Cat#11067-030), and this was added to a 96-well plate at a rate of 200 μL per well. Approximately 1 × 10⁶ positive hybridoma cells screened by the fusion were added. 5 The cell suspension was added to the first row of a 96-well plate at a density of 100 cells / mL at a rate of 300 μL per well and thoroughly mixed. 100 μL of the cell suspension was taken from the first row and added to the second row, thoroughly mixed, then another 100 μL was taken and added to the next row, repeating the above steps until the last row was reached, and the mixture was allowed to stand for 15 minutes. Cells were observed and counted under a microscope, and a volume corresponding to 100 cells was added to 20 mL of the basic medium, which was then mixed uniformly at a rate of 200 μL per well and seeded. After 2 days, the cells were observed under a microscope, and monoclonal wells were identified and labeled. When the cell confluence in each well reached 50% or more, detection was performed using the high-throughput FACS screening method, target-positive wells were removed, and the resulting cell clones were cryopreserved.

[0122] The positive control antibody used in this invention is MGA271, also known as Enoblituzumab (derived from MacroGenics US20160264672A1).

[0123] Example 2. Production of Chimeric Antibodies Using molecular biology techniques, the light and heavy chain gene sequences of the antibody were extracted from the hybridoma-positive clone obtained in Example 1 and used to construct a human-mouse chimeric antibody.

[0124] 1. Hybridoma sequencing Newly cultured approximately 5 x 10 6 RNA was extracted from individual hybridoma cells, and cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). The steps were as follows:

[0125] Preparation of reaction system I in Table 3

[0126] [Table 8] After incubation at 65°C for 5 minutes, the mixture was rapidly cooled on ice. A total of 20 μL of reaction system I was added to the following reverse transcription system (Table 4).

[0127] [Table 9] After slow and uniform mixing, reverse transcription and translation were performed according to the conditions of 42°C for 60 minutes followed by 95°C for 5 minutes, and then the mixture was cooled on ice to obtain cDNA.

[0128] After ligating the cDNA to a T vector, the heavy chain and light chain variable regions of the cDNA-amplified antibody were obtained by PCR using the Mighty TA-cloning Kit reagent kit (Takara). The PCR reaction system is shown in Table 5.

[0129] [Table 10] The PCR reaction conditions are shown in Table 6.

[0130] [Table 11] 4.5 μL of the PCR product obtained by the aforementioned PCR reaction was taken, 0.5 μL of pMD20-T vector (Takara) and 5 μL of Ligation Mighty Mix (Takara) were added, and the mixture was gently and uniformly mixed. The reaction was carried out at 37°C for 2 hours to obtain the linked product.

[0131] Transformed cells: 5 μL of the obtained ligation product was added to E. coli TOP10-receptor cells (Tiangen Biochemical Technology (Beijing) Co., Ltd.), mixed uniformly, and incubated on ice for 30 minutes. After a 90-second heat shock at 42°C, the cells were rapidly cooled on ice for 2 minutes. 900 μL of LB medium (Bioeng Biotechnology (Shanghai) Co., Ltd.) was added to the EP tube, and the cells were cultured at 37°C for 1 hour in a 220 rpm shaker. The cells were centrifuged at 3000 g for 2 minutes, 800 μL of the supernatant was aspirated and removed, and the remaining cells were resuspended in the medium and coated onto ampicillin-resistant plates. The cells were cultured overnight at 37°C, clones were selected, and sequencing was performed.

[0132] 2. Construction of Chimeric Antibodies PCR amplified the VH and VL regions of the anti-B7H3 antibody produced by the sequenced hybridoma cells of Example 1. Upstream and downstream primer sequences are shown in Tables 7 and 8.

[0133] [Table 12] After mixing in the aforementioned proportions, Primer Mix 1 was obtained for subsequent PCR amplification of VH.

[0134] [Table 13] After mixing in the aforementioned proportions, Primer Mix 2 was obtained for subsequent PCR amplification of VL.

[0135] The PCR systems are shown in Table 9.

[0136] [Table 14] The PCR amplification product was isolated by gel cutting.

[0137] Homologous recombination reaction: Homologous recombination systems are shown in Table 10.

[0138] [Table 15] The reaction was carried out at 37°C for 30 minutes to obtain the recombinant product. The recombinant product was transformed into the TOP10 receptor, a monoclonal was selected and sequenced, and a clone containing the plasmid with the correct insertion direction was selected as the positive clone. By saving the positive clone, the recombinant plasmid for the chimeric antibody was obtained. A certain amount of the recombinant plasmid was extracted and prepared to express the antibody.

[0139] The present invention yields a total of two chimeric antibodies (Ch19A2 and Ch20G5), the CDR sequence, light chain variable region sequence and heavy chain variable region sequence thereof are the same as the corresponding sequences of hybridoma cells in Tables A and B, and the preferred light chain and heavy chain amino acid sequences of the chimeric antibodies are shown in Table C.

[0140] 3. Expression and purification of chimeric antibodies Depending on the required transfection volume, HEK293 cells (Invitrogen) are passaged, and the cell density is increased to 1.5 × 10⁶ the day before transfection. 6 The concentration was adjusted to 10 cells / mL. On the day of transfection, the cell density was approximately 3 × 10⁶. 6 The cell density was 100 cells / mL. The recombinant expression plasmid constructed above was added to 1 / 10 (v / v) Opti-MEM medium (Gibco product number: 31985-070) of the final volume as a transfection buffer, mixed uniformly, and filtered through a 0.22 μm filter head to prepare for use. An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid from the previous step (mass ratio of plasmid to PEI: 1:3), mixed uniformly, and incubated at room temperature for 10 minutes to obtain a DNA / PEI mixture. The DNA / PEI mixture was gently injected into HEK293 cells and mixed uniformly. After incubation for 24 hours at 37°C and 8% CO2, the cells were supplemented with VPA (Sigma, product number: P4543-100G) with a final concentration of 2 mM and a 2% (v / v) feed solution (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone), and the culture was continued for 6 days.

[0141] After cell culture, the cell culture medium was centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected. The supernatant was purified using a pre-packed Hitrap Mabselect Sure column (GE, 11-0034-95) according to the manufacturer's instructions, and the concentration was measured. 100 μg of the purified protein was taken, the concentration was adjusted to 1 mg / mL, and the protein purity was measured using a gel filtration chromatography column SW3000 (TOSOH product number: 18675). The results showed that a high-purity chimeric antibody was obtained. Example 3. Measurement of the binding kinetics of the chimeric antibody and antigen of the present invention using biolayer interference technology. The equilibrium dissociation constant (KD) of the antibody of the present invention, which binds to human B7H3, was measured using biolayer interferometry (ForteBio). ForteBio affinity was measured according to a conventional method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p270-8).

[0142] In short, AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were equilibrated offline in analytical buffer for 30 minutes, then detected online for 60 seconds to establish a baseline. The purified antibodies obtained above were then loaded online onto the AHQ sensor (ForteBio) and the ForteBio affinity was measured. Furthermore, the antibody-loaded sensors were exposed to antigens (including human 4Ig-B7H3, human 2Ig-B7H3 (ACRO, product number B73-H52E2), and cynomolgus monkey B7H3 (SINO BIOLOGICAL, product number 90806-C02H-50)), and then the sensors were transferred to analytical buffer to measure the dissociation rate. The KD values ​​were analyzed using ForteBio analysis software.

[0143] The antibody affinity detection results are shown in Table 11.

[0144] [Table 16] The affinity data mentioned above indicates that the chimeric antibody obtained from hybridomas exhibits good affinity for human B7H3 protein and maintains very high affinity for cynomolgus monkey B7H3. Compared to the control group MGA271, the antibody in this study exhibits higher affinity.

[0145] Example 4. Humanization of chimeric antibodies The chimeric antibody obtained in Example 2 was humanized according to a conventional method. The humanized antibodies obtained in this way were hz20G5 and hz19A2, and their CDR sequences, light chain variable region sequences, heavy chain variable region sequences, and amino acid sequences of the light and heavy chains are shown in Tables A to C.

[0146] Example 5. ForteBio measurement of the affinity of humanized antibodies to antigens. As described in Example 3, the affinity of the humanized antibodies obtained in Example 4 to the antigens (human B7H3 and cynomolgus monkey B7H3) was measured using the ForteBio assay method and expressed as the equilibrium dissociation constant (KD). The results are shown in Table 12.

[0147] [Table 17] Table 12 shows that the humanized antibody still has high affinity for the antigen B7H3, and together with the corresponding chimeric antibody, the equilibrium dissociation constant K for the antigen B7H3 is similar. D It was found that it possesses [the characteristic]. Furthermore, the humanized antibody obtained in this application has a higher binding antigen affinity compared to the control antibody MGA271, and in particular, the affinity of the antibody in this application to human 2Ig-B7-H3 was 20 to 100 times higher than that of MGA271. Example 6. Binding of humanized antibodies to human and cynomolgus monkey B7H3 overexpressing CHO-S cells. To verify whether the antibody of the present invention can bind to antigens expressed on the surface of cells, flow cytometry technology was used to detect the binding of the humanized antibody of the present invention to cells overexpressing human B7H3 and cynomolgus monkey B7H3.

[0148] Construction of cells that overexpress B7H3 ExpiCHO TM Using the Expression System Kit (Invitrogen, catalog number: A29133), the following procedure was performed according to the manufacturer's instructions: cDNA encoding human 4Ig-B7H3 (uniprot: Q5ZPR3, SEQ ID NO: 32), human 2Ig-B7H3 (uniprot: Q5ZPR3-2, SEQ ID NO: 33), and cynomolgus monkey B7H3 (NCBI: XP_015308534.1, SEQ ID NO: 34) was cloned into the pCHO1.0 vector (Invitrogen), and then transfected into CHO-S cells to generate CHOS-hB7H3-4Ig, CHOS-hB7H3-2Ig, and CHOS-cyno B7H3, which are CHO-S cells overexpressing human 4Ig-B7H3, human 2Ig-B7H3, and cynomolgus monkey B7H3, respectively.

[0149] Simply put, 1) CHOS-hB7H3-4Ig, CHOS-hB7H3-2Ig, and CHOS-cyno B7H3 cells in PBS solution. 2 × 10 6 The antibody was diluted to cells / mL, 100 μL was added to each well of a U-bottom 96-well plate, and the antibody diluted with a 3-fold gradient was added.

[0150] 2) The mixture was incubated on ice for 30 minutes. The cells were centrifuged at 400 g for 5 minutes, the supernatant was removed, and the cells were washed with PBS solution to remove unbound antibodies. 100 μL of anti-human Fc antibody (SouthernBiotech) conjugated to PE diluted at 1:200 was added to each well, and the cells were incubated on ice under light-shielded conditions for 30 minutes. The cells were centrifuged at 400 g for 5 minutes, and the supernatant was removed. The cells were washed twice with PBS to remove unbound PE conjugated anti-human Fc antibody. The cells were resuspended in 100 μL of PBS, and antibody binding to the cells was detected by FACS.

[0151] The detection results are shown in Figure 1. For cells overexpressing human 4Ig-B7H3, the overall affinity of the humanized antibody of this invention is equivalent to that of the positive control MGA271. For cells overexpressing human 2Ig-B7H3, the affinity of the hz19A2 antibody is equivalent to that of MGA271, but the affinity of hz 20G5 is clearly higher than that of MGA271. It was found that the antibody of this invention exhibits significantly improved antigen-binding ability at the cellular level. Example 7. Antibody-dependent cell-mediated cytotoxicity (ADCC) In this study, we investigated the effect of the obtained antibody on eliminating tumor cells via the ADCC effect. In this study, we used Promega's Jurkat-ADCCNF-AT luciferase effector cell line (hereinafter abbreviated as ADCC effector cells) to detect the activation status of the NF-AT signaling pathway, thereby detecting the ADCC activity of the antibody. The experimental procedure was specifically as follows.

[0152] 1) Cell preparation CHO-hB7H3-4Ig cells and ADCC effector cells were counted. The supernatant was removed by centrifugation, the cells were washed twice with PBS, and resuspended in detection medium (Gibco 1640 medium with 5% low IgG serum). ADCC effector cells were counted at 1 × 10⁶. 7 The solution was adjusted to have a concentration of cells / mL, and CHO-hB7H3-4Ig cells reached 1 × 10⁶ cells / mL. 6The concentration was adjusted to have cells / mL. Two types of cells were mixed in a 1:1 ratio, and the final ratio of ADCC effector cells to CHO-hB7H3-4Ig cells was 10:1.

[0153] 2) Seeding: Mixed cells were seeded into a 96-well plate at a rate of 100 μL per well, and 50 μL of cells were added to the first well.

[0154] 3) The antibodies of the present invention were added sequentially in various concentration gradients, with the final concentration in the first well being 30 nM, followed by a 3-fold dilution with a total of 10 gradients.

[0155] 4) Incubated in a 37°C incubator for 7 hours.

[0156] 5) After 7 hours, the 96-well plate was removed, and 100 μL of thawed Luciferase test reagent was added to each well. The plate was incubated at room temperature for 20 minutes. Detection was performed using a microplate reader. Concentration-dependent curves were fitted using GraphPad software.

[0157] The detection results are shown in Figure 2. Both the humanized antibody and the chimeric antibody obtained in this application can effectively activate the NF-AT signal, which is a downstream signaling pathway for ADCC activation. As a result, the antibodies in this application have excellent ADCC-killing ability. Furthermore, the humanized antibody obtained in this application has ADCC activity equivalent to that of the corresponding chimeric antibody. Example 8. In vivo antitumor effect of the antibody molecule of the present invention In this example, the in vivo antitumor activity of the anti-B7H3 antibody molecule obtained in this invention was studied in a tumor-bearing mouse model.

[0158] For the experiment, we used SPF-grade female CB-17-SCID mice (18g-20g) purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., with the certificate of conformity number NO.1100112011025061.

[0159] A375 cells (ATCC, CRL-1619) were regularly subcultured for subsequent in vivo experiments. Cells were collected by centrifugation, and the A375 cells were dispersed in PBS (1×) and divided into 2.5 × 10⁶ cells. 7 A cell suspension with a cell concentration of 100 cells / mL was prepared. On day 0, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right ventral region of CB-17 SCID mice to establish an A375 tumor-bearing mouse model.

[0160] On day 0 after inoculation of tumor cells, all mice were randomly divided into groups (8 mice per group), and administered to each group on days 0, 4, 7, and 11 after inoculation. The dosage, administration method, and corresponding antibodies are shown in Table 13.

[0161] [Table 18] Tumor volume was detected in each mouse 5 days after vaccination, and tumor volume and body weight were monitored twice a week until 14 days later. On 14 days after vaccination, the relative tumor-to-graft (TGI) rate was calculated using the following formula: TGI% = 100% * (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group pre-treatment tumor volume).

[0162] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor are measured using calipers, and the tumor volume is calculated using the formula V = L * W. 2 It was calculated according to / 2.

[0163] The tumor suppression rates are shown in Figure 3a and Table 14. On day 14 after vaccination, the tumor suppression rates of the humanized antibodies hz19A2 and hz20G5 were 62.4% and 46.0%, respectively, compared to the h-IgG1 control group. This indicates that the humanized anti-B7H3 antibodies (hz19A2 and hz20G5) obtained in this application have excellent antitumor activity.

[0164] [Table 19] Furthermore, in this experiment, changes in the mice's body weight were also monitored, and as shown in Figures 3b to 3c, there was no significant difference in body weight between the experimental group and the control group of mice throughout the entire administration period. The present invention includes the following embodiments: <Aspect 1> An antibody or antigen-binding fragment that binds to B7-H3, 1) HCDR1, HCDR2, and HCDR3 contained in VH as indicated by sequence number 16, and LCDR1, LCDR2, and LCDR3 contained in VL as indicated by sequence number 17, 2) HCDR1, HCDR2 and HCDR3 contained in VH indicated by Sequence ID No. 18, and LCDR1, LCDR2 and LCDR3 contained in VL indicated by Sequence ID No. 19, 3) HCDR1, HCDR2, and HCDR3 included in VH indicated by SEQ ID NO: 20, and LCDR1, LCDR2, and LCDR3 included in VL indicated by SEQ ID NO: 21, or 4) Including HCDR1, HCDR2, and HCDR3 contained in VH as shown in Sequence ID No. 22, and LCDR1, LCDR2, and LCDR3 contained in VL as shown in Sequence ID No. 23, An antibody or its antigen-binding fragment. <Aspect 2> An antibody or antigen-binding fragment that binds to B7-H3, comprising a heavy chain variable region VH and / or a light chain variable region VL, wherein, (i) The VH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1 or 8, HCDR2 comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 2, 7, 9 and 14, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or 10. and / or (ii) The VL includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 includes or consists of the amino acid sequence shown in any one of SEQ ID NOs: 4, 11, and 15; LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NOs: 5 or 12; and LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NOs: 6 or 13. An antibody or its antigen-binding fragment. <Aspect 3> It includes three complementarity determination regions HCDR of the heavy chain variable region and three complementarity determination regions LCDR of the light chain variable region, where, 1) HCDR1 contains or consists of the amino acid sequence shown in Sequence ID No. 1. HCDR2 contains or consists of the amino acid sequence shown in Sequence ID No. 2. HCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 3. LCDR1 contains or consists of the amino acid sequence shown in Sequence ID No. 4. LCDR2 contains or consists of the amino acid sequence shown in Sequence ID No. 5. LCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 6. 2) HCDR1 contains or consists of the amino acid sequence shown in Sequence ID No. 1. HCDR2 contains or consists of the amino acid sequence shown in Sequence ID No. 7. HCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 3. LCDR1 contains or consists of the amino acid sequence shown in Sequence ID No. 4. LCDR2 contains or consists of the amino acid sequence shown in Sequence ID No. 5. LCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 6. 3) HCDR1 contains or consists of the amino acid sequence shown in Sequence ID No. 8. HCDR2 contains or consists of the amino acid sequence shown in Sequence ID No. 9. HCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 10. LCDR1 contains or consists of the amino acid sequence shown in Sequence ID No. 11. LCDR2 contains or consists of the amino acid sequence shown in Sequence ID No. 12. LCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 13, or 4) HCDR1 contains or consists of the amino acid sequence shown in Sequence ID No. 8. HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 14. HCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 10. LCDR1 contains or consists of the amino acid sequence shown in Sequence ID No. 15. LCDR2 contains or consists of the amino acid sequence shown in Sequence ID No. 12. LCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 13. The antibody or its antigen-binding fragment according to Embodiment 2. <Aspect 4> It includes a heavy chain variable region VH and / or a light chain variable region VL, where, (a) The heavy chain variable region VH is (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one of sequence numbers 16, 18, 20, and 22, and comprising or including the corresponding CDR sequence of said sequence, (ii) containing or consisting of the amino acid sequence shown in any one of sequence numbers 16, 18, 20, and 22, or (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the amino acid sequence represented by any one of SEQ ID NOs. 16, 18, 20, and 22, wherein the amino acid mutations do not occur in the CDR region. and / or (b) The light chain variable region VL is (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one of sequence numbers 17, 19, 21, and 23, and comprising the corresponding CDR sequence of said sequence, (ii) containing or consisting of the amino acid sequence shown in any one of sequence numbers 17, 19, 21, and 23, or (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the amino acid sequence represented by any one of SEQ ID NOs. 17, 19, 21, and 23, wherein the amino acid mutations do not occur in the CDR region. An antibody or antigen-binding fragment according to any one of embodiments 1 to 3. <Aspect 5> 1) A heavy chain variable region VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16, and a light chain variable region VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 17, 2) A heavy chain variable region VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 19, 3) A heavy chain variable region VH containing or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 20, and a light chain variable region VL containing or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 21, or 4) A heavy chain variable region VH containing or consisting of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 22, and a light chain variable region VL containing or consisting of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 23 The antibody or antigen-binding fragment according to embodiment 4, comprising: <Pattern 6> An antibody or antigen-binding fragment according to any one of embodiments 1 to 5, comprising a heavy chain variable region and a light chain variable region selected from the following: 1) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 16, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 17. 2) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 19. 3) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 20, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 21, or 4) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 22, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 23. <Aspect 7> Including heavy chains and / or light chains, where, (a) The heavy chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one of sequence numbers 24, 26, 28, and 30, and comprising or consisting of an amino acid sequence that includes the corresponding CDR sequence of said sequence, (ii) containing or consisting of the amino acid sequence shown in any one of sequence numbers 24, 26, 28, and 30, or (iii) an amino acid sequence having one or more (preferably 20 or fewer or 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) with respect to the amino acid sequence represented by any one of SEQ ID NOs: 24, 26, 28, and 30, wherein the amino acid mutations do not occur in the CDR region of the heavy chain, and more preferably, the amino acid mutations do not occur in the variable region of the heavy chain. and / or (b) The light chain is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by any one of sequence numbers 25, 27, 29, and 31, and comprising or consisting of an amino acid sequence that includes the corresponding CDR sequence of said sequence, (ii) containing or consisting of the amino acid sequence shown in any one of sequence numbers 25, 27, 29, and 31, or (iii) an amino acid sequence having one or more (preferably 20 or fewer or 10 or fewer, more preferably 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) with respect to the amino acid sequence represented by any one of SEQ ID NOs. 25, 27, 29, and 31, wherein the amino acid mutations do not occur in the CDR region of the light chain, and more preferably, the amino acid mutations do not occur in the variable region of the light chain. An antibody or antigen-binding fragment according to any one of embodiments 1 to 6. <Aspect 8> 1) A heavy chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 24, and a light chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 25. 2) A heavy chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 26, and a light chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 27. 3) A heavy chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 28, and a light chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 29, or 4) A heavy chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 30, and a light chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 31. The antibody or antigen-binding fragment according to embodiment 7, comprising: <Aspect 9> 1) A heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 24, and a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 25, 2) A heavy chain containing or consisting of the amino acid sequence shown in Sequence ID No. 26, and a light chain containing or consisting of the amino acid sequence shown in Sequence ID No. 27, 3) A heavy chain containing or consisting of the amino acid sequence shown in Sequence ID No. 28, and a light chain containing or consisting of the amino acid sequence shown in Sequence ID No. 29, or 4) A heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 30, and a light chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 31, The antibody or antigen-binding fragment according to embodiment 8, comprising: <Aspect 10> The antibody is an antibody or antigen-binding fragment in the form of IgG1, IgG2, IgG3, or IgG4, preferably an antibody in the form of IgG1, and more preferably an antibody or antigen-binding fragment in the form of human IgG1 Fc region, according to any one of embodiments 1 to 9. <Aspect 11> The antibody or its antigen-binding fragment according to any one of embodiments 1 to 10, wherein the antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody. <Aspect 12> The antigen-binding fragment is an antibody fragment selected from single-chain antibodies such as Fab, Fab', Fab'-SH, Fv, and scFv, (Fab')2 fragments, single-domain antibodies, bispecific antibodies (dAb), or linear antibodies. An antibody or antigen-binding fragment according to any one of embodiments 1 to 11. <Aspect 13> Encoding an anti-B7-H3 antibody or its antigen-binding fragment according to any one of the above embodiments, Isolated nucleic acids. <Aspect 14> A vector comprising nucleic acid as described in embodiment 13, Preferably, the vector is an expression vector. vector. <Aspect 15> A host cell comprising the nucleic acid described in embodiment 13 or the vector described in embodiment 14, Preferably, the host cell is a prokaryotic or eukaryotic cell, more preferably an Escherichia coli cell, a mammalian cell (e.g., 293 cells or CHO cells), or another cell suitable for the production of antibodies or their antigen-binding fragments. host cell. <Aspect 16> A method for producing an anti-B7-H3 antibody or its antigen-binding fragment, The method comprises culturing host cells as described in embodiment 15 under conditions suitable for the expression of nucleic acids encoding the antibody or antigen-binding fragment described in any one of embodiments 1 to 12, optionally isolating the antibody or antigen-binding fragment, and optionally further isolating the anti-B7-H3 antibody or antigen-binding fragment from the host cells. Manufacturing method. <Aspect 17> An anti-B7-H3 antibody or its antigen-binding fragment, manufactured by the method described in Embodiment 16. <Aspect 18> An immune complex comprising an antibody or its antigen-binding fragment as described in any one of embodiments 1 to 12 or 17, conjugated with a therapeutic or diagnostic agent. <Aspect 19> The present invention comprises an antibody or antigen-binding fragment thereof as described in any one of embodiments 1 to 12 and 17, or an immune complex as described in embodiment 18, and optionally a medicinal auxiliary material. Pharmaceutical composition. <Aspect 20> The use of an antibody or antigen-binding fragment according to any one of embodiments 1 to 12 and 17, an immune complex according to embodiment 18, or a pharmaceutical composition according to embodiment 19 in the preparation of a drug for treating and / or diagnosing cancer or tumor, wherein the tumor is a solid tumor. use. <Aspect 21> In the subject, a method for preventing or treating diseases or conditions associated with B7-H3, such as cancer, The method comprises administering to the subject an effective amount of an anti-B7-H3 antibody or its antigen-binding fragment according to any one of embodiments 1 to 12 and 17, an immune complex according to embodiment 18, or a pharmaceutical composition according to embodiment 19. method. <Aspect 22> A method for detecting B7-H3 in a sample, (a) Contacting the sample with an anti-B7-H3 antibody or its antigen-binding fragment as described in any one of embodiments 1 to 12 and 17, or with an immune complex as described in embodiment 18, (b) Detection of the antibody or its antigen-binding fragment, or a complex formed by the immune complex and B7-H3, This includes, and in some cases, the antibody is labeled in a detectable manner. method.

Claims

1. It includes three complementarity determination regions (HCDRs) in the heavy chain variable region and three complementarity determination regions (LCDRs) in the light chain variable region, where, 1) HCDR1 contains or consists of the amino acid sequence shown in Sequence ID No.

1. HCDR2 contains or consists of the amino acid sequence shown in Sequence ID No.

7. HCDR3 contains or consists of the amino acid sequence shown in Sequence ID No.

3. LCDR1 contains or consists of the amino acid sequence shown in Sequence ID No.

4. LCDR2 contains or consists of the amino acid sequence shown in Sequence ID No.

5. LCDR3 contains or consists of the amino acid sequence shown in Sequence ID No.

6. 2) HCDR1 contains or consists of the amino acid sequence shown in Sequence ID No.

1. HCDR2 contains or consists of the amino acid sequence shown in Sequence ID No.

2. HCDR3 contains or consists of the amino acid sequence shown in Sequence ID No.

3. LCDR1 contains or consists of the amino acid sequence shown in Sequence ID No.

4. LCDR2 contains or consists of the amino acid sequence shown in Sequence ID No.

5. LCDR3 contains or consists of the amino acid sequence shown in Sequence ID No.

6. 3) HCDR1 contains or consists of the amino acid sequence shown in Sequence ID No.

8. HCDR2 contains or consists of the amino acid sequence shown in Sequence ID No.

9. HCDR3 contains or consists of the amino acid sequence shown in Sequence ID No.

10. LCDR1 contains or consists of the amino acid sequence shown in Sequence ID No.

11. LCDR2 contains or consists of the amino acid sequence shown in Sequence ID No.

12. LCDR3 contains or consists of the amino acid sequence shown in Sequence ID No. 13, or 4) HCDR1 contains or consists of the amino acid sequence shown in Sequence ID No.

8. HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO:

14. HCDR3 contains or consists of the amino acid sequence shown in Sequence ID No.

10. LCDR1 contains or consists of the amino acid sequence shown in Sequence ID No.

15. LCDR2 contains or consists of the amino acid sequence shown in Sequence ID No.

12. LCDR3 contains or consists of the amino acid sequence shown in Sequence ID No.

13. An antibody or its antigen-binding fragment that binds to B7-H3.

2. 1) A heavy chain variable region VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 22, and a light chain variable region VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 23, 2) A heavy chain variable region VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 18, or comprising the amino acid sequence said, and a light chain variable region VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 19, or comprising the amino acid sequence said, 3) A heavy chain variable region VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 20, and a light chain variable region VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 21, or the amino acid sequence comprising the light chain variable region VL, 4) A heavy chain variable region VH containing or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 16, and a light chain variable region VL containing or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 17 The antibody or antigen-binding fragment according to claim 1, comprising:

3. The antibody or antigen-binding fragment according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region selected from the following: 1) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 22, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 23, 2) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 19, 3) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 20, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO: 21, or 4) A heavy chain variable region VH containing or consisting of the amino acid sequence shown in SEQ ID NO: 16, and a light chain variable region VL containing or consisting of the amino acid sequence shown in SEQ ID NO:

17.

4. 1) A heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 26, and a light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 27, 2) A heavy chain comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 30, and a light chain comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

31. 3) A heavy chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 28, and a light chain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 29, or 4) A heavy chain comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 24, and a light chain comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

25. An antibody or antigen-binding fragment according to any one of claims 1 to 3, comprising:

5. 1) A heavy chain containing or consisting of the amino acid sequence shown in Sequence ID No. 26, and a light chain containing or consisting of the amino acid sequence shown in Sequence ID No. 27, 2) A heavy chain containing or consisting of the amino acid sequence shown in Sequence ID No. 30, and a light chain containing or consisting of the amino acid sequence shown in Sequence ID No. 31, 3) A heavy chain containing or consisting of the amino acid sequence shown in Sequence ID No. 28, and a light chain containing or consisting of the amino acid sequence shown in Sequence ID No. 29, or 4) A heavy chain containing or consisting of the amino acid sequence shown in Sequence ID No. 24, and a light chain containing or consisting of the amino acid sequence shown in Sequence ID No. 25, The antibody or antigen-binding fragment according to claim 4, comprising:

6. The antibody is an antibody or antigen-binding fragment in the form of IgG1, IgG2, IgG3, or IgG4, preferably an antibody in the form of IgG1, and more preferably comprising a human IgG1 Fc region, according to any one of claims 1 to 5.

7. The antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

8. The antigen-binding fragment is an antibody fragment selected from single-chain antibodies such as Fab, Fab', Fab'-SH, Fv, and scFv, a (Fab')2 fragment, a bispecific antibody (dAb), or a linear antibody. The antibody or antigen-binding fragment according to any one of claims 1 to 7.

9. Encoding the anti-B7-H3 antibody or its antigen-binding fragment according to any one of claims 1 to 8, Isolated nucleic acids.

10. A vector comprising the nucleic acid described in claim 9.

11. The vector according to claim 10, wherein the vector is an expression vector.

12. A host cell comprising the nucleic acid described in claim 9 or the vector described in claim 10 or 11.

13. The host cell is an E. coli cell, a mammalian cell, or another cell suitable for the production of antibodies or their antigen-binding fragments. The host cell according to claim 12.

14. The host cell according to claim 13, wherein the mammalian cell is a 293 cell or a CHO cell.

15. A method for producing an anti-B7-H3 antibody or its antigen-binding fragment, The method comprises culturing host cells according to any one of claims 12 to 14 under conditions suitable for the expression of nucleic acids encoding the antibody or antigen-binding fragment according to any one of claims 1 to 8, optionally isolating the antibody or antigen-binding fragment, and optionally further isolating the anti-B7-H3 antibody or antigen-binding fragment from the host cells. Manufacturing method.

16. An immune complex comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, conjugated with a therapeutic or diagnostic agent.

17. The present invention comprises an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or an immune complex according to claim 16, and optionally a medicinal auxiliary material. Pharmaceutical composition.

18. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, an immune complex according to claim 16, or a pharmaceutical composition according to claim 17, used for the preparation of a drug for treating and / or diagnosing cancer or tumor, wherein the tumor is a solid tumor.

19. A method for detecting B7-H3 in a sample, (a) Contacting the sample with the anti-B7-H3 antibody or its antigen-binding fragment according to any one of claims 1 to 8, or the immune complex according to claim 16, (b) To detect the antibody or its antigen-binding fragment, or a complex formed by the immune complex and B7-H3, This includes, and in some cases, the antibody is labeled in a detectable manner. method.

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