Anti-B7-H3 Antibodies and Uses Thereof

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] 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 also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof, host cells comprising said nucleic acids, and related uses. The present invention further relates to therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof. [Background technology]

[0002] B7-H3 (also called CD276) is a type I transmembrane glycoprotein, highly similar in structure to PD-L1, and both belong to the B7 / CD28 superfamily. B7-H3 is widely expressed in lymphoid tissues and non-lymphoid organs at the transcriptional level (RNA), but the protein expression of B7-H3 is very limited, mainly expressed in activated dendritic cells, monocytes, T lymphocytes, B lymphocytes and Nk lymphocytes, and the expression level in other normal tissues is very low. In recent years, it has been found that B7-H3 is 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 is closely related to survival, prognosis or tumor level. In addition to being highly expressed in tumors, B7-H3 may have a function similar to the T cell inhibitory signal mediated by PD-L1. B7-H3 has been proposed to have costimulatory and co-inhibitory functions, depending on tumor specificity, microenvironmental factors, and signal strength. In addition to functioning as an immunomodulator, B7-H3 has been implicated in promoting cancer metastasis and angiogenesis.

[0003] Since B7-H3 expression is largely restricted to tumors, it is a highly important tumor-associated antigen and can be used as a potential broad-spectrum immunotherapy target. Summary of the Invention

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

[0005] In one aspect, the present invention provides novel antibodies or antigen-binding fragments thereof that bind to the B7-H3 molecule.

[0006] In some embodiments, the anti-B7-H3 antibodies of the invention (i) binds to human and cynomolgus monkey B7-H3 with high affinity; (ii) effectively binds to B7-H3 on the cell surface; (iii) effectively binds soluble B7-H3; (iv) effectively activates the ADCC effect; (v) effectively inhibiting or reducing the growth and progression of tumors in the body; It has one or more of the above characteristics.

[0007] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH is (i) the three complementarity determining regions (CDRs) contained in the VH of any antibody shown in Table B; or (ii) the 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 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) relative to the VH sequence of any of the antibodies shown in Table B, wherein said amino acid mutations do not occur in the CDR regions; or (iv) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VH sequence of any antibody shown in Table B and containing the corresponding CDRs of said sequence.

[0008] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL is (i) the three complementarity determining regions (CDRs) contained in the VL of any antibody shown in Table B; or (ii) the 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 no more than 10, more preferably no more than 5, no more than 4, no more than 3, no more than 2, no more than 1) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the VL sequence of any of the antibodies shown in Table B, wherein said amino acid mutations do not occur in the CDR regions; or (iv) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VL sequence of any antibody shown in Table B and containing the corresponding CDRs of said sequence.

[0009] In some embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region, VH, and / or a light chain variable region, VL, wherein: (a) the VH is (i) the three complementarity determining regions (CDRs) contained in the VH of any antibody shown in Table B; or (ii) the 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 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) relative to the VH sequence of any of the antibodies shown in Table B, wherein said amino acid mutations do not occur in the CDR regions; or (iv) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VH sequence of any antibody shown in Table B and containing the corresponding CDRs of said sequence; and / or (b) the VL is (i) the three complementarity determining regions (CDRs) contained in the VL of any antibody shown in Table B; or (ii) the 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 no more than 10, more preferably no more than 5, no more than 4, no more than 3, no more than 2, no more than 1) amino acid mutations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) relative to the VL sequence of any of the antibodies shown in Table B, wherein said amino acid mutations do not occur in the CDR regions; or (iv) a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VL sequence of any antibody 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, and comprises the HCDR1, HCDR2 and HCDR3 sequences of a heavy chain variable region set forth in any one of SEQ ID NOs: 16, 18, 20 and 22, and / or the LCDR1, LCDR2 and LCDR3 sequences of one of the light chain variable regions set forth in any one of SEQ ID NOs: 17, 19, 21 and 23, or a variant of a combination of the above CDR sequences.

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

[0012] In some embodiments, the present invention provides an anti-B7-H3 antibody or 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 shown in SEQ ID NO:22, and the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO:23.

[0013] In some embodiments, the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, 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 NO: 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 comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 4, 11 and 15, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5 or 12, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6 or 13.

[0014] In some embodiments, the present invention provides 1) HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:1; HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:2; HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:3; LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:4; LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:5; LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:6; 2) HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:1; HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:3; LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:4; LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:5; LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:6; 3) HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:8; HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9, HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10; LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11; LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12; LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13; 4) HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:8; HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10; LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15; LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 12; LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13; The present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof comprising:

[0015] In some embodiments, the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, 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) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 16, 18, 20 and 22 and the corresponding CDR sequences of said sequences; or (ii) comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 16, 18, 20 and 22; 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 amino acid sequence shown in any one of SEQ ID NOs: 16, 18, 20, and 22, and preferably, the amino acid mutations do not occur in the CDR regions; and / or (b) the light chain variable region VL is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 17, 19, 21 and 23, and comprising the corresponding CDR sequences of said sequences; (ii) comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 17, 19, 21 and 23; 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) relative to the amino acid sequence shown in any one of SEQ ID NOs: 17, 19, 21 and 23, and preferably, the amino acid mutations do not occur in the CDR region.

[0016] In some embodiments, the present invention provides 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 to 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 to 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 to 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 to the amino acid sequence shown in SEQ ID NO: 19; 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 to 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 to the amino acid sequence shown in SEQ ID NO: 21; 4) 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 to 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 to the amino acid sequence shown in SEQ ID NO: 23; The present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof comprising:

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

[0018] In some embodiments, the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof comprising a heavy chain and / or a light chain, wherein: (a) the heavy chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 24, 26, 28 and 30 and including the corresponding CDR sequences of said sequences; (ii) comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 24, 26, 28 and 30; or (iii) an amino acid sequence having one or more (preferably 20 or less or 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) relative to the amino acid sequence shown in any one of SEQ ID NOs: 24, 26, 28, and 30, preferably, the amino acid mutations do not occur in the CDR region of the heavy chain, more preferably, the amino acid mutations do not occur in the heavy chain variable region; and / or (b) the light chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence set forth in any one of SEQ ID NOs: 25, 27, 29 and 31 and including the corresponding CDR sequences of said sequences; (ii) comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 25, 27, 29 and 31; or (iii) An amino acid sequence having one or more (preferably 20 or less or 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) relative to the amino acid sequence shown in any one of SEQ ID NOs: 25, 27, 29 and 31, and preferably, the amino acid mutations do not occur in the light chain CDR region, and more preferably, the amino acid mutations do not occur in the light chain variable region.

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

[0020] In some embodiments, the present invention provides 1) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:24, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:25; 2) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:26, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:27; 3) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:28, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:29; 4) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 30, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 31; The present invention provides an antibody or antigen-binding fragment thereof that binds to B7-H3, comprising the compound of formula (I) or (II).

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

[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 a preferred embodiment, the anti-B7-H3 antibody is a humanized antibody. The anti-B7-H3 antibody of the present invention also includes an antibody fragment thereof, preferably an antibody fragment selected from Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain antibody (such as scFv), single domain antibody, bispecific antibody (dAb) or linear antibody.

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

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

[0026] In some embodiments, the present invention provides immunoconjugates and pharmaceutical compositions comprising anti-B7-H3 antibodies or antigen-binding fragments thereof.

[0027] In some embodiments, the present invention further provides the use of an anti-B7-H3 antibody or antigen-binding fragment thereof, immunoconjugate or pharmaceutical composition in the preparation of a medicament for preventing and / or treating a B7-H3-related disease or condition (such as a tumor).

[0028] In some embodiments, the present invention further provides a method for preventing and / or treating a B7-H3-related disease or condition (such as a tumor), the method comprising administering to a subject an effective amount of an antibody or antigen-binding fragment thereof, immunoconjugate or pharmaceutical composition that binds to B7-H3 of the present invention.

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

[0030] In another aspect, the present invention further relates to a method for diagnosing a tumor expressing a B7-H3 molecule in a subject, the method comprising: (a) obtaining a sample from the subject; (b) contacting the sample with an antibody or antigen-binding fragment thereof described in the present invention; and (c) detecting in the sample a complex formed between the antibody or antigen-binding fragment thereof and the B7-H3 molecule. [Brief description of the drawings]

[0031] Preferred embodiments of the invention, which are described in detail below, are better understood when read in conjunction with the following drawings: For the purposes of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, but it should be understood that the invention is not limited to the precise arrangements and instrumentalities 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. [Diagram 2] 1 shows the results of detection of antibody ADCC activity. [Diagram 3] 3 shows the antitumor results of the antibody in vivo, where FIG. 3a shows the change in tumor volume in tumor-bearing mice, and FIGS. 3b and 3c show the change in body weight in tumor-bearing mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] I. Definition Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, forms, or reagents described herein, as they may be modified. In addition, the terms used in the present invention are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the claims. Unless otherwise defined, technical and scientific terms used in the present specification have the same meaning as commonly understood by those of ordinary skill in the art.

[0033] The following definitions will be used to interpret the specification, and where appropriate, terms used in the singular may also include the plural and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0034] The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values ​​from 5% less than the number or numerical value specified as the lower limit to 5% more than the number or numerical value specified as the upper limit.

[0035] The term "and / or," when used in conjunction with two or more options, should be understood to refer to any one of the options or any two or more of the options.

[0036] The term "comprise" or "comprise" means including the element, integer or step, but not excluding any other element, integer or step. When the term "containing" or "comprise" is used herein, it also includes combinations of the other elements, integers or steps, unless otherwise specified. For example, when it is referred to as "comprising" an antibody variable region of a certain sequence, it is intended to include an antibody variable region consisting of that specific sequence.

[0037] The term "antibody" is used herein in the broadest sense and includes a number of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (such as bispecific antibodies), single chain antibodies, complete antibodies or antibody fragments that exhibit the desired antigen-binding activity. Complete antibodies usually contain at least two full-length heavy chains and two full-length light chains, although in some cases they may contain fewer chains, e.g., naturally occurring antibodies in camels may contain only heavy chains.

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

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

[0040] The term "Fc region" as used herein defines the C-terminal region of an immunoglobulin heavy chain, said region comprising at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the Fc region of a human IgG heavy chain typically extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain, while the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region follows the sequence shown in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th According to the EU numbering system (also known as the EU Index), as described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0041] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies usually have a similar structure, in which each domain contains four conserved framework regions (FRs) and three complementarity determining regions (see, for example, Kindt et al. Kuby Immunology, 6 th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain can also confer antigen-binding specificity. Furthermore, the VH or VL domain of an antibody that binds to a particular antigen can be used to screen a library of complementary VL or VH domains, respectively, to isolate antibodies that bind to said antigen; see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0042] "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" (herein used interchangeably with hypervariable region "HVR") are regions in an antibody variable domain that are hypervariable in sequence and form structurally determined rings ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are numbered sequentially from the N-terminus and are usually referred to as CDR1, CDR2 and CDR3. The CDRs located in the heavy chain variable domain of an antibody are also referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located in the light chain variable domain of an antibody are referred to as LCDR1, LCDR2 and LCDR3. For a given light or heavy chain variable region amino acid sequence, its CDR sequences can be determined by various methods known in the art, for example, Kabat complementarity determining regions (CDRs) are the most common ones 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)). Meanwhile, Chothia refers to the location of the structural rings (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 the analysis of available complex crystal structures. According to different CDR definition schemes, the residues of each HVR / CDR in these CDRs are as follows:

[0043] [Table 1] The CDRs are CDR sequences that map to Kabat residue positions based on the Kabat numbering system as follows:

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

[0045] In one embodiment, HCDR1 of an antibody of the invention is bounded by the AbM rules, and HCDR2, HCDR3 and LCDR are bounded by the Kabat rules, e.g., as shown in Table A below.

[0046] A CDR may be determined by having the same Kabat numbering position as the sequence of a reference CDR (eg, any one of the exemplary CDRs of the invention).

[0047] Unless otherwise specified, in the present invention, when referring to residue positions in an 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 term "CDR" or "CDR sequence" includes a CDR sequence determined by any one of the above methods.

[0049] However, it should be recognized that there may be differences in the boundaries of the CDRs of the variable regions of the same antibody obtained based on different allocation systems. That is, there will be differences in the CDR sequences of the variable regions of the same antibody defined by different allocation systems. Thus, when an antibody is defined by a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences include the specific CDR sequences, but where a different approach (e.g., rules or combinations of different allocation systems) is used and such CDR boundaries differ from the specific CDR boundaries defined in the present invention.

[0050] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, despite the different CDRs of different antibodies, the number of amino acid positions in the CDR that are directly involved in binding to the antigen is limited. The minimum overlapping region can be determined to provide a "minimum binding unit" for antigen binding by at least two of the Kabat, Chothia, AbM, Contact and North methods. The minimum binding unit may be a subpart of one of the CDRs. As known to those skilled in the art, the structure of the antibody and protein folding can determine the residues of the remaining part of the CDR sequence. Thus, the present invention contemplates variants of any of the CDRs provided herein. For example, in a variant of one CDR, the amino acid residues of the minimum binding unit may be left as they are, but the remaining CDR residues defined according to Kabat or Chothia may be replaced with conservative amino acid residues.

[0051] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which some cytotoxic cells (e.g., NK cells, neutrophils, macrophages) specifically bind to antigen-bearing target cells by binding to secreted immunoglobulins at Fc receptors (FcR) present on these cells, and kill the target cells with cytotoxins. The cells that mediate ADCC are mainly NK cells, which express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, or the ADCC activity of a target molecule can be assessed in vivo, for example, in an animal model. The examples herein provide exemplary assays for assessing ADCC activity.

[0052] The term "functional Fc region" refers to an Fc region that has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of a cell surface receptor (e.g., B cell receptor, BCR), and the like. Such effector functions are generally determined by the association of an Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, by a number of assays disclosed herein.

[0053] The term "therapeutic agent" as used herein includes any substance effective in the prevention or treatment of tumors (such as cancer), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs or immunomodulatory agents.

[0054] The term "immunomodulatory agent" as used herein refers to a natural or synthetic active agent or drug that suppresses or modulates the immune response, which may be a humoral or cellular response.

[0055] The term "effective amount" refers to an amount or dosage that will produce the desired effect in a patient in need of treatment or prevention after administration of one or more doses of the antibody or fragment thereof or complex or composition of the present invention to the patient. For the purpose of treatment or prevention, the "effective amount" can be divided into a "therapeutically effective amount" and a "prophylactically effective amount". The effective amount can be easily determined by the attending physician who is skilled in the art, taking into account various factors such as, for example, the mammalian species, volume, age, health status, and the specific disease involved, the extent or severity of the disease, the response of the individual patient, the specific antibody administered, the form of administration, the bioavailability characteristics of the administration formulation, the selected administration schedule, the application of a combination therapy, etc.

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

[0057] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be exactly identical to the parent cell in nucleic acid content and may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected from the primary transformed cell.

[0058] The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0059] The term "humanized antibody" refers to an antibody that connects an antigen-binding site derived from another mammalian species, such as a mouse germ line, to a human immunoglobulin sequence. Humanized antibodies are chimeric molecules that are usually prepared using recombinant techniques, allowing additional framework region modifications within the human framework sequence. The antigen-binding site may comprise a complete variable domain fused to a constant region, or may comprise only the complementarity determining regions grafted into the appropriate framework sequence within the variable domain. In some embodiments, a humanized antibody comprises at least one, typically two, of substantially all of the variable domain, in which all or substantially all of the CDRs (e.g., six CDRs) correspond to portions derived from a non-human antibody, and all or substantially all of the FRs correspond to portions derived from a human antibody. A humanized antibody optionally comprises at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0060] The term "immunoconjugate" refers to an antibody conjugated with one or more other substances, including, but not limited to, a cytotoxic agent or a label.

[0061] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (e.g., a polynucleotide probe or an antibody) and facilitates detection by the reagent to which it is conjugated or fused. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, when labeled by enzyme catalysis, may catalyze the chemical alteration of a substrate compound or composition that is detectable. The term is intended to include direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody by a fluorescently labeled secondary antibody, and the use of end-labeling of a DNA probe with biotin that is detectable with a fluorescently labeled streptavidin protein.

[0062] The term "individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, 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 is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0064] "An isolated nucleic acid encoding an anti-B7-H3 antibody or antigen-binding fragment thereof" refers to one or more nucleic acid molecules encoding an antibody heavy or light chain (or antigen-binding fragment thereof), including such nucleic acid molecules in a single vector or separate vectors, and including such nucleic acid molecules present in one or more locations within a host cell.

[0065] Calculate sequence identity between sequences as follows.

[0066] To determine the percentage identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then 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 this position.

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

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

[0069] Additionally or alternatively, the nucleic acid and protein sequences described herein can be used as "query sequences" to perform a search against public databases, e.g., to identify other family member sequences or related sequences.

[0070] The term "auxiliary pharmaceutical material" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete or incomplete)), excipient, vector, stabilizer, etc., administered with an active substance.

[0071] The term "pharmaceutical composition" refers to a composition that is present in a form that effectively activates the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0072] As used herein, "treatment" refers to relieving, interrupting, slowing, ameliorating, arresting, reducing, or reversing the progression or severity of an existing symptom, condition, medical condition, or disease.

[0073] As used herein, "prevention" includes the inhibition of the onset or progression of a disease, condition, or symptom associated with a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a prevention program. In general, in the context of cancer, the term "prevention" refers to the administration of a drug before symptoms or symptoms associated with cancer arise, particularly before cancer occurs in a subject at risk of suffering from cancer.

[0074] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures and vectors integrated into the genome of a host cell into which it is introduced. Some vectors are capable of directing the expression of a nucleic acid to which it is operably linked. 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 source of the tissue or cell sample may be solid tissue (such as fresh, frozen and / or preserved organ or tissue samples, biopsy or aspirate samples), blood or any blood component, bodily fluid (such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites) or interstitial fluid), or cells from any stage of pregnancy or development in a subject. II. Antibodies Unless otherwise noted, 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 of 28 amino acids, an extracellular and transmembrane region of 217 amino acids, and a cytoplasmic domain of 45 amino acids, with a molecular weight of approximately 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 sequence in humans.

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

[0077] In some embodiments, the anti-B7-H3 antibodies, or antigen-binding fragments thereof, of the invention bind to B7-H3 (such as human or cynomolgus B7-H3) with sufficient affinity to exhibit an equilibrium dissociation constant (K D ) binds to B7-H3, and D ≦1μM, ≦100nM, ≦10nM, ≦1nM, ≦0.1nM, ≦0.01nM, or ≦0.001nM (e.g., 10 -7 M or less, e.g. 10 -7 M~10 -10 In some embodiments, the B7-H3 is human or cynomolgus B7-H3. In some embodiments, the antibody binding affinity is measured using a light coherence bioassay, e.g., in a light coherence bioassay, the antibody is about 1×10 -7 K below M 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 binds to human B7-H3.

[0078] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to B7-H3 expressed on the surface of a cell.

[0079] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can induce an ADCC effect. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can inhibit and / or reduce the growth and / or volume of tumors in vivo.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof that binds to B7-H3 of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein 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 mutations described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid mutations described herein are amino acid substitutions, preferably conservative substitutions.

[0082] In a preferred embodiment, the amino acid mutations according to the present invention occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid mutations according to the present invention occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0083] In some embodiments, substitution is conservative substitution.Conservative substitution refers to one amino acid being replaced by another amino acid of the same kind, for example, one acidic amino acid being replaced by another acidic amino acid, one basic amino acid being replaced by another basic amino acid, or one neutral amino acid being replaced by another neutral amino acid.Exemplary substitutions are as shown in the table below.

[0084] [Table 2] In some embodiments, the substitutions are made in the CDR regions of the antibody. Generally, the resulting variant has a modification (e.g., improvement) in certain biological properties (e.g., improved affinity) relative to the parent antibody and / or has substantially retained certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody.

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

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

[0087] In certain embodiments, it may be necessary to produce cysteine ​​engineered antibodies, e.g., "ThioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. For example, cysteine ​​engineered antibodies can be generated 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 non-protein moieties that are known and readily available in the art. Moieties suitable for inducing antibody activity 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 homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. III. Nucleic Acids of the Invention, Vectors Containing Them, and Host Cells The present invention provides a nucleic acid encoding any of the anti-B7-H3 antibodies or antigen-binding fragments thereof described above. It further provides a vector comprising the nucleic acid. In one embodiment, the vector is an expression vector.

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

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

[0091] Using methods known in the art, these polynucleotide sequences can be generated by solid phase DNA synthesis from scratch or by PCR to derive sequences encoding antibodies or antigen-binding fragments thereof that bind B7-H3.

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

[0093] In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing the 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 functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, 5,840,523, and Charlton, Methods in Molecular Biology, vol 248 (edited by BKC Lo, Humana Press, Totowa, NJ, 2003), p245-254, Expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial cell paste in a 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 producing antibodies or antigen-binding fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi and yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains in which the glycosylation pathway has already been "humanized" produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006). Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells may be used as hosts. For example, mammalian cell lines modified to be adapted for suspension growth can be used. Other examples of mammalian host cell lines that can be used include the SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (293HEK or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)). Other mammalian host cell lines that can be used 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 generally described, for example, in Yazaki & Wu, Methods in Molecular Biology, vol 248 (BKC Lo, ed., Humana Press, Totowa, NJ), p255-268 (2003). IV. Production and Purification of Antibody Molecules of the Invention In one embodiment, the invention provides a method for producing an anti-B7-H3 antibody or fragment thereof (preferably an antigen-binding fragment), the method comprising culturing the host cell under conditions suitable for expression of a nucleic acid encoding the antibody or fragment thereof (preferably an antigen-binding fragment), and optionally isolating the antibody or fragment thereof. In some embodiments, the method further comprises isolating the anti-B7-H3 antibody or fragment thereof from the host cell. V. Multispecific antibodies In a further aspect, the invention provides multispecific (including bispecific) antibody molecules that specifically bind to B7-H3. In one embodiment, in a multispecific antibody, an antibody of the invention (or antigen-binding fragment thereof) forms a first binding specificity for B7-H3.

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

[0096] For multispecific antibodies and their production, see, for example, 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. VI. Immune complex In some embodiments, the invention provides an immunoconjugate produced by conjugating an antibody of the invention to a heterologous molecule. In some embodiments, the heterologous molecule is a therapeutic or diagnostic agent, such as a cytotoxic agent or a chemotherapeutic agent. A cytotoxic agent includes any agent that is toxic to cells. Examples of cytotoxic agents suitable for generating immunoconjugates are known in the art.

[0097] Linkers can be used to covalently link various solids of the conjugate. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a "cleavable linker" that facilitates the release of the polypeptide after delivery to the target site. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Research 52 (1992) 127-131; US ​​5,208,020) can be used.

[0098] In some embodiments, the antibodies of the present invention can be conjugated to diagnostic or detectable reagents. Such conjugates can monitor or predict the onset, formation, progression and / or severity of a disease or condition as part of a clinical testing procedure (e.g., to confirm the efficacy of a particular therapy). Such diagnosis and detection can be achieved by coupling the antibody to a detectable reagent, which includes, but is not limited to, various enzymes, such as but not limited to horseradish peroxidase, prosthetic groups, such as but not limited to streptavidin / biotin and avidin / biotin, fluorescent materials, luminescent materials, radioactive materials, and various positron-emitting metals and non-radioactive paramagnetic metal ions used in positron emission imaging.

[0099] In some embodiments, the immunoconjugates are used to prevent or treat tumors. In some embodiments, the tumor is a cancer. VII. Pharmaceutical Compositions, Pharmaceutical Formulations and Combination Products The present invention further includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising anti-B7-H3 antibodies or immunoconjugates thereof or multispecific antibodies, and compositions comprising polynucleotides encoding anti-B7-H3 antibodies or immunoconjugates thereof or multispecific antibodies. These compositions may also contain suitable pharmaceutical auxiliary materials, such as pharma- ceutically acceptable vectors known in the art, pharma- ceutically acceptable excipients including buffers, as necessary.

[0100] Pharmaceutical compositions or formulations of the present invention may further be combined with one or more other active ingredients, said active ingredients having complementary activities necessary for the treatment of a particular indication, preferably that do not adversely affect each other.

[0101] Thus, in one aspect, the invention also provides a drug combination product. In one embodiment, the combination product comprises an antibody, immunoconjugate or multispecific antibody of the invention and a second therapeutic agent in the same pharmaceutical composition or formulation preparation. In another embodiment, the combination product comprises an antibody, immunoconjugate or multispecific antibody of the invention and a second therapeutic agent separately contained in different pharmaceutical compositions or formulations. The second therapeutic agent can be administered prior to, simultaneously with (e.g., in the same formulation or a different formulation), or after administration of the antibody of the invention.

[0102] The pharmaceutical compositions, formulations and combination products of the invention can be provided as products for the treatment, prevention and / or diagnosis of diseases and / or conditions described herein. The products can include a container and a label or instructions. Suitable containers include, for example, bottles, syringes, IV infusion bags, and the like. The containers can be made of a variety of materials, such as glass or plastic. In one embodiment, the products can include (a) a first container containing the antibody or antibody fragment, immunoconjugate or multispecific antibody of the invention, and optionally (b) a second container containing a second therapeutic agent. In addition, the products can also include other materials desirable from a commercial and user standpoint, including buffers, pharma- ceutically acceptable diluents such as sterile water for injection, needles, syringes, injection pumps, and the like. VIII. Application According to one aspect of the present invention, there is provided a method for preventing and / or treating a B7-H3-related disease or condition (such as cancer), comprising administering to a subject an effective amount of an anti-B7-H3 antibody or antigen-binding fragment thereof, immunoconjugate, or pharmaceutical composition of the present invention.

[0103] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human. In one embodiment, the subject is suffering from or at risk of suffering from a disease as described herein. In some embodiments, the subject is undergoing or has undergone other treatments, such as chemotherapy and / or radiation therapy. According to another aspect, the present invention provides the use of an anti-B7-H3 antibody or antigen-binding fragment thereof, immunoconjugate or pharmaceutical composition in the production or preparation of a medicament for preventing and / or treating a B7-H3-related disease or condition as referred to herein.

[0104] In some embodiments, the antibody or antibody fragment or immunoconjugate or composition or product of the invention is capable of delaying the onset of a disease condition and / or symptoms associated with the disease condition.

[0105] The antibody of the present invention (and pharmaceutical compositions or immunoconjugates comprising same, any additional therapeutic agents) may be administered by any suitable method, including parenteral, pulmonary, intranasal, and, for localized treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending in part on whether administration is short-term or long-term, administration may be by any suitable route, including injection, e.g., intravenous or subcutaneous injection. As used herein, various administration schedules are included, but are not limited to, a single dose or multiple doses at multiple times, a bolus dose, or a pulse infusion.

[0106] When the antibodies of the invention are used to prevent or treat disease, their appropriate dosage (whether administered alone or in combination with one or more additional therapeutic agents) will be determined by the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatment history, the patient's clinical history, the response to the antibody, and the judgment of the attending physician. The antibodies are suitably administered to the patient in a single treatment or over a series of treatments.

[0107] In the methods of the invention, a composition, multispecific antibody or immunoconjugate of the invention can be administered in place of an antibody or antigen-binding portion of the invention, or, alternatively, in these methods, a composition, multispecific antibody or immunoconjugate of the invention can be further administered in addition to an antibody or antigen-binding portion of the invention. IX. METHODS AND COMPOSITIONS FOR DIAGNOSIS AND DETECTION In some embodiments, any of the anti-B7-H3 antibodies or antigen-binding fragments provided herein can be used to detect the presence of B7-H3 in a biological sample. In one embodiment, the antibody or antigen-binding fragment is used for diagnosis or auxiliary diagnosis of a disease based on the result of detection. The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods include immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody molecule-complexed magnetic beads, ELISA measurement 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 tissues. In some embodiments, the biological sample is derived from a hyperproliferative lesion or a cancerous lesion.

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

[0109] In some embodiments, methods are provided for treating a B7-H3-associated disease or condition (such as a cancer or tumor) comprising administering to a subject a therapeutically effective amount of an anti-B7-H3 antibody, hi another embodiment, the method further comprises administering to the subject one or more other therapies.

[0110] In one embodiment, the anti-B7-H3 antibody is used to select subjects suitable for treatment with the anti-B7-H3 antibody, e.g., B7-H3 is a biomarker for selecting said subjects. In one embodiment, the antibody of the present invention can be used to diagnose tumors, e.g., to evaluate (e.g., monitor) the treatment or progression, diagnosis and / or staging of a disease described herein in a subject. In one embodiment, the anti-B7-H3 antibody of the present application is used to determine the presence or absence of cancer cells in various tissues (e.g., ovarian, lung, breast, prostate, kidney, pancreas, thyroid, brain, etc.). 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 from solid tumors into the circulating blood, and the circulating B7-H3 antigen can be the 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. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels and radioactive labels) and moieties that are indirectly detected, for example, by enzyme-catalyzed reactions or molecular interactions, such as enzymes and ligands. X. Exemplary Anti-B7-H3 Antibodies of the Invention

[0112] [Table 3]

[0113] [Table 4]

[0114] [Table 5-1] [Table 5-2] [Table 5-3] Working Example The following examples are provided to aid in understanding the present invention. The examples are not intended to and should not be construed as limiting the scope of the present invention in any manner, and those skilled in the art can make various modifications based on the description of the present specification.

[0115] Unless expressly indicated to the contrary, the practice of the present invention employs methods of routine chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology within the skill of the art.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 special publications such as: 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 QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology. Example 1. Preparation of hybridoma cells immunized animal According to conventional methods, recombinant human 4Ig-B7H3 protein (sequence number 32) (SINO BIOLOGICAL, product number 11188-H08H) was used to immunize Bal b / c mice (Beijing Weitong Lihua), and the recombinant human 4Ig-B7H3 protein (50ug per mouse) was homogeneously mixed with an equal volume of TiterMax (Sigma, product number T2684-1ML) adjuvant, and then subcutaneously injected once every two weeks, for a total of five immunizations.

[0116] Cell fusion After the serum titer met the requirements, according to conventional methods, the mouse spleen was removed to prepare a B lymphocyte suspension, which was then mixed with SP2 / 0 myeloma cells (ATCC, CRL-1581) at a ratio of 1:2 to 1:1, followed by electrofusion. The cells after fusion were transferred from the electrode dish to a 50 mL centrifuge tube, and the cells were diluted with screening medium (the composition of the preparation is shown in Table 1) to prepare a cell suspension (concentration: 1 × 10 4 Cells / mL ~ 2 x 10 4 Cells were cultured for 10 days (or more) to obtain a total cell number (cells / mL). 100 μL of cell suspension was added per well to a 96-well plate. Fresh screening medium was replaced on the 5th day after fusion. Depending on the cell growth status, positive clones were detected by flow cytometry (FACS) after 10 days (or more) of culture.

[0117] [Table 6] High throughput screening hybridoma cells Hybridoma cells that specifically expressed anti-B7H3 antibodies were screened by flow cytometry (FACS). Briefly, CHO cells that expressed human B7H3 (CHO-huB7H3) were counted and 1 × 10 6The hybridoma 96-well plate was diluted to 100 μL per well, and then 100 μL was added to each well of the U-bottom 96-well plate, centrifuged at 500 g for 5 min, and the cell medium was removed. Then, each culture supernatant of the hybridoma 96-well plate and the positive control antibody (MGA271) were added to the U-shaped plate containing the CHO cells, and the cells were resuspended at 100 μL per well and left on ice for 30 min. The cells were further centrifuged at 500 g for 5 min to remove the supernatant, and then the cells were washed once with PBS solution. The cells were centrifuged at 500 g for 5 min to remove the PBS solution. Each well was added with 100 μL of FITC-labeled secondary antibody of anti-mouse Fab (diluted in PBS solution at 1:500), and 100 μL of FITC-labeled secondary antibody of anti-human Fab was added to the positive control antibody culture well. The cells were incubated on ice in the dark for 30 min, centrifuged at 500 g for 5 min to remove the supernatant, and the cells were washed once with PBS solution. The cells were then resuspended in 50 μL of PBS solution, detected by FACS, and positive clones were screened to obtain.

[0118] The obtained positive clones were rescreened in the same manner as described above using CHO cells (CHO-cynoB7H3) expressing cynomolgus monkey B7H3 (sequence number 34), and a total of two hybridoma cell lines, 19A2 and 20G5, which bind to both human B7H3 and monkey B7H3, were obtained.

[0119] The affinity of the two resulting hybridoma cell lines for the antigen was measured using biofilm thin layer interferometry technology (ForteBio), 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 clones.

[0121] The specific steps are as follows: The HAT in the screening medium was replaced with HT (Gibco, Cat#11067-030) to obtain a basal medium, which was added to a 96-well plate at 200 μL per well. The positive hybridoma cells screened by the fusion were added at approximately 1 × 10 5 The cells were added to the first row of a 96-well plate at a density of 100 cells / mL at 300 μL per well and mixed thoroughly and evenly. 100 μL of the cell suspension was taken from the first row and added to the second row, and after thorough and uniform mixing, 100 μL was taken and added to the next row, and the above steps were repeated until the last row and left to stand for 15 min. Observed under a microscope and counted, a volume corresponding to 100 cells was added to 20 mL of the above basic medium, and mixed uniformly and seeded at 200 μL per well. After 2 days, the cells were observed under a microscope and the monoclonal wells were judged and labeled. When the cell confluence of each well reached 50% or more, it was detected using the high-throughput FACS screening method, the target-positive wells were taken out, and the obtained cell cloning was cryopreserved.

[0122] The positive control antibody used in the present invention is MGA271, also called Enoblituzumab (from MacroGenics US20160264672A1).

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

[0124] 1. Sequencing of hybridomas Freshly cultured approximately 5 × 10 6 RNA was extracted from each hybridoma cell line and cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) in the following steps.

[0125] Table 3 Preparation of reaction system I

[0126] [Table 8] After incubation at 65° C. for 5 min, the mixture was quickly cooled on ice. Reaction system I was added to the following reverse transcription system (Table 4) in a total of 20 μL.

[0127] [Table 9] After gentle mixing, reverse transcription and translation were carried out according to the following conditions: 42°C for 60 min, then 95°C for 5 min, and then cooled on ice to obtain cDNA.

[0128] After the cDNA was ligated into the T vector, the heavy and light chain variable regions of the cDNA-amplified antibody were obtained by PCR using the Mighty TA-cloning Kit reagent kit (Takara), respectively. 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 PCR reaction was taken, and 0.5 μL of pMD20-T vector (Takara) and 5 μL of Ligation Mighty Mix (Takara) were added thereto, gently mixed to homogenity, and reacted at 37° C. for 2 hours to obtain a ligation product.

[0131] Transformed cells: 5 μL of the resulting ligation product was added to E. coli TOP10 competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd.), mixed uniformly, and then incubated on ice for 30 min. After a 90 s heat shock at 42 °C, the cells were quickly cooled on ice for 2 min, and the EP tube was supplemented with 900 μL of LB medium (Shanghai Biotechnology Co., Ltd.) and cultured at 37 °C for 1 h in a shaker at 220 rpm. The cells were centrifuged at 3000 g for 2 min, 800 μL of the supernatant was aspirated, and the remaining medium was used to resuspend the cells and coat them on an ampicillin-resistant plate. After overnight culture at 37 °C, clones were selected and sequenced.

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

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

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

[0135] The PCR system is shown in Table 9.

[0136] [Table 14] The PCR amplified products were isolated by gel cutting.

[0137] Homologous recombination reaction: The homologous recombination system is shown in Table 10.

[0138] [Table 15] The recombinant product was obtained by reacting at 37°C for 30 min. The recombinant product was transformed into the TOP10 receptor, monoclonals were selected and sequenced, and clones containing the plasmid with the correct insert direction were selected as positive clones. The positive clones were then stored to obtain the recombinant plasmid for the chimeric antibody. A certain amount of the recombinant plasmid was extracted and prepared for antibody expression.

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

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

[0141] After cell culture, the cell culture solution was centrifuged at 13000 rpm for 20 min, the supernatant was collected, and the supernatant was purified using a prepacked column Hitrap Mabselect Sure (GE, 11-0034-95) according to the manufacturer's instructions, and the concentration was measured. 100 μg of purified protein was taken, the concentration was adjusted to 1 mg / mL, and the purity of the protein was measured using a gel filtration chromatography column SW3000 (TOSOH product number: 18675). The results showed that a highly pure chimeric antibody was obtained. Example 3. Measurement of binding kinetics between the chimeric antibody of the present invention and an antigen by biolayer interference technology The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human B7H3 was measured using Biolayer Interferometry Technology (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] Briefly, AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were equilibrated offline for 30 min with analysis buffer, then detected online for 60 s to establish baseline, and the purified antibodies obtained above were loaded online onto AHQ sensors (ForteBio) to measure ForteBio affinity. Furthermore, the antibody-loaded sensors were exposed to antigens (including human 4Ig-B7H3, human 2Ig-B7H3 (ACRO, product no. B73-H52E2) and cynomolgus monkey B7H3 (SINO BIOLOGICAL, product no. 90806-C02H-50)), and then the sensors were transferred to analysis buffer to measure dissociation rates. 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 showed that the chimeric antibody obtained by the hybridoma had good affinity for human B7H3 protein and maintained very high affinity for cynomolgus monkey B7H3. Compared to the control MGA271, the antibody in this study had 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 thus obtained are hz20G5 and hz19A2, and their CDR sequences, light chain variable region and heavy chain variable region sequences, and light and heavy chain amino acid sequences are shown in Tables A to C.

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

[0147] [Table 17] From Table 12, the humanized antibody still has high affinity for the antigen B7H3, and together with the corresponding chimeric antibody, has the same equilibrium dissociation constant K D Furthermore, the humanized antibody obtained in the present application had higher binding affinity to the antigen compared to the control antibody MGA271, and in particular, the affinity of the antibody of the present application for human 2Ig-B7-H3 was improved by 20 to 100 times compared to MGA271. Example 6. Binding of humanized antibodies to CHO-S cells overexpressing human and cynomolgus B7H3 To verify whether the antibodies of the present invention can bind to antigens expressed on the surface of cells, flow cytometry techniques were used to detect the binding of the humanized antibodies of the present application to cells overexpressing human B7H3 and cynomolgus monkey B7H3.

[0148] Construction of cells overexpressing B7H3 ExpiCHO TM The following operations were performed using the Expression System Kit (Invitrogen, Cat. No.: A29133) according to the manufacturer's instructions: cDNAs encoding human 4Ig-B7H3 (uniprot: Q5ZPR3, SEQ ID NO: 32), human 2Ig-B7H3 (uniprot: Q5ZPR3-2, SEQ ID NO: 33) and cynomolgus B7H3 (NCBI: XP_015308534.1, SEQ ID NO: 34) were cloned into 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 B7H3.

[0149] In simple terms, 1) 2 × 10 CHOS-hB7H3-4Ig, CHOS-hB7H3-2Ig and CHOS-cyno B7H3 cells were cultured in PBS solution. 6 The antibody was diluted to 5000 cells / mL and 100 μL was added to each well of a U-bottom 96-well plate, with a 3-fold gradient of diluted antibody.

[0150] 2) The mixture was incubated on ice for 30 minutes. After centrifugation at 400g for 5 minutes, the supernatant was removed, and the cells were washed with PBS solution to remove unbound antibodies. 100 μL of PE-conjugated anti-human Fc antibody (SouthernBiotech) diluted 1:200 was added to each well and incubated on ice for 30 minutes in the dark. After centrifugation at 400g for 5 minutes, 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 the 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 the present application 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 obviously higher than that of MGA271. It was found that the antibody of the present application has a significantly improved antigen binding ability at the cellular level. Example 7. Antibody-dependent cell-mediated cytotoxicity (ADCC) In this example, we investigated the effect of the obtained antibody in eliminating tumor cells through the ADCC effect. In this study, we used Promega's Jurkat-ADCC NF-AT luciferase effector cell line (hereinafter referred to as ADCC effector cell) to detect the activation status of NF-AT signaling, thereby detecting the ADCC activity of the antibody. The specific experimental procedure is as follows:

[0152] 1) Cell preparation The cells of 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 (1640 medium (Gibco) with 5% low IgG serum) to count 1 × 10 ADCC effector cells. 7 Adjust CHO-hB7H3-4Ig cells to have a concentration of 1 x 10 cells / mL. 6The two types of cells were mixed at a ratio of 1:1, and the final ratio of ADCC effector cells to CHO-hB7H3-4Ig cells was 10:1.

[0153] 2) Seeding: The mixed cells were seeded into a 96-well plate at 100 μL per well, and the first well was supplemented with 50 μL of cells.

[0154] 3) Various concentration gradients of antibodies of the present invention were added in sequence, with the final concentration in the first well being 30 nM, then diluted 3-fold for a total gradient of 10.

[0155] 4) Incubate 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. Incubated at room temperature for 20 minutes. Detection was performed using a microplate reader. Concentration-dependence curves were fitted using GraphPad software.

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

[0158] In the experiments, SPF grade female CB-17-SCID mice (18 g~20 g) purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. were used, with the qualification certificate number NO.1100112011025061.

[0159] A375 cells (ATCC, CRL-1619) were subcultured periodically for subsequent in vivo experiments. Cells were collected by centrifugation, and A375 cells were dispersed in PBS (1x) and diluted to 2.5x10 7 A cell suspension with a cell concentration of cells / mL was prepared. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank area of ​​CB-17 SCID mice to establish the A375 tumor-bearing mouse model.

[0160] On day 0 after inoculation with tumor cells, all mice were randomly divided into groups (8 mice per group), and the mice were dosed on days 0, 4, 7, and 11 after inoculation, respectively. The doses, administration methods, and corresponding antibodies are shown in Table 13.

[0161] [Table 18] The tumor volume of each mouse was detected on the 5th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week until the 14th day. On the 14th day after inoculation, the relative tumor inhibition rate (TGI%) was calculated, and the calculation formula was: TGI% = 100% * (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group).

[0162] Measurement of tumor volume: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using calipers, and tumor volume was calculated using the formula V = L * W 2 The calculation is based on / 2.

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

[0164] [Table 19] Furthermore, in this experiment, changes in the body weight of the mice were also monitored, and the results, as shown in Figures 3b to 3c, showed that there was no significant difference in the body weight of the mice in the experimental and control groups during the entire administration period.

Claims

1. The heavy chain variable region comprises three complementarity determining regions HCDR and three complementarity determining regions LCDR of the light chain variable region, 1) HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:1; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:7; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:3; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:4; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5; LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:6; 2) HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:1; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:2; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:3; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:4; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5; LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:6; 3) HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:11; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12; LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13; or 4) HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12; LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13; An antibody or an antigen-binding fragment thereof.

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

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

17.

4. 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 to 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 to 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 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to 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 to the amino acid sequence shown in SEQ ID NO: 31; 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 to 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 to 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 to 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 to the amino acid sequence shown in SEQ ID NO:25; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3,

5. 1) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:26, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:27; 2) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 30, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 31; 3) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:28, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:29; or 4) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:24, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:25; The antibody or antigen-binding fragment thereof of claim 4 .

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

7. The antibody or antigen-binding fragment thereof 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 a single chain antibody such as Fab, Fab', Fab'-SH, Fv, scFv, (Fab')2 fragment, single domain antibody, bispecific antibody (dAb) or linear antibody, An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. A gene encoding the anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. Isolated nucleic acid.

10. A vector comprising the nucleic acid of claim 9.

11. The vector described in claim 10, wherein the vector is an expression vector.

12. A host cell comprising a nucleic acid according to claim 9 or a vector according to claim 10 or 11.

13. The host cell is an E. coli cell, a mammalian cell, or other cell suitable for producing an antibody or an antigen-binding fragment thereof. The host cell of claim 12.

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

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

16. An anti-B7-H3 antibody or antigen-binding fragment thereof produced by the method of claim 15.

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

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 16, or the immunoconjugate according to claim 17, and optionally a pharmaceutical auxiliary material. Pharmaceutical compositions.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 16, the immunoconjugate according to claim 17, or the pharmaceutical composition according to claim 18, for use in the preparation of a medicament for treating and / or diagnosing cancer or a tumor, wherein the tumor is a solid tumor.

20. 1. A method for detecting B7-H3 in a sample, comprising: (a) contacting the sample with an anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and 16, or an immune complex according to claim 17; (b) detecting a complex formed by the antibody or antigen-binding fragment thereof, or the immune complex, and B7-H3; and and optionally, said antibody is detectably labeled. method.

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