WO2022135467A1 - 抗b7-h3抗体及其用途 - Google Patents

抗b7-h3抗体及其用途 Download PDF

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WO2022135467A1
WO2022135467A1 PCT/CN2021/140449 CN2021140449W WO2022135467A1 WO 2022135467 A1 WO2022135467 A1 WO 2022135467A1 CN 2021140449 W CN2021140449 W CN 2021140449W WO 2022135467 A1 WO2022135467 A1 WO 2022135467A1
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amino acid
acid sequence
seq
antibody
sequence shown
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PCT/CN2021/140449
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English (en)
French (fr)
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李莉
付凤根
倪海晴
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信达生物制药(苏州)有限公司
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Priority to EP21909462.0A priority Critical patent/EP4269435A1/en
Priority to CN202180086116.XA priority patent/CN117098548A/zh
Priority to AU2021407023A priority patent/AU2021407023A1/en
Priority to KR1020237023796A priority patent/KR20230124959A/ko
Priority to CA3203257A priority patent/CA3203257A1/en
Priority to US18/258,576 priority patent/US20240043540A1/en
Priority to JP2023539061A priority patent/JP2024500512A/ja
Publication of WO2022135467A1 publication Critical patent/WO2022135467A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Definitions

  • the present invention relates to novel antibodies and antigen-binding fragments thereof that specifically bind to B7-H3 and compositions containing the antibodies or antigen-binding fragments thereof. Furthermore, the present invention relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof, host cells comprising the nucleic acids, and related uses. Furthermore, the present invention relates to the therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.
  • B7-H3 (also known as CD276) is a type I transmembrane glycoprotein, very similar in structure to PD-L1, and belongs 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, On B lymphocytes and Nk lymphocytes, the expression level is very low in other normal tissues.
  • 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. Expression is strongly correlated with survival, prognosis or tumor grade.
  • B7-H3 may have functions similar to PD-L1-mediated T cell inhibitory signaling. It has been proposed that B7-H3 has co-stimulatory and co-suppressive functions depending on tumor specificity, microenvironmental factors and signal strength. In addition to its role as an immunomodulator, B7-H3 has been implicated in enhancing cancer metastasis and angiogenesis.
  • B7-H3 expression is mainly restricted to tumors, B7-H3 is a very important tumor-associated antigen and can be a target for potential broad-spectrum immunotherapy.
  • the present invention provides anti-B7-H3 antibody and its encoding gene and application.
  • the present inventors obtained the anti-human B7-H3 antibody of the present invention with high affinity and specificity through hybridoma screening, chimeric antibody construction and humanization.
  • the present invention provides a novel antibody or antigen-binding fragment thereof that binds to the B7-H3 molecule.
  • the anti-B7-H3 antibodies of the invention have one or more of the following properties:
  • the anti-B7-H3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH comprises
  • amino acid changes preferably amino acids
  • amino acid changes compared to the VH sequence of any of the antibodies listed in Table B substitutions, more preferably conservative substitutions of amino acids
  • an anti-B7-H3 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises:
  • amino acid changes preferably amino acids
  • amino acid changes compared to the VL sequence of any of the antibodies listed in Table B substitutions, more preferably conservative substitutions of amino acids
  • 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
  • amino acid changes preferably amino acids
  • amino acid changes compared to the VH sequence of any of the antibodies listed in Table B substitutions, more preferably conservative substitutions of amino acids
  • VL comprises:
  • amino acid changes preferably amino acids
  • amino acid changes compared to the VL sequence of any of the antibodies listed in Table B substitutions, more preferably conservative substitutions of amino acids
  • the present invention provides an antibody or antigen-binding fragment thereof that binds B7-H3, comprising: HCDR1 of the heavy chain variable region set forth in any one of SEQ ID NOs: 16, 18, 20 or 22, 2 and 3 sequences, and/or the LCDR1, 2 and 3 sequences of one of the light chain variable regions shown in any one of SEQ ID NOs: 17, 19, 21 or 23, or a variant of a combination of said CDR sequences .
  • the present invention provides an antibody or antigen-binding fragment thereof that binds B7-H3, comprising the 3 complementarity determining regions HCDR of the heavy chain variable region, and the 3 complementarity determining regions of the light chain variable region LCDR, wherein HCDR1 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NO: 1 or 8 and HCDR2 comprises the amino acid set forth in any one of SEQ ID NO: 2, 7, 9 or 14
  • the sequence or consists of the amino acid sequence, HCDR3 comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 3 or 10, and LCDR1 comprises any one of SEQ ID NO: 4, 11 or 15
  • LCDR2 comprises or consists of the amino acid sequence shown in any one of SEQ ID NO:5 or 12
  • LCDR3 comprises any one of SEQ ID NO:6 or 13
  • the invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof that binds a B7-H3 molecule, comprising a heavy chain variable region VH and/or a light chain variable region VL, wherein,
  • VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 16
  • the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 17;
  • VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 18, and the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 19;
  • the VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 20
  • the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 21;
  • 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.
  • 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,
  • the VH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 1 or 8; HCDR2 comprises SEQ ID NO: 2, 7, 9 , the amino acid sequence shown in any one of 14 or consists of the sequence; HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 3 or 10 or consists of the sequence;
  • VL comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence shown in any one of SEQ ID NO:4, 11 or 15; LCDR2 comprises SEQ ID NO:5 or The amino acid sequence shown in any one of 12 or consists of the sequence; LCDR3 comprises or consists of the amino acid sequence shown in any one of SEQ ID NO: 6 or 13.
  • the invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof comprising:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the sequence
  • 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;
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the sequence
  • 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;
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 8, or consists of the sequence
  • 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;
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 8, or consists of the sequence
  • 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 which comprises or consists 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 a heavy chain variable region VH and/or a light chain variable region VL, wherein,
  • (ii) comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 16, 18, 20 or 22; or
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR region;
  • (ii) comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 17, 19, 21 or 23; or
  • amino acid sequence of amino acid changes preferably amino acid substitutions, more preferably amino acid conservative substitutions, preferably, the amino acid changes do not occur in the CDR regions.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof comprising
  • amino acid sequence comprising 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
  • the heavy chain variable region VH and comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or The light chain variable region VL of the amino acid sequence of 99% identity;
  • amino acid sequence comprising 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
  • the heavy chain variable region VH and comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or The light chain variable region VL of the amino acid sequence of 99% identity;
  • amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 20
  • the heavy chain variable region VH and comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or The light chain variable region VL of the amino acid sequence of 99% identity;
  • amino acid sequence comprising 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
  • the heavy chain variable region VH and comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or The light chain variable region VL of the amino acid sequence of 99% identity.
  • the present invention provides an antibody or antigen-binding fragment thereof that binds B7-H3, comprising
  • the invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof comprising a heavy chain and/or a light chain, wherein
  • amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, the amino acid changes The changes do not occur in the variable region of the heavy chain;
  • (ii) comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 25, 27, 29 or 31; or
  • amino acid sequence of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region of the light chain, more preferably, the amino acid changes Changes did not occur in the light chain variable region.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof comprising
  • the present invention provides an antibody or antigen-binding fragment thereof that binds B7-H3, comprising
  • a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 30 or composed thereof, and a light chain comprising the amino acid sequence shown in SEQ ID NO: 31 or composed thereof.
  • the anti-B7-H3 antibody of the invention is an antibody in the IgGl format, an antibody in the IgG2 format, an antibody in the IgG3 format, or an antibody in the IgG4 format; preferably, the anti-B7-H3 antibody is an antibody in the IgGl format.
  • the anti-B7-H3 antibody is a monoclonal antibody.
  • the anti-B7-H3 antibody is a chimeric antibody, and in preferred embodiments, the anti-B7-H3 antibody is a humanized antibody.
  • Anti-B7-H3 antibodies of the invention also encompass antibody fragments thereof, preferably antibody fragments selected from the group consisting of: Fab, Fab', Fab'-SH, F(ab') 2 , Fv, single chain antibodies (eg scFv) , single domain antibodies, diabodies (dAbs) or linear antibodies.
  • the present invention provides an isolated nucleic acid encoding an anti-B7-H3 antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector.
  • the invention provides a method of making an anti-B7-H3 antibody or antigen-binding fragment thereof, the method comprising culturing a host cell of the invention under conditions suitable for expression of a nucleic acid encoding the invention .
  • the present invention provides anti-B7-H3 antibodies and antigen-binding fragments thereof prepared by the methods described above.
  • the present invention provides immunoconjugates and pharmaceutical compositions comprising anti-B7-H3 antibodies or antigen-binding fragments thereof.
  • the present invention also provides the use of anti-B7-H3 antibodies or antigen-binding fragments thereof, immunoconjugates or pharmaceutical compositions in the preparation for the prevention and/or treatment of B7-H3-related diseases or disorders (eg, tumors) ) in the drug application.
  • B7-H3-related diseases or disorders eg, tumors
  • the present invention also provides a method of preventing and/or treating a B7-H3-related disease or disorder (eg, tumor), the method comprising administering to a subject an effective amount of a B7-H3-binding antibody of the present invention or an antigen-binding fragment, immunoconjugate or pharmaceutical composition thereof.
  • a B7-H3-related disease or disorder eg, tumor
  • the present invention also relates to a method for detecting B7-H3 molecules in a sample, the method comprising (a) contacting the antibody or antigen-binding fragment thereof of the present invention with the sample; and (b) detecting whether the antibody or antigen thereof is formed in the sample The complex of the binding fragment with the B7-H3 molecule.
  • the present invention also 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) binding the antibody or antigen thereof of the present invention contacting the fragment with the sample; and (c) detecting whether a complex of the antibody or antigen-binding fragment thereof and the B7-H3 molecule is formed in the sample.
  • Figure 3 Anti-tumor results of antibodies in vivo, in which Figure 3a: tumor volume changes in tumor-bearing mice, Figure 3b-3c: body weight changes in tumor-bearing mice
  • antibody is used herein in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (eg, , bispecific antibodies), single chain antibodies, whole antibodies or antibody fragments thereof exhibiting the desired antigen-binding activity.
  • An intact antibody will generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, eg, antibodies naturally occurring in camels may contain only heavy chains.
  • antigen-binding fragment refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds the antigen to which the intact antibody binds.
  • antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies (dAbs); linear antibodies; single chain antibodies (eg, scFv); single domains Antibodies (single domain antibodies); antigen-binding fragments of bivalent or bispecific antibodies; camelid antibodies; and other fragments that exhibit the desired ability to bind antigen (eg, B7-H3).
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively).
  • KD equilibrium dissociation constant
  • kdis and kon association rate constant
  • Affinity can be measured by common methods known in the art.
  • One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region generally extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed . Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in antibody binding to an antigen.
  • the variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (see, e.g., Kindt et al. Kuby Immunology, 6th ed ., WH Freeman and Co. p. 91 (2007)).
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • antibodies that bind a particular antigen can be isolated using the VH or VL domains from antibodies that bind to the antigen by screening libraries of complementary VL or VH domains, respectively, see, eg, Portolano et al., J. Immunol. 150:880 -887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • CDR regions or “CDRs” or “hypervariable regions” (used interchangeably herein with hypervariable regions “HVR”), are the variable domains of antibodies that are highly variable in sequence And form structurally defined loops ("hypervariable loops") and/or regions containing antigen-contacting residues ("antigen contact points").
  • the CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are numbered sequentially from the N-terminus and are commonly referred to as CDR1, CDR2 and CDR3.
  • the CDRs located within the variable domains of antibody heavy chains are also referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3.
  • its CDR sequence can be determined using various protocols well known in the art, for example, Kabat complementarity determining regions (CDRs) are determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • AbM CDRs are a compromise between Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. The residues of each of these CDRs HVR/CDR are described below according to different CDR determination schemes.
  • the CDRs may also be CDR sequences located at the following Kabat residue positions according to the Kabat numbering system:
  • Positions 24-36 or 24-34 in VL (LCDR1), Positions 46-56 or 50-56 (LCDR2), and Positions 89-97 or 89-96 (LCDR3); and Positions 26-35 in VH or 27-35B (HCDR1), positions 50-65 or 49-65 (HCDR2), and positions 93-102, 94-102 or 95-102 (HCDR3).
  • HCDR1 of an antibody of the invention is bounded by the AbM rules
  • HCDR2, HCDR3 and LCDR are bounded by the Kabat rules, eg, as shown in Table A below.
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • a residue position in an antibody variable region refers to the numbering system according to the Kabat ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) numbered positions.
  • the CDR boundaries of the variable regions of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined by the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells eg, NK cells, neutrophils, and macrophages
  • the secreted immunoglobulins enable these cytotoxic effector cells to specifically bind antigen-bearing target cells, followed by a cytotoxic form of cytotoxicity that kills the target cells.
  • NK cells the primary cells mediating ADCC, express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • an in vitro ADCC assay can be performed, or the ADCC activity of a molecule of interest can be assessed in vivo, eg, in an animal model.
  • An exemplary assay for assessing ADCC activity is provided in the Examples herein.
  • the term "functional Fc region” refers to an Fc region that possesses the "effector functions" of a native sequence Fc region.
  • exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; Such effector functions generally require the association of an Fc region with a binding domain, such as an antibody variable domain, and can be assessed using a variety of assays, such as those disclosed herein.
  • therapeutic agent encompasses any substance that is effective in preventing or treating tumors (eg, cancer), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulatory agents .
  • immunomodulator refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response.
  • the immune response can be a humoral response or a cellular response.
  • an “effective amount” refers to an amount or dose of an antibody or fragment or conjugate or composition of the invention which, after administration to the patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.
  • an “effective amount” can be distinguished as a “therapeutically effective amount” and a “prophylactically effective amount”.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by taking into account a variety of factors such as the species, size, age and general health of the mammal, the specific disease involved, the degree or severity of the disease, the individual patient response, the particular antibody administered, the mode of administration, the bioavailability characteristics of the administered formulation, the chosen dosing regimen, and the use of any concomitant therapy.
  • an effective amount of a B7-H3 antibody of the invention preferably inhibits a measurable parameter (eg, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, compared to a control.
  • a measurable parameter eg, tumor growth rate, tumor volume, etc.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the original primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell.
  • 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, eg, an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody .
  • humanized antibody refers to an antibody in which an antigen-binding site derived from the germline of another mammalian species, such as a mouse, is attached to human immunoglobulin sequences.
  • a humanized antibody is a chimeric molecule, usually prepared using recombinant techniques, that can have additional framework region modifications within the human framework sequences.
  • the antigen binding site may comprise the entire variable domain fused to the constant region, or only the complementarity determining regions grafted onto suitable framework sequences in the variable domain.
  • a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDRs (eg, 6 CDRs) correspond to those of the non-human antibody, And all or substantially all of the FRs correspond to those of the human antibody.
  • a humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody eg, a non-human antibody refers to an antibody that has been humanized.
  • immunoconjugate is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused.
  • the label can itself be detectable (eg, a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, can catalyze a detectable chemical change of a substrate compound or composition.
  • the term is intended to encompass direct labeling of a probe or antibody by coupling (ie, 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 primary antibodies using fluorescently labeled secondary antibodies and end-labeling of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin.
  • mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, , mice and rats).
  • domestic animals eg, cattle, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and rats
  • rodents eg, mice and rats.
  • the individual or subject is a human.
  • isolated antibody is an antibody that has been separated from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity, such as by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) HPLC) determined.
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography eg, ion exchange or reversed phase
  • 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), contained in a single vector or separate vectors such nucleic acid molecules in , as well as such nucleic acid molecules present at one or more locations in the host cell.
  • the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred set of parameters (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • pharmaceutical adjuvant refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active substance is administered.
  • composition refers to a composition that is in a form that allows the biological activity of the active ingredients contained therein to be effective and does not contain additional ingredients.
  • treating refers to slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or progression of a disease or disorder or symptoms of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for preventive regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • tissue or cell samples refers to a collection of tissue or cell samples obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue (eg from a fresh, frozen and/or preserved organ or tissue sample or biopsy or biopsy); blood or any blood component; bodily fluids (such as cerebrospinal fluid, amniotic fluid ( amniotic fluid), peritoneal fluid (ascites), or interstitial fluid); cells from any time of pregnancy or development in a subject.
  • B7-H3 is a type I transmembrane glycoprotein belonging to a member of the B7/CD28 superfamily and is similar in sequence to the extracellular domain of PD-L1.
  • B7-H3 has 316 amino acids, including a putative signal peptide consisting of 28 amino acids, an extracellular domain consisting of 217 amino acids, a transmembrane region and a cytoplasmic domain consisting of 45 amino acids, molecular weight About 45-66kDa.
  • B7-H3 In humans, due to exon duplication, the extracellular structure of B7-H3 can be an IgV-IgC-like domain (2Ig-B7-H3), or an IgV-IgC-IgV-IgC-like domain (4Ig-B7- H3).
  • the sequence of cynomolgus B7-H3 shares approximately 90% homology with its human counterpart.
  • anti-B7-H3 antibody refers to an antibody, or antigen-binding fragment thereof, capable of Sufficient affinity to bind B7-H3 protein.
  • the antibodies can be used as diagnostic and/or therapeutic agents targeting B7-H3.
  • an anti-B7-H3 antibody or antigen-binding fragment thereof of the invention binds B7-H3 (eg, human or cynomolgus monkey B7-H3) with sufficient affinity, eg, with the following equilibrium dissociation constant ( KD ) binds to B7-H3 with a K D ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (eg, below 10 ⁇ 7 M, eg, 10 ⁇ 7 M to 10 -10 M).
  • KD equilibrium dissociation constant
  • the B7-H3 is human or cynomolgus monkey B7-H3.
  • antibody binding affinity is determined using biolight interferometry, eg, in biolight interferometry, the antibody is measured at about 1 x 10-7 M or less, about 5 x 10-8 M or less, About 1x10-8M or less, about 5x10-9M or less KD , about 1x10-9M or less KD , about 1x10-10M or less K D , binds to human B7-H3.
  • the antibodies or antigen-binding fragments thereof of the invention bind to cell surface expressed B7-H3.
  • the antibodies or antigen-binding fragments thereof of the invention can induce ADCC effects. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention can inhibit and/or reduce tumor growth and/or volume in vivo.
  • the B7-H3 binding 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 the VH and VL comprise a variable region selected from the group consisting of A combination of the 6 CDRs shown in Table A.
  • VH heavy chain variable region
  • VL light chain variable region
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain.
  • substitutions are conservative substitutions.
  • Conservative substitutions refer to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another neutral amino acid replacement. Exemplary permutations are shown in the following table:
  • the substitutions occur in the CDR regions of the antibody.
  • the variant obtained has a modification (eg, improvement) in certain biological properties (eg, increased affinity) relative to the parent antibody and/or will have certain biological properties that are substantially retained of the parent antibody.
  • exemplary substitutional variants are affinity matured antibodies.
  • the antibodies provided herein are altered to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When an antibody contains an Fc region, the carbohydrate attached to it can be varied. In some applications, modifications to remove unwanted glycosylation sites are useful, such as removal of fucose moieties to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fc region variants can include human Fc region sequences (eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • human Fc region sequences eg, human IgGl, IgG2, IgG3, or IgG4 Fc regions
  • amino acid modifications eg, substitutions
  • Fc variants see US Pat. No. 7,332,581, US Pat. No. 6,737,056, US Pat. No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001), USA Patent No.
  • cysteine-engineered antibodies eg, "thioMAbs”
  • cysteine residues of the antibody are replaced with cysteine residues.
  • Cysteine engineered antibodies can be generated as described, eg, in US Pat. No. 7,521,541.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • the present invention provides nucleic acids encoding any of the above anti-B7-H3 antibodies or antigen-binding fragments thereof.
  • a vector comprising the nucleic acid is also provided.
  • the vector is an expression vector.
  • the present invention also provides host cells comprising the nucleic acid or the vector.
  • the host cell is eukaryotic.
  • the host cell is selected from E. coli cells, mammalian cells (eg, CHO cells or 293 cells), or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • the host cell is prokaryotic.
  • the host cell is selected from Escherichia coli.
  • each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences because of codon degeneracy.
  • polynucleotide sequences can be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of sequences encoding antibodies or antigen-binding fragments thereof that bind B7-H3 using methods well known in the art.
  • one or more vectors comprising the nucleic acids of the invention are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • a host cell comprising the vector.
  • Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required.
  • the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • fungal and yeast strains in which the glycosylation pathway has been "humanized” result in antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).
  • Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines engineered for growth in suspension can be used.
  • Other examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (293HEK or 293 cells, eg, Graham et al., J. Gen Virol. 36:59 (1977) described in) etc.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • the present invention provides a method of making an anti-B7-H3 antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises in a method suitable for expressing an antibody or fragment thereof (preferably an antigen-binding fragment) encoding the The host cell is cultured under conditions for nucleic acid, and the antibody or fragment thereof is optionally isolated. In a certain embodiment, the method further comprises recovering the anti-B7-H3 antibody or fragment thereof from the host cell.
  • the present invention provides multispecific (including bispecific) antibody molecules that specifically bind B7-H3.
  • the antibody of the invention (or antigen-binding fragment thereof) forms a first binding specificity for B7-H3.
  • the binding specificity is provided by the "binding site” or "antigen binding site” of the antibody (the region of the antibody molecule that actually binds to the antigen).
  • 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).
  • a "multispecific" antibody is an antibody having at least two antigen-binding sites, each of which may be associated with a different expression of the same antigen. or bind to different epitopes of different antigens.
  • the invention provides immunoconjugates produced by conjugating an antibody of the invention to a heterologous molecule.
  • the heterologous molecule is, for example, a therapeutic or diagnostic agent, such as a cytotoxic or chemotherapeutic agent.
  • Cytotoxic agents include any agent that is detrimental to cells. Examples of cytotoxic agents suitable for forming immunoconjugates are known in the art.
  • Linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers.
  • the linker is a "cleavable linker" that facilitates release of the polypeptide after delivery to the target site.
  • acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers can be used (Chari et al. Cancer Research 52(1992) 127-131; US 5,208,020) .
  • the antibodies of the invention may be conjugated to a diagnostic or detectable agent.
  • a diagnostic or detectable agent can be used to monitor or predict the onset, development, progression and/or severity of a disease or disorder as part of a clinical assay (e.g., to determine the efficacy of a particular therapy).
  • diagnosis and detection can be accomplished by conjugating antibodies to detectable agents including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase; prosthetic groups, such as, but not limited to, streptavidin. Avidin/biotin and avidin/biotin; fluorescent substances; luminescent substances; radioactive substances; and positron emitting and nonradioactive paramagnetic metal ions used in various positron emission imaging procedures.
  • the immunoconjugate is used to prevent or treat tumors.
  • the tumor is cancer.
  • compositions comprising an anti-B7-H3 antibody or an immunoconjugate or multispecific antibody thereof and a composition comprising an anti-B7-H3 antibody or an immunoconjugate or multispecific antibody encoding the same
  • compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • compositions or formulations of the present invention may also be combined with one or more other active ingredients desired for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other Element.
  • the present invention also provides pharmaceutical combinations.
  • the combination product comprises an antibody, immunoconjugate or multispecific antibody of the invention and a second therapeutic agent formulated in the same pharmaceutical composition or formulation.
  • the combination product comprises the antibody, immunoconjugate or multispecific antibody of the invention and the second therapeutic agent contained separately in different pharmaceutical compositions or formulations.
  • the second therapeutic agent can be administered before, simultaneously (eg, in the same formulation or in a different formulation), or after administration of the antibody of the invention.
  • the pharmaceutical compositions, formulations and combination products of the present invention can be provided in articles of manufacture for the treatment, prevention and/or diagnosis of the diseases and/or disorders described herein.
  • the article of manufacture may contain a container and a label or package insert. Suitable containers include, for example, bottles, syringes, IV bags, and the like. Containers can be made from a variety of materials such as glass or plastic.
  • the article of manufacture may comprise (a) a first container containing an antibody or antibody fragment, immunoconjugate or multispecific antibody of the invention; and optionally (b) a first container containing a second therapeutic agent Two containers.
  • the articles of manufacture may also contain other materials desirable from a commercial and user standpoint, including buffers, pharmaceutically acceptable diluents, such as sterile water for injection, needles, syringes, syringe pumps, and the like.
  • One aspect of the present invention provides a method of preventing and/or treating a B7-H3-related disease or disorder (eg, cancer), comprising administering to a subject an effective amount of an anti-B7-H3 antibody or antigen-binding fragment thereof of the present invention, immune Conjugates or pharmaceutical compositions.
  • a B7-H3-related disease or disorder eg, cancer
  • the subject may be a mammal, eg, a primate, preferably a higher primate, eg, a human.
  • the subject has or is at risk of having a disease described herein.
  • the subject has received or has received other treatments, such as chemotherapy treatment and/or radiation therapy.
  • the present invention provides the use of an anti-B7-H3 antibody or antigen-binding fragment, immunoconjugate or pharmaceutical composition thereof in the manufacture or manufacture of a medicament for the prevention and/or treatment of B7 as referred to herein -H3-related disease or condition.
  • an antibody or antigen-binding fragment or immunoconjugate or composition or product of the invention delays the onset of the disorder and/or symptoms associated with the disorder.
  • the antibodies of the invention can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal, Also, intralesional administration if required for local treatment.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • administration may be by any suitable route, eg, by injection, eg, intravenously or subcutaneously.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • an antibody of the invention when used alone or in combination with one or more other therapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease , whether the antibody is administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to the patient in a single treatment or over a series of treatments.
  • compositions, multispecific antibodies or immunoconjugates of the present invention may be administered in place of the antibodies or antigen-binding portions of the present invention.
  • a composition, multispecific antibody or immunoconjugate of the present invention may be further administered.
  • any of the anti-B7-H3 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of B7-H3 in a biological sample.
  • the detection results are used for diagnosis or auxiliary diagnosis of diseases.
  • the term "detection" includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg RT-PCR).
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the biological sample is from a hyperproliferative or cancerous lesion.
  • the present invention provides methods and kits for the detection of B7-H3 in biological samples.
  • the B7-H3 is human B7-H3 or cynomolgus monkey B7-H3.
  • the method comprises contacting a biological sample with an anti-B7-H3 antibody as described herein under conditions that allow binding of the antibody to B7-H3, and detecting whether the anti-B7-H3 antibody and B7-H3 are formed H3 complex.
  • the method can be in vitro or in vivo.
  • the sample is from a cancer patient.
  • the sample can be a tissue biopsy, tissue section, body fluid such as blood, plasma, or serum.
  • a method of treating a B7-H3-related disease or disorder comprising: administering to a subject a therapeutically effective amount of an anti-B7-H3 antibody.
  • the method further comprises administering to the subject one or more other therapies.
  • an anti-B7-H3 antibody is used to select subjects suitable for treatment with an anti-B7-H3 antibody, eg, wherein B7-H3 is a biomarker for selection of said subjects.
  • the antibodies of the invention can be used to diagnose tumors, eg, to evaluate (eg, monitor) a subject for treatment or progression of a disease described herein, diagnosis and/or staging thereof.
  • the presence or absence of cancer cells in various tissues is determined by using the anti-B7-H3 antibodies of the present application.
  • the anti-B7-H3 antibody of the present application can be used to determine the presence or absence of cancer cells released from solid tumors into the circulating blood and the level of B7-H3, and the circulating B7-H3 antigen can be an intact B7-H3 molecule , or a fragment thereof.
  • the detection method is carried out, for example, by the FACS method.
  • labeled anti-B7-H3 antibodies include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.
  • the spleen of the mouse is extracted to prepare a B lymphocyte suspension, which is then mixed with SP2/0 myeloma cells (ATCC, CRL-1581) in a ratio of 1:2 to 1:1. Electrofusion was performed after mixing. Transfer the fused cells from the electrode dish into a 50ml centrifuge tube, and dilute the cells with screening medium (the composition is shown in Table 1) to obtain a cell suspension (concentration 1-2 ⁇ 10 4 cells/ml). Add 100 ⁇ l of cell suspension to each well of a 96-well plate. Fresh selection medium was replaced on day 5 after fusion. According to the cell growth state, after culturing for 10 days (or longer), perform flow cytometry (FACS) detection to screen positive clones.
  • FACS flow cytometry
  • Hybridoma cells specifically expressing anti-B7H3 antibody were screened by flow cytometry (FACS). Briefly, CHO cells expressing human B7H3 (CHO-huB7H3) were counted, diluted to 1 ⁇ 10 6 cells/ml, then 100 ⁇ l was added to each well of a U-bottom 96-well plate, and the cell culture was removed by centrifugation at 500 g for 5 min. base. Then, each culture supernatant and positive control antibody (MGA271) in the above hybridoma 96-well plate were added to the U-shaped plate containing CHO cells and the cells were resuspended, 100 ⁇ l per well, and let stand on ice for 30 min.
  • FACS flow cytometry
  • the supernatant was removed by centrifugation at 500 g for 5 min, and the cells were washed once with PBS solution.
  • the PBS solution was removed by centrifugation at 500 g for 5 min.
  • 100 ⁇ l of anti-mouse Fab FITC-labeled secondary antibody (diluted 1:500 in PBS solution) was added to each well, and 100 ⁇ l of anti-human Fab FITC-labeled secondary antibody was added to the positive control antibody culture well. Incubate on ice for 30 min in the dark, centrifuge at 500 g for 5 min to remove the supernatant, and wash the cells once with PBS solution. Afterwards, the cells were resuspended in 50 ⁇ l PBS solution for FACS detection, and positive clones were obtained by screening.
  • the obtained positive clone was re-screened by the CHO cell (CHO-cynoB7H3) expressing cynomolgus monkey B7H3 (SEQ ID NO: 34), and 2 strains combined with both human B7H3 and monkey B7H3 were obtained.
  • Hybridoma cells 19A2 and 20G5.
  • the affinity of the obtained two hybridoma cell lines to the antigen was measured by biofilm thin-layer interferometry (ForteBio). The obtained KD values are shown in Table 2.
  • the specific steps are as follows: replace the HAT in the screening medium with HT (Gibco, Cat#11067-030) to obtain a basal medium, and add 200 ⁇ l per well to a 96-well plate.
  • the positive hybridoma cells screened by the above fusion were added to the first row of a 96-well plate at a density of about 1 ⁇ 10 5 cells/ml, 300ul per well, and mixed well. Take 100 ⁇ l of the cell suspension in the first row and add it to the second row. After fully mixing, take 100 ⁇ l and add it to the next row. Repeat the above steps until the last row, and let it stand for 15 minutes.
  • the positive control antibody used in the present invention is MGA271, also known as Enoblituzumab (derived from MacroGenics US20160264672A1).
  • the antibody light and heavy chain gene sequences were extracted from the hybridoma positive clones obtained in Example 1, and human-mouse chimeric antibodies were constructed using them.
  • Reaction 1 was added to the following reverse transcription system (Table 4) in a total amount of 20 ⁇ l:
  • the cDNA was connected to the T vector, and then the Mighty TA-cloning Kit (Takara) was used to amplify the heavy and light chain variable regions of the antibody from the obtained cDNA by PCR.
  • the PCR reaction system is shown in Table 5.
  • OVH2 CAGGTRCAGCTGAAGSAGTCAGG (SEQ ID NO: 40) 10.7 OVH3 GAKGTGCAGCTTCAGCAGTCRGG (SEQ ID NO: 41) 8.9 OVH5 GAVGTGAWGCTGGTGGAGTCTGR (SEQ ID NO: 42) 7.1 OVH11 GAAGTGCAGCTGTTGGAGACTGG (SEQ ID NO: 43) 3.6 OVH14 GAGGTTCAGCTGCAGCAGTCTGK (SEQ ID NO: 44) 16.1 OVH15 CAGGTTCACCTACAACAGTCTGG (SEQ ID NO:45) 3.5 REVESE-6 CTGAGGARACGGTGACCG (SEQ ID NO: 46) 6 REVESE-4 CTGAGGAGACTGTGAGAGWGGT (SEQ ID NO: 47) 4 REVESE-2-1 CTGAGGAGACGGTGACTGAGGT (SEQ ID NO: 48) 2 REVESE-2-2 CTGCAGAGACAGTGACCAGAGT (SEQ ID NO: 49) 2 water qs
  • Primer Mix 1 was obtained for subsequent PCR amplification of VH.
  • VL Light chain variable region
  • Primer Mix 2 was obtained for subsequent PCR amplification of VL.
  • the PCR system is shown in Table 9.
  • the PCR amplification product was recovered by cutting the gel.
  • the homologous recombination system is shown in Table 10.
  • the reaction was carried out at 37°C for 30 min to obtain the recombinant product.
  • the recombinant products were transformed into TOP10 competent cells, and single clones were picked for sequencing.
  • the clones containing the plasmids with the correct insertion direction were selected as positive clones, and the positive clones were saved, thereby obtaining the recombinant plasmids of chimeric antibodies.
  • a certain amount of recombinant plasmid was prepared by extraction for antibody expression.
  • the present invention obtains a total of 2 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 hybridoma cells in Table A-B, the chimeric antibodies are preferably The amino acid sequences of the light and heavy chains are shown in Table C.
  • HEK293 cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1.5 x 106 cells/ml the day before transfection. The cell density on the day of transfection was approximately 3 x 106 cells/ml. Take Opti-MEM medium (Gibco product number: 31985-070) with a final volume of 1/10 (v/v) as the transfection buffer, add the recombinant expression plasmid constructed above, mix well, and filter with a 0.22 ⁇ m filter for use .
  • Opti-MEM medium Gibco product number: 31985-070
  • PEI polyethyleneimine
  • the cell culture medium was centrifuged at 13000 rpm for 20 min, the supernatant was collected, and the supernatant was purified with a prepacked column Hitrap Mabselect Sure (GE, 11-0034-95) according to the manufacturer's instructions, and the concentration was determined. Take 100 ⁇ g of purified protein, adjust the concentration to 1 mg/mL, and use gel filtration chromatography column SW3000 (TOSOH product number: 18675) to measure the protein purity. The results show that a high-purity chimeric antibody has been obtained.
  • Example 3 Determination of the binding kinetics of the chimeric antibody of the present invention to the antigen by thin-layer interference technology
  • the equilibrium dissociation constant (KD) of the antibody of the present invention binding to human B7H3 was determined by biofilm thin-layer interferometry (ForteBio). ForteBio affinity assays were performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).
  • AMQ Pall, 1506091
  • AHQ Pall, 1502051
  • positive control detection sensors were equilibrated off-line in assay buffer for 30 minutes, followed by on-line detection for 60 seconds to establish a baseline
  • Purified antibodies obtained as described above were loaded online onto an AHQ sensor (ForteBio) for ForteBio affinity measurements.
  • the sensors with loaded antibodies were then exposed to antigens (including human 4Ig-B7H3, human 2Ig-B7H3 (ACRO, cat. no. B73-H52E2), and cynomolgus monkey B7H3 (SINO BIOLOGICAL, cat. no. Transfer to assay buffer for off rate measurements.
  • KD values were analyzed using ForteBio analysis software.
  • the chimeric antibody we obtained through hybridoma has a good affinity for human B7H3 protein, and also maintains a high affinity for cynomolgus monkey B7H3.
  • the antibody of this study has a higher affinity.
  • the chimeric antibody obtained in Example 2 was humanized according to a conventional method.
  • humanized antibodies hz20G5, hz19A2, their CDR sequences, light chain variable region and heavy chain variable region sequences, amino acid sequences of light chain and heavy chain are shown in Tables A-C.
  • Example 4 As described in Example 3, the affinity of the humanized antibodies obtained in Example 4 for binding to antigens (human B7H3 and cynomolgus monkey B7H3) was determined using the ForteBio assay, expressed as equilibrium dissociation constant (KD). The results are shown in Table 12.
  • the humanized antibody still has a high affinity for the antigen B7H3, and it has an equilibrium dissociation constant K D comparable to the corresponding chimeric antibody for the antigen B7H3.
  • the humanized antibody obtained in the present application has higher binding antigen affinity than the control antibody MGA271, especially for human 2Ig-B7-H3, and the affinity of the antibody of the present application is 20-100 times higher than that of MGA271.
  • Example 6 Humanized antibodies bind to CHO-S cells overexpressing human and cynomolgus B7H3
  • cDNAs were cloned into pCHO1.0 vector (Invitrogen) and then transfected into CHO-S cells, resulting in overexpression CHO-S cells of human 4Ig-B7H3, human 2Ig-B7H3 and cynomolgus monkey B7H3: CHOS-hB7H3-4Ig, CHOS-hB7H3-2Ig, CHOS-cyno B7H3.
  • Fig. 1 The detection results are shown in Fig. 1.
  • the overall affinity of the antibody of the present invention is comparable to that of the positive control MGA271.
  • the affinity of the hz19A2 antibody was comparable to that of MGA271, while the affinity of hz 20G5 was significantly higher than that of MGA271. It can be seen that the antibody of the present application exhibits a significantly improved antigen-binding ability at the cellular level.
  • ADCC effector cells Promega's Jurkat-ADCCNF-AT luciferase effector cell line
  • the antibodies of the present invention with different concentration gradients were added in sequence: the final concentration of the initial well was 30 nM, and then three-fold dilution was carried out, for a total of 10 gradients.
  • Example 8 In vivo anti-tumor effect of the antibody molecule of the present application
  • the anti-tumor effect of the anti-B7H3 antibody molecule obtained in the present application was studied in a tumor-bearing mouse model.
  • mice (18-20 g) with SPF grade were used, which were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the certificate number was NO.1100112011025061.
  • A375 cells (ATCC, CRL-1619) were routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation, and the A375 cells were dispersed with PBS (1 ⁇ ) to prepare a cell suspension with a cell concentration of 2.5 ⁇ 10 7 cells/ml.
  • the A375 tumor-bearing mouse model was established by subcutaneously inoculating 0.2 ml of the cell suspension into the right abdominal area of CB-17SCID mice on day 0.
  • mice On the 0th day of inoculation with tumor cells, all mice were randomly divided into groups (8 mice in each group), and the mice were administered on the 0th, 4th, 7th, and 11th days after inoculation, respectively, with the dosage, the way of administration and the corresponding antibody As shown in Table 13.
  • TGI% relative tumor inhibition rate
  • TGI% 100%*(tumor volume of control group-tumor volume of treatment group)/(tumor volume of control group-tumor volume of control group before administration).
  • Tumor inhibition rate (%) h-IgG1, 10mg/kg 389.52 N/A hz19A2, 10mg/kg 194.82 62.4 hz20G5, 10mg/kg 246.05 46.0

Abstract

涉及特异性结合B7-H3的新型抗体及其抗原结合片段和含有所述抗体或其抗原结合片段的组合物。涉及编码所述抗体或其抗原结合片段的核酸、包含所述核酸的宿主细胞，以及相关用途。此外，涉及这些抗体或其抗原结合片段的治疗和诊断用途。

Description

抗B7-H3抗体及其用途 技术领域
本发明涉及特异性结合B7-H3的新型抗体及其抗原结合片段和含有所述抗体或其抗原结合片段的组合物。此外，本发明涉及编码所述抗体或其抗原结合片段的核酸、包含所述核酸的宿主细胞，以及相关用途。此外，本发明涉及这些抗体或其抗原结合片段的治疗和诊断用途。
背景技术
B7-H3(又称为CD276)是一种I型跨膜糖蛋白，和PD-L1结构非常类似，同属于B7/CD28超家族。B7-H3在转录水平上(RNA)广泛表达于淋巴组织和非淋巴器官，但是B7-H3的蛋白表达却非常的局限，主要表达在激活的树突状细胞、单核细胞、T淋巴细胞、B淋巴细胞、Nk淋巴细胞上，而在其他正常组织中表达量则非常低。最近发现B7-H3在多种实体瘤上高表达，例如在肺癌，胃癌，胰腺癌，前列腺癌，肾癌，卵巢癌，子宫内膜癌，结直肠癌，肝癌和乳腺癌中高表达，其过度表达与生存，预后或肿瘤分级密切相关。除了在肿瘤上高表达之外，B7-H3可能有类似PD-L1介导的T细胞抑制信号的功能。有人提出B7-H3具有共刺激和共抑制功能，这取决于肿瘤特异性，微环境因素和信号强度。除了其作为免疫调节剂的作用，B7-H3与增强癌症的转移和血管生成有关。
由于B7-H3表达主要限于肿瘤，因此，B7-H3是一个非常重要的肿瘤相关抗原，可以作为潜在广谱免疫疗法的靶点。
发明内容
本发明提供了抗B7-H3抗体及其编码基因与应用。本发明人通过杂交瘤筛选、嵌合抗体构建、人源化，获得了具有高亲和力和特异性的本发明抗人B7-H3抗体。
在一个方面，本发明提供了一种新的结合B7-H3分子的抗体或其抗原结合片段。
在一些实施方案中，本发明的抗B7-H3抗体具有以下一个或多个特性：
(i)以高亲和力与人和食蟹猴B7-H3结合；
(ii)与细胞表面的B7-H3有效结合；
(iii)与可溶性B7-H3有效结合；
(iv)有效激活ADCC效应；
(v)有效抑制或减缓体内肿瘤的生长和发展。
在一些实施方案中，本发明的抗B7-H3抗体或其抗原结合片段包含重链可变区(VH)，其中所述VH包含
(i)表B所列任一抗体的VH中所含的三个互补决定区域(CDR)；或
(ii)表A所列任一抗体的三个重链互补决定区域(CDR)；或
(iii)与表B所列任一抗体的VH序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，所述氨基酸改变不发生在CDR区中；或
(iv)与表B所列任一抗体的VH序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性，且包含所述序列的相应CDR的序列。
在一些实施方案中，本发明的抗B7-H3抗体或其抗原结合片段包含轻链可变区(VL)，其中所述VL包含：
(i)表B所列任一抗体的VL中所含的三个互补决定区域(CDR)；或
(ii)表A所列任一抗体的三个轻链互补决定区域(CDR)；或
(iii)与表B所列任一抗体的VL序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，所述氨基酸改变不发生在CDR区中；或
(iv)与表B所列任一抗体的VL序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性，且包含所述序列的相应CDR的序列。
在一些实施方案中，本发明的抗B7-H3抗体或其抗原结合片段包含重链可变区VH和/或轻链可变区VL，其中
(a)所述VH包含
(i)表B所列任一抗体的VH中所含的三个互补决定区域(CDR)；或
(ii)表A所列任一抗体的三个重链互补决定区域(CDR)；或
(iii)与表B所列任一抗体的VH序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，所述氨基酸改变不发生在CDR区中；或
(iv)与表B所列任一抗体的VH序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性，且包含所述序列的相应CDR的序列；
和/或
(b)所述VL包含：
(i)表B所列任一抗体的VL中所含的三个互补决定区域(CDR)；或
(ii)表A所列任一抗体的三个轻链互补决定区域(CDR)；或
(iii)与表B所列任一抗体的VL序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，所述氨基酸改变不发生在CDR区中；或
(iv)与表B所列任一抗体的VL序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性，且包含所述序列的相应CDR的序列。
在一些实施方案中，本发明提供了结合B7-H3的抗体或其抗原结合片段，包含：SEQ ID NO:16、18、20或22中任一项所示的重链可变区的HCDR1、2和3序列，和/或SEQ ID NO:17、19、21或23中任一项所示的轻链可变区之一的LCDR1、2和3序列，或所述CDR序列组合的变体。
在另一些实施方案中，本发明提供了结合B7-H3的抗体或其抗原结合片段，其包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中HCDR1包含SEQ ID NO:1或8中任一项所示的氨基酸序列或由所述氨基酸序列组成，HCDR2包含SEQ ID NO:2、7、9或14中任一项所示的氨基酸序列或由所述氨基酸序列组成，HCDR3包含SEQ ID NO:3或10中任一项所示的氨基酸序列或由所述氨基酸序列组成，LCDR1包含SEQ ID NO:4、11或15中任一项所示的氨基酸序列或由所述氨基酸序列组成，LCDR2包含SEQ ID NO:5或12中任一项所示的氨基酸序列或由所述氨基酸序列组成，LCDR3包含SEQ ID NO:6或13中任一项所示的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，本发明提供了结合B7-H3分子的抗B7-H3抗体或其抗原结合片段，其包含重链可变区VH和/或轻链可变区VL，其中，
1)所述VH包含SEQ ID NO:16所示的VH中所含的HCDR1、HCDR2和HCDR3，并且所述VL包含SEQ ID NO:17所示的VL中所含的LCDR1、LCDR2和LCDR3；
2)所述VH包含SEQ ID NO:18所示的VH中所含的HCDR1、HCDR2和HCDR3，并且 所述VL包含SEQ ID NO:19所示的VL中所含的LCDR1、LCDR2和LCDR3；
3)所述VH包含SEQ ID NO:20所示的VH中所含的HCDR1、HCDR2和HCDR3，并且所述VL包含SEQ ID NO:21所示的VL中所含的LCDR1、LCDR2和LCDR3；或
4)所述VH包含SEQ ID NO:22所示的VH中所含的HCDR1、HCDR2和HCDR3，并且所述VL包含SEQ ID NO:23所示的VL中所含的LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明提供了抗B7-H3抗体或其抗原结合片段，其包含重链可变区VH和/或轻链可变区VL，其中，
(i)所述VH包含HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:1或8中任一项所示的氨基酸序列或由所述序列组成；HCDR2包含SEQ ID NO:2、7、9、14中任一项所示的氨基酸序列或由所述序列组成；HCDR3包含SEQ ID NO:3或10中任一项所示的氨基酸序列或由所述序列组成；
和/或
(ii)其中所述VL包含LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO:4、11或15中任一项所示的氨基酸序列或由所述序列组成；LCDR2包含SEQ ID NO:5或12中任一项所示的氨基酸序列或由所述序列组成；LCDR3包含SEQ ID NO:6或13中任一项所示的氨基酸序列或由所述序列组成。
在一些实施方案中，本发明提供了抗B7-H3抗体或其抗原结合片段，其包含：
1)HCDR1，其包含SEQ ID NO:1所示的氨基酸序列，或由所述序列组成；
HCDR2，其包含SEQ ID NO:2所示的氨基酸序列，或由所述序列组成；
HCDR3，其包含SEQ ID NO:3所示的氨基酸序列，或由所述序列组成；
LCDR1，其包含SEQ ID NO:4所示的氨基酸序列，或由所述序列组成；
LCDR2，其包含SEQ ID NO:5所示的氨基酸序列，或由所述序列组成；
LCDR3，其包含SEQ ID NO:6所示的氨基酸序列，或由所述序列组成；
2)HCDR1，其包含SEQ ID NO:1所示的氨基酸序列，或由所述序列组成；
HCDR2，其包含SEQ ID NO:7所示的氨基酸序列，或由所述序列组成；
HCDR3，其包含SEQ ID NO:3所示的氨基酸序列，或由所述序列组成；
LCDR1，其包含SEQ ID NO:4所示的氨基酸序列，或由所述序列组成；
LCDR2，其包含SEQ ID NO:5所示的氨基酸序列，或由所述序列组成；
LCDR3，其包含SEQ ID NO:6所示的氨基酸序列，或由所述序列组成；
3)HCDR1，其包含SEQ ID NO:8所示的氨基酸序列，或由所述序列组成；
HCDR2，其包含SEQ ID NO:9所示的氨基酸序列，或由所述序列组成；
HCDR3，其包含SEQ ID NO:10所示的氨基酸序列，或由所述序列组成；
LCDR1，其包含SEQ ID NO:11所示的氨基酸序列，或由所述序列组成；
LCDR2，其包含SEQ ID NO:12所示的氨基酸序列，或由所述序列组成；
LCDR3，其包含SEQ ID NO:13所示的氨基酸序列，或由所述序列组成；
4)HCDR1，其包含SEQ ID NO:8所示的氨基酸序列，或由所述序列组成；
HCDR2，其包含SEQ ID NO:14所示的氨基酸序列，或由所述序列组成；
HCDR3，其包含SEQ ID NO:10所示的氨基酸序列，或由所述序列组成；
LCDR1，其包含SEQ ID NO:15所示的氨基酸序列，或由所述序列组成；
LCDR2，其包含SEQ ID NO:12所示的氨基酸序列，或由所述序列组成；
LCDR3，其包含SEQ ID NO:13所示的氨基酸序列，或由所述序列组成。
在一些实施方案中，本发明提供了抗B7-H3抗体或其抗原结合片段，其包含重链可变区VH和/或轻链可变区VL，其中，
(a)重链可变区VH
(i)包含与SEQ ID NO:16、18、20或22中任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成，且包含所述序列的相应CDR序列；或者
(ii)包含SEQ ID NO:16、18、20或22中任一项所示的氨基酸序列或由其组成；或者
(iii)包含与SEQ ID NO:16、18、20或22中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中；
和/或
(b)轻链可变区VL
(i)包含与SEQ ID NO:17、19、21或23中任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成，且包含所述序列的相应CDR序列；
(ii)包含SEQ ID NO:17、19、21或23中任一项所示的氨基酸序列或由其组成；或者
(iii)包含与SEQ ID NO:17、19、21或23中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换， 更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明提供了抗B7-H3抗体或其抗原结合片段，其包含
1)包含与SEQ ID NO:16所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区VH，和包含与SEQ ID NO:17所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区VL；
2)包含与SEQ ID NO:18所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区VH，和包含与SEQ ID NO:19所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区VL；
3)包含与SEQ ID NO:20所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区VH，和包含与SEQ ID NO:21所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区VL；
4)包含与SEQ ID NO:22所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链可变区VH，和包含与SEQ ID NO:23所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链可变区VL。
在一些实施方案中，本发明提供了结合B7-H3的抗体或其抗原结合片段，其包含
1)包含SEQ ID NO:16所示的氨基酸序列或由其组成的重链可变区VH，和包含SEQ ID NO:17所示的氨基酸序列或由其组成的轻链可变区VL；
2)包含SEQ ID NO:18所示的氨基酸序列或由其组成的重链可变区VH，和包含SEQ ID NO:19所示的氨基酸序列或由其组成的轻链可变区VL；
3)包含SEQ ID NO:20所示的氨基酸序列或由其组成的重链可变区VH，和包含SEQ ID NO:21所示的氨基酸序列或由其组成的轻链可变区VL；
4)包含SEQ ID NO:22所示的氨基酸序列或由其组成的重链可变区VH，和包含SEQ ID NO:23所示的氨基酸序列或由其组成的轻链可变区VL。
在一些实施方案中，本发明提供了抗B7-H3抗体或其抗原结合片段，其包含重链和/或轻 链，其中
(a)重链
(i)包含与SEQ ID NO:24、26、28或30中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性且包含所述序列相应CDR序列的氨基酸序列或由其组成；
(ii)包含SEQ ID NO:24、26、28或30中任一项所示的氨基酸序列或由其组成；或者
(iii)包含与SEQ ID NO:24、26、28或30中任一项所示的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中；
和/或
(b)轻链
(i)包含与SEQ ID NO:25、27、29或31中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性且包含所述序列相应CDR序列的氨基酸序列或由其组成；
(ii)包含SEQ ID NO:25、27、29或31中任一项所示的氨基酸序列或由其组成；或者
(iii)包含与SEQ ID NO:25、27、29或31中任一项所示的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中。
在一些实施方案中，本发明提供了抗B7-H3抗体或其抗原结合片段，其包含
1)包含与SEQ ID NO:24所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链，和包含与SEQ ID NO:25所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链；
2)包含与SEQ ID NO:26所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链，和包含与SEQ ID NO:27所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链；
3)包含与SEQ ID NO:28所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链，和包含与SEQ ID NO:29所示的氨 基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链；
4)包含与SEQ ID NO:30所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的重链，和包含与SEQ ID NO:31所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列的轻链。
在一些实施方案中，本发明提供了结合B7-H3的抗体或其抗原结合片段，其包含
1)包含SEQ ID NO:24所示氨基酸序列或由其组成的重链，和包含SEQ ID NO:25所示氨基酸序列或由其组成的轻链；
2)包含SEQ ID NO:26所示氨基酸序列或由其组成的重链，和包含SEQ ID NO:27所示氨基酸序列或由其组成的轻链；
3)包含SEQ ID NO:28所示氨基酸序列或由其组成的重链，和包含SEQ ID NO:29所示氨基酸序列或由其组成的轻链；
4)包含SEQ ID NO:30所示氨基酸序列或由其组成的重链，和包含SEQ ID NO:31所示氨基酸序列或由其组成的轻链。
在一些实施方案中，本发明的抗B7-H3抗体是IgG1形式的抗体、IgG2形式的抗体、IgG3形式的抗体或IgG4形式的抗体；优选地，抗B7-H3抗体是IgG1形式的抗体。
在一些实施方案中，抗B7-H3抗体是单克隆抗体。
在一些实施方案中，抗B7-H3抗体是嵌合抗体，在优选实施方案中，抗B7-H3抗体是人源化的抗体。本发明的抗B7-H3抗体还涵盖其抗体片段，优选地选自以下的抗体片段：Fab、Fab’、Fab’-SH、，F(ab’) 2、Fv、单链抗体(例如scFv)、单结构域抗体、双抗体(dAb)或线性抗体。
在一些实施方案中，本发明提供了编码抗B7-H3抗体或其抗原结合片段的分离的核酸，包含所述核酸的载体，包含所述核酸或所述载体的宿主细胞。
在一些实施方案中，本发明提供了制备抗B7-H3抗体或其抗原结合片段的方法，所述方法包括在适于表达编码本发明所述的核酸的条件下培养本发明所述的宿主细胞。在另一个实施方案中，本发明提供了由上述方法制备的抗B7-H3抗体及其抗原结合片段。
在一些实施方案中，本发明提供了包含抗B7-H3抗体或其抗原结合片段的免疫缀合物和药物组合物。
在一些实施方案中，本发明还提供了利用抗B7-H3抗体或其抗原结合片段、免疫缀合物或药物组合物在制备用于预防和/或治疗B7-H3相关疾病或病症(例如肿瘤)的药物中的应用。
在一些实施方案中，本发明还提供了预防和/或治疗B7-H3相关疾病或病症(例如肿瘤)的方法，所述方法包括向受试者施用有效量的本发明结合B7-H3的抗体或其抗原结合片段、免疫缀合物或药物组合物。
本发明还涉及检测样品中B7-H3分子的方法，所述方法包括(a)使本发明所述的抗体或其抗原结合片段与样品接触；以及(b)检测样品中是否形成抗体或其抗原结合片段与B7-H3分子的复合物。
另一个方面，本发明还涉及诊断受试者表达B7-H3分子的肿瘤的方法，所述方法包括(a)获取受试者的样品；(b)使本发明所述的抗体或其抗原结合片段与样品接触；以及(c)检测样品中是否形成抗体或其抗原结合片段与B7-H3分子的复合物。
附图说明
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1.抗体结合过表达人B7H3的CHOS细胞的FACS检测结果
图2：抗体ADCC活性检测结果
图3：抗体体内抗肿瘤结果，其中图3a：荷瘤小鼠的肿瘤体积变化，图3b-3c：荷瘤小鼠体重变化
具体实施方式
I.定义
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”意指当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其它要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“抗体”在本文中以最广意义使用并且涵盖多种抗体结构物，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人源化抗体、嵌合抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体或其显示出所需的抗原结合活性的抗体片段。完整抗体通常将包含至少两条全长重链和两条全长轻链，但在某些情况下可包括较少的链，例如骆驼中天然存在的抗体可仅包含重链。
术语“抗原结合片段”(在本文中可与“抗体片段”和“抗原结合部分”互换使用)，指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗原结合片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’) 2；双抗体(diabodies,dAb)；线性抗体；单链抗体(例如scFv)；单结构域抗体(单域抗体)；双价或双特异性抗体的抗原结合片段；骆驼科抗体；和表现出所需的结合抗原(例如B7-H3)能力的其它片段。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。在某些实施方案中，人IgG重链Fc区通常从Cys226或Pro230延伸至重链的羰基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5 th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的框架区(FR)和三个互补决定区(参见，例如，Kindt等Kuby Immunology，6 th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域分别筛选互补VL或VH结构域的文库来分离结合所述抗原的抗体，参见，例如，Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature 352：624-628(1991)。
“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变结构域中在序列上高度可变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR从N-端开始顺序编号，通常称作CDR1、CDR2和CDR3。位于抗体重链可变结构域内的CDR也称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR则称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，可以采用本领域公知的多种方案确定其CDR序列，例如，Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。而Chothia指的是结构环的位置(Chothia和Lesk，J.Mol.Biol.196:901-917(1987))。AbM CDR是Kabat CDR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用。“接触性”(Contact)CDR基于对可获得的复杂晶体结构的分析。根据不同的CDR确定方案，这些CDR中的每一个HVR/CDR的残基如下所述。
Figure PCTCN2021140449-appb-000001
CDR也可以是根据Kabat编号系统位于如下Kabat残基位置的CDR序列：
VL中的位置24-36或24-34(LCDR1)，位置46-56或50-56(LCDR2)，和位置89-97或89-96位置(LCDR3)；和VH中的位置26-35或27-35B(HCDR1)，位置50-65或49-65(HCDR2)，和位置93-102、94-102或95-102(HCDR3)。
在一个实施方案中，本发明抗体的HCDR1通过AbM规则确定边界，HCDR2、HCDR3及LCDR通过Kabat规则确定边界，例如下文表A所示。
CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,第5版，Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
然而，应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
术语“抗体依赖性细胞介导的细胞毒性”或“ADCC”指其中结合到某些细胞毒性细胞(例如NK细胞，嗜中性粒细胞和巨噬细胞)上存在的Fc受体(FcR)上的分泌型免疫球蛋白使得这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后用细胞毒素杀死靶细胞的细胞毒性形式。介导ADCC的主要细胞NK细胞只表达FcγRIII，而单核细胞表达FcγRI，FcγRII和FcγRIII。为了评估目的分子的ADCC活性，可进行体外ADCC测定法，或者可在体内评估目的分子的ADCC活性，例如在动物模型中。本文实施例中提供了用于评估ADCC活性的一种例示性测定法。
术语“功能性Fc区”指这样的Fc区，其拥有天然序列Fc区的“效应器功能”。例示性的“效应器功能”包括C1q结合；CDC；Fc受体结合；ADCC；吞噬作用；细胞表面受体(例如B细胞受体；BCR)下调等。此类效应器功能一般要求Fc区与结合结构域(例如抗体可变域)联 合，而且可以使用多种测定法来评估，例如本文所公开的那些。
本文所述的术语“治疗剂”涵盖在预防或治疗肿瘤(例如癌症)中有效的任何物质，包括化疗剂、细胞毒性剂、疫苗、其它抗体、抗感染活性剂、小分子药物或免疫调节剂。
本文使用的术语“免疫调节剂”指抑制或调节免疫应答的天然或合成活性剂或者药物。免疫应答可以是体液应答或细胞应答。
术语“有效量”指本发明的抗体或其片段或缀合物或组合物这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。针对治疗或预防的目的，可以将“有效量”区分为“治疗有效量”和“预防有效量”。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素而容易地确定：诸如哺乳动物的物种、大小、年龄和一般健康、涉及的具体疾病、疾病的程度或严重性、个体患者的应答、施用的具体抗体、施用模式、施用制剂的生物利用率特征、选择的给药方案、和任何伴随疗法的使用。
在一个实施方式中，相比较于对照，有效量的本发明B7-H3抗体优选地抑制可度量参数(例如肿瘤生长率，肿瘤体积等)至少约20％、更优选地至少约40％。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括最初原代转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
术语“嵌合抗体”是指可变区序列源自一物种、恒定区序列源自另一物种的抗体，例如，其中可变区序列源自小鼠抗体、恒定区序列源自人抗体的抗体。
术语“人源化抗体”是指将源自其他哺乳动物物种例如小鼠种系的抗原结合位点接到人免疫球蛋白序列上的抗体。人源化抗体是一种嵌合分子，该嵌合分子通常使用重组技术制备，可以在人构架序列内进行额外的构架区修饰。抗原结合位点可以包含融合到恒定区的完整可变域，或仅包含嫁接到可变域中的合适框架序列上的互补决定区。在一些实施方案中，人源化抗体将包含基本上所有的至少一个、通常两个可变结构域，其中所有或基本上所有的CDR(例如，6个CDR)对应于非人抗体的那些，并且所有或基本上所有的FR对应于人抗体的那些。人源化抗体任选可以包含至少一部分的来源于人抗体的抗体恒定区。抗体(例如非人抗体)的“人源化形式”是指已经进行了人源化的抗体。
术语“免疫缀合物”是与一个或多个其它物质(包括但不限于细胞毒性剂或标记)缀合的抗体。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗 体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体以及通过与直接标记的另一种试剂反应来间接标记探针或抗体。间接标记的实例包括使用荧光标记的二级抗体进行的一级抗体的检测和具有生物素的DNA探针的末端标记，使得其可以用荧光标记的链霉抗生素蛋白来检测。
术语“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
术语“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。
“分离的编码抗B7-H3抗体或其抗原结合片段的核酸”是指一个或多个核酸分子，其编码抗体重链或轻链(或其抗原结合片段)，包括在单一载体或分开的载体中的这样的核酸分子，以及存在于宿主细胞中的一个或多个位置处的这样的核酸分子。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码 空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其它家族成员序列或相关序列。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
术语“受试者/患者样品”指从患者或受试者得到的组织或细胞样品集合。组织或细胞样品的来源可以是实体组织(例如来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品)；血液或任何血液组分；体液(诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液)；来自受试者的妊娠或发育任何时间的细胞。
II.抗体
除非另外说明，术语“B7-H3”、“B7H3”和“CD276”在本文中可互换使用。B7-H3是属于B7/CD28超家族成员的I型跨膜糖蛋白，与PD-L1的胞外结构域在序列上相似。B7-H3具有316个氨基酸，其中包含一个推定的由28个氨基酸组成的信号肽，一个217个氨基酸组成的的胞外区以及一个跨膜区和一个45个氨基酸组成的胞质结构域，分子量约为45-66kDa。在人体中，由于外显子复制，B7-H3的胞外结构可以为IgV-IgC样结构域(2Ig-B7-H3)，或者为IgV-IgC-IgV-IgC样结构域(4Ig-B7-H3)。食蟹猴B7-H3的序列与其人类对应物具有约90％的同源性。
本文所用的术语“抗B7-H3抗体”、“抗B7-H3”、“B7-H3抗体”或“抗B7-H3的抗体”是指这样的抗体，所述抗体或其抗原结合片段能够以足够的亲合力结合B7-H3蛋白。所述抗体可以用作靶向B7-H3中的诊断剂和/或治疗剂。
在一些实施方案中，本发明的抗B7-H3抗体或其抗原结合片段以足够的亲和力结合B7-H3(例如人或食蟹猴B7-H3)，例如，以下述平衡解离常数(K D)与B7-H3结合，所述K D≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM(例如10 -7M以下，例如10 -7M至10 -10M)。在一些实施方案中，B7-H3为人或食蟹猴B7-H3。在一些实施方案中，抗体结合亲和力是使用生物光干涉测量测定的，例如在生物光干涉测量中，抗体以大约1×10 -7M或更低、大约5×10 -8M或更低、大约1×10 -8M或更低、大约5×10 -9M或更低的K D、大约1×10 -9M或更低的K D、大约1×10 -10M或更低的K D，与人B7-H3结合。
在一些实施方案中，本发明的抗体或其抗原结合片段结合细胞表面表达的B7-H3。
在一些实施方案中，本发明的抗体或其抗原结合片段可以诱导ADCC效应。在一些实施方案中，本发明的抗体或其抗原结合片段可以抑制和/或减小体内肿瘤的生长和/或体积。
在一些实施方案中，本发明的结合B7-H3的抗体或其抗原结合片段包含重链可变区(VH)和/或轻链可变区(VL)，其中所述VH和VL包含选自表A所示的6个CDR的组合。
在本发明的一个实施方案中，本文所述的氨基酸改变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域。
在一些实施方案中，置换为保守性置换。保守置换是指一个氨基酸经相同类别内的另一氨基酸置换，例如一个酸性氨基酸经另一酸性氨基酸置换，一个碱性氨基酸经另一碱性氨基酸置换，或一个中性氨基酸经另一中性氨基酸置换。示例性的置换如下表所示：
原始残基 示例性置换 优选的保守氨基酸置换
Ala(A) Val、Leu、Ile Val
Arg(R) Lys、Gln、Asn Lys
Asn(N) Gln、His、Asp、Lys、Arg Gln
Asp(D) Glu、Asn Glu
Cys(C) Ser、Ala Ser
Gln(Q) Asn、Glu Asn
Glu(E) Asp、Gln Asp
Gly(G) Ala Ala
His(H) Asn、Gln、Lys、Arg Arg
Ile(I) Leu、Val、Met、Ala、Phe、正亮氨酸 Leu
Leu(L) 正亮氨酸、Ile、Val、Met、Ala、Phe Ile
Lys(K) Arg、Gln、Asn Arg
Met(M) Leu、Phe、Ile Leu
Phe(F) Trp、Leu、Val、Ile、Ala、Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val、Ser Ser
Trp(W) Tyr、Phe Tyr
Tyr(Y) Trp、Phe、Thr、Ser Phe
Val(V) Ile、Leu、Met、Phe、Ala、正亮氨酸 Leu
在某些实施方案中，置换发生在抗体的CDR区。通常，获得的变体相对于亲本抗体在某些生物学特性方面(例如，增加的亲和力)具有修饰(例如，改善)和/或将具有亲本抗体的基本上保留的某些生物学特性。示例性置换变体是亲和力成熟抗体。
在某些实施方案中，本文中所提供的抗体经改变以增加或降低抗体经糖基化的程度。对抗体的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。当抗体包含Fc区时，可以改变附着于其的糖类。在一些应用中，除去不想要的糖基化位点的修饰是有用的，例如除去岩藻糖模块以提高抗体依赖性细胞介导的细胞毒性细胞毒性(ADCC)功能(参见Shield等(2002)JBC277:26733)。在其它应用中，可以进行半乳糖苷化修饰以修饰补体依赖性细胞毒性(CDC)。
在某些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包括在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgGl、IgG2、IgG3或IgG4Fc区)。关于Fc变体的实例参见美国专利号7,332,581，美国专利号6,737,056，美国专利号6,737,056；WO 2004/056312和Shields等人，J.Biol.Chem.9(2):6591-6604(2001)，美国专利号6,194,551、WO 99/51642和Idusogie等人J.Immunol.164:4178-4184(2000)，美国专利号7,371,826，Duncan&Winter,Nature 322:738-40(1988)；美国专利号5,648,260；美国专利号5,624,821；和WO 94/29351。
在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。可以如，例如美国专利号7,521,541中所述地生成半胱氨酸改造的抗体。
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其它非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二烷、聚-1,3,6-三烷、乙烯/马来酸酐共聚物、聚 氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
III.本发明的核酸以及包含其的载体和宿主细胞
本发明提供编码以上任何抗B7-H3抗体或其抗原结合片段的核酸。还提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。
本发明还提供了包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自大肠杆菌细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。在一个实施方案中，宿主细胞选自大肠杆菌。
如本领域技术人员明了的，因为密码子简并性，每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。
可以采用本领域熟知的方法，通过从头固相DNA合成或通过PCR诱变编码结合B7-H3的抗体或其抗原结合片段的序列，产生这些多核苷酸序列。
在一个实施方案中，提供包含本发明核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。
在一个实施方案中，提供包含所述载体的宿主细胞。用于克隆或表达编码抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，抗体可在细菌中产生，特别当不需要糖基化和Fc效应子功能时。对于抗体片段和多肽在细菌中的表达，见，例如，美国专利号5,648,237，5,789,199和5,840,523，还见Charlton，Methods in Molecular Biology，卷248(B.K.C.Lo，编辑，Humana Press，Totowa，NJ，2003)，第245-254页，其描述抗体片段在大肠杆菌中的表达。在表达后，抗体可以从可溶级分中的细菌细胞糊状物分离，并且可以进一步纯化。
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是关于编码抗体的载体的合适克隆或表达宿主。例如，糖基化途径已经进行“人源化”的真菌和酵母菌株导致产生具有部分或完全人糖基化模式的抗体。参见Gerngross，Nat.Biotech.22：1409-1414(2004)，和Li等，Nat.Biotech.24：210-215(2006)。适于表达糖基化抗体的宿主细胞也衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用哺乳动物宿主细胞系的其它实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(293HEK或293细胞，如例如Graham等，J.Gen Virol.36：59(1977)中所描述的)等。其它有用的哺乳动物宿主细胞系包 括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞(Urlaub等，Proc.Natl.Acad.Sci.USA 77：216(1980))；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。关于适合产生抗体的某些哺乳动物宿主细胞系的综述见例如Yazaki和Wu，Methods in Molecular Biology，卷248(B.K.C.Lo，ed.，Humana Press，Totowa，NJ)，第255-268页(2003)。
IV.本发明的抗体分子的生产和纯化
在一个实施方案中，本发明提供制备抗B7-H3抗体或其片段(优选抗原结合片段)的方法，其中所述方法包括在适于表达编码所述抗体或其片段(优选抗原结合片段)的核酸的条件下培养所述宿主细胞，以及任选地分离所述抗体或其片段。在某个实施方案中，所述方法还包括从宿主细胞回收抗B7-H3抗体或其片段。
V.多特异性抗体
在再一方面，本发明提供特异性地结合B7-H3的多特异性(包括双特异性)抗体分子。在一个实施方案中，在多特异性抗体中，本发明的抗体(或其抗原结合片段)形成针对B7-H3的第一结合特异性。
在一个实施方案中，结合特异性由抗体的“结合位点”或“抗原结合位点”(抗体分子中与抗原实际结合的区域)提供。在一个优选的实施方案中，抗原结合位点由抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)组成的VH/VL对构成。因此，在一个实施方案中，“多特异性”抗体是具有至少两个抗原结合位点的抗体，所述至少两个抗原结合位点中的每一个抗原结合位点可以与相同抗原的不同表位或与不同抗原的不同表位结合。
有关多特异性抗体及其制备，可以参见例如WO 2009/080251、WO 2009/080252、WO 2009/080253、WO 2009/080254、WO 2010/112193、WO 2010/115589、WO 2010/136172、WO 2010/145792和WO 2010/145793中的描述。
VI.免疫缀合物
在一些实施方案中，本发明提供了通过将本发明抗体缀合于异源分子而产生的免疫缀合物。在一些实施方案中，异源分子例如治疗剂或诊断剂，如细胞毒性剂或化疗剂。细胞毒性剂包括任何对细胞有害的药剂。适合于形成免疫缀合物的细胞毒性剂的例子是本领域中已知的。
可以使用接头来共价连接缀合物的不同实体。适宜的接头包括化学接头或肽接头。有利地的是，接头是利于多肽在递送至靶位点后释放的“可裂解接头”。例如，可以使用酸不稳定性接头、肽酶敏感性接头、光不稳定性接头、二甲基接头或含二硫化物的接头(Chari等，Cancer Research 52(1992)127-131；US 5,208,020)。
在一些实施方案中，本发明的抗体可以与诊断剂或可检测剂缀合。这类缀合物可以作为临 床检验方法的部分(如确定特定疗法的效力)，用于监测或预测疾病或病症的发作、形成、进展和/或严重性。可以通过将抗体与可检测剂偶联实现这类诊断和检测，所述可检测剂包括但不限于多种酶，如但不限于辣根过氧化物酶；辅基，如但不限于链霉亲和素/生物素和抗生物素蛋白/生物素；荧光物质；发光物质；放射性物质；和用于各种正电子发射成像术中的正电子发射金属和非放射性顺磁金属离子。
在一些实施方案中，所述免疫缀合物用于预防或治疗肿瘤。在一些实施方案中，肿瘤为癌症。
VII.药物组合物、药物制剂和组合产品
本发明还包括包含抗B7-H3抗体或其免疫缀合物或多特异性抗体的组合物(包括药物组合物或药物制剂)和包含编码抗B7-H3抗体或其免疫缀合物或多特异性抗体的多核苷酸的组合物。这些组合物还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
本发明的药物组合物或制剂还可以与一种或多种其它活性成分组合，所述活性成分是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。
因此，在一个方面，本发明也提供了药物组合产品。在一个实施方案中，所述组合产品包含配制在相同药物组合物或制剂中的本发明抗体、免疫缀合物或多特异性抗体与第二治疗剂。在另一实施方案中，所述组合产品包含分开包含在不同的药物组合物或制剂中的本发明的抗体、免疫缀合物或多特异性抗体与第二治疗剂。第二治疗剂可以在本发明抗体施用之前、同时(例如在相同制剂中或在不同制剂中)、或之后施用。
本发明的药物组合物、制剂和组合产品可以在制品中提供，用于本文所述的疾病和/或病症的治疗、预防和/或诊断。制品可以包含容器和标签或包装说明书。合适的容器包括例如瓶、注射器、IV输液袋等。容器可以由玻璃或塑料等多种材料制成。在一个实施方案中，制品可以包含(a)容纳有本发明抗体或抗体片段、免疫缀合物或多特异性抗体的第一容器；和任选地(b)容纳有第二治疗剂的第二容器。此外，制品也可以包含从商业和用户角度看期望的其它材料，包括缓冲液、可药用稀释剂，如无菌注射用水、针头、注射器、注射泵等。
VIII应用
本发明一方面提供了预防和/或治疗B7-H3相关疾病或病症(例如癌症)的方法，包括向受试者施用有效量的本发明的抗B7-H3的抗体或其抗原结合片段、免疫缀合物或药物组合物。
受试者可以是哺乳动物，例如，灵长类，优选地，高级灵长类，例如，人类。在一个实施方案中，受试者患有本文所述疾病或具有患有本文所述疾病的风险。在某些实施方案中，受试 者接受或已经接受过其它治疗，例如化疗治疗和/或放射疗法。在其它方面，本发明提供抗B7-H3抗体或其抗原结合片段、免疫缀合物或药物组合物在生产或制备药物中的应用，所述药物用于预防和/或治疗本文提及的B7-H3相关疾病或病症。
在一些实施方案中，本发明的抗体或其抗原结合片段或免疫缀合物或组合物或产品会延迟病症和/或与病症相关的症状的发作。
本发明的抗体(以及包含其的药物组合物或免疫缀合物，以及任何另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的抗体的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、所述抗体是以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
在上述本发明方法中，可以替代本发明抗体或抗原结合部分，施用本发明的组合物、多特异性抗体或免疫缀合物。或者，在这些方法中，除了施用本发明抗体或抗原结合部分，还可以进一步施用本发明的组合物、多特异性抗体或免疫缀合物。
IX.用于诊断和检测的方法和组合物
在某些实施方案中，本文中提供的任何抗B7-H3抗体或其抗原结合片段可以用于检测B7-H3在生物样品中的存在。在一个实施方案中，基于检测结果用于对疾病的诊断或者辅助诊断。术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是血、血清或生物来源的其它液体样品。在某些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自过度增生性或癌性病灶。
在一个实施方案中，本发明提供检测生物样品中B7-H3的方法和试剂盒。在某些实施方案中，B7-H3是人B7-H3或食蟹猴B7-H3。在某些实施方案中，所述方法包括将生物样品与如本文所述的抗B7-H3抗体在允许抗体与B7-H3结合的条件下接触，并检测是否形成抗B7-H3抗体和B7-H3的复合物。该方法可以是体外或体内方法。在一些实施方案中，样品来自癌症 患者。所述样品可以是组织活检物、组织切片、体液例如血液、血浆、或血清。
在一些实施方案中，提供了一种治疗B7-H3相关疾病或病症(例如癌症或肿瘤)的方法，所述方法包括：向受试者施用治疗有效量的抗B7-H3抗体。在另一个实施方案中，所述方法还包括向受试者施用一种或多种其它疗法。
在一个实施方案中，抗B7-H3抗体被用于选择适合利用抗B7-H3抗体治疗的受试者，例如其中B7-H3是用于选择所述受试者的生物标记物。在一个实施方案中，可以使用本发明抗体诊断肿瘤，例如评价(例如，监测)对象中本文所述疾病的治疗或进展、其诊断和/或分期。在一个实施方案中，通过采用本申请的抗B7-H3抗体来确定多种组织(例如卵巢、肺、乳腺、前列腺、肾、胰脏、甲状腺、脑等组织)中是否存在癌细胞。采用本申请的抗B7-H3抗体可以用来确定从实体瘤释放进入循环血液中的癌细胞的存在与否以及B7-H3的水平，循环中的B7-H3抗原可以是完整的B7-H3分子，或者是其片段。检测方法例如通过FACS方法进行。
在某些实施方案中，提供标记的抗B7-H3抗体。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。
X.本发明的示例性抗B7-H3抗体
表A本发明示例抗体的CDR的氨基酸序列
Figure PCTCN2021140449-appb-000002
表B.本发明示例抗体的重链可变区(VH)和轻链可变区(VL)
Figure PCTCN2021140449-appb-000003
表C.本发明部分示例序列
Figure PCTCN2021140449-appb-000004
Figure PCTCN2021140449-appb-000005
Figure PCTCN2021140449-appb-000006
Figure PCTCN2021140449-appb-000007
实施例
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围，根据本申请说明书的描述，本领域技术人员可以进行多种修改。
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。这些方法的描述可以参见，例如，Sambrook等人，Molecular Cloning:A Laboratory Manual(第3版，2001)；Sambrook等人，Molecular Cloning:A Laboratory Manual(第2版,1989)；Maniatis等人，Molecular Cloning:A Laboratory Manual(1982)；Ausubel等人，Current Protocols in Molecular Biology(John Wiley和Sons,2008年7月更新)；Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates和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 Translation(B.Hames&S.Higgins,Eds.,1984)；Perbal,A Practical Guide to Molecular Cloning(1984)；Harlow和Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1998)Current Protocols in Immunology Q.E.Coligan,A.M.Kruisbeek,D.H.Margulies,E.M.Shevach和W.Strober,eds.,1991)；Annual Review of Immunology；以及期刊专著如Advances in Immunology。
实施例1、杂交瘤细胞的制备
免疫动物
根据常规方法，使用重组的人4Ig-B7H3蛋白(SEQ ID NO:32)(SINO BIOLOGICAL，货号11188-H08H)免疫Bal b/c小鼠(北京维通利华)，将重组人4Ig-B7H3蛋白(每只小鼠50ug)与TiterMax(Sigma,货号T2684-1ML)佐剂等体积混匀后，每两周皮下注射一次，共免疫5次。
细胞融合
当血清效价满足要求后，按照常规方法，摘取小鼠的脾脏制备B淋巴细胞悬液，然后与SP2/0骨髓瘤细胞(ATCC,CRL-1581)以1:2～1:1的比例混合后进行电融合。将融合后的细胞从电极皿中转移入50ml离心管中，用筛选培养基(配置组成如表1所示)稀释细胞获得细胞 悬液(浓度1～2×10 4个细胞/ml)。向96孔板中每孔加入100μl细胞悬液。融合后第5天更换新鲜筛选培养基。根据细胞生长状态，培养10天(或更久)后进行流式细胞仪(FACS)检测，筛选阳性克隆。
表1：筛选培养基
Figure PCTCN2021140449-appb-000008
高通量筛选杂交瘤细胞
通过流式细胞仪(FACS)筛选特异性表达抗B7H3抗体的杂交瘤细胞。简言之，将表达人B7H3的CHO细胞(CHO-huB7H3)计数，稀释至1×10 6个细胞/ml，然后向U型底96孔板各孔中加入100μl，500g离心5min，去除细胞培养基。之后将上述杂交瘤96孔板中的各个培养上清和阳性对照抗体(MGA271)分别加入含有CHO细胞的U型板中并重悬细胞，每孔100μl，冰上静置30min。再500g离心5min以去除上清，然后用PBS溶液洗细胞1次。500g离心5min以去除PBS溶液。向每孔加入100μl抗鼠Fab的FITC标记的二抗(1:500稀释于PBS溶液中)，向阳性对照抗体培养孔中加入100μl抗人Fab的FITC标记的二抗。冰上避光孵育30min，500g离心5min以去除上清，PBS溶液洗细胞1次。之后用50μl PBS溶液重悬细胞，进行FACS检测，筛选获得阳性克隆。
将获得的阳性克隆，采用表达食蟹猴B7H3(SEQ ID NO:34)的CHO细胞(CHO-cynoB7H3)，通过上述同样的方法进行复筛，共获得2株与人B7H3和猴B7H3都结合的杂交瘤细胞：19A2和20G5。
采用生物膜薄层干涉测定技术(ForteBio)测定所获得的2株杂交瘤细胞株与抗原的亲和力，获得的KD值如表2所示。
表2：ForteBio亲和力测定结果
Figure PCTCN2021140449-appb-000009
阳性杂交瘤细胞的亚克隆
根据细胞结合以及亲和力测定的结果，将上述克隆进行亚克隆。
具体步骤如下：将筛选培养基中的HAT更换成HT(Gibco,Cat#11067-030)获得基础培养基，每孔200μl加入96孔板。将上述融合筛选出的阳性杂交瘤细胞，按约1×10 5个细胞/ml的密度，每孔300ul加入到96孔板第一排中，充分混匀。取第1排细胞悬液100μl加入第2排，充分混匀后取100μl加入下一排，重复上述步骤，直至最后一排，静置15min。显微镜下观察计数，取100个细胞对应的体积加入20ml如上所述的基础培养基中，并混匀铺板，每孔200μl。两天后显微镜下观察，判断并标记出单克隆孔。待每孔细胞汇合度达到50％以上，采用上述高通量的FACS筛选方法进行检测，挑出目标阳性孔，将获得的细胞克隆冻存。
本发明所用的阳性对照抗体为MGA271，也称作Enoblituzumab(来源于MacroGenics US20160264672A1)。
实施例2.嵌合抗体的制备
利用分子生物学技术，对实施例1中获得的杂交瘤阳性克隆进行抗体轻重链基因序列的调取，并利用其构建人鼠嵌合抗体。
1. 杂交瘤测序
提取新鲜培养的约5×10 6个杂交瘤细胞的RNA，利用PrimeScript II 1st Strand cDNA Synthesis Kit(Takara)反转录获得cDNA。步骤如下：
配制表3的反应体系I
表3：
Figure PCTCN2021140449-appb-000010
65℃温育5min后，迅速置冰上冷却。将反应体系I加入到下列反转录体系(表4)中，总量为20μl：
表4：反转录体系
Figure PCTCN2021140449-appb-000011
缓慢混匀后按下列条件进行反转录翻译：42℃60min→95℃5min，然后放冰上冷却，获得cDNA。
将cDNA连接到T载体，之后采用Mighty TA-cloning Kit试剂盒(Takara)，通过PCR分别从获得的cDNA扩增抗体的重链和轻链可变区，PCR反应体系如表5所示。
表5：
Figure PCTCN2021140449-appb-000012
PCR反应条件如表6所示。
表6：
Figure PCTCN2021140449-appb-000013
取4.5μl上述PCR反应获得的PCR产物，加入0.5μl pMD20-T载体(Takara)，5μl Ligation Mighty Mix(Takara)，轻轻混匀，于37℃反应2h，获得连接产物。
转化细胞：
将5μl获得的连接产物加入到大肠杆菌TOP10感受态细胞(天根生化科技(北京)有限公司)中，混匀后冰上孵育30min。42℃热激90s后迅速冰上冷却2min，向EP管中补加900μl LB培养基(生工生物工程(上海)股份有限公司)，37℃，220rpm摇床培养1h。3000g离心2min，吸除800μl上清，用剩余的培养基将菌体重悬并涂布在氨苄青霉素抗性的平板上。于37℃培养过夜，挑克隆测序。
2.构建嵌合抗体
PCR扩增已经测序的实施例1中杂交瘤细胞产生的抗B7H3抗体VH及VL区：上下游引物序列见表7及表8。
表7.小鼠抗体重链可变区(VH)引物(Primer Mix 1)
引物名称 序列(5’-3’) 比例(％)
OVH1 SAGGTCCAGCTGCAGCAGYYTGG(SEQ ID NO:39) 28.6
OVH2 CAGGTRCAGCTGAAGSAGTCAGG(SEQ ID NO:40) 10.7
OVH3 GAKGTGCAGCTTCAGCAGTCRGG(SEQ ID NO:41) 8.9
OVH5 GAVGTGAWGCTGGTGGAGTCTGR(SEQ ID NO:42) 7.1
OVH11 GAAGTGCAGCTGTTGGAGACTGG(SEQ ID NO:43) 3.6
OVH14 GAGGTTCAGCTGCAGCAGTCTGK(SEQ ID NO:44) 16.1
OVH15 CAGGTTCACCTACAACAGTCTGG(SEQ ID NO:45) 3.5
REVESE-6 CTGAGGARACGGTGACCG(SEQ ID NO:46) 6
REVESE-4 CTGAGGAGACTGTGAGAGWGGT(SEQ ID NO:47) 4
REVESE-2-1 CTGAGGAGACGGTGACTGAGGT(SEQ ID NO:48) 2
REVESE-2-2 CTGCAGAGACAGTGACCAGAGT(SEQ ID NO:49) 2
水   qs
按上述比例混合后，获得Primer Mix 1用于后续VH的PCR扩增。
表8.小鼠抗体的轻链可变区(VL)引物(Primer Mix 2)：
Figure PCTCN2021140449-appb-000014
按上述比例混合后，获得Primer Mix 2用于后续VL的PCR扩增。
PCR体系如表9所示。
表9：
Figure PCTCN2021140449-appb-000015
*对于VH链扩增，应用Primer Mix 1；对于VL链扩增，应用Primer Mix 2。
切胶回收PCR扩增产物。
同源重组反应：
同源重组体系如表10所示。
表10：
Figure PCTCN2021140449-appb-000016
37℃反应30min，获得重组产物。重组产物转化TOP10感受态，并挑取单克隆测序，选择包含插入方向正确的质粒的克隆作为阳性克隆，保存阳性克隆，由此获得嵌合抗体的重组质粒。抽提制备一定量的重组质粒，用于抗体的表达。
本发明共获得2个嵌合抗体(Ch19A2和Ch20G5)，其CDR序列、轻链可变区和重链可变区序列与表A-B中杂交瘤细胞的相应序列相同，所述嵌合抗体优选的轻链和重链的氨基酸序列在表C中示出。
3.嵌合抗体的表达和纯化
根据所需转染体积传代HEK293细胞(Invitrogen)，转染前一天将细胞密度调整至1.5×10 6个细胞/ml。转染当天细胞密度约为3×10 6个细胞/ml。取终体积1/10(v/v)的Opti-MEM培养基(Gibco货号：31985-070)作为转染缓冲液，加入上述构建的重组表达质粒，混匀，用0.22μm的滤头过滤备用。加合适的聚乙烯亚胺(PEI)(Polysciences，23966)到上一步的质粒中(质粒与PEI的质量比例为1:3)，混匀后室温孵育10min，获得DNA/PEI混合物。将DNA/PEI混合物轻柔倒入HEK293细胞并混匀，在37℃，8％CO 2的条件下培养24h后，补加终浓度为2mM的VPA(Sigma，货号：P4543-100G)，及2％(v/v)的Feed溶液(1g/L Phytone Peptone+1g/L Difco Select Phytone)，继续培养6天。
在细胞培养后，细胞培养液以13000rpm离心20min，收集上清，根据制造商的说明书，用预装柱Hitrap Mabselect Sure(GE,11-0034-95)纯化上清液，并测定浓度。取100μg纯化后蛋白，调整浓度至1mg/mL，使用凝胶过滤色谱柱SW3000(TOSOH货号：18675)测定蛋白质纯度，结果表明获得了高纯度的嵌合抗体。
实施例3生物膜薄层干涉技术测定本发明的嵌合抗体与抗原的结合动力学
采用生物膜薄层干涉测定技术(ForteBio)测定本发明抗体结合人B7H3的平衡解离常数(KD)。ForteBio亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):第270-8页)进行。
简言之，AMQ(Pall，1506091)(用于样品检测)或AHQ(Pall，1502051)(用于阳性对照检测)传感器在分析缓冲液中线下平衡30分钟，然后线上检测60秒建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器(ForteBio)上进行ForteBio亲和测量。再将具有加载的抗体的传感器暴露于抗原(包括人4Ig-B7H3、人2Ig-B7H3(ACRO，货号B73-H52E2)及食蟹猴B7H3(SINO BIOLOGICAL，货号90806-C02H-50)，之后将传感器转移至分析缓冲液用于解离速率测量。使用ForteBio分析软件分析KD值。
抗体亲和力的检测结果如表11所示：
表11.ForteBio检测抗原抗体结合的亲和力(平衡解离常数KD)
Figure PCTCN2021140449-appb-000017
从上述亲和力数据可以看出，我们通过杂交瘤获得的嵌合抗体，与人B7H3蛋白具有良好的亲和力，并且对食蟹猴B7H3也保持很高的亲和力。与对照组的MGA271相比，本研究的抗体有更高的亲和力。
实施例4嵌合抗体的人源化
根据常规方法，将实施例2得到的嵌合抗体进行人源化。由此获得人源化抗体：hz20G5,hz19A2，其CDR序列、轻链可变区和重链可变区序列，轻链和重链的氨基酸序列参见表A-C。
实施例5 ForteBio测定人源化抗体与抗原的亲和力
如实施例3所述，采用ForteBio测定法测定实施例4获得的人源化抗体结合抗原(人B7H3和食蟹猴B7H3)的亲和力，以平衡解离常数(KD)表示。结果如表12所示。
表12.ForteBio检测抗原抗体结合的亲和力常数(M)
Figure PCTCN2021140449-appb-000018
Figure PCTCN2021140449-appb-000019
从表12可以看出：人源化后的抗体对抗原B7H3依然具有高的亲和力，其与相应的嵌合抗体具有针对抗原B7H3相当的平衡解离常数K D。此外，本申请获得的人源化抗体比对照抗体MGA271具有更高的结合抗原亲和力，尤其针对人2Ig-B7-H3，本申请的抗体的亲和力相比较于MGA271提高了20-100倍。
实施例6人源化抗体结合过表达人和食蟹猴B7H3的CHO-S细胞
为了验证本发明的抗体是否可以和细胞表面表达的抗原结合，利用流式细胞技术检测了本申请人源化抗体和过表达人B7H3和食蟹猴B7H3的细胞的结合情况。
过表达B7H3的细胞构建
使用ExpiCHO TM Expression System Kit(Invitrogen，目录号：A29133)，根据制造商的说明书实施如下操作：将编码人4Ig-B7H3(uniprot:Q5ZPR3,SEQ ID NO:32),人2Ig-B7H3(uniprot:Q5ZPR3-2,SEQ ID NO:33)和食蟹猴B7H3(NCBI:XP_015308534.1,SEQ ID NO:34)的cDNA克隆到pCHO1.0载体(Invitrogen)上，之后转染CHO-S细胞，产生过表达人4Ig-B7H3，人2Ig-B7H3和食蟹猴B7H3的CHO-S细胞：CHOS-hB7H3-4Ig,CHOS-hB7H3-2Ig，CHOS-cyno B7H3。
简言之：
1)用PBS溶液稀释CHOS-hB7H3-4Ig,CHOS-hB7H3-2Ig，CHOS-cyno B7H3细胞至2×10 6个/ml，向U型底96孔板各孔中加入100μl，加入三倍梯度稀释的抗体。
2)将上述混合物在冰上孵育30分钟。400g离心5分钟，去除上清，用PBS溶液洗涤细胞，移除未结合的抗体。每孔加入100μl 1:200稀释的PE缀合的抗人Fc抗体(SouthernBiotech)，冰上避光孵育30分钟。400g离心5分钟，去除上清。用PBS洗涤细胞2次，移除未结合的PE缀合的抗人Fc抗体。用100μl PBS重悬细胞，通过FACS检测抗体与细胞的结合。
检测结果如图1所示，对于过表达人4Ig-B7H3的细胞，本申请人源化抗体总体上的亲和力与阳性对照MGA271相当。对于过表达人2Ig-B7H3的细胞，hz19A2抗体的亲和力与MGA271相当，而hz 20G5的亲和力明显高于MGA271。可见，本申请的抗体在细胞水平上体现出了显著提升的抗原结合能力。
实施例7抗体依赖细胞介导的细胞毒作用(ADCC)
本实施例研究了所获得抗体介导ADCC效应进而清除肿瘤细胞的作用。本研究使用 Promega的Jurkat-ADCCNF-AT luciferase效应细胞株(以下简称ADCC效应细胞)，通过检测NF-AT信号的激活情况，从而检测抗体的ADCC活性。具体实验过程如下：
1)细胞准备
对细胞CHO-hB7H3-4Ig和ADCC效应细胞进行细胞计数。离心去上清，细胞用PBS洗涤两次，用检测培养基(5％low IgG血清的1640培养基(Gibco)重悬，调整ADCC效应细胞浓度为1×10 7个/ml，调整CHO-hB7H3-4Ig细胞浓度为1×10 6个/ml。将两种细胞按照1:1混合，最终ADCC效应细胞与CHO-hB7H3-4Ig细胞的比为10:1。
2)铺板：将混合后的细胞铺96孔板，每孔100ul，第一孔补加50μl细胞。
3)依次加入本发明的不同浓度梯度的抗体：起始孔终浓度为30nM，之后三倍稀释，共10个梯度。
4)37℃培养箱中孵育7小时。
5)7小时后，取出96孔板，每孔加入解冻的Luciferase测试试剂100ul。室温孵育20分钟。用酶标仪检测。用GraphPad软件拟合浓度依赖的曲线。
检测结果如图2所示，本申请获得的人源化抗体和嵌合抗体均可以有效激活NF-AT信号，该信号是ADCC激活的下游信号通路，由此本申请的抗体具有优良的ADCC杀伤能力。此外，本申请获得的人源化抗体与相应的嵌合抗体的ADCC活性相当。
实施例8：本申请抗体分子的体内抗肿瘤作用
本实施例在荷瘤小鼠模型中研究了本申请所获得的抗B7H3抗体分子的体内抗肿瘤作用。
实验采用SPF等级的雌性C.B-17-SCID小鼠(18-20g)，购自北京维通利华实验动物技术有限公司，合格证编号为NO.1100112011025061。
将A375细胞(ATCC，CRL-1619)进行常规传代培养用于后续体内实验。离心收集细胞，以PBS(1×)分散A375细胞，制备成细胞浓度为2.5×10 7个/ml的细胞悬液。在第0天取0.2ml细胞悬液皮下接种至C.B-17SCID小鼠右侧腹部区域中来建立A375荷瘤小鼠模型。
接种肿瘤细胞第0天将所有小鼠进行随机分组(每组8只小鼠)，分别在接种后第0、4、7、11天给药小鼠，给药剂量，给药方式以及相应抗体如表13所示。
表13.
组别 给药剂量 给药方式
h-IgG1 10mg/kg 腹腔注射
hz19A2 10mg/kg 腹腔注射
hz20G5 10mg/kg 腹腔注射
接种第5天后检测各只小鼠瘤体积，每周2次监测小鼠瘤体积与体重，监测至14天后结束。接种后第14天计算相对肿瘤抑制率(TGI％)，计算公式如下：
TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。
肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按公式V＝L*W 2/2进行计算。
肿瘤抑制率结果如图3a和表14所示：在接种后第14天，与h-IgG1对照组对比，人源化抗体hz19A2和hz20G5对肿瘤的抑制率分别为62.4％、46.0％。由此表明，本申请获得的人源化抗B7H3抗体(hz19A2和hz20G5)具有优良的抗肿瘤作用。
表14.第14天肿瘤抑制率
组别 肿瘤体积(mm 3) 肿瘤抑制率(％)
h-IgG1,10mg/kg 389.52 N/A
hz19A2,10mg/kg 194.82 62.4
hz20G5,10mg/kg 246.05 46.0
此外，本实验还监测了小鼠的体重变化，结果如图3b-3c所示，在整个给药期间，实验组和对照组小鼠的体重无显著差异。

Claims (22)

  1. 结合B7-H3的抗体或其抗原结合片段，其包含
    1)如SEQ ID NO:16所示的VH中所含的HCDR1、HCDR2和HCDR3，以及如SEQ ID NO:17所示的VL中所含的LCDR1、LCDR2和LCDR3；
    2)如SEQ ID NO:18所示的VH中所含的HCDR1、HCDR2和HCDR3，以及如SEQ ID NO:19所示的VL中所含的LCDR1、LCDR2和LCDR3；
    3)如SEQ ID NO:20所示的VH中所含的HCDR1、HCDR2和HCDR3，以及如SEQ ID NO:21所示的VL中所含的LCDR1、LCDR2和LCDR3；或
    4)如SEQ ID NO:22所示的VH中所含的HCDR1、HCDR2和HCDR3，以及如SEQ ID NO:23所示的VL中所含的LCDR1、LCDR2和LCDR3。
  2. 结合B7-H3的抗体或其抗原结合片段，其包含重链可变区VH和/或轻链可变区VL，其中，
    (i)所述VH包含HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:1或8中任一项所示的氨基酸序列或由所述氨基酸序列组成；HCDR2包含SEQ ID NO:2、7、9、14中任一项所示的氨基酸序列或由所述氨基酸序列组成；HCDR3包含SEQ ID NO:3或10中任一项所示的氨基酸序列或由所述氨基酸序列组成；
    和/或
    (ii)其中所述VL包含LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO:4、11或15中任一项所示的氨基酸序列或由所述氨基酸序列组成；LCDR2包含SEQ ID NO:5或12中任一项所示的氨基酸序列或由所述氨基酸序列组成；LCDR3包含SEQ ID NO:6或13中任一项所示的氨基酸序列或由所述氨基酸序列组成。
  3. 根据权利要求2所述的抗体或其抗原结合片段，其包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
    1)HCDR1，其包含SEQ ID NO:1所示的氨基酸序列或由所述氨基酸序列组成；
    HCDR2，其包含SEQ ID NO:2所示的氨基酸序列或由所述氨基酸序列组成；
    HCDR3，其包含SEQ ID NO:3所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR1，其包含SEQ ID NO:4所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR2，其包含SEQ ID NO:5所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR3，其包含SEQ ID NO:6所示的氨基酸序列或由所述氨基酸序列组成；
    2)HCDR1，其包含SEQ ID NO:1所示的氨基酸序列或由所述氨基酸序列组成；
    HCDR2，其包含SEQ ID NO:7所示的氨基酸序列或由所述氨基酸序列组成；
    HCDR3，其包含SEQ ID NO:3所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR1，其包含SEQ ID NO:4所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR2，其包含SEQ ID NO:5所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR3，其包含SEQ ID NO:6所示的氨基酸序列或由所述氨基酸序列组成；
    3)HCDR1，其包含SEQ ID NO:8所示的氨基酸序列或由所述氨基酸序列组成；
    HCDR2，其包含SEQ ID NO:9所示的氨基酸序列或由所述氨基酸序列组成；
    HCDR3，其包含SEQ ID NO:10所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR1，其包含SEQ ID NO:11所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR2，其包含SEQ ID NO:12所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR3，其包含SEQ ID NO:13所示的氨基酸序列或由所述氨基酸序列组成；或
    4)HCDR1，其包含SEQ ID NO:8所示的氨基酸序列或由所述氨基酸序列组成；
    HCDR2，其包含SEQ ID NO:14所示的氨基酸序列或由所述氨基酸序列组成；
    HCDR3，其包含SEQ ID NO:10所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR1，其包含SEQ ID NO:15所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR2，其包含SEQ ID NO:12所示的氨基酸序列或由所述氨基酸序列组成；
    LCDR3，其包含SEQ ID NO:13所示的氨基酸序列或由所述氨基酸序列组成。
  4. 权利要求1至3中任一项的抗体或其抗原结合片段，其包含重链可变区VH和/或轻链可变区VL，其中，
    (a)重链可变区VH
    (i)包含与SEQ ID NO:16、18、20或22中任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同一性的氨基酸序列或由其组成，且包含所述序列的相应CDR序列；或者
    (ii)包含SEQ ID NO:16、18、20或22中任一项所示的氨基酸序列或由其组成；或者
    (iii)包含与SEQ ID NO:16、18、20或22中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由其组成，优选地，所述氨基酸改变不发生在CDR区中；
    和/或
    (b)轻链可变区VL
    (i)包含与SEQ ID NO:17、19、21或23中任一项所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由其组成，且包含所述序列的相应CDR序列；
    (ii)包含SEQ ID NO:17、19、21或23中任一项所示的氨基酸序列或由其组成；或者
    (iii)包含与SEQ ID NO:17、19、21或23中任一项所示的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由其组成，优选地，所述氨基酸改变不发生在CDR区中。
  5. 根据权利要求4所述的抗体或其抗原结合片段，其包含
    1)包含与SEQ ID NO:16所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链可变区VH，和包含与SEQ ID NO:17所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链可变区VL；
    2)包含与SEQ ID NO:18所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链可变区VH，和包含与SEQ ID NO:19所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链可变区VL；
    3)包含与SEQ ID NO:20所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链可变区VH，和包含与SEQ ID NO:21所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链可变区VL；或
    4)包含与SEQ ID NO:22所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链可变区VH，和包含与SEQ ID NO:23所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链可变区VL。
  6. 权利要求1至5中任一项的抗体或其抗原结合片段，其包含选自以下的重链可变区和轻链可变区
    1)包含SEQ ID NO:16所示的氨基酸序列或由其氨基酸组成的重链可变区VH，和包含SEQ ID NO:17所示的氨基酸序列或由其氨基酸组成的轻链可变区VL；
    2)包含SEQ ID NO:18所示的氨基酸序列或由其氨基酸组成的重链可变区VH，和包含SEQ ID NO:19所示的氨基酸序列或由其氨基酸组成的轻链可变区VL；
    3)包含SEQ ID NO:20所示的氨基酸序列或由其氨基酸组成的重链可变区VH，和包含SEQ ID NO:21所示的氨基酸序列或由其氨基酸组成的轻链可变区VL；或
    4)包含SEQ ID NO:22所示的氨基酸序列或由其氨基酸组成的重链可变区VH，和包含SEQ ID NO:23所示的氨基酸序列或由其氨基酸组成的轻链可变区VL。
  7. 权利要求1至6中任一项的抗体或其抗原结合片段，其包含重链和/或轻链，其中
    (a)重链
    (i)包含与SEQ ID NO:24、26、28或30中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性且包含所述序列相应CDR序列的氨基酸序列或由其组成；
    (ii)包含SEQ ID NO:24、26、28或30中任一项所示的氨基酸序列或由其组成；或者
    (iii)包含与SEQ ID NO:24、26、28或30中任一项所示的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由其组成，优选地，所述氨基酸改变不发生在重链的CDR区中，更优选地，所述氨基酸改变不发生在重链可变区中；
    和/或
    (b)轻链
    (i)包含与SEQ ID NO:25、27、29或31中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性且包含所述序列相应CDR序列的氨基酸序列或由其组成；
    (ii)包含SEQ ID NO:25、27、29或31中任一项所示的氨基酸序列或由其组成；或者
    (iii)包含与SEQ ID NO:25、27、29或31中任一项所示的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由其组成，优选地，所述氨基酸改变不发生在轻链的CDR区中，更优选地，所述氨基酸改变不发生在轻链可变区中。
  8. 根据权利要求7所述的抗体或其抗原结合片段，其包含
    1)包含与SEQ ID NO:24所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链，和包含与SEQ ID NO:25所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链；
    2)包含与SEQ ID NO:26所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链，和包含与SEQ ID NO:27所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链；
    3)包含与SEQ ID NO:28所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链，和包含与SEQ ID NO:29所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、 97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链；
    4)包含与SEQ ID NO:30所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的重链，和包含与SEQ ID NO:31所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成的轻链。
  9. 根据权利要求8所述的抗体或其抗原结合片段，其包含
    1)包含SEQ ID NO:24所示氨基酸序列或由所述氨基酸序列组成的重链，和包含SEQ ID NO:25所示氨基酸序列或由所述氨基酸序列组成的轻链；
    2)包含SEQ ID NO:26所示氨基酸序列或由所述氨基酸序列组成的重链，和包含SEQ ID NO:27所示氨基酸序列或由所述氨基酸序列组成的轻链；
    3)包含SEQ ID NO:28所示氨基酸序列或由所述氨基酸序列组成的重链，和包含SEQ ID NO:29所示氨基酸序列或由所述氨基酸序列组成的轻链；
    4)包含SEQ ID NO:30所示氨基酸序列或由所述氨基酸序列组成的重链，和包含SEQ ID NO:31所示氨基酸序列或由所述氨基酸序列组成的轻链。
  10. 权利要求1-9中任一项的抗体或其抗原结合片段，其中所述抗体是IgG1,IgG2,IgG3或IgG4形式的抗体或其抗原结合片段，优选是IgG1形式的抗体，更优选包含人IgG1 Fc区。
  11. 权利要求1-10中任一项的抗体或其抗原结合片段，其中所述抗体是单克隆抗体、嵌合抗体或人源化抗体。
  12. 权利要求1-11中任一项的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体例如scFv、(Fab’)2片段、单结构域抗体、双抗体(dAb)或线性抗体。
  13. 一种分离的核酸，其编码前述权利要求任一项的抗B7-H3抗体或其抗原结合片段。
  14. 一种载体，其包含权利要求13的核酸，优选地所述载体是表达载体。
  15. 一种宿主细胞，其包含权利要求13的核酸或权利要求14的载体，优选地，所述宿主细胞是原核的或真核的，更优选是大肠杆菌细胞、哺乳动物细胞(例如293细胞或CHO细胞)或适用于制备抗体或其抗原结合片段的其它细胞。
  16. 制备抗B7-H3抗体或其抗原结合片段的方法，所述方法包括在适于表达编码前述权利要求1-12中任一项的抗体或其抗原结合片段的核酸的条件下培养权利要求15的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述抗B7-H3抗体或其抗原结合片段。
  17. 由权利要求16所述的方法制备的抗B7-H3抗体或其抗原结合片段。
  18. 免疫缀合物，其包含与治疗剂或诊断剂缀合的前述权利要求1-12或17中任一项的抗体或其抗原结合片段。
  19. 药物组合物，其包含前述权利要求1-12或17中任一项的抗体或其抗原结合片段或权利要求18的免疫缀合物，以及任选地药用辅料。
  20. 权利要求1-12或17中任一项的抗体或其抗原结合片段或权利要求18的免疫缀合物或权利要求19的药物组合物在制备用于治疗和/或诊断癌症或者肿瘤的药物中的用途，优选地，所述肿瘤为实体瘤。
  21. 在受试者中预防或治疗与B7-H3相关疾病或病症例如癌症的方法，所述方法包括向所述受试者施用有效量的前述权利要求1-12或17中任一项的抗B7-H3抗体或其抗原结合片段、或权利要求18的免疫缀合物、或权利要求19的药物组合物。
  22. 检测样品中B7-H3的方法，所述方法包括
    (a)将样品与前述权利要求1-12或17中任一项的抗B7-H3抗体或其抗原结合片段、或权利要求18的免疫缀合物接触；和
    (b)检测所述抗体或其抗原结合片段或免疫缀合物和B7-H3间的复合物的形成；任选地，抗体是被可检测地标记的。
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