WO2021027674A1 - 一种含有抗体的肿瘤治疗剂的开发和应用 - Google Patents

一种含有抗体的肿瘤治疗剂的开发和应用 Download PDF

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WO2021027674A1
WO2021027674A1 PCT/CN2020/107351 CN2020107351W WO2021027674A1 WO 2021027674 A1 WO2021027674 A1 WO 2021027674A1 CN 2020107351 W CN2020107351 W CN 2020107351W WO 2021027674 A1 WO2021027674 A1 WO 2021027674A1
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antibody
amino acid
variant
acid sequence
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王景坤
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康诺亚生物医药科技（成都）有限公司
上海苓樾生物医药科技有限公司
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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]

Definitions

  • the present disclosure relates to an antibody that recognizes B7-H3 protein and its manufacturing method and application.
  • B7-H3 also known as CD276, is a type I transmembrane protein and belongs to the B7-CD28 superfamily.
  • the extracellular region of B7-H3 contains two tandem IgV-IgC domains (4Ig-B7-H3, namely IgV-IgC-IgV-IgC) (CollinsM, 2005), and mouse B7-H3 (2Ig-B7-H3 ) Shows similar functions (HofmeyerK, 2008).
  • B7-H3 as a costimulatory molecule, had a synergistic effect on the proliferation of CD4 and CD8 T cells.
  • B7-H3 is an inhibitory molecule.
  • B7-H3 can inhibit T cell proliferation mediated by antibodies against TCR or allogeneic antigen presenting cells (Suh WK, 2003; Veenstra RG, 2015), this inhibitory effect may be controlled by NFAT, NFkB and AP-1 factors (Hofmeyer KA, 2007).
  • Human B7-H3 protein has two types: membrane-bound and soluble.
  • Membrane-bound B7-H3 protein is mainly distributed on the surface of tumor cells. It is also found in the nucleus, transporters and exosomes inside the cell (Ingebrigtsen VA, 2012; Flem-Karlsen K, 2017). Soluble B7-H3 is hydrolyzed from the cell membrane by proteases, and high levels of soluble B7-H3 can be detected in the serum of tumor patients, suggesting that B7-H3 can be used as a biomarker (Xie C, 2016).
  • B7-H3 mRNA transcription fragments can be detected in a variety of normal tissues (Collins M, 2005), evidence shows that the expression of B7-H3 protein levels is strictly regulated and is absent or extremely low on the cell surface of normal tissues (YiKH, 2009). ). On the contrary, the expression of B7-H3 protein is up-regulated on the surface of tumor cells and is positively correlated with the severity of the disease (Zang X, 2007; Sun Y, 2006; Tekle C, 2012; Wang L, 2013). In non-small cell lung cancer, elevated B7-H3 protein expression is negatively correlated with patient prognosis (Lou Y, 2016; Danilova L, 2016).
  • B7-H3 is highly expressed on neuroblastoma, gastric cancer, ovarian cancer, prostate cancer and cultured tumor stem cells (Modak S, 2001; Zang X, 2010). B7-H3 may be used by tumors as an immune escape route (Hofmeyer K, 2008). In human hepatocellular carcinoma, the expression of B7-H3 is related to the inhibition of T cell proliferation and the reduction of interferon- ⁇ expression (Sun TW, 2012). In mouse pancreatic cancer models, B7-H3 blockade leads to increased CD8+ T cell infiltration and significant anti-tumor effects (Yamato I, 2009).
  • B7-H3 is also expressed in tumor-associated epithelial cells, and usually high expression levels correspond to high degrees of deterioration. Some studies have used B7-H3 expression levels as breast cancer diagnostic indicators (Bachawal SV, 2015). Recent studies have applied the B7-H3 monoclonal antibody to 212Pb for the treatment of ovarian cancer. Preclinical studies have proved that the B7-H3 monoclonal antibody can simultaneously target tumors and tumor vascular endothelial cells, showing preliminary efficacy and good safety. (Kasten BB, 2017). In addition, the B7-H3 antibody coupled with pyrrolobenzodiazepine also acts on B7-H3-positive tumor cells and tumor vascular endothelial system (Seaman S, 2017).
  • B7-H3 in the tumor vascular endothelial system may contribute to the formation of the microenvironment before tumor metastasis and promote metastasis. It has been reported that soluble B7-H3 mediates the expression of vascular endothelial growth factor in tumor cells (Xie C, 2016) .
  • the present inventors immunized mice with recombinant human B7-H3 protein, and obtained multiple high-affinity antibodies that recognize human B7-H3 recombinant protein, which bind to the IgC domain or IgV domain of B7-H3, respectively.
  • the antibody of the present invention can bind to B7-H3 protein with high specificity, has high affinity, and mediates cell killing effect, and can be used for the diagnosis and treatment of gastric cancer, pancreatic cancer and other malignant tumors.
  • the present invention further constructs a bispecific antibody between the humanized B7-H3 antibody and the humanized CD3 antibody, and the bispecific antibody can effectively kill tumor cells expressing B7-H3.
  • the present disclosure provides antibodies or antigen binding portions thereof that bind to human B7-H3 protein.
  • the present disclosure provides a bispecific antibody or antigen binding portion thereof that binds human B7-H3 protein and human CD3.
  • the present disclosure provides a nucleic acid molecule encoding the antibody or antigen binding portion thereof according to the aforementioned aspect.
  • the present disclosure provides a vector containing the nucleic acid of the aforementioned aspect.
  • the present disclosure provides a cell containing the vector of the aforementioned aspect.
  • the antibody or antigen binding portion thereof according to any of the preceding aspects, wherein the antibody or antigen binding portion thereof is humanized.
  • the present disclosure provides a pharmaceutical composition or kit comprising the antibody or antigen-binding portion or nucleic acid encoding thereof according to any one of the foregoing aspects and a pharmaceutically acceptable carrier.
  • a method for treating cancer which comprises the following steps: administering to the mammal a therapeutically effective amount of the antibody or antigen-binding fragment or nucleic acid molecule or carrier or cell or pharmaceutical composition of any of the foregoing aspects to the mammal.
  • FIG. 1 Detection of human B7-H3/CHO transient cells by flow cytometry.
  • CHO-B7-H3-4Ig and CHO-B7-H3-IgC cells express full-length B7-H3 (B7-H3-4Ig) and IgC domains (B7-H3-IgC) in the proximal membrane region of B7-H3, FACS
  • the results showed that CHO-B7-H3-4Ig and CHO-B7-H3-IgC transiently transduced cells showed moderate levels of B7-H3 or B7-H3-IgC expression respectively.
  • the gray peak was the isotype antibody control and the black line was B7.
  • Figure 4 FACS screening of antibodies that bind to B7-H3 + cells.
  • the gray peak is the negative control, and the black peak is the antibody bound to B7-H3 + cells
  • antibodies that specifically bind to B7-H3 and antigen-binding fragments thereof.
  • a monoclonal anti-B7-H3 antibody that specifically binds to human B7-H3, wherein the anti-B7-H3 antibody includes a variant of the parent antibody.
  • antibodies that specifically bind to B7-H3 e.g., human B7-H3.
  • an anti-B7-H3 antibody comprising one or more modifications in amino acid residues (for example, 5-13 amino acid substitutions in the framework region of the heavy chain variable region), and no Compared with the modified parent antibody, it maintains the affinity to the antigen.
  • this article also provides a bispecific antibody and an antigen-binding fragment thereof, containing a first protein-binding functional region capable of specifically binding to B7-H3 protein, and a second protein-binding functional region capable of specifically binding to CD3 protein .
  • bispecific antibodies and antigen-binding fragments thereof that specifically bind to human B7-H3 and human CD3.
  • bispecific antibodies that specifically bind to B7-H3 e.g., human B7-H3.
  • -H3 e.g., human B7-H3
  • CD3 e.g., human CD3 anti-B7-H3/CD3 bispecific antibody.
  • an anti-B7-H3/CD3 bispecific antibody comprising one or more modifications in amino acid residues (e.g., 5-13 amino acid substitutions in the framework region of the heavy chain variable region ), compared with the parent antibody without the modification, it maintains the affinity to the antigen.
  • the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where an integer is required, the term refers to within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.
  • the phrase "substantially identical" can be understood as showing at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains with 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequence the term can be understood as showing that the reference nucleic acid sequence is at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. %, 99% or higher sequence identity nucleotide sequence.
  • Sequence "identity” or “identity” has the art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule.
  • identity is well known to the skilled person (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)) ).
  • substitution type variant is a variant in which at least one amino acid residue in the natural sequence is removed and a different amino acid is inserted into the same position.
  • the substitution may be single, in which only one amino acid in the molecule is substituted; or may be multiple, in which two or more amino acids in the same molecule are substituted. Multiple substitutions can be located at consecutive positions.
  • an amino acid may be substituted by multiple residues, where such variants include both substitutions and insertions.
  • An “insertion type” variant is a variant in which one or more amino acids are inserted at a specific position immediately adjacent to a natural sequence. Immediately adjacent to the amino acid means to be attached to the ⁇ -carboxy or ⁇ -amino functional group of the amino acid.
  • a “deletion type” variant is a variant in which one or more amino acids in the natural amino acid sequence have been removed. Usually, deletion variants have one or two amino acids deleted in a specific region of the molecule.
  • variable domains of antibodies refers to certain parts of related molecules that have broad sequence differences between antibodies and are used for specific recognition and binding of specific antibodies against their specific targets.
  • variability is not evenly distributed throughout the variable domains of antibodies.
  • the variability is concentrated in three segments called complementarity determining regions (CDRs; namely CDR1, CDR2, and CDR3) or hypervariable regions, which are all located in the variable domains of the light chain and the heavy chain.
  • CDRs complementarity determining regions
  • FR framework sequences.
  • Each variable domain of the natural heavy chain and light chain includes four FR regions, which mainly adopt a ⁇ -sheet configuration.
  • the CDRs of each chain are usually connected by FR regions adjacent to each other, and with the help of CDRs from other chains, it helps to form the antibody target binding site (epitope or determinant) (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)).
  • the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified.
  • a CDR may have the ability to specifically bind to cognate epitopes.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any part of a full-length antibody that is less than full-length but contains at least a portion of the variable region (eg, one or more CDR and/or one or more antibody binding sites), and thus retain the binding specificity and at least part of the specific binding capacity of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment that includes an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, and synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragment may include multiple chains linked together, for example by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into the antibody framework (for example, by replacing the corresponding region), obtains an antibody that immunospecifically binds (ie, exhibits a Ka of at least or at least about 10 7 -10 8 M-1) antigen .
  • “Functional fragments” or “anti-B7-H3 antibody analogs” are fragments or analogs that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction.
  • a functional fragment generally has the same meaning as an "antibody fragment", and in the case of an antibody, it may refer to a fragment that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv , F ab , F (ab')2 and so on.
  • the "F v "fragment is composed of a dimer (V H- VL dimer) formed by a non-covalent combination of a variable domain of a heavy chain and a variable domain of a light chain.
  • V H- VL dimer dimer
  • the three CDRs of each variable domain interact to determine the target binding site on the surface of the VH- VL dimer, as in the case of intact antibodies.
  • the six CDRs confer the target binding specificity of a complete antibody.
  • even a single variable domain or half of the F v comprising only three CDRs specific for a target), still having the ability to recognize and bind target.
  • Bispecific antibody refers to the ability of antibodies and/or antigen-binding molecules to specifically bind to two different antigenic determinants, usually, bispecific antibodies and/or antigen-binding molecules Contains two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antibody and/or antigen binding molecule can simultaneously bind to two antigenic determinants, especially two antigenic determinants expressed on two different cells.
  • monoclonal antibody refers to a population of the same antibody, meaning that each individual antibody molecule in the monoclonal antibody population is the same as other antibody molecules. This characteristic is in contrast to the characteristic of the polyclonal population of antibodies, which contains antibodies with multiple different sequences.
  • Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117) .
  • monoclonal antibodies can be produced by immortalizing B cells, for example by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with viruses such as EBV.
  • Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibodies.
  • hybridomas refers to a cell or cell line (usually myeloma or lymphoma cell) produced by fusing antibody-producing lymphocytes and antibody-non-producing cancer cells.
  • hybridomas can proliferate and continue to be supplied to produce specific monoclonal antibodies. Methods for generating hybridomas are known in the art (see, for example, Harlow & Lane, 1988).
  • hybridodoma or “hybridoma cell”
  • it also includes subclones and progeny cells of the hybridoma.
  • a full-length antibody has two full-length heavy chains (for example, VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region
  • VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4 and VL-CL full-length light chains
  • VL-CL full-length light chains
  • chimeric antibody refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as where the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody Of antibodies.
  • Humanized antibody refers to a non-human (e.g., mouse) antibody form, which is a chimeric immunoglobulin, immunoglobulin chain or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or Other antigen-binding subsequences of antibodies) contain minimal sequences derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody), wherein the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and ability ( Donor antibody) such as mouse, rat or rabbit CDR residue replacement.
  • CDR complementarity determining region
  • PCR-mediated mutations can be introduced to introduce mutations, and their impact on antibody binding or other functional properties can be assessed using the in vitro or in vivo tests described herein. Usually, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, there are usually no more than one or two mutations in the CDR. Therefore, the humanized antibody of the present invention also encompasses antibodies that contain one or two amino acid mutations in the CDR.
  • CDR refers to a complementarity-determining region, and it is known that each heavy chain and light chain of an antibody molecule has 3 CDRs. CDR is also called hypervariable region, and exists in the variable region of each heavy chain and light chain of an antibody, and has a very high variability site in the primary structure of the CDR.
  • the CDR of the heavy chain is represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the heavy chain
  • CDR of the light chain is represented by CDR1, CDR2, CDR3 derived from the amino terminal of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.
  • epitope refers to any epitope on the antigen to which the paratope of an antibody binds.
  • Epitope determinants usually contain chemically active surface typing of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka (or 1 ⁇ 10 -7 M or 1 ⁇ 10 7 M-1 or 1 ⁇ 10 8 M-1 or more).
  • Ka affinity constant
  • Kd dissociation constant
  • the affinity constant can be determined by standard kinetic methods of antibody reaction, for example, immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11: 54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also US Patent No. 7,229,619 describing an exemplary SPR and ITC method for calculating the binding affinity of antibodies).
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • the term "competition" with respect to antibodies means that the first antibody or antigen-binding fragment thereof binds an epitope in a manner sufficiently similar to that of the second antibody or antigen-binding fragment thereof, whereby the first antibody and its cognate epitope The binding result is detectably reduced in the presence of the second antibody compared to the absence of the second antibody.
  • the first antibody can inhibit the binding of the second antibody to its epitope, without the second antibody inhibiting the binding of the first antibody to its respective epitope.
  • each antibody can detectably inhibit the binding of the other antibody to its cognate epitope or ligand, whether to the same, higher or lower degree, the antibodies are said to "cross-compete” with each other for binding Their respective epitopes.
  • Competitive and cross-competitive antibodies are both included in the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (for example, steric hindrance, conformational changes, or binding to a common epitope or fragments thereof), those skilled in the art will be aware of such competing and/or cross-competitive antibodies based on the teachings provided by the present invention It is covered in the present invention and can be used in the method disclosed in the present invention.
  • operably linked with respect to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide, so that the promoter regulates or mediates transcription of the nucleic acid.
  • Constant sequence modifications of the sequences described in the sequence listing described herein are also provided, that is, nucleotide and amino acid sequence modifications that do not eliminate the binding of an antibody encoded by a nucleotide sequence or containing an amino acid sequence to an antigen.
  • conservative sequence modifications include conservative nucleotide and amino acid substitutions, and nucleotide and amino acid additions and deletions.
  • modifications can be introduced into the sequence listing described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative sequence modifications include conservative amino acid substitutions, in which amino acid residues are replaced with amino acid residues with similar side chains. The families of amino acid residues with similar side chains are already defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • side chains with no electrical polarity e.g., Amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan)
  • amino acids with non-polar side chains e.g., alanine, valine Acid, leucine, isoleucine, proline, phenylalanine, methionine
  • amino acids with ⁇ -branched side chains e.g.
  • the predicted non-essential amino acid residue in the anti-B7-H3 antibody is preferably replaced by another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (for example, see Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10)) : 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).
  • mutations can be randomly introduced along all or a part of the coding sequence of the anti-B7-H3 antibody by, for example, saturation mutagenesis, and the resulting modified anti-B7-H3 antibody can be screened for improved binding activity .
  • expression refers to the process of producing a polypeptide through the transcription and translation of polynucleotides.
  • the expression level of the polypeptide can be evaluated by any method known in the art, including, for example, a method of measuring the amount of the polypeptide produced from the host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell used to receive, maintain, replicate, and amplify a vector.
  • the host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • the host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include but are not limited to CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell.
  • the vector includes those vectors into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced by restriction digestion and ligation.
  • the vectors also include those containing nucleic acid encoding a polypeptide.
  • the vector is used to introduce the nucleic acid encoding the polypeptide into the host cell, to amplify the nucleic acid or to express/display the polypeptide encoded by the nucleic acid.
  • the vector usually remains free, but can be designed to integrate the gene or part of it into the chromosome of the genome.
  • Vectors of artificial chromosomes are also considered, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
  • vectors also include “viral vectors” or “viral vectors”.
  • the vector of the virus is an engineered virus that is operably linked to a foreign gene to transfer the foreign gene (as a vehicle or shuttle) into the cell.
  • expression vector includes a vector capable of expressing DNA that is operably linked to a regulatory sequence capable of affecting the expression of such DNA fragments, such as a promoter region. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which when introduced into an appropriate host cell, results in the expression of the cloned DNA. Appropriate expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and/or prokaryotic cells, and expression vectors that remain free or that are integrated into the host cell genome.
  • treating an individual suffering from a disease or disease condition means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and/or cure. Prevention refers to preventing underlying diseases and/or preventing symptoms from worsening or disease progression. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided and the composition provided herein.
  • therapeutic effect means the effect resulting from the treatment of an individual, which changes, generally improves or ameliorates the symptoms of a disease or disease condition, or cures the disease or disease condition.
  • prophylactically effective dose or “prophylactically effective dose” refers to the amount of a substance, compound, material, or composition containing the compound that will have the expected preventive effect when administered to a subject, for example, to prevent or delay disease or symptoms To reduce the occurrence or recurrence of diseases or symptoms.
  • a completely preventive effective dose does not have to occur by administering one dose, and may only occur after administering a series of doses. Therefore, the prophylactically effective amount can be administered in one or more applications.
  • the term "patient” refers to a mammal, such as a human.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises an amino acid sequence SEQ ID NO: 5-7, 15-17, 25-27, 35-37, 45-47 , 55-57, 65-67, 75-77, 85-87, 95-97, 100-102, 105-107, 110-112, 115-117 or any variant of heavy chain CDR, and/or selected From the amino acid sequence SEQ ID NO: 10-12, 20-22, 30-32, 40-42, 50-52, 60-62, 70-72, 80-82, 90-92, 120-122, 125-127 , 130-132 or any variant of the light chain CDR.
  • the antibody or antigen-binding portion thereof which comprises an amino acid sequence SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 100, 105, 110, 115 or
  • the heavy chain CDR1 of any variant thereof is selected from the amino acid sequence SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 101, 106, 111, 116 or any variant thereof
  • the heavy chain CDR2 is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 102, 107, 112, 117 or any variant thereof; and / Or light chain CDR1 selected from amino acid sequence SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 120, 125, 130 or any variant thereof, selected from amino acid sequence SEQ ID NO :11, 21, 31, 41, 51, 61, 71, 81, 91, 121, 126,
  • the antibody or antigen-binding portion thereof which comprises an amino acid sequence SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 99, 104, 109, 114 , 218 or the heavy chain variable region of any variant thereof, and/or selected from the amino acid sequence SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 89, 119, 124, 129, 220 Or the light chain variable region of any variant thereof.
  • a nucleic acid molecule encoding an antibody or an antigen-binding portion thereof comprising a nucleic acid molecule selected from SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 103, 108, 113 , 118 or any variant of the antibody heavy chain nucleic acid sequence, and/or selected from SEQ ID NO: 13, 23, 33, 43, 53, 63, 73, 83, 93, 123, 128, 133 or any of its variants Body's antibody light chain nucleic acid sequence.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain selected from the group consisting of amino acid sequence SEQ ID NO: 94, 99, 104, 109, 114 or any variant thereof.
  • Variable region, and/or light chain variable region selected from amino acid sequence SEQ ID NO: 119, 124, 129 or any variants thereof.
  • the present disclosure relates to an antibody or antigen binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 94 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 119 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 94 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 129 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 99 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 119 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 99 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 124 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 99 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 129 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 104 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 119 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 104 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 124 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 109 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 119 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 109 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 124 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 109 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 129 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 114 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 119 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 114 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 124 or the light chain variable region of any variant thereof.
  • the present disclosure relates to an antibody or antigen-binding portion thereof that binds to human B7-H3, which comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 114 or any variant thereof, and/or is selected from The amino acid sequence SEQ ID NO: 129 or the light chain variable region of any variant thereof.
  • the present disclosure provides an antibody or antigen binding portion thereof that binds to human B7-H3, which comprises an amino acid sequence SEQ ID NO: 95-97, 100-102, 105-107, 110-112,
  • the antibody or antigen-binding portion thereof that binds to human B7-H3 has at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • the present disclosure also relates to a bispecific antibody or antigen-binding fragment thereof, which includes:
  • the antibody comprises a heavy chain CDR selected from the amino acid sequence of SEQ ID NO: 115-117 or any variant thereof, and/or a light chain CDR selected from the amino acid sequence of SEQ ID NO: 125-127 or any variant thereof;
  • the two protein functional region is an anti-CD3 antibody or an antigen-binding fragment thereof, wherein the anti-CD3 antibody comprises a heavy chain CDR selected from the amino acid sequence SEQ ID NO: 159-161 or any variant thereof, and/or selected from Amino acid sequence SEQ ID NO: 194-196 or the light chain CDR of any variant.
  • the first protein functional region comprises a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO: 115 or any variant thereof, and is selected from the amino acid sequence SEQ ID NO:
  • the heavy chain CDR2 of 116 or any variant thereof is selected from the heavy chain CDR3 of the amino acid sequence SEQ ID NO: 117 or any variant thereof; and/or the light chain is selected from the amino acid sequence SEQ ID NO: 125 or any variant thereof
  • CDR1 is selected from the light chain CDR2 of the amino acid sequence SEQ ID NO: 126 or any variant thereof, is selected from the light chain CDR3 of the amino acid sequence SEQ ID NO: 127 or any variant thereof
  • the second protein functional region includes The heavy chain CDR1 of the amino acid sequence of SEQ ID NO: 159 or any variant thereof is selected from the heavy chain CDR2 of the amino acid sequence of SEQ ID NO: 160 or any of its variants, which is selected from the amino acid sequence of SEQ ID NO: 161 or
  • the first protein functional region comprises a heavy chain variable region selected from the amino acid sequence SEQ ID NO: 218 or any variant thereof, and/or is selected from amino acids The light chain variable region of the sequence SEQ ID NO: 220 or any variant thereof;
  • the second protein functional region comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO: 222 or any variant thereof, and/or A light chain variable region selected from the amino acid sequence SEQ ID NO: 224 or any variant thereof.
  • the first protein functional region comprises a heavy chain constant region selected from the amino acid sequence SEQ ID NO: 219, 226, 228 or any variant thereof, and/or The light chain constant region selected from the amino acid sequence SEQ ID NO: 221 or any variant thereof;
  • the second protein functional region comprises a heavy chain constant selected from the amino acid sequence SEQ ID NO: 223, 227, 229 or any variant thereof Region, and/or a light chain constant region selected from the amino acid sequence SEQ ID NO: 225 or any variant thereof.
  • An antibody or antigen-binding portion thereof that binds to CD3, comprising a weight selected from the group consisting of amino acid sequence SEQ ID NO: 138, 143, 148, 153, 158, 163, 168, 173, 178, 183, 222 or any variant thereof Chain variable region, and/or light chain variable region selected from the amino acid sequence SEQ ID NO: 188, 193, 198, 203, 208, 213, 224 or any variant thereof.
  • the antibody or antigen-binding portion thereof that binds to human B7-H3 has at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • a vector containing a nucleic acid as in any one of the foregoing is provided.
  • a cell containing a vector as in any one of the preceding aspects is a cell containing a vector as in any one of the preceding aspects.
  • a pharmaceutical composition comprising the antibody or antigen-binding portion or nucleic acid encoding thereof according to any one of the foregoing aspects and a pharmaceutically acceptable carrier.
  • a method for treating cancer which comprises the following steps: administering to the mammal a therapeutically effective amount of the antibody or antigen-binding fragment or nucleic acid molecule or carrier or cell or pharmaceutical composition of any one of the foregoing aspects to the mammal.
  • the cancer is gastric cancer.
  • the antibody kills cancer cells through ADCC or CDC effects.
  • the antibody is coupled to other drugs, such as labeled or having Cytotoxic conjugate.
  • the present disclosure also includes kits.
  • the kit includes the antibodies of the present disclosure, fragments, homologs, derivatives thereof, etc., such as labeled or cytotoxic conjugates, and instructions for use of the antibody , Conjugates that kill specific types of cells, etc.
  • the instructions may include instructions for using antibodies, conjugates, etc. in vitro, in vivo, or ex vivo.
  • Antibodies can be in liquid form or solid, and are usually lyophilized.
  • the kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. Consider the combination of reagents packaged in predetermined amounts and instructions for their use, such as for therapeutic use or for performing diagnostic assays.
  • the kit may include the substrate and cofactors required for the enzyme (for example, a substrate precursor that provides a detectable chromophore or fluorophore).
  • the substrate and cofactors required for the enzyme for example, a substrate precursor that provides a detectable chromophore or fluorophore.
  • other additives such as stabilizers, buffers (for example, blocking buffer or lysis buffer), etc. may also be included.
  • the relative amount of multiple reagents can be changed to provide a concentrate of the reagent solution, which provides user flexibility, space saving, reagent saving, etc.
  • These reagents can also be provided in dry powder form, usually in a lyophilized form, including excipients, which can provide a reagent solution with an appropriate concentration when dissolved.
  • the present invention also includes multivalent antibodies, including bispecific anti-B7-H3 antibodies, which have effector molecules, atoms or other substances with diagnostic or therapeutic functions connected to them.
  • the antibody may have a radioactive diagnostic label or a radioactive cytotoxic atom or a metal or cytotoxic substance such as a ricin chain, which is linked to it for in vivo diagnosis or treatment of cancer.
  • antibodies of the present invention can also be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
  • the present invention also provides a composition
  • a composition comprising the anti-B7-H3 antibody of the present invention or a fragment thereof, the antibody is conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, which is in the art Normal practice.
  • composition refers to a preparation of various preparations.
  • the preparation containing a therapeutically effective amount of the multivalent antibody is in a sterile liquid solution, liquid suspension or lyophilized form, and optionally contains stabilizers or excipients.
  • cancer refers to or describes the physiological condition of mammals, especially humans, whose typical feature is unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • the antibody of the present invention can be used as a composition administered alone, or can be used in combination with other active agents.
  • the therapeutic agents according to the described embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc.
  • suitable pharmaceutically acceptable carriers excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc.
  • suitable formulations can be found in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), especially Chapter 87 by Blaug, Seymour .
  • These formulations include for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) vector (e.g.
  • Lipofectin TM DNA conjugates, anhydrous absorption pulp, oil in water And water-in-oil emulsion, emulsion polyethylene glycol (polyethylene glycol of various molecular weight), semi-solid gel and semi-solid mixture containing polyethylene glycol.
  • emulsion polyethylene glycol polyethylene glycol of various molecular weight
  • semi-solid gel and semi-solid mixture containing polyethylene glycol.
  • the antibody can be used as a therapeutic agent.
  • agents will generally be used to treat, alleviate, and/or prevent diseases or pathologies associated with abnormal B7-H3 expression, activity, and/or signaling in a subject.
  • Standard methods can be used to identify subjects, such as human patients suffering from (or at risk or developing) diseases or disorders related to abnormal B7-H3 expression, activity, and/or signaling, such as cancer or other neoplastic disorders Implement a treatment plan.
  • An antibody preparation preferably an antibody preparation with high specificity and high affinity for its target antigen, is administered to a subject and will usually have an effect due to its binding to the target.
  • the administered antibody can eliminate or inhibit or hinder the expression, activity, and/or signaling function of the target (for example, B7-H3).
  • the administered antibody can eliminate or inhibit or hinder the binding of the target (such as B7-H3) to the endogenous ligand to which it naturally binds.
  • the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with B7-H3 expression, activity, and/or signaling.
  • antibodies with heavy and light chain CDRs can be administered to the subject.
  • the disease or disorder associated with abnormal B7-H3 expression may be cancer.
  • diseases or disorders associated with abnormal B7-H3 expression, activity, and/or signal transduction include hematological cancers and/or solid tumors.
  • Hematological cancers include, for example, leukemia, lymphoma and myeloma.
  • certain forms of leukemia include acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); myeloproliferative Disease/neoplastic (MPDS); and myelodysplastic syndrome.
  • lymphoma examples include Hodgkin’s lymphoma, low-grade and aggressive non-Hodgkin’s lymphoma, Burkitt’s lymphoma, and follicular lymphoma (small cell and Large cells).
  • myeloma include multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or Bence-Jones myeloma.
  • Solid tumors include, for example, breast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostate tumors, melanomas, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, and kidney tumors.
  • Symptoms associated with cancer and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain, frequent local inflammation, loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue, and abdomen Symptoms such as abdominal pain, dysentery or constipation.
  • antibodies against B7-H3 can be used in methods known in the art related to the localization and/or quantification of B7-H3 (for example, for the determination of B7-H3 and/or B7-H3 in appropriate physiological samples).
  • an antibody specific for B7-H3 or its derivatives, fragments, analogs or homologues, comprising an antibody-derived antigen-binding domain is used as a pharmacologically active compound (hereinafter Known as "therapeutics").
  • B7-H3 polypeptides can be isolated by standard techniques such as immunoaffinity, chromatography or immunoprecipitation using antibodies specific for B7-H3.
  • Antibodies against B7-H3 protein (or fragments thereof) can be used to detect proteins in biological samples.
  • B7-H3 can be detected in biological samples as part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen. Coupling (ie, physically connecting) the antibody to the detectable substance may facilitate detection.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/ Biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine aminofluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include Lu Minol; Examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H.
  • the antibody according to the present disclosure can be used as a reagent for detecting the presence of B7-H3 or its protein fragment in a sample.
  • the antibody comprises a detectable label.
  • the antibody is a polyclonal antibody, or more preferably a monoclonal antibody. Use whole antibodies or fragments thereof (e.g. Fab, scFv or F(ab') 2 ).
  • labeling with respect to an antibody is intended to include directly labeling the antibody by coupling (ie, physically connecting) a detectable substance to the antibody, and indirectly labeling the antibody by reaction with another reagent that is directly labeled.
  • Examples of indirect labeling include detecting the first antibody using a fluorescently-labeled second antibody, and end-labeling the antibody with biotin to enable detection with fluorescently-labeled streptavidin.
  • bio sample is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in the subject. Therefore, the term “biological sample” used includes blood and fractions or components in blood, including serum, plasma, or lymph.
  • the detection method of the described embodiment can be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo.
  • in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization.
  • In vitro detection techniques for analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence.
  • In vitro detection techniques for analyte genomic DNA include Southern hybridization. The procedure for performing immunoassays is described in, for example, "ELISA: Theory and Practice: Methods in Molecular Biology", Volume 42, JRCrowther (Editor) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.
  • the in vivo detection technology of analyte protein includes the introduction of labeled anti-analyte protein antibodies into the subject.
  • the antibody can be labeled with a radioactive label, and then the presence and location of the radioactive label in the subject can be detected by standard imaging techniques.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • the principles and considerations involved in preparing such compositions and guidelines for selecting components are well known in the art, for example, see Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th Edition (Alfonso R. Gennaro et al. Edit) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide AndProtein Drug Delivery(AdvancesIncience Parent , Volume 4), 1991, M. Dekker, New York.
  • compositions generally include an antibody and a pharmaceutically acceptable carrier.
  • antibody fragments When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein may be preferred.
  • peptide molecules that retain the ability to bind the target protein sequence can be designed.
  • Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)).
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. compatible with drug administration .
  • Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard bibliography in the field, which is incorporated herein by reference.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as immobilized oils can also be used.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except for the incompatibility of any conventional media or reagents with the antibody, its use in the composition is envisaged.
  • the pharmaceutical composition of the described embodiments is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (eg inhalation), transdermal (ie topical), transmucosal, and rectal administration.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solutions, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; Antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate Or phosphate, and an agent that regulates osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injection use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.
  • suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability is achieved. It must be stable under manufacturing and storage conditions and must be able to prevent the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.
  • suitable fluidity can be maintained.
  • the prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars, polyols (such as mannitol, sorbitol), sodium chloride.
  • Prolonged absorption of the composition for injection can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.
  • a sterile injectable solution can be prepared by incorporating the antibody in the required amount in a suitable solvent having one or a combination of the ingredients listed above (as required), and then filtering and sterilizing.
  • dispersions are prepared by incorporating the antibody into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above.
  • the preparation methods are vacuum drying and freeze-drying to obtain powders containing active ingredients and any additional desired ingredients, which are derived from the aforementioned sterile filtered solutions of these ingredients .
  • the compound is delivered as an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant, such as a gas such as carbon dioxide.
  • a suitable propellant such as a gas such as carbon dioxide.
  • transmucosal or transdermal administration penetrants suitable for penetrating the barrier are used in the formulation.
  • penetrants are generally generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration.
  • Transmucosal administration can be achieved by using nasal sprays or suppositories.
  • one or more of the antibodies can be formulated as an ointment, ointment, gel, or cream as commonly known in the art.
  • the compounds can also be prepared in the form of suppositories (for example, with a conventional suppository base such as cocoa butter or other glycerides) or retention enemas for transrectal delivery.
  • suppositories for example, with a conventional suppository base such as cocoa butter or other glycerides
  • retention enemas for transrectal delivery.
  • the antibody can be prepared with a carrier that prevents it from being rapidly eliminated by the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems.
  • a sustained/controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be obvious to those skilled in the art.
  • dosage unit form refers to a physically separable unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined amount calculated in combination with the required pharmaceutical carrier to produce the desired therapeutic effect One or more of said antibodies.
  • the specifications of the dosage unit form of the embodiment are indicated by and directly depend on the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the limitations inherent in the field of formulation of such antibodies for treating individuals.
  • the pharmaceutical composition can be placed in a container, package, or dispenser together with instructions for administration.
  • compositions described herein may also contain more than one of the antibodies according to the specific situation to be treated, preferably those with complementary activities but not negatively affecting each other.
  • the composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • cytotoxic agent such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • cytokine cytokine
  • chemotherapeutic agent chemotherapeutic agent
  • growth inhibitory agent such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent.
  • Such molecules are suitably present in combination in an amount effective for the intended purpose. For example, they can be co-existing in the kit, or co-existing in use.
  • one or more of the antibodies can be administered in combination therapy, that is, with other agents such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders And inflammatory disease).
  • therapeutic agents which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders And inflammatory disease.
  • combined herein refers to the administration of agents substantially simultaneously, simultaneously or sequentially. If administered sequentially, when the second compound is initially administered, the first of the two compounds is still preferably detected at an effective concentration at the treatment site.
  • “combination” can also mean that the antibody of the present invention and other therapeutic agents are included in the kit.
  • the combination therapy may include one or more antibodies described herein and one or more additional therapeutic agents (e.g., one or more cytokines and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , Enzyme inhibitors, and/or cytotoxins or cell growth inhibitors, as described in more detail below) co-formulated and/or co-administered.
  • additional therapeutic agents e.g., one or more cytokines and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors , Enzyme inhibitors, and/or cytotoxins or cell growth inhibitors, as described in more detail below.
  • Such combination therapy can advantageously utilize lower doses of administered therapeutic agents, thus avoiding possible toxicity or complications associated with various monotherapy.
  • plasmid pMD18-B7-H3 (Sino Biological Inc.) containing the human B7-H3 gene cDNA sequence (SEQ ID NO:1) as the template, the forward primer 5'-CTGAGAGGTGCCAGATGTATGCTGCGTCGGCGGGGC-3' and the reverse primer 5'- TCCGCCTCCGCCGCTAGCTGTCATAGGCTGCCCTGTG-3', PCR amplified human B7-H3 extracellular region fragment (Met1-Thr461), the amplified fragment was digested with BspQI and cloned into the eukaryotic expression plasmid system (pSect). HEK293E cells were transfected with this plasmid. After 6 days, the culture supernatant was collected and purified by nickel column affinity chromatography to obtain the recombinant protein of human B7-H3 extracellular domain (SEQ ID NO: 2, the result is shown in Figure 1) .
  • Example 2 Cell lines expressing full length of human B7-H3 or IgC domain
  • B7-H3 is widely and highly expressed in tumor cell lines, and the common cell line HEK293 cells also highly express B7-H3 (Shi J, 2016), so in preliminary screening, B7-H3 recombinant protein and HEK293 cells can be used interchangeably.
  • B7-H3 full-length (B7-H3-4Ig) or IgC (B7-H3-IgC) was constructed to construct eukaryotic expression plasmids and transiently transfected into CHO cells.
  • the voltage of the electroporator is set to 300V
  • the capacitance is 950 ⁇ F
  • the constructed eukaryotic expression plasmid containing the human B7-H3 full-length sequence (SEQ ID NO: 1) and B7-H3 IgC sequence (SEQ ID NO: 3) is electroporated Into the cells, the cells were inoculated in complete medium after transfection, and the expression of B7-H3 on the cell surface was detected 48 hours after transfection (the results are shown in Figure 2).
  • mice spleen and lymph nodes were taken and ground in DMEM to obtain a B cell suspension. After mixing an appropriate amount of B cell suspension and SP2/0, the cells were fused using an electrofusion instrument. The fused cells were cultured at 5% CO 2 in DMEM complete medium containing HAT at 37°C.
  • the 50 ⁇ L reaction system add 1 ⁇ L of cDNA, 5 ⁇ L of 10 ⁇ PCR buffer, 1 ⁇ L of forward and reverse primers, 1 ⁇ L of dNTP, 25mmol MgCl 2 1 ⁇ L, 39 ⁇ L of H 2 O, 1 ⁇ L of Taq enzyme, and pre-denaturation at 95°C for 10 minutes. Enter the temperature cycle and perform PCR amplification.
  • the reaction conditions were denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 15 seconds, a total of 30 cycles, and then holding at 72°C for 10 minutes.
  • the obtained candidate positive clone heavy chain and light chain variable region sequences are:
  • the heavy chain variable region is cloned into a vector containing the human heavy chain constant region to express a complete IgG1 heavy chain in mammalian cells.
  • the light chain variable region is cloned into a vector containing the human light chain constant region to express the complete kappa light chain in mammalian cells.
  • Example 5 ADCC killing activity experiment of chimeric antibody on target cells
  • the antibodies were diluted with RPMI-1640 medium to 1 ⁇ g/mL, and diluted by 10 times, a total of 5 gradients.
  • Collect NK92MI-CD16a cells centrifuge at 300g for 10 minutes, wash them with PBS, centrifuge and resuspend them in RPMI-1640 complete medium to a cell density of 2 ⁇ 10 5 /mL.
  • the Calu-6 cells were collected, centrifuged at 300g for 10 minutes, washed with PBS, centrifuged and resuspended in RPMI-1640 complete medium to a cell density of 1 ⁇ 10 6 /mL.
  • NK92MI-CD16a cells to a 96-well U-shaped plate, 50 ⁇ L/well, add 50 ⁇ L/well of diluted antibody, 37°C, 5% CO 2 incubator and incubate for 30 minutes. Then Calu-6 cells were added to a 96-well U-shaped plate, 50 ⁇ L/well, centrifuged at 1000 rpm for 1 minute, and then placed in a 37°C, 5% CO 2 incubator for 24 hours. Thirty minutes before the test, add 2 ⁇ L of cell lysate to the maximum release hole of Calu-6 cells and put it back into the incubator to continue culturing.
  • Target cell spontaneous well including target cell suspension, RPMI 1640 culture medium
  • target cells including target cell suspension, RPMI 1640 culture medium, cell lysate
  • CHO-B7-H3-4Ig cells Take the transiently transfected CHO-B7-H3-4Ig cells, CHO-B7-H3-IgC cells and the negative control CHO cells, add 5 ⁇ 10 4 cells/100 ⁇ L to a U-shaped 96-well plate, centrifuge at 1100 rpm for 3 minutes, and discard Clear, pat the cells gently, add 50 ⁇ L of antibody to each well, and incubate at 4°C for 1 hour. After the incubation, add 180 ⁇ L 0.5% BSA to each well to wash 3 times, add 30 ⁇ L/well of secondary antibody AlexaFluro647 anti-human IgG (Jackson ImmunoResearch, catalog number: 109-606-170), and incubate at 4°C for 40 minutes.
  • secondary antibody AlexaFluro647 anti-human IgG Jackson ImmunoResearch, catalog number: 109-606-170
  • FIG. 7 shows that each antibody can specifically bind to CHO-B7-H3-4Ig cells, but only the monoclonal antibodies 6E4, 15C11 and 24A11 can specifically bind to CHO-B7-H3-IgC cells, suggesting that it is relative to other monoclonal antibodies.
  • the cloned antibodies, 6E4, 15C11 and 24A11 bind to the B7-H3 IgC domain closer to the cell membrane.
  • mAb15C11 mouse antibody light chain is mouse IMGT_mVK_6_14, select The human IMGT_hVK_4_59 with the highest homology to the framework region was used for CDR transplantation.
  • the human IGKJ2*01 with the highest homology was selected for FM4; the mouse-derived antibody heavy chain was IGHV1-76.
  • the human germline gene IMGT_hVH_1_18 was selected for CDR transplantation, and FM4 was selected for homology. The most sexual person IGHJ6*01.
  • a computer was used to conduct homology modeling at the same time to analyze the CDR region and its surrounding framework amino acid sequence, to avoid the selected human germline gene to cause the molecular surface charge or hydrophobic region to be concentrated; at the same time, by calculating the electrostatic force, van der Waals force, and hydrophobicity And the entropy value, analyze the key amino acid individuals that may interact with B7-H3 and maintain the spatial structure in the gene sequence of each positive monoclonal antibody, and design back mutation sites on this basis.
  • a total of different heavy chain variants and light chain variants were designed and synthesized. After the light and heavy chains were synthesized in full sequence, they were cloned into the eukaryotic expression vector containing the constant region of antibody kappa chain Ckappa or the constant region of human IgG1 CH1-CH3. After the light and heavy chain plasmids are combined and paired, they are transfected into HEK293.6E cells, expressed at 37 degrees Celsius for 5-6 days, and the culture supernatant is collected and purified by a Protein A column.
  • the humanized antibody heavy chain/light chain variable region sequence is as follows:
  • the 96-well microtiter plate was coated with human B7-H3 or cynomolgus B7-H3, and incubated at 37 degrees Celsius for 60 minutes. Then the solution in the well was discarded, washed 3 times with washing buffer, and blocked with PBS solution containing 2% BSA for 60 minutes.
  • Figure 9 shows that the humanized variant h15C11-VH5/Vk2 has the same affinity for the chimeric antibody 6E4 and the parent 15C11, and can be both high The affinity binds to human and cynomolgus monkey B7-H3 recombinant proteins (Table 2).
  • the antibodies were diluted with RPMI-1640 medium to 1 ⁇ g/mL, and diluted by 10 times, a total of 5 gradients.
  • Collect NK92MI-CD16a cells centrifuge at 300g for 10 minutes, wash them with PBS, centrifuge and resuspend them in RPMI-1640 complete medium to a cell density of 2 ⁇ 10 5 /mL.
  • the Calu-6 cells were collected, centrifuged at 300g for 10 minutes, washed with PBS, centrifuged and resuspended in RPMI-1640 complete medium to a cell density of 1 ⁇ 10 6 /mL.
  • NK92MI-CD16a cells to a 96-well U-shaped plate, 50 ⁇ L/well, add 50 ⁇ L/well of diluted antibody, 37°C, 5% CO 2 incubator and incubate for 30 minutes. Then Calu-6 cells were added to a 96-well U-shaped plate, 50 ⁇ L/well, centrifuged at 1000 rpm for 1 minute, and then placed in a 37°C, 5% CO 2 incubator for 24 hours. Thirty minutes before the test, add 2 ⁇ L of cell lysate to the maximum release hole of Calu-6 cells and put it back into the incubator to continue culturing.
  • Killing rate (%) (experimental hole-target cell spontaneous hole) / (target cell maximum release hole-target cell spontaneous hole) ⁇ 100%
  • Target cell spontaneous well including target cell suspension, RPMI 1640 culture medium
  • target cells including target cell suspension, RPMI 1640 culture medium, cell lysate
  • Figure 11 shows that the 15C11 humanized variant retains ADCC activity on B7-H3 + HEK293 cells compared to the parent 15C11.
  • Murine hybridoma CD3 antibody (EMBO J.1985.4(2):337-344; J.Immunol.1986,137(4):1097-100; J.Exp.Med.1991,174:319-326; J. Immunol. 1991, 147(9): 3047-52) Recognize the recombinant human CD3 ⁇ protein (SEQ ID NO: 134) expressed in HEK293 cells, and also recognize the cynomolgus CD3 ⁇ recombinant protein (SEQ ID NO: 135), its sequence as follows:
  • Amino acid sequence of light chain of anti-CD3 mouse monoclonal antibody Mab01 (SEQ ID NO: 136):
  • Amino acid sequence of heavy chain of anti-CD3 mouse monoclonal antibody Mab01 (SEQ ID NO: 137):
  • IMGT_hVL7-43 which has the highest homology with the light chain framework region of anti-CD3 mouse monoclonal antibody, for light chain CDR transplantation
  • FM4 selects human IGLJ3*02 with the highest homology
  • FM4 uses human IGHJ4*01 with the highest homology.
  • a computer was used to conduct homology modeling, analyze the CDR region and its surrounding framework amino acid sequence, and design back mutation sites on this basis.
  • the sequence of the heavy chain/light chain variable region of the anti-CD3 murine monoclonal antibody humanized antibody is as follows:
  • the light and heavy chain variable region genes of monoclonal antibody h15C11-VH5/Vk2 and anti-CD3 humanized antibody (Table 3) were cloned into the expression vector, and the four plasmids were simultaneously transfected into HEK293.6E cells. After 6 days, they were collected and cultured. The supernatant was purified by Protein A affinity chromatography to obtain the B7-H3/CD3 bispecific antibody. Three different bispecific antibodies were constructed, the corresponding sequence numbers are shown in Table 4, and the epitope sequences corresponding to the bispecific antibodies are shown in Table 5.
  • the 96-well microplate is coated with human B7-H3 or human CD3 ⁇ protein, and incubated at 37 degrees Celsius for 60 minutes. Then the solution in the well was discarded, washed 3 times with washing buffer, and blocked with PBS solution containing 2% BSA for 60 minutes.
  • Construct-1 has the highest affinity
  • Construct-4 has the second highest affinity
  • Construct-7 has the weakest affinity.
  • Figure 15 shows that all three B7-H3/CD3 bispecific antibodies can bind to human CD3 protein. Among them, Construct-1 has the highest affinity, Construct-4 has the second highest affinity, and Construct-7 has the weakest affinity.
  • the separated PBMC was mixed with 1 ⁇ PBS larger than 3 times its volume, centrifuged at 4°C, 1300rpm for 10 minutes, the supernatant was aspirated, and then 10mL 1 ⁇ PBS was added to resuspend the count.
  • PBMC Place the PBMC in a 37°C 5% CO 2 incubator for 2 hours. Centrifuge at 1300 rpm for 10 minutes, remove the supernatant, and resuspend in pre-warmed 2.5% FBS/RPMI 1640 medium. Adjust the density to 2 ⁇ 10 6 cells/mL, 100 ⁇ L/well, that is, 2 ⁇ 10 5 cells/well. Add 50 ⁇ L of diluted Calu-6 cells or HEK293 cells to the U-shaped 96-well plate, that is, 2 ⁇ 10 4 cells/well. Then add the diluted antibody (from 40 ⁇ g/mL, 10 times dilution 6 gradients) 50 ⁇ L/well, and incubate for 24 hours at 37°C in a 5% CO 2 incubator.
  • diluted antibody from 40 ⁇ g/mL, 10 times dilution 6 gradients
  • Killing rate (%) (experimental hole-target cell spontaneous hole) / (target cell maximum release hole-target cell spontaneous hole) ⁇ 100%
  • Target cell spontaneous hole including target cell suspension, RPMI 1640 culture medium
  • target cells including target cell suspension, RPMI 1640 culture medium, cell lysate
  • Figure 16 shows that all three B7-H3/CD3 bispecific antibodies can mediate the TDCC effect.
  • FIG. 17 shows that the three B7-H3/CD3 bispecific antibodies can all induce the activation of CD4 + and CD8 + T cells, and all have a dose-response relationship.

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Abstract

本发明公开了一种识别B7-H3蛋白的抗体、一种识别B7-H3蛋白和CD3的双特异性抗体，及其制造方法和应用。

Description

一种含有抗体的肿瘤治疗剂的开发和应用
本申请要求于2019年08月14日向中华人民共和国知识产权局提交的申请号为201910747678.9的中国发明专利申请的优先权。在此通过引用方式将其全部内容以其整体并入本文。
技术领域
本公开涉及一种识别B7-H3蛋白的抗体及其制造方法和应用。
背景技术
B7-H3也称为CD276，是一种I型跨膜蛋白，属于B7-CD28超家族。B7-H3胞外区包含两个串联的IgV-IgC结构域(4Ig-B7-H3，即IgV-IgC-IgV-IgC)(CollinsM，2005)，与鼠源B7-H3(2Ig-B7-H3)显示具有类似的功能(HofmeyerK，2008)。最初人们认为B7-H3作为一种共刺激分子，对CD4和CD8T细胞的增殖具有协同刺激作用，在TCR信号的存在下，选择性地增强干扰素的产生(Chapoval，AI.2001；Zhang，GB.2004)。然而，近年来的多数研究结果表明，B7-H3是一种抑制性分子，如B7-H3可抑制由抗TCR或同种异体抗原呈递细胞的抗体介导的T细胞增殖(Suh WK，2003；Veenstra RG，2015)，这种抑制作用可能通过NFAT、NFkB和AP-1因子来控制(Hofmeyer KA，2007)。
人B7-H3蛋白具有膜结合型和可溶性两种，膜结合型B7-H3蛋白主要分布于肿瘤细胞表面，在细胞内部的细胞核、运输小体和外泌体等也有发现(Ingebrigtsen VA，2012；Flem-Karlsen K，2017)。可溶性B7-H3由蛋白酶从细胞膜上水解下来，肿瘤患者血清中可以检测到高水平的可溶性B7-H3，提示B7-H3可以作为生物标志物(Xie C，2016)。尽管B7-H3的mRNA转录片段可以在多种正常组织检测到(Collins M，2005)，但证据显示B7-H3蛋白水平的表达受到严格调控，在正常组织细胞表面缺失或极低(YiKH，2009)。相反，B7-H3蛋白在肿瘤细胞表面表达上调，且与疾病严重程度正相关(Zang X，2007；Sun Y，2006；Tekle C，2012；Wang L，2013)。在非小细胞肺癌中，B7-H3蛋白的表达升高与患者预后呈现负相关(Lou Y，2016；Danilova L，2016)。有报道B7-H3在成神经细胞瘤、胃癌、卵巢癌、前列腺癌和培养的肿瘤干细胞上高表达(Modak S，2001；Zang X，2010)。B7-H3可能被肿瘤利用作为免疫逃避途径(Hofmeyer K，2008)，在人肝细胞癌中，B7-H3表达与T细胞增殖抑制和降低干扰素-γ表达有关(Sun TW，2012)，在小鼠胰腺癌模型中，B7-H3阻断导致CD8+T细胞浸润增加和明显的抗肿瘤作用(Yamato I，2009)。
B7-H3在肿瘤相关上皮细胞也有表达，通常高表达水平对应恶化程度高，有研究将B7-H3的表达水平作为乳腺癌诊断指标(Bachawal SV，2015)。最近有研究将B7-H3单克隆抗体标记212Pb，应用于卵巢癌治疗，临床前研究证明了B7-H3单克隆抗体可同时靶向肿瘤和肿瘤血管内皮细胞，显示出初步疗效和良好的安全性(Kasten BB，2017)。另外，偶联pyrrolobenzodiazepine的B7-H3抗体也同时作用于B7-H3阳性肿瘤细胞和肿瘤血管内皮系统(Seaman S，2017)。B7-H3在肿瘤血管内皮系统中的高表达可能有助于肿瘤转移前微环境的形成并促进转移，有报道可溶性B7-H3介导肿瘤细胞中血管内皮生长因子的表达(Xie C，2016)。
发明内容
本发明人以重组人B7-H3蛋白免疫小鼠，得到了多株识别人B7-H3重组蛋白的高亲和力抗体，分别结合于B7-H3的IgC结构域或IgV结构域。本发明抗体能够高特异性地结合B7-H3蛋白，其具有很高的亲和力，并且介导细胞杀伤作用，从而可以用于胃癌、胰腺癌等一些恶性肿瘤的诊断和治疗。本发明进一步将人源化的B7-H3抗体与人源化CD3抗体构建双特异抗体，该双特异抗体可有效杀伤表达B7-H3的肿瘤细胞。
在一方面，本公开提供了结合人B7-H3蛋白的抗体或其抗原结合部分。
在一方面，本公开提供了结合人CD3蛋白的抗体或其抗原结合部分。
在一方面，本公开提供了结合人B7-H3蛋白和人CD3的双特异性抗体或其抗原结合部分。
在一方面，本公开提供了编码如前述方面的抗体或其抗原结合部分的核酸分子。
在一方面，本公开提供了含有前述方面的核酸的载体。
在一方面，本公开提供了含有前述方面的载体的细胞。
根据前述任一方面的抗体或其抗原结合部分，其中所述抗体或其抗原结合部分是人源化的。
在一方面，本公开提供了包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和药学上可接受的载体的药物组合物或试剂盒。
治疗癌症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物。
前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中癌症的药物或试剂盒中的用途。
附图说明
图1、HEK293细胞重组表达的人B7-H3胞外区蛋白
图2、流式细胞术检测人B7-H3/CHO瞬转细胞。CHO-B7-H3-4Ig和CHO-B7-H3-IgC细胞表面分别表达全长B7-H3(B7-H3-4Ig)和B7-H3近膜区IgC结构域(B7-H3-IgC)，FACS结果显示，CHO-B7-H3-4Ig和CHO-B7-H3-IgC瞬转细胞表面分别有B7-H3或B7-H3-IgC中等水平的表达，其中灰色峰为同型抗体对照，黑线为B7-H3抗体检测到的阳性细胞或HA抗体检测到的B7-H3-IgC阳性细胞
图3、ELISA筛选B7-H3阳性杂交瘤
图4、FACS筛选结合B7-H3 +细胞的抗体。其中灰色峰为阴性对照，黑色峰为与B7-H3 +细胞结合的抗体
图5、嵌合抗体对HEK293细胞的ADCC活性
图6、嵌合抗体对Calu-6细胞的ADCC活性
图7、抗B7-H3抗体的结合表位
图8、抗B7-H3抗体人源化后与重组蛋白的结合
图9、抗B7-H3人源化抗体的种属交叉反应
图10、抗B7-H3人源化抗体与HEK293的结合
图11、15C11人源化抗体对HEK293细胞的ADCC活性
图12、抗CD3人源化抗体与人CD3的结合
图13、抗CD3人源化抗体与Jurkat细胞的结合
图14、B7-H3/CD3双特异抗体与人B7-H3蛋白的结合
图15、B7-H3/CD3双特异抗体与人CD3蛋白的结合
图16、B7-H3/CD3双特异抗体介导的T细胞杀伤作用
图17、B7-H3/CD3双特异抗体对T细胞的活化作用
具体实施方式
I.定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在一个方面，本文提供的是特异性结合B7-H3的抗体(例如，单克隆抗体)及其抗原结合片段。在具体的方面，本文提供的是特异性结合人B7-H3的单克隆抗B7-H3抗体，其中所述抗B7-H3抗体包括亲本抗体的变体。在具体的方面，本文提供的是特异性结合B7-H3(例如，人B7-H3)的抗体。在特定的方面，本文提供的是包含一个或更多个氨基酸残基中的修饰的抗B7-H3抗体(例如，重链可变区的框架区中5-13个氨基酸取代)，与没有所述修饰的亲本抗体相比，其保持与抗原的亲和力。
在一个方面，本文还提供一种双特异性抗体及其抗原结合片段，含有能够特异性结合B7-H3蛋白的第一蛋白结合功能区，以及能够特异性结合CD3蛋白的第二蛋白结合功能区。在具体的方面，本文提供的是特异性结合人B7-H3和人CD3的双特异性抗体及其抗原结合片段，在具体的方面，本文提供的是特异性结合B7-H3(例如，人B7-H3)和CD3(例如，人CD3)的抗B7-H3/CD3双特异性抗体。在特定的方面，本文提供的是包含一个或更多个氨基酸残基中的修饰的抗B7-H3/CD3双特异性抗体(例如，重链可变区的框架区中5-13个氨基酸取代)，与没有所述修饰的亲本抗体相比，其保持与抗原的亲和力。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核苷酸序列。
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。(参见，例如：Computational Molecular Biology，Lesk，A.M.，ed.，Oxford University Press，New York，1988；Biocomputing：Informatics and Genome Projects，Smith，D.W.，ed.，Academic Press，New York，1993；Computer Analysis of Sequence Data，Part I，Griffin，A.M.，and Griffin，H.G.，eds.，Humana Press，New Jersey，1994；Sequence Analysis in Molecular Biology，von Heinje，G.，Academic Press，1987；and Sequence Analysis Primer，Gribskov，M.and Devereux，J.，eds.，M Stockton Press，New York，1991)。虽然存在许多测量两个多核苷酸 或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo，H.&Lipman，D.，SIAM J Applied Math 48：1073(1988))。
“取代型”变体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变体。所述取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变体包括取代和插入二者。“插入型”变体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。“缺失型”变体是天然氨基酸序列中一个或多个氨基酸被除去的变体。通常情况下，缺失型变体在其分子的特定区域内有一个或两个氨基酸被缺失。
就抗体的可变结构域而言，术语“可变”系指抗体之间有广泛序列差异的相关分子的某些部分，且被用于针对其特异靶的特定抗体的特异识别和结合。但是，可变性在抗体的整个可变结构域内不是均匀分布的。可变性集中在被称为互补决定区域(CDRs；即CDR1、CDR2和CDR3)或超变区的三个区段，它们均位于轻链和重链的可变结构域内。可变结构域内保守程度更高的部分被称为构架(FR)区或构架序列。天然重链和轻链的每个可变结构域均包括四个FR区，其主要采用β-折叠构型，它们籍三个CDRs连接起来，CDRs形成环，所述环连接β-折叠结构并在某些情形下形成部分的β-折叠结构。每条链的CDRs通常被FR区在邻近连接起来，并且借助于来自其它链的CDR，有助于抗体靶结合位点(表位或决定簇)的形成(参看Kabat等人Sequences of Proteins of Immunological Interest，NationalInstitute of Health，Bethesda，MD(1987))。正如本文所使用，免疫球蛋白氨基酸残基的编号是依据Kabat等人的免疫球蛋白氨基酸残基编号系统而进行的，除非另有说明。一个CDR可具有特异结合关联表位的能力。
如本文所用，抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab′、F(ab’)2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd’片段以及其他片段，包括修饰的片段(参见，例如，Methods in Molecular Biology，Vol 207：Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25，Kipriyanov)。所述片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约10 7-10 8M-1的Ka)抗原的抗体。“功能片段”或“抗B7-H3抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段″含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如F v、F ab、F (ab′)2等等。“F v”片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二聚体(V H-V L二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定V H-V L二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的F v的一半)，仍可具有识别和结合靶的能力。
如本文中所用，术语“双特异性”(Bispecific antibody，BsAb)指抗体和/或抗原结合分子能够特异性结合两种不同的抗原性决定簇，通常，双特异性抗体和/或抗原结合分子包含两种抗原结合位点，其中每种特异于不同的抗原性决定簇。在某些实施方案中，所述双特异性抗体和/或抗原结合分子能够同时结合两种抗原决定簇，特别是在两种不同的细胞上表达的两种抗原性决定簇。
如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备(Smith et al.(2004)J.Clin.Pathol.57，912-917；和Nelson et al.，J Clin Pathol(2000)，53，111-117)。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
如本文中所用，术语“杂交瘤”或“杂交瘤细胞”指由融合产抗体的淋巴细胞和不产抗体的癌细胞而产生的细胞或细胞系(通常为骨髓瘤或淋巴瘤细胞)。如本领域普通技术人员所知的，杂交瘤可增殖并持续供应产生特定单克隆抗体。用于产生杂交瘤的方法为本领域已知的(见例如，Harlow&Lane，1988)。当提及术语“杂交瘤”或“杂交瘤细胞”时，其还包括杂交瘤的亚克隆和后代细胞。
如本文所用，全长抗体是具有两条全长重链(例如VH-CH1-CH2-CH3或VH-CH1-CH2-CH3-CH4)和两条全长轻链(VL-CL)和铰链区的抗体，例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab′、F(ab′)2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本发明所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
如本文所用，术语“CDR”指互补决定区(complementarity-determining region)，已知抗体分子的每个重链和轻链具有3个CDR。CDR也称作高变区，且存在于抗体的每个重链和轻链的可变区中，在CDR的一级结构中具有非常高的变异性位点。本说明书中，重链的CDR由来自重链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，轻链的CDR由来自轻链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示。这些位点在三级结构中彼此临近，并决定抗体所结合的抗原的特异性。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×10 7M-1或1x10 8M-1或更大的亲和常数Ka(或者1x10 -7M或1×10 -8M或更低的解离常数(Kd))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)(Rich and Myszka(2000)Curr.Opin.Biotechnol 11：54；Englebienne(1998)Analyst.123：1599)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定(参见，例如，Paul，ed.，Fundamental Immunology，2nd ed.，Raven Press，New York，pages 332-336(1989)；还参见描述用于计算抗体的结合亲和力的示例性SPR和ITC方法的美国专利第7,229,619号)。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购(参见，BiaCore 2000，Biacore AB，Upsala，Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27：335)。
如本文所用，关于抗体的术语“竞争”是指第一抗体或其抗原结合片段以与第二抗体或其抗原结合片段足够相似的方式结合一个表位，由此第一抗体与其关联表位的结合结果在存在第二抗体的条件下与不存在第二抗体的条件下相比可检测地降低。或者，在第二抗体与其表位的结合在存在第一抗体条件下也可检测地降低的情况中，可以但不必需是这种情况。也就是说，第一抗体可以抑制第二抗体与其表位的结合，而不用第二抗体抑制第一抗体与其各自表位的结合。然而，在每个抗体均可检测地抑制另一抗体与其关联表位或配体的结合的情况中，无论是相同、更高或更低程度，所述抗体被称为彼此“交叉竞争”结合其各自的表位。竞争及交叉竞争抗体均涵盖在本发明中。无论这种竞争或交叉竞争发生的机制如何(例如位阻、构象改变或者结合共同表位或其片段)，本领域技术人员基于本发明提供的教导将意识到这种竞争和/或交叉竞争抗体涵盖在本发明中且可用于本发明揭示的方法中。
如本文所用，术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。如本文所使用，术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链，且可为cDNA。
如本文所用，分离的核酸分子是从存在于核酸分子的天然来源中的其他核酸分子分离的核酸分子。诸如cDNA分子的“分离的”核酸分子可以在通过重组技术制备时基本上不含其他细胞物质或培养基，或者在化学合成时基本上不含化学前体或其他化学成分。本文所提供的示例性分离的核酸分子包括编码所提供的抗体或抗原结合片段的分离的核酸分子。
如本文所用，关于核酸序列、区域、元件或结构域的“可操作地连接”表示核酸区域互相功能相关。例如，启动子可以可操作地连接至编码多肽的核酸，从而所述启动子调控或介导所述核酸的转录。
亦提供本文所述序列表中所述序列的“保守序列修饰”，即不消除由核苷酸序列编码或含有氨基酸序列的抗体与抗原的结合的核苷酸及氨基酸序列修饰。这些保守序列修饰包括保守核苷酸及氨基酸取代以及核苷酸及氨基酸添加及缺失。例如，可通过本领域已知的标准技术(例如定点诱变及PCR介导的诱变)将修饰引入本文所述的序列表中。保守序列修饰包括保守氨基酸取代，其中氨基酸残基被替换为具有类似侧链的氨基酸残基。具有类似侧链的氨基酸残基的家族是本领域中已有定义的。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电极性侧链的氨基酸(例如 甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、具有β分枝侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及具有芳香族侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，抗B7-H3抗体中的预测的非必需氨基酸残基优选被来自同一侧链家族的另一氨基酸残基替代。鉴定不消除抗原结合的核苷酸及氨基酸保守取代的方法为本领域所熟知(例如，参见Brummell等人，Biochem.32：1180-1187(1993)；Kobayashi等人，Protein Eng.12(10)：879-884(1999)；及Burks等人，Proc.Natl.Acad.Sci.USA94：412-417(1997))。
作为另一选择，在另一实施方案中，可通过例如饱和诱变沿抗B7-H3抗体编码序列的全部或一部分随机引入突变，且可针对改良的结合活性筛选所得经修饰抗B7-H3抗体。
如本文所用，“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合片段以及本文所提供的组合物的任何药学用途。
如本文所用，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
如本文所用，“预防有效量”或“预防有效剂量”指在施用于对象时会具有预期的预防效果的物质、化合物、材料或包含化合物的组合物的量，例如，防止或延迟疾病或症状的发生或复发，减少疾病或症状发生或复发的可能性。完全预防有效剂量不必通过施用一个剂量发生，并且可以仅在施用一系列剂量之后发生。因此，预防有效量可以在一次或多次施用中施用。
如本文中所使用的，术语“患者”是指哺乳动物，例如人。
II.具体实施方式
在一方面，本公开提供了结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：5-7、15-17、25-27、35-37、45-47、55-57、65-67、75-77、85-87、95-97、100-102、105-107、110-112、115-117或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：10-12、20-22、30-32、40-42、50-52、60-62、70-72、80-82、90-92、120-122、125-127、130-132或任何变体的轻链CDR。
根据前一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：5、15、25、35、45、55、65、75、85、95、100、105、110、115或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO：6、16、26、36、46、56、66、76、86、96、101、106、111、116或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO：7、17、27、37、47、57、67、77、87、97、102、107、112、117或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO：10、20、30、40、50、60、70、80、90、120、125、130或其任何变体的轻链CDR1，选自氨基 酸序列SEQ ID NO：11、21、31、41、51、61、71、81、91、121、126、131或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO：12、22、32、42、52、62、72、82、92、122、127、132或其任何变体的轻链CDR3。
根据前述任一方面的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：4、14、24、34、44、54、64、74、84、94、99、104、109、114、218或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：9、19、29、39、49、59、69、79、89、119、124、129、220或其任何变体的轻链可变区。
编码根据前述任一方面的抗体或其抗原结合部分的核酸分子，其包含选自SEQ ID NO：8、18、28、38、48、58、68、78、88、98、103、108、113、118或其任何变体的抗体重链核酸序列，和/或选自SEQ ID NO：13、23、33、43、53、63、73、83、93、123、128、133或其任何变体的抗体轻链核酸序列。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：94、99、104、109、114或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：119、124、129或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：94或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：119或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：94或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：124或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：94或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：129或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：99或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：119或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：99或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：124或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：99或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：129或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：104或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：119或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：104或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：124或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：104或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：129或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：109或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：119或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：109或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：124或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：109或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：129或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：114或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：119或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：114或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：124或其任何变体的轻链可变区。
在一方面，本公开涉及一种结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：114或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：129或其任何变体的轻链可变区。
在一个具体实施方式中，本公开提供了结合人B7-H3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：95-97、100-102、105-107、110-112、115-117或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：120-122、125-127、130-132、或任何变体的轻链CDR。
一种结合人B7-H3的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
(1)分别包含SEQ ID NO：95-97的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
(2)分别包含SEQ ID NO：95-97的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
(3)分别包含SEQ ID NO：95-97的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列；
(4)分别包含SEQ ID NO：100-102的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
(5)分别包含SEQ ID NO：100-102的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
(6)分别包含SEQ ID NO：100-102的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列；
(7)分别包含SEQ ID NO：105-107的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
(8)分别包含SEQ ID NO：105-107的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
(9)分别包含SEQ ID NO：105-107的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列；
(10)分别包含SEQ ID NO：110-112的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
(11)分别包含SEQ ID NO：110-112的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
(12)分别包含SEQ ID NO：110-112的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列；
(13)分别包含SEQ ID NO：115-117的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
(14)分别包含SEQ ID NO：115-117的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
(15)分别包含SEQ ID NO：115-117的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列。
结合人B7-H3的抗体或其抗原结合部分，其与前述任一方面的抗体或其抗原结合部分具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性。
另一方面，本公开还涉及一种双特异性抗体或其抗原结合片段，其包括：
结合B7-H3蛋白的第一蛋白功能区，和结合CD3的第二蛋白功能区；所述第一蛋白功能区为抗B7-H3的抗体或其抗原结合片段，其中，所述抗B7-H3的抗体包含选自氨基酸序列SEQ ID NO：115-117或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：125-127或任何变体的轻链CDR；所述第二蛋白功能区为抗CD3的抗体或其抗原结合片段，其中，所述抗CD3的抗体包含选自氨基酸序列SEQ ID NO：159-161或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：194-196或任何变体的轻链CDR。
根据前一方面的双特异性抗体或其抗原结合部分，所述第一蛋白功能区包含选自氨基酸序列SEQ ID NO：115或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO：116或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO：117或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO：125或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO：126或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO：127或其任何变体的轻链CDR3；所述第二蛋白功能区包含选自氨基酸序列SEQ ID NO：159或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO：160或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO：161或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO：194或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO：195或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO：196或其任何变体的轻链CDR3。
根据前一方面的双特异性抗体或其抗原结合部分，所述第一蛋白功能区包含选自氨基酸序列SEQ ID NO：218或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：220或其任何变体的轻链可变区；所述第二蛋白功能区包含选自氨基酸序列SEQ ID NO：222或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：224或其任何变体的轻链可变区。
根据前一方面的双特异性抗体或其抗原结合部分，所述第一蛋白功能区包含选自氨基酸序列SEQ ID NO：219、226、228或其任何变体的重链恒定区，和/或选自氨基酸序列SEQ ID NO：221或其任何变体的轻链恒定区；所述第二蛋白功能区包含选自氨基酸序列SEQ ID NO：223、227、229或其任何变体的重链恒定区，和/或选自氨基酸序列SEQ ID NO：225或其任何变体的轻链恒定区。
一种结合CD3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：138、143、148、153、158、163、168、173、178、183、222或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：188、193、198、203、208、213、224或其任何变体的轻链可变区。
结合人B7-H3的抗体或其抗原结合部分，其与前述任一方面的抗体或其抗原结合部分具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性。
编码如前述任一方面的抗体或其抗原结合部分的核酸分子，或与其具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核酸分子。
含有如前述任一方面的核酸的载体。
含有如前述任一方面的载体的细胞。
包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和药学上可接受的载体的药物组合物。
治疗癌症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物。
前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中癌症的药物中的用途。
根据前述任一方面，任选地，所述癌症是胃癌，任选地，所述抗体通过ADCC或CDC效应杀死癌细胞，任选地，所述抗体偶联其他药物，例如带标记或具有细胞毒性的缀合物。
在一方面，本公开还包括试剂盒，例如所述试剂盒包括本公开的抗体、其片段、同源物、其衍生物等，例如带标记或具有细胞毒性的缀合物，以及抗体使用说明书、杀死特定类型细胞的缀合物等等。该说明书可包括在体外、体内或离体使用抗体、缀合物等的指导。抗体可以是液体形式或固体，通常是冻干的。该试剂盒可包含其它适宜的试剂，如缓冲液、重构溶液以及为了预定用途的其它必要成分。考虑了以预定量包装好的试剂组合与用于其用途的说明书，所述用途例如用于治疗用途或用于进行诊断测定。当抗体是带标记的时，例如用酶标记的，那么该试剂盒可包括底物和酶所需的辅因子(例如提供可检测生色团或荧光团的底物前体)。此外，其它添加剂，如稳定剂、缓冲液(例如封闭缓冲液或裂解缓冲液)等也可包括在内。多种试剂的相对量可以改变而提供试剂溶液的浓缩物，这就提供了用户灵活性、节省空间、节省试剂等。这些试剂也可以干粉形式提供，通常是冻干形式，包括赋形剂，它在溶解时可提供具有适当浓度的试剂溶液。
前述任一方面的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于制备介导细胞表面B7-H3内吞的试剂中的用途。
本发明还包含多价抗体，包括双特异抗B7-H3抗体，其具有与之相连的诊断或治疗功能的效应分子、原子或其它物质。例如，抗体可具有放射性诊断标签或放射性细胞毒性原子或金属或细胞毒性物质如蓖麻毒蛋白链，与之相连用于癌症的体内诊断或治疗。
此外，本发明的抗体还可用于免疫测定、纯化方法以及其它用到免疫球蛋白或其片段的方法。此类用途在本领域为人所熟知。
相应地，本发明还提供包含本发明的抗B7-H3的抗体或其片段的组合物，所述抗体方便地和药学上可接受的载体、稀释剂或赋形剂组合，这是本领域的常规做法。
本发明所使用的术语″药物组合物″系指多种制备物的制剂。含有治疗有效量的多价抗体的制剂为无菌液体溶液、液体悬浮剂或冻干形式，任选地包含稳定剂或赋形剂。
本发明所使用的术语“癌症”系指或描述了哺乳动物尤其是人的生理状况，其典型特点是细胞无调控地生长。癌症的实例包括但不限于癌(carcinoma)、淋巴瘤、母细胞瘤、肉瘤和白血病。
本发明的抗体可以作为单独施用的组合物使用，或可与其它活性剂联合使用。
应当理解，根据所述实施方案的治疗剂将与合适的药学上可接受的载体、赋形剂、以及其它被掺入制剂中以提供改善的转移、递送、耐受性等的试剂一同施用。大量适当的制剂可见于所有药物化学工作者已知的药典中：Remington′s Pharmaceutical Sciences(第15版，Mack Publishing Company，Easton，Pa.(1975))，特别是其中Blaug、Seymour的第87章。这些制剂包括例如粉末、糊剂、膏剂、凝胶剂、蜡、油、脂质、含脂质(阳离子或阴离子)载体(例如Lipofectin TM)、DNA缀合物、无水吸浆、水包油和油包水乳液、乳液聚乙二醇(各种分子量的聚乙二醇)、半固态凝胶以及含有聚乙二醇的半固态混合物。任何前述混合物均可适用于根据本发明的治疗或疗法，条件是制剂中的活性成分不被制剂灭活并且制剂在生理学上是相容的并耐受给药途径。
在一个实施方案中，可将所述抗体用作治疗剂。此类试剂将通常用于治疗、缓解和/或预防受试者的与异常B7-H3表达、活性和/或信号传导相关的疾病或病理。可使用标准方法通过鉴定受试者，例如患有(或处于风险或发展)与异常B7-H3表达、活性和/或信号传导相关的疾病或障碍，例如癌症或其它赘生性障碍的人患者来实施治疗方案。将抗体制剂，优选对其靶抗原有高特异性和高亲和性的抗体制剂施用给受试者并且将通常因其与靶标结合而产生效应。施用的抗体可消除或抑制或妨碍靶标(例如B7-H3)的表达、活性和/或信号传导功能。施用的抗体可消除或抑制或妨碍靶标(例如B7-H3)与其所天然结合的内源性的配体结合。例如，抗体与靶标结合并调节、阻断、抑制、减少、拮抗、中和、或以其它方式妨碍B7-H3表达、活性和/或信号传导。在一些实施方案中，为治疗与异常B7-H3表达相关的疾病或障碍，可将具有重链和轻链CDR的抗体施用给受试者。在一个实施方案中，与异常B7-H3表达相关的疾病或障碍可为癌症。
作为非限制性示例，与异常B7-H3表达、活性和/或信号传导相关的疾病或障碍包括血液学癌症和/或实体瘤。血液学癌症包括例如白血病、淋巴瘤和骨髓瘤。作为非限制性示例，某些形式的白血病包括急性淋巴细胞性白血病(ALL)；急性髓性白血病(AML)；慢性淋巴细胞性白血病(CLL)；慢性髓细胞性白血病(CML)；骨髓增生性疾病/赘生物(MPDS)；和骨髓增生异常综合征。作为非限制性示例，某些形式的淋巴瘤包括霍奇金氏淋巴瘤、低度恶性和侵袭性非霍奇金氏淋巴瘤、伯基特淋巴瘤、以及滤泡性淋巴瘤(小细胞和 大细胞)。作为非限制性示例，某些形式的骨髓瘤包括多发性骨髓瘤(MM)、巨细胞性骨髓瘤、重链骨髓瘤和轻链或Bence-Jones骨髓瘤。实体瘤包括例如乳腺肿瘤、卵巢肿瘤、肺肿瘤、胰腺肿瘤、前列腺肿瘤、黑色素瘤、结直肠肿瘤、肺肿瘤、头颈肿瘤、膀胱肿瘤、食管肿瘤、肝肿瘤和肾瘤。
与癌症及其它赘生性障碍相关的症状包括例如发炎、发烧、全身不适、发烧、疼痛、经常局部发炎、食欲不振、体重减轻、浮肿、头痛、疲乏、皮疹、贫血、肌无力、肌肉疲劳和腹部症状例如腹痛、痢疾或便秘。
在另一个实施方案中，针对B7-H3的抗体可用于本领域中已知的与B7-H3定位和/或定量相关的方法(例如，用于测定适当生理样品中的B7-H3和/或B7-H3的水平，用于诊断方法，用于蛋白成像等等)。在一个给定实施方案中，对B7-H3或其衍生物、片段、类似物或同系物具有特异性的、包含源于抗体的抗原结合结构域的抗体，被用作药物学活性化合物(下文称为“治疗剂”)。
在另一个实施方案中，可通过标准技术例如免疫亲和、色谱或免疫沉淀，使用对B7-H3具有特异性的抗体来分离B7-H3多肽。针对B7-H3蛋白质的抗体(或其片段)可用于检测生物样品中的蛋白质。在一些实施方案中，在生物样品中可检测B7-H3作为临床测试过程的一部分，例如，用于确定给定治疗方案的功效。将抗体偶联(即物理连接)到可检测物省可有利于检测。可检测物质的示例包括各种酶、辅基、荧光材料、发光材料、生物发光材料和放射性材料。合适的酶的示例包括辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶；合适的辅基复合物的示例包括链霉亲和素/生物素和亲和素/生物素；合适的荧光材料的示例包括伞形酮、荧光素、异硫氰酸荧光素、罗丹明、二氯三嗪氨荧光素、丹磺酰氯或藻红蛋白；发光材料的一个示例包括鲁米诺；生物发光材料的示例包括荧光素酶、荧光素和水母蛋白，并且合适放射性材料的示例包括 125I、 131I、 35S或 3H。
在另一个实施方案中，根据本公开的抗体可用作检测样品中B7-H3或其蛋白质片段存在的试剂。在一些实施方案中，抗体包含可检测标记。抗体为多克隆抗体，或更优选单克隆抗体。使用完整的抗体或其片段(例如Fab、scFv或F(ab′) 2)。关于抗体的术语“标记”旨在包括通过将可检测物质偶联(即物理连接)到该抗体来直接标记该抗体，以及通过与直接标记的另一种试剂反应来间接标记该抗体。间接标记的示例包括使用荧光标记的第二抗体检测第一抗体，以及用生物素进行末端标记抗体，以便能够用荧光标记的链霉亲和素进行检测。术语“生物样品”旨在包括从受试者分离的组织、细胞和生物学流体，以及受试者体内存在的组织、细胞和流体。因此，使用的术语“生物样品”包括血液和血液中的级分或组分，包括血清、血浆、或淋巴液。换言之，所述实施方案的检测方法可用于在体外及体内检测生物样品中的分析物mRNA、蛋白质或基因组DNA。例如，分析物mRNA体外检测技术包括Norhtern杂交和原位杂交。分析物蛋白质体外检测技术包括酶联免疫吸附测定(ELISA)、Western印迹、免疫沉淀、以及免疫荧光。分析物基因组DNA体外检测技术包括Southern杂交。用于进行免疫测定的过程描述于例如“ELISA：Theory and Practice：Methods in Molecular Biology”，第42卷，J.R.Crowther(编辑)Human Press，Totowa，N.J.，1995；“Immunoassay”，E.Diamandis和T.Christopoulus，Academic Press，Inc.，San Diego，Calif.，1996；以及“Practice and Theory of Enzyme Immunoassays”，P.Tijssen，Elsevier Science Publishers，Amsterdam，1985。此外，分析物蛋白质的体内检测技术包括向受试者体内导入标记的抗分析物蛋白抗体。例如，可以用放射性标记标记抗体，然后可以通过标准成像技术检测受试者体内该放射性标记物的存在和位置。
可将本文所述抗体和其衍生物、片段、类似物和同系物掺入适于施用的药物组合物中。制备此类组合物所涉及的原理和考虑事项以及选择组分的指南在本领域中是熟知的，例如参见Remington′s Pharmaceutical Sciences：The Science And Practice Of Pharmacy第19版(Alfonso R.Gennaro等人编辑)Mack Pub.Co.，Easton，Pa.：1995；Drug Absorption Enhancement：Concepts，Possibilities，Limitations，And Trends，Harwood Academic Publishers，Langhorne，Pa.，1994；以及Peptide And Protein Drug Delivery(Advances In Parenteral Sciences，第4卷)，1991，M.Dekker，New York。
此类组合物通常包含抗体和药学上可接受的载体。当使用抗体片段时，与靶蛋白结合结构域特异性结合的最小抑制片段可为优选的。例如，基于抗体的可变区序列，可以设计保留结合靶蛋白质序列能力的肽分子。此类肽可化学合成和/或通过重组DNA技术产生(参见例如Marasco等人，Proc.Natl.Acad.Sci.USA，90：7889-7893(1993))。
如本文所用，术语“药学上可接受的载体”旨在包括与药物给药相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延缓剂等。合适的药学上可接受的载体描述于最新版的Remington′s Pharmaceutical Sciences中，这是本领域的标准参考书目，其以引用方式并入本文。此类载体或稀释剂的优选示例包括但不限于水、盐水、林格氏溶液、葡萄糖溶液和5％的人血清白蛋白。也可以使用脂质体和非水性载体，例如固定化油。将此类介质和试剂用于药物活性物质是本领域熟知的。除去任何常规的介质或试剂与抗体不相容之外，设想其在组合物中的用途。
将所述实施方案的药物组合物配制成与其预期施用途径相容。给药途径的示例包括肠胃外，例如静脉内、皮内、皮下、经口(例如吸入)、经皮(即局部的)、经粘膜和直肠给药。用于肠胃外、皮内或皮下施用的溶液或悬浮液可包括以下组分：注射用无菌稀释剂例如水、盐溶液、固定油、聚乙二醇类、甘油、丙二醇或其它合成溶剂；抗细菌剂，例如苄醇或对羟基苯甲酸甲酯；抗氧化剂，例如抗坏血酸或亚硫酸氢钠；螯合剂，例如 乙二胺四乙酸(EDTA)；缓冲剂，例如乙酸盐、柠檬酸盐或磷酸盐、以及调节渗透压的试剂，例如氯化钠或右旋糖。pH可用酸或碱进行调节，例如盐酸或氢氧化钠。可将肠胃外制剂包装在安瓿、一次性注射器或玻璃或塑料制多剂量小瓶内。
适于注射用途的药物组合物包括无菌水性溶液(在此是水溶性的)或分散体以及用于即时制备无菌注射液或分散体的无菌粉末。对于静脉内施用，合适的药学上可接受的载体包括生理盐水、抑菌水、Cremophor EL TM(BASF，Parsippany，N.J.)或磷酸盐缓冲盐水(PBS)。在所有情况下，组合物必须是无菌的并且应当为流动性达到易于注射的程度。其在制造和储存条件下必须是稳定的并且必须能防止微生物例如细菌和真菌的污染作用。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)的溶剂或分散介质，及其适宜的混合物。例如通过利用涂层例如卵磷脂，在分散体情况下维持所需颗粒尺寸，以及利用表面活性剂，可以保持适宜的流动性。对微生物作用的防止可以通过各种抗细菌剂和抗真菌剂例如对羟基苯甲酸酯、氯代丁醇、苯酚、抗坏血酸、硫柳汞等来实现。在许多情况下，将优选在组合物中包含等渗剂，例如糖、多元醇(诸如甘露糖醇、山梨醇)、氯化钠。注射用组合物的延长吸收可通过在所述组合物中包含延缓吸收的试剂例如单硬脂酸铝和明胶来达到。
根据需要，可以通过将抗体以所需量掺入具有上文所列成分中的一种或组合(按需要)的合适溶剂中来制备无菌注射溶液，然后过滤消毒。一般来讲，通过将抗体掺入含有碱性分散介质和上文所列那些中的所需其它成分的无菌载体中来制备分散体。就用于制备无菌注射溶液的无菌粉末而言，制备方法是获得粉末的真空干燥和冷冻干燥，该粉末包含活性成分和任何另外的期望成分，它们来自前述的这些成分的无菌过滤溶液。
对于吸入给药，从包含合适推进剂如二氧化碳等气体的加压容器或分配器或者喷雾器以气溶胶喷雾形式递送化合物。
还可以通过经粘膜或透皮方式全身给药。对于经粘膜或透皮给药，在制剂中使用适于渗透屏障的渗透剂。此类渗透剂通常在本领域是通常所知的，并且包括如用于经粘膜给药的去污剂、胆盐和夫西地酸衍生物。经粘膜给药可以通过使用喷鼻剂或栓剂来实现。对于透皮给药，可将一种或多种所述抗体配制成如本领域通常所知的膏剂、软膏、凝胶、或霜膏。
还可将化合物以栓剂(例如，具有常规栓剂基质，如可可脂或其它甘油酯)或滞留性灌肠剂形式进行制备以用于经直肠递送。
在一个实施方案中，所述抗体可用防止其不被身体迅速消除的载体制备，例如缓释/控释制剂，包括植入体和微胶囊化递送体系。可使用可生物降解、可生物相容的聚合物，例如乙烯-乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。用于制备此类制剂的方法对于本领域技术人员而言是显而易见的。
尤其有利的是以剂量单位形式配制肠胃外组合物以易于施用和剂量的一致性。如本文所用，剂量单位形式是指用于待治疗的受试者，适合作为单位剂量的物理上可分离的单位；每个单位含有经计算与所需药物载体结合产生期望治疗效果的预定量的一种或多种所述抗体。所述实施方案的剂量单位形式的规格由以下指示并直接取决于：抗体的独特特征和待实现的具体治疗效果，和用于治疗个体的此类抗体的调配领域中固有的局限性。
所述药物组合物可与给药说明书一起放于容器、包装、或分配器中。
本文所述制剂还可根据要治疗的具体情况而包含多于一种所述抗体，优选具有互补活性但对彼此无负面影响的那些。另选地或除此之外，组合物可例如包含增强其功能的试剂，诸如细胞毒素试剂、细胞因子、化学治疗剂、或生长抑制剂。此类分子以对预期目的有效的量适当地联合存在。例如，可以在试剂盒中联合存在，也可以在使用中联合存在。
在一个实施方案中，一种或多种所述抗体可在联合治疗中施用，即与其它试剂例如治疗剂(其可用于治疗病理学病症或障碍，例如各种形式的癌症、自身免疫性障碍和炎性疾病)联合。术语“联合”在本文中是指将试剂基本上同步地，同时地或顺次地给予。如果顺次给予，则在开始施用第二种化合物时，两种化合物中的第一种仍优选在治疗位点处以有效浓度被检测到。在一种情况下，“联合”也可以是在试剂盒中同时包含本发明的抗体和其他治疗剂。
例如，联合治疗可包含本文所述一种或多种抗体与一种或多种附加治疗剂(例如一种或多种细胞因子和生长因子抑制剂、免疫抑制剂、抗炎剂、代谢抑制剂、酶抑制剂、和/或细胞毒素或细胞生长抑制剂，如下更详述的)共同配制和/或共同施用。此类联合治疗可有利地利用较低剂量的施用的治疗剂，因而避免了与各种单一疗法相关的可能毒性或并发症。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本发明的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
实施例
实施例1、B7-H3重组蛋白的制备
以含有人B7-H3基因cDNA序列(SEQ ID NO：1)的质粒pMD18-B7-H3(Sino Biological Inc.)为模板，利用正向引物5’-CTGAGAGGTGCCAGATGTATGCTGCGTCGGCGGGGC-3’，反向引物5’- TCCGCCTCCGCCGCTAGCTGTCATAGGCTGCCCTGTG-3’，PCR扩增人B7-H3胞外区片段(Met1-Thr461)，扩增片段经BspQI酶切后，克隆到真核表达质粒系统(pSect)中。以此质粒转染HEK293E细胞，6天后，收集培养基上清液，通过镍柱亲和层析纯化得到人B7-H3胞外区重组蛋白(SEQ ID NO：2，结果如图1所示)。
实施例2、表达人B7-H3全长或IgC结构域的细胞株
B7-H3在肿瘤细胞系中广泛高表达，常见细胞系HEK293细胞也高度表达B7-H3(Shi J，2016)，因此在初步筛选中，可交替运用B7-H3重组蛋白和HEK293细胞。为进一步确定候选抗体结合特异性，将B7-H3全长(B7-H3-4Ig)或IgC(B7-H3-IgC)构建真核细胞表达质粒，瞬时转染CHO细胞。
提前16-24小时，将CHO-k1细胞铺入10cm细胞培养皿中，使转染时汇合度达90％左右，收集5×10 6个细胞，PBS洗3遍后重悬至Opti-MEM培养基中，电转仪电压设置为300V，电容950μF，将构建的含人B7-H3全长序列(SEQ ID NO：1)、B7-H3 IgC序列(SEQ ID NO：3)的真核表达质粒电转至细胞中，转染后细胞接种于完全培养基中，转染48小时后检测细胞表面B7-H3表达(检测结果如图2所示)。
实施例3、抗人B7-H3单克隆抗体的制备
1)免疫小鼠
将400μL浓度为1mg/mL的人B7-H3重组蛋白作为抗原与等体积免疫佐剂(Titermax佐剂，Sigma-Aldrich)混合，取6只6周龄雌性Balb/c小鼠(北京维通利华)进行皮下免疫。在初次免疫以后，每两周进行一次相同剂量的加强免疫。
2)细胞融合与杂交瘤细胞筛选
在融合前三天用10μg/100μL/只人B7-H3重组蛋白，进行尾静脉冲击免疫。三天后取小鼠脾脏、淋巴结，在DMEM中研磨后得到B细胞悬浮液，取适量的B细胞混悬液和SP2/0混合后，采用电融合仪进行细胞融合。将融合后的细胞在含有HAT的DMEM完全培养基中置于5％CO 2，37℃条件下培养。
实施例4、抗人B7-H3抗体的筛选和基因克隆
1)杂交瘤细胞酶联免疫吸附实验(ELISA)筛选
在96孔酶标板上包被1μg/mL的人B7-H3重组蛋白，4℃过夜。以含有2％脱脂牛奶的PBS封闭后，加入单克隆杂交瘤细胞培养上清，室温孵育60分钟；用PBST和PBS各洗3遍后，加入100μL/孔HRP标记的抗小鼠IgG Fc二抗，室温孵育60分钟。用PBST和PBS各洗三遍后，加入100μL TMB底物37℃显色10分钟后，以50μL 2M硫酸溶液终止反应，并在450nm波长处读取吸光度。筛选出156个分泌的抗体可结合B7-H3(部分结果如图3所示，所有筛选结果如表1所示)。
2)细胞筛选抗体B7-H3特异性抗体(FACS)
在96孔U型板每孔中加入1x10 5B7-H3 +HEK293细胞，取50μL杂交瘤上清加入孔中，与细胞混合孵育1小时，用0.5％BSA洗2次，加50μL/孔Alexa Fluro647标记的羊抗鼠IgG Fc二抗，4℃孵育45分钟。用0.5％BSA洗2次后，加入PBS重悬细胞，上流式细胞仪检测。结果显示，从ELISA筛选得到的阳性克隆中，6E4、14A7、15C11、21H2、24A11、31F3、41C10、42B5和53E9以高亲和力结合HEK293细胞(结果如图4所示，同型对照抗体以阴影表示，实线为杂交瘤上清；所有杂交瘤筛选结果总结如表1所示)。
表1、杂交瘤上清与人B7-H3蛋白和稳转细胞的结合
Figure PCTCN2020107351-appb-000001
3)杂交瘤抗体基因的克隆
将阳性单克隆细胞1000rpm离心，收集细胞，并以Trizol提取总RNA，以此为模板，合成第一链cDNA后，以第一链cDNA为后续模板扩增杂交瘤细胞所对应的可变区DNA序列。在50μL反应体系中，分别加入cDNA1μL，10×PCR缓冲液5μL，正向及反向引物各1μL，dNTP 1μL，25mmol MgCl 2 1μL，H 2O 39μL，Taq酶1μL，95℃预变性10分钟，进入温度循环，进行PCR扩增。反应条件为94℃变性1分钟，58℃退火1分钟，72℃延伸15秒，共30个循环，然后72℃保温10分钟。经测序后，得到的候选阳性克隆重链和轻链可变区序列分别为：
Figure PCTCN2020107351-appb-000002
Figure PCTCN2020107351-appb-000003
Figure PCTCN2020107351-appb-000004
Figure PCTCN2020107351-appb-000005
Figure PCTCN2020107351-appb-000006
4)抗B7-H3嵌合抗体的表达
加上述重链和轻链可变区序列片段PCR扩增，将重链可变区克隆入含有人重链恒定区的载体，以在哺乳动物细胞中表达完整的IgG1重链。类似地，将轻链可变区克隆入含有人轻链恒定区的载体，以在哺乳动物细胞中表达完整的kappa轻链。经测序正确后转染入HEK293.6E哺乳动物细胞中，IgG1经表达分泌入培养基中，合并收集上清，过滤后纯化。采用ProteinA层析纯化IgG，将洗脱蛋白超滤浓缩，通过分光光度法测定IgG的浓度，采用SDS-PAGE分析IgG的纯度。
实施例5、嵌合抗体对靶细胞的ADCC杀伤活性实验
将抗体分别用RPMI-1640培养基稀释至1μg/mL，10倍稀释共5个梯度。收集NK92MI-CD16a细胞，300g离心10分钟，PBS洗一遍，离心后重悬至RPMI-1640完全培养基中，至细胞密度为2×10 5/mL。收集Calu-6细胞，300g离心10分钟，PBS洗一遍，离心后重悬至RPMI-1640完全培养基中，至细胞密度为1×10 6/mL。将NK92MI-CD16a细胞加入96孔U型板，50μL/孔，加入稀释后的抗体50μL/孔，37℃，5％CO 2培养箱培养30分钟。再将Calu-6细胞加入96孔U型板，50μL/孔，1000rpm离心1分钟后，再次置于37℃，5％CO 2培养箱培养24小时。在检测前30分钟，向Calu-6细胞最大释放孔加入2μL细胞裂解液，放回培养箱继续培养。1000rpm离心3分钟，取50μL上清至黑色酶标板，50μL/孔加入LDH检测底物(提前溶解放置室温)，轻轻震荡混匀，10分钟后加入终止缓冲液25μL/孔，震荡10秒，选择发射波长560nm，吸收波长590nm检测荧光值，根据荧光值计算相应的杀伤率。图5显示嵌合抗体对B7-H3阳性的HEK293细胞的杀伤效果明显，ADCC杀伤具有抗体浓度依赖。图6显示嵌合抗体对B7-H3阳性的Calu-6细胞同样具有抗体浓度依赖的ADCC杀伤效果。杀伤率(％)＝(实验孔-靶细胞自发孔)/(靶细胞最大释放孔-靶细胞自发孔)×100％
实验孔(含靶细胞悬液、效应细胞悬液、待测样品)
靶细胞自发孔(含靶细胞悬液、RPMI 1640培养液)
靶细胞最大释放孔(含靶细胞悬液、RPMI 1640培养液、细胞裂解液)
实施例6、抗人B7-H3抗体的结合表位
取瞬时转染的CHO-B7-H3-4Ig细胞、CHO-B7-H3-IgC细胞和阴性对照CHO细胞，以5×10 4细胞/100μL加入U型96孔板，1100rpm离心3分钟，弃上清，轻轻拍散细胞，每孔加入50μL抗体，4℃孵育1小时。孵育结束后，每孔加入180μL 0.5％BSA洗3次，加30μL/孔二抗AlexaFluro647抗人IgG(Jackson ImmunoResearch，货号：109-606-170)，4℃孵育40分钟。孵育结束后，每孔加入180μL 0.5％BSA洗3次，最后每孔重悬于50μL PBS进行流式细胞仪(iQue Screener)检测(结果如图7所示)。图7显示各抗体均可与CHO-B7-H3-4Ig细胞特异性结合，但其中仅单克隆抗体6E4、15C11和24A11抗体可与CHO-B7-H3-IgC细胞特异性结合，提示相对其他单克隆抗体，6E4、15C11和24A11结合更靠近细胞膜的B7-H3 IgC结构域。
实施例7、Anti-B7-H3抗体的人源化
首先把鼠抗体15C11可变区序列与人抗体序列进行比对，找出适合的同源性高的人胚系基因序列进行抗体决定簇CDR移植：mAb15C11鼠源抗体轻链为小鼠IMGT_mVK_6_14，选择与其框架区同源性最高的人IMGT_hVK_4_59进行CDR移植，FM4选用同源性最高的人IGKJ2*01；鼠源抗体重链为IGHV1-76，选择人胚 系基因IMGT_hVH_1_18进行CDR移植，FM4选用同源性最高的人IGHJ6*01。随后同时利用计算机进行同源建模，分析CDR区及其周边的框架氨基酸序列，避免选择的人胚系基因造成分子表面电荷或疏水区域集中分布；同时，通过计算静电力、范德华力、亲疏水性和熵值，分析各阳性单克隆抗体基因序列中可能与B7-H3作用以及维护空间构架的关键氨基酸个体，在此基础上设计回复突变位点。
设计共得到不同的重链变体和轻链变体，将轻、重链分别进行全序列合成后，克隆到含有抗体kappa链恒定区Ckappa或人IgG1恒定区CH1-CH3的真核表达载体，将轻、重链质粒进行组合配对后，转染HEK293.6E细胞，37摄氏度表达5-6天，收取培养上清，通过Protein A柱进行纯化。
人源化抗体重链/轻链可变区序列如下：
Figure PCTCN2020107351-appb-000007
Figure PCTCN2020107351-appb-000008
实施例8、Anti-B7-H3人源化抗体的生物学活性检测
1)人源化抗体的亲和力测定
96孔酶标板包被人B7-H3或食蟹猴B7-H3，37摄氏度恒温孵育60分钟。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有2％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗3次后100μL每孔加入不同稀释倍数的抗体，37摄氏度孵育1小时，经洗涤缓冲液冲洗三次后，用洗涤缓冲液以1∶5000倍稀释HPR标记的goat anti-human IgG Fc，室温孵育1小时，经洗涤缓冲液冲洗3次后，加入100μL TMB底物溶液显色，37摄氏度反应10分钟后，以50μL 2M的硫酸溶液终止反应并在450nm处读出吸光度。图8显示部分人源化变体仍保留与B7-H3重组蛋白的高亲和力，图9显示人源化变体h15C11-VH5/Vk2与嵌合抗体6E4及母本15C11亲和力相当，可以同时以高亲和力结合人和食蟹猴B7-H3重组蛋白(表2)。
表2、人源化B7-H3单克隆抗体的亲和力
EC50(nM) 人B7-H3 食蟹猴B7-H3
6E4 0.389 0.881
15C11 0.287 0.325
h15C11-VH5/Vk2 0.215 0.229
2)抗体与细胞表面B7-H3的结合
取对数生长期的HEK293细胞，3％BSA封闭30分钟以5×10 4细胞/100μL铺U型96孔板，1100rpm离心3分钟，弃上清，轻轻拍散细胞，每孔加入50μL梯度稀释的抗体(抗体浓度从30μg/mL起，3倍稀释5个梯度)，4℃孵育1小时。孵育结束后，每孔加入180μL 0.5％BSA洗3次，加30μL/孔二抗AlexaFluro647抗人IgG(Jackson ImmunoResearch，货号：109-606-170)，4℃孵育45分钟。孵育结束后，每孔加入180μL 0.5％BSA洗3次，最后每孔重悬于50μL PBS进行iQue(Intellicyt，USA)检测，结果如图10所示，抗B7-H3人源化变体h15C11-VH5/Vk1和h15C11-VH5/Vk2保留了嵌合抗体15C11与HEK293细胞的亲和力。
3)Anti-B7-H3人源化抗体对靶细胞的ADCC杀伤活性
将抗体分别用RPMI-1640培养基稀释至1μg/mL，10倍稀释共5个梯度。收集NK92MI-CD16a细胞，300g离心10分钟，PBS洗一遍，离心后重悬至RPMI-1640完全培养基中，至细胞密度为2×10 5/mL。收集Calu-6细胞，300g离心10分钟，PBS洗一遍，离心后重悬至RPMI-1640完全培养基中，至细胞密度为1×10 6/mL。将NK92MI-CD16a细胞加入96孔U型板，50μL/孔，加入稀释后的抗体50μL/孔，37℃，5％CO 2培养箱培养30分钟。再将Calu-6细胞加入96孔U型板，50μL/孔，1000rpm离心1分钟后，再次置于37℃，5％CO 2培养箱培养24小时。在检测前30分钟，向Calu-6细胞最大释放孔加入2μL细胞裂解液，放回培养箱继续培养。1000rpm离心3分钟，取50μL上清至黑色酶标板，50μL/孔加入LDH检测底物(提前溶解放置室温)，轻轻震荡混匀，10分钟后加入终止缓冲液25μL/孔，震荡10秒，选择发射波长560nm，吸收波长590nm检测荧光值。根据荧光值计算相应的杀伤率。(结果如图11所示)。杀伤率(％)＝(实验孔-靶细胞自发孔)/(靶细胞最大释放孔-靶细胞自发孔)×100％
实验孔(含靶细胞悬液、效应细胞悬液、待测样品)
靶细胞自发孔(含靶细胞悬液、RPMI 1640培养液)
靶细胞最大释放孔(含靶细胞悬液、RPMI 1640培养液、细胞裂解液)
图11显示15C11人源化变体与母本15C11相比，保留对B7-H3 +HEK293细胞的ADCC活性。
实施例9、CD3抗体的人源化
鼠源杂交瘤CD3抗体(EMBO J.1985.4(2)：337-344；J.Immunol.1986，137(4)：1097-100；J.Exp.Med.1991，174：319-326；J.Immunol.1991，147(9)：3047-52)识别HEK293细胞中重组表达的人CD3εγ蛋白(SEQ ID NO：134)，同时也识别食蟹猴CD3εγ重组蛋白(SEQ ID NO：135)，其序列如下：
抗CD3鼠单抗Mab01轻链氨基酸序列(SEQ ID NO：136)：
Figure PCTCN2020107351-appb-000009
抗CD3鼠单抗Mab01重链氨基酸序列(SEQ ID NO：137)：
Figure PCTCN2020107351-appb-000010
选择与抗CD3鼠单抗轻链框架区同源性最高的人IMGT_hVL7-43进行轻链CDR移植，FM4选用同源性最高的人IGLJ3*02；选择人胚系基因IMGT_hVH3-73进行重链CDR移植，FM4选用同源性最高的人IGHJ4*01。随后同时利用计算机进行同源建模，分析CDR区及其周边的框架氨基酸序列，在此基础上设计回复突变位点。
设计共得到不同的重链变体和轻链变体(表3)，将轻、重链变体分别进行全序列合成后，分别克隆到含有抗体轻链恒定区Clambda或含有人IgG4恒定区CH1-CH3的真核表达载体，制备高纯度质粒后，将轻、重链质粒进行组合配对，共转染HEK293.6E细胞，37摄氏度表达5-6天，收取培养上清，通过Protein A柱进行纯化。
抗CD3鼠单抗人源化抗体重链/轻链可变区的序列如下：
表3：抗CD3鼠单抗的人源化
Figure PCTCN2020107351-appb-000011
ELISA亲和力测定
96孔酶标板包被人CD3εγ蛋白，4摄氏度恒温孵育过夜。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有2％牛奶的PBS溶液封闭60分钟。用洗涤缓冲液洗3次后100μL每孔加入不同稀释倍数的抗体，37摄氏度孵育1小时，经洗涤缓冲液冲洗三次后，用0.5％BSA以1∶5000倍稀释HPR标记的goat anti-human IgG Fc，室温 孵育1小时，经洗涤缓冲液冲洗3次后，加入100μL TMB底物溶液显色，37摄氏度反应10分钟后，以50μL 2M的硫酸溶液终止反应并在450nm处读出吸光度(结果如图12所示)。图12显示与人CD3εγ蛋白结合的抗CD3人源化抗体组合。
细胞(Jurkat)亲和力测定
取对数生长期的Jurkat细胞，3％BSA封闭30分钟以5×10 4细胞/100μL铺U型96孔板，1100rpm离心3分钟，弃上清，轻轻拍散细胞，每孔加入50μL梯度稀释的抗体(抗体浓度从30μg/mL起，3倍稀释5个梯度)，4℃孵育1小时。孵育结束后，每孔加入180μL 0.5％BSA洗3次，加30μL/孔二抗AlexaFluro647抗人IgG(Jackson ImmunoResearch，货号：109-606-170)，4℃孵育45分钟。孵育结束后，每孔加入180μL 0.5％BSA洗3次，最后每孔重悬于50μL PBS进行iQue(Intellicyt，USA)检测(结果如图13所示)。图13显示与Jurkat细胞结合的抗CD3人源化抗体组合。
实施例10、双特异性抗体表达载体的构建
将单克隆抗体h15C11-VH5/Vk2和抗CD3人源化抗体(表3)的轻重链可变区基因分别克隆到表达载体中，4种质粒同时转染HEK293.6E细胞，6天后，收集培养基上清液，通过Protein A亲和层析纯化得到B7-H3/CD3双特异性抗体。构建了三种不同的双特异抗体，相应的序列编号见表4，双特异抗体对应的抗原决定簇序列见表5。
表4：双特异抗体序列编号
Figure PCTCN2020107351-appb-000012
表5：双特异抗体对应的抗原决定簇序列
Figure PCTCN2020107351-appb-000013
实施例11、Anti-B7-H3双特异性抗体的亲和力测定
96孔酶标板包被人B7-H3或人CD3εγ蛋白，37摄氏度恒温孵育60分钟。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有2％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗3次后100μL每孔加入不同稀释倍数的抗体，37摄氏度孵育1小时，经洗涤缓冲液冲洗三次后，用洗涤缓冲液以1∶5000倍稀释HPR标记的goat anti-human IgG Fc，室温孵育1小时，经洗涤缓冲液冲洗3次后，加入100μL TMB底物溶液显色，37摄氏度反应10分钟后，以50μL 2M的硫酸溶液终止反应并在450nm处读出吸光度(结果如图14、图15所示)。图14显示三种B7-H3/CD3双特异抗体均能与人B7-H3蛋白结合，其中构建体-1亲和力最高，构建体-4亲和力次之，构建体-7亲和力最弱。图15显示三种B7-H3/CD3双特异抗体均能与人CD3蛋白结合，其中构建体-1亲和力最高，构建体-4亲和力次之，构建体-7亲和力最弱。
实施例12、Anti-B7-H3双特异性抗体在Calu-6细胞上的T细胞介导的细胞杀伤(TDCC)
取30mL新鲜抽取血液加入至50mL离心管中，与15mL 1×PBS混和均匀，另取一个50mL离心管加入20mL Ficoll-Paque Plus，然后轻轻加入上述稀释的30mL新鲜血液铺于Ficoll-Paque Plus表面，将其置于20℃，2000rpm离心30分钟。离心结束后，小心用一次性吸管将离心管中最上层血清吸除，吸取由上至下的四层液体中的第二层白膜层(即PBMC)至50mL离心管，每管10mL。将分离取得的PBMC加入大于其3倍体积的1×PBS混匀，于4℃，1300rpm离心10分钟，将上清液吸除后，再加入10mL 1×PBS重悬计数。
将PBMC置于37℃5％CO 2培养箱中培养2小时。1300rpm离心10分钟，去上清，重悬于已预热的2.5％FBS/RPMI 1640培养基中，调整密度至2×10 6细胞/mL，100μL/孔，即2×10 5细胞/孔铺至U型96孔板中，加入50μL稀释好的Calu-6细胞或HEK293细胞，即2×10 4细胞/孔。然后加入稀释好的抗体(从40μg/mL起，10倍稀释6个梯度)50μL/孔，37℃5％CO 2培养箱培养24小时。在检测前30分钟，向靶细胞最大释放孔加入2μL裂解液(10×)，放回继续培养。1000rpm离心3分钟，取50μL上清至黑色酶标板，加入LDH检测底物50μL/ 孔，10分钟后加入终止液25μL/孔终止反应，震荡10秒，选择发射波长560nm，吸收波长590nm检测荧光值。根据荧光值计算相应的杀伤率。(结果如图16所示)。杀伤率(％)＝(实验孔-靶细胞自发孔)/(靶细胞最大释放孔-靶细胞自发孔)×100％
实验孔(含靶细胞悬液、效应细胞悬液、待测样品)
靶细胞自发孔(含靶细胞悬液、RPMI 1640培养液)
靶细胞最大释放孔(含靶细胞悬液、RPMI 1640培养液、细胞裂解液)
图16显示三种B7-H3/CD3双特异抗体均能介导TDCC效应。
同时通过流式细胞术检测双特异性抗体介导T细胞杀伤诱导CD4 +和CD8 +T细胞活化标志物CD25和CD69的剂量-反应关系(结果如图17A-17D所示)。图17显示三种B7-H3/CD3双特异抗体均能诱导CD4 +和CD8 +T细胞活化，且均有剂量-反应关系。

Claims (16)

  1. 一种结合人B7-H3蛋白的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：5-7、15-17、25-27、35-37、45-47、55-57、65-67、75-77、85-87、95-97、100-102、105-107、110-112、115-117或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：10-12、20-22、30-32、40-42、50-52、60-62、70-72、80-82、90-92、120-122、125-127、130-132或任何变体的轻链CDR。
  2. 根据权利要求1的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：5、15、25、35、45、55、65、75、85、95、100、105、110、115或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO：6、16、26、36、46、56、66、76、86、96、101、106、111、116或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO：7、17、27、37、47、57、67、77、87、97、102、107、112、117或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO：10、20、30、40、50、60、70、80、90、120、125、130或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO：11、21、31、41、51、61、71、81、91、121、126、131或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO：12、22、32、42、52、62、72、82、92、122、127、132或其任何变体的轻链CDR3。
  3. 根据权利要求1或2的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：4、14、24、34、44、54、64、74、84、94、99、104、109、114、218或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：9、19、29、39、49、59、69、79、89、119、124、129、220或其任何变体的轻链可变区。
  4. 根据前述权利要求任一项的抗体或其抗原结合部分，其包含选自下列的重链和轻链的CDR组合：
    (1)分别包含SEQ ID NO：95-97的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
    (2)分别包含SEQ ID NO：95-97的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
    (3)分别包含SEQ ID NO：95-97的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列；
    (4)分别包含SEQ ID NO：100-102的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
    (5)分别包含SEQ ID NO：100-102的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
    (6)分别包含SEQ ID NO：100-102的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列；
    (7)分别包含SEQ ID NO：105-107的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
    (8)分别包含SEQ ID NO：105-107的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
    (9)分别包含SEQ ID NO：105-107的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列；
    (10)分别包含SEQ ID NO：110-112的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
    (11)分别包含SEQ ID NO：110-112的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
    (12)分别包含SEQ ID NO：110-112的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列；
    (13)分别包含SEQ ID NO：115-117的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：120-122的轻链CDR1、CDR2及CDR3序列；
    (14)分别包含SEQ ID NO：115-117的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：125-127的轻链CDR1、CDR2及CDR3序列；
    (15)分别包含SEQ ID NO：115-117的重链CDR1、CDR2及CDR3序列，和/或分别包含SEQ ID NO：130-132的轻链CDR1、CDR2及CDR3序列。
  5. 一种结合CD3的抗体或其抗原结合部分，其包含选自氨基酸序列SEQ ID NO：138、143、148、153、158、163、168、173、178、183、222或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：188、193、198、203、208、213、224或其任何变体的轻链可变区。
  6. 一种双特异性抗体或其抗原结合片段，其包括：
    结合B7-H3蛋白的第一蛋白功能区，和
    结合CD3蛋白的第二蛋白功能区；
    其中：
    所述第一蛋白功能区为根据权利要求1-4任一项的抗B7-H3的抗体或其抗原结合片段，优选地，所述抗B7-H3的抗体包含选自氨基酸序列SEQ ID NO：115-117或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：125-127或任何变体的轻链CDR；和/或
    所述第二蛋白功能区为抗CD3的抗体或其抗原结合片段，其中，所述抗CD3的抗体包含选自氨基酸序列SEQ ID NO：159-161或其任何变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：194-196或任何变体的轻链CDR。
  7. 根据权利要求6的双特异性抗体或其抗原结合部分，所述第一蛋白功能区包含选自氨基酸序列SEQ ID NO：115或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO：116或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO：117或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO：125或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO：126或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO：127或其任何变体的轻链CDR3；和/或
    所述第二蛋白功能区包含选自氨基酸序列SEQ ID NO：159或其任何变体的重链CDR1，选自氨基酸序列SEQ ID NO：160或其任何变体的重链CDR2，选自氨基酸序列SEQ ID NO：161或其任何变体的重链CDR3；和/或选自氨基酸序列SEQ ID NO：194或其任何变体的轻链CDR1，选自氨基酸序列SEQ ID NO：195或其任何变体的轻链CDR2，选自氨基酸序列SEQ ID NO：196或其任何变体的轻链CDR3。
  8. 根据权利要求6或7的双特异性抗体或其抗原结合部分，所述第一蛋白功能区包含选自氨基酸序列SEQ ID NO：218或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：220或其任何变体的轻链可变区；和/或
    所述第二蛋白功能区包含选自氨基酸序列SEQ ID NO：138、143、148、153、158、163、168、173、178、183、222或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：188、193、198、203、208、213、224或其任何变体的轻链可变区；优选包含选自氨基酸序列SEQ ID NO：222或其任何变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：224或其任何变体的轻链可变区。
  9. 根据前述权利要求6-8任一项的双特异性抗体或其抗原结合部分，所述第一蛋白功能区包含选自氨基酸序列SEQ ID NO：219、226、228或其任何变体的重链恒定区，和/或选自氨基酸序列SEQ ID NO：221或其任何变体的轻链恒定区；
    所述第二蛋白功能区包含选自氨基酸序列SEQ ID NO：223、227、229或其任何变体的重链恒定区，和/或选自氨基酸序列SEQ ID NO：225或其任何变体的轻链恒定区。
  10. 根据前述权利要求任一项的抗体或其抗原结合部分，所述抗体或其抗原结合部分是人源化的。
  11. 编码根据前述权利要求任一项的抗体或其抗原结合部分的核酸分子，或与其具有至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核酸分子，任选地，所述核酸分子包含选自SEQ ID NO：8、18、28、38、48、58、68、78、88、98、103、108、113、118或其任何变体的抗体重链核酸序列，和/或选自SEQ ID NO：13、23、33、43、53、63、73、83、93、123、128、133或其任何变体的抗体轻链核酸序列。
  12. 含有权利要求11的核酸的载体。
  13. 含有权利要求12的载体的细胞或试剂盒。
  14. 含有前述权利要求1-10任一项的抗体或其抗原结合部分，或权利要求11的核酸，权利要求12的载体，或权利要求13的细胞或试剂盒的药物组合物。
  15. 前述权利要求1-10任一项的抗体或其抗原结合部分，或权利要求11的核酸，权利要求12的载体，或权利要求13的细胞或试剂盒，或权利要求14的药物组合物在治疗哺乳动物中癌症的用途，任选地，所述癌症是胃癌，任选地，所述抗体通过ADCC或CDC效应杀死癌细胞，任选地，所述抗体偶联其他细胞毒性药物。
  16. 前述权利要求1-10任一项的抗体或其抗原结合部分，或权利要求11的核酸，权利要求12的载体，或权利要求13的细胞或试剂盒，或权利要求14的药物组合物在制备用于治疗哺乳动物中癌症的药物或试剂盒中的用途，任选地，所述癌症是胃癌，任选地，所述抗体通过ADCC或CDC效应杀死癌细胞，任选地，所述抗体偶联其他细胞毒性药物。
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