WO2019024911A1 - B7h3抗体-药物偶联物及其医药用途 - Google Patents

B7h3抗体-药物偶联物及其医药用途 Download PDF

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WO2019024911A1
WO2019024911A1 PCT/CN2018/098480 CN2018098480W WO2019024911A1 WO 2019024911 A1 WO2019024911 A1 WO 2019024911A1 CN 2018098480 W CN2018098480 W CN 2018098480W WO 2019024911 A1 WO2019024911 A1 WO 2019024911A1
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antibody
seq
cancer
chain variable
variable region
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French (fr)
Inventor
顾津明
叶鑫
杨柳青
梁金栋
蒋贵阳
陶维康
张连山
应华
张玲
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Priority to CN201880004425.6A priority Critical patent/CN109963591B/zh
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to a B7H3 antibody-drug conjugate and its use in medicine, further, the present invention relates to a B7H3 antibody-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and the aforementioned coupling Or a pharmaceutical composition thereof, or a pharmaceutically acceptable salt or solvate thereof, and a use thereof for the manufacture of a medicament for the treatment of a B7H3-mediated disease or condition; especially for the preparation of an anticancer drug.
  • the T cell-mediated immune response plays an extremely important role in the body's anti-tumor process, and the activation and proliferation of T cells requires not only the antigen signal recognized by the TCR, but also the second signal provided by the costimulatory molecule.
  • the B7 family of molecules belongs to the co-stimulatory molecule immunoglobulin superfamily. More and more studies have shown that this family of molecules plays an important regulatory role in the normal immune function and pathological state of the body.
  • B7H3 is a member of the B7 family and belongs to the type I transmembrane protein, which contains a signal peptide at the amino terminus, an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region and a Cytoplasmic tail region containing 45 amino acids (Tissue Antigens. 2007 Aug; 70(2): 96-104).
  • IgV immunoglobulin-like variable region
  • IgC constant region
  • B7H3a and B7H3b in B7H3.
  • the extracellular domain of B7H3a is composed of two immunoglobulin domains of IgV-IgC, also known as 2IgB7H3, and the extracellular domain of B7H3b is composed of four immunoglobulin domains of IgV-IgC-IgV-IgC, also known as 4IgB7H3.
  • B7H3 protein is not expressed or expressed in normal tissues and cells, but is highly expressed in various tumor tissues, and is closely related to tumor progression, patient survival and prognosis. It has been reported clinically that B7H3 is among many cancer types, especially in non-small cell lung cancer, kidney cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. Overexpression (Lung Cancer. 2009 Nov; 66(2): 245-249; Clin Cancer Res. 2008 Aug 15; 14(16): 5150-5157).
  • B7H3 is considered a new tumor marker and potential therapeutic target
  • Phage display technology is the fusion of a foreign protein or polypeptide with a phage coat protein to express a foreign protein on the surface of the phage.
  • the phage antibody library is an antibody library established by combining phage display technology, PCR amplification technology and protein expression technology using a comprehensive technical means.
  • the biggest advantage of the phage antibody library is the preparation of fully humanized antibodies by three processes that mimic in vivo immunization without in vivo immunization.
  • the phage antibody library has the following advantages: 1 to achieve the unification of genotype and phenotype.
  • the experimental method is simple and rapid, and the traditional antibody production method by hybridoma technology takes several months, and the antibody library technology takes only a few weeks. 2
  • the expression is a fully human antibody, and the molecular weight is small, mainly expressed in the form of active fragments Fab, scFV, and has obvious advantages in tissue penetrating power compared with intact antibodies.
  • Antibody-conjugates link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds, making full use of the specificity of antibodies for normal cell and tumor cell surface antigen binding.
  • ADCs Antibody-conjugates
  • ADC drugs have been used in clinical or clinical studies, such as Kadcyla, which is an ADC drug that targets Her2's trastuzumab and DM1.
  • Kadcyla is an ADC drug that targets Her2's trastuzumab and DM1.
  • patent reports targeting antibodies against B7H3 and ADC drugs such as WO2008100934, WO2012147713, WO2014061277, WO2015184203, WO2016044383.
  • WO2008100934 targeting antibodies against B7H3 and ADC drugs
  • WO2012147713, WO2014061277, WO2015184203, WO2016044383 there are still no ADC drugs for B7H3 targets listed for clinical treatment research. Therefore, the development of new B7H3 target ADC drugs has broad prospects.
  • a therapeutic agent using the antibody ADC drug as an active ingredient is provided by screening a highly active and highly stable anti-human B7H3 fully human antibody ADC drug.
  • the present invention provides an antibody-drug conjugate represented by the formula (A) or a pharmaceutically acceptable salt or solvent compound thereof,
  • D is a cytotoxic drug
  • L 1 , L 2 are joint units
  • y is a number selected from 1-8, preferably a number selected from 2-4; y may be a decimal or an integer;
  • Ab is a B7H3 antibody or antigen-binding fragment thereof, which binds to human B7H3, which is a monoclonal antibody or antigen-binding fragment thereof selected from any one of the following (a) to (c):
  • Antibody heavy chain variable region HCDR region sequences as shown in SEQ ID NOs: 10, 11 and 12 amino acid sequences; and antibody light chain variable region LCDR region sequences: as shown in SEQ ID NOs: 13, 14 and 15 amino acid sequences ;
  • Antibody heavy chain variable region HCDR region sequences as shown in SEQ ID NOs: 16, 17 and 18 amino acid sequences; and antibody light chain variable region LCDR region sequences: as shown in SEQ ID NOs: 19, 20 and 21 amino acid sequences ;
  • Antibody heavy chain variable region HCDR region sequences as shown in SEQ ID NO: 30, 31 and 32 amino acid sequences; and antibody light chain variable region LCDR region sequences: as shown in SEQ ID NOs: 33, 34 and 35 amino acid sequences .
  • a monoclonal antibody comprising HCDR1, HCDR2, HCDR3 of the heavy chain variable region of the antibody as shown in the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively; and amino acid sequences of SEQ ID NOs: 13, 14 and 15 LCDR1, LCDR2, LCDR3 of the light chain variable region of the indicated antibody;
  • a monoclonal antibody comprising HCDR1, HCDR2, HCDR3 of the heavy chain variable region of the antibody shown in the amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively; and amino acid sequences of SEQ ID NOs: 19, 20 and 21 LCDR1, LCDR2, LCDR3 of the light chain variable region of the indicated antibody;
  • the antibody-drug conjugate of the formula (A) or a pharmaceutically acceptable salt or solvent compound thereof is provided.
  • D is a cytotoxic drug
  • L 1 , L 2 are joint units
  • y is a number selected from 1-8, preferably a number selected from 2-4;
  • Ab is a monoclonal antibody or antigen-binding fragment thereof that competes for binding to human B7H3 with a B7H3 antibody or antigen-binding fragment thereof as defined above.
  • the antibody-drug conjugate of the formula (A), wherein the Ab is a recombinant antibody is a recombinant antibody.
  • the antibody-drug conjugate of the formula (A), wherein the light and heavy chain FR region sequences on the light and heavy chain variable regions of the Ab are derived from Human germline light and heavy chain sequences or mutant sequences thereof.
  • the antibody-drug conjugate of the formula (A), wherein the Ab comprises a monoclonal antibody or antigen thereof selected from any one of the following (1) to (7) Combine fragments:
  • a monoclonal antibody comprising the antibody heavy chain variable region of SEQ ID NO: 6; and the antibody light chain variable region of SEQ ID NO: 7;
  • a monoclonal antibody comprising the antibody heavy chain variable region of SEQ ID NO: 36; and the antibody light chain variable region of SEQ ID NO: 38;
  • a monoclonal antibody comprising the antibody heavy chain variable region of SEQ ID NO: 36; and the antibody light chain variable region of SEQ ID NO: 39;
  • a monoclonal antibody comprising the antibody heavy chain variable region of SEQ ID NO: 37; and the antibody light chain variable region of SEQ ID NO: 38;
  • a monoclonal antibody comprising the antibody heavy chain variable region of SEQ ID NO: 37; and the antibody light chain variable region of SEQ ID NO: 39.
  • An h1702 antibody which is a full-length antibody consisting of the heavy chain sequence of SEQ ID NO: 22 and the light chain sequence of SEQ ID NO: 23,
  • An h1703 antibody which is a full-length antibody consisting of the heavy chain sequence of SEQ ID NO: 24 and the light chain sequence of SEQ ID NO: 25,
  • An h1702-DS antibody which is a full-length antibody consisting of the heavy chain sequence of SEQ ID NO: 22 and the light chain sequence of SEQ ID NO:
  • h1704-3 antibody which is a full length antibody consisting of the heavy chain sequence shown by SEQ ID NO: 40 and the light chain sequence shown by SEQ ID NO:41.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2, single chain antibody (scFv) , a dimerized V region (diabody), a disulfide-stabilized V region (dsFv), and an antigen-binding fragment of a CDR-containing peptide.
  • the antibody-drug conjugate of the formula (A) wherein the cytotoxic drug is selected from the group consisting of a toxin, a chemotherapeutic drug, an antibiotic, a radioisotope, and a nucleolase.
  • the antibody-drug conjugate of the formula (A) wherein the cytotoxic drug is selected from the group consisting of DM1, DM3, DM4, MMAF and MMAE.
  • the antibody-drug conjugate of the formula (A) wherein the cytotoxic drug is selected from the group consisting of:
  • the antibody-drug conjugate of the formula (A) is a compound of the formula I or a pharmaceutically acceptable salt or solvent compound thereof,
  • L 1 , L 2 are joint units
  • y is a number selected from 1-8, preferably a number selected from 2-4;
  • Ab is a B7H3 antibody or antigen-binding fragment thereof as defined above, or a monoclonal antibody or antigen-binding fragment thereof that competes for binding to human B7H3 with a B7H3 antibody or antigen-binding fragment thereof as defined above.
  • X 1 is selected from a hydrogen atom, a halogen, a hydroxyl group, a cyano group, an alkyl group, an alkoxy group, and a cycloalkyl group;
  • X 2 is selected from the group consisting of alkyl, cycloalkyl and heterocyclic;
  • n is an integer selected from 0 to 5, preferably 1, 2 or 3; and S is a sulfur atom.
  • X 3 is an alkyl group, and the alkyl group optionally further is taken from a substituent selected from the group consisting of halogen, hydroxyl and cyano
  • n is an integer selected from 0 to 5, preferably 1, 2 or 3.
  • the antibody-drug conjugate of the formula (A) wherein the cytotoxic drug is linked to the linker L1 provides the compound:
  • the antibody-drug conjugate of the formula (A) is an antibody-drug conjugate of the formula (II):
  • L 2 is a linker unit
  • y is a number selected from 1-8, preferably a number selected from 2-4;
  • Ab is a B7H3 antibody or antigen-binding fragment thereof as defined above, or a monoclonal antibody or antigen-binding fragment thereof that competes for binding to human B7H3 with a B7H3 antibody or antigen-binding fragment thereof as defined above.
  • the antibody-drug conjugate of the formula (A) is an antibody-drug conjugate of the formula (III):
  • L 1 is a joint unit
  • y is a number selected from 1-8, preferably a number selected from 2-4;
  • Ab is a B7H3 antibody or antigen-binding fragment thereof as defined above, or a monoclonal antibody or antigen-binding fragment thereof that competes for binding to human B7H3 with a B7H3 antibody or antigen-binding fragment thereof as defined above.
  • the antibody-drug conjugate of the formula (A), or a pharmaceutically acceptable salt thereof includes but is not limited to:
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate of the formula (A) of the present invention or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable Excipient, diluent or carrier.
  • the present invention further relates to an antibody-drug conjugate of the formula (A), or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same, which is prepared for the treatment of a human B7H3-positive cell Use in a medicament for a disease; preferably, wherein said use is in the use of a medicament for the treatment of a B7H3 high expression cancer.
  • the present invention further relates to an antibody-drug conjugate represented by the general formula (A), or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same, for use in the preparation of a medicament for treating a disease
  • the disease is selected from the group consisting of a therapeutic glioma (non-limiting example is a human brain astrocytoma), human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, Bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, Promoting connective tissue proliferative small round cell tumor, ependymoma,meaning tumor, extraosseous mucinous sarcoma, bone fiber dysplasia, bone fibrosis,
  • the invention further relates to a method of treating a disease comprising administering to a patient in need thereof a therapeutically effective amount of the antibody-drug conjugate of the formula (A), or a pharmaceutically acceptable salt or solvent thereof, a compound, or a pharmaceutical composition comprising the same, selected from the group consisting of human brain astroglioma, human pharyngeal carcinoma, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder Cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, promotion Connective tissue proliferative small round cell tumor, ependymoma,meaning tumor, extraosseous mucinous sarcoma, bone fiber dysplasia, osteofibrous dysplasia, gallbladder or cholangiocarcino
  • FIG. 1 Binding of antibodies to U87MG cells
  • FIG. 1 Endocytic effect of different antibodies on U87MG cells
  • Figure 3 Endocytic effect of different ADC h1702-3024, h1703-3024 on U87MG cells of the present invention
  • Figure 4 Different ADCs h1702-cys-3024, h1702DS-cys-3024 and
  • Figure 5 Effect of h1702-3024 on nude mice U87MG xenografts
  • the "antibody” as used in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or in accordance with the numbering rules of kabat and chothia (HCDR1).
  • the antibody of the present invention includes a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.
  • murine antibody is in the present invention a monoclonal antibody to human B7H3 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the B7H3 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine B7H3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , a ⁇ chain or a variant thereof, or further comprising a murine IgG1, IgG2 The heavy chain constant region of IgG3 or a variant thereof.
  • recombinant antibody includes “chimeric antibodies”, “humanized antibodies” and “fully human antibodies”.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the murine variable region gene is cloned.
  • the human constant region gene is ligated into a chimeric gene, inserted into an expression vector, and finally expressed in a eukaryotic or prokaryotic system.
  • the antibody light chain of the B7H3 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the B7H3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or an amino acid mutation
  • An IgGl, IgG2 or IgG4 variant (such as a YTE mutation or a back mutation).
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of murine CDR sequences into human antibody variable region frameworks, ie different types of human germline antibodies An antibody produced in a framework sequence. It is possible to overcome the heterologous reaction induced by the chimeric antibody by carrying a large amount of the mouse protein component.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc.
  • humanized antibodies of the invention also include humanized antibodies that are further affinity matured by phage display.
  • the CDR sequence of the mouse in the B7H3 humanized antibody is selected from the group consisting of SEQ ID NO: 8-13 or 14-19;
  • the human antibody variable region framework is designed and selected, wherein The sequence of the heavy chain FR region on the variable region of the heavy chain of the antibody, derived from the combined sequence of the human germline heavy chain IGHV1-18*01 and hjh4.1 or the combined sequence of IGHV3-7*01 and hjh4.1;
  • the light chain FR region sequence on the variable region of the antibody light chain is derived from the combined sequence of the human germline light chain IGKV1-33*01 and hjk4.1 or the combined sequence of IGKV1-39*01 and hjk2.1.
  • the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
  • the CDR graft can attenuate the affinity of the B7H3 antibody or antigen-binding fragment thereof to the antigen due to framework residues that are in contact with the antigen. Such interactions can be the result of high mutations in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids to the framework of humanized antibodies.
  • Amino acid residues involved in antigen binding from a non-human B7H3 antibody or antigen-binding fragment thereof can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be related.
  • the sequence can be compared to a subtype consensus sequence or a consensus sequence of a murine sequence with a high percent similarity.
  • Rare framework residues are thought to be the result of high somatic mutations and thus play an important role in binding.
  • Fully human antibody or “fully human antibody”, also known as “full human monoclonal antibody”, has variable and constant regions of the antibody that are human, removing immunogenicity and toxic side effects.
  • monoclonal antibodies has gone through four phases: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies.
  • Related technologies for the preparation of fully human antibodies include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody production technology (transgenic mouse) and single B cell antibody preparation technology.
  • the "complete human antibody” of the present invention uses a phage display technique to obtain an antibody variable region, which can be further recombined with an antibody constant region to obtain an intact antibody.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, B7H3). It has been shown that fragments of full length antibodies can be utilized for antigen binding function of antibodies.
  • binding fragment contained in the term "antigen-binding fragment" of an antibody examples include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab') 2 fragment, including a divalent fragment of two Fab fragments joined by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of a single arm VH and VL domain of the antibody (v) a single domain or dAb fragment (Ward et al, (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) A combination of two or more separate CDRs, optionally joined by a synthetic linker.
  • CDR complementarity determining region
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using a recombinant method such that they are capable of producing a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chains referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • the antigen binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the antigen-binding fragment of the present invention includes Fab, F(ab')2, Fab', single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv), and Peptides of CDRs, etc.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity in a fragment obtained by treating an IgG antibody molecule with a protease papain (cleaving an amino acid residue at position 224 of the H chain), wherein the N-terminal side of the H chain About half of the entire L chain is bound by a disulfide bond.
  • the Fab of the present invention can be produced by treating a monoclonal antibody of the present invention which specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with papain. Furthermore, the Fab can be produced by inserting a DNA encoding a Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express a Fab.
  • F(ab')2 is an antibody obtained by digesting the lower portion of two disulfide bonds in the IgG hinge region with an enzyme pepsin, having an molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions linked at the hinge position. Fragment.
  • the F(ab')2 of the present invention can be produced by treating a monoclonal antibody of the present invention which specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with pepsin. Further, the F(ab') 2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above F(ab')2.
  • the Fab' of the present invention can be produced by treating the F(ab')2 of the present invention which specifically recognizes B7H3 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting a DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express Fab'.
  • single-chain antibody single-chain Fv
  • scFv single-chain Fv
  • scFv antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • scFv molecules can have the general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variant thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present invention are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996). , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the scFv of the present invention can be produced by obtaining a cDNA encoding VH and VL of a monoclonal antibody of the present invention which specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof, and constructs a DNA encoding scFv.
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express an scFv.
  • a diabody is an antibody fragment in which an scFv is dimerized, and is an antibody fragment having a bivalent antigen-binding activity.
  • the two antigens may be the same or different.
  • the diabody of the present invention can be produced by obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present invention which specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs a cDNA encoding scFv.
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector such that the amino acid sequence of the peptide linker is 8 residues or less in length, and then the expression vector is introduced into a prokaryote or eukaryote In order to express double antibodies.
  • dsFv is obtained by linking a polypeptide in which one of amino acid residues in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues.
  • the amino acid residue substituted with a cysteine residue can be selected based on a three-dimensional structure prediction of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).
  • the dsFv of the present invention can be produced by obtaining a cDNA encoding VH and VL of a monoclonal antibody which specifically recognizes human B7H3 of the present invention and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof, and constructs a cDNA encoding dsFv.
  • DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express dsFv.
  • a peptide comprising a CDR is constructed by one or more regions of a CDR comprising a VH or VL. Peptides comprising a plurality of CDRs can be joined directly or via a suitable peptide linker.
  • the CDR-containing peptide of the present invention can be produced by constructing the DNA encoding the CDRs of the VH and VL of the monoclonal antibody of the present invention which specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express the peptide.
  • the CDR-containing peptide can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of a CDR applies only to the light chain variable domain LCDR1, LCDR2, and LCDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3), as well as the heavy chain variable domain.
  • HCDR2 and HCDR3 CDR H2, CDR H3 or H2, H3.
  • antibody framework refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain that does not have a CDR.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a B7H3 molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • KD affinity
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • an antibody of the invention binds B7H3 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M or 10 -10 M or less, for example, if a surface is used Plasma resonance (SPR) techniques were measured in a BIACORE instrument.
  • SPR Plasma resonance
  • a monoclonal antibody that competes with a B7H3 antibody for binding to human B7H3 means that the monoclonal antibody of the present invention competes for recognition of the same epitope (also referred to as an antigenic determinant) or part of the same epitope on the extracellular region of human B7H3. And an antibody that binds to the epitope.
  • An antibody that binds to the same epitope as the B7H3 antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human B7H3 recognized by the B7H3 antibody of the present invention.
  • nucleic acid molecule refers to a DNA molecule and an RNA molecule.
  • the nucleic acid molecule may be single stranded or double stranded, but is preferably a double stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to the coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (for example, a bacterial vector having an origin of replication of bacteria and an episomal mammalian vector) or can be integrated into the genome of the host cell after introduction into the host cell, thereby The host genome is replicated together (eg, a non-episomal mammalian vector).
  • a mouse can be immunized with human B7H3 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human B7H3. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, for example a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any degree of clinical right measurement.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount" effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. While embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in ameliorating the symptoms of each target disease, any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living being, cell or human, depending on the situation.
  • Endogenous refers to a substance produced in a cell, organism or human body, depending on the circumstances.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared x 100.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • the present invention relates to a method for immunodetection or assay of B7H3, a reagent for immunodetection or assay of B7H3, a method for immunodetection or assay of cells expressing B7H3, and a diagnosis for diagnosing a disease associated with B7H3-positive cells
  • An agent comprising the monoclonal antibody or antibody fragment of the present invention which specifically recognizes human B7H3 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof as an active ingredient.
  • the method for detecting or measuring the amount of B7H3 may be any known method.
  • it includes immunodetection or assay methods.
  • the immunodetection or assay method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody.
  • immunoassay or assay methods include radioactive substance labeling immunological antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods Wait.
  • the above-mentioned diseases associated with B7H3-positive cells can be diagnosed by detecting or measuring cells expressing B7H3 with the monoclonal antibody or antibody fragment of the present invention.
  • a known immunodetection method can be used, and immunoprecipitation, fluorescent cell staining, immunohistochemical staining, or the like is preferably used. Further, a fluorescent antibody staining method or the like using the FMAT8100HTS system (Applied Biosystem) can be used.
  • a living sample for detecting or measuring B7H3 is not particularly limited as long as it has a possibility of including cells expressing B7H3, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, Tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody of the present invention or an antibody fragment thereof may further contain a reagent for performing an antigen-antibody reaction or an agent for detecting a reaction, depending on a desired diagnostic method.
  • Agents for performing antigen-antibody reactions include buffers, salts, and the like.
  • the reagents for detection include reagents commonly used in immunoassays or assay methods, such as labeled secondary antibodies that recognize the monoclonal antibodies, antibody fragments or conjugates thereof, substrates corresponding to the labels, and the like.
  • cytotoxic drug refers to a chemical molecule that has a strong disruption to its normal growth in tumor cells.
  • cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to lack of specificity, while killing tumor cells, it also causes apoptosis of normal cells, leading to serious side effects.
  • radioisotopes eg, radioisotopes of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu
  • chemotherapeutic drugs eg, chemotherapeutic drugs, toxins such as bacteria, fungi Small molecule toxins or enzymatically active toxins of plant or animal origin, including fragments and/or variants thereof.
  • toxin refers to small molecular toxins and derivatives thereof derived from bacteria, fungi, plants or animals, including maytansinoids and derivatives thereof (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and Derivatives such as MMAF, MMAE, 3024 (WO 2016/127790 A1, Compound 7), diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modeccin, ⁇ - Aspergillus (sarcin), Aleutites fordii toxic protein, dianthin toxic protein, Phytolaca americana toxic protein (PAPI, PAPII and PAP-S), Momordica charantia inhibition , curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, Phenomycin, enomycin, and trichothecenes.
  • maytansinoids and derivatives thereof such as
  • MMAF, MMAE are auristatin derivatives, see US2005/0238649 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124, the structural formula is as follows:
  • the auristatin/dorlastatin drug module such as MMAF and its derivatives can be prepared using the methods described in US2005-0238649A1 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124.
  • the auristatin/dorlastatin drug module such as MMAE and its derivatives can be prepared using the method described in Doronina et al. (2003) Nat. Biotech. 21:778-784.
  • the drug-linker modules MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF and MC-vc-PAB-MMAE can be conveniently synthesized by conventional methods, for example, Doronina et al. (2003) Nat. Biotech. 21: 778-784 And as described in U.S. Patent Application Publication No. US 2005/0238649 A1, which is then coupled to the antibody of interest.
  • chemotherapeutic drug is a chemical compound used in the treatment of tumors.
  • examples of chemotherapy drugs include alkylating agents, such as Thiotepa (Thiotepa); cyclophosphamide (cyclosphamide) (CYTOXAN TM); alkyl sulfonates such as busulfan aliphatic (busulfan), where English C Shu (improsulfan) piperazine and poise Piposulfan; aziridine such as benaodopa, carboquone, meturedopa and uredopa; aziridine and methylamelamine included Altretamine, triethylenemelamine, triethylenephosphoramide, triethylene thiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil , naphthalene mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, oxychloride mustard; melphalan, mel
  • anti-hormonal agents that modulate or inhibit the effects of hormones on tumors, such as anti-estrogen preparations including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and faremis (Fareston); and antiandrogen preparations such as flutamide, nilutamide ( Nilutamide), bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogen preparations including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and faremis (Fareston)
  • antiandrogen preparations such as flutamide, nil
  • linker unit in the present invention is L1 and L2, and refers to a chemical structural fragment or bond which is covalently linked at one end to the antibody and to the cytotoxic drug at the other end.
  • drug loading refers to the average number of cytotoxic drugs loaded on each ligand in the molecule, and can also be expressed as the ratio of the amount of drug to the amount of antibody.
  • the range of drug loading can be per ligand (Pc) linkage.
  • y may be limited by the number of attachment sites.
  • the cytotoxic drug is coupled via a linker unit to the ⁇ -amino group of the N-terminal amino and/or lysine residue of the ligand, typically in a coupling reaction that is conjugated to the antibody.
  • the number of drug molecules will be less than the theoretical maximum.
  • the loading of antibody cytotoxic drug conjugates can be controlled by the following non-limiting methods, including:
  • carrier refers to a system which changes the manner in which the drug enters the body and the distribution in the body, controls the release rate of the drug, and delivers the drug to the targeted organ.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and increase bioavailability.
  • Polymeric surfactants which can be used as carriers, can be self-assembled due to their unique amphiphilic structure to form aggregates of various forms, such as micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules while having good permeability to the membrane and can serve as an excellent drug carrier.
  • excipient is an addition to a pharmaceutical preparation other than the main drug, and may also be referred to as an excipient.
  • excipients such as adhesives, fillers, disintegrants, lubricants in tablets; semi-solid preparation ointments, matrix parts in creams; preservatives, antioxidants, flavoring agents, fragrances, and liquids in liquid preparations Solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants and the like can be referred to as excipients.
  • the term "diluent” is also known as a filler and its primary use is to increase the weight and volume of the tablet. The addition of the diluent not only ensures a certain volume, but also reduces the dose deviation of the main component and improves the compression moldability of the drug. When the drug of the tablet contains an oily component, it is necessary to add an absorbent to absorb the oily substance so as to maintain a "dry" state to facilitate tableting.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oily phase.
  • the injection or microemulsion can be injected into the bloodstream of the patient by a local injection.
  • the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of the invention.
  • a continuous intravenous delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous pump.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration.
  • the suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent.
  • sterile fixed oils may conveniently be employed as a solvent or suspension medium.
  • the invention also relates to methods of treating diseases associated with human B7H3-positive cells, particularly in the treatment of cancer and inflammation.
  • the amino acid sequence of the antigen and the protein for detection of the present invention was designed using the human B7H3 shown in SEQ ID NO: 1 as a template for the B7H3 of the present invention.
  • the following B7H3 antigens are not specifically described as human B7H3.
  • B7H3 (SEQ ID NO: 1):
  • the double-crossed line is the signal peptide (Signal peptide: 1–28);
  • the cross-hatched portion is the extracellular domain of B7H3 (Extracellular domain: 29-466), wherein 29-139 is an Ig-like V-type 1 domain, and 145-238 is an Ig-like C2-type 1 domain; 243-357 Is an Ig-like V-type 2 domain, 363-456 is an Ig-like C2-type 2 domain;
  • the dotted line portion is a transmembrane domain (Transmembrane domain: 467-487);
  • the italicized portion is the intracellular region (Cytoplasmic domain: 488-534).
  • the double-crossed line is the signal peptide (Signal peptide: 1–28);
  • the horizontal line portion is the extracellular domain of B7H3 (Extracellular domain: 29-248), wherein 29-139 is an Ig-like V-type domain, and 145-238 is an Ig-like C2-type domain;
  • the dotted line portion is the transmembrane domain portion (Transmembrane domain: 249-269);
  • the italic part is the intracellular domain (Cytoplasmic domain: 270-316).
  • the cross-hatched portion is the B7H3 extracellular region; the italic portion is the His-tag marker.
  • the cross-hatched portion is the B7H3 extracellular region; the italic portion is the His-tag marker.
  • the cross-hatched portion is the B7H3 extracellular region; the italic portion is the His-tag marker.
  • B cells were isolated using human PBMC, spleen, and lymph node tissues, and RNA was extracted to construct a natural single-stranded phage antibody library (capacity 3.2 ⁇ 10 10 ).
  • the constructed natural single-stranded phage library was packaged to form phage particles, and then sieved by a liquid phase method, and the phage was combined with the biotinylated B7H3 liquid phase, and then separated by streptavidin magnetic beads.
  • biotinylated human B7H3 and organisms were used, respectively.
  • the primed mouse B7H3 was subjected to alternate panning.
  • the first round was sieved with 2 ⁇ g/ml biotinylated human B7H3
  • the second round was sieved with 2 ⁇ g/ml biotinylated mouse B7H3
  • the third round was 0.5 ⁇ g/ The ml biotinylated human B7H3 was subjected to panning.
  • mouse B7H3 and 1% BSA add phage supernatant diluted in 1:1 blocking buffer, and finally detect with anti-M13 HRP; ELISA test OD450 value greater than 0.5, and ELISA OD450 value combined with human and mouse B7H3
  • Two clones with an ELISA OD450 value of 1% BSA combined with a ratio greater than 2.0 were sequenced to obtain 9 specific sequences.
  • the 9 specific sequences obtained by phage library screening were constructed by ELISA binding assay and the binding of the two antibodies was strong, which were h1702 and h1703, respectively.
  • the process of constructing a complete monoclonal antibody is as follows:
  • primers were designed to construct VH/VK/VL gene fragments of each single-chain antibody sequence.
  • the heavy light chain variable regions of h1702 and h1703 were obtained.
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • the antibody variable region was then homologously recombined with the constant region gene (CH1-FC/CL) fragment to construct the intact antibody VH-CH1-FC/VK-CL/VL-CL.
  • H1702 light chain amino acid sequence Lamada (SEQ ID NO: 23)
  • amino acid mutation of the light chain sequence of h1702 was specifically mutated to the light chain (SEQ ID NO: 23).
  • the N-terminal first amino acid residue Q was replaced by D, and the deletion mutation C-terminal was first. Amino acid residue S to obtain a more stable and uniform monoclonal antibody h1702-DS.
  • the heavy chain sequence of the h1702-DS after mutation modification is SEQ ID NO: 22, and the light chain amino acid sequence is as follows: (SEQ ID NO: 26).
  • the plasmids expressing the light and heavy heavy chains of the antibodies were transfected into HEK293E cells at a ratio of 1.5:1. After 6 days, the expression supernatants were collected, and the impurities were removed by high-speed centrifugation and purified by Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. The protein of interest was eluted with an acidic eluent of pH 3.0 - pH 3.5 and neutralized with 1 M Tris-HCl, pH 8.0-9.0. The eluted sample was appropriately concentrated, and further purified by PBS-balanced gel chromatography Superdex 200 (GE) to remove the aggregate, collect the monomer peak, and equilibrate the device.
  • PBS-balanced gel chromatography Superdex 200 GE
  • the human B7-H3 (h-B7H3-Fc) sequence encoding the huFc tag was synthesized by Integrated DNA Technology (IDT) (the above B7-H3 recombinant proteins were all designed by the present invention) and cloned into the pTT5 vector (Biovector). .
  • IDT Integrated DNA Technology
  • the recombinant B7-H3 protein is purified by conventional techniques in the art after expression in 293T cells. The purified protein can be used to immunize mice to obtain antibodies.
  • Anti-human B7H3 monoclonal antibodies are produced by immunizing mice.
  • the experiment used Swiss Webster white mice, female, 6 weeks old (Charles River). Feeding environment: SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%.
  • the immunizing antigen is the Fc-tagged human B7H3 recombinant protein (huB7H3-Fc).
  • Titermax Sigma Lot Num: T2684
  • Titermax was used as an adjuvant.
  • the ratio of antigen to adjuvant (titermax) was 1:1, and the antigen was emulsified and inoculated for 0, 21, 35, 49, and 63 days.
  • IP intraperitoneal
  • mice with high antibody titers in serum and titers tended to plate and spleen lymphocytes and myeloma cells Sp2/0 cells were optimized using an electrofusion procedure ( CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • CHO-S cells were compared to blank CHO-S cells to exclude non-specific binding antibody hybridoma strains, and screened by flow sorting to select two hybridomas that bind to the recombinant protein and also bind to the cell expressing antigen.
  • the cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and then sequenced, and finally the sequence of the mouse antibody m1704 was obtained.
  • the heavy and light chain variable region sequences of murine mAb m1704 are as follows:
  • the optimized CDR region has the following sequence:
  • the heavy and light chain variable regions of the murine antibody were cloned into the pTT vector plasmid (Biovector) containing the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively, and then transiently transfected into HEK293 cells to obtain anti-B7-
  • the chimeric antibody of H3 was purified by conventional techniques and used.
  • Humanization of murine anti-human B7-H3 monoclonal antibodies was performed as disclosed in many literatures in the art. Briefly, a human constant domain is used in place of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody.
  • the present invention humanizes the antibody m1704.
  • the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from the human kappa light chain gene human germline light chain template IGkV1-33, and the human germline heavy chain framework region is derived from the human heavy chain template IGHV3-23.
  • the CDR region of the murine antibody m1704 was transplanted onto the selected corresponding humanized template, the humanized variable region was replaced, and then recombined with the IgG constant region (preferably the heavy chain was IgG1 and the light chain was ⁇ ). Then, based on the three-dimensional structure of the murine antibody, the residues which have an important interaction with the CDRs and the residues of the CDRs, and the residues which have an important influence on the conformation of VL and VH are subjected to back mutation and the CDR regions are The chemically labile amino acid residues were optimized, and antibodies assembled from the following humanized light and heavy chain variable region sequences were designed and tested.
  • the final humanized h1704-3 antibody molecule (using the VH1 heavy chain variable region and the VL2 light chain variable region) was selected by expression test and the number of back mutations, the heavy and light chain sequences of which are SEQ ID NO: 40 and 41 are shown.
  • a cDNA fragment was synthesized based on the gene sequences of the above humanized antibody light and heavy chains, and inserted into a pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20).
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2 and placed in a CO 2 incubator for incubation 4- 5 days.
  • the expressed antibody is recovered by centrifugation and then subjected to antibody purification to obtain a humanized antibody protein of the present invention.
  • Examples 4 to 11 are the preparation processes of the related ADCs of the present invention.
  • the antibodies (h1702, h1703) of Examples 4 to 7 were prepared by coupling a drug (MMAF or 3024) having a linking unit to its lysine, and the antibodies of Examples 8 to 11 were passed (Examples 8 to 11) H1702, h1704-3, h1702DS) A thiol group on cysteine, which is reacted with a drug (3024) having a linking unit to prepare an ADC.
  • MMAF or 3024 MMAF or 3024
  • the reaction solution was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water at pH 6.5, containing 0.001 M EDTA) to remove unreacted thioacetic acid S-(3-carbonylpropyl).
  • the ester and sodium cyanoborohydride obtained the title product 1b in PBS buffer (about 5.5 mL), and concentrated by ultrafiltration to about 2.5 ml to carry out the next reaction.
  • reaction solution was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water, pH 6.5, containing 0.001 M EDTA) to give the title product h1702-MMAF in PBS (0.21 mg/mL, 14.5 mL), stored frozen at 4 °C.
  • the reaction solution was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water at pH 6.5, containing 0.001 M EDTA) to remove unreacted thioacetic acid S-(3-carbonylpropyl).
  • the ester and sodium cyanoborohydride obtained the title product 2b in PBS buffer (about 5.5 mL), and concentrated by ultrafiltration to about 2.5 ml to carry out the next reaction.
  • reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water at pH 6.5, containing 0.001 M EDTA) to give the title product h1703-MMAF in PBS buffer (0.20 mg/mL, 13.0 mL), stored frozen at 4 °C.
  • the reaction solution was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water at pH 6.5, containing 0.001 M EDTA) to remove unreacted thioacetic acid S-(3-carbonylpropyl).
  • the ester and sodium cyanoborohydride obtained the title product 3b in PBS buffer (about 14.5 mL), and concentrated by ultrafiltration to about 7.5 ml for the next reaction.
  • reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water at pH 6.5, containing 0.001 M EDTA) to give the title product h1702-3024 in PBS buffer (1.35 mg/mL, 27.5 mL), stored frozen at 4 °C.
  • the reaction solution was purified by desalting on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water at pH 6.5, containing 0.001 M EDTA) to remove unreacted thioacetic acid S-(3-carbonylpropyl).
  • the ester and sodium cyanoborohydride obtained the title product 4b in PBS buffer (about 14.0 mL), and concentrated by ultrafiltration to about 7.5 ml to carry out the next reaction.
  • reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water, pH 6.5, containing 0.001 M EDTA) to give the title product h1703-3024 in PBS (1.20 mg/mL, 28.0 mL), stored frozen at 4 °C.
  • reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water, pH 6.5, containing 0.001 M EDTA) to give the title product in PBS buffer (6.85 mg/mL, 7.5 mL). Store frozen at 4 °C.
  • reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water, pH 6.5, containing 0.001 M EDTA) to give the title product in PBS (6.75 mg/mL, 7.8 mL) Store frozen at 4 °C.
  • reaction solution was subjected to desalting purification on a Sephadex G25 gel column (elution phase: 0.05 M in PBS buffered water, pH 6.5, containing 0.001 M EDTA) to give the title product in PBS (6.65 mg/mL, 2.7 mL) Store frozen at 4 °C.
  • Test Example 1 ELISA binding assay
  • Human B7H3 protein (2Ig/4Ig) was diluted to a concentration of 1 ⁇ g/ml with PBS (Sigma, P4417-100TAB) in pH 7.4, and added to a 96-well microtiter plate (Corning, CLS3590-100EA) in a volume of 100 ⁇ l/well. Place at 16 ° C overnight for 16-20 hours. After discarding the liquid, 120 ⁇ l/well of a 5% skim milk (bright skimmed milk powder) blocking solution diluted with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20) was added, and the mixture was incubated at 37 ° C for 2 hours for blocking.
  • PBS Sigma, P4417-100TAB
  • CLS3590-100EA 96-well microtiter plate
  • the secondary antibody (Jackson Immuno Research, 109-005-008) was incubated for 1 hour at 37 °C. After washing the plate 4 times with PBST, add 100 ⁇ l/well TMB chromogenic substrate (KPL, 52-00-03), incubate for 3-5 min at room temperature, and add 100 ⁇ l/well 1 M H 2 SO 4 to stop the reaction with NOVOStar.
  • the instrument reads the absorbance at 450 nm and calculates the binding EC50 value of the antibody to the antigen. The results are shown in Table 2.
  • mouse B7H3 (Cat.#1397-B3-050/CF, R&D) was used for in vitro binding to different species of B7H3. Combined detection.
  • B7H3 protein was diluted to a concentration of 1 ⁇ g/ml with PBS (Sigma, P4417-100TAB) buffer of pH 7.4, added to a 96-well microtiter plate at a volume of 100 ⁇ l/well, and placed at 4 °C. Stay 16-20 hours overnight.
  • the Biacore, GE instrument was used to determine the affinity of the anti-B7H3 antibody, and the anti-B7H3-ADC and human 2Ig-B7H3 antigens, and the various antigens of the human 4Ig-B7H3 antigen.
  • the biosensor chip Protein A (Cat.#29127556, GE) was used to affinity capture a certain amount of the antibody to be tested/ ADC to be tested, and then flowed through the surface of the chip through a series of concentration gradients of human 2Ig-B7H3 antigen (Cat.# 1949-B3-050/CF, R&D), human 4Ig-B7H3 antigen (Cat. #11188-H08H, Sino Biological), the reaction signal was detected in real time using a Biacore instrument (Biacore T200, GE) to obtain binding and dissociation curves. After completion of each cycle dissociation, the biochip was washed and regenerated with a glycine-hydrochloric acid regeneration solution (pH 1.5) (Cat. #BR-1003-54, GE). The buffer used in the experiment was HBS-EP buffer solution (pH 7.4) (Cat. #BR-1001-88, GE).
  • the endocytosis effect of the antibody was evaluated based on the fluorescence signal of the intracellular antibody or different ADCs according to the fluorescence signal strength.
  • B7H3 antibody/ADC and APC anti-human IgG Fc Biolegend, 409306 were mixed in ice at a molar ratio of 1:2 and incubated on ice for 15 minutes.
  • the antibody mixture was incubated with 2 ⁇ 10 5 U87MG cells on ice for 30 minutes, then the excess antibody was washed away, and then the cells were transferred to a medium pre-warmed at 37 ° C, and incubated at 37 ° C for 0, 15, 30, 60 respectively. And 120 minutes.
  • the cells were centrifuged and the cells were resuspended in the antibody eluate for 7 minutes at room temperature, the antibody eluate was washed away, and the intracellular fluorescence signal was read using BD Verse, see Figure 2, Figure 3, Figure 4.
  • the results showed that the endocytosis effect of ADC on U87MG cells was comparable to that of naked resistance.
  • the time of blood collection was: 5 min, 8 h, 24 h (day 2), day 3, day 5, day 8, day 11 and day 15 of the first day of blood collection. 200 ⁇ L each (corresponding to taking 100 ⁇ L of serum); the collected blood samples were allowed to stand at room temperature for half an hour until agglutination, and then centrifuged at 10,000 ⁇ g for 10 minutes at 4 °C. The supernatant was collected and immediately stored at -80 ° C.
  • the B7H3 antibody concentration in the serum was measured by ELISA, and PK analysis was performed. The results are shown in Table 5.
  • Test drug Analyte Mode of administration T 1/2 (mean ⁇ SD, h) H1702-3024 Total ADC IV (3mg/kg) 97 ⁇ 15 h1702DS-cys-3024 Total ADC IV (3mg/kg) 98.20 ⁇ 10.16 H1704-3-cys-3024 Total ADC IV (3mg/kg) 65.20 ⁇ 4.18
  • DSC was used to detect the thermal stability of different antibodies, and the thermal stability of different buffer systems under different pH conditions was compared.
  • Exemplary buffer systems corresponding to different pHs were 10 mM PB (pH 7) and 10 mM Acetate (pH 5.2).
  • the sample was replaced with the corresponding buffer, and the sample concentration was controlled at about 1 mg/ml, and detection was performed using a MicroCal* VP-Capillary DSC (Malvern).
  • each sample and the blank buffer were degassed by a vacuum degasser for 1 to 2 min. Add 400 ⁇ l of sample or blank buffer to each well of the sample plate (the instrument load is 300 ⁇ l).
  • the purity of the sample was monitored by SEC-HPLC to investigate the periodic stability at a certain concentration.
  • Exemplary conditions such as controlling the sample concentration at about 40-50 mg/ml in PBS (pH 7.4) system and pH 5.2 acetic acid/sucrose system Compare the stability of different antibodies at, for example, -80 °C for repeated freezing and thawing and storage at 4 ° C, 30 ° C, 40 ° C for one month.
  • the purity of the antibody was examined using a Xbridge protein BEH SEC 200A (Waters) HPLC column. After one month of investigation, both h1702 and h1703 showed good stability. The results are shown in Table 8.
  • the solution of 6.0 was ultrafiltered twice, and 3 ⁇ L of 0.25 mg/mL trypsin (trypsin) was added, and the mixture was hydrolyzed overnight at 37 ° C in a water bath.
  • trypsin trypsin
  • the Agilent 6530Q-TOF was subjected to LC-MS detection analysis, and the potential modification sites were subjected to mass spectrometry (the results are shown in Table 9).
  • the results show that the h1702 and h1703 involved in the present invention have no significant deamidation, oxidation or isomerization. The trend indicates that the molecule has good physical and chemical stability.
  • U87MG cells (Catalyst Cell Bank, Catalog #TCHu138) were cultured in EMEM medium containing 10% FBS, passaged 2 to 3 times a week, and passaged 1:2 or 1:5. At the time of passage, the medium was aspirated, the cell layer was washed with 5 ml of 0.25% trypsin, then the trypsin was aspirated, and the cells were digested in an incubator for 3 to 5 minutes, and the cells were resuspended by adding fresh medium.
  • the sample to be tested was diluted to 50 mM with PBS or DMSO, and diluted to 10 concentrations in 3 folds to set blank and control wells.
  • 10 ⁇ l of a compound solution formulated to a gradient concentration was added to 90 ⁇ l of fresh medium. Further, 10 ⁇ l of the above drug-containing medium solution was added to the plate.
  • the plates were incubated for 3 days in the incubator (37 ° C, 5% CO 2 ).
  • 100 ⁇ l of CellTiter-Glo reagent was added to each well, and allowed to stand at room temperature for 5-10 min in the dark, and the chemiluminescence signal value was read in Victor 3, and the data was processed using GraphPad software.
  • the measured IC50 values are shown in Table 10.
  • the ADC obtained by naked anti-coupling toxin has a good killing effect on U87MG cells.
  • ZR-75-1 cells (ATCC, Catalog #CRL-1500) were cultured in RPMI-1640 medium containing 10% FBS, passaged 2 to 3 times a week, and passaged 1:4 or 1:6. At the time of passage, the medium was aspirated, the cell layer was washed with 5 mL of 0.25% trypsin, then the trypsin was aspirated, the cells were digested in an incubator for 3 to 5 minutes, and the cells were resuspended in fresh medium.
  • the sample to be tested was diluted to 50 mM with PBS or DMSO, and diluted to 10 concentrations in 3 folds to set blank and control wells.
  • 10 ⁇ l of a compound solution formulated to a gradient concentration was added to 90 ⁇ l of fresh medium. Further, 10 ⁇ l of the above drug-containing medium solution was added to the plate.
  • the plates were incubated for 6 days in the incubator (37 ° C, 5% CO 2 ).
  • 100 ⁇ L of CellTiter-Glo reagent was added to each well, and the chemiluminescence signal value was read in Victor 3 at room temperature for 5-10 min, and the data was processed using GraphPad software.
  • the measured IC50 values are shown in Table 11.
  • the ADC obtained by naked anti-coupling toxin has a good killing effect on ZR-75-1 cells.
  • Detroit 562 cells (ATCC, Catalog #CCL-138) were cultured in EMEM medium containing 10% FBS, passaged 2 to 3 times a week, and passaged 1:4 or 1:6. At the time of passage, the medium was aspirated, the cell layer was washed with 5 mL of 0.25% trypsin, then the trypsin was aspirated, the cells were digested in an incubator for 3 to 5 minutes, and the cells were resuspended in fresh medium. 90 ⁇ L of the cell suspension was added to a 96-well cell culture plate at a density of 2.2 ⁇ 10 4 cells/mL, and only 100 ⁇ L of 10% FBS EMEM medium was added to the periphery of the 96-well plate. The plates were incubated in an incubator for 24 hours (37 ° C, 5% CO 2 ).
  • the sample to be tested was diluted to 50 mM with PBS or DMSO, and diluted to 10 concentrations in 3 folds to set blank and control wells.
  • 10 ⁇ l of a compound solution formulated to a gradient concentration was added to 90 ⁇ l of fresh medium. Further, 10 ⁇ l of the above drug-containing medium solution was added to the plate.
  • the plates were incubated for 6 days in the incubator (37 ° C, 5% CO 2 ).
  • 100 ⁇ L of CellTiter-Glo reagent was added to each well, and the chemiluminescence signal value was read in Victor 3 at room temperature for 5-10 min, and the data was processed using GraphPad software.
  • the measured IC50 values are shown in Table 12.
  • the ADC obtained by naked anti-coupling toxin has a good killing effect on Detroit 562 cells.
  • the ADC obtained by naked anti-coupling toxin has a good killing effect on U87MG, ZR-75-1 and Detroit562 cells.
  • Test Example 10 Evaluation of the efficacy of ADC on human brain astroglioma U87MG nude mice xenografts
  • Relative volume (RTV) VT / V0
  • Tumor inhibition rate (%) (CRTV-TRTV) / CRTV (%)
  • V0 and VT are the tumor volume at the beginning of the experiment and at the end of the experiment.
  • CRTV and TRTV were the control group (blank) at the end of the experiment and the relative tumor volume of the experimental group.
  • Vs blank *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001
  • the blank group and the 1mpk group had larger tumor volume at 26 days, so the blank and 1mpk groups were sacrificed at D26.
  • the tumor growth was still slow after the withdrawal of h1702DS-cys-3024 (3mpk) group.
  • the tumor inhibition effect is the best. There was no significant difference in the inhibition rate between h1702DS-cys-3024 (3mpk) and h1704-3-cys-3024 (3mpk) at D26 and D36 (P>0.05).
  • Vs blank *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001
  • Test Example 11 Evaluation of the efficacy of ADC on human pharyngeal carcinoma pleural fluid metastatic cell Detroit 562 nude mice xenografts
  • mice Female, 6-7 weeks, were subcutaneously inoculated with human pharyngeal carcinoma pleural fluid metastatic cell Detroit 562 cells. On the tenth day after inoculation of the cells, the animals were randomly divided into groups (D0), 8 rats in each group, and the intraperitoneal injection was started once a week for 3 times, and the tumor volume and body weight were measured 2-3 times per week. .
  • the tumor volume (V) is calculated as:
  • Relative volume (RTV) VT / V0
  • Tumor inhibition rate (%) (CRTV-TRTV) / CRTV (%)
  • V0 and VT are the tumor volume at the beginning of the experiment and at the end of the experiment.
  • CRTV and TRTV were the control group (blank) at the end of the experiment and the relative tumor volume of the experimental group.
  • the tumor inhibition rate of the test antibody ADC was: h1702DS-cys-3024 ADC (1mpk) inhibition rate reached 39.22% (P ⁇ 0.05); h1702DS-cys-3024 ADC (3mpk) inhibition rate It reached 70.50% (P ⁇ 0.001) (see Table 16).
  • the animals in each group had normal body weight during the course of the drug.
  • Vs blank *p ⁇ 0.05, ***p ⁇ 0.001
  • Test Example 12 Physical stability of the B7H3 ADC
  • the thermal stability of different antibody ADCs was compared.
  • the exemplary buffer systems corresponding to different pHs were 10 mM PBS (pH 7.4) and 10 mM Acetate (pH 5.5). ).
  • the sample was replaced with the corresponding buffer, and the sample concentration was controlled at about 1 mg/ml, and detection was performed using a MicroCal* VP-Capillary DSC (Malvern).
  • each sample and the blank buffer were degassed by a vacuum degasser for 1 to 2 min. Add 400 ⁇ l of sample or blank buffer to each well of the sample plate (the instrument load is 300 ⁇ l).
  • the purity of the sample was monitored by SEC-HPLC.
  • the stability was determined under certain conditions. For example, the sample concentration was controlled at about 50 mg/ml in PBS ( The pH 7.4) system and the pH 5.5 acetic acid/sucrose system (referred to as the 559 system) were compared for stability of different antibodies at 40 ° C for 28 days.
  • the purity of the antibody was examined using a Xbridge protein BEH SEC 200A (Waters) HPLC column.
  • the h1702DS-cys-3024 ADC showed good stability after 28 days of investigation. The results are shown in FIG.

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Abstract

一种B7H3抗体-药物偶联物及其医药用途。具体而言，B7H3抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，以及包含前述偶联物或其药学上可接受的盐或溶剂化合物的药物组合物，以及其制备用于治疗B7H3介导的疾病或病症的药物中的用途；尤其在制备抗癌药物中的用途。

Description

B7H3抗体-药物偶联物及其医药用途 技术领域
本发明涉及B7H3抗体-药物偶联物及其在医药上的应用，进一步地，本发明涉及B7H3抗体-细胞毒性药物偶联物或其药学上可接受的盐或溶剂化合物，以及包含前述偶联物或其药学上可接受的盐或溶剂化合物的药物组合物，以及其制备用于治疗B7H3介导的疾病或病症的药物中的用途；尤其在制备抗癌药物中的用途。
背景技术
T细胞介导的免疫反应在机体抗肿瘤过程中发挥着极其重要的作用，而T细胞的活化和增殖不仅需要TCR识别的抗原信号，还需要共刺激分子提供的第二信号。B7家族分子属于共刺激分子免疫球蛋白超家族，越来越多的研究表明，该家族分子在机体正常免疫功能和病理状态下均发挥了重要的调节作用。
B7H3是B7家族的成员之一，属于Ⅰ型跨膜蛋白，包含氨基端的一个信号肽，一个细胞外的免疫球蛋白样可变区(IgV)和恒定区(IgC)、一个跨膜区和一个含有45个氨基酸的胞质尾区(Tissue Antigens.2007 Aug；70(2):96-104)。目前，B7H3主要存在2种剪切体，B7H3a和B7H3b。B7H3a胞外段由IgV-IgC 2个免疫球蛋白结构域组成，又称为2IgB7H3，而B7H3b胞外段由IgV-IgC-IgV-IgC 4个免疫球蛋白结构域组成，又称为4IgB7H3。
B7H3蛋白质在正常组织、细胞中不表达或极低表达，却高表达于多种肿瘤组织，并与肿瘤的进展、患者的生存及预后密切相关。临床上已经报道，B7H3在许多癌症类型中、特别是在非小细胞肺癌、肾癌、泌尿道上皮癌、结直肠癌、前列腺癌、多形性胶质母细胞瘤、卵巢癌和胰腺癌中过表达(Lung Cancer.2009 Nov；66(2):245-249；Clin Cancer Res.2008 Aug 15；14(16):5150-5157)。此外，也有文献报道，在前列腺癌中，B7H3的表达强度与临床病理学恶性(诸如肿瘤体积、前列腺外侵袭或Gleason评分)正相关，且也与癌症进展相关(Cancer Res.2007 Aug 15；67(16):7893-7900)。类似地，在多形性胶质母细胞瘤中，B7H3的表达与无事件存活负相关，且在胰腺癌中，B7H3的表达与淋巴结转移和病理学进展相关。因此，B7H3被认为是一种新的肿瘤标志物和潜在的治疗靶点
目前，已有针对B7H3靶点的治疗策略用于临床前研究，如靶向小鼠B7H3的抗体会增强瘤内的浸润性的CD8-阳性的T细胞和抑制肿瘤生长(Mod Pathol.2010 Aug；23(8):1104-1112)。此外，WO 2008/066691专利显示，识别B7H3变体B7H3a的抗体会对腺癌表现出体内抗肿瘤作用。在临床研究中，一种鼠源的B7H3抗体与放射性I 131的偶联药物可显著抑制患者成神经母细胞瘤的生长[J Neufooocol  97(3):409-l 8(2010]。但目前在研的项目都是鼠源抗体经人源化改造的人源化抗体，而人源化抗体在免疫时存在免疫原性相对较高的问题，在人体应用时是一个不利的因素。
噬菌体展示技术(phage display technology)是将外源蛋白质或多肽与噬菌体外壳蛋白融合表达，从而将外源蛋白表达在噬菌体的表面。噬菌体抗体库是将噬菌体展示技术、PCR扩增技术、蛋白表达技术相结合的一项运用综合技术手段所建立起来的抗体库。
噬菌体抗体库最大的优点是不经体内免疫，模拟体内抗体生成的三个过程而制备出完全人源化抗体。除此之外，噬菌体抗体库还具有以下优势：①实现了基因型与表型的统一。此外，实验方法简单、快速，传统的通过杂交瘤技术抗体产生方法需历经数月，而抗体库技术只需短短几周的时间。②表达的是完全人源抗体，且分子量小，主要以活性片段Fab、scFV的形式表达，与完整抗体相比在组织穿透力方面都有明显优势。③筛选容量大，杂交瘤技术是在上千个克隆内筛选，抗体库技术可以对百万甚至亿万个分子选择。筛选到的抗体种类越多。④用途广泛，采用了原核表达系统，当大规模生产时优势更加明显(Curr Opin Biotechnol.2002 Dec；13(6):598-602；Immunotechnology,2013,48(13)48(13):63-73)。
抗体-药物偶联物(antibody drug conjugate,ADC)把单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性物质的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体-药物偶联物能更精准地结合肿瘤细胞并降低将对正常细胞的影响。
目前已有多种ADC药物被用于临床或临床研究，如Kadcyla，是靶向Her2的曲妥珠单抗与DM1形成的ADC药物。同时，也有靶向B7H3的抗体及ADC药物的专利报道，如WO2008100934、WO2012147713、WO2014061277、WO2015184203、WO2016044383。但仍没有B7H3靶点的ADC药物上市或用于临床治疗研究，因此，开发新的B7H3靶点的ADC药物具有广阔的前景。
发明内容
本发明的目的是提供与B7H3的胞外区的氨基酸序列或三维结构结合的单克隆抗体与细胞毒性物质偶联的ADC药物。通过筛选高活性和高稳定性的抗人B7H3全人抗体ADC药物，提供使用所述抗体ADC药物作为活性成分的治疗剂。
本发明提供一种通式(A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，
Ab-(L 2-L 1-D) y        (A)
其中：
D是细胞毒性药物；
L 1，L 2是接头单元；
y为选自1-8的数，优选为选自2-4的数；y可以为小数或整数；
Ab为B7H3抗体或其抗原结合片段，其与人B7H3结合，所述B7H3抗体或其抗原结合片段是选自下面(a)至(c)中任一种的单克隆抗体或其抗原结合片段：
(a)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：
抗体重链可变区HCDR区序列：如SEQ ID NO：10、11和12氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：13、14和15氨基酸序列所示；
(b)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：
抗体重链可变区HCDR区序列：如SEQ ID NO：16、17和18氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：19、20和21氨基酸序列所示；
(c)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：
抗体重链可变区HCDR区序列：如SEQ ID NO：30、31和32氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：33、34和35氨基酸序列所示。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述B7H3抗体或其抗原结合片段是选自下面(a)至(c)中任一种的单克隆抗体或其抗原结合片段：
(a)单克隆抗体，其包含分别如SEQ ID NO：10、11和12氨基酸序列所示抗体重链可变区的HCDR1，HCDR2，HCDR3；和如SEQ ID NO：13、14和15氨基酸序列所示抗体轻链可变区的LCDR1，LCDR2，LCDR3；
(b)单克隆抗体，其包含分别如SEQ ID NO：16、17和18氨基酸序列所示抗体重链可变区的HCDR1，HCDR2，HCDR3；和如SEQ ID NO：19、20和21氨基酸序列所示抗体轻链可变区的LCDR1，LCDR2，LCDR3；
(c)单克隆抗体，，其包含分别如SEQ ID NO：30、31和32氨基酸序列所示抗体重链可变区的HCDR1，HCDR2，HCDR3；和如SEQ ID NO：33、34和35氨基酸序列所示抗体轻链可变区的LCDR1，LCDR2，LCDR3。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，
Ab-(L 2-L 1-D) y        (A)
其中：
D是细胞毒性药物；
L 1，L 2是接头单元；
y为选自1-8的数，优选为选自2-4的数；
Ab为与如上所定义的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab是重组抗体。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab是人源的重组抗体或其抗原结合片段。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab的轻链和重链可变区上的轻链和重链FR区序列分别来源于人种系轻链和重链序列或其突变序列。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab的恒定区包括来源于人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选人源IgG1重链恒定区；和来源于人源κ、λ链或其变体的轻链恒定区，优选人源κ链轻链恒定区。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab含有SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:36或SEQ ID NO:37所示的重链可变区或其变体；所述变体是在SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:36或SEQ ID NO:37所示的重链可变区序列上具有1-10个氨基酸替换的序列。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab含有SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:38或SEQ ID NO:39所示的轻链可变区或其变体；所述变体是在SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:38或SEQ ID NO:39所示的轻链可变区序列上具有1-10个氨基酸替换的序列。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab含有选自下面(1)至(7)中任一种的单克隆抗体或其抗原结合片段：
(1)单克隆抗体，其包含SEQ ID NO:6所示的抗体重链可变区；和SEQ ID NO:7所示抗体轻链可变区；
(2)单克隆抗体，其包含SEQ ID NO:8所示的抗体重链可变区；和SEQ ID NO:9所示抗体轻链可变区；
(3)单克隆抗体，其包含SEQ ID NO:28所示的抗体重链可变区；和SEQ ID NO:29所示抗体轻链可变区；
(4)单克隆抗体，其包含SEQ ID NO:36所示的抗体重链可变区；和SEQ ID NO:38所示抗体轻链可变区；
(5)单克隆抗体，其包含SEQ ID NO:36所示的抗体重链可变区；和SEQ ID  NO:39所示抗体轻链可变区；
(6)单克隆抗体，其包含SEQ ID NO:37所示的抗体重链可变区；和SEQ ID NO:38所示抗体轻链可变区；
(7)单克隆抗体，其包含SEQ ID NO:37所示的抗体重链可变区；和SEQ ID NO:39所示抗体轻链可变区。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述Ab为全长抗体，其进一步包括人抗体恒定区；其中所述的全长抗体选自：
h1702抗体，其是由SEQ ID NO:22所示的重链序列和SEQ ID NO:23所示的轻链序列组成的全长抗体，
h1703抗体，其是由SEQ ID NO:24所示的重链序列和SEQ ID NO:25所示的轻链序列组成的全长抗体，
h1702-DS抗体，其是由SEQ ID NO:22所示的重链序列和SEQ ID NO:26所示的轻链序列组成的全长抗体，
或者h1704-3抗体，其是由SEQ ID NO:40所示的重链序列和SEQ ID NO:41所示的轻链序列组成的全长抗体。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中细胞毒性药物选自毒素、化疗药物、抗生素、放射性同位素和核溶酶。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中细胞毒性药物选自DM1、DM3、DM4、MMAF和MMAE。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中细胞毒性药物选自：
Figure PCTCN2018098480-appb-000001
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其为式I所示化合物或其药学上可接受的盐或溶剂化合物，
Figure PCTCN2018098480-appb-000002
其中：
L 1，L 2是接头单元；
y为选自1-8的数，优选为选自2-4的数；
Ab为如上所定义的B7H3抗体或其抗原结合片段、或与如上所定义的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中L 2如以下通式L 2所示：
Figure PCTCN2018098480-appb-000003
其中
X 1选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
X 2选自烷基、环烷基和杂环基；
m为选自0-5的整数，优选为1、2或3；S为硫原子。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中L 1如以下通式(L 1)所示：
Figure PCTCN2018098480-appb-000004
其中
X 3为烷基，任选所述的烷基进一步被选自卤素、羟基和氰基的取代基所取
代；
n为选自0-5的整数，优选为1、2或3。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其中细胞毒性药物经与连接物L1连接后，得到化合物：
Figure PCTCN2018098480-appb-000005
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其为通式(II)所示的抗体-药物偶联物：
Figure PCTCN2018098480-appb-000006
其中，L 2是接头单元；
y为选自1-8的数，优选为选自2-4的数；
Ab为如上所定义的B7H3抗体或其抗原结合片段、或与如上所定义的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，其为通式(III)所示的抗体-药物偶联物：
Figure PCTCN2018098480-appb-000007
其中，L 1是接头单元；
y为选自1-8的数，优选为选自2-4的数；
Ab为如上所定义的B7H3抗体或其抗原结合片段、或与如上所定义的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。
在一个优选的实施方案中，如通式(A)所示的抗体-药物偶联物，或其可药用盐，包括但不限于：
Figure PCTCN2018098480-appb-000008
Figure PCTCN2018098480-appb-000009
Figure PCTCN2018098480-appb-000010
本发明进一步涉及一种药物组合物，其包含如本发明通式(A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，和一种或多种可药用的赋形剂、稀释剂或载体。
本发明进一步涉及通式(A)所示的抗体-药物偶联物，或其药学上可接受的盐或溶剂化合物，或包含其的药物组合物，在制备用于治疗与人B7H3阳性细胞相关的疾病的药物中的用途；优选的，其中所述的用途，其在于制备用于治疗B7H3高表达癌症的药物中的用途。
本发明进一步涉及通式(A)所示的抗体-药物偶联物，或其药学上可接受的盐或溶剂化合物，或包含其的药物组合物在制备用于治疗疾病的药物中的用途，所述疾病选自治疗胶质瘤(非限制性实施例为人脑星形胶质母细胞瘤)、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、宫颈癌、软骨肉瘤、脊索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促结缔组织增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤/恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、乳头状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、前列腺癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌和子宫癌。
本发明进一步涉及一种治疗疾病的方法，该方法包括向需要其的患者施用治疗有效剂量的该通式(A)所示的抗体-药物偶联物，或其药学上可接受的盐或溶剂化合物，或包含其的药物组合物，所述疾病选自人脑星形胶质母细胞瘤、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、宫颈癌、软骨肉瘤、脊 索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促结缔组织增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤/恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、乳头状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、前列腺癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌和子宫癌。
附图说明
图1：抗体与U87MG细胞的结合力；
图2：不同抗体在U87MG细胞上的内吞效果；
图3：本发明不同ADC h1702-3024，h1703-3024在U87MG细胞上的内吞效果；
图4：本发明不同ADC h1702-cys-3024，h1702DS-cys-3024和
h1704-3-cys-3024，及其相应抗体在U87MG细胞上的内吞效果；
图5：h1702-3024对裸鼠U87MG移植瘤的疗效；
图6：h1702-3024对U87MG裸鼠体重的影响。
图7：h1702DS-cys-3024和h1704-3-cys-3024的稳定性结果
具体实施方式
一.术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本发明所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人B7H3的单克隆抗体。制备时用B7H3抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源B7H3抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“重组抗体”包括“嵌合抗体”、“人源化抗体”和“完全人源抗体”。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本发明一个优选的实施方案中，所述的B7H3嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的B7H3嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(如YTE突变或回复突变)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网 www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。在本发明 一个优选的实施方案中，所述的B7H3人源化抗体中鼠的CDR序列选自SEQ ID NO:8-13或14-19；人的抗体可变区框架经过设计选择，其中所述抗体重链可变区上的重链FR区序列，来源于人种系重链IGHV1-18*01和hjh4.1的组合序列或IGHV3-7*01和hjh4.1的组合序列；其中所述抗体轻链可变区上的轻链FR区序列，来源于人种系轻链IGKV1-33*01和hjk4.1的组合序列或IGKV1-39*01和hjk2.1的组合序列。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
CDR的移植可由于与抗原接触的构架残基而导致产生的B7H3抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人B7H3抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查鼠单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
术语“完全人源抗体”或“全人抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。全人源抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。本发明中的“完全人抗体”采用噬菌体显示技术获得抗体可变区，可与抗体恒定区进一步重组获得完整抗体。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，B7H3)的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术 语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
本发明的抗原结合片段包括Fab、F(ab')2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)、包含CDR的肽等。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
本发明的Fab可以通过用木瓜蛋白酶处理本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
本发明的F(ab')2可以通过用胃蛋白酶处理本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab')2。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本发明的Fab'可以通过用还原剂例如二硫苏糖醇处理本发明的特异性识别B7H3并与胞外区的氨基酸序列或其三维结构结合的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本发明的scFv可以通过以下步骤来生产：获得本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
双抗体是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
本发明的双抗体可以通过以下步骤来生产：获得本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering,7,697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本发明的dsFv可以通过以下步骤来生产：获得获得本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
包含CDR的肽是通过包含VH或VL的CDR中的一个或多个区域而构成的。包含多个CDR的肽可以被直接相连或经由适合的肽接头相连。
本发明的包含CDR的肽可以通过以下步骤来生产：构建本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的CDR的编码DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达所述肽。也可以通过化学合成方法例如Fmoc方法或tBoc方法来生产所述包含CDR的肽。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的LCDR1、LCDR2和LCDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的HCDR2和HCDR3(CDR H2、CDR H3或H2、H3)。
本文中使用的术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位 (例如，B7H3分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合。
术语"KD"或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本发明的抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合B7H3，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
术语“与B7H3抗体竞争结合人B7H3的单克隆抗体”是指与本发明的单克隆抗体竞争识别人B7H3的胞外区上的相同表位(也称为抗原决定簇)或相同表位的一部分并与所述表位结合的抗体。与本发明的B7H3抗体结合相同表位的抗体是指识别并结合于本发明的B7H3抗体识别的人B7H3的氨基酸序列的抗体。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人B7H3或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科 (Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人B7H3特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本发明的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽 的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
此外，本发明涉及用于免疫检测或测定B7H3的方法、用于免疫检测或测定B7H3的试剂、用于免疫检测或测定表达B7H3的细胞的方法和用于诊断与B7H3阳性细胞相关的疾病的诊断剂，其包含本发明的特异性识别人B7H3并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体或抗体片段作为活性成分。
在本发明中，用于检测或测定B7H3的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与B7H3阳性细胞相关的疾病可以通过用本发明的单克隆抗体或抗体片段检测或测定表达B7H3的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本发明中，对用于检测或测定B7H3的活体样品没有特别限制，只要它具有包含表达B7H3的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本发明的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
术语“细胞毒性药物”是指在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语意在包括放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，化疗药物，毒素如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，包括其片段和/或变体。
术语“毒素”指来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4，auristatin F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790 A1，化合物7)，白喉 毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。
MMAF、MMAE为澳瑞他汀衍生物，参见US2005/0238649及Doronina等(2006)Bioconjugate Chem.17:114-124，结构式如下：
Figure PCTCN2018098480-appb-000011
具体而言，澳瑞他汀/多拉司他汀药物模块诸如MMAF及其衍生物可以使用US2005-0238649A1及Doronina等(2006)Bioconjugate Chem.17:114-124中记载的方法来制备。澳瑞他汀/多拉司他汀药物模块诸如MMAE及其衍生物可以使用Doronina等(2003)Nat.Biotech.21:778-784中记载的方法来制备。可以通过常规方法方便地合成药物-接头模块MC-MMAF、MC-MMAE、MC-vc-PAB-MMAF和MC-vc-PAB-MMAE，例如Doronina等(2003)Nat.Biotech.21:778-784及美国专利申请公开号US2005/0238649A1中所记载的，然后将它们偶联至感兴趣的抗体。
本发明中所述药物2852，参见WO 2016/127790 A1(化合物实施例7)，其结构式如下：
Figure PCTCN2018098480-appb-000012
通过该发明中所述方法(化合物实施例8)或所属领域人员所能推定的方法，合成药物-接头模块(本发明中所述的3024)后，其结构式如下：
Figure PCTCN2018098480-appb-000013
术语“化疗药物”是在肿瘤治疗中使用的化学化合物。化疗药物实例包括烷化剂，如噻替哌(thiotepa)；环磷酰胺(cyclosphamide)(CYTOXAN TM)；烷基磺酸脂如白消安(busulfan)，英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶(aziridine)如苯并多巴(benaodopa)，卡波醌(carboquone)，美妥替哌(meturedopa)和尿烷亚胺(uredopa)；氮丙啶和methylamelamine包括六甲蜜胺(altretamine)，三亚胺嗪(triethylenemelamine)，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)；氮芥(nitrogen mustards)如苯丁酸氮芥，萘氮芥，胆磷酰胺(cholophosphamide)，雌氮芥(estramustine)，异环磷酰胺(ifosfamide)，氮芥(mechlorethamine)，盐酸氧氮芥；左旋苯丙氨酸氮芥(melphalan)，新氮芥(novembichin)，胆甾醇苯乙酸氮芥，松龙苯芥(prednimustine)，曲磷胺(trofosfamide)，尿嘧啶氮芥；亚硝基脲(nitrosureas)如亚硝基脲氮芥(carmustine)，氯脲菌素(chlorozotocin)，福莫司汀(fotemustine)，洛莫司汀(lomustine)，尼莫司汀(nimustine)，雷莫司汀(ranimustine)；抗生素如阿克拉霉素，放线菌素，authramycin，重氮丝氨酸，博来霉素，放线菌素C(cactinomycin)，加利车霉素(calicheamicin)，carabicin,洋红霉素(chromomycin)，嗜癌素(carzinophilin)，色霉素，放线菌素D，柔红菌素(daunorubicin)，地托比星(detorubicin)，6-重氮-5-氧-L-正亮氨酸，阿霉素(doxorubicin)，表阿霉素(epirubicin)，依索比星(esorubicin)，伊达比星(idarubicin)，发波霉素(marcellomycin)，丝裂霉素，霉酚酸，诺加霉素(nogalamycin)，橄榄霉素(olivomycin)，培洛霉素(peplomycin)，potfiromycin,嘌呤霉素，三铁阿霉素(quelamycin)，罗多比星(rodorubicin)，链黑菌素；链脲霉素(streptozocin)，杀结核菌素，乌苯美司(ubenimex)，净司他丁(zinostatin)，佐柔比星(zorubicin)；抗代谢药如氨甲蝶吟，5-氟尿嘧啶(5-FU)；叶酸类似物如二甲叶酸(denopterin)，氨甲蝶呤，蝶罗呤，三甲曲沙(trimetrexate)；喋吟类似物氟达拉滨(f1udarabine)，6-巯基蝶呤，硫咪蝶呤，硫鸟蝶呤；嘧啶类似物如安西他滨(ancitabine)，阿扎胞苷(azacitidine)，6-氮尿苷，卡莫氟(carmofur)，阿糖胞苷，双脱氧尿苷，去氟氧尿苷(doxitluridine),依诺他滨(enocitabine)，氟尿苷，5-FU；雄激素类如二甲睾酮(calusterone),丙酸甲雄烷酮(dromostanolong propionate)，环硫雄醇(epitiostanol)，美雄氨(mepitiostane)，睾内酯(testolactone)；抗肾上腺类如氨鲁米特(aminoglutethimide)，米托坦(mitotane)，曲洛司坦(trilostane)；叶酸补充剂如frolinic acid；醋葡内脂；醛磷酰胺糖苷(aldophosphamideglycoside)；氨基乙酰丙酸(aminolevulinic acid)；安吖啶(amsacrine)；bestrabucil；比生群(biasntrene)；依达曲沙(edatraxate)；defofamine；秋水仙胺；地吖醌(diaziquone)；elfomithine；依利醋铵(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖(lentinan)；氯尼达明(lonidamine)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌达醇(mopidamol)；硝呋旦(nitracrine)；喷司他丁(pintostatin)；phenamet；吡柔比星(pirarubicin)；鬼臼树酸(podophyllinic acid)；2-乙基酰肼；丙卡巴肼(procarbazine)；
Figure PCTCN2018098480-appb-000014
雷佐生(razoxane)；西索菲兰(sizofiran)；锗螺胺 (spirogermanium)；细交链孢菌酮酸；三亚胺醌；2，2'，2"-三氯二乙胺(trichlorrotriethylamine)；乌拉坦(urethan)；长春碱酰胺；达卡巴嗪(dacarbazine)；甘露醇氮芥；二溴甘露醇(mitobronitol)；二溴卫矛醇；哌溴烷坑(pipobroman)；gacytosine；阿拉伯糖苷("Ara-C")；环磷酰胺；三胺硫磷(thiotepa)；紫杉烷，如紫杉醇(
Figure PCTCN2018098480-appb-000015
Bristol-Myers Squibb Oncology,Princeton,NJ)和docetaxel(
Figure PCTCN2018098480-appb-000016
Rhone-Poulenc Rorer,Antony,France)；苯丁酸氮芥；吉西他滨(gemcitabine)；6-硫代鸟嘌呤；巯基嘌呤；氨甲蝶呤；铂类似物如顺铂和卡铂；长春花碱；铂；依托泊甙(etoposide)(VP-16)；异环磷航胶；丝裂霉素C；米托蒽醌；长春新碱；长春瑞宾(vinorelbine)；新霉酰胺(navelbine)；novantrone；替尼泊甙(teniposide)；柔红霉素；氨基蝶呤；xeloda；伊拜磷酸盐(ibandronate)；CPT-11；拓扑异构酶抑制剂RFS2000；二氟甲基鸟氨酸(DMFO)；维甲酸esperamicins；capecitabine；以及上述任何物质的可药用盐，酸或衍生物。此定义还包括能调节或抑制激素对肿瘤的作用的抗激素制剂，如抗雌激素制剂包括他莫昔芬(tamoxifen)，雷洛昔芬(raloxifene)，芳香酶抑制剂4(5)-咪唑，4-羟基他莫昔芬，曲沃昔芬(trioxifene),keoxifene，LY117018,onapristone，和托瑞米芬(Fareston)；和抗雄激素制剂如氟他氨(flutamide)，尼鲁米特(nilutamide)，bicalutamide，亮丙瑞林(leuprolide)和戈舍瑞林(goserelin)；和上述任何物质的可药用盐，酸或衍生物。
术语“接头单元”在本发明中为L1和L2，指一端与抗体共价连接而另一端与细胞毒性药物相连的化学结构片段或键。
术语“载药量”是指分子中每个配体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个配体(Pc)连接1-8个细胞毒性药物，在本发明的实施方式中，载药量表示为y，可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物品均数量。
在本发明中，y可能受连接位点数量的限制。本发明的一个实施方式中，细胞毒性药物通过接头单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制抗体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
术语“载体”用于本发明的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包 载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本发明化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。
本发明还涉及治疗人B7H3阳性细胞相关的疾病的方法，特别是在治疗癌症和炎症中的用途。
二.实施例与测试例
以下结合实施例进一步描述本发明，但这些实施例及测试例并非限制着本发明的范围。本发明实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.B7H3抗原及检测用蛋白的制备
以SEQ ID NO:1所示人B7H3作为本发明B7H3的模板，设计本发明涉及的抗原及检测用蛋白的氨基酸序列。以下B7H3抗原未特殊说明的均指人B7H3。
1.1人B7H3全长氨基酸序列：B7H3(SEQ ID NO：1)：
Figure PCTCN2018098480-appb-000017
Figure PCTCN2018098480-appb-000018
注释：
双横线部分为信号肽(Signal peptide:1–28)；
划横线部分为B7H3胞外区(Extracellular domain：29-466)，其中29-139为Ig-样V-型1结构域，145–238为Ig-样C2-型1结构域；243-357为Ig-样V-型2结构域，363–456为Ig-样C2-型2结构域；
点划线部分为跨膜区部分(Transmembrane domain:467-487)；
斜体部分为胞内区(Cytoplasmic domain:488-534)。
1.2鼠B7H3全长氨基酸序列(SEQ ID NO：2)
Figure PCTCN2018098480-appb-000019
注释：
双横线部分为信号肽(Signal peptide:1–28)；
划横线部分为B7H3胞外区(Extracellular domain：29-248)，其中29–139为Ig-样V-型结构域，145–238为Ig-样C2-型结构域；
点划线部分为跨膜区部分(Transmembrane domain:249-269)；
斜体部分为胞内区(Cytoplasmic domain:270-316)。
1.3筛选及检测用人B7H3抗原(SEQ ID NO：3)
为商业化产品(R&D cat#1949-B3-050/CF，简称2Ig-B7H3)，序列如下：
Figure PCTCN2018098480-appb-000020
注释：划横线部分为B7H3胞外区；斜体部分为His-tag标记。
1.4检测用人B7H3抗原(SEQ ID NO：4)
为商业化产品(SinoBiological cat#11188-H08H，简称4Ig-B7H3)，序列如下：
Figure PCTCN2018098480-appb-000021
Figure PCTCN2018098480-appb-000022
注释：划横线部分为B7H3胞外区；斜体部分为His-tag标记。
1.5筛选及检测用鼠B7H3抗原(SEQ ID NO：5)
为商业化产品(R&D cat#1397-B3-050/CF)，序列如下：
Figure PCTCN2018098480-appb-000023
注释：划横线部分为B7H3胞外区；斜体部分为His-tag标记。
实施例2.完全人源抗体的制备
2.1阳性序列的筛选
利用人PBMC、脾脏、淋巴结组织分离B细胞，并提取RNA，构建天然单链噬菌体抗体库(库容3.2×10 10)。将构建的天然单链噬菌体文库经过包装形成噬菌体颗粒后，采用液相法进行淘筛，噬菌体与生物素化的B7H3液相结合，再采用链霉亲和素磁珠分离。为了获得可分别与人B7H3(R&D cat#1949-B3-050/CF)和鼠B7H3(R&D cat#1397-B3-050/CF)交叉结合的阳性序列，分别采用生物素化的人B7H3和生物素化的鼠B7H3进行交替淘筛，首轮采用2μg/ml生物素化的人B7H3进行淘筛，第二轮采用2μg/ml生物素化的鼠B7H3进行淘筛，第三轮采用0.5μg/ml生物素化的人B7H3进行淘筛，经三轮淘筛后，挑取500个单克隆包装成噬菌体，用于噬菌体ELISA测试。分别测试单克隆噬菌体与人B7H3(R&D cat#1949-B3-050/CF)和鼠B7H3(R&D cat#1397-B3-050/CF)的结合活性：ELISA板上分别包被1μg/ml人B7H3或鼠B7H3以及1％BSA，加入1:1封闭缓冲液稀释的噬菌体上清，最后用anti-M13 HRP检测；将ELISA测试到的OD450值大于0.5，以及结合人和鼠B7H3的ELISA OD450值除以结合1％BSA的ELISA OD450值的两个比值均大于2.0的克隆进行测序，得到9个特异性序列。
2.2完整单克隆抗体的构建
噬菌体库筛选得到的9个特异性序列构建完整抗体后通过ELISA结合实验确定其中2个抗体结合力强，分别是h1702和h1703。对其完整单克隆抗体构建的过程如下：
根据测序得到的单链抗体序列，设计引物PCR搭建各单链抗体序列的VH/VK/VL基因片段。获得h1702和h1703的重轻链可变区。
>h1702重链可变区序列
Figure PCTCN2018098480-appb-000024
                                                      SEQ ID NO：6
>h1702轻链可变区序列
Figure PCTCN2018098480-appb-000025
                                                      SEQ ID NO：7
>h1703重链可变区序列
Figure PCTCN2018098480-appb-000026
                                                     SEQ ID NO：8
>h1703轻链可变区序列
Figure PCTCN2018098480-appb-000027
                                                      SEQ ID NO：9
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
其中各抗体轻重链中CDR序列如表1所示。
表1 各重链及轻链CDR区序列
Figure PCTCN2018098480-appb-000028
抗体可变区再与恒定区基因(CH1-FC/CL)片段进行同源重组，构建完整抗体VH-CH1-FC/VK-CL/VL-CL。
构建的完整全长抗体h1702、h1703序列如下：
h1702：
h1702重链(IgG1)氨基酸序列：(SEQ ID NO：22)
Figure PCTCN2018098480-appb-000029
h1702轻链氨基酸序列：Lamada(SEQ ID NO：23)
Figure PCTCN2018098480-appb-000030
h1703：
h1703重链(IgG1)氨基酸序列：(SEQ ID NO：24)
Figure PCTCN2018098480-appb-000031
h1703轻链氨基酸序列：Kappa(SEQ ID NO：25)
Figure PCTCN2018098480-appb-000032
为进一步提高抗体的稳定性，对h1702的轻链序列进行氨基酸突变，具体突变为轻链(SEQ ID NO：23)N端第一个氨基酸残基Q替代为D，缺失突变C端第一个氨基酸残基S，以获得更加稳定和均一的单克隆抗体h1702-DS。
突变修饰后的h1702-DS的重链序列为SEQ ID NO：22，轻链氨基酸序列如下：(SEQ ID NO：26)。
Figure PCTCN2018098480-appb-000033
2.3全人抗体的表达与纯化
分别表达抗体轻重链的质粒以1.5:1的比例转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl,pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
B7H3抗体-药物偶联物制备实施例
实施例3、鼠源抗体的制备
3.1抗原的制备
编码带huFc标签的人B7-H3(h-B7H3-Fc)序列由Integrated DNA Technology(IDT)公司合成(以上B7-H3重组蛋白均由本发明设计模版序列)，分别克隆到pTT5载体上(Biovector)。重组的B7-H3蛋白在293T细胞表达后，通过所属领域的常规技术进行纯化。纯化的蛋白可用于小鼠免疫获得抗体。
h-B7H3-Fc的序列:
Figure PCTCN2018098480-appb-000034
3.2抗体的制备
抗人B7H3单克隆抗体通过免疫小鼠产生。实验用Swiss Webster白小鼠，雌性，6周龄(Charles River公司)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周， 12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。免疫抗原为带Fc标签的人B7H3重组蛋白(huB7H3-Fc)。用Titermax(sigma Lot Num：T2684)为佐剂。抗原与佐剂(titermax)比例为1:1，抗原乳化后进行接种，时间为第0、21、35、49、63天。第0天腹膜内(IP)注射15μg+爪垫(footpad)25/只的乳化后抗原。21,35,49,63天腹膜内(IP)注射15μg+爪垫(footpad)15/只的乳化后抗原,在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射15μg+爪垫(footpad)15/只的生理盐水配制的抗原溶液。于第42,56,70天进行血检，用ELISA及FACS方法检测小鼠血清，确定小鼠血清中的抗体滴度。在第5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，采用优化的电融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2018098480-appb-000035
CRL-8287 TM)进行融合得到杂交瘤细胞。
融合后的杂交瘤细胞培养7-14天后，取培养基上清，使用B7-H3重组蛋白，ELISA实验对杂交瘤上清进行抗体筛选，得到的阳性抗体株进一步使用稳转表达B7-H3的CHO-S细胞，对比空白CHO-S细胞以排除非特异性结合抗体杂交瘤株，用流式分选方法进行筛选，从而选定两株结合重组蛋白且也结合细胞表达抗原的杂交瘤。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA并反转录(PrimeScript TM Reverse Transcriptase，Takara#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增后测序，最终得到鼠源抗体m1704的序列。
鼠单抗m1704的重链和轻链可变区序列如下：
m1704重链
Figure PCTCN2018098480-appb-000036
                                                       SEQ ID NO：28
m1704轻链
Figure PCTCN2018098480-appb-000037
                                                       SEQ ID NO：29
为了提高抗体的稳定性，对上述鼠抗的CDR序列进行优化设计。优化后的CDR区具有如下序列：
名称 序列 编号
1704-HCDR1 RYGMS SEQ ID NO：30
1704-HCDR2 ISSGGGSIYYPDTVKG SEQ ID NO：31
1704-HCDR3 TRHYLLFEMDY SEQ ID NO：32
1704-LCDR1 KASQNVNTAVA SEQ ID NO：33
1704-LCDR2 SASNRYT SEQ ID NO：34
1704-LCDR3 QQYSSSLT SEQ ID NO：35
将鼠抗的重链和轻链可变区分别克隆进入含人IgG1重链恒定区和κ轻链恒定区的pTT载体质粒(Biovector)，然后瞬转转染入HEK293细胞，得到了抗B7-H3的嵌合抗体，按常规技术方法纯化后备用。
3.3小鼠抗体的人源化
鼠源抗人B7-H3单克隆抗体人源化如本领域许多文献公示的方法进行。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种抗体序列，本发明将抗体m1704进行人源化。
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同源性高的人种系模板。其中人类种系轻链框架区来自人κ轻链基因人种系轻链模板IGkV1-33，人类种系重链框架区来自人重链模版IGHV3-23
将鼠源抗体m1704的CDR区移植到选择好的相应人源化模板上，替换人源化可变区，再与IgG恒定区(优选重链为IgG1，轻链为κ)重组。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并对CDR区化学不稳定氨基酸残基优化，设计并检测了由如下人源化轻重链可变区序列组合而成的抗体。
h1704VH1(SEQ ID NO:36)：
Figure PCTCN2018098480-appb-000038
h1704VH2(SEQ ID NO:37)：
Figure PCTCN2018098480-appb-000039
h1704VL1(SEQ ID NO:38)：
Figure PCTCN2018098480-appb-000040
h1704VL2(SEQ ID NO:39)：
Figure PCTCN2018098480-appb-000041
  h1704VL1 h1704VL2
h1704VH1 h1704-1 h1704-3
h1704VH2 h1704-2 h1704-4
经表达测试和回复突变数量对比，选择出最终的人源化h1704-3抗体分子(使用VH1重链可变区和VL2轻链可变区)，其重链和轻链序列如SEQ ID NO:40和41所示。
h1704-3抗体重链(IgG1)序列：
Figure PCTCN2018098480-appb-000042
                                                       SEQ ID NO：40
h1704-3抗体轻链(Kappa)序列：
Figure PCTCN2018098480-appb-000043
                                                       SEQ ID NO：41
根据以上各人源化抗体轻链和重链的基因序列合成cDNA片段，插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences,Inc.Cat.No.23966)以1:2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。表达的抗体通过离心回收后，进行抗体纯化，得到本发明的人源化抗体蛋白。
B7H3抗体-药物偶联物制备实施例
以下实施例4至实施例11为本发明相关ADC的制备过程。其中实施例4至实施例7中抗体(h1702、h1703)通过在其赖氨酸上偶联带有连接单元的药物(MMAF或3024)反应，制备ADC；实施例8至实施例11通过抗体(h1702、h1704-3、h1702DS)半胱氨酸上的巯基,与带有连接单元的药物(3024)反应制备ADC。
实施例4：B7H3-h1702-L2-MC-MMAF(h1702-MMAF)的制备
Figure PCTCN2018098480-appb-000044
将硫代乙酸S-(3-羰基丙基)酯(0.10mg，0.75μmol)，溶解于0.2mL乙腈溶液，备用；向抗体B7H3-h1702，PH＝4.3的乙酸/乙酸钠缓冲液(2.5mg/ml，2.0mL，0.034umol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加0.1mL的氰基硼氢化钠(3.4mg,53μmol)的水溶液，于25℃下振荡反应2小时。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯以及氰基硼氢化钠，得到标题产物1b的PBS缓冲溶液(约5.5mL)，超滤离心浓缩至约2.5ml进行下一步反应。
第二步
向1b的PBS缓冲溶液(2.5mL)中加入0.07mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，得到标题产物B7H3-h1702单抗-丙硫醇1c的PBS缓冲溶液(浓度0.84mg/ml，5.0mL)。
第三步
将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸MC-MMAF(0.32mg，0.34μmol)溶解于0.5mL乙腈中，加入B7H3-h1702单抗-丙硫醇PBS缓冲溶液1c(0.84mg/mL，5.0mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物h1702-MMAF的PBS缓冲液(0.21mg/mL，14.5mL)，于4℃冷冻储存。
Q-TOF LC/MS计算平均值：y＝1.76。
实施例5：B7H3-h1703-L2-MC-MMAF(h1703-MMAF)的制备
Figure PCTCN2018098480-appb-000045
将硫代乙酸S-(3-羰基丙基)酯(0.11mg，0.82μmol)，溶解于0.2mL乙腈溶液，备用；向抗体B7H3-h1703，PH＝4.3的乙酸/乙酸钠缓冲液(2.5mg/ml，2.0mL，0.034umol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加0.1mL的氰基硼氢化钠(3.4mg,53μmol)的水溶液，于25℃下振荡反应2小时。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯以及氰基硼氢化钠，得到标题产物2b的PBS缓冲溶液(约5.5mL)，超滤离心浓缩至约2.5ml进行下一步反应。
第二步
向2b的PBS缓冲溶液(2.5mL)中加入0.07mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，得到标题产物B7H3-h1703单抗-丙硫醇2c的PBS缓冲溶液(浓 度0.82mg/ml，5.0mL)。
第三步
将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-苯丙酸MC-MMAF(0.32mg，0.34μmol)溶解于0.5mL乙腈中，加入B7H3-h1703单抗-丙硫醇PBS缓冲溶液2c(0.82mg/mL，5.0mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物h1703-MMAF的PBS缓冲液(0.20mg/mL，13.0mL)，于4℃冷冻储存。
Q-TOF LC/MS计算平均值：y＝2.29。
实施例6：B7H3-h1702-L2-3024(h1702-3024)的制备
Figure PCTCN2018098480-appb-000046
将硫代乙酸S-(3-羰基丙基)酯(0.45mg，3.38μmol)，溶解于0.8mL乙腈溶液，备用；向抗体B7H3-h1702，pH＝4.3的乙酸/乙酸钠缓冲液(5.0mg/ml，8.0mL，0.270μmol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加0.1mL的氰基硼氢化钠(12.8mg,0.2mmol)的水溶液，于25℃下振荡反应2小时。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯 以及氰基硼氢化钠，得到标题产物3b的PBS缓冲溶液(约14.5mL)，超滤离心浓缩至约7.5ml进行下一步反应。
第二步
向3b的PBS缓冲溶液(7.5mL)中加入0.20mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，得到标题产物B7H3-h1702单抗-丙硫醇3c的PBS缓冲溶液(浓度2.84mg/ml，14.0mL)。
第三步
将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸化合物3024(2.6mg，2.7μmol)溶解于1.4mL乙腈中，加入B7H3-h1702单抗-丙硫醇PBS缓冲溶液3c(2.84mg/mL，14.0mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物h1702-3024的PBS缓冲液(1.35mg/mL，27.5mL)，于4℃冷冻储存。
Q-TOF LC/MS计算平均值：y＝2.05。
实施例7：B7H3-h1703-L2-3024(h1703-3024)的制备
Figure PCTCN2018098480-appb-000047
Figure PCTCN2018098480-appb-000048
将硫代乙酸S-(3-羰基丙基)酯(0.50mg，3.78μmol)，溶解于0.8mL乙腈溶液，备用；向抗体B7H3-h1703，pH＝4.3的乙酸/乙酸钠缓冲液(5.0mg/ml，8.0mL，0.270umol)加入上述预制的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液，然后滴加0.1mL的氰基硼氢化钠(12.8mg,0.2mmol)的水溶液，于25℃下振荡反应2小时。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，除去未反应的硫代乙酸S-(3-羰基丙基)酯以及氰基硼氢化钠，得到标题产物4b的PBS缓冲溶液(约14.0mL)，超滤离心浓缩至约7.5ml进行下一步反应。
第二步
向4b的PBS缓冲溶液(7.5mL)中加入0.20mL的2.0M盐酸羟胺溶液，加毕，置于水浴振荡器，于25℃下振荡反应30分钟，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)进行纯化，得到标题产物B7H3-h1703单抗-丙硫醇4c的PBS缓冲溶液(浓度2.64mg/ml，14.5mL)。
第三步
将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸化合物3024(2.6mg，2.7μmol)溶解于1.45mL乙腈中，加入B7H3-h1703单抗-丙硫醇PBS缓冲溶液6c(2.64mg/mL，14.5mL)中，置于水浴振荡器中，于25℃下振荡反应4小时后停止反应。
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物h1703-3024的PBS缓冲液(1.20mg/mL，28.0mL)，于4℃冷冻储存。
Q-TOF LC/MS计算平均值：y＝1.62。
实施例8.B7H3-h1702-cys-3024(h1702-cys-3024)的制备
Figure PCTCN2018098480-appb-000049
在37℃条件下，向抗体h1702，pH＝6.5的0.05M的PBS缓冲水溶液(10.0mg/ml，5.0mL，0.333umol)加入配置好的0.01mM的三(2-羧乙基)膦(TCEP)的10mM水溶液0.07mL(0.7umol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；
将反应液用水浴降温至25℃，再将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸SHR3024(2.6mg，2.7μmol)溶解于0.5mL乙腈中，加入到降温至25℃的抗体h1702(含有TCEP)中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应；
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物的PBS缓冲液(6.85mg/mL，7.5mL)，于4℃冷冻储存。
HIC-HPLC计算平均值：y＝3.5。
实施例9.B7H3-h1704-3-cys-3024(h1704-3-cys-3024)的制备
Figure PCTCN2018098480-appb-000050
在37℃条件下，向抗体B7H3-h1704-3，PH＝6.5的0.05M的PBS缓冲水溶液(10.0mg/ml，5.0mL，0.333umol)加入配置好的0.01mM的三(2-羧乙基)膦(TCEP) 的10mM水溶液0.075mL(0.75umol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；
将反应液用水浴降温至25℃，再将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸化合物3024(2.6mg，2.7μmol)溶解于0.5mL乙腈中，加入到降温至25℃的抗体B7H3-h1704-3(含有TCEP)中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应；
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物的PBS缓冲液(6.75mg/mL，7.8mL)，于4℃冷冻储存。
HIC-HPLC计算平均值：y＝3.4。
实施例10.B7H3-h1702DS-cys-3024(h1702DS-cys-3024)的制备
Figure PCTCN2018098480-appb-000051
在37℃条件下，向抗体H1702-DS，pH＝6.5的0.05M的PBS缓冲水溶液(10.0mg/ml，2.0mL，0.133umol)加入配置好的0.01mM的三(2-羧乙基)膦(TCEP)的10mM水溶液0.028mL(0.28umol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；
将反应液用水浴降温至25℃，再将化合物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氢-1H-吡咯-1-基)-N-甲基己酰胺)-3-甲基丁酰胺)-N,3-二甲基丁酰胺)-3-甲氧基-5-甲基庚酰基)-2-氮杂双环[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酰胺)-3-(2-氟苯基)丙酸化合物3024(1.02mg，1.1μmol)溶解于0.2mL乙腈中，加入到降温至25℃的抗体h1702-DS(含有TCEP)中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应；
将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物的PBS缓冲液(6.65mg/mL，2.7mL)，于4℃冷冻储存。
HIC-HPLC计算平均值：y＝3.55。
以下用测试方法验证本发明抗体性能及有益效果。
测试例1.ELISA结合实验
为检测筛选到的B7H3抗体，及相应的不同B7H3-ADC对于人不同形式B7H3的体外结合能力，人2Ig-B7H3(Cat.#1949-B3-050/CF，R&D)和人4Ig-B7H3(Cat.#11188-H08H，Sino Biological)被用于进行体外结合检测。
用pH7.4的PBS(Sigma，P4417-100TAB)缓冲液将人B7H3蛋白(2Ig/4Ig)稀释至1μg/ml浓度，以100μl/孔的体积加入96孔酶标板(Corning，CLS3590-100EA)中，于4℃放置过夜16-20小时。弃去液体后，加入用PBST缓冲液(PH7.4PBS含0.05％Tween-20)稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液120μl/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板4次后，加入100μl/孔初始浓度为1μM的相应B7H3抗体(或B7H3-ADC)，用PBST缓冲液倍比稀释8个梯度，置于37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板4次，加入100μl/孔用PBST稀释(1:4000)的HRP标记的羊抗人IgG(山羊抗人IgG)Fcγ片段特异性的二抗(Jackson Immuno Research,109-005-008)，37℃孵育1小时。用PBST洗板4次后，加入100μl/孔TMB显色底物(KPL，52-00-03)，于室温孵育3-5min，加入100μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算抗体对抗原的结合EC50值，结果见表2。
表2.不同抗体及抗体ADC与人2Ig-B7H3及4Ig-B7H3抗原的结合力
EC50 人2Ig-B7H3(nM) 人4Ig-B7H3(nM)
h1702 0.11 0.16
h1703 10.28 2.10
h1702-3024 0.17 0.26
h1703-3024 15.07 4.97
h1702-MMAF 0.16 0.18
h1703-MMAF 2.36 3.08
h1702-cys-3024   0.29
h1702DS-cys-3024   0.30
h1704-3-cys-3024   0.09
结论：针对人2Ig-B7H3及人4Ig-B7H3,ADC与裸抗在ELISA实验上具有类似的结合力，即ADC标记后未降低结合力。
测试例2.与不同种属B7H3的交叉结合实验
为检测筛选到的B7H3抗体，及相应的不同B7H3-ADC，对于不同种属来源的B7H3的体外结合能力，小鼠B7H3(Cat.#1397-B3-050/CF，R&D)被用于进行体外结合检测。
用pH7.4的PBS(Sigma，P4417-100TAB)缓冲液将不同种属B7H3蛋白(mouse B7H3)稀释至1μg/ml浓度，以100μl/孔的体积加入96孔酶标板中，于4℃放置过夜16-20小时。弃去液体后，加入用PBST缓冲液(PH7.4PBS含0.05％Tween-20)稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液120μl/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板4次后，加入100μl/孔初始浓度为1μM的相应B7H3抗体(或B7H3-ADC)，用PBST缓冲液倍比稀释8个梯度，置于37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板4次，加入100μl/孔用PBST稀释(1:4000)的HRP标记的山羊抗人IgG，Fcγ片段特异性的二抗(Jackson Immuno Research,109-005-008)，37℃孵育1小时。用PBST洗板4次后，加入100μl/孔TMB显色底物(KPL,52-00-03)，于室温孵育3-5min，加入100μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算抗体对抗原的结合EC50值(结果见表3)。
表3.不同抗体与鼠B7H3抗原的结合力
EC50 鼠B7H3(nM)
h1702 18.12
h1703 86.68
h1702-3024 130.8
h1703-3024 115.3
h1702-MMAF 58.95
h1703-MMAF 115.6
结果显示，h1702和h1703，及不同ADC与鼠B7H3结合力相对较弱，显示出两个单克隆抗体具有良好的人B7H3结合特异性。
测试例3.Biacore抗体亲和力实验
用Biacore，GE仪器测定抗B7H3抗体，及抗B7H3-ADC和人2Ig-B7H3抗原，人4Ig-B7H3抗原各种抗原之间的反应亲和力。
用生物传感芯片Protein A(Cat.#29127556，GE)亲和捕获一定量的待测抗体/待测ADC，然后于芯片表面流经一系列浓度梯度下的人2Ig-B7H3抗原(Cat.#1949-B3-050/CF，R&D)、人4Ig-B7H3抗原(Cat.#11188-H08H，Sino Biological)、利用Biacore仪器(Biacore T200，GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用甘氨酸-盐酸再生溶液(pH 1.5)(Cat.#BR-1003-54，GE)将生物芯片洗净再生。实验中用到的缓冲液为HBS-EP缓冲溶液(pH 7.4)(Cat.#BR-1001-88，GE)。
实验得到的数据用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型进行拟合，从而得出亲和力数值。实验结果见表4。
表4.不同抗体/ADC和各种抗原之间的反应亲和力(单位：M)
抗体 人2Ig-B7H3 人4Ig-B7H3
h1702 7.97E-7 8.55E-9
h1703 4.48E-7 -
h1702-MMAF 4.52E-7  
h1702-3024 5.80E-7 1.05E-8
h1703-MMAF 1.93E-7  
h1703-3024 2.19E-7 结合极弱
h1702-cys-3024   1.12E-8
h1702DS-cys-3024   1.06E-8
h1704-3-cys-3024   7.78E-10
结论：针对与人2Ig-B7H3，人4Ig-B7H3在Biacore实验上的亲和力测试表明，ADC与裸抗的亲和力相似。
测试例4.体外细胞结合实验
本实验通过检测细胞表面抗体的荧光信号，根据荧光信号强弱来评价抗体的结合。将10μg一抗与2×10 5个U87MG细胞在冰上孵育30分钟后洗掉多余的抗体。将细胞与APC抗人IgG Fc(Biolegend，409306)在室温孵育30分钟，洗掉多余抗体后使用BD Verse读取细胞表面的荧光信号(结果见图1)。
结果表明：h1702与细胞表面的B7H3抗原具有很强的结合能力。
测试例5.体外细胞内吞实验
本实验通过检测细胞内抗体或不同ADC的荧光信号，根据荧光信号强弱来评价抗体的內吞效果。将B7H3抗体/ADC和APC抗人IgG Fc(Biolegend，409306)以1:2的摩尔比例混合在冰上孵育15分钟。将抗体混合物与2×10 5个U87MG细胞在冰上孵育30分钟后洗掉多余的抗体，然后将细胞转入预温37℃的培养基中，在37℃分别孵育0，15，30，60和120分钟。离心细胞并将细胞重悬在抗体洗脱液中室温孵育7分钟，洗掉抗体洗脱液，使用BD Verse读取细胞内荧光信号，见图2，图3，图4。结果可见ADC在U87MG细胞上的内吞效果与裸抗相当。
测试例6.SD大鼠T1/2评价
SD大鼠4只，雌雄各半，12/12小时光/暗调节，温度24±3℃恒温，湿度50-60％，自由进食饮水。购自杰思捷实验动物有限公司。实验当天对SD大鼠分别尾静脉注射受试药物B7H3抗体/ADC，给药剂量为3mg/kg，注射体积5ml/kg。
取血时间点为：第1天给药后5min、8h、24h(第2天)，第3天，第5天，第8天，第11天，第15天，于大鼠眼底静脉取血，每次200μL(相当于取血清100μL)；收集的血样在室温下置放半小时至凝集，然后4℃下10000×g离心10分钟。收集上清，立即放置-80℃贮存。
用ELISA检测血清中的B7H3抗体浓度，进行PK分析，结果见表5。
表5.B7H3抗体在SD大鼠中的T 1/2
受试药物 给药方式 T 1/2(平均值±SD,h)
h1702 IV(3mg/kg) 185±17
结果表明，本发明h1702在大鼠体内的半衰期约为185h(7.7天)。
用ELISA检测血清中的B7H3抗体ADC的浓度，进行PK分析，结果见表6
表6.B7H3抗体偶联物在SD大鼠中的T1/2
受试药物 分析物 给药方式 T 1/2(平均值±SD,h)
h1702-3024 总ADC(total ADC) IV(3mg/kg) 97±15
h1702DS-cys-3024 总ADC(total ADC) IV(3mg/kg) 98.20±10.16
h1704-3-cys-3024 总ADC(total ADC) IV(3mg/kg) 65.20±4.18
结果表明，大鼠静脉给予3mg/kg受试抗体偶联物h1702-3024，h1702DS-cys-3024,h1704-3-cys-3024后，总ADC(total ADC)在大鼠体内的半衰期约为97h(4.04天)，98h(4.08天)，65h(2.71天)。
测试例7.B7H3抗体的物理稳定性
利用DSC检测不同抗体的热稳定性，比较了不同的缓冲体系不同pH条件下的热稳定性情况，不同pH对应的示例性缓冲体系如10mM PB(pH7)，10mM Acetate(pH5.2)。将样品置换到对应缓冲液中，控制样品浓度在1mg/ml左右，利用MicroCal*VP-Capillary DSC(Malvern)进行检测。检测前，将各个样品及空白缓冲液用真空脱气器脱气1～2min。样品板每个孔加入400μl样品或空白缓冲液(仪器上样量为300μl)。最后两对孔板分别加入14％Decon 90和ddH 2O，以备清洗用，样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到100℃结束，扫描速率60℃/h。具体结果如表7所示，在几个测试体系中h1702、h1703均表现了较好的热稳定性。
表7.不同抗体的DSC实验结果
Figure PCTCN2018098480-appb-000052
通过SEC-HPLC监测样品纯度考察一定浓度条件下周期性稳定性，示例性的条件比如将样品浓度控制在约40-50mg/ml，在PBS(pH7.4)体系及pH5.2醋酸/蔗糖体系中比较不同抗体在比如-80℃反复冻融及4℃、30℃、40℃保存一个月的稳定性情况。利用Xbridge protein BEH SEC 200A(Waters)HPLC柱子检测抗体纯度，通过一个月的考察，h1702、h1703均表现了良好的稳定性，结果如表8所示。
表8.不同抗体的周期稳定性结果
样品 4℃/月/纯度 30℃/月/纯度 40℃/月/纯度
h1702/醋酸 99.25％ 98.68％ 97.85％
h1702/PBS 99.21％ 98.07％ 96.34％
h1703/醋酸 99.31％ 99.04％ 98.38％
h1703/PBS 99.18％ 98.56％ 96.99％
结果显示，h1702和h1703在醋酸和PBS缓冲液中均显示出良好的周期稳定性。
测试例8.B7H3抗体的化学稳定性
抗体制备后化学修饰是导致产品稳定性的常见问题之一，尤其是CDR区域的部分氨基酸高度脱酰胺、氧化或者异构化修饰一般选择尽量避免或者突变降低。取500μg待测抗体溶于500μl pH 7.4的PBS中，40℃水浴；分别于0、10、20天取样，用于酶解实验。将100μg不同时间点取样的样品溶于100μl 0.2M His-HCl，8M Gμa-HCl，pH 6.0溶液中，加3μl 0.1g/mL DTT，50℃水浴1小时，后用0.02M His-HCl，pH 6.0的溶液超滤两次，加入3μL 0.25mg/mL的胰蛋白酶(trypsin),37℃水浴酶解过夜。Agilent 6530Q-TOF进行LC-MS检测分析，对潜在的修饰位点进行质谱分析(结果见表9)，结果显示本发明中涉及的h1702、h1703均无明显的脱酰胺、氧化或者异构化加剧趋势，提示分子良好的理化稳定性。
表9.不同抗体的化学稳定性
Figure PCTCN2018098480-appb-000053
测试例9：细胞增殖实验
本实验通过检测细胞内ATP含量，根据IC50大小评价B7H3-ADC对U87MG细胞和ZR-75-1细胞增殖的抑制效果。
1、对U87MG细胞增殖的抑制效果
U87MG细胞(中科院细胞库，Catalog#TCHu138)培养在含10％FBS的EMEM培养基中，一周传代2～3次，传代比列1:2或1:5。传代时，吸掉培养基，用5ml 0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。在96孔细胞培养板中加入90μL的细胞悬液，密度为4×10 4细胞/ml，培养基为10％FBS的DMEM，96孔板外围只加入100μl 10％FBS的DMEM培养基。将培养板在培养箱培养24小时(37℃，5％CO 2)。
将待测样品用PBS或DMSO稀释成50mM，并以3倍依次稀释成10个浓度，设置成空白和对照的孔。取10μl配制成梯度浓度的化合物溶液加入到90μl新鲜培养基中。再向培养板中加入10μl上述含药物的培养基溶液。将培养板在培养箱孵育3天(37℃，5％CO 2)。在96孔细胞培养板中，每孔加入100μl CellTiter-Glo试剂，室温避光放置5-10min，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。测得的IC50值见表10。
表10.不同ADC在U87MG细胞上的增殖实验结果
化合物 IC50(nM) 最大抑制(％)
IgG >500 -0.7
h1702 >500 3.9
h1703 >500 2.4
2852 298.4 50.9
h1702-3024 14.63 49.4
h1703-3024 22.58 54.4
MMAF 1477 55.9
h1702-MMAF 20.09 46.4
h1703-MMAF 37.29 55.8
结论：裸抗偶联毒素得到的ADC在U87MG细胞上具有很好的杀伤作用。
2、对ZR-75-1细胞增殖的抑制效果
ZR-75-1细胞(ATCC，Catalog#CRL-1500)培养在含10％FBS的RPMI-1640培养基中，一周传代2～3次，传代比列1:4或1:6。传代时，吸掉培养基，用5mL0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。在96孔细胞培养板中加入90μL的细胞悬液，密度为2.8×10 4细胞/mL，96孔板外围只加入100μL 10％FBS的RPMI-1640培养基。将培养板在培养箱培养24小时(37℃，5％CO 2)。
将待测样品用PBS或DMSO稀释成50mM，并以3倍依次稀释成10个浓度，设置成空白和对照的孔。取10μl配制成梯度浓度的化合物溶液加入到90μl新鲜培养基中。再向培养板中加入10μl上述含药物的培养基溶液。将培养板在培养箱孵育6天(37℃，5％CO 2)。在96孔细胞培养板中，每孔加入100μL CellTiter-Glo试剂，室温避光放置5-10min，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。测得的IC50值见表11。
表11.不同ADC在ZR-75-1细胞上的增殖实验结果
Figure PCTCN2018098480-appb-000054
Figure PCTCN2018098480-appb-000055
结论：裸抗偶联毒素得到的ADC在ZR-75-1细胞上具有很好的杀伤作用。
3、对Detroit 562细胞增殖的抑制效果
Detroit 562细胞(ATCC，Catalog#CCL-138)培养在含10％FBS的EMEM培养基中，一周传代2～3次，传代比列1:4或1:6。传代时，吸掉培养基，用5mL 0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。在96孔细胞培养板中加入90μL的细胞悬液，密度为2.2×10 4细胞/mL，96孔板外围只加入100μL 10％FBS的EMEM培养基。将培养板在培养箱培养24小时(37℃，5％CO 2)。
将待测样品用PBS或DMSO稀释成50mM，并以3倍依次稀释成10个浓度，设置成空白和对照的孔。取10μl配制成梯度浓度的化合物溶液加入到90μl新鲜培养基中。再向培养板中加入10μl上述含药物的培养基溶液。将培养板在培养箱孵育6天(37℃，5％CO 2)。在96孔细胞培养板中，每孔加入100μL CellTiter-Glo试剂，室温避光放置5-10min，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。测得的IC50值见表12。
表12.不同ADC在Detroit 562细胞上的增殖实验结果
化合物 IC50(nM) 最大抑制(％)
IgG >500 6.4
h1702DS >500 4.55
2852 133.8 87.99
h1702DS-3024 20.78 85.92
结论：裸抗偶联毒素得到的ADC在Detroit 562细胞上具有很好的杀伤作用。
4、以上述方法对h1702DS，h1704-3抗体，及其相应的ADC:h1702-cys-3024,h1702DS-cys-3024,h1704-3-cys-3024测试对U87MG,ZR-75-1及Detroit 562细胞的增殖抑制效果，结果如下表13：
Figure PCTCN2018098480-appb-000056
Figure PCTCN2018098480-appb-000057
结论：裸抗偶联毒素得到的ADC在U87MG,ZR-75-1和Detroit562细胞上具有很好的杀伤作用。
体内活性生物学评价
测试例10：ADC对人脑星形胶质母细胞瘤U87MG裸小鼠移植瘤的疗效评价
一、测试方法
实验用BALB/cA-nude裸小鼠，雌性，6-7周，购自上海西普尔·必凯实验动物有限责任公司(合格证号：SCXK(沪)2008－0016)。饲养环境：SPF级。裸小鼠皮下接种人脑星形胶质母细胞瘤U87MG细胞(中科院，货号TCHu138)，接种细胞后第十天(肿瘤平均体积122mm 3)，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2－3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b2
其中a、b分别表示长、宽。
相对体积(RTV)＝VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。CRTV、TRTV分别为实验结束时的对照组(空白)及实验组的相对肿瘤体积。
二、测试对象
h1702-3024 ADC(1mpk,3mpk,10mpk)；
h1702DS-cys-3024 ADC(1mpk,3mpk)；
h1704-3-cys-3024 ADC(1mpk,3mpk)；
空白组(空白)：PH7.4的PBS缓冲液
三、抗体ADC的抑瘤效果
1)观察至给药开始后第26天(D26)时，受试抗体h1702-3024 ADC(1mpk,3mpk, 10mpk)的抑瘤率分别为33.68％，99.13％，100％；见图5，与对照组相比均有显著差异。给药第21天时，对照组由于肿瘤过大，个别鼠体重下降明显，并于给药第26天时，对照组有两只鼠因肿瘤太大死亡。其他各组均体重正常，见图6，表14，给药过程中未出现小鼠死亡，提示h1702-3024各给药剂量没有明显毒副作用。
h1702-3024各剂量间有一定的剂量依赖关系，在3mpk的时候已经达到95％以上的抑瘤率，10mpk达到100％的抑瘤率。
表14.给药抗体h1702-3024对荷瘤裸鼠U87MG移植瘤的疗效
Figure PCTCN2018098480-appb-000058
vs空白：*p<0.05，**p<0.01，***p<0.001
2)对受试抗体h1702DS-cys-3024 ADC，h1704-3-cys-3024 ADC抑瘤效果实验结果见表15。结果显示，腹腔注射给药1次/周，共给药3次，观察至D26时，受试ADC h1702DS-cys-3024(1mpk)的抑瘤率达到22.99％；h1702DS-cys-3024(3mpk)的抑瘤率达到98.16％；h1704-3-cys-3024(1mpk)的抑瘤率达到31.05％；h1704-3-cys-3024(3mpk)的抑瘤率达到94.83％；1mpk的3个受试抗体组与空白组相比都无显著差异(P>0.05)，3mpk的3个受试抗体组与空白组相比均有极显著差异(P＜0.001)。
给药过程中各组动物体重正常,提示ADC无明显毒副作用。
空白组和1mpk各组在26天时肿瘤体积较大，因此D26时将空白和1mpk各组处死，其余组观察至D36时，h1702DS-cys-3024(3mpk)组停药后肿瘤生长仍然较慢，抑瘤效果最好。h1702DS-cys-3024(3mpk)和h1704-3-cys-3024(3mpk)在D26和D36时的抑瘤率组间比较无明显差异(P＞0.05)。
表15.给药抗体对荷瘤裸鼠U87MG移植瘤的疗效(D26)
Figure PCTCN2018098480-appb-000059
vs空白：*p<0.05，**p<0.01，***p<0.001
测试例11：ADC对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效评价
一、测试方法
实验用BALB/cA-nude裸小鼠，雌性，6-7周，皮下接种人咽头癌胸水转移细胞Detroit 562细胞。接种细胞后第十天，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2－3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b2
其中a、b分别表示长、宽。
相对体积(RTV)＝VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。CRTV、TRTV分别为实验结束时的对照组(空白)及实验组的相对肿瘤体积。
二、测试对象
h1702DS-cys-3024 ADC(1mpk,3mpk)；
空白组(空白)：PH7.4的PBS缓冲液
三、抗体ADC的抑瘤效果
观察至D35时，受试抗体ADC抑瘤率分别是：h1702DS-cys-3024 ADC(1mpk)的抑瘤率达到39.22％(P<0.05)；h1702DS-cys-3024 ADC(3mpk)的抑瘤率达到70.50％(P<0.001)(见表16)。药过程中各组动物体重正常。
表16.给药抗体对荷瘤裸鼠Detroit 562移植瘤的疗效
Figure PCTCN2018098480-appb-000060
vs空白：*p<0.05，***p<0.001
稳定性评价
测试例12：B7H3 ADC的物理稳定性
利用DSC检测不同抗体ADC的热稳定性，比较了不同的缓冲体系不同pH条件下的热稳定性情况，不同pH对应的示例性缓冲体系如10mM PBS(pH7.4)，10mM Acetate(pH5.5)。将样品置换到对应缓冲液中，控制样品浓度在1mg/ml左右，利用MicroCal*VP-Capillary DSC(Malvern)进行检测。检测前，将各个样品及空白缓冲液用真空脱气器脱气1～2min。样品板每个孔加入400μl样品或空白缓冲液(仪器上样量为300μl)。最后两对孔板分别加入14％Decon 90和ddH 2O，以备清洗用， 样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到100℃结束，扫描速率60℃/h。具体结果如表17所示，在几个测试体系中h1702-3024、h1703-3024均表现了较好的热稳定性。
表17.不同ADC的DSC实验结果
Figure PCTCN2018098480-appb-000061
测试例13：ADC的稳定性
通过SEC-HPLC监测样品(h1702DS-cys-3024 ADC,h1704-3-cys-3024 ADC)纯度考察一定浓度条件下稳定性，示例性的条件比如将样品浓度控制在约50mg/ml，在PBS(pH7.4)体系及pH5.5醋酸/蔗糖体系(代称559体系)中比较不同抗体在40℃保存28天的稳定性情况。利用Xbridge protein BEH SEC 200A(Waters)HPLC柱子检测抗体纯度，通过28天的考察，h1702DS-cys-3024 ADC表现了良好的稳定性，结果如图7所示。
结果显示，h1702DS-cys-3024在醋酸和PBS缓冲液中均显示出良好的稳定性。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本发明的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (22)

  1. 一种通式(A)所示的抗体-药物偶联物，
    Ab-(L 2-L 1-D) y         (A)
    其中：
    D是细胞毒性药物；
    L 1，L 2是接头单元；
    y为选自1-8的数，优选为选自2-4的数；
    Ab为B7H3抗体或其抗原结合片段，其与人B7H3结合，所述B7H3抗体或其抗原结合片段是选自下面(a)至(c)中任一种的单克隆抗体或其抗原结合片段：
    (a)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：
    抗体重链可变区HCDR区序列：如SEQ ID NO：10、11和12氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：13、14和15氨基酸序列所示；
    (b)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：
    抗体重链可变区HCDR区序列：如SEQ ID NO：16、17和18氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：19、20和21氨基酸序列所示；
    (c)单克隆抗体，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：
    抗体重链可变区HCDR区序列：如SEQ ID NO：30、31和32氨基酸序列所示；和抗体轻链可变区LCDR区序列：如SEQ ID NO：33、34和35氨基酸序列所示。
  2. 一种通式(A)所示的抗体-药物偶联物，
    Ab-(L 2-L 1-D) y         (A)
    其中：
    D是细胞毒性药物；
    L1，L2是接头单元；
    y为选自1-8的数，优选为选自2-4的数；
    Ab为与权利要求1中所述的B7H3抗体或其抗原结合片段竞争结合人B7H3的单克隆抗体或其抗原结合片段。
  3. 如权利要求1或2所述的抗体-药物偶联物，其中所述Ab是重组抗体。
  4. 如权利要求3所述的抗体-药物偶联物，其中所述Ab的恒定区和/或框架区是来源于人的重组抗体或其抗原结合片段。
  5. 如权利要求4所述的抗体-药物偶联物，其中所述Ab的轻链和重链可变区上的轻链FR区和重链FR区序列分别来源于人种系轻链和重链序列或其突变序列；其中所述的恒定区包括来源于人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选人源IgG1重链恒定区；和来源于人源κ、λ链或其变体的轻链恒定区。
  6. 如权利要求5所述的抗体-药物偶联物，其中所述Ab含有SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:36或SEQ ID NO:37所示的重链可变区或其变体；所述变体是在SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:36或SEQ ID NO:37所示的重链可变区序列上具有1-10个氨基酸替换的序列；
    和含有SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:38或SEQ ID NO:39所示的轻链可变区或其变体；所述变体是在SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:38或SEQ ID NO:39所示的轻链可变区序列上具有1-10个氨基酸替换的序列。
  7. 如权利要求5所述的抗体-药物偶联物，其中所述Ab含有选自下面(1)至(7)中任一种的单克隆抗体或其抗原结合片段：
    (1)单克隆抗体，其包含SEQ ID NO:6所示的抗体重链可变区；和SEQ ID NO:7所示抗体轻链可变区；
    (2)单克隆抗体，其包含SEQ ID NO:8所示的抗体重链可变区；和SEQ ID NO:9所示抗体轻链可变区；
    (3)单克隆抗体，其包含SEQ ID NO:28所示的抗体重链可变区；和SEQ ID NO:29所示抗体轻链可变区；
    (4)单克隆抗体，其包含SEQ ID NO:36所示的抗体重链可变区；和SEQ ID NO:38所示抗体轻链可变区；
    (5)单克隆抗体，其包含SEQ ID NO:36所示的抗体重链可变区；和SEQ ID NO:39所示抗体轻链可变区；
    (6)单克隆抗体，其包含SEQ ID NO:37所示的抗体重链可变区；和SEQ ID NO:38所示抗体轻链可变区；
    (7)单克隆抗体，其包含SEQ ID NO:37所示的抗体重链可变区；和SEQ ID NO:39所示抗体轻链可变区。
  8. 如权利要求1-7中任一项所述的抗体-药物偶联物，其中所述Ab为全长抗体，其进一步包括人抗体恒定区；其中所述的全长抗体选自：
    h1702抗体，其是由SEQ ID NO:22所示的重链序列和SEQ ID NO:23所示的轻链序列组成的全长抗体，
    h1703抗体，其是由SEQ ID NO:24所示的重链序列和SEQ ID NO:25所示的轻链序列组成的全长抗体，
    h1702-DS抗体，其是由SEQ ID NO:22所示的重链序列和SEQ ID NO:26所示的轻链序列组成的全长抗体，和
    h1704-3抗体，其是由SEQ ID NO:40所示的重链序列和SEQ ID NO:41所示的轻链序列组成的全长抗体。
  9. 如权利要求1-7中任一项所述的抗体-药物偶联物，其中所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
  10. 如权利要求1-9任一项所述的抗体-药物偶联物，其中所述细胞毒性药物选自毒素、化疗药物、抗生素、放射性同位素和核溶酶。
  11. 如权利要求10所述的抗体-药物偶联物，其中所述细胞毒性药物选自DM1、DM3、DM4、MMAF和MMAE。
  12. 如权利要求10所述的抗体-药物偶联物，其中所述细胞毒性药物选自：
    Figure PCTCN2018098480-appb-100001
  13. 如权利要求10所述的抗体-药物偶联物，其为式I所示化合物或其药学上可接受的盐或溶剂化合物，
    Figure PCTCN2018098480-appb-100002
    其中：
    L 1，L 2是接头单元；
    y为选自1-8的数，优选为选自2-4的数；
    Ab为权利要求1-9中任一项所述的B7H3抗体或其抗原结合片段。
  14. 如权利要求13所述的抗体-药物偶联物，其中L 2如以下通式L 2所示：
    Figure PCTCN2018098480-appb-100003
    其中
    X 1选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    X 2选自烷基、环烷基和杂环基；
    m为选自0-5的整数，优选为1、2或3；S为硫原子。
  15. 如权利要求14所述的抗体-药物偶联物，其中L 1如以下通式(L 1)所示：
    Figure PCTCN2018098480-appb-100004
    其中
    X 3为烷基，任选所述的烷基进一步被选自卤素、羟基和氰基的取代基所取代；
    n为选自0-5的整数，优选为1、2或3。
  16. 如权利要求13所述的抗体-药物偶联物，其为通式(II)所示的抗体-药物偶联物：
    Figure PCTCN2018098480-appb-100005
  17. 如权利要求13所述的抗体-药物偶联物，其为通式(III)所示的抗体-药物偶联物：
    Figure PCTCN2018098480-appb-100006
  18. 如权利要求1或2所述的抗体-药物偶联物，其选自如下化合物：
    Figure PCTCN2018098480-appb-100007
  19. 一种药物组合物，其包含如权利要求1-18任一项所述的抗体-药物偶联物或所述抗体-药物偶联物药学上可接受的盐或溶剂化合物，和一种或多种可药用的赋形剂、稀释剂或载体。
  20. 如权利要求1-18任一项所述的抗体-药物偶联物或如权利要求19所述的药物组合物在制备用于治疗与人B7H3相关的疾病的药物中的用途。
  21. 如权利要求20所述的用途，其在于制备用于治疗B7H3高表达癌症的药物中的用途。
  22. 如权利要求1-18任一项所述的抗体-药物偶联物或如权利要求19所述的药物组合物在制备用于治疗疾病的药物中的用途，所述疾病选自人脑星形胶质母细胞瘤、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、宫颈癌、软骨肉瘤、脊索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促结缔组织增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤/恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、乳头状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、前列腺癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌和子宫癌。
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