WO2019024911A1 - Conjugué anticorps anti-b7h3-médicament et son utilisation médicale - Google Patents

Conjugué anticorps anti-b7h3-médicament et son utilisation médicale 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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PCT/CN2018/098480
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English (en)
Chinese (zh)
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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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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN201880004425.6A priority Critical patent/CN109963591B/zh
Publication of WO2019024911A1 publication Critical patent/WO2019024911A1/fr

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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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

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.

Abstract

L'invention concerne un conjugué anticorps anti-B7H3-médicament et son utilisation médicale. En particulier, l'invention concerne un conjugué anticorps anti-B7H3-médicament cytotoxique ou un sel ou solvate pharmaceutiquement acceptable de celui-ci, une composition pharmaceutique comprenant le conjugué précité ou le sel ou solvate pharmaceutiquement acceptable de celui-ci, et son utilisation dans la préparation d'un médicament pour le traitement d'une maladie ou d'une affection médiée par B7H3, notamment dans la préparation de médicaments anticancéreux.
PCT/CN2018/098480 2017-08-04 2018-08-03 Conjugué anticorps anti-b7h3-médicament et son utilisation médicale WO2019024911A1 (fr)

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WO2021190586A1 (fr) * 2020-03-25 2021-09-30 江苏恒瑞医药股份有限公司 Conjugué d'anticorps anti-b7h3 et d'analogue d'exatecan et utilisation pharmaceutique de celui-ci
WO2022200525A1 (fr) 2021-03-26 2022-09-29 Innate Pharma Protéines multi-spécifiques comprenant un site de liaison à nkp46, un site de liaison à un antigène tumoral fusionné à une cytokine pour la liaison à des cellules nk
WO2022258691A1 (fr) 2021-06-09 2022-12-15 Innate Pharma Protéines multispécifiques se liant à nkg2d, récepteur de cytokine, antigène tumoral et cd16a
WO2022258678A1 (fr) 2021-06-09 2022-12-15 Innate Pharma Protéines multispécifiques se liant à nkp30, un récepteur de cytokine, un antigène tumoral et cd16a
WO2022258662A1 (fr) 2021-06-09 2022-12-15 Innate Pharma Protéines multispécifiques se liant à nkp46, récepteur de cytokine, antigène tumoral et cd16a
WO2023236949A1 (fr) * 2022-06-07 2023-12-14 映恩生物制药（苏州）有限公司 Conjugué anticorps-médicament anti-b7h4 et son utilisation

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