WO2022262859A1 - Anticorps humanisé anti-msln humain et son utilisation - Google Patents

Anticorps humanisé anti-msln humain et son utilisation Download PDF

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WO2022262859A1
WO2022262859A1 PCT/CN2022/099508 CN2022099508W WO2022262859A1 WO 2022262859 A1 WO2022262859 A1 WO 2022262859A1 CN 2022099508 W CN2022099508 W CN 2022099508W WO 2022262859 A1 WO2022262859 A1 WO 2022262859A1
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antibody
antigen
binding fragment
cells
msln
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PCT/CN2022/099508
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Chinese (zh)
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邵小慧
刘雷
杨翠青
曹卓晓
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to CN202280037440.7A priority Critical patent/CN117412991A/zh
Publication of WO2022262859A1 publication Critical patent/WO2022262859A1/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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present disclosure relates to the field of antibodies, in particular to an anti-human MSLN humanized antibody and applications thereof.
  • MSLN Mesothelin
  • Mesothelin is a differentiation antigen present on normal mesothelial cells, and can be expressed in mesothelial cells of normal pleura, pericardium and peritoneum. Limited expression in normal tissues, but MSLN was found to be expressed in 90% of epithelioid malignant pleural mesothelioma cells, 69% of lung adenocarcinoma cells, 60% of breast cancer cells, 46% of esophageal cancer cells, pancreatic tumor cells and ovarian cancer cells. Therefore, MSLN may become an important target for cancer therapy.
  • the MSLN gene is located on chromosome 16p13.3, the full length of the gene is 8kb, the cDNA size is 2138bp, contains an open reading frame of 1884bp, 17 exons, and encodes 628 amino acids.
  • the MSLN gene encodes a 71kDa precursor protein.
  • the MSLN precursor protein is anchored on the cell membrane by glycophosphatidylinositol (GPI), and can be hydrolyzed by furin into two parts: the N-terminal soluble protein with a molecular weight of 31 kDa, called megakaryocyte enhancer factor ( megakaryocyte-potentiating factor, MPF) and a cell surface glycoprotein with a molecular weight of 40kDa, which is a mature MSLN (Chang K et al., Proc Natl Acad Sci US A.1996; 93(1):136-140; Manzanares et al., Hepatol Commun. 2017;2(2):155-172).
  • GPI glycophosphatidylinositol
  • mice The biological function of mesothelin has not been fully elucidated.
  • researchers have studied mice that knocked out the MSLN gene and found that the mice showed no abnormalities in development, reproduction, and blood cell counts, indicating that it did not affect the normal growth and development of mice. (Bera TK et al., Mol Cell Biol. 2000; 20(8):2902-2906).
  • MSLN The abnormal expression of MSLN plays an important role in the proliferation, differentiation, adhesion and drug resistance of tumor cells.
  • Overexpression of MSLN can activate multiple signaling pathways of NF- ⁇ B (nuclear factor kappa-light-chain-enhancer of activated B cells), MAPK (mitogen-activated protein kinase) and PI3K (Phosphoinositide 3-kinases), thereby inducing cell apoptosis Promoting cell proliferation, migration and metastasis by inducing the activation and expression of MMP7 (matrix metalloproteinase 7, matrix metalloproteinase-7) and MMP9 (matrix metalloproteinase 9, matrix metalloproteinase-9).
  • MSLN can block paclitaxel-induced tumor cell apoptosis and increase cancer cell resistance to drugs by simultaneously activating PI3K/AKT (Protein Kinase B, PKB) and MAPK/ERK (extracellular regulated protein kinases) signaling pathways (Bharadwaj U et al. Mol Cancer. 2011; 10:106; Cheng WF et al. Br J Cancer. 2009; 100(7):1144-1153).
  • PI3K/AKT Protein Kinase B, PKB
  • MAPK/ERK extracellular regulated protein kinases
  • Drug development targeting MSLN includes immunotoxins, vaccines, chimeric monoclonal antibodies, ADCs and CAR-Ts.
  • Antibody drugs mainly mediate tumors through antibody neutralization, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and binding of antibodies to effector molecules (toxins or inhibitors). Apoptosis or inhibition of tumor cell proliferation, targeted killing of tumor cells.
  • the present disclosure provides an anti-human MSLN humanized antibody, a nucleic acid encoding the same, a method for preparing the antibody, a pharmaceutical composition containing the antibody, and related uses of the pharmaceutical composition for treating tumors.
  • the present disclosure provides a humanized antibody or antigen-binding fragment that specifically binds human MSLN, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising a complementarity determining Regions CDR1-3 and framework regions FR1-4:
  • the CDR1-3 has the sequence shown in SEQ ID NO: 3-5 respectively, the FR1-3 has the FR1-3 derived from the human germline IGHV3-11*05, and the FR4 has the sequence derived from FR4 of IGHJ3*01; or
  • the CDR1-3 has the sequence shown in SEQ ID NO: 6-8 respectively, the FR1-3 has the FR1-3 derived from IGHV3-30*01, and the FR4 has the sequence derived from IGHJ3*01 FR4.
  • the heavy chain variable region comprises a sequence selected from (1) or (2):
  • a sequence having at most 90% identity preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; or
  • the heavy chain variable region has the following characteristics, wherein the position numbers of amino acids are determined by IMGT rules:
  • Digit 1 is E or Q; and/or,
  • Position 52 is W or L; and/or,
  • Position 54 is A or T; and/or,
  • the 55th position is Y or T; and/or,
  • the 87th bit is L or V
  • the heavy chain variable region has the following characteristics, wherein the position numbers of amino acids are determined by IMGT rules:
  • the 40th position is G, the 42nd position is Y, the 52nd position is L, the 54th position is A, and the 55th position is T; or,
  • the 40th is G
  • the 42nd is Y
  • the 49th is E
  • the 50th is R
  • the 52nd is L
  • the 54th is A
  • the 55th is T
  • the 87th is V .
  • the heavy chain variable region has the following characteristics, wherein the numbering of amino acid positions is determined by IMGT rules:
  • Position 42 is V or Y; and/or,
  • the 66th position is Y or K; and/or,
  • the 86th position is T or I; and/or,
  • the 95th position is R or K; and/or,
  • the 96th bit is A or P;
  • the heavy chain variable region has the following properties, wherein the amino acid position numbering is determined by the IMGT rules:
  • the 40th position is A, the 42nd position is Y and the 52nd position is A; or,
  • the equilibrium dissociation constant KD of the antibody or antigen-binding fragment binding to human MSLN is less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M or 1E -12M.
  • the antibody or antigen-binding fragment also binds monkey MSLN, preferably, the equilibrium dissociation constant KD of the antibody or antigen-binding fragment binding to monkey MSLN is less than 1E-7M, 1E-8M, 1E -9M, 1E-10M, 1E-11M or 1E-12M.
  • the antibody or antigen-binding fragment includes or does not include an antibody heavy chain constant region; optionally, the antibody heavy chain constant region can be selected from human, alpaca, mouse, rat , rabbit or sheep; alternatively, the heavy chain constant region of the antibody may be selected from IgG, IgM, IgA, IgE or IgD, and the IgG may be selected from IgG1, IgG2, IgG3 or IgG4; alternatively, the heavy chain The chain constant region may be selected from an Fc region, a CH3 region, a heavy chain constant region without a CH1 fragment or a complete heavy chain constant region; preferably, the heavy chain constant region is a human Fc region; preferably, the antibody or antigen binding Fragments are heavy chain antibodies.
  • the antibody heavy chain constant region can be selected from human, alpaca, mouse, rat , rabbit or sheep; alternatively, the heavy chain constant region of the antibody may be selected from IgG, IgM, IgA, IgE or IgD,
  • the antibody or antigen-binding fragment is also coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, cytotoxic agents or immunomodulators, the indicated
  • the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from alkaloids, methotrexate methotrexate, doxorubicin, taxanes or toxin compounds.
  • the antibody or antigen-binding fragment is also linked with another functional molecule, and the functional molecule can be selected from one or more of the following: signal peptide, protein tag or cytokine; preferably Preferably, the cytokine may be selected from IL-2, IL-6, IL-12, IL-15, IL-21, IFN or TNF-alpha.
  • the present disclosure provides a multispecific antibody comprising the antibody or antigen-binding fragment described in the first aspect; preferably, the multispecific antibody further comprises an MSLN-specific binding Antigens other than those described in the first aspect or antibodies or antigen-binding fragments that bind to MSLN epitopes different from those of the antibody or antigen-binding fragment of the first aspect.
  • the antigens other than MSLN can be selected from: CD3, preferably CD3 ⁇ ; CD16, preferably CD16A; CD32B; PD-1; PD-2; PD-L1; VEGF; NKG2D; CD19; CD20 ; CD40; CD47; 4-1BB; CD137; EGFR; EGFRvIII; TNF-alpha; CD33; HER2; HER3; HSA; CD5; CD27; EphA2; EpCAM; MUC1; MUC16; CEA; Claudin18.2; Folate receptor; Claudin6 ; WT1; NY-ESO-1; MAGE3; ASGPR1 or CDH16.
  • the multispecific antibody can be bispecific, trispecific or tetraspecific, and the multispecific antibody can be bivalent, tetravalent or hexavalent.
  • the present disclosure provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the The extracellular antigen-binding domain comprises an antibody or antigen-binding fragment optionally selected from the first aspect.
  • CAR chimeric antigen receptor
  • the present disclosure provides an immune effector cell expressing the chimeric antigen receptor described in the third aspect, or comprising a nucleic acid fragment encoding the chimeric antigen receptor described in the third aspect;
  • the immune effector cells are selected from T cells, NK cells (natural killer cell), NKT cells (natural killer T cell), DNT cells (double negative T cell), monocytes, macrophages, dendritic cells or mast cells, the T cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
  • the present disclosure provides an isolated nucleic acid fragment capable of encoding the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, or the chimeric antigen receptor of the third aspect.
  • the present disclosure provides a vector comprising the isolated nucleic acid fragment of the fifth aspect.
  • the present disclosure provides a host cell comprising the vector described in the sixth aspect above; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the present disclosure also provides a method for preparing an antibody or an antigen-binding fragment, or a multispecific antibody, the method comprising culturing the cells described in the seventh aspect above, and isolating the cells under suitable conditions Expressed antibodies or antigen-binding fragments, or isolated multispecific antibodies expressed by said cells.
  • the present disclosure also provides a method for preparing immune effector cells, the method comprising introducing the nucleic acid fragment encoding the CAR described in the third aspect into the immune effector cells, optionally, the method further includes Enabling the immune effector cells to express the CAR described in the third aspect.
  • the present disclosure also provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment optionally selected from the first aspect, or the multispecific antibody selected from the second aspect.
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition also includes an additional antineoplastic agent.
  • the present disclosure provides an antibody or antigen-binding fragment optionally selected from the first aspect, or a multispecific antibody optionally selected from the second aspect, or the immune effector described in the fourth aspect
  • the present disclosure also provides a method for preventing and/or treating tumors, comprising administering an effective amount of the antibody or antigen-binding fragment optionally selected from the first aspect, or any Selected from the multispecific antibody described in the second aspect, or the immune effector cell described in the fourth aspect, or the nucleic acid fragment described in the fifth aspect, or the carrier described in the sixth aspect; or the eighth and ninth aspects
  • the product obtained by the method; or the pharmaceutical composition described in the tenth aspect comprising administering an effective amount of the antibody or antigen-binding fragment optionally selected from the first aspect, or any Selected from the multispecific antibody described in the second aspect, or the immune effector cell described in the fourth aspect, or the nucleic acid fragment described in the fifth aspect, or the carrier described in the sixth aspect.
  • the tumor is preferably mesothelioma, lung cancer, breast cancer, esophageal cancer, pancreatic cancer, ovarian cancer or pleural cancer; more preferably epithelioid malignant pleural mesothelioma, lung adenocarcinoma.
  • the present disclosure also provides the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect, or the immune effector cell described in the fourth aspect, or the antibody described in the fifth aspect.
  • the nucleic acid fragment described above, or the carrier described in the sixth aspect, or the product prepared by the method described in the eighth or ninth aspect, or the pharmaceutical composition described in the tenth aspect is used to prevent and/or treat tumors; the tumor Mesothelioma, lung cancer, breast cancer, esophageal cancer, pancreatic cancer, ovarian cancer or pleural cancer are preferred; epithelioid malignant pleural mesothelioma, lung adenocarcinoma are more preferred.
  • the present disclosure provides a kit comprising the antibody or antigen-binding fragment optionally selected from the first aspect, or the multispecific antibody optionally selected from the second aspect, or the fourth aspect
  • the present disclosure provides a method for inhibiting the proliferation or migration of cells expressing MSLN in vitro, under the condition that a complex can be formed between the antibody or antigen-binding fragment optionally selected from the first aspect and MSLN, The cells are contacted with an antibody or antigen-binding fragment optionally selected from the first aspect.
  • the present disclosure provides a method for detecting the expression of MSLN, by allowing the cells to interact with Optionally contacting an antibody or antigen-binding fragment from the first aspect.
  • the term “optional” or “optionally” means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where such event or circumstance occurs or does not occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that antibody heavy chain variable regions may but need not be present; when present, there may be 1, 2 or 3.
  • MSLN refers to Mesothelin (MSLN), which is a differentiation antigen present on normal mesothelial cells and can be expressed in mesothelial cells of normal pleura, pericardium and peritoneum. The expression in normal tissues is limited, but MSLN was found to be highly expressed in epithelioid malignant pleural mesothelioma, lung adenocarcinoma, breast cancer, esophageal cancer, pancreatic tumor and ovarian cancer.
  • MSLN includes MSLN proteins of any human and non-human animal species, and specifically includes human MSLN as well as MSLN of non-human mammals.
  • the term "specifically binds” means that an antigen-binding molecule (eg, an antibody) specifically binds an antigen and substantially the same antigen with high affinity, typically, but does not bind an unrelated antigen with high affinity. Affinity is usually reflected in an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity.
  • KD equilibrium dissociation constant
  • high affinity usually refers to having about 10 -7 M or lower, about 10 -8 M or lower, about 1 ⁇ 10 -9 M or lower, about 1 ⁇ 10 -10 M or lower, KD of 1 ⁇ 10 -11 M or lower or 1 ⁇ 10 -12 M or lower.
  • KD KD/Ka, where Kd represents the dissociation rate and Ka represents the on-rate.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antibody refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g. bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), antibody conjugates) As well as antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments).
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • IgM, IgD, IgG, IgA, and IgE immunoglobulins
  • their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not comprise light chains, for example, antibodies produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca (The heavy-chain antibodies (HCAbs) produced by Vicugna pacos) and the new immunoglobulin antigen receptor (Ig new antigen receptor, IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs heavy-chain antibodies
  • Ig new antigen receptor Ig new antigen receptor
  • an antibody fragment refers to one or more antibody fragments that retain the ability to specifically bind a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • An antibody fragment may be a Fab, F(ab') 2 , scFv, SMIP, diabody, triabody, affibody, Nanobody, aptamer or domain antibody.
  • binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fd fragments consisting of VH and CH1 domains; (ii) dAb fragments or VHH consisting of VH domains; (iii) Isolated complementarity determining regions (CDRs); (iv) heavy chain antibody fragments consisting of VHH and CH2, CH3; and (v) combinations of two or more isolated CDRs, which may optionally be synthesized from Joint connection.
  • CDRs complementarity determining regions
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as whole antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • heavy chain antibody refers to an antibody that lacks the light chains of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • the term “nanobody” refers to the natural heavy chain antibody that lacks the light chain in camels, and its variable region can be cloned to obtain a single domain antibody consisting of only the variable region of the heavy chain, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Very domain of heavy chain of heavy chain antibody
  • VHH domain and “nanobody” and “single domain antibody” (single domain antibody, sdAb) have the same meaning and are used interchangeably, and refer to the variable domain of a cloned heavy chain antibody. region, to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest fully functional antigen-binding fragment. Usually, after obtaining the heavy chain antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation by the method by which the antibody was produced.
  • multispecific means having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen. Binding of different epitopes.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different targets or epitopes to which an antibody/antigen binding molecule can bind.
  • valence refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • an “antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , guanaco, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).
  • chimeric antibody refers to an antibody that has variable sequences derived from immunoglobulins of one source organism (such as rat, mouse, rabbit, or alpaca) as well as those derived from a different organism.
  • the constant region of an (eg, human) immunoglobulin e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; Gillies et al., 1985 J Immunol Methods 125:191-202; incorporated by reference above and into this article.
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.
  • the term "fully human antibody” refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, “fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region refers to the region of an antibody heavy or light chain that is involved in making the antibody bind to an antigen
  • “heavy chain variable region” is used interchangeably with “VH” and “HCVR”
  • “light chain “Variable region” and “VL” and “LCVR” are used interchangeably.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably and generally refer to the hypervariable region (HVR) of the variable region of the heavy chain (VH) or the variable region of the light chain (VL), which is due to the In terms of spatial structure, it can form a precise complementarity with the antigenic epitope, so it is also called complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • LCDR light chain variable region
  • frame region or "FR region” are used interchangeably and refer to those amino acid residues in an antibody heavy chain variable region or light chain variable region other than the CDRs.
  • FR region usually consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference).
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the chain (or its variable region);
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and second CDR of the light chain variable region (VL), respectively
  • IMGT numbering system generally refers to a numbering system based on The international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat . Immunol. 27:55-77, 2003.
  • IMGT ImMunoGeneTics information system
  • the term “heavy chain constant region” refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to The variable domains of antibodies have more conserved amino acid sequences.
  • the "heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment”, the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • typical "heavy chain constant region fragments" can be selected from Fc or CH3 domains.
  • the term "light chain constant region” refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc region is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage whereby one or more, especially one or two amino acids are excised from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include cleavage variants of the full-length heavy chain.
  • the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.
  • the IgG Fc region includes IgG CH2 and IgG CH3 domains, optionally, on this basis, it can also include a complete or partial hinge region, but does not include a CH1 domain.
  • the "CH2 domain" of a human IgG Fc region generally extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, the carbohydrate chain is attached to the CH2 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.
  • a "CH3 domain" comprises the stretch of residues in the Fc region that is C-terminal to the CH2 domain (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG).
  • the CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. having a "knob” ("knob”, knob) introduced in one of its chains and a correspondingly introduced “cavity” in the other chain thereof (CH3 domain of a "hole”; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • a variant CH3 domain e.g. having a "knob” ("knob”, knob) introduced in one of its chains and a correspondingly introduced “cavity” in the other chain thereof (CH3 domain of a "hole”; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • such variant CH3 domains can be used to facilitate heterodimerization of two non-identical antibody heavy chains.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Described in Institutes of Health, Bethesda, MD, 1991.
  • germline or “germline sequence” refers to the sequence of sequences encoded by the unrearranged (unrearranged) immunoglobulin V, D and/or J regions, or portions thereof, present in the genomic DNA of an organism. amino acid sequence. Germline represents the basic genetic information transmitted from parent to sibling prior to any functional rearrangement in the coding sequence of the gene. Non-germline gene products are produced in the case of antibody homologous recombination of individual germline genes encoding variable (V), diversity (D, heavy chain-specific) and joining genes (J); VDJ for heavy chain , and VJ for the light chain.
  • V variable
  • D diversity
  • J heavy chain-specific
  • J joining genes
  • the term "derived from a specified sequence” refers to the origin of the sequence.
  • the derivative sequence derived from a specific starting sequence has an amino acid sequence substantially identical to the starting sequence or a part of the same amino acid sequence.
  • the "derived sequence” and the “starting sequence” have at least 80%, 85%, 90% or 95% identity.
  • conservative amino acid generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • conservative substitutions for each other are examples of amino acids that are considered conservative substitutions for each other:
  • percent (%) sequence identity and “percent (%) identity” are used interchangeably and refer to the sequence after aligning sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity ( For example, for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and non-homologous sequences may be ignored for comparison purposes), after the amino acid (or nucleotide) of the candidate sequence ) residues are identical to the amino acid (or nucleotide) residues of the reference sequence.
  • alignment can be achieved in a number of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAi) software.
  • a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% to 100% sequence identity.
  • the length of a candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence .
  • chimeric antigen receptor refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and specifically bind an antigen comprising at least (1) an extracellular antigen binding domain , such as the variable heavy or light chain of an antibody, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs are able to redirect T cells and other immune effector cells to a target of choice, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen-binding domain.
  • antibody conjugate refers to a couple/conjugate formed by chemically bonding an antibody molecule to another molecule either directly or through a linker.
  • ADC antibody-drug conjugate
  • the "another molecule” may be selected from a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a cytotoxic agent or an immunomodulator, and the tracer is selected from a radiological contrast agent, Paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agents are selected from the group consisting of alkaloids, methotrexate, anthracyclines (doxorubicin) or taxanes (taxanes).
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies of the present disclosure in vitro and/or in vivo, eg, in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified.
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see e.g.
  • An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • vector includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses or other suitable replicons (eg, viral vectors).
  • DNA vectors eg, plasmids
  • RNA vectors eg, viruses or other suitable replicons
  • viral vectors eg, viral vectors.
  • a variety of vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
  • Expression vectors of the present disclosure contain polynucleotide sequences together with additional sequence elements, eg, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells.
  • vectors that can be used to express the antibodies and antibody fragments of the present disclosure include plasmids that contain regulatory sequences, such as promoter and enhancer regions, that direct transcription of the gene.
  • Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA resulting from transcription of the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosomal entry sites (IRES), and polyadenylation signal sites to direct the efficient transcription of genes carried on the expression vector.
  • Expression vectors of the present disclosure may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourthricin.
  • the step of transforming host cells with recombinant DNA described in this disclosure can be performed by conventional techniques well known to those skilled in the art.
  • the obtained transformants can be cultured by conventional methods and express the polypeptides encoded by the genes of the present disclosure.
  • the medium used in the culture can be selected from various conventional media according to the host cells used.
  • the host cells are cultured under conditions suitable for the growth of the host cells.
  • the term "pharmaceutical composition” refers to a preparation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unsuitable for the subject to which the pharmaceutical composition is administered. Additional ingredients of acceptable toxicity.
  • the terms "subject”, “subject” and “patient” refer to an organism receiving treatment for a particular disease or condition, such as cancer or an infectious disease, as described herein.
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the bovid family (such as domestic cattle, bison, buffalo, elk, and yaks), cattle, sheep, horses, and bison.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology in the subject being treated, such as a cell proliferative disorder (such as cancer or infectious disease).
  • a cell proliferative disorder such as cancer or infectious disease.
  • Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • the term "effective amount” refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. "Effective amount” also refers to an amount of a compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • FIG. 1 Amino acid numbering (IMGT) of huNB149-95-graft and huNB149-70-graft;
  • Fig. 3. detects the binding activity of human MSLN-FL-his protein and anti-MSLN antibody for ELISA
  • Figure 6A is the FACS result of detecting the expression level of HEK293T-monkey MSLN cells with NB149 antiserum;
  • Figure 6B is the FACS detection of the binding reaction of the control antibody to HEK293T-monkey MSLN cells
  • 7A-7C are ELISA detection of the binding reaction of humanized antibody and human MSLN full-length protein
  • 8A to 8C are FACS detection of the binding reaction of the humanized antibody to HEK293T-human MSLN cells
  • 9A-9C are FACS detection of the binding reaction of the humanized antibody to OVCAR3 cells.
  • Figures 10A to 10C are ELISA detection of the binding reaction of the humanized antibody to the full-length monkey MSLN protein
  • 11A-11C are FACS detection of the binding reaction of humanized antibody to HEK293T-monkey MSLN cells.
  • Alpaca (Alpaca, code: NB149) was immunized with human MSLN (Glu296-Gly580)-Fc protein (purchased from Acro, product number: MSN-H5253). Alpaca peripheral blood was collected, PBMCs were isolated, total RNA was extracted, and reverse transcribed into cDNA. The nucleic acid fragment encoding the variable region of the single domain antibody was amplified by nested PCR, cloned into a phage display vector, and electrotransformed into Escherichia coli electroporation competent cell TG1. A phage display library of single domain antibody against human MSLN was constructed and tested. After multiple rounds of panning, positive clones of anti-human MSLN single domain antibody were screened from the library. The VHH sequence information and CDRs information of the positive clones were obtained by sequencing and sequence analysis, see Table 1 for details.
  • the VHH sequences of NB149-95 and NB149-70 were recombined into the human IgG1 Fc expression vector to obtain recombinant plasmids.
  • the construction of the plasmid and the expression and purification of the antibody were completed by Taizhou Baiying Biotechnology Co., Ltd., and the purified chimeric antibodies NB149-95 VHH-hFc and NB149-70 VHH-hFc were obtained.
  • the human IgG1 Fc sequence information comes from WO1997000319A2, as follows:
  • NB149-95 VHH-hFc and NB149-70 VHH-hFc bind to human MSLN protein and cells expressing human MSLN protein, and have good specific binding activity to cells expressing monkey MSLN protein.
  • VHH-hFc and His-tagged human MSLN protein were further detected by Biacore technology, and the specific detection methods included: using a Protein A chip ( GE Healthcare; 29-127-558) captures anti-human MSLN VHH-hFc.
  • Sample and running buffer was HBS-EP+(10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25°C.
  • the sample block was set to 16°C. Both were pretreated with running buffer.
  • IGHV3-11*05 and IGHJ3*01 were selected as the humanization of alpaca antibody NB149-95
  • IGHV3-30*01 and IGHJ3*01 were selected as the humanized heavy chain template for alpaca-derived antibody NB149-70.
  • the CDRs of alpaca antibodies were grafted into the FRs of their human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the FR region sequence of the humanized antibody were back-mutated to the corresponding amino acids of the alpaca antibody to ensure the original affinity.
  • the humanized antibody VHs designed by the above methods are shown in Table 3 and Table 4, respectively, and the specific sequence information is shown in Table 5.
  • the numbering of amino acid residues and the CDR region of the antibody in this example are determined and annotated by the IMGT numbering system. For example, the amino acid numbering of huNB149-95-graft and huNB149-70-graft is shown in FIG. 1 .
  • S40G means that the 40th position is mutated from S to G, and so on for the rest, where the number of amino acid residues is determined by IMGT.
  • H40A means that the 40th position is mutated from H to A, and the rest can be deduced by analogy, where the number of amino acid residues is determined by IMGT.
  • the underlined area is the CDR region, and the rest is the framework region, and the border with characters indicates the position of the mutation.
  • the MSLN humanized antibody is expressed in the form of VHH-his. Antibody expression and purification was undertaken by Taizhou Baiying Biotechnology Co., Ltd. The purified humanized antibody was tested for protein concentration, purity, and endotoxin (Lonza kit), and the results are shown in Table 6. The results showed that, except for huNB149-95-H4, the purity of which was lower than 90%, the endotoxin concentration Within 5.0EU/mg, the purity of other antibodies was >98%, and the endotoxin concentration was within 1.0EU/mg.
  • Both the positive control antibody and the negative control antibody were constructed by Taizhou Baiying Biotechnology Co., Ltd. and the production and purification of antibodies were completed.
  • NB149-95 SEQ ID NO: 1 to construct an antibody in the form of VHH-his, named NB149-95-his.
  • the sequence of Amatuximab comes from the patent US20140127237A1, which is in the form of hIgG1 and named Tab142.
  • the YP223 sequence comes from the patent US20150252118A1, which is constructed as a rabbit IgG1 antibody and named Tab020.
  • Negative control The isotype negative control of the MSLN humanized antibody is an irrelevant antibody m971 that does not bind to the MSLN protein.
  • the sequences of the heavy chain variable region and light chain variable region are from the patent US 8591889B, constructed as VH-(G4S)3-VL -his form, named Tab084.
  • the isotype negative control of Amatuximab is the antibody against Hen Egg Lysozyme chicken egg lysozyme (purchased from Baiying, product number: B117901), named hIgG1.
  • MSLN protein has three IgG-like domains extracellularly, of which Region1 (R1) is located at the farthest membrane end, and Region3 (R3) is located at the proximal membrane end.
  • Region1 Region1
  • R3 Region3
  • the nucleotide sequence containing the amino acid sequence Glu296-Gly580 (MSLN-FL) of the extracellular region encoding human MSLN protein (NCBI: AAH09272.1) was cloned into the pTT5 vector (completed by General Biosystems (Anhui) Co., Ltd.) and followed by The established standard molecular biology method prepares the plasmid, and the specific method is referred to Sambrook, J., Fritsch, EF, and Maniatis, T. (1989).
  • HEK293E cells purchased from Suzhou Yiyan Biotechnology Co., Ltd.
  • PI Protein Engineering Company
  • FreeStyle TM 293 Thermofisher scientific, catalog number: 12338018
  • the culture supernatant was loaded onto a nickel ion affinity chromatography column HisTrap TM Excel (GE Healthcare, product number: GE17-3712-06), and an ultraviolet (UV) detector was used to monitor changes in ultraviolet absorbance (A280nm).
  • UV ultraviolet
  • the tagged human MSLN protein was dialyzed with PBS phosphate buffer (pH 7.4) at 4°C overnight.
  • the dialyzed protein was sterile-filtered at 0.22 microns and stored at -80°C to obtain purified human MSLN extracellular region protein.
  • the target bands of samples detected by SDS-PAGE reducing gel and non-reducing gel are shown in Figure 2.
  • Human MSLN-FL-his protein has binding activity with the control antibody, which is consistent with the binding characteristics of Tab142 (Amatuximab) reported in the literature. It shows that the human MSLN protein with binding activity has been prepared.
  • the nucleotide sequence encoding the full-length amino acid sequence of human MSLN was cloned into pcDNA3.1 vector and a plasmid was prepared.
  • HEK293T cell line purchased from ATCC
  • plasmid 3000Transfection Kit
  • use rabbit anti-human MSLN antibody (Tab020) and goat anti-rabbit IgG Fab antibody cell signaling, product number: 4414S
  • were used to sort positive monoclonal cells on a flow cytometer FACSAria II purchasedd from BD Biosciences to a 96-well plate, and placed at 37°C.
  • the nucleotide sequence encoding the full-length amino acid sequence of monkey MSLN (NCBI: XP_028696439.1) was cloned into the pcDNA3.1 vector and a plasmid was prepared.
  • HEK293T cell line (purchased from ATCC) was transfected with plasmid ( 3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015), and selectively cultured in DMEM/F12 medium containing 5 ⁇ g/mL puromycin and 10% (w/w) fetal bovine serum for 2 weeks, with limited Subcloning was carried out in a 96-well culture plate by the dilution method, and cultured at 37° C., 5% (v/v) CO 2 .
  • Figure 6B shows that the positive control antibody Tab142 has cross-binding activity with HEK293T-monkey-MSLN (see Example 4.1 for the specific method), which again verifies that HEK293T-monkey-MSLN can be used in subsequent experiments.
  • human MSLN protein purchased from Acro, product number: MSN-H5253
  • PBS fetal bovine serum
  • 96-well ELISA 96-well ELISA
  • the plate was sealed with a plastic film and incubated overnight at 4°C.
  • the plate was washed twice with PBST, and the blocking solution [PBS+2% (w/w) BSA] was added to block at room temperature for 2 hours. Pour off the blocking solution, wash the plate twice with PBST, and add the test antibody or control antibody with an initial concentration of 100nM and a 1:10 gradient dilution at 50 ⁇ l/well.
  • FACS buffer PBS+2% fetal calf serum
  • Example 5.2 The preparation of detection cells and antibody to be tested and the detection method refer to Example 5.2.
  • the results are shown in Figures 9A-9C and Tables 14-15, huNB149-95-H6, huNB149-95-H7, huNB149-95-H8 and huNB149-70-H6 had a better correlation with OVCAR3 endogenous cells expressing human MSLN protein. binding activity, and no binding activity to A431 cells that do not express human MSLN protein. The binding activities of the remaining humanized antibodies were weakened to varying degrees, or even completely lost.
  • the monkey MSLN full-length protein (purchased from Biointron, catalog number: 20210308A031) was diluted with PBS to a final concentration of 2 ⁇ g/mL, and ELISA was performed according to the method in Example 5.1 Detection and data analysis. The results are shown in Figures 10A to 10C and Table 16-17. Humanized huNB149-95-H6, huNB149-95-H7, huNB149-95-H8, huNB149-70-H6 and huNB149-70-H8 have effects on monkey MSLN protein The binding activity is well retained.
  • HEK293T-monkey MSLN cells and HEK293T cells were collected, and FACS detection and data analysis were performed according to the method in Example 5.2.
  • the analysis results are shown in Figures 11A-11C and Tables 18-19, wherein Tab084 is a negative control; NB149-95-his is a positive control.
  • the results showed that humanized antibodies huNB149-95-H6, huNB149-95-H7, huNB149-95-H8, huNB149-70-H6 and huNB149-70-H8 retained good binding to recombinant cells expressing monkey MSLN protein active.
  • the BIAcore 8K instrument was used to detect the binding strength of the antibody to the antigen by the anti-human antibody capture method.
  • the Anti-Human IgG antibody was immobilized on the CM5 chip (Cytiva; 29-1496-03) by amino coupling method, and the HBS -EP+pH7.4 (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (Cytiva; BR-1006-69) as the mobile phase, after mixing NHS and EDC, activate the chip for about 600 seconds, and use 10mM acetic acid Dilute Anti-Human IgG antibody to 15 ⁇ g/mL with sodium pH 5.0, inject for 420 seconds, and finally block the remaining activation sites with ethanolamine.
  • the affinity of the antibody to the antigen was determined by a multi-cycle kinetic method.
  • human MSLN-Fc protein purchased from Acro, catalog number: MSN-H5253
  • MSN-H5253 human MSLN-Fc protein
  • 3M MgCl 2 where the mobile phase is HBS-EP+pH7.4, the flow rate is 30 ⁇ L/min, the regeneration time is 30 seconds, and the detection temperature 25°C.
  • the surface plasmon resonance (SPR) method was used to determine the affinity between the MSLN humanized antibody and the monkey MSLN protein (purchased from Biointron, product number: 20210308A031), and the experimental method was referred to Example 7.1.
  • the binding rate (Kd), dissociation rate (Ka) and binding affinity (KD) of the MSLN humanized antibody to the monkey MSLN protein are shown in Table 21.

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Abstract

La présente invention concerne un anticorps humanisé anti-MSLN humain et son utilisation. En particulier, la présente invention concerne un anticorps humanisé anti-MSLN humain, un acide nucléique codant pour celui-ci, un procédé de préparation d'un anticorps, une composition pharmaceutique contenant l'anticorps, et l'utilisation associée de la composition pharmaceutique pour traiter une tumeur.
PCT/CN2022/099508 2021-06-18 2022-06-17 Anticorps humanisé anti-msln humain et son utilisation WO2022262859A1 (fr)

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