WO2022267936A1 - Anticorps spécifiquement lié à ceacam5 glycosylé - Google Patents

Anticorps spécifiquement lié à ceacam5 glycosylé Download PDF

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WO2022267936A1
WO2022267936A1 PCT/CN2022/098746 CN2022098746W WO2022267936A1 WO 2022267936 A1 WO2022267936 A1 WO 2022267936A1 CN 2022098746 W CN2022098746 W CN 2022098746W WO 2022267936 A1 WO2022267936 A1 WO 2022267936A1
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antibody
seq
amino acid
antigen
deletions
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牟男
于跃
杜靓
袁纪军
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上海吉倍生物技术有限公司
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    • 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
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
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    • 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
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    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6853—Carcino-embryonic antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates to antibodies. More specifically, this application relates to the preparation and application of a monoclonal antibody specifically binding to glycosylated CEACAM5, and its humanized antibody.
  • CEA antigen (CEACAM5) in human colon cancer tissue extracts, and then detected free CEA antigen in the peripheral blood of patients with colorectal cancer and other cancers.
  • CEA antigen is widely involved in cell adhesion, cell differentiation, cell proliferation and cell survival.
  • CEA antigen is widely and highly expressed in digestive tract cancers, such as colorectal cancer, esophageal cancer, gastric cancer, etc. It has been used as a tumor marker for decades.
  • CEACAM5 (CD66e, Carcinoembryonic antigen related cell adhesion molecule 5) is a carcinoembryonic antigen belonging to the CEA family.
  • the CEA family includes 12 genes, all of which are located on chromosome 19q13;
  • CEACAM5 is the gene with the largest molecular weight in the CEA family, expressed in the cell membrane (GPI anchor) and will be shed into the peripheral blood to become free CEA; it consists of 702 amino acids (N- A1-B1-A2-B2-A3-B3 domains in tandem), with a molecular weight of about 77KDa; the recombinantly expressed or purified CEACAM5 protein in ascites of colorectal cancer patients has a molecular weight of about 180KDa, indicating that CEACAM5 is highly glycosylated in cells.
  • CEA family antigens exist in three forms: transmembrane, GPI-anchored and secreted, and their extracellular domains are composed of immunoglobulin domains.
  • the extracellular region includes N-terminal domain, A1-3 domain and B1-3 domain formed in series.
  • CEACAM1, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM18, CEACAM20, and CEACAM21 contain N-terminal and AB domains;
  • CEACAM3, CEACAM4, and CEACAM19 only contain N-terminal domains;
  • CEACAM16 is a secreted protein.
  • CEACAM family proteins, such as CEACAM1, CEACAM3, CEACAM5, CEACAM6, and CEACAM8 have high homology, and their tissue distribution is different.
  • CEACAM1 is mainly distributed in urothelium and intestinal epithelium
  • CEACAM6 is mainly distributed in alveoli and bile ducts
  • CEACAM8 is mainly distributed in Bone marrow and hematopoietic system.
  • CEACAM5 is widely and highly expressed in many cancers, such as digestive tract cancer (colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer), lung cancer, breast cancer, etc., and is an ideal tumor-associated antigen target (TAA, Tumor Associated Antigen).
  • TAA tumor-associated antigen target
  • the development of drugs targeting CEACAM5 is mainly concentrated in the field of antibody discovery, and the sequence homology of the CEA family is relatively high. Therefore, screening specific CEACAM5-targeting antibodies may reduce side effects in clinical use, thereby increasing the therapeutic window and improving drug efficacy.
  • the invention provides a humanized antibody specifically binding to CEACAM5, which can be applied to the treatment of tumors with high expression of CEACAM5.
  • One aspect of the present invention provides a monoclonal antibody antibody or antigen-binding fragment thereof against CEACAM5, which specifically binds to the N-A1-B1 domain of CEACAM5 and does not bind to A2-B2 and A3-B3 domain.
  • a monoclonal antibody of the invention comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: CDR-H1 shown in SEQ ID NO: 1 or a substitution, deletion or addition of one or several amino acids thereto (for example, 1, 2 or 3 amino acids Substitution, deletion or addition) sequence, CDR-H2 shown in SEQ ID NO: 2 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids compared thereto) substitution, deletion or addition), and CDR-H3 shown in SEQ ID NO: 3 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) compared therewith.
  • the light chain variable region comprises: CDR-L1 shown in SEQ ID NO: 4 or a substitution, deletion or addition of one or several amino acids compared thereto (such as 1 A, 2 or 3 amino acid substitutions, deletions or additions) sequence, CDR-L2 shown in SEQ ID NO: 5 or with one or several amino acid substitutions, deletions or additions (such as 1 , 2 or 3 amino acid substitutions, deletions or additions), and the CDR-L3 shown in SEQ ID NO: 6 or with one or several amino acid substitutions, deletions or additions (such as 1 , 2 or 3 amino acid substitutions, deletions or additions); or
  • the heavy chain variable region comprises: CDR-H1 shown in SEQ ID NO: 7 or a substitution, deletion or addition of one or several amino acids thereto (for example, 1, 2 or 3 amino acids substitution, deletion or addition), the sequence of CDR-H2 shown in SEQ ID NO: 8, or a substitution, deletion or addition of one or several amino acids compared thereto (such as 1, 2 or 3 amino acids) substitution, deletion or addition), and the CDR-H3 shown in SEQ ID NO: 9 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids compared thereto) Substitution, deletion or addition) sequence; at the same time, the light chain variable region comprises: CDR-L1 shown by SEQ ID NO: 10 or a substitution, deletion or addition of one or several amino acids compared thereto (such as 1 a, 2 or 3 amino acid substitutions, deletions or additions), the CDR-L2 shown in SEQ ID NO: 11 or therewith one or several amino acid substitutions, deletions or additions (such as
  • the monoclonal antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the polypeptide shown in SEQ ID NO: 13 or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions) or addition), while the light chain variable region comprises the polypeptide shown in SEQ ID NO: 14 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions); or
  • the heavy chain variable region comprises the polypeptide shown in SEQ ID NO: 15 or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions) or addition), while the light chain variable region comprises the polypeptide shown in SEQ ID NO: 16 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions).
  • substitution described in any one of said SEQ ID NO: 1-12 is a conservative substitution
  • the CDRs of any one of said SEQ ID NOs: 1-12 are defined according to the Kabat numbering system.
  • the antibodies of the invention are humanized antibodies.
  • the present invention provides a humanized antibody or an antigen-binding fragment thereof that specifically binds CEACAM5, wherein the humanized antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the polypeptide shown in SEQ ID NO: 17 or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions) or addition), while the light chain variable region comprises the polypeptide shown in SEQ ID NO: 18 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions); or
  • the heavy chain variable region comprises the polypeptide shown in SEQ ID NO: 19 or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions) or addition), while the light chain variable region comprises the polypeptide shown in SEQ ID NO: 20 or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions or additions).
  • substitution described in any one of the SEQ ID NO: 17-20 is a conservative substitution
  • the CDRs of any one of said SEQ ID NOs: 17-20 are defined according to the Kabat numbering system.
  • the present invention provides a humanized antibody or an antigen-binding fragment thereof that specifically binds CEACAM5, wherein the antibody or an antigen-binding fragment thereof comprises a constant region derived from human immunoglobulin or a variant thereof;
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived e.g. , up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions or added or any combination thereof; and/or
  • the light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence from which it is derived e.g. , up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions or addition or any combination thereof;
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region;
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) shown in SEQ ID NO: 17 or SEQ ID NO: 19;
  • VH heavy chain variable region
  • said light chain constant region is a kappa light chain constant region
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) shown in SEQ ID NO: 18 or SEQ ID NO: 20.
  • VL light chain variable region
  • the present invention provides a humanized antibody specifically binding to CEACAM5 or an antigen-binding fragment thereof, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide bond-linked Fv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', scFv, scFv dimer, camelized single chain domain antibody, diabody, ds diabody, nanobody, Single domain antibody (sdAb), bivalent domain antibody; and/or, the antibody is murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide bond-linked Fv, BsFv, dsFv, (dsFv)2, dsFv-dsFv', scFv,
  • the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is labeled detectably, such as enzymes (such as horseradish peroxidase), radionuclides, fluorescent dyes , luminescent substances (such as chemiluminescent substances) or biotin.
  • enzymes such as horseradish peroxidase
  • radionuclides such as horseradish peroxidase
  • fluorescent dyes such as fluorescent dyes
  • luminescent substances such as chemiluminescent substances
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds CEACAM5.
  • the invention relates to an expression vector comprising a nucleic acid molecule encoding an antibody that specifically binds CEACAM5 as disclosed herein.
  • the invention relates to host cells comprising the expression vectors disclosed herein.
  • the invention in another aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one antibody that specifically binds CEACAM5 disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises an additional pharmaceutically active agent;
  • the additional pharmaceutically active agent is a drug with antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses;
  • a drug with antitumor activity such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses;
  • said antibody or antigen-binding fragment thereof, bispecific or multispecific molecule or immunoconjugate and said additional pharmaceutically active agent are provided as separate components or as components of the same composition.
  • the present invention provides a chimeric antigen receptor comprising the antigen-binding domain of the humanized antibody or an antigen-binding fragment thereof;
  • the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of the humanized antibody or antigen-binding fragment thereof;
  • said antigen binding domain is a scFv
  • said antigen-binding receptor comprises said humanized antibody or an antigen-binding fragment thereof;
  • said antigen binding receptor is expressed by immune effector cells (eg T cells).
  • immune effector cells eg T cells.
  • the present invention provides an isolated nucleic acid molecule encoding said chimeric antigen receptor.
  • the present invention provides a vector comprising the isolated nucleic acid molecule; preferably, it is used for preparing chimeric antigen receptor T cells.
  • the present invention provides a host cell comprising the isolated nucleic acid molecule or the vector;
  • the host cell is an immune effector cell (eg, T cell or NK cell);
  • an immune effector cell eg, T cell or NK cell
  • the host cell is a chimeric antigen receptor T cell (CAR-T).
  • CAR-T chimeric antigen receptor T cell
  • the present invention provides a method for reducing the expression level of CEACAM5 on the cell surface, which comprises combining said cells with said humanized antibody or its antigen-binding fragment, or said pharmaceutical composition, or said The chimeric antigen receptor, or the contacting of the host cell, reduces the expression level of CEACAM5 on the surface of the cell; wherein the cell expresses CEACAM5 on its surface;
  • the cells are tumor cells expressing CEACAM5.
  • the present invention provides a method for inhibiting the growth of tumor cells expressing CEACAM5 and/or killing the tumor cells, which comprises combining the tumor cells with an effective amount of the humanized antibody or antigen-binding fragment thereof, or the The pharmaceutical composition, or the chimeric antigen receptor, or the host cell is contacted.
  • the present invention relates to a method for preparing an antibody that specifically binds CEACAM5, comprising expressing in a host cell a nucleic acid sequence encoding an antibody that specifically binds CEACAM5 as disclosed herein and isolating the specific binding antibody from the host cell.
  • the present invention provides the use of the antibody of the present invention in the preparation of a medicament for treating cancers that highly express CEACAM5, wherein the cancers that highly express CEACAM5 are selected from colorectal cancer, gastric cancer, pancreatic cancer, and esophageal cancer , lung or breast cancer.
  • the present invention provides a method for treating cancers that highly express CEACAM5, comprising administering the antibody of the present invention to a subject in need thereof, wherein the cancers that highly express CEACAM5 are selected from colorectal cancer, gastric cancer , pancreatic, esophageal, lung, or breast cancer.
  • the subject is a mammal, such as a human;
  • the method further comprises administering additional drugs with antitumor activity, such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses;
  • antitumor activity such as alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses;
  • the method further comprises administering an additional antineoplastic therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative therapy.
  • an additional antineoplastic therapy such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative therapy.
  • the present invention also provides an antibody-drug conjugate, which contains:
  • the drug is a toxin, preferably monomethylauristatin (monomethylauristatin), calicheamicin, maytansinoids, or a combination thereof; more preferably selected from: monomethylauristatin-E ( MMAE), monomethylauristatin-D (MMAD), monomethylauristatin-F (MMAF), or a combination thereof.
  • monomethylauristatin monomethylauristatin
  • calicheamicin maytansinoids
  • MMAF monomethylauristatin-F
  • the present invention provides the use of the antibody-drug conjugate in the preparation of medicines.
  • the present invention provides a bispecific antibody, which comprises a domain specifically binding to CEACAM5 and a domain specifically binding to CD3, CD28 or 41BB,
  • structural domain specifically binding to CEACAM5 comprises: CDR-H1 shown in SEQ ID NO:1; CDR-H2 shown in SEQ ID NO:2, CDR-H3 shown in SEQ ID NO:3, SEQ ID NO: CDR-L1 set forth in 4, CDR-L2 set forth in SEQ ID NO:5, and CDR-L3 set forth in SEQ ID NO:6; or
  • CDR-H1 shown in SEQ ID NO:7 CDR-H2 shown in SEQ ID NO:8, CDR-H3 shown in SEQ ID NO:9, CDR-L1 shown in SEQ ID NO:10, SEQ ID CDR-L2 shown in NO:11, and CDR-L3 shown in SEQ ID NO:12.
  • the present invention also provides the use of the bispecific antibody in the preparation of medicines.
  • Monoclonal antibodies can be prepared as follows. Injected mice or other suitable host animals are first immunized with the immunogen (adding an adjuvant if necessary).
  • the immunogen or adjuvant is usually injected subcutaneously at multiple points or intraperitoneally.
  • the immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host.
  • the adjuvant can be Freund's adjuvant or MPL-TDM, etc.
  • lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body. In addition, lymphocytes can also be obtained by in vitro immunization.
  • the target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996).
  • a suitable fusion agent such as PEG
  • the preferred myeloma cells should have the characteristics of high fusion rate, stable antibody secretion ability, and sensitivity to HAT medium.
  • the culture medium of growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens.
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the affinity of mAbs can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107:220 (1980).
  • the target cell line can be limited by the criteria described in (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). Subcloning by dilution.
  • a suitable culture medium can be DMEM or RPMI-1640, etc.
  • hybridoma cells can also be grown in animals in the form of ascites tumors.
  • Monoclonal antibodies secreted by subcloned cells can be purified from cell culture fluid, isolated from ascites or serum.
  • Monoclonal antibodies can also be obtained through genetic engineering and recombination techniques.
  • the DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells by using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody to carry out PCR amplification. Insert the obtained DNA molecule into the expression vector, then transfect host cells (such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin), and culture under appropriate conditions, Antibodies of interest can be obtained recombinantly expressed.
  • host cells such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin
  • a tumor cell line with high expression of CEACAM5 (such as Lovo) is used to immunize mice to obtain antibodies that recognize natural CEACAM5 antigen.
  • a “humanized antibody” is an antibody comprising one or both of a humanized VH domain and a humanized VL domain.
  • the one or more immunoglobulin constant regions need not be present, but if present, are derived entirely or substantially from human immunoglobulin constant regions.
  • Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor” antibody are grafted into human "recipient” antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557).
  • the recipient antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline sequence.
  • the human acceptor sequence can be chosen such that the variable region framework has a high degree of sequence identity to the donor sequence to match typical patterns and other criteria between the acceptor and donor CDRs.
  • a humanized antibody is one whose CDRs are derived wholly or substantially from the donor antibody and variable region framework sequences and whose constant regions, if present, are derived wholly or substantially from human antibody sequences.
  • a humanized heavy chain typically has all three CDRs derived from the heavy chain and heavy chain variable region framework sequences of the donor antibody and substantially from human heavy chain variable region framework and constant region sequences. Chain constant region (if present).
  • a humanized light chain typically has all three CDRs derived entirely or substantially from the light chain and light chain variable region framework sequences of the donor antibody and light chains substantially derived from the human light chain variable region framework and constant region sequences. Chain constant region (if present).
  • the CDRs in a humanized antibody are substantially derived from the corresponding CDRs in a non-human antibody.
  • variable region framework sequences of the antibody chains or the constant regions of the antibody chains, respectively are substantially derived from humans Variable region framework sequences or human constant regions.
  • humanized antibodies can also be composed of fewer than all six CDRs (e.g., at least 3 CDRs) from a mouse antibody. 1, 4 or 5) CDR composition (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol.36 : 1079-1091, 1999; Tamura et al., Journal of Immunology, 164: 1432-1441, 2000).
  • CDR composition e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol.36 : 1079-1091, 1999; Tamura et al., Journal of Immunology, 164: 1432-1441, 2000).
  • a CDR in a humanized antibody is "substantially from the corresponding CDRs in a non-human antibody.
  • the CDRs of the humanized VH or VL domain span all three CDRs with respect to the corresponding non-human VH or VL CDRs. No more than six (eg, no more than five, no more than four, no more than three, no more than two, or no more than one) amino acid substitutions (preferably conservative substitutions).
  • variable region framework sequences of antibody VH or VL domains or immunoglobulin constant region are "substantially derived from" human VH or VL framework sequences or human constant regions, respectively.
  • all portions of a humanized antibody, except the CDRs will usually be derived wholly or substantially from corresponding portions of native human immunoglobulin sequences.
  • antibody refers to an immunoglobulin molecule, usually composed of two pairs of polypeptide chains, each pair having one light chain and one heavy chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.Mol.Biol.196:901-917; Definition by Chothia et al. (1989) Nature 342:878-883.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • antibody when the term “antibody” is referred to, it includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for Specific binding to an antigen, which is also referred to as an "antigen-binding moiety". See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, N.Y.
  • antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Antigen-binding fragments of antibodies can be obtained from a given antibody using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and can be used in the same way as for the intact Antigen-binding fragments of antibodies are screened for specificity in the same manner as antibodies.
  • mAb and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a population of highly homogeneous antibody molecules, that is, excluding natural mutations that may occur spontaneously , a group of identical antibody molecules. mAbs are highly specific for a single epitope on an antigen. Compared with monoclonal antibodies, polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens. Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (see, for example, U.S.P 4,816,567).
  • monoclonal antibodies can be prepared as follows. Injected mice or other suitable host animals are first immunized with the immunogen (adding an adjuvant if necessary).
  • the immunogen or adjuvant is usually injected subcutaneously at multiple points or intraperitoneally.
  • the immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host.
  • the adjuvant can be Freund's adjuvant or MPL-TDM, etc.
  • lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body.
  • lymphocytes can also be obtained by in vitro immunization.
  • the target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996).
  • a suitable fusion agent such as PEG
  • the hybridoma cells prepared above can be inoculated to grow in a suitable culture medium, which preferably contains one or more substances capable of inhibiting the growth of unfused, parental myeloma cells.
  • HGPRT hypoxanthine guanine phosphotransferase
  • adding substances such as hypoxanthine, aminopterin, and thymine (HAT medium) to the culture medium will inhibit HGPRT- Defective cell growth.
  • HAT medium hypoxanthine, aminopterin, and thymine
  • the preferred myeloma cells should have the characteristics of high fusion rate, stable antibody secretion ability, and sensitivity to HAT medium.
  • murine myeloma is the first choice for myeloma cells, such as MOP-21 or MC-11 mouse tumor derivative strain (THE Salk Institute Cell Distribution Center, San Diego, Calif.
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the affinity of mAbs can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107:220 (1980).
  • the target cell line can be limited by the criteria described in (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). Subcloning by dilution.
  • a suitable culture medium can be DMEM or RPMI-1640, etc.
  • hybridoma cells can be grown in animals in the form of ascites tumors.
  • Monoclonal antibodies secreted by subcloned cells can be purified from cell culture fluid, isolated from ascites or serum.
  • Monoclonal antibodies can also be obtained through genetic engineering and recombination techniques.
  • the DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells by using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody to carry out PCR amplification. Insert the obtained DNA molecule into the expression vector, then transfect host cells (such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin), and culture under appropriate conditions, Antibodies of interest can be obtained recombinantly expressed.
  • host cells such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin
  • chimeric antibody refers to an antibody whose light chain and/or heavy chain are partially derived from one antibody (which may be derived from a specific species or belong to a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains Binding activity to target antigen (U.S.P 4,816,567to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • human antibody refers to a humanized antibody in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by the CDR regions of a non-human antibody (donor antibody)
  • the donor antibody may be a non-human (for example, mouse, rat or rabbit) antibody with desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, so as to further improve or optimize the performance of the antibody.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or an antibody.
  • An “epitope” is also referred to in the art as an "antigenic determinant”.
  • Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrate or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes 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, which may be "linear of” or “conformational”. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • epitope peptide refers to a peptide segment on an antigen that can serve as an epitope.
  • an epitope peptide alone is capable of being specifically recognized/bound by an antibody directed against the epitope.
  • carrier protein refers to a protein that can act as a carrier for an epitope peptide, i.e., it can insert an epitope peptide at a specific position (e.g., inside the protein, N-terminal or C-terminal) , so that the epitope peptide can be presented, so that the epitope peptide can be recognized by antibodies or the immune system.
  • carrier proteins are well known to those skilled in the art and include, for example, the HPV L1 protein (epitope peptides may be inserted between amino acids 130-131 or between amino acids 426-427 of the protein, see Slupetzky ,K.
  • HBV core antigen the 79th part of the protein can be replaced with an epitope peptide -81 amino acid, see Koletzki, D., et al.HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein[J].Biol Chem,1999,380:325-333), Woodchuck hepatitis virus core protein (amino acids 79-81 of the protein can be replaced with epitope peptides, see Sab
  • CRM197 protein epitope peptides can be linked to the N- or C-terminus of the protein or fragments thereof.
  • a linker eg, a flexible or rigid linker
  • the epitope peptide and the carrier protein can be used to facilitate folding of both.
  • Antibodies can be competitively screened for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to an antigen (eg, influenza virus hemagglutinin protein). A high-throughput method to obtain antibodies binding to the same epitope based on their cross-competition is described in International Patent Application WO 03/48731. Thus, antibodies and antigen-binding fragments thereof (ie, antigen-binding portions) that compete with the monoclonal antibodies of the invention for binding to the same epitope on the influenza virus hemagglutinin protein can be obtained using routine techniques known to those skilled in the art.
  • an antigen eg, influenza virus hemagglutinin protein
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (K D ) of 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and antigen.
  • Figure 1 shows the binding results of recombinant chimeric antibody to CEACAM5 recombinant protein.
  • Figure 2 shows the binding of recombinant chimeric antibody to CEACAM5 domain.
  • Figure 3 shows the binding of recombinant chimeric antibody to LS174T cells and KATO3 cells highly expressing CEACAM5.
  • Figure 4 shows the binding of humanized antibodies to recombinant CEACAM5 antigen.
  • Figure 5 shows the binding of humanized antibody pairs to LS174T cells highly expressing CEACAM5.
  • SEQ ID NO: describe 1 M22 heavy chain variable region CDR1 2 M22 heavy chain variable region CDR2 3 M22 heavy chain variable region CDR3 4 M22 light chain variable region CDR1 5 M22 light chain variable region CDR2 6 M22 light chain variable region CDR3 7 M25 heavy chain variable region CDR1 8 M25 heavy chain variable region CDR2 9 M25 heavy chain variable region CDR3 10 M25 light chain variable region CDR1 11 M25 light chain variable region CDR2 12 M25 light chain variable region CDR3 13 M22 heavy chain variable region 14 M22 light chain variable region
  • a tumor cell line expressing CEACAM5 was used to immunize mice to prepare monoclonal antibodies.
  • the Lovo cell line ATCC CCL-229 which highly expresses CEACAM5
  • Anti-CEACAM5 antibody hMN14 (Immunomedics, Phase 2 drug) was used to Lovo cells, expressed through recombinant, the sequence is as follows
  • the Lovo 6-1 clone was knocked out of the CEACAM5 gene by the CRISPR method, and the Lovo CEACAM5 KO cell line (hereinafter referred to as the Lovo CEA KO cell line) was screened.
  • the vector carrying CRISPR and sgRNA was packaged into a lentiviral vector (CEACAM5 KO1-3), and transduced into Lovo 6-1 cells. After transduction, CEACAM5 expression (MN14 antibody) was detected by FACS, and MN14 binding was detected by mouse Fc-APC secondary antibody .
  • the results showed that CEACAM5 KO1-3 could knock out CEACAM5, and the knockout efficiency of CEACAM5 KO3 carrier was higher, and the negative population of CEACAM5 was clearer.
  • the sequences of the three sgRNAs targeting CEACAM5 are as follows:
  • Lovo cells and Lovo CEA KO cells at 10 4 per well in a 96-well plate for overnight culture, add 1-10 ⁇ l of the hybridoma supernatant to the Lovo/Lovo CEA KO cell culture plate, incubate for 1 hour, and discard the supernatant
  • Add anti-mouse Fc-FITC fluorescent secondary antibody after incubation for 1 hour, discard the supernatant, add DPBS solution containing 2% BSA, read and analyze the fluorescent signal and FITC staining area in Celigo.
  • the picked hybridoma clones were subjected to hybridoma sequencing according to a standard hybridoma sequencing method to obtain the heavy chain and light chain variable regions (VH and VL) of the picked clones.
  • VH and VL were synthesized by whole gene synthesis and linked to the human IgG1 and kappa chain constant regions, and the heavy chain and light chain sequences were linked to the pcDNA3.4 vector for transient expression in the 293 system and protein A/G purification.
  • the resulting chimeric recombinant antibody was subjected to ultrafiltration for buffer replacement with PBS solution.
  • the sequencing results are shown in the table below.
  • the CEACAM5 molecule was split according to its extracellular domain (A1-B1-A2-B2-A3-B3), and A1-B1-His Tag, A2-B2-His Tag, and A3-B3-His Tag were respectively constructed for expression Vector, and after being expressed in the 293 system, it was purified using a Ni column, and the sequence is shown in the table below:
  • LS174T cells and KATO3 cells were cultured in RPMI1640 medium containing 10% FBS. After the cells were digested with trypsin, they were centrifuged and resuspended in DPBS solution (FACS buffer, 4 °C) containing 2% BSA.
  • DPBS solution FACS buffer, 4 °C
  • CEA family Gene ID mRNA sequence CEACAM1 634 NM_001712.5 CEACAM3 1084 NM_001815.5 CEACAM5 1048 NM_004363.6 CEACAM6 4680 NM_002483.7 CEACAM8 1088 NM_001816.4
  • cM22hIgG1 and cM25hIgG1 antibodies to the above cells at a final concentration of 5ug/ml, namely RKO, RKO-CEA1, RKO-CEA3, RKO-CEA5, RKO-CEA6, RKO-CEA8, and perform cell binding FACS determination.
  • the positive percentage of antibody binding to the corresponding cell line the result is as follows:
  • VH and VL regions of the above antibodies were linked to the human IgG1 Fc region and the kappa constant region, and the heavy and light chain sequences of the antibody were inserted into the pcDNA3.4 vector, transiently expressed in 293 cells, and purified by protein A or G.
  • LS174T cells (high expression of CEACAM5; ATCC, CL-188) were cultured in RPMI1640 medium containing 10% FBS, digested with trypsin, centrifuged and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C ), add 5 ⁇ 10 5 /100 ⁇ l/well into a U-bottom 96-well plate, and add antibody diluted in a gradient concentration, incubate at 4°C for 1 hour, discard the supernatant after centrifugation; add 100 ⁇ l containing anti-human IgG Fc-APC secondary antibody was incubated at 4°C for 1 hour, washed once with FACS buffer, resuspended in 200 ⁇ l FACS buffer, and the fluorescence signal value was read on BD CantoII. The result is shown in Figure 5.
  • hM22.9hIgG1, hM25.6hIgG1, hAb796hIgG1, hPR1A3hIgG1, and hIgG Ctrl were dye-labeled with pHAb amino-conjugated dyes (labeled antibodies are endocytosed in a low-pH environment, and excitation light at 532nm and 560nm can detect excitation fluorescence).
  • pHAb amino-conjugated dyes labeled antibodies are endocytosed in a low-pH environment, and excitation light at 532nm and 560nm can detect excitation fluorescence.
  • Add the above antibody to MKN45 cells to a final concentration of 10 ⁇ g/ml, and incubate at 37°C for 24 hours. After the cells are washed, read the fluorescence value of the PE channel to detect the endocytosis of the labeled antibody.

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Abstract

La présente invention concerne un anticorps spécifiquement lié à CEACAM5, la préparation d'un anticorps humanisé associé et son utilisation.
PCT/CN2022/098746 2021-06-23 2022-06-14 Anticorps spécifiquement lié à ceacam5 glycosylé WO2022267936A1 (fr)

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