WO2020244526A1 - 一种抗ceacam5的单克隆抗体及其制备方法和用途 - Google Patents

一种抗ceacam5的单克隆抗体及其制备方法和用途 Download PDF

Info

Publication number
WO2020244526A1
WO2020244526A1 PCT/CN2020/094045 CN2020094045W WO2020244526A1 WO 2020244526 A1 WO2020244526 A1 WO 2020244526A1 CN 2020094045 W CN2020094045 W CN 2020094045W WO 2020244526 A1 WO2020244526 A1 WO 2020244526A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
seq
binding fragment
cell
Prior art date
Application number
PCT/CN2020/094045
Other languages
English (en)
French (fr)
Inventor
莫世甫
徐伟
Original Assignee
上海吉倍生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海吉倍生物技术有限公司 filed Critical 上海吉倍生物技术有限公司
Priority to AU2020289301A priority Critical patent/AU2020289301A1/en
Priority to CN202080040435.2A priority patent/CN113906052B/zh
Priority to CA3142635A priority patent/CA3142635A1/en
Priority to KR1020227000006A priority patent/KR20220016943A/ko
Priority to US17/596,183 priority patent/US20220242968A1/en
Priority to JP2021572365A priority patent/JP2022536114A/ja
Priority to EP20818729.4A priority patent/EP3981793A4/en
Priority to BR112021024544A priority patent/BR112021024544A2/pt
Publication of WO2020244526A1 publication Critical patent/WO2020244526A1/zh

Links

Images

Classifications

    • 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
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464482—Carcinoembryonic antigen [CEA]
    • 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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/50—Colon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This application belongs to the field of biological immunity technology, and specifically relates to monoclonal antibodies and antigen-binding fragments thereof that can specifically bind to human carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5). This application also relates to the preparation method and use of the antibody and its antigen-binding fragment.
  • CEACAM5 human carcinoembryonic antigen cell adhesion molecule 5
  • CEACAM human carcinoembryonic antigen cell adhesion molecule
  • CEACAM protein is located outside the cell membrane, among which CEACAM1, CEACAM3 and CEACAM4 are connected to the cell membrane through a hydrophobic transmembrane domain; CEACAM5-8 is connected to the cell membrane through glycosyl phosphoryl inositol. These extracellular domains usually serve as adhesion molecules between cells (e.g., epithelium, endothelium, dendrites, and leukocytes).
  • CEACAM involves a variety of cell functions. Based on the adhesion function between cells, it regulates cell growth and differentiation through signal transduction, and plays an important role in insulin homeostasis, angiogenesis and immune regulation. Members of the CEACAM gene family are involved in a variety of pathophysiological roles, including as receptors for microbial pathogens. They play an important role in carcinogenesis, especially in cancer detection, progression and metastasis.
  • CEACAM5 (referred to as CEA, also known as CD66e) is a glycoprotein with a molecular weight of about 180kDa. CEACAM5 contains 7 domains connected to the cell membrane via glycosylphosphatidylinositol (GPI) anchors.
  • GPI glycosylphosphatidylinositol
  • the 7 domains include a single N-terminal Ig variable domain and 6 domains (A1-B1-A2-B2-A3-B3) homologous to the Ig constant domain.
  • CEACAM5 was originally thought to be a protein expressed in fetal tissues, and has now been identified in several normal adult tissues. Overexpression of CEACAM5 has been observed in many types of cancer. For example, CEACAM5 can be detected in the blood of colon cancer patients. Moreover, further studies have determined that its overexpression is associated with many malignant tumors, usually with poor prognosis. In prostate cancer and colorectal cancer, the overexpression of CEACAM5 has been shown to be a tumor biomarker.
  • CEACAM5/CEACAM6 have also been found to be overexpressed in a variety of malignant tumors, such as breast, pancreas, ovarian, colon, lung, and gastric gland tumors, and are related to tumor invasion and metastasis.
  • malignant tumors such as breast, pancreas, ovarian, colon, lung, and gastric gland tumors
  • CEACAM5 binds to its receptor CEAr, and their interaction leads to the activation and production of pro-inflammatory cytokines, mainly IL-1, IL-6, IL-10 and TNF- ⁇ .
  • pro-inflammatory cytokines mainly IL-1, IL-6, IL-10 and TNF- ⁇ .
  • these cytokines change the microenvironment of hepatocytes and Kupffer cells, as well as their interaction with sinusoidal cells. These interactions not only affect tumor cells or other liver cells, but also seem to promote the vitality of CSC and other circulating tumor cells in the cerebrospinal fluid.
  • CEACAM5 being used as a tumor marker
  • an immunological assay to measure the elevated levels of CEACAM5 in the blood of cancer patients has been clinically used for the prognosis and control of cancer, and more importantly, CEACAM5 has become Potentially useful tumor-associated antigens for targeted therapy.
  • One method uses anti-CEACAM5 antibodies to trigger the lytic activity of immune cells, especially through antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), to eliminate tumor cells expressing CEACAM5; the other method is through anti- CEACAM5 antibodies or antibody fragments are conjugated with effector molecules such as drugs, toxins, radionucleotides, immunomodulators or cytokines to specifically target tumor cells expressing CEACAM5, thereby exerting the therapeutic effect of the effector molecules.
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • CEACAM5 is more overexpressed in some solid tumors such as colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatocellular tumor, breast cancer and thyroid cancer
  • current research focuses on the antigen recognition ability of anti-CEACAM5 antibodies.
  • CEACAM5 will help to inhibit tumor metastasis.
  • antibodies with strong specificity to CEACAM5 can better avoid toxic and side effects caused by antibody off-target.
  • CEACAM1, CEACAM3, and CEACAM8 are widely distributed in the human immune system and bone marrow, such as neutrophils.
  • Specific CEACAM5 and/or CEACAM6 antibodies can reduce the possible side effects of drugs and increase the treatment window.
  • antibody in this application includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that can bind to a specific antigen.
  • a natural intact antibody contains two heavy chains and two light chains. Each heavy chain is composed of a variable region and first, second and third constant regions; each light chain is composed of a variable region and a constant region.
  • the mammalian heavy chain can be divided into ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , and the mammalian light chain can be divided into ⁇ or ⁇ .
  • the antibody has a "Y" shape, and the neck of the Y-shaped structure is composed of the second and third constant regions of two heavy chains, which are joined by disulfide bonds.
  • Each arm of the "Y" type structure includes the variable region and the first constant region of one heavy chain, which is combined with the variable region and constant region of one light chain.
  • the variable regions of the light and heavy chains determine antigen binding.
  • the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDR).
  • the CDRs of the light chain (L) include VLCDR1, VLCDR2 and VLCDR3, and the CDRs of the heavy chain (H) include VHCDR1, VHCDR2, and VHCDR3.
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed in this application can be named or identified by Kabat, Chothia or Al-Lazikani nomenclature.
  • the three CDRs are separated by side continuous parts called framework regions (FR), which are more highly conserved than CDRs and form a scaffold to support hypervariable loops.
  • FR framework regions
  • the constant regions of the heavy and light chains have nothing to do with antigen binding, but have multiple effector functions.
  • Antibodies can be divided into several categories based on the amino acid sequence of the constant region of the heavy chain. According to whether they contain alpha, delta, epsilon, gamma, and mu heavy chains, antibodies can be divided into five main categories or isoforms: IgA, IgD, IgE, IgG, and IgM.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • antigen-binding fragment in this application refers to an antibody fragment formed by an antibody portion containing one or more CDRs or any other antibody fragment that binds to an antigen but does not have a complete antibody structure.
  • antigen-binding fragments include, but are not limited to, such as diabody, Fab, Fab', F(ab') 2 , Fv fragments, disulfide bond stabilized Fv fragments (dsFv), (dsFv) 2 , Bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized bifunctional antibody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), bivalent single chain antibody (BsFv), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies and bivalent domain antibodies.
  • the antigen-binding fragment can bind to the same antigen as the parent antibody.
  • the antigen-binding fragment may contain one or more CDRs from a specific human antibody, which are grafted into the framework regions from one or more different human antibodies.
  • the "Fab" fragment of an antibody refers to the part of the antibody molecule that is composed of a light chain (including the variable region and the constant region) and the variable region and part of the constant region of a heavy chain through disulfide bonds.
  • the "Fab'" fragment refers to a Fab fragment containing a part of the hinge region.
  • F(ab') 2 refers to the dimer of Fab.
  • the Fc segment of an antibody is responsible for a variety of different effector functions such as ADCC and CDC, but does not participate in antigen binding.
  • the "Fv” segment of an antibody refers to the smallest antibody fragment that contains a complete antigen binding site.
  • the Fv fragment consists of a variable region of a light chain and a variable region of a heavy chain.
  • Single-chain Fv antibody or “scFv” refers to an engineered antibody formed by connecting the variable region of the light chain and the variable region of the heavy chain directly or through a peptide chain (Huston JS et al., Proc Natl Acad Sci USA, 85: 5879 (1988)).
  • Single-chain antibody Fv-Fc or “scFv-Fc” refers to an engineered antibody composed of scFv linked to the Fc segment of a certain antibody.
  • variable region of heavy chain antibodies ( VH domain) is the smallest known antigen-binding unit produced by acquired immunity (Koch-Nolte F. et al., FASEB J. 21 (13): 3490-8. Epub (2007)).
  • Nanobody refers to an antibody fragment, which consists of a VH domain from a heavy chain antibody and two constant regions CH2 and CH3.
  • Diabody includes a small antibody fragment with two antigen binding sites, where the fragment contains a VH domain and a VL domain connected on the same polypeptide chain (see, Holliger P. et al., Proc Natl Acad Sci U S A.90(14): 6444-8 (1993); EP404097; W093/11161).
  • the adaptor between the two domains is so short that the two domains on the same chain cannot pair with each other, which forces the two domains to pair with the complementary domain of the other chain to form two antibody binding sites.
  • These two antibody binding sites can target the same or different antigens (or epitopes).
  • Domain antibody refers to an antibody fragment containing only one heavy chain variable region or one light chain variable region.
  • two or more VH domains are covalently joined by a polypeptide linker to form a bivalent domain antibody.
  • the two VH domains of a bivalent domain antibody can target the same or different antigens.
  • (dsFv) 2 contains three peptide chains: two VH genes are connected by a polypeptide linker, and are connected to two VL groups by disulfide bonds.
  • the "bispecific ds bifunctional antibody” contains VL1-VH2 (connected by a polypeptide linker) and VH1-VL2 (also connected by a polypeptide linker), and the two pass between VH1 and VL1. Sulfur bonding.
  • Double-specific dsFv or “dsFv-dsFv” contains three polypeptide chains: VH1-VH2 groups, where the heavy chains of the two are connected by a polypeptide linker (such as a long elastic linker) and are respectively connected by a disulfide bond Combining with VL1 and VL2 groups, each pair of heavy and light chains paired by disulfide bonds has a different antigen specificity.
  • a polypeptide linker such as a long elastic linker
  • the "scFv dimer” is a bivalent bifunctional antibody or a bivalent single chain antibody (BsFv), containing two dimerized VH-VL (connected by a polypeptide linker) group, wherein The VH of one group and the VL of the other group cooperate to form two binding sites, which can target the same antigen (or epitope) or different antigens (or epitope).
  • the "scFv dimer” is a bispecific bifunctional antibody that contains VL1-VH2 (connected by a polypeptide linker) and VH1-VL2 (connected by a polypeptide linker), wherein VH1 and VL1 cooperates, VH2 and VL2 cooperate, and each cooperative pair has a different antigen specificity.
  • fully human when applied to an antibody or antigen-binding fragment, means that the antibody or antigen-binding fragment has a certain amino acid sequence or consists of the amino acid sequence, and the amino acid sequence corresponds to a human Or amino acid sequences of antibodies produced by human immune cells or derived from non-human sources such as transgenic non-human animals using human antibody libraries, or other sequences encoding human antibodies.
  • fully human antibodies do not contain amino acid residues (especially antigen-binding residues) derived from non-human antibodies.
  • humanized when applied to antibodies or antigen-binding fragments, means to include CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable ) Antibody or antigen-binding fragment. Since the humanized antibody or antigen-binding fragment has reduced immunogenicity, it can be used as a human therapeutic agent in certain embodiments.
  • the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.
  • the humanized antibody or antigen-binding fragment consists essentially of human sequences, except that the CDR sequences are non-human.
  • the human-derived FR region may include the same amino acid sequence as the human antibody from which it is derived, or it may include some amino acid changes, for example, no more than 10,9,8,7,6,5. , 4, 3, 2 or 1 amino acid change.
  • the amino acid change may exist only in the FR region of the heavy chain, only in the FR region of the light chain, or in both chains.
  • the humanized antibody includes human FR1-3 and human JH and JK.
  • chimeric used in this application refers to having a part of a heavy chain and/or light chain derived from one species, and the remaining part of the heavy chain and/or light chain is derived from an antibody or antigen of a different species. Fragment.
  • a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal such as a mouse.
  • CEACAM5 Carcinoembryonic antigen cell adhesion molecule 5
  • CD66e also known as CD66e; see, for example, AAA51967.1/GI: 180223, 702 amino acids
  • CEACAM5 contains 7 Ig-like domains, which include a single N-terminal Ig variable domain and 6 domains (A1-B1-A2-B2-A3-B3) homologous to the Ig constant domain.
  • CEACAM is connected to the cell membrane via the carboxy-terminal glycosylphosphatidylinositol (GPI) anchor.
  • GPI carboxy-terminal glycosylphosphatidylinositol
  • the “specific binding” or “specific binding” in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen.
  • the antibody or antigen-binding fragment thereof of the present application specifically binds to human and/or monkey carcinoembryonic antigen cell adhesion molecule 5, and its binding affinity (KD) is ⁇ 10 -6 M.
  • KD in this application refers to the ratio of the dissociation rate to the binding rate (koff/kon), which can be measured by surface plasmon resonance, for example, using an instrument such as Biacore.
  • the “selective binding” in the present application means that the antibody or antigen-binding fragment of the present application specifically binds to CEACAM5 protein, but does not substantially bind or binds to other CEACAM proteins at a significantly lower level, such as CEACAM1 protein, CEACAM3 Protein, CEACAM7 protein.
  • the heavy chain constant region of the antibody described in this application is of human IgG1 type. In some embodiments, the light chain constant region of the antibody described in this application is a kappa chain. In some embodiments, the heavy chain and light chain constant regions of the antibodies described in this application are human IgG1 and kappa chain, respectively.
  • conservative substitution when “conservative substitution” is used in an amino acid sequence, it means that one amino acid residue is replaced with another amino acid residue of a side chain with similar physical and chemical properties. For example, between hydrophobic side chain amino acid residues (such as Met, Ala, Val, Leu and Ile), neutral hydrophilic side chain residues (such as Cys, Ser, Thr, Asn and Gln), acidic side chain residues Conservative substitutions are made between groups (such as Asp, Glu), between basic side chain amino acids (such as His, Lys, and Arg), or between aromatic side chain residues (such as Trp, Tyr, and Phe). It is known in the art that conservative substitutions usually do not cause significant changes in the conformational structure of the protein, and therefore can retain the biological activity of the protein.
  • hydrophobic side chain amino acid residues such as Met, Ala, Val, Leu and Ile
  • neutral hydrophilic side chain residues such as Cys, Ser, Thr, Asn and Gln
  • amino side chain residues Conservative substitution
  • percent sequence identity when used for amino acid sequence (or nucleic acid sequence), it means that after sequence alignment is performed, and intervals are introduced when necessary to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence, with reference The percentage of amino acid (or nucleic acid) residues with the same sequence as the amino acid (or nucleic acid) residues of the candidate sequence. The conservative substitutions of the amino acid residues may or may not be considered the same residues.
  • the sequences can be aligned by tools disclosed in the art to determine the percent sequence identity of the amino acid (or nucleic acid) sequence. Those skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the needs of the comparison, for example, by selecting a suitable algorithm.
  • T cells as used in this application include CD4 + T cells, CD8 + T cells, T helper 1 T cells, T helper 2 T cells, T helper 17 T cells, and suppressor T cells.
  • Effective function refers to the biological activity of the Fc region of an antibody binding to its effectors such as C1 complex and Fc receptor.
  • exemplary effector functions include complement-dependent cytotoxicity (CDC) induced by the interaction of the antibody with C1q on the C1 complex, antibody-dependent cell-mediated cells induced by the binding of the Fc region of the antibody to the Fc receptor on the effector cell Toxicity (ADCC) and phagocytosis.
  • Cancer or “cancerous condition” in this application refers to any medical condition that is mediated by the growth, proliferation or metastasis of tumor or malignant cells and causes solid tumors and non-solid tumors such as leukemia.
  • Tumor in this application refers to the solid substance of tumors and/or malignant cells.
  • Treatment or “therapy” of a certain condition includes preventing or alleviating a certain condition, reducing the speed of the rise or development of a certain condition, reducing the risk of developing a certain condition, and preventing or delaying the development of symptoms related to a certain condition , Reduce or terminate the symptoms related to a certain condition, produce a complete or partial reversal of a certain condition, cure a certain condition, or a combination of the above.
  • treatment or “therapy” can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumor or malignant cells, or some combination of the above.
  • treatment or “therapy” includes removing all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, preventing or delaying the development of the tumor, or some combination of the above.
  • the “separated” material has been artificially changed from its natural state. If a certain "separated” substance or component appears in nature, it has been changed or deviated from its original state, or both.
  • a polynucleotide or polypeptide naturally occurring in a living animal has not been isolated, but if these polynucleotides or polypeptides and the substances that coexist in their natural state are sufficiently separated and exist in a sufficiently pure state, they can Think of it as being “separated”.
  • the purity of antibodies and antigen-binding fragments is at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, which can be determined by electrophoresis methods (such as SDS-PAGE, isoelectric focusing). , Capillary electrophoresis), or chromatography (such as ion exchange chromatography or reverse phase HPLC) to determine.
  • electrophoresis methods such as SDS-PAGE, isoelectric focusing.
  • Capillary electrophoresis Capillary electrophoresis
  • chromatography such as ion exchange chromatography or reverse phase HPLC
  • vectors refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted and the protein can be expressed.
  • Vectors can be used to transform, transduce or transfect host cells so that the genetic material elements they carry can be expressed in the host cells.
  • vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 bacteriophage, and animal viruses.
  • the types of animal viruses used as vectors include retroviruses (including lentivirus, adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuole virus (such as SV40)).
  • retroviruses including lentivirus, adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuole virus ( Such as SV40)).
  • the vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain an origin of replication site.
  • the carrier may also include components to assist its entry into cells, including but not limited to, viral particles, liposomes, or protein coats.
  • host cell refers to a cell that guides the introduction of exogenous polynucleotides and/or vectors.
  • the host cells described in this application include, but are not limited to, prokaryotic cells such as E. coli or subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells , COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • chimeric antigen receptor is abbreviated as CAR, which refers to a cell surface receptor that can recognize a specific antigen (such as a tumor antigen), and it contains an extracellular domain capable of recognizing a specific antigen (for example, an antibody that recognizes and binds to a specific antigen).
  • Antigen-binding fragments) and intracellular domains also called intracellular signal transduction regions, such as the delta chain of CD3 or the intracellular part of Fc ⁇ RI ⁇ capable of transmitting extracellular signals to the inside of the cell.
  • CAR-T cells T cells that carry and express such chimeric antigen receptors are called CAR-T cells, which can recognize and bind specific antigens and cells expressing the specific antigens (such as tumor cells) through the extracellular domain, and pass intracellular
  • the signal transduction function of the domain activates the immune response, releases a large number of various effectors, and efficiently kills cells expressing the specific antigen (such as tumor cells), thereby exerting a therapeutic effect (such as treating tumors).
  • one of the main objectives of this application is to provide an anti-CEACAM5 antibody with stronger specificity and better selectivity.
  • the application also provides the preparation method and application of the antibody, and the anti-CEACAM5 antibody of the application can be used to detect and/or treat tumors.
  • the present application provides an antibody or antigen-binding fragment thereof that specifically binds to CEACAM5 protein, the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • CDRs 3 complementarity determining regions
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 1, or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acid substitutions, deletions Or add) sequence,
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 2, or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acid substitutions, deletions) Or add) sequence, and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 3, or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acid substitutions, deletions) Or add) sequence;
  • VL The light chain variable region (VL) comprising the following 3 complementarity determining regions (CDRs):
  • VL CDR1 which consists of the following sequence: SEQ ID NO: 4, or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acid substitutions, deletions) Or add) sequence,
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 5, or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acid substitutions, deletions) Or add) sequence, and
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 6, or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions) Or add) sequence.
  • substitutions described in any one of (i)-(vi) are conservative substitutions.
  • the CDRs described in any one of (i) to (vi) are defined according to the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof includes the following 3 heavy chain CDRs: the sequence is SEQ ID NO: 1 or SEQ ID NO: VH CDR1, the sequence is SEQ ID NO: 2 or SEQ The VH CDR2 of ID NO: 21, the sequence of VH CDR3 of SEQ ID NO: 3; and/or, the following 3 light chain CDRs: the sequence of VL CDR1 of SEQ ID NO: 4, the sequence of SEQ ID NO: 5 or SEQ VL CDR2 of ID NO: 22, the sequence is VL CDR3 of SEQ ID NO: 6.
  • the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 1, VH CDR2 as shown in SEQ ID NO: 2, and VH CDR2 as shown in SEQ ID VH CDR3 shown in NO: 3; and/or, the following 3 light chain CDRs: VL CDR1 shown in SEQ ID NO: 4, VL CDR1 shown in SEQ ID NO: 5, and VL CDR2 shown in SEQ ID NO: 6 VL CDR3 shown.
  • the antibody or antigen-binding fragment thereof includes the following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO: 20, VH CDR2 as shown in SEQ ID NO: 21, and VH CDR2 as shown in SEQ ID VH CDR3 shown in NO: 3; and/or, the following 3 light chain CDRs: VL CDR1 shown in SEQ ID NO: 4, VL CDR1 shown in SEQ ID NO: 22, and VL CDR2 shown in SEQ ID NO: 6 VL CDR3 shown.
  • the heavy chain CDRs and light chain CDRs are defined according to the Kabat numbering system.
  • the present application provides an antibody or antigen-binding fragment thereof that specifically binds to CEACAM5 protein, the antibody or antigen-binding fragment thereof comprises:
  • CDR1 contained in the light chain variable region (VL) shown in SEQ ID NO: 8, or compared with it, has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 Amino acid substitution, deletion or addition) sequence;
  • CDR3 contained in the light chain variable region (VL) shown in SEQ ID NO: 8, or compared with it, has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 Amino acid substitution, deletion or addition) sequence.
  • the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined by the Kabat, IMGT, or Chothia numbering system. In certain embodiments, the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region
  • sequence shown in SEQ ID NO: 7 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, at least 99%, or 100% sequence identity;
  • VL The light chain variable region (VL), which comprises an amino acid sequence selected from:
  • sequence shown in SEQ ID NO: 8 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, A sequence with at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain framework region sequence and/or a light chain framework region sequence derived from a human immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain framework region sequence encoded by a human heavy chain germline gene, and/or a light chain framework region sequence encoded by a human light chain germline gene.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 19 And a VL comprising the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown in SEQ ID NO: 7 and VL having the sequence shown in SEQ ID NO: 8. In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown in SEQ ID NO: 18 and VL having the sequence shown in SEQ ID NO: 8. In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown in SEQ ID NO: 16 and VL having the sequence shown in SEQ ID NO: 17. In certain exemplary embodiments, the antibody or antigen-binding fragment thereof includes: VH having the sequence shown in SEQ ID NO: 19 and VL having the sequence shown in SEQ ID NO: 17.
  • the antibody or antigen-binding fragment thereof of the present application may comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin or a variant thereof, which has one or more sequences than the sequence from which it is derived.
  • a substitution, deletion or addition of amino acids or any combination thereof e.g., at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions or additions, or any combination thereof; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof).
  • the heavy chain of the antibody or antigen-binding fragment thereof of the present application comprises the heavy chain constant region (CH) of human immunoglobulin or a variant thereof, which has a One or more amino acid substitutions, deletions or additions or any combination thereof (for example, 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, one, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); and/or,
  • CH heavy chain constant region
  • the light chain of the antibody or antigen-binding fragment thereof of the present application comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof, which has compared with the sequence from which it is derived
  • CL light chain constant region
  • One or more amino acid substitutions, deletions or additions or any combination thereof for example, 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, one, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof).
  • the variant of the heavy chain constant region may have conservative substitutions of one or more amino acids (for example, 1, 2, 3, 4 or Conservative substitution of 5 amino acids).
  • the variant of the light chain constant region may have conservative substitutions of one or more amino acids (e.g., 1, 2, 3, 4, or 4) compared to the sequence from which it is derived. Conservative substitution of 5 amino acids).
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.
  • the light chain constant region is a kappa light chain constant region. In certain embodiments, the light chain constant region is a human kappa light chain constant region.
  • the antibody or antigen-binding fragment thereof of the present application comprises the heavy chain constant region (CH) shown in SEQ ID NO: 9; and/or, the light chain constant shown in SEQ ID NO: 11 District (CL).
  • CH heavy chain constant region
  • CL light chain constant
  • the antigen-binding fragment is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).
  • the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.
  • the antibody or antigen-binding fragment thereof of the present application has one or more activities selected from the following:
  • CEACAM5 protein such as colon cancer cells, gastric cancer cells
  • the antibody or antigen-binding fragment thereof of the present application is labeled.
  • the antibody or antigen-binding fragment thereof bears a detectable label, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or Biotin.
  • a detectable label such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or Biotin.
  • the application provides exemplary anti-CEACAM5 antibodies UM05-5L and hUM05-3.
  • CDR sequences can be modified to include one or more amino acid substitutions, thereby obtaining improved biological activity such as increased binding affinity to human carcinoembryonic antigen cell adhesion molecule 5.
  • phage display technology can be used to produce and express antibody variant libraries (such as Fab or FcFv variants), and then screen for antibodies that have affinity with CEACAM5.
  • computer software can be used to simulate the binding of the antibody to CEACAM5 and identify the amino acid residues on the antibody that form the binding interface. The substitution of these residues can be avoided to prevent a decrease in binding affinity, or these residues can be targeted for substitution to form a stronger binding.
  • at least one (or all) substitutions in the CDR sequence are conservative substitutions.
  • the antibody or antigen-binding fragment includes one or more CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity, and at the same time retains its parent antibody similar or even higher binding affinity with CEACAM5 And sex.
  • the parent antibody has substantially the same sequence, but its corresponding CDR sequence has 100% sequence identity with the sequence listed in SEQ ID NO: 1-6.
  • the antibody or antigen-binding fragment described in the present application can specifically bind to CEACAM5 with a binding affinity (KD) of ⁇ 10 -7 M, which can be measured by surface plasmon resonance.
  • the binding affinity value can be expressed by the KD value, which is calculated by the ratio of the dissociation rate to the binding rate (koff/kon) when the binding of the antigen and the antigen-binding molecule reaches equilibrium.
  • the antigen binding affinity e.g., KD
  • KD can be appropriately determined by a suitable method known in the art, for example, a plasmon resonance binding method including the use of an instrument such as Biacore.
  • the antibody or antigen-binding fragment described in this application binds to CEACAM5 at an EC50 (ie, half binding concentration) of 1 ng/mL-10 ⁇ g/mL.
  • the binding of antibodies or antigen-binding fragments to CEACAM5 can be determined by methods known in the art, such as sandwich methods such as ELISA, Western blot, FACS or other binding tests.
  • the antibody to be tested ie, the primary antibody
  • the unbound antibody is washed away, and a labeled secondary antibody is introduced, which can bind to the primary antibody and therefore can Bound secondary antibody is detected.
  • immobilized carcinoembryonic antigen cell adhesion molecule 5 the detection can be performed on a microplate reader, or when cells expressing CEACAM5 are used, FACS analysis can be used for the detection.
  • the antibody or antigen-binding fragment described in this application binds to CEACAM5 at an EC50 (ie, an effective concentration of 50%) of 10 ng/mL-10 ⁇ g/mL (determined using FACS analysis).
  • the antibody is specific for CEACAM5.
  • the antibody optionally does not bind to CEACAM1, CEACAM3, CEACAM7, CEACAM8, and binds to CEACAM6 with a binding affinity that is significantly lower than that of CEACAM5.
  • the antibodies described in this application can be used in combination with immunogenic substances, such as tumor cells, purified tumor antigens, cells transfected with encoding immunostimulatory factors, and tumor vaccines.
  • immunogenic substances such as tumor cells, purified tumor antigens, cells transfected with encoding immunostimulatory factors, and tumor vaccines.
  • the anti-CEACAM5 antibody and its antigen-binding fragment can be included in combination therapy, including standard chemotherapy and radiotherapy, target-based small molecule therapy, and other emerging immune checkpoint modulator therapy.
  • the antibodies and antigen-binding fragments thereof can be used as the basic molecules of antibody-drug conjugates, bispecific or multivalent antibodies.
  • the antibodies and antigen-binding fragments described in this application are camelized single chain domain antibodies, diabodies, scFv, scFv dimers, BsFv, dsFv, ( dsFv) 2 , dsFv-dsFv', Fv fragment, Fab, Fab', F(ab') 2 , ds diabody, nanobody, domain antibody or bivalent domain antibody.
  • the antibodies described herein include immunoglobulin constant regions.
  • the immunoglobulin constant region includes a heavy chain and/or light chain constant region.
  • the heavy chain constant region includes CH1, CH1-CH2 or CH1-CH3 regions.
  • the immunoglobulin constant region may further include one or more modifications to obtain desired properties.
  • the constant region can be modified to reduce or eliminate one or more effector functions, enhance FcRn receptor binding, or introduce one or more cysteine residues.
  • the antibody and antigen-binding fragment thereof further comprise a conjugate.
  • the antibodies or antigen-binding fragments thereof in this application can be linked to a variety of conjugates (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JMCruse and RELewis, Jr. (eds.), Carger Press , New York (1989)).
  • conjugates can be connected to the antibody or antigen-binding fragment by other methods such as covalent binding, affinity binding, intercalation, coordinate binding, complexation, binding, mixing or addition.
  • the antibodies and antigen-binding fragments disclosed in the present application can be engineered to contain specific sites other than the epitope binding portion, and these sites can be used to bind one or more conjugates.
  • such sites may contain one or more reactive amino acid residues, such as cysteine residues and histidine residues, to assist covalent attachment to the conjugate.
  • the antibody may be attached to the conjugate indirectly, or through another conjugate.
  • the antibody or antigen-binding fragment thereof can bind to biotin and then indirectly bind to a second conjugate, which is linked to avidin.
  • the conjugate may be a detectable label, a pharmacokinetic modification part, a purified part or a cytotoxic part.
  • detectable labels can include fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin or Texas red), enzyme substrate labels (e.g. horseradish peroxidase, alkaline phosphatase) , Luciferase, glucoamylase, lysozyme, sugar oxidase or ⁇ -D galactosidase), stable isotope or radioisotope, chromophore part, digoxin, biotin/avidin, DNA molecule Or gold for testing.
  • the conjugate may be a pharmacokinetic modification moiety such as PEG, which helps extend the half-life of the antibody.
  • the conjugate may be a purified part such as magnetic beads.
  • the "cytotoxic moiety" can be any agent that is harmful to cells or may damage or kill cells.
  • cytotoxic part examples include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, acetophane bromide, imitrine, mitomycin, etopoxib, tenipokan, vincristine, Vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracisin dione, mitoxantrone, mitomycin, actinomycin D, l-dehydrotestosterone, glucocorticoid, Procaine, tetracaine, lidocaine, procylol, puromycin and its analogues, antimetabolites (for example, methotrexate, 6-mercaptopurine, 6-mercaptoguanine, arabin Cytidine, 5-fluorouracil dacarbazine), alkylating agents (such as nitrogen mustard, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
  • the antibody of the present application can be prepared by various methods known in the art, for example, obtained by genetic engineering recombination technology.
  • DNA molecules encoding the antibodies of the present application or antigen-binding fragments thereof can be obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into the expression vector and then transfected into the host cell. Then, the transfected host cell is cultured under specific conditions, and the antibody or antigen-binding fragment thereof of the application is expressed.
  • the antigen-binding fragment of the present application can be obtained by hydrolyzing a complete antibody molecule (see Morimoto et al., J.Biochem.Biophys.Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium.
  • the preparation method of anti-CEACAM5 antibody or its antigen binding provided in this application includes the following steps:
  • mice immunized with the CHO cell line overexpressing carcinoembryonic antigen cell adhesion molecule 5 as the antigen were used to extract splenocytes and fused with the mouse myeloma cell line SP2/0-AG14 to obtain expression Hybridoma cell lines of anti-CEACAM5 antibodies or antigen-binding fragments thereof;
  • step 3 Provide an expression vector, the expression vector comprising the gene cloned in step 2) and an expression control sequence operatively connected to the gene;
  • the present application provides a hybridoma cell line used in the above preparation method.
  • a human-mouse hybridoma secreting a specific anti-CEACAM5 antibody was prepared, and the heavy chain and light chain sequences of the antibody (100% human genes) were cloned using molecular biology technology to construct an anti-human carcinoembryonic antigen cell adhesion Molecule 5 is a human monoclonal antibody that is expressed and produced by CHO cells. These antibodies have stronger binding capacity and specificity as drugs compared with existing antibodies.
  • the application provides an isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, or the variable region of the heavy chain and/or the variable region of the light chain thereof.
  • the nucleic acid molecule comprises the nucleotide coding sequence shown in SEQ ID NO: 14 and/or SEQ ID NO: 15.
  • the application provides a vector (for example, a cloning vector or an expression vector), which comprises the isolated nucleic acid molecule as described above.
  • a vector for example, a cloning vector or an expression vector
  • the vectors of the present application are plasmids, cosmids, bacteriophages and the like.
  • the vector contains the first nucleotide sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present application, and/or the light source encoding the antibody or antigen-binding fragment thereof of the present application.
  • the second nucleotide sequence of the chain variable region In some embodiments, the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.
  • the present application provides a host cell comprising the isolated nucleic acid molecule or vector as described above.
  • the host cell is a mammalian cell.
  • the host cell is a human, murine, sheep, horse, dog, or cat cell.
  • the host cell is a Chinese hamster ovary cell.
  • a method for preparing the antibody or antigen-binding fragment thereof of the present application comprises culturing the host cell as described above under conditions that allow expression of the antibody or antigen-binding fragment thereof, and obtaining The antibody or antigen-binding fragment thereof is recovered from the host cell culture.
  • the application also provides a bispecific or multispecific molecule, which comprises the antibody or antigen-binding fragment thereof.
  • the bispecific or multispecific molecule specifically binds CEACAM5, and additionally specifically binds one or more other targets.
  • the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity for a second target (e.g., a second antibody).
  • the application also provides an immunoconjugate, which comprises the antibody or antigen-binding fragment thereof and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.
  • the therapeutic agent is selected from cytotoxic agents.
  • the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • the amino acid sequence of the antibody and its antigen-binding fragment described in the present application can be converted into the corresponding DNA coding sequence. Due to the degeneracy of the genetic code, the DNA sequence obtained by conversion may not be completely identical, while the encoded protein sequence remains unchanged.
  • vectors including polynucleotides encoding the antibodies and antigen-binding fragments thereof can be introduced into host cells for cloning (amplification of DNA) or gene expression.
  • the antibodies and antigen-binding fragments thereof can be prepared by homologous recombination methods known in the art.
  • a variety of carriers are available.
  • Vector components usually include, but are not limited to, two or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer sequences, promoters (for example: SV40, CMV, EF-1a) and Transcription termination sequence.
  • the vector system includes mammalian, bacterial, yeast systems, etc., and will include plasmids such as but not limited to pALTER, pBAD, pcDNA, pCal, pL, pELpGEMEX, pGEX, pCLpCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2 and other vectors that can be obtained from laboratories or are commercially available.
  • Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses).
  • Vectors including polynucleotides encoding the antibodies and antigen-binding fragments thereof can be introduced into host cells for cloning or gene expression.
  • the host cells suitable for cloning or expressing the DNA in the vector in this application are prokaryotic cells, yeast or the above-mentioned higher eukaryotic cells.
  • Prokaryotic cells suitable for the purposes of this application include eubacteria, such as Gram-negative bacteria or Gram-positive bacteria, such as Enterobacteriaceae (such as Escherichia coli), Enterobacter, Erwinia, Klebsiella , Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, and Bacillus such as Bacillus subtilis and Bacillus licheniformis , Pseudomonas such as Peptidobacterium aeruginosa and Streptomyces.
  • Enterobacteriaceae such as Escherichia coli
  • Enterobacter Erwinia
  • Klebsiella Proteus
  • Salmonella such as Salmonella typhimurium
  • Serratia such as Serratia marcescens
  • Shigella Shigella
  • Bacillus such as Bacillus subtilis and Bacillus licheniformis
  • eukaryotic microorganisms such as filamentous fungi or yeast can also be used as host cells to clone or express vectors encoding antibodies.
  • Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism.
  • Kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC12424), Kluyveromyces bulgaricus (ATCC16045), Kluyveromyces weichii (ATCC24178), Kluyveromyces (ATCC56500), Kluyveromyces fruit fly (ATCC36906), Kluyveromyces thermotolera and Kluyveromyces marxianus: Yarrowia lipolytica (EP402226); Pichia pastoris (EP183070); Candida: Trichoderma reesei (EP244234); Alternaria; Western Schwann yeast, such as Western Schwann yeast; and filamentous Fungi, such as Neurospora, Penicillium, Curvularia, and Aspergillus, such as Aspergillus undulatus and Aspergillus
  • the host cells suitable for expressing glycosylated antibodies or antigen-binding fragments thereof provided in this application are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • a variety of baculoviral strains and their variants as well as the corresponding permissive insect host cells have been discovered, which come from hosts such as the following: Spodoptera frugiperda (caterpillar), Aegypti mosquitoes (mosquitoes) ), Aedes albopictus (mosquito), Drosophila melanogaster (Drosophila) and silkworm.
  • virus strains used for transfection are publicly available, such as Autographa californica nuclear polyhedrosis virus and the Bm-5 variant of Bombyx mori nuclear polyhedrosis virus. These viruses can be used in this application, especially Used to transfect Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potatoes, soybeans, petunia, tomatoes and tobacco can also be used as hosts.
  • spinal cells tissue culture
  • mammalian host cells include SV40 transformed monkey kidney cell line CV1 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al.,]. Gen Virol. 36: 59 (1977)); Baby hamster kidney cells (B blood, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77 : 4216 (1980)); mouse Sertoli cells (TM4, Mather JP, Biol.Reprod.
  • the host cell is a 293F cell.
  • the host cells used to produce the antibodies and antigen-binding fragments thereof in this application can be cultured in a variety of media well-known in the art.
  • the culture medium may also contain any other necessary additives in appropriate concentrations known in the art.
  • the conditions of the medium such as temperature, pH and similar conditions, are the conditions previously used for selecting host cells for expression, and are well known to those of ordinary skill.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, the particulate remains of the host cells or lysed fragments are first removed, for example, by centrifugation or ultrasound. Carter et al., Bio/Technology 10:163-167 (1992) describes a method for separating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is melted in the presence of uranyl acetate (pH 3.5), EDTA and toluene sulfonate fluoride (PMSF) for more than 30 minutes. Centrifuge to remove cell debris.
  • uranyl acetate pH 3.5
  • EDTA and toluene sulfonate fluoride
  • a commercially available protein concentration filter such as Iamicon or Millipore Pellicon ultrafiltration unit, is usually first used to concentrate the supernatant of the expression system.
  • protease inhibitors such as PMSF can be added to inhibit protein degradation, and antibiotics to prevent the growth of accidental contaminants.
  • the antibodies produced from the cells can be purified by purification methods, such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography column, ammonium sulfate precipitation, salting out, and affinity chromatography, where Affinity chromatography is the preferred purification technique.
  • purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography column, ammonium sulfate precipitation, salting out, and affinity chromatography, where Affinity chromatography is the preferred purification technique.
  • the type of the antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether protein A is suitable as an affinity ligand.
  • Protein A can be used to purify antibodies based on human ⁇ 1, ⁇ 2 or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 13 (1983)).
  • Protein G is suitable for all mouse-derived isoforms and human ⁇ 3 (Guss et al., EMBO J. 5: 1567 1575 (1986)).
  • Agarose is the most commonly used affinity ligand attachment matrix, but other matrices can also be used.
  • Mechanically stable substrates such as controlled pore glass or poly(styrene)benzene can achieve faster flow rates and shorter processing times than with agarose. If the antibody contains a CH3 domain, it can be purified with Bakerbond ABX.TM resin (J.T.Baker, Phillipsburg, N.J.).
  • Protein purification techniques can also be determined according to the antibodies that need to be obtained, such as fractionation in ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resins (such as polyasparagus Acid column), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation.
  • the mixture containing the antibody of interest and impurities can be processed by low pH hydrophobic interaction chromatography, using an elution buffer with a pH of about 2.5-4.5, preferably at low salt concentrations (e.g., From about 0 to 0.25M salt concentration).
  • the application provides a pharmaceutical composition, which contains the antibody or antigen-binding fragment of the application, the bispecific or multispecific molecule of the application, or the immunoconjugate of the application, and a pharmaceutically acceptable Accepted carriers and/or excipients.
  • the pharmaceutical composition may also include additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • a drug with anti-tumor activity such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • the antibody or antigen-binding fragment, bispecific or multispecific molecule or immunoconjugate and the additional pharmaceutically active agent are used as separate components or as components of the same composition provide.
  • the antibody or antigen-binding fragment thereof of the present application and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.
  • the application further provides a pharmaceutical composition comprising the antibody and one or more pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier used in the pharmaceutical composition disclosed in this application may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antimicrobial substance, etc. Osmotic substances, buffers, antioxidants, anesthetics, suspending agents/dispersants, integrating agents, diluents, adjuvants, adjuvants or non-toxic auxiliary substances, other components known in the art or multiple combinations of the above.
  • Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, coloring agents, emulsifiers or stabilizers such as Sugar and cyclodextrin.
  • Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl Anisole, butylated hydroxytoluene and/or propyl gallate.
  • Including one or more antioxidants such as methionine in a composition containing the antibodies disclosed in this application can reduce the oxidation of the antibodies. The reduction in oxidation can prevent or reduce the decrease in binding affinity, thereby improving antibody stability and extending shelf life.
  • the pharmaceutically acceptable carrier may include, for example, an aqueous medium such as sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, or glucose and lactate.
  • Grid injection non-aqueous medium such as: plant-derived fixed oil, cotton seed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacterial or fungal inhibitory concentrations, isotonic agents such as sodium chloride or glucose, Buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose Or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), integration reagents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraace
  • the antibacterial agent as a carrier can be added to the pharmaceutical composition in a multi-dose container, which includes phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methyl and propyl parabens, Thiimerosal, chloroanisole and chloroanisole.
  • Suitable excipients may include, for example, water, salt, glucose, glycerol or ethanol.
  • Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or sodium acetate, sorbitan laurate, triethanolamine oleate, or cyclodextrin. substance.
  • the pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder.
  • Oral preparations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate and the like.
  • the pharmaceutical composition is formulated as an injectable composition.
  • injectable pharmaceutical compositions can be prepared in any conventional form, for example, liquid solvents, suspensions, emulsifiers, or solid forms suitable for producing liquid solvents, suspensions, or emulsifiers.
  • Injection preparations may include currently used sterile and/or pyrogen-free solutions, sterile dry solubles combined with solvents before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, Sterile dry insoluble products that are combined with the medium before use, and sterile and/or pyrogen-free emulsions.
  • the solvent can be an aqueous phase or a non-aqueous phase.
  • the unit-dose injection preparation is packaged in an ampoule, a tube, or a syringe with a needle. It is known in the art that all preparations for injection administration should be sterile and pyrogen-free.
  • a sterile lyophilized powder can be prepared by dissolving the antibody or antigen-binding fragment thereof disclosed in the present application in a suitable solvent.
  • the solvent may contain a kind of other pharmacological components that can improve the stability of the powder or the reconstituted solution prepared from the powder, or improve the powder or the reconstituted solution.
  • Applicable excipients include, but are not limited to, water, glucose, tribitol, fructose, corn syrup, xylitol, glycerol, glucose, brown sugar or other applicable substances.
  • the solvent may contain a buffer, such as citrate buffer, sodium phosphate or potassium phosphate buffer, or other buffers known to those skilled in the art. In one embodiment, the pH of the buffer is neutral.
  • each tube can contain a single dose or multiple doses of the anti-CEACAM5 antibody or antigen-binding fragment or combination thereof.
  • the filling amount in each tube can be slightly higher than that required for each dose or multiple doses (for example, 10% excess), so as to ensure accurate sampling and accurate administration.
  • the lyophilized powder can be stored under appropriate conditions, such as in the range of about 4°C to room temperature.
  • the lyophilized powder is re-dissolved with water for injection to obtain a preparation for injection administration.
  • the lyophilized powder can be added to sterile pyrogen-free water or other suitable liquid carrier for reconstitution.
  • the precise amount is determined by the selected therapy and can be determined based on empirical values.
  • the antibody or antigen-binding fragment thereof of the present application can be derivatized, for example, linked to another molecule (for example, another polypeptide or protein).
  • another molecule for example, another polypeptide or protein.
  • the derivatization (eg, labeling) of the antibody or its antigen-binding fragment will not adversely affect its binding to CEACAM5. Therefore, the antibodies or antigen-binding fragments thereof of the present application are also intended to include such derivatized forms.
  • the antibody or antigen-binding fragment thereof of the present application can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (for example, to form Bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment to another molecule (for example, avidin or polyhistidine tag).
  • the antibodies or antigen-binding fragments thereof of the present application can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or sugar groups. These groups can be used to improve the biological properties of antibodies, for example to increase serum half-life.
  • the present application provides an immunoconjugate comprising the antibody or antigen-binding fragment thereof described herein and a therapeutic agent linked to the antibody or antigen-binding fragment thereof;
  • the therapeutic agent is selected from cytotoxic agents
  • the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination;
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the antibody or antigen-binding fragment thereof of the present application can specifically bind to CEACAM5 protein, and basically does not bind to CEACAM1, CEACAM3, CEACAM7, CEACAM8 protein, but only weakly binds to CEACAM6 protein. Therefore, the antibody of the present application or its Antigen-binding fragments have higher specificity and accuracy in detection.
  • the present application provides a kit, which includes the antibody of the present application or the antigen-binding fragment thereof, or the conjugate of the present application.
  • the antibody or antigen-binding fragment thereof bears a detectable label, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or Biotin.
  • a detectable label such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance) or Biotin.
  • the kit further includes a second antibody, which specifically recognizes the antibody or antigen-binding fragment thereof.
  • the second antibody further includes a detectable label, such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.
  • a detectable label such as an enzyme (such as horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.
  • the kit of the present application may further include reagents for allowing the corresponding detectable label to be detected.
  • the detectable label when the detectable label is an enzyme, the kit may also include a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD) for horseradish peroxidase, tetramethyl Aniline (TMB), ABTS or luminol compounds, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase.
  • OPD o-phenylenediamine
  • TMB tetramethyl Aniline
  • ABTS tetramethyl Aniline
  • p-NPP p-nitrophenyl phosphate
  • AMPPD p-nitrophenyl phosphate
  • the detectable label when the detectable label is a chemiluminescence reagent (for example, acridine ester compound), the kit may further include a pre-excitation
  • the application also provides the use of the antibody or antigen-binding fragment thereof in preparing a kit for detecting whether a tumor can be treated by anti-tumor therapy targeting CEACAM5.
  • the antibody or antigen-binding fragment thereof bears a detectable label.
  • the CEACAM5 is human CEACAM5.
  • the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyn
  • CHL
  • kits including the antibody or antigen-binding fragment thereof.
  • the kit is used to detect the presence or level of CEACAM5 in a biological sample.
  • the biological sample may include cells or tissues.
  • the kit includes an antibody or antigen-binding fragment thereof conjugated to a detectable label.
  • the kit includes an unlabeled antibody, and further includes a labeled secondary antibody capable of binding to the unlabeled antibody.
  • the kit may further include instructions for use and packaging separating each component in the kit.
  • the antibody is connected to a substrate or instrument for sandwich assay such as ELISA or immunochromatographic assay.
  • a substrate or instrument for sandwich assay such as ELISA or immunochromatographic assay.
  • Suitable substrates or instruments can be, for example, microplates and test papers.
  • the present application also provides a chimeric antigen receptor, which comprises the antigen-binding domain of the antibody or antigen-binding fragment thereof.
  • the antigen-binding domain comprises the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof.
  • the antigen binding domain is a scFv.
  • the antigen binding receptor comprises an antigen binding fragment of the antibody.
  • the antigen binding receptor is expressed by immune effector cells (eg, T cells).
  • immune effector cells eg, T cells
  • the present application also provides an isolated nucleic acid molecule, which encodes the chimeric antigen receptor.
  • the application also provides a vector, which contains the isolated nucleic acid molecule; in some embodiments, it is used to prepare chimeric antigen receptor T cells.
  • the application also provides a host cell, which contains the isolated nucleic acid molecule or vector;
  • the host cell is an immune effector cell (for example, T cell or NK cell);
  • an immune effector cell for example, T cell or NK cell
  • the host cell is a chimeric antigen receptor T cell (CAR-T).
  • CAR-T chimeric antigen receptor T cell
  • CEACAM5 In many malignant tumors, the overexpression of CEACAM5 can be used as a tumor biomarker. Therefore, measuring CEACAM5 in the blood of patients can be used for the prognosis and control of cancer, and CEACAM5 targeted therapy has also become a potential cancer treatment method.
  • the antibodies of the present application (for example, UM05-5L and hUM05-3) can bind to CEACAM5 protein with high affinity and specificity, and have ADCC activity, which can inhibit tumor growth and kill tumor cells.
  • the present application also 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 antibody or antigen thereof. Binding fragment, or said bispecific or multispecific molecule, or said immunoconjugate, or said pharmaceutical composition, or said chimeric antigen receptor, or said host cell contact .
  • the application also provides a method for reducing the expression level of CEACAM5 on the cell surface, which comprises combining the cell with the antibody or antigen-binding fragment thereof, or the bispecific or Multispecific molecules, or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the host cell contact, so that the expression level of CEACAM5 on the cell surface is reduced; Wherein, the cell expresses CEACAM5 on its surface.
  • the cell is a tumor cell expressing CEACAM5.
  • the application also provides a method for preventing and/or treating tumors in a subject (such as a human), the method comprising administering to a subject in need thereof an effective amount of the The antibody or antigen-binding fragment thereof, or the bispecific or multispecific molecule, or the immunoconjugate, or the pharmaceutical composition, or the chimeric antigen receptor, or the The host cell.
  • the tumor expresses CEACAM5.
  • the tumor involves tumor cells that express CEACAM5.
  • the CEACAM5 is expressed on the surface of the tumor cells.
  • the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyn
  • CHL
  • the subject is a mammal, such as a human.
  • the method further comprises administering another drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • anti-tumor activity such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • the method further includes administration of additional anti-tumor therapy, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.
  • additional anti-tumor therapy such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.
  • the application also provides the antibody or antigen-binding fragment thereof, or the bispecific or multispecific molecule, or the immunoconjugate, or the pharmaceutical composition, Or the use of the chimeric antigen receptor, or the host cell, in the preparation of a medicament for the prevention and/treatment of tumors in a subject (such as a human).
  • the medicament further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • a drug with anti-tumor activity such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.
  • the tumor expresses CEACAM5.
  • the tumor involves tumor cells that express CEACAM5; preferably, the CEACAM5 is expressed on the surface of the tumor cells.
  • the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyn
  • CHL
  • the subject is a mammal, such as a human.
  • this application also provides a method for detecting the presence or amount of CEACAM5 (such as human CEACAM5) in a sample, which includes the following steps:
  • the antibody or antigen-binding fragment thereof bears a detectable label.
  • the CEACAM5 is human CEACAM5.
  • the present application also provides a method for detecting whether a tumor can be treated by anti-tumor therapy targeting CEACAM5, which includes the following steps:
  • the antibody or antigen-binding fragment thereof bears a detectable label.
  • the CEACAM5 is human CEACAM5.
  • the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, Melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cells Cancer and other hematological malignancies, such as classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell-rich lymphoma, EBV-positive and negative PTLD and EBV-related Diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyn
  • CHL
  • the application also provides a method of treatment, including administering a therapeutically effective amount of the antibody described in the application to a subject in need thereof.
  • the therapeutically effective dose of the antibody provided in this application depends on a variety of factors known in the art, such as weight, age, past medical history, current treatment, the subject’s health status and the potential for cross infection, allergies, hypersensitivity and side effects, and administration Drug route and extent of tumor development. Those skilled in the art (such as doctors or veterinarians) can proportionally reduce or increase the dosage according to these or other conditions or requirements.
  • the antibodies provided herein can be administered at a therapeutically effective dose of between about 0.01 mg/kg and about 100 mg/kg. In some embodiments, the antibody is administered at a dose of about 50 mg/kg or less. In some embodiments, the administered dose is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less. kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less.
  • a certain dose can be administered at multiple intervals, such as once a day, twice or more a day, twice or more a month, once a week, once every two weeks, once every three weeks, once a month or every two Once a month or more. In certain embodiments, the dose administered can vary with the course of treatment. For example, in certain embodiments, the initial dose administered may be higher than the subsequent dose administered. In some embodiments, the administered dose is adjusted according to the response of the subject during the course of treatment.
  • the dosage regimen can be adjusted to achieve the optimal response (such as treatment response). For example, it can be administered in a single dose or in multiple divided doses over a period of time.
  • the antibodies disclosed in this application can be administered by administration methods known in the art, such as injection administration (eg, subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection) or non-injection.
  • injection administration eg, subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection
  • non-injection eg, oral administration, nasal administration, sublingual administration, rectal administration, or topical administration.
  • the antibody can be used to treat diseases related to its molecular mechanism, including tumors and cancers, such as non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreas Cancer, stomach cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma , Granuloma fungoides (mycosis fungoids), Merkel cell carcinoma and other hematological malignancies, such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cell/histiocytic B-cell lymphoma, EBV-positive and negative PTLD and EBV-related diffuse large B-cell lymphoma (DLBCL), plasmablastic lymph
  • the application further provides methods for using the antibodies.
  • the application provides a method of treating a condition or disorder related to the antibody mechanism in an individual, including administering a therapeutically effective amount of the antibody described herein.
  • the antibodies disclosed in this application can be administered alone or in combination with one or more other therapeutic means or substances.
  • the antibodies disclosed in the present application can be combined with chemotherapy, radiotherapy, cancer treatment surgery (such as tumor resection), antiviral drugs, one or more antiemetic drugs or other chemotherapy-induced complications, or any other therapy for Combination of cancer or virus treatment substances.
  • when the antibodies disclosed in this application are used in combination with one or more therapeutic substances they can be administered simultaneously with the one or more therapeutic substances.
  • the antibodies can be administered simultaneously as part of the same pharmaceutical composition. However, antibodies that are "combined" with other therapeutic substances do not need to be administered simultaneously or in the same composition as the therapeutic substance.
  • the meaning of “combination” in this application also includes that an antibody administered before or after another therapeutic substance is considered to be “combined” with the therapeutic substance, even if the antibody and the second substance are administered in different ways Administration.
  • other therapeutic substances used in combination with the antibodies disclosed in this application can be administered by referring to the product instructions of the other therapeutic substances, or refer to the surgeon's desk reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)), or refer to other methods known in the art.
  • the therapeutic substance can induce or enhance an immune response against cancer.
  • tumor vaccines can be used to induce immune responses to certain tumors or cancers.
  • Cytokine therapy can be used to increase the presentation of tumor antigens to the immune system.
  • examples of cytokine therapy include but are not limited to interferons such as interferon alpha, beta and gamma, colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF, interleukins such as IL-1, IL-1a , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor necrosis factors such as TNF - ⁇ and TNF- ⁇ .
  • interferons such as interferon alpha, beta and gamma
  • colony stimulating factors such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF
  • interleukins
  • Reagents that inactivate immunosuppressive targets can also be used, such as PD-1 antibodies, TGF- ⁇ inhibitors, IL-10 inhibitors, and Fas ligand inhibitors.
  • Another group of reagents includes those that activate the immune response to tumors or cancer cells, for example, those that increase T cell activation (such as T cell costimulatory signaling pathways such as CTLA-4, ICOS, OX40, 4-1BB, etc.), And those that improve dendritic cell function and antigen presentation.
  • the monoclonal antibodies of the present application can bind to CEACAM5 protein or cells expressing CEACAM5 protein with high specificity and selectivity, and they do not substantially bind to CEACAM1, CEACAM3, CEACAM7, and CEACAM8 proteins. Binding, only weakly bound to CEACAM6 protein.
  • the antibody UM05-5L also has ADCC activity.
  • the UM05-5L-CAR constructed based on the antibody UM05-5L can be expressed on the surface of human T cells, can recognize tumor cells in vivo, secrete cytokines, and has a strong tumor suppressing effect. Therefore, the monoclonal antibodies of the present application (such as UM05-5L and hUM05-3 antibodies) have high clinical application value.
  • Figure 1 shows the overexpression factor of the CHO cell line overexpressing CEACAM5 constructed in this application; compared with conventional CHO cells, the overexpression factor of the CHO cell line constructed in this application is 904 times.
  • Figure 2 shows the results of the ELISA experiment of the binding of antibody UM05-5L to CEACAM5 protein.
  • Figure 3 shows the detection results of the binding ability of antibody UM05-5L to LOVO cells.
  • Figures 4a-4b show the ADCC reporter gene activity of antibody UM05-5L; among them, Figure 4a shows the ADCC reporter gene activity of the positive control (Erbitux antibody) and the negative control, and Figure 4b shows the ADCC reporter gene activity of UM05-5L.
  • Figures 5a-5b show the results of flow cytometry;
  • Figure 5a is the expression efficiency of control T cells (Mock T), and
  • Figure 5b is the expression efficiency of UM05-5L-CAR.
  • Figure 6 shows the secretion of cytokines IFN ⁇ (left panel) and IL-2 (right panel) after UM05-5L-CAR-T is mixed with tumor cells.
  • Figure 7 shows the killing results of UM05-5L-CAR-T cells on tumor cells KATO3.
  • Figure 8 shows the expansion results of UM05-5L-CAR-T cells stimulated by tumor cells LS174T.
  • Figure 9 shows the results of UM05-5L-CAR-T inhibiting tumors in mice.
  • Figure 10 shows the release of IFN ⁇ in mice receiving UM05-5L-CAR-T.
  • Figure 11 shows the detection results of the binding ability of the humanized antibody hUM05-3 to LS174T cells.
  • Figure 12 shows the detection results of the binding ability of antibodies UM05-5L, UM05-5L C47W, hUM05-3 and hUM05-3 W47C to LS174T cells.
  • the molecular biology experimental methods and immunoassay methods used in this application basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compiled Molecular Biology Experiment Guide, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes are used in accordance with the conditions recommended by the product manufacturer. If specific conditions are not indicated in the examples, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer.
  • the reagents or instruments used without the manufacturer's indication are all conventional products that are commercially available.
  • Example 1 Obtaining of monoclonal antibodies against human CEACAM5 protein
  • This application has constructed a CHO cell line overexpressing CEACAM5 protein.
  • the plasmid of CEACAM5 (Beijing Yiqiao Shenzhou Technology Co., Ltd., HG11077-UT) was transfected into CHO cell line (ATCC) and expressed.
  • the plasmid is resistant to Hygromycin, so cells stably transfected with the plasmid can be stably passaged in a medium containing Hygromycin. Pick the cells and measure the expression of CEACAM5.
  • the CHO-CEACAM5 cells detected by FACS have a 904-fold overexpression of CEACAM5.
  • mice Six 5-8 weeks old Balb/c mice (Shanghai Slack) were immunized with CHO-CEACAM5 cells and CEACAM5 protein purified from tumor patients (L2C01001) according to the immunization schedule in Table 2.
  • Intraperitoneal injection CHO-CEACAM5 cells CFA Day 26 Intraperitoneal injection CHO-CEACAM5 cells IFA Day 49 Intraperitoneal injection CHO-CEACAM5 cells IFA Day 91 Intraperitoneal injection CEACAM5 protein Gerbu(MM3001) Day 104 Intraperitoneal injection CEACAM5 protein Gerbu(MM3001) 105th day Intraperitoneal injection CEACAM5 protein Gerbu(MM3001) Day 106 Intraperitoneal injection CEACAM5 protein Gerbu(MM3001)
  • spleen cells of the immunized mice were fused with SP2/0-AG14 cells (ATCC) for hybridoma cell fusion, and an appropriate amount of the fused cells were plated on a 96-well plate. About 10 days after fusion, the supernatant of each well was taken, and the binding activity of mouse antibodies secreted by hybridoma cells to human CEACAM5 was detected by ELISA.
  • SP2/0-AG14 cells ATCC
  • Hybridoma cells ELISA OD value LOVO FACS combination 1-2C5 1.0690 12.1 1-9G3 1.0390 7.01 3-2F3 1.4480 11 3-6F5 1.3520 6.51 3-8C10 2.3030 137 3-8G8 1.9270 82.9 2A10A5 2.392 / 2A10C4 2.434 /
  • the hybridoma cell 3-8C10 with better binding activity was selected for sequencing.
  • an antibody was obtained, named UM05-5, using the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), pages 647-669) determined the CDR sequence of the UM05-5 antibody, as shown in Table 4.
  • UM05-5L a human-mouse chimeric antibody was designed and expressed and named UM05-5L.
  • VH and VL sequences encoding the mouse antibody are respectively linked to the constant region sequence encoding the human IgG1 heavy chain (the amino acid sequence is shown in SEQ ID NO: 9, and the nucleotide sequence is shown in SEQ ID NO: 10. )
  • the constant region sequence of the kappa chain (the amino acid sequence is shown in SEQ ID NO: 11, and the nucleotide sequence is shown in SEQ ID NO: 12) to obtain the human-mouse chimeric antibody UM05-5L.
  • the nucleotide sequences encoding the heavy chain and light chain of the antibody were cloned into the mammalian cell expression vector pcDNA3.4.
  • the heavy chain expression vector and the light chain expression vector were transfected into HEK293 cells with Lipofectamine 2000 transfection reagent (Invitrogen) at a molar ratio of 2:1, and cultured at 37° C. and 5% carbon dioxide for 7 days. Collect the supernatant of the culture medium, and use Protein A affinity chromatography to purify the antibody in the supernatant.
  • the purified antibody was dialyzed with PBS solution, freeze-dried and concentrated, and stored at -20°C.
  • the human CEACAM5 protein solution with a concentration of 1 ⁇ g/mL was coated on a 96-well high-affinity plate at 100 ⁇ L/well, and shaken overnight at 4°C. On the next day, it was washed 3 times with 300 ⁇ L PBST (Tween20: 0.5 ⁇ ), and then blocked with 100 ⁇ L/well of 5% BSA/PBS for 1 hour, and shaken at room temperature. Wash 3 times with 300 ⁇ L PBST. Use PBS to prepare serial dilutions of antibody samples. Add 100 ⁇ L/well to a 96-well plate and shake at room temperature for 1 hour. Wash 3 times with 300 ⁇ L PBST.
  • Flow cytometry was used to detect the binding of antibodies to LOVO tumor cells that naturally express human CEACAM5 protein or cells overexpressing different CEACAM proteins. To put it simply, first collect the cells, wash them with PBS, count them, and dilute to 2*10 6 /ml cell suspension; add 10 ⁇ l of antibody working solution to 100 ⁇ l cell suspension, incubate at 4°C for 30 min in the dark; wash twice with PBS Then, add the corresponding fluorescent-labeled secondary antibody Goat-anti-Human IgG (H+L) (Invitrogen), incubate at 4°C for 30 minutes in the dark, wash twice with PBS, suspend it in 400 ⁇ l FACS buffer, and detect the antibody and cells by FACS The combination of circumstances.
  • Goat-anti-Human IgG H+L
  • SW620-CEACAM1 is a cell overexpressing CEACAM1 protein
  • SW620-CEACAM3 is a cell overexpressing CEACAM3 protein
  • CHO-CEACAM6 is a cell overexpressing CEACAM6 protein
  • CHO-CEACAM7 is a cell overexpressing CEACAM7 protein
  • CHO-CEACAM8 is a cell overexpressing CEACAM6 protein.
  • the chimeric antibody UM05-5L has particularly outstanding selectivity: it binds to CEACAM5 with high affinity, but has no obvious binding to CEACAM1, CEACAM3, CEACAM7, CEACAM8, and only weakly binds to CEACAM6. Combine.
  • ordinary antibodies such as the control antibody UM05-8
  • CEACAM5 can bind to CEACAM1, CEACAM3, CEACAM6, CEACAM7, and CEACAM8 at a higher level, and are not selective.
  • the LOVO cells were seeded on a 96-well cell culture plate at a density of 20,000 cells/well, and incubated overnight at 37°C and 5% CO 2 .
  • the antibody UM05-5L and the positive control antibody Erbitux were prepared in the culture medium at 20 ⁇ g/ml, and were diluted by 3 times to 8 concentrations.
  • the effector cells Jurkat/ADC (Nanjing Nuoxin Biotechnology Co., Ltd.) were plated into LOVO cell wells at 120,000 cells/30 ⁇ L/well, and incubated at 37° C., 5% CO 2 for 16-20 hours. After the incubation, the Bright-Glo kit (Promega, cat.E2620) was used to detect the expression level of luciferase in the effector cells. The luciferase level represents the degree of ADCC activation of effector cells.
  • Figure 4a shows the ADCC reporter gene activity of the positive control Erbitux antibody and the negative control
  • Figure 4b shows the ADCC reporter gene activity of UM05-5L. Based on the above results, it can be seen that the ADCC activity of UM05-5L is very strong, with an EC50 of 0.85 ⁇ g/mL.
  • anti-CEACAM5 single-chain antibody as CAR-T recognition antibody, and use one or more 41BB, CD28, OX40 and other costimulatory components to carry out different CEA-CAR Design the structure and construct the corresponding lentiviral plasmid to generate lentivirus that can infect cells and express the corresponding CAR.
  • the lentivirus can be used to infect T cells isolated from the peripheral blood of tumor patients to produce CAR-T cells expressing corresponding CAR receptors on the cell membrane surface. This CAR-T cell can effectively recognize and kill tumor cells expressing CEACAM5.
  • the single-chain antibody UM05-5L scFv (in the order of VH-G4S3-VL) shown in SEQ ID NO: 13 is connected with the sequence CD8 ⁇ -CD137-CD3 ⁇ -p2A-tEGFR to construct a chimeric antigen receptor CAR, and then The nucleotide sequence encoding the CAR was cloned into a lentiviral vector (Jike Gene, GV401), and the vector was named UM05-5L-CAR.
  • the expression cassette is EF1a promoter-CAR-2A-tEGFR-WPRE.
  • the UM05-5L-CAR lentiviral vector and packaging plasmid were transiently transfected into 293T cells for 16 hours, the medium was changed, and the culture was continued for 24-48 hours.
  • the medium supernatant (including lentivirus) was collected and stored at -80°C.
  • the results are shown in Figure 5.
  • UM05-5L-CAR Figure 5b
  • UM05-5L-CAR-T cells and CEACAM5-expressing tumor cells such as KATO3 and LS174T
  • a medium control that is, only UM05-5L-CAR-T cells
  • a CEACAM5 negative cell control that is, use UM05 -5L-CAR-T cells and cells that do not express CEACAM5, such as RKO cells
  • the release of IFN ⁇ and IL2 in the supernatant is detected.
  • UM05-5L-CAR-T can recognize the cell line expressing CEACAM5 and release the cytokines IFN ⁇ and IL-2.
  • Example 8 In vivo drug efficacy experiment of LS174T tumor model
  • LS174T cells (ATCC CL-187) were inoculated subcutaneously in NSG mice (Biocytometer) at a rate of 1 ⁇ 10 7 per mouse. When the tumor volume reached 200-400 mm 3 , PBS control and unmodified were administered via the tail vein. T cell control (Mock T) or UM05-5L CAR-T, the doses given are 100 ⁇ l, 1 ⁇ 10 7 cells, 1 ⁇ 10 7 tEGFR+ cells, record the changes in tumor volume and analyze the release of IFN ⁇ in peripheral blood . The results are shown in Figure 9 and Figure 10. UM05-5L recognized LS174T tumor cells in mice and released a large amount of IFN ⁇ , resulting in strong tumor suppression.
  • the UM05-5L antibody was humanized, and a humanized antibody was obtained, which was named hUM05-3.
  • the specific sequence is shown in Table 6.
  • the antibody preparation was carried out as described in Example 2.
  • the nucleotide sequences encoding SEQ ID NO: 16 and SEQ ID NO: 17 were synthesized, cloned into an expression vector and transiently expressed in HEK293 cells. After culturing, the culture supernatant was collected and subjected to Protein A affinity chromatography. Method to purify the antibody in the supernatant. The purified antibody was dialyzed with PBS solution, freeze-dried and concentrated, and stored at -20°C.
  • LS174T cells tumor cells with high CEACAM5 expression
  • the LS174T cells were cultured in RPMI1640 medium containing 10% FBS.
  • the LS174T cells were trypsinized with TrypLE, centrifuged, and resuspended in DPBS solution (FACS buffer, 4°C) containing 2% BSA; then, at a concentration of 5 ⁇ 10 5 /100 ⁇ l/well, they were added to U Type the bottom 96-well plate, and add the antibody of concentration gradient, incubate at 4°C for 1 hour. After incubation, centrifuge and discard the supernatant; add 100 ⁇ l of anti-human IgG Fc-APC secondary antibody to each well, and incubate at 4°C for 1 hour. After incubation, the cells were washed once with FACS buffer, resuspended in 200 ⁇ l FACS buffer, and read the fluorescence signal value in BD C6 plus.
  • the 47th amino acid (Cys) of the VH of the human-mouse chimeric antibody UM05-5L was mutated to Trp, and the mutant antibody was named UM05-5L C47W; the humanized antibody hUM05-3 was the 47th amino acid of the VH (Ie Trp) is mutated to Cys, and the mutated antibody is named hUM05-3 W47C.
  • the variable region sequences of the mutant antibodies are shown in Table 7. As described in Example 10, the ability of the antibody to bind to LS174T cells was tested. The experimental results are shown in Figure 12 and Table 8. The results showed that the human-mouse chimeric antibody UM05-5L and the humanized antibody hUM05-3 can bind to cells expressing CEACAM5 before and after the mutation.

Abstract

公开了能特异性结合人癌胚抗原细胞粘附分子5(CEACAM5)的单克隆抗体及其抗原结合片段，还公开了基于所述抗体的嵌合抗原受体，以及所述抗体及抗原结合片段的制备方法和治疗CEACAM5相关肿瘤的用途。

Description

一种抗CEACAM5的单克隆抗体及其制备方法和用途 技术领域
本申请属于生物免疫技术领域，具体涉及能特异性结合人癌胚抗原细胞粘附分子5(CEACAM5)的单克隆抗体及其抗原结合片段。本申请还涉及所述抗体及其抗原结合片段的制备方法和用途。
背景技术
人癌胚抗原细胞粘附分子(CEACAM)基因家族早在上世纪60年代就被发现，包括位于19号染色体(q13.1-13.3之间)的35个基因/伪基因，在这其中有21个编码蛋白质。CEACAM属于免疫球蛋白超家族黏附分子，其结构域高度糖基化，通常包括1-2个免疫球蛋白可变区样结构域(N domain)和0-6个免疫球蛋白恒定区样结构域。CEACAM蛋白位于细胞膜外，其中CEACAM1、CEACAM3和CEACAM4通过疏水跨膜结构域与细胞膜连接；CEACAM5-8则通过糖基磷酰肌醇连接到细胞膜上。这些细胞外域通常作为细胞(例如上皮、内皮、树突状和白细胞)间的粘附分子。
CEACAM涉及多种细胞功能，以细胞间的黏附功能为基础，通过信号转导调节细胞生长和分化，并在胰岛素稳态、血管生成及免疫调节中发挥重要作用。CEACAM基因家族成员参与各种各样的病理生理作用，包括作为微生物病原体的受体。它们在致癌中起重要作用，特别是在癌症检测、进展和转移中。CEACAM5(简称为CEA，又称为CD66e)是具有约180kDa分子量的糖蛋白。CEACAM5含有经由糖基磷脂酰肌醇(GPI)锚与细胞膜连接的7个结构域。7个结构域包括单一N端Ig可变域和与Ig恒定域同源的6个结构域(A1-B1-A2-B2-A3-B3)。CEACAM5最初被认为是在胎儿组织中表达的蛋白质，现在已经在几种正常成年组织中被鉴定出来。CEACAM5的过量表达在许多类型的癌症中被观察到。例如，在结肠癌患者的血液中可以检测到CEACAM5。并且，进一步研究确定了它的过量表达与许多恶性肿瘤，通常是不良预后相关。在前列腺癌和结直肠癌中，CEACAM5的过度表达被证明可以作为肿瘤生物标志物。
此外，在多种恶性肿瘤，如乳房、胰腺、卵巢、结肠、肺和胃腺肿瘤中CEACAM5/CEACAM6亦被发现呈过度表达，并且与肿瘤的侵袭性和转移有关。在肝转移的起始过程中，CEACAM5与其受体CEAr结合，它们的相互作用导致促炎细胞因子的活化和产生，主要是IL-1、IL-6、IL-10和TNF-α。总之，这些细胞因子改变肝细胞和Kupffer 细胞的微环境，以及它们与肝窦细胞的相互作用。这些相互作用不仅影响肿瘤细胞或其他肝细胞，似乎也促进了脑脊液中CSC和其他循环肿瘤细胞的活力。
基于此，除了已知的CEACAM5被用作肿瘤标记，测量癌症患者血液中升高的CEACAM5的免疫学测定法已在临床上用于癌症的预后和控制之外，更重要的是，CEACAM5已成为用于靶向治疗的潜在有用的肿瘤相关抗原。已经报道的使用CEACAM5靶向免疫治疗癌症主要有2种主要方法。一种方法使用抗CEACAM5抗体引发免疫细胞的溶解活性，特别是通过抗体依赖性细胞毒性(ADCC)或补体依赖性细胞毒性(CDC)，以消除表达CEACAM5的肿瘤细胞；另一种方法是通过抗CEACAM5抗体或抗体片段与例如药物、毒素、放射性核苷酸、免疫调节剂或细胞因子等效应分子缀合，特异性靶向表达CEACAM5的肿瘤细胞，从而发挥效应分子的治疗作用。鉴于CEACAM5更多地过量表达于一些诸如结肠直肠癌、胰腺癌、肺癌、胃癌、肝细胞瘤、乳腺癌和甲状腺癌等实体肿瘤中，因此当前的研究集中在抗CEACAM5抗体的抗原识别能力。
总之，当前的研究表明，靶向CEACAM5的治疗方法将有助于抑制肿瘤的转移过程。特别地，对CEACAM5具有较强特异性的抗体，能够更好的避免抗体脱靶而造成的毒副作用。例如CEACAM1，CEACAM3，CEACAM8广泛分布于人免疫系统及骨髓，例如中性粒细胞等，特异性的CEACAM5和/或CEACAM6抗体，可降低药物可能存在的副作用，提高治疗窗口。
基于此，当前对亲和力更高，特异性更强的抗CEACAM抗体存在需求。
发明内容
在本申请中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本申请，下面提供相关术语的定义和解释。
术语定义
本申请中的"抗体"一词包括任意可结合某特定抗原的免疫球蛋白、单克隆抗体、多克隆抗体、多特异性抗体或双特异性(双价)抗体。一个天然的完整抗体包含两条重链和两条轻链。每条重链由一可变区和第一、第二、第三恒定区组成；每条轻链由一可变区和一恒定区组成。哺乳动物的重链可分为α、δ、ε、γ和μ，哺乳动物的轻链可分为λ或κ。抗 体呈"Y"型，Y型结构的颈部由两条重链的第二和第三恒定区组成，其通过二硫键结合。"Y"型结构的每条臂包括其中一条重链的可变区和第一恒定区，其与一条轻链的可变区和恒定区结合。轻链和重链的可变区决定抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)。轻链(L)的CDR包含VLCDR1、VLCDR2、VLCDR3，重链(H)的CDR包含VHCDR1、VHCDR2、VHCDR3。本申请中公开的抗体和抗原结合片段的CDR边界可通过Kabat，Chothia或Al-Lazikani命名法命名或识别。(Al-Lazikani，B.，Chothia,C.，Lesk，A.M.，J.Mol.Biol.，273(4)：927(1997)；Chothia，C.等，J.Mol.Biol.，186(3)：651-63(1985)；Chothia，C.and Lesk，A.M.，J.Mol.Biol.，196：901(1987)；Chothia，C.等，Nature，342(6252)：877-83(1989)；Kabat，E.A.等，National Institutes of Health，Bethesda，Md.(1991))。其中，三个CDR由被称为框架区(FR)的侧面连续部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。重链和轻链的恒定区与抗原结合无关，但具有多种效应功能。抗体依据重链恒定区的氨基酸序列可以分成几类。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分类或异构体：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。
本申请中的"抗原结合片段"一词，指由含有一个或多个CDR的抗体部分或者任何其它结合抗原但不具有完整抗体结构的抗体片段所形成的一种抗体片段。抗原结合片段的例子包括，但不限于，如双功能抗体(diabody)、Fab、Fab'、F(ab') 2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv) 2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双功能抗体(ds diabody)、单链抗体分子(scFv)、scFv二聚体(双价的双功能抗体)、双价单链抗体(BsFv)、多特异性抗体、骆驼化单域抗体(camelized single domain antibody)、纳米抗体、域抗体和双价域抗体。抗原结合片段可以与母体抗体结合相同的抗原。在某些实施方式中，抗原结合片段可以含有来自某特定人抗体的一个或多个CDR，移接至来自一个或多个不同人抗体的框架区。
抗体的"Fab"片段是指由一条轻链(包括可变区和恒定区)和一条重链的可变区和部分恒定区经二硫键结合起来的那部分抗体分子。
"Fab’"片段是指包含了部分绞链区的Fab片段。
"F(ab') 2"指的是Fab的二聚体。
抗体的Fc段负责多种不同的效应功能如ADCC和CDC，但不参与抗原的结合。
抗体的"Fv"段指的是含有完整抗原结合位点的最小抗体片段。Fv片段由一条轻链的可变区和一条重链的可变区组成。
"单链Fv抗体"或"scFv"是指由轻链可变区与重链可变区直接相连或通过一个肽链连接而成的工程抗体(Huston JS等，Proc Natl Acad Sci USA，85：5879(1988))。
"单链抗体Fv-Fc"或"scFv-Fc"是指由连接到某抗体Fc段的scFv组成的工程抗体。
"骆驼化单域抗体(Camelized single domain antibody)"，"重链抗体"或"HCAb(Heavy-chain-only antibodies，HCAb)"都是指含有两个VH域而不含有轻链的抗体(Riechmann L.和Muyldermans S.，J Immunol Methods.231(1-2)：25-38(1999)；Muyldermans S.，J Biotechnol.74(4)：277-302(2001)；W094/04678；W094/25591；U.S.Patent No.6,005，079)。重链抗体最初从驼科(骆驼、单峰驼和美洲驼)衍生得到。虽然缺失轻链，骆驼化抗体(camelized antibodies)有确证的抗原结合全部功能(Hamers Casterman C.等，Nature 363(6428)：446-8(1993)；Nguyen VK.等，"Heavy-chain antibodies in Camelidae：a case of evolutionary innovation，Immunogenetics.54(1)：39-47(2002)；Nguyen VK.等，Immunology.109(1)：93-101(2003))。重链抗体的可变区(VH域)是己知的最小的获得性免疫产生的抗原结合单位(Koch-Nolte F.等，FASEB J.21(13)：3490-8.Epub(2007))。
"纳米抗体"是指一种抗体片段，其由一个来自重链抗体的VH域和两个恒定区CH2和CH3组成。
"双功能抗体(diabody)"包括带有两个抗原结合位点的小抗体片段，其中该片段含有在同一条多肽链上相连的VH域和VL域(请参见，Holliger P.等，Proc Natl Acad Sci U S A.90(14)：6444-8(1993)；EP404097；W093/11161)。两个域之间衔接物很短，使同一条链上的两个域不能互相配对，从而迫使两个域与另一条链的互补域配对，形成两个抗体结合位点。这两个抗体结合位点可靶向结合相同或不同的抗原(或抗原表位)。
"域抗体"是指仅含有一条重链可变区或一条轻链可变区的抗体片段。在某些情况下，两个或多个VH域由一个多肽衔接物共价结合并形成双价域抗体。双价域抗体的两个VH域可靶向作用于相同或不同的抗原。
在某些实施方式中，"(dsFv) 2"含有三条肽链：两个VH基因间通过一条多肽衔接物相连，并通过二硫键与两个VL基团结合。
在某些实施方式中"双特异性ds双功能抗体"含有VL1-VH2(由一个多肽衔接物相连)和VH1-VL2(也是由一个多肽衔接物相连)，两者在VH1和VL1间通过二硫键结合。
"双特异性dsFv"或"dsFv-dsFv"含有三条多肽链：VH1-VH2基团，其中两者的重链通过多肽衔接物(如：长的弹性衔接物)相连，并通过二硫键分别与VL1和VL2基团结合，每对通过二硫键配对的重链轻链具有不同的抗原特异性。
在某些实施方式中，"scFv二聚体"是双价双功能抗体或双价单链抗体(BsFv)，含有二聚化的两个VH-VL(由多肽衔接物连接)基团，其中一个基团的VH与另一个基团的VL协作形成两个结合位点，这两个结合位点可靶向结合相同抗原(或抗原表位)或不同抗原(或抗原表位)。在另一些实施方式中，"scFv二聚体"是双特异性双功能抗体，含有相互连接的VL1-VH2(由多肽衔接物连接)和VH1-VL2(由多肽衔接物连接)，其中VH1和VL1协作，VH2和VL2协作，且每个协作的配对具有不同的抗原特异性。
本申请中使用的术语"全人源"当用于抗体或抗原结合片段时，是指所述抗体或抗原结合片段具有某氨基酸序列或由所述氨基酸序列组成，所述氨基酸序列对应于由人或人免疫细胞生产的、或从例如利用人源抗体库的转基因非人动物等非人来源衍生的抗体的氨基酸序列，或者其他编码人源抗体的序列。在某些实施方式中，全人源抗体不包含来源于非人抗体的氨基酸残基(特别是抗原结合残基)。
本申请中使用的术语"人源化"当用于抗体或抗原结合片段时，是指包括来源于非人动物的CDR、来源于人的FR区，以及来源于人的恒定区(当适用时)的抗体或抗原结合片段。由于人源化的抗体或抗原结合片段具有降低的免疫原性，其在某些实施方式中可用作人的治疗剂。在一些实施方式中，所述非人动物是哺乳动物例如小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠。在一些实施方式中，所述人源化抗体或抗原结合片段除了CDR序列是非人源的以外，基本上全部由人源序列组成。在一些实施方式中，所述来源于人的FR区可以包括与其来自的人源抗体相同的氨基酸序列，或其可以包括一些氨基酸改变，例如，不超过10，9、8、7、6、5、4、3、2或1个氨基酸改变。在一些实施方式中，该氨基酸改变可以仅存在于重链FR区、仅存在于轻链FR区或同时存在于两个链中。在一些优选实施方式中，所述人源化抗体包括人源FR1-3和人源JH和JK。
本申请中使用的术语"嵌合"是指具有来源于一种物种的重链和/或轻链的一部分，和所述重链和/或轻链其余部分来源于不同物种的抗体或抗原结合片段。在一个示例性的例子中，嵌合抗体可以包括来源于人的恒定区和来源于非人动物例如小鼠的可变区。
术语“癌胚抗原细胞粘附分子5”(CEACAM5，简称为CEA，又称为CD66e；参见例如，AAA51967.1/GI：180223，702个氨基酸)是具有约180kDa分子量的糖蛋白。CEACAM5含有7个Ig-like结构域，这7个结构域包括单一N端Ig可变域和与Ig恒定 域同源的6个结构域(A1-B1-A2-B2-A3-B3)。CEACAM经由羧基端的糖基磷脂酰肌醇(GPI)锚与细胞膜连接。
本申请中的"特异性结合"或"特异性的结合"是指两分子间的非随机结合反应，如抗体和抗原间的反应。在某些实施方式中，本申请的抗体或其抗原结合片段与人和/或猴癌胚抗原细胞粘附分子5特异性结合，并且其结合亲和力(KD)≤10 -6M。本申请中的KD是指解离速度与结合速度的比值(koff/kon)，可通过表面等离子共振的方法测定，例如使用如Biacore的仪器。
本申请中的"选择性结合"是指，本申请的抗体或其抗原结合片段与CEACAM5蛋白特异性结合，但基本上不结合或者以显著更低的水平结合其他CEACAM蛋白，例如CEACAM1蛋白，CEACAM3蛋白，CEACAM7蛋白。
在某些实施方式中，本申请所述的抗体的重链恒定区为人IgG1型。在某些实施方式中，本申请所述的抗体的轻链恒定区为κ链。在某些实施方式中，本申请所述的抗体的重链和轻链恒定区分别为人IgG1和κ链。
在本申请中当"保守置换"用于氨基酸序列时，是指将一个氨基酸残基用另一个具有相似理化性质的侧链的氨基酸残基替代。例如，可以在疏水侧链氨基酸残基间(例如Met、Ala、Val、Leu和Ile)、中性亲水侧链残基间(例如Cys、Ser，Thr、Asn和Gln)、酸性侧链残基间(例如Asp、Glu)、碱性侧链氨基酸间(例如His、Lys和Arg)或芳香侧链残基间(例如Trp、Tyr和Phe)进行保守置换。本领域己知保守置换通常不会引起蛋白构象结构的显著变化，因此能够保留蛋白质的生物活性。
当"百分比序列同一性"用于氨基酸序列(或核酸序列)时，是指在进行序列比对，并且必要时引入间隔使相同氨基酸(或核酸)数目达到最多后，在候选序列中，与参比序列相同的氨基酸(或核酸)残基占所述候选序列的氨基酸(或核酸)残基的百分比。所述氨基酸残基的保守置换可以认为或可以不认为是相同残基。可以通过本领域公开的工具对序列进行比对以确定氨基酸(或核酸)序列的百分比序列同一性。本领域技术人员可以使用所述工具的默认参数或根据比对的需要适当调整参数，例如通过挑选合适的算法。
本申请中使用的“T细胞”包括CD4 +T细胞、CD8 +T细胞、T辅助1型T细胞、T辅助2型T细胞、T辅助17型T细胞和抑制性T细胞。
本申请中使用的"效应功能"是指抗体的Fc区与其效应器例如C1复合物和Fc受体结合的生物活性。示例性的效应功能包括抗体与C1复合物上的C1q相互作用诱导的补体依 赖性细胞毒性(CDC)、抗体的Fc区与效应细胞上的Fc受体结合诱导的抗体依赖性细胞介导的细胞毒性(ADCC)以及吞噬。
本申请中的“癌症”或“癌病症”是指任何由肿瘤或恶性细胞生长、增殖或转移所介导，并引发实体瘤和非实体瘤如白血病的医学状况。本申请中的"肿瘤"是指肿瘤和/或恶性细胞的实体物质。
对某种状况的"治疗"或"疗法"包括预防或减轻某种状况，降低某种状况兴起或发展的速度，减少发展出某种状况的风险，预防或延迟与某种状况相关的症状发展，减少或终止与某种状况相关的症状，产生某种状况的完全或部分的逆转，治愈某种状况，或以上的组合。对于癌症来说，"治疗"或"疗法"可以指抑制或减缓肿瘤或恶性细胞生长，繁殖，或转移，或以上的某些组合。对于肿瘤来说，"治疗"或"疗法"包括清除全部或部分的肿瘤，抑制或减缓肿瘤生长和转移，预防或延缓肿瘤的发展，或以上的某些组合。
"被分离"的物质已经经人工由自然状态改变。如果自然界中出现某种"被分离"的物质或成分，那么其已经被改变或脱离其原始状态，或二者均有发生。例如，某一活体动物体内天然存在的多核苷酸或多肽是未被分离的，但如果这些多核苷酸或多肽与之在天然状态下共存的物质足够分离并以足够纯的状态存在，则可以认为是"被分离"。在某些实施方式中，抗体和抗原结合片段的纯度为至少90％、93％、95％、96％、97％、98％、99％，其可由电泳方法(如SDS-PAGE、等电聚焦、毛细管电泳)，或色谱法(如离子交换色谱法或反相HPLC)确定。
本申请中"载体"是指，可将编码某蛋白的多核苷酸操作性地插入其中并使该蛋白获得表达的一种运载工具。载体可用于转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。举例来说，载体包括：质粒、噬菌粒、柯斯质粒、人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1衍生的人工染色体(PAC)、噬菌体如λ噬茵体或M13噬菌体，以及动物病毒等。用作载体的动物病毒种类有逆转录病毒(包括慢病毒、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40))。载体可含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还可包括协助其进入细胞的成分，包括但不限于，病毒颗粒、脂质体或蛋白外壳。
本申请中"宿主细胞"是指导入外源多核苷酸和/或载体的细胞。本申请所述的宿主细胞包括但不限于，原核细胞例如大肠杆菌或枯草菌，真菌细胞例如酵母细胞或曲霉菌，昆 虫细胞例如S2果蝇细胞或Sf9，或者动物细胞例如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞。
本申请中"嵌合抗原受体"简称CAR，是指可以识别特定抗原(例如肿瘤抗原)的细胞表面受体，其包含能够识别特定抗原的胞外域(例如，识别并结合特定抗原的抗体的抗原结合片段)和能够将细胞外的信号传递到细胞内部的胞内域(也称为胞内信号转导区，例如CD3的δ链或FcεRIγ的胞内部分)。携带并表达此类嵌合抗原受体的T细胞即被称为CAR-T细胞，其能够通过胞外域识别并结合特定抗原以及表达所述特定抗原的细胞(例如肿瘤细胞)，并通过胞内域的信号转导作用，激活免疫应答，释放大量的多种效应因子，高效杀伤表达所述特定抗原的细胞(例如肿瘤细胞)，从而发挥治疗作用(例如治疗肿瘤)。
基于现有技术的不足，本申请的主要目的之一在于，提供一种特异性更强、选择性更好的抗CEACAM5抗体。本申请还提供了所述抗体的制备方法和用途，本申请的抗CEACAM5抗体可以用于检测和/或治疗肿瘤。
本申请的抗体
在一个方面，本申请提供了一种特异性结合CEACAM5蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
和/或，
(b)包含下述3个互补决定区(CDRs)的轻链可变区(VL)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:4，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:5，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:6，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，(i)-(vi)任一项中所述的置换为保守置换。
在某些实施方案中，(i)-(vi)任一项中所述的CDR根据Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：如下3个重链CDRs：序列为SEQ ID NO：1或SEQ ID NO:20的VH CDR1，序列为SEQ ID NO：2或SEQ ID NO:21的VH CDR2，序列为SEQ ID NO：3的VH CDR3；和/或，如下3个轻链CDRs：序列为SEQ ID NO：4的VL CDR1，序列为SEQ ID NO：5或SEQ ID NO:22的VL CDR2，序列为SEQ ID NO：6的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含如下3个重链CDRs：如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:2所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:4所示的VL CDR1、如SEQ ID NO:5所示的VL CDR2、如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述抗体或其抗原结合片段包含如下3个重链CDRs：如SEQ ID NO:20所示的VH CDR1、如SEQ ID NO:21所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:4所示的VL CDR1、如SEQ ID NO:22所示的VL CDR2、如SEQ ID NO:6所示的VL CDR3。
在某些实施方案中，所述的重链CDR和轻链CDR根据Kabat编号系统定义。
在一个方面，本申请提供了一种特异性结合CEACAM5蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(i)如SEQ ID NO:7所示的重链可变区(VH)中含有的CDR1，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
(ii)如SEQ ID NO:7所示的重链可变区(VH)中含有的CDR2，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；和
(iii)如SEQ ID NO:7所示的重链可变区(VH)中含有的CDR3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；和/或，
(iv)如SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR1，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
(v)如SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR2，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；和
(vi)如SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat、IMGT或Chothia编号系统定义。在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO：7所示的序列；
(ii)与SEQ ID NO：7所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO：7所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO：8所示的序列；
(v)与SEQ ID NO：8所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO：8所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的重链框架区序列和/或轻链框架区序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：人重链胚系基因编码的重链框架区序列，和/或人轻链胚系基因编码的轻链框架区序列。
在某些示例性实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:7、SEQ ID NO:16、SEQ ID NO:18或SEQ ID NO:19所示的序列的VH和包含如SEQ ID NO:8或SEQ ID NO:17所示的序列的VL。
在某些示例性实施方案中，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:7所示的序列的VH和具有如SEQ ID NO:8所示的序列的VL。在某些示例性实施方案中，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:18所示的序列的VH和具有如SEQ ID NO:8所示的序列的VL。在某些示例性实施方案中，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:16所示的序列的VH和具有如SEQ ID NO:17所示的序列的VL。在某些示例性实施方案中，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:19所示的序列的VH和具有如SEQ ID NO:17所示的序列的VL。
本申请的抗体或其抗原结合片段可以包含来源于哺乳动物(例如，鼠或人)免疫球蛋白的恒定区序列或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)。
在某些实施方案中，本申请的抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；和/或，
在某些实施方案中，本申请的抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)。
在一些实施方案中，所述重链恒定区(CH)的变体与其所源自的序列相比可以具有一个或多个氨基酸的保守置换(例如1个，2个，3个，4个或5个氨基酸的保守置换)。
在一些实施方案中，所述轻链恒定区(CL)的变体与其所源自的序列相比可以具有一个或多个氨基酸的保守置换(例如1个，2个，3个，4个或5个氨基酸的保守置换)。
在某些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。在某些实施方案中，所述重链恒定区是人IgG1、IgG2、IgG3或IgG4重链恒定区。
在某些实施方案中，所述轻链恒定区是κ轻链恒定区。在某些实施方案中，所述轻链恒定区是人κ轻链恒定区。
在某些示例性实施方案中，本申请的抗体或其抗原结合片段包含SEQ ID NO:9所示的重链恒定区(CH)；和/或，SEQ ID NO:11所示的轻链恒定区(CL)。
在某些实施方案中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)和单域抗体(sdAb)。
在某些实施方案中，所述抗体为鼠源抗体、嵌合抗体、人源化抗体、双特异性抗体或多特异性抗体。
本申请的抗体或其抗原结合片段具有选自下列的一种或多种活性：
(1)特异性结合CEACAM5蛋白或表达CEACAM5蛋白的细胞；
(2)基本上不与CEACAM1、CEACAM3、CEACAM7、CEACAM8蛋白或表达CEACAM1、CEACAM3、CEACAM7、CEACAM8蛋白的细胞结合；
(3)与CEACAM6蛋白或表达CEACAM6蛋白的细胞仅具有微弱的结合，其结合亲和力显著低于与CEACAM5蛋白或表达CEACAM5蛋白的细胞的结合亲和力；
(4)具有ADCC活性；
(5)能够抑制或杀伤表达CEACAM5蛋白的肿瘤细胞(例如结肠癌细胞，胃癌细胞)，具有治疗肿瘤的活性。
在某些实施方案中，本申请的抗体或其抗原结合片段带有标记。在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
在某些实施方式中，本申请提供了示例性的抗CEACAM5抗体UM05-5L和hUM05-3。
本领域技术人员应理解可以将前述CDR序列进行修饰以包含一个或更多氨基酸的取代，由此得到提高的生物学活性例如提高的与人癌胚抗原细胞粘附分子5的结合亲和性。例如，可以利用噬菌体展示技术生产并表达抗体变体库(例如Fab或FcFv变体)，随后筛选与CEACAM5有亲和性的抗体。另一个例子中，可以用计算机软件模拟所述抗体与CEACAM5的结合并鉴别抗体上形成结合界面的氨基酸残基。可以避免这些残基的替代以防止结合亲和性降低，或可以靶向这些残基进行替代以形成更强的结合。在某些实施方式中，CDR序列中的至少一个(或全部)取代是保守替代。
在某些实施方式中，所述抗体或抗原结合片段包括一个或多个CDR序列，这些序列具有与SEQ ID NO:1-6的序列至少80％(例如至少85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)的序列同一性，并且同时保留了与其亲本抗体相似或甚至更高的与CEACAM5的结合亲和性。所述亲本抗体具有基本相同的序列，但其相应的CDR序列与SEQ ID NO:1-6所列的序列具有100％序列同一性。
在一些实施方式中，本申请所述的抗体或抗原结合片段能够以≤10 -7M的结合亲和性(KD)与CEACAM5特异性结合，其可通过表面等离子共振法测量。结合亲和性值可以用KD值表示，其通过当抗原和抗原结合分子的结合达到平衡时的解离速率与结合速率的比值(koff/kon)计算得到。所述抗原结合亲和性(例如KD)可以通过本领域己知的适宜方法适宜地确定，例如包括使用仪器如Biacore的等离子共振结合法。
在某些实施方式中，本申请所述抗体或抗原结合片段与CEACAM5以1ng/mL-10μg/mL的EC50(即半数结合浓度)结合。抗体或抗原结合片段与CEACAM5的结合可以通过本领域己知的方法如夹心法如ELISA，Western印迹，FACS或其他结合试验测定。在示例性的例子中，将待测抗体(即一抗)与固定化的CEACAM5或表达CEACAM5的细胞结合，随后洗掉未结合抗体，引入标记的二抗，其能够与一抗结合，因此能够检测出结合的二抗。当使用固定化的癌胚抗原细胞粘附分子5时，可在酶标仪板上进行所述检测，或当使用表达CEACAM5的细胞时，可使用FACS分析进行所述检测。
在某些实施方式中，本申请所述的抗体或抗原结合片段以10ng/mL-10μg/mL(使用FACS分析测定)的EC50(即50％的有效浓度)与CEACAM5结合。
所述抗体是CEACAM5特异性的。在某些实施方式中，所述抗体任选地不与CEACAM1、CEACAM3、CEACAM7、CEACAM8结合，且以显著低于与CEACAM5的结合亲和性结合CEACAM6。
在一些实施方式中，本申请所述的抗体能与具有免疫原性的物质联用，如肿瘤细胞、纯化的肿瘤抗原和用编码免疫刺激因子转染的细胞、肿瘤疫苗。此外，所述抗CEACAM5抗体和其抗原结合片段可以包括在联用治疗中，包括标准化学疗法和放射疗法、基于靶点的小分子疗法、其他新兴免疫检查点调节剂疗法。在一些实施方式中，所述抗体和其抗原结合片段可以用作抗体-药物缀合物、双特异性或多价抗体的基础分子。
在一些实施方式中，本申请所述的抗体和其抗原结合片段是骆驼化单域抗体(camelized single chain domain antibody)、双功能抗体(diabody)、scFv、scFv二聚体、BsFv、dsFv、(dsFv) 2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab') 2、ds双功能抗体(ds diabody)、纳米抗体、域抗体或双价域抗体。
在一些实施方式中，本申请所述的抗体包括免疫球蛋白恒定区。在一些实施方式中，免疫球蛋白恒定区包括重链和/或轻链恒定区。所述重链恒定区包括CH1、CH1-CH2或CH1-CH3区。在一些实施方式中，免疫球蛋白恒定区可以进一步包括一个或多个修饰以获得所需的性质。例如，可以将所述恒定区修饰，以降低或消除一种或多种效应功能，增强FcRn受体结合或引入一个或多个半胱氨酸残基。
在某些实施方式中，所述抗体及其抗原结合片段进一步包含缀合物。可以设想，本申请中的抗体或其抗原结合片段可与多种缀合物连接(见例如"Conjugate Vaccines"、Contributions to Microbiology and Immunology、J.M.Cruse and R.E.Lewis、Jr.(eds.)、Carger Press、New York(1989))。这些缀合物可以通过共价结合、亲和结合、嵌入、同等结合(coordinate binding)、络合、结合、混合或加入等其他方式与所述抗体或抗原结合片段连接。在某些实施方式中，本申请公开的抗体和抗原结合片段可以通过工程的方法使其含有表位结合部分以外的特定位点，这些位点可用来结合一种或多种缀合物。例如，这样的位点可包含一种或多种反应性氨基酸残基，例如半胱氨酸残基和组氨酸残基，用于协助与结合物的共价连接。在某些实施方式中，抗体可间接连于缀合物，或通过另一个缀合物相连。例如，所述抗体或其抗原结合片段可结合生物素，然后间接结合第二个缀合物，其与亲和素相连。所述缀合物可以是可检测的标记、药代动力学修饰部分、纯化部分或细胞毒性部分。可检测的标记的例子可以包括荧光标记(例如荧光素、罗丹明、丹酰、藻红蛋白或德克萨斯红)、酶底物标记物(例如辣根过氧化物酶、碱性磷酸酶、荧光素酶、葡糖淀粉酶、溶菌酶、糖氧化酶或β-D半乳糖昔酶)、稳定同位素或者放射性同位素、发色团部分、地高辛、生物素/亲和素、DNA分子或金以进行检测。在某些实施方式中，所述缀合物可以是药代动力学修饰部分如PEG，其帮助延长抗体的半衰期。其他适宜的聚合物 包括例如羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、乙二醇/丙二醇共聚物等。在某些实施方式中，所述缀合物可以是纯化部分例如磁珠。"细胞毒性部分"可以是对细胞有害的或可能损坏或杀死细胞的任何试剂。细胞毒性部分的示例包括，但不限于，紫杉醇、细胞松弛素B、短杆菌肽D、溴化乙绽、吐根碱、丝裂霉素、依托泊昔、替尼泊甘、长春新碱、长春碱、秋水仙碱、阿霉素、柔红霉素、二羟基炭疽菌素二酮、米托蒽醌、光神霉素、放线菌素D、l-去氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普茶洛尔、嘌呤霉素及其类似物、抗代谢物(例如，甲氨喋呤、6-巯基嘌呤、6-巯鸟嘌呤、阿糖胞苷、5氟尿嘧啶达卡巴)、烷化剂(例如氮芥、塞替派苯丁酸氮芥、美法仑、卡莫司汀(BSNU)和洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲霉素、丝裂霉素C和顺-二氯二胺铂(DDP)顺铂)、蒽环类抗生素(例如柔红霉素(以前的道诺霉素)和阿霉素)、抗生素(例如更生霉素(以前称为放线菌素)、博来霉素、光神霉素和氨茵霉素(AMC))以及抗有丝分裂剂(例如长春新碱和长春碱)。
核酸分子和重组方法
本申请的抗体可以以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，可通过化学合成或PCR扩增获得编码本申请抗体或其抗原结合片段的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本申请的抗体或其抗原结合片段。
本申请的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’) 2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’) 2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
在某些具体的实施方案中，本申请提供的抗CEACAM5抗体或其抗原结合的制备方法包括以下步骤：
1)以过表达癌胚抗原细胞粘附分子5的CHO细胞株为抗原免疫的Balb/c小鼠为材料，提取脾细胞，与小鼠骨髓瘤细胞系SP2/0-AG14融合，获得可表达抗CEACAM5抗体或其抗原结合片段的杂交瘤细胞株；
2)在由步骤1)得到的杂交瘤细胞株中克隆表达抗CEACAM5抗体或其抗原结合片段的基因；
3)提供表达载体，所述表达载体包含步骤2)所克隆得到的基因以及与所述基因操作性相连的表达调控序列；
4)用步骤3)所述的表达载体转化宿主细胞；
5)培养步骤4)得到的宿主细胞；
6)分离纯化得到单克隆抗体。
在另一方面，本申请提供了用于上述制备方法的杂交瘤细胞株。
本申请制备了分泌特异性抗CEACAM5抗体的人-鼠杂交瘤，利用分子生物学技术克隆了该抗体的重链和轻链序列(百分之百人的基因)，构建了抗人癌胚抗原细胞粘附分子5人源单克隆抗体，并由CHO细胞来表达生产抗体。这些抗体作为药物与现有抗体比较，具有更强的结合能力和特异性。
在另一个方面，本申请提供了一种分离的核酸分子，其包含编码本申请的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。在某些实施方案中，所述核酸分子包含如SEQ ID NO:14和/或SEQ ID NO:15所示的核苷酸编码序列。
在另一个方面，本申请提供了一种载体(例如克隆载体或表达载体)，其包含如上所述的分离的核酸分子。在某些实施方案中，本申请的载体是质粒，粘粒，噬菌体等。
在某些实施方案中，所述载体包含编码本申请的抗体或其抗原结合片段的重链可变区的第一核苷酸序列，和/或编码本申请的抗体或其抗原结合片段的轻链可变区的第二核苷酸序列。在某些实施方案中，所述第一核苷酸序列和第二核苷酸序列被提供在相同或不同的载体上。
在另一个方面，本申请提供了一种宿主细胞，其包含如上所述的分离的核酸分子或载体。在某些实施方案中，所述宿主细胞是哺乳动物细胞。在某些实施方案中，所述宿主细胞是人、鼠、羊、马、狗或猫的细胞。在某些实施方案中，所述宿主细胞是中国仓鼠卵巢细胞。
在另一个方面，提供了制备本申请的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
在另一个方面，本申请还提供了双特异性或多特异性分子，其包含所述的抗体或其抗原结合片段。在某些实施方案中，所述双特异性或多特异性分子特异性结合CEACAM5，并且额外地特异性结合一个或多个其他靶标。在某些实施方案中，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
在另一个方面，本申请还提供了免疫缀合物，其包含所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂。在某些实施方案中，所述治疗剂选自细胞毒剂。在某些实施方案中，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合。在某些实施方案中，所述免疫缀合物是抗体-药物偶联物(ADC)。
使用本领域公知的遗传工程学技术，可以将本申请所述抗体及其抗原结合片段的氨基酸序列转换成相应的DNA编码序列。由于遗传密码的简并性，转换所得的DNA序列可以不完全一致，而编码的蛋白序列保持不变。
使用本领域公知的重组技术，可以将包括编码所述抗体及其抗原结合片段的多核苷酸的载体引入宿主细胞用于克隆(扩增DNA)或基因表达。在另一实施方式中，所述抗体及其抗原结合片段可通过本领域公知的同源重组的方法制得。多种载体可供选择。载体组分通常包括，但不限于，以下的二种或多种：信号序列、复制起始点、一种或多种标记基因、增强序列、启动子(例如：SV40、CMV、EF-1a)和转录终止序列。
在一些实施方式中，所述载体系统包括哺乳动物、细菌、酵母系统等，并将包括质粒例如但不限于pALTER、pBAD、pcDNA、pCal、pL、pELpGEMEX、pGEX、pCLpCMV、pEGFP、pEGFT，pSV2、pFUSE、pVITRO，pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS420、pLexA、pACT2等其他可从实验室获得或市售的载体。适宜的载体可以包括质粒或病毒载体(例如复制缺陷型逆转录病毒、腺病毒和腺相关病毒)。
可以将包括编码所述抗体及其抗原结合片段的多核苷酸的载体引入宿主细胞用于克隆或基因表达。本申请中适用于克隆或表达所述载体中的DNA的宿主细胞为原核细胞、酵母或上述高级真核细胞。适用于本申请用途的原核细胞包括真细菌，如革兰氏阴性菌或 革兰氏阳性菌，例如肠杆菌科(如大肠杆菌)，肠杆菌属，欧文氏菌属，克雷白氏杆菌属，变形杆菌属，沙门氏菌属，如，鼠伤寒沙门(氏)杆菌，沙雷氏菌属，如粘质沙雷氏菌，以及志贺氏菌属，及杆菌属如枯草芽孢杆菌和地衣芽孢杆菌，假单胞菌如绿肽杆菌和链霉菌。
除了原核细胞以外，真核微生物如丝状真菌或酵母也可作宿主细胞克隆或表达编码抗体的载体。酿酒酵母，或面包酵母是最常用的低等真核宿主微生物。但是，许多其他属、种和株都比较常用且在本申请中适用，如粟酒裂殖酵母；克鲁维酵母属宿主如，乳酸克鲁维酵母、脆壁克鲁维酵母(ATCC12424)、保加利亚克鲁维酵母(ATCC16045)、魏氏克鲁维酵母(ATCC24178)、克鲁雄酵母(ATCC56500)、果蝇克鲁维酵母(ATCC36906)、耐热克鲁维酵母和马克斯克鲁维酵母：解脂耶氏酵母(EP402226)；巴斯德毕赤酵母(EP183070)；假丝酵母：里氏木霉(EP244234)；链孢霉；西方许旺酵母，如：西方许旺酵母；和丝状真菌，如：脉孢菌、青霉菌、弯颈霉和曲霉菌，如：钩巢曲霉和黑曲霉。
本申请中提供的适用于表达糖基化抗体或其抗原结合片段的宿主细胞由多细胞生物衍生得到。无脊椎细胞的实例包括植物和昆虫细胞。己发现多种杆状病毒株(baculoviral strains)及其变体以及对应的许可性昆虫宿主细胞(permissive insect host cells)，来自于诸如以下的宿主：草地夜蛾(毛虫)、埃及斑蚊(蚊子)、白纹伊蚊(蚊子)、黑腹果蝇(果蝇)及家蚕。多种用于转染的病毒株为公众可得，例如苜蓿银纹夜蛾核型多角体病毒和家蚕核型多角体病毒的Bm-5变种，这些病毒都可在本申请中使用，特别是用于转染草地夜蛾细胞。棉花、玉米、土豆、大豆、矮牵牛花、西红柿和烟草的植物细胞培养也可用作宿主。
但是，最感兴趣的是脊椎细胞，且脊椎细胞的培养(组织培养)己经成为常规操作。可用的哺乳动物宿主细胞实例有，SV40转化的猴肾细胞CV1系(COS-7，ATCC CRL 1651)；人胚胎肾细胞系(293或悬浮培养的293细胞亚克隆，Graham et al.,].Gen Virol.36：59(1977))；幼地鼠肾细胞(B血，ATCC CCL 10)；中国仓鼠卵巢细胞/-DHFR(CHO，Urlaub et al.，Proc.Natl.Acad.Sci.USA 77：4216(1980))；小鼠睾丸支持细胞(TM4，Mather J.P.,Biol.Reprod.23:243-252(1980))；猴肾细胞(CV1ATCC CCL 70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC CCL 2)；犬肾细胞(MDCK，ATCC CCL 34)；布法罗大鼠肝细胞(BRL 3A,ATCC CRL 1442)；人肺细胞(W138，ATCC CCL75)；人肝细胞(Hep G2,HB 8065)；小鼠乳腺瘤(MMT 060562,ATCC CCL51)；TRI细胞(Mather等，Annals N.Y.Acad.Sci.383：44-68(1982))； MRC 5细胞；FS4细胞；及人肝癌细胞系HepG2)。在某些优选的实施方式中，所述宿主细胞是293F细胞。
用上述的可产生所述抗体及其抗原结合片段的表达或克隆载体转化宿主细胞，并将其在常规的营养培养基中培养，所述营养培养基经修饰后适宜于诱导启动子、选择转化细胞或扩增编码目的序列的基因。
本申请中用于产生所述抗体及其抗原结合片段的宿主细胞可在多种本领域公知的培养基中培养。所述培养基还可含有本领域公知的适当浓度的任何其他必要的添加剂。所述培养基的条件，如温度、pH值等类似条件，为选择用于表达的宿主细胞此前所使用的条件，为普通技术人员所熟知。
在使用重组技术时，所述抗体可在胞内、壁膜空间生成，或直接分泌到培养基中。如果所述抗体在胞内生成，首先除去宿主细胞或裂解片断的颗粒残骸，例如，可通过离心或超声的方法。Carter et al.,Bio/Technology 10：163-167(1992)描述了将分泌到大肠杆菌壁膜空间的抗体分离的方法。简要地说，在醋酸铀(pH 3.5)、EDTA和苯甲磺酣氟(PMSF)存在的条件下化开细胞糊(cell paste)约30分钟以上。离心除去细胞碎片。如所述抗体分泌到培养基中，则通常首先使用市售的蛋白浓度过滤器，如IAmicon或Millipore Pellicon ultrafiltration unit，浓缩该表达系统的上清液。在任何前述的步骤中都可加入蛋白酶抑制剂如PMSF以抑制蛋白降解，以及抗生素以防止偶然污染物的生长。
从所述细胞中制得的抗体可采用纯化方法进行纯化，例如羟磷灰石色谱、凝胶电泳、透析、DEAE-纤维素离子交换色谱柱、硫酸铵沉淀、盐析以及亲和色谱，其中亲和色谱为优选的纯化技术。所述抗体的种类以及所述抗体中存在任何免疫球蛋白的Fc结构域决定了蛋白A作为亲和配体是否适合。蛋白A可用于纯化基于人γ1、γ2或γ4重链的抗体(Lindmark et al.，J.Immunol.Meth.62：13(1983))。蛋白G适用于所有鼠源异构体和人γ3(Guss et al.,EMBO J.5：1567 1575(1986))。琼脂糖是最常用的亲和配体附着基质，但也可选用其他基质。机械力稳定的基质如可控孔度玻璃或聚(苯乙烯)苯与用琼脂糖相比可实现更快的流速和更短的处理时间。如该抗体含有CH3结构域，则可用Bakerbond ABX.TM树脂进行纯化(J.T.Baker，Phillipsburg，N.J.)。也可根据需要获得的抗体确定其他蛋白纯化的技术，如离子交换柱中的分馏、乙醇沉淀、反相HPLC、硅胶色谱、基于阴离子或阳离子交换树脂的肝素琼脂糖凝胶色谱(如聚天冬氨酸柱)、层析聚焦、SDS-PAGE、以及硫酸铵沉淀。
在任意初步纯化步骤之后，可用低pH疏水相互作用色谱的方法处理含有感兴趣的抗体和杂质的混合物，用pH约2.5-4.5的洗脱缓冲液，优选地在低盐浓度下进行(例如，从约0到0.25M盐浓度)。
药物组合物及治疗用途
在另一个方面，本申请提供了一种药物组合物，其含有本申请的抗体或其抗原结合片段、本申请的双特异性或多特异性分子或者本申请的免疫缀合物以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述药物组合物还可以包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，所述抗体或其抗原结合片段、双特异性或多特异性分子或免疫缀合物与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。本申请的抗体或其抗原结合片段与所述另外的药学活性剂可以同时、分开或相继施用。
本申请进一步提供了包括所述抗体的药物组合物和一个或多个药学上可接受的载体。
用在本申请公开的药物组合物中的药用可接受载剂可包括，例如，药用可接受的液体、凝胶或固体载剂、水相介质、非水相介质、抗微生物物质、等渗物质、缓冲液、抗氧剂、麻醉剂、悬浮剂/分散剂、整合剂、稀释剂、佐剂、辅料或无毒辅助物质，其他本领域公知的组分或以上的多种组合。
适用的组分可包括，例如，抗氧剂、填充剂、粘合剂、崩解剂、缓冲液、防腐剂、润滑剂、矫味剂、增稠剂、着色剂、乳化剂或稳定剂例如糖和环糊精。适用的抗氧剂可包括，例如，甲硫氨酸、抗坏血酸、EDTA、硫代硫酸钠、铂、过氧化氢酶、柠檬酸、半胱氨酸、巯基甘油、巯基乙酸、巯基山梨醇、丁基甲基茴香醚、丁基化羟基甲苯和/或没食子酸丙酯。在一种含有本申请公开的抗体的组合物中包括一种或多种抗氧剂如甲硫氨酸，可将降低所述抗体的氧化。对氧化作用的减少可防止或减少结合亲和力的降低，从而提高抗体稳定性并延长保质期。
进一步的说，药用可接受的载剂可包括，例如，水相介质如氯化钠注射液、林格氏液注射液、等渗葡萄糖注射液、无菌水注射液、或葡萄糖和乳酸林格注射液、非水介质例如：植物来源的不挥发性油、棉花子油、玉米油、芝麻油、或者花生油、细菌抑制或真菌抑制浓度下的抗菌物质、等渗剂如氯化钠或葡萄糖、缓冲液如磷酸盐或枸橼酸盐缓冲液，抗氧化剂如硫酸氢钠，局部麻醉剂如盐酸普鲁卡因，助悬剂和分散剂如羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮，乳化剂如聚山梨醇酯80(吐温-80)、整合试剂如EDTA(乙二胺四乙酸)或EGTA(乙二醇双(2-氨基乙基醚)四乙酸)、乙醇、聚乙二醇、丙二醇、氢氧化钠、盐酸、柠檬酸或乳酸。作为载剂的抗菌剂可加入多次剂量容器中的药物组合物中，其包括酚类或甲酚、汞制剂、苯甲醇、氯代丁醇、甲基和丙基对羟基苯甲酸酯、噻汞撒、氯苯甲烷铵和氯苯乙铵。适用的辅料可包括，例如，水、盐、葡萄糖、甘油或乙醇。适用的无毒辅助物质可包括，例如，乳化剂、pH值缓冲剂、稳定剂、增溶剂，或者醋酸钠、去水山梨糖醇月桂酸酯、三乙醇胺油酸酯或者环糊精之类的物质。
所述药物组合物可以是液体溶液、悬浮液、乳剂、丸剂、胶囊、片剂、持续释放制剂或粉末。口服制剂可以包括标准载体如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、聚乙烯吡咯烷酮、糖精钠、纤维素、碳酸镁等。
在某些实施方式中，所述药物组合物被制剂成可注射的组合物。可注射的药物组合物可以任何常规的形式制备，例如，液体溶剂、悬浮剂、乳化剂或适用于产生液体溶剂、悬浮剂或乳化剂的固体形式。注射制剂可包括现用的无菌和/或无热原溶液、使用前现与溶剂结合的无菌干燥的可溶物，如冻干粉，包括皮下片、注射即用的无菌悬浮剂、使用前现与介质结合的无菌干燥不溶产品，和无菌和/或无热原的乳剂。溶剂可以为水相或非水相。
在某些实施方式中，单位剂量的注射制剂包装在一个安瓿、一支管或一支带有针的针筒中。本领域悉知，所有注射给药的制剂应为无菌无热原。
在某些实施方式中，通过将本申请公开的抗体或其抗原结合片段溶解于某适当的溶剂中可制备无菌冻干的粉末。所述溶剂可含有一种可提高粉或由粉末制得的重组溶液的稳定性，或改善粉末或重组溶液的其他药理组分。适用的辅料包括，但不限于，水、葡萄糖、三梨糖醇、果糖、玉米糖浆、木糖醇、甘油、葡萄糖、黑糖或其他适用的物质。溶剂可含有缓冲液，如枸橼酸缓冲液、磷酸钠或磷酸钾缓冲液或其他本技术熟练人员公知的缓冲液，在一种实施方式中，缓冲液的pH为中性。在本领域公知的标准条件下进行对所述溶液进行随后的过滤除菌，然后冻干制得理想的制剂。在一种实施方式中，将所得的溶剂分装至小管中冻干。每支小管可容纳单次剂量或多次剂量的所述抗CEACAM5抗体或其抗原结 合片段或其组合物。每支小管中的装入量可略微高于每次剂量所需或多次剂量所需(例如10％过量)，从而保证取样精确和给药精确。冻干粉可在适当的条件下储存，如在约4℃到室温范围。
用注射用水将冻干粉重溶得到用于注射给药的制剂。在一种实施方式中，可将冻干粉加至无菌无热原水或其他适用的液体载剂中重溶。精确的量由选择的疗法决定，可根据经验值决定。
抗体衍生化和免疫缀合物
本申请的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会不利影响其对CEACAM5的结合。因此，本申请的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本申请的抗体或其抗原结合片段功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。此外，本申请的抗体或其抗原结合片段还可以用化学基团进行衍生，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。这些基团可用于改善抗体的生物学特性，例如增加血清半衰期。
因此，在一个方面，本申请提供一种免疫缀合物，其包含本申请所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂；
在某些实施方案中，所述治疗剂选自细胞毒剂；
在某些实施方案中，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合；
优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
试剂盒及检测用途
本申请的抗体或其抗原结合片段能够特异性结合CEACAM5蛋白，并且基本上不与CEACAM1、CEACAM3、、CEACAM7、CEACAM8蛋白结合，仅与CEACAM6蛋白有较弱的结合，因此，本申请的抗体或其抗原结合片段在检测中具有更高的特异性和准确性。
因此，在另一个方面，本申请提供了一种试剂盒，其包括本申请的抗体或其抗原结合片段、或本申请的缀合物。
在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
在某些实施方案中，所述试剂盒还包括第二抗体，其特异性识别所述的抗体或其抗原结合片段。
在某些实施方案中，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
在某些实施方案中，本申请的试剂盒可以进一步包含用于使相应可检测的标记被检测到的试剂。例如，当所述可检测的标记为酶时，所述试剂盒还可以包含相应酶的显色底物，例如用于辣根过氧化物酶的邻苯二胺(OPD)、四甲基联苯胺(TMB)、ABTS或鲁米诺类化合物，或用于碱性磷酸酶的对硝基苯磷酸酯(p-NPP)或AMPPD。例如当所述可检测的标记为化学发光试剂(例如吖啶酯类化合物)时，所述试剂盒还可以包含用于化学发光的预激发液和/或激发液。
在另一个方面，本申请还提供了所述的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测肿瘤是否能够通过靶向CEACAM5的抗肿瘤疗法来治疗。
在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记。
在某些实施方案中，所述CEACAM5是人CEACAM5。
在某些实施方案中，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
本申请提供了包括所述抗体或其抗原结合片段的试剂盒。在一些实施方式中，所述试剂盒用于检测在生物样品中的CEACAM5的存在情况或水平。所述生物样品可以包括细胞或组织。
在一些实施方式中，所述试剂盒包括与可检测标记缀合的抗体或其抗原结合片段。在一些实施方式中，所述试剂盒包括未标记的抗体，并进一步包括能够与未标记的抗体结合标记的二抗。所述试剂盒可以进一步包括使用说明和在试剂盒中将每个组件分隔开的包装。
在一些实施方式中，所述抗体与底物或仪器连接用于夹心测定如ELISA或免疫色谱测定。适用的底物或仪器可以是例如微孔板和试纸。
嵌合抗原受体
在另一个方面，本申请还提供了嵌合抗原受体，其包含所述的抗体或其抗原结合片段的抗原结合结构域。
在某些实施方案中，所述抗原结合结构域包含所述的抗体或其抗原结合片段的重链可变区和轻链可变区。
在某些实施方案中，所述抗原结合结构域是scFv。
在某些实施方案中，所述抗原结合受体包含所述的抗体的抗原结合片段。
在某些实施方案中，所述抗原结合受体由免疫效应细胞(例如T细胞)所表达。
在另一个方面，本申请还提供了分离的核酸分子，其编码所述的嵌合抗原受体。
在另一个方面，本申请还提供了载体，其包含所述的分离的核酸分子；在某些实施方案中，其用于制备嵌合抗原受体T细胞。
在另一个方面，本申请还提供了宿主细胞，其包含所述的分离的核酸分子或载体；
优选地，所述宿主细胞是免疫效应细胞(例如，T细胞或NK细胞)；
优选地，所述宿主细胞是嵌合抗原受体T细胞(CAR-T)。
检测方法与治疗方法
在许多恶性肿瘤中，CEACAM5的过度表达可以作为肿瘤生物标志物。因此，测量患者血液中的CEACAM5可用于癌症的预后和控制，CEACAM5的靶向治疗也成为潜在的癌症治疗方法。而本申请的抗体(例如，UM05-5L和hUM05-3)能够高亲和、高特异的结合CEACAM5蛋白，并具有ADCC活性，能够抑制肿瘤生长，杀灭肿瘤细胞。
因此，在另一个方面，本申请还提供了一种抑制表达CEACAM5的肿瘤细胞生长和/或杀伤所述肿瘤细胞的方法，其包括将所述肿瘤细胞与有效量的所述的抗体或其抗原结合片段，或所述的双特异性或多特异性分子，或所述的免疫缀合物，或所述的药物组合物，或所述的嵌合抗原受体，或所述的宿主细胞接触。
在另一个方面，本申请还提供了一种用于降低CEACAM5在细胞表面的表达水平的方法，其包括将所述细胞与所述的抗体或其抗原结合片段，或所述的双特异性或多特异性分子，或所述的免疫缀合物，或的药物组合物，或所述的嵌合抗原受体，或所述的宿主细胞接触，使得CEACAM5在所述细胞表面的表达水平降低；其中，所述细胞在其表面表达CEACAM5。
在某些实施方案中，所述细胞是表达CEACAM5的肿瘤细胞。
在另一个方面，本申请还提供了一种用于在受试者(例如人)中预防和/或治疗肿瘤的方法，所述方法包括向有此需要的受试者施用有效量的所述的抗体或其抗原结合片段，或所述的双特异性或多特异性分子，或所述的免疫缀合物，或所述的药物组合物，或所述的嵌合抗原受体，或所述的宿主细胞。
在某些实施方案中，所述肿瘤表达CEACAM5。
在某些实施方案中，所述肿瘤涉及表达CEACAM5的肿瘤细胞。在某些实施方案中，所述CEACAM5在所述肿瘤细胞表面上表达。
在某些实施方案中，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在某些实施方案中，所述方法还包括施用另外的具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，所述方法还包括施用另外的抗肿瘤疗法，例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗。
在另一个方面，本申请还提供了所述的抗体或其抗原结合片段，或所述的双特异性或多特异性分子，或所述的免疫缀合物，或所述的药物组合物，或所述的嵌合抗原受体，或所述的宿主细胞，在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/治疗肿瘤。
在某些实施方案中，药物还包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，所述肿瘤表达CEACAM5。
在某些实施方案中，所述肿瘤涉及表达CEACAM5的肿瘤细胞；优选地，所述CEACAM5在所述肿瘤细胞表面上表达。
在某些实施方案中，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在另一个方面，本申请还提供了一种检测CEACAM5(例如人CEACAM5)在样品中的存在或其量的方法，其包括以下步骤：
(1)将所述样品与所述的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与CEACAM5之间复合物的形成或检测所述复合物的量。
在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记。
在某些实施方案中，所述CEACAM5是人CEACAM5。
在另一个方面，本申请还提供了一种用于检测肿瘤是否能够通过靶向CEACAM5的抗肿瘤疗法来治疗的方法，其包括以下步骤：
(1)将含有所述肿瘤细胞的样品与所述的抗体或其抗原结合片段接触；
(2)检测所述抗体或其抗原结合片段与CEACAM5之间复合物的形成。
在某些实施方案中，所述抗体或其抗原结合片段带有可检测的标记。
在某些实施方案中，所述CEACAM5是人CEACAM5。
在某些实施方案中，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
本申请还提供了治疗方法，包括将治疗有效量的本申请所述的抗体施用给需要其的受试者。
本申请中提供的抗体的治疗有效剂量依赖于本领域公知的多种因素，例如体重、年龄、过往病史、现用治疗、对象的健康状况和交叉感染的潜力、过敏、超敏和副作用，以及给药途径和肿瘤发展的程度。本领域熟练人员(例如医生或兽医)可根据这些或其它条件或要求按比例降低或升高剂量。
在某些实施方式中，本申请提供的抗体可在治疗有效剂量约0.01mg/kg到约100mg/kg之间给药。在某些实施方式中，所述抗体以约50mg/kg或更少的剂量给药，在某些实施方式中，给药剂量为10mg/kg或更少、5mg/kg或更少、1mg/kg或更少、0.5mg/kg或更少或0.1mg/kg或更少。某特定剂量可在多个间隔给药，例如每天一次、每天两次或更多、每月两次或更多、每周一次、每两周一次、每三周一次、每月一次或每两月或更多月一次。在某些实施方式中，给药剂量可随治疗进程变化。例如，在某些实施方式中，初始给药剂量可比后续给药剂量高。在某些实施方式中，给药剂量在治疗进程中根据给药对象的反应进行调整。
给药方案可通过调整达到最优反应(如治疗反应)。例如，可进行单剂量给药或在一段时间分多个分隔的剂量给药。
本申请中公开的抗体可通过本领域公知的给药方式给药，例如注射给药(如，皮下注射、腹腔注射、静脉注射，包括静脉滴注，肌肉注射或皮内注射)或非注射给药(如，口服给药、鼻腔给药、舌下给药、直肠给药或外用给药)。
在某些实施方式中，所述抗体可用于治疗与其分子机制相关的病症，包括肿瘤和癌症，例如非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
使用方法
本申请进一步提供了使用所述抗体的方法。
在一些实施方式中，本申请提供了在个体中治疗与所述抗体机制相关的状况或病症的方法，包括施用治疗有效量的本申请所述的抗体。
本申请公开的抗体可单独给药或与一种或多种其他治疗手段或物质联合给药。例如，本申请公开的抗体可与化疗、放疗、癌症治疗手术(如肿瘤切除术)、抗病毒药物、一种或多种抗呕吐药或其他化疗导致的并发症的疗法、或任何其他用于癌症或病毒的治疗物质进行联用。在某些这样的实施方式中，本申请公开的抗体与一种或多种治疗物质联用时，可与所述的一种或多种治疗物质同时给药，在某些这样的实施方式中，所述的抗体可作为同一个药物组合物的一部分同时给药。但是，与其他治疗物质"联用"的抗体不需要同时给药或与该治疗物质在同一组合物中给药。本申请中"联用"的含义还包括在另一个治疗物质之前或之后给药的抗体也被认为是与该治疗物质"联用"，即使所述抗体与第二种物质通过不同给药方式给药。在可能的情况下，与本申请公开的抗体联用的其他治疗物质可参照该其他治疗物质的产品说明书的方法用药，或参照、外科医生的案头参考书2003(Physicians' Desk Reference，57th Ed；Medical Economics Company；ISBN：1563634457；第57版(2002年11月))，或参照其他本领域公知的方法。
在某些实施方式中，所述治疗物质能够诱导或增强针对癌症的免疫反应。例如，肿瘤疫苗可以用于诱导对某些肿瘤或癌症的免疫应答。细胞因子治疗可以用于提高将肿瘤抗原向免疫系统的递呈。细胞因子治疗的示例包括但不限于干扰素如干扰素α、β和γ，集落剌激因子如巨噬细胞CSF、粒细胞巨噬细胞CSF和粒细胞CSF，白介素如IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7，IL-8、IL-9、IL-10、IL-11和IL-12，肿瘤坏死因子如TNF-α和TNF-β。还可以使用灭活免疫抑制目标的试剂，如PD-1抗体、TGF-β抑制剂、IL-10抑制剂和Fas配体抑制剂。另一组试剂包括激活针对肿瘤或癌细胞的免疫响应的那些试剂，例如，提高T细胞激活(如T细胞共刺激信号通路如CTLA-4、ICOS、OX40、4-1BB等通路)的那些，以及提高树突细胞功能和抗原递呈的那些。
以下实施例旨在更好地说明本申请，且不应理解为限制本申请的范围。所有下述的特定组合物、材料和方法，其整体或部分都在本申请的范围内。这些特定的组合物、材料和方法不是为了限制本申请，而只是为说明在本申请的范围内的特定的实施方式。本领域熟练技术人员可不添加创造性及不偏离本申请范围而开发出等同的组合物、材料和方法。应理解，在对本申请的方法作出的多种改动可以仍然包括在本申请范围内。发明人意在将这样的变动包括在本申请的范围内。
序列信息
本申请所涉及的部分序列的信息如下面的表1所示。
表1.部分序列的信息
Figure PCTCN2020094045-appb-000001
Figure PCTCN2020094045-appb-000002
Figure PCTCN2020094045-appb-000003
有益效果
本申请的单克隆抗体(例如UM05-5L、hUM05-3抗体)能够以高特异性和高选择性与CEACAM5蛋白或表达CEACAM5蛋白的细胞结合，并且其基本不与CEACAM1、CEACAM3、CEACAM7、CEACAM8蛋白结合，仅与CEACAM6蛋白有较弱的结合。同时，抗体UM05-5L还具有ADCC活性。基于抗体UM05-5L所构建的UM05-5L-CAR能够表达于人T细胞表面，在体内能够识别肿瘤细胞，分泌细胞因子，具有强烈的抑制肿瘤作用。因此，本申请的单克隆抗体(例如UM05-5L、hUM05-3抗体)较高的临床应用价值。
附图说明
图1显示了本申请构建的过表达CEACAM5的CHO细胞株的过表达倍数；与常规的CHO细胞相比，本申请构建的CHO细胞株过表达倍数达904倍。
图2显示了抗体UM05-5L与CEACAM5蛋白结合的ELISA实验结果图。
图3显示了抗体UM05-5L与LOVO细胞结合能力的检测结果。
图4a-4b显示了抗体UM05-5L的ADCC报告基因活性；其中，图4a显示了阳性对照(Erbitux抗体)和阴性对照的ADCC报告基因活性，图4b显示了UM05-5L的ADCC报告基因活性。
图5a-5b显示了流式细胞的检测结果；图5a为对照T细胞(Mock T)的表达效率，图5b为UM05-5L-CAR的表达效率。
图6显示了UM05-5L-CAR-T与肿瘤细胞混合后细胞因子IFNγ(左图)和IL-2(右图)的分泌结果。
图7显示了UM05-5L-CAR-T细胞对肿瘤细胞KATO3的杀伤结果。
图8显示了UM05-5L-CAR-T细胞经肿瘤细胞LS174T刺激后的扩增结果。
图9显示了UM05-5L-CAR-T在小鼠体内对肿瘤的抑制结果。
图10显示了接受UM05-5L-CAR-T的小鼠体内IFNγ的释放情况。
图11显示了人源化抗体hUM05-3与LS174T细胞结合能力的检测结果。
图12显示了抗体UM05-5L、UM05-5L C47W、hUM05-3和hUM05-3 W47C与LS174T细胞结合能力的检测结果。
具体实施方式
现参照下列意在举例说明本申请(而非限定本申请)的实施例来描述本申请。
除非特别指明，本申请中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本申请，且不意欲限制本申请所要求保护的范围。
实施例1：抗人CEACAM5蛋白的单克隆抗体的获得
本申请构建了过表达CEACAM5蛋白的CHO细胞株。简言之，将CEACAM5的质粒(北京义翘神州科技有限公司，HG11077-UT)转染入CHO细胞株(ATCC)并表达。该质粒带有潮霉素(Hygromycin)抗性，因此稳定转染具有该质粒的细胞可以在含有Hygromycin的培养基中稳定传代。挑取细胞并测量CEACAM5的表达量，如图1所示，经FACS检测得到的CHO-CEACAM5细胞，其CEACAM5的过表达倍数达904倍。
用CHO-CEACAM5细胞和从肿瘤病人中提纯的CEACAM5蛋白(上海领潮生物，L2C01001)按表2的免疫方案对6只5-8周龄Balb/c mice小鼠(上海斯莱克)进行免疫。
表2.小鼠免疫方案
日期 操作 抗原 佐剂
第1天 腹腔注射免疫 CHO-CEACAM5细胞 CFA
第26天 腹腔注射免疫 CHO-CEACAM5细胞 IFA
第49天 腹腔注射免疫 CHO-CEACAM5细胞 IFA
第91天 腹腔注射免疫 CEACAM5蛋白 Gerbu(MM3001)
第104天 腹腔注射免疫 CEACAM5蛋白 Gerbu(MM3001)
第105天 腹腔注射免疫 CEACAM5蛋白 Gerbu(MM3001)
第106天 腹腔注射免疫 CEACAM5蛋白 Gerbu(MM3001)
免疫结束后第二天，取免疫小鼠的脾细胞与SP2/0-AG14细胞(ATCC)进行杂交瘤细胞融合，并取适量融合后的细胞铺至96孔板。融合后10天左右取各孔上清，以ELISA法检测杂交瘤细胞分泌的小鼠抗体与人CEACAM5的结合活性。
进一步的，取ELISA检测得到的强阳性孔中的上清，以流式细胞术检测与LOVO细胞(中国科学院上海细胞生物学研究所)的结合活性，得到了具有较高LOVO细胞结合活性的杂交瘤细胞。将这部分细胞进行亚克隆获得单克隆细胞。表3所示的是部分杂交瘤细胞的检测数据。
表3.杂交瘤细胞与CEACAM的结合结果
杂交瘤细胞 ELISA OD值 LOVO FACS结合
1-2C5 1.0690 12.1
1-9G3 1.0390 7.01
3-2F3 1.4480 11
3-6F5 1.3520 6.51
3-8C10 2.3030 137
3-8G8 1.9270 82.9
2A10A5 2.392 /
2A10C4 2.434 /
选择其中结合活性较好的杂交瘤细胞3-8C10进行测序，经测序获得了一株抗体，命名为UM05-5，使用Kabat编号系统(Kabat等，Sequences of Proteins of Immunological Interest，第五版，Public Health Service，美国国立卫生研究院，贝塞斯达、马里兰州(1991)，第647-669页)确定了UM05-5抗体的CDR序列，具体如表4所示。
表4.UM05-5可变区序列
Figure PCTCN2020094045-appb-000004
实施例2：人鼠嵌合抗体的制备
根据表4中UM05-5的序列，设计并表达了人鼠嵌合抗体，将其命名为UM05-5L。简言之，将编码小鼠抗体的VH和VL序列分别连接至编码人IgG1重链的恒定区序列(其 氨基酸序列如SEQ ID NO:9，核苷酸序列如SEQ ID NO:10所示，)和κ链的恒定区序列(其氨基酸序列如SEQ ID NO:11，核苷酸序列如SEQ ID NO:12所示)，得到人鼠嵌合抗体UM05-5L。
将编码抗体重链、轻链的核苷酸序列分别克隆到哺乳动物细胞表达载体pcDNA3.4上。将重链表达载体和轻链表达载体按2：1的摩尔比用Lipofectamine 2000转染试剂(Invitrogen)转染入HEK293细胞，并在37℃、5％二氧化碳条件下培养7天。收集培养液上清，并用Protein A亲和层析法提纯上清中的抗体。纯化后的抗体经PBS溶液透析和冷冻干燥浓缩后，保存于-20℃。
实施例3：抗体与CEACAM5蛋白的结合
将浓度为1μg/mL的人CEACAM5蛋白溶液以100μL/well包被96孔高亲和力板，4℃，振荡过夜。第二天先以300μL PBST(Tween20：0.5‰)洗涤3次，之后用100μL/well的5％BSA/PBS封闭1小时，室温振荡。300μL PBST洗涤3次。用PBS配制抗体样品的梯度稀释溶液。以100μL/孔加入96孔板，室温振荡1小时。300μL PBST洗涤3次。配制二抗羊抗人(goat anti-human)IgG HRP溶液，以100μL/孔加入96孔板，室温振荡30min。300μL PBST洗涤4次。加入100μL/孔TMB，显色20min。加入100μL/孔0.6N H 2SO 4，终止显色，检测OD450nm。经检测，结果如图2所示，嵌合抗体UM05-5L与CEACAM5蛋白结合的EC50为105.3ng/mL。
实施例4：抗体与表达CEACAM5的细胞的结合
用流式细胞仪(FACS)检测抗体与天然表达人CEACAM5蛋白的LOVO肿瘤细胞或过表达不同CEACAM蛋白细胞的结合情况。简单来说，首先收集细胞，PBS洗涤一遍后计数，稀释成2*10 6/ml细胞悬液；分别取10μl抗体工作液加入100μl细胞悬液中，4℃避光孵育30min；PBS洗涤2次后，加入对应的荧光标记二抗Goat-anti-Human IgG(H+L)(Invitrogen)，4℃避光孵育30min，PBS洗涤2次后，以400μl FACS buffer悬起，以FACS检测抗体与细胞的结合情况。
检测了嵌合抗体UM05-5L和对照抗体UM05-8(本实验室通过杂交瘤法自制的抗CEACAM5抗体)的上清在浓度为3μg/mL时与不同细胞的结合情况。其中，SW620-CEACAM1为过表达CEACAM1蛋白的细胞，SW620-CEACAM3为过表达CEACAM3蛋白的细胞，CHO-CEACAM6为过表达CEACAM6蛋白的细胞， CHO-CEACAM7为过表达CEACAM7蛋白的细胞，CHO-CEACAM8为过表达CEACAM8蛋白的细胞。以抗体与LOVO细胞(其表达CEACEM5)的结合活性(即，FACS的检测值)为100％作为基准，计算抗体与不同细胞(其表达不同CEACAM蛋白)的相对结合活性，检测结果如表5所示。
表5.抗体与LOVO细胞和过表达不同CEACAM蛋白的细胞的结合情况
细胞名称 UM05-5L UM05-8
LOVO 100.00％ 100.00％
SW620-CEACAM1 2.01％ 120.47％
SW620-CEACAM3 1.29％ 91.56％
CHO-CEACAM6 26.60％ 69.61％
CHO-CEACAM7 0.38％ 13.71％
CHO-CEACAM8 0.40％ 61.60％
从表5中可以看到，嵌合抗体UM05-5L具有尤为突出的选择性：其以高亲和力结合CEACAM5，但与CEACAM1、CEACAM3、CEACAM7、CEACAM8均无明显结合，且与CEACAM6仅有较弱的结合。而普通的抗体(如对照抗体UM05-8)，在与CEACAM5结合的同时，能够以较高水平与CEACAM1、CEACAM3、CEACAM6、CEACAM7、CEACAM8结合，不具有选择性。
进一步的，我们用FACS检测了嵌合抗体UM05-5L与天然表达人CEACAM5的LOVO肿瘤细胞的EC50。结果如图3所示，嵌合抗体UM05-5L与LOVO细胞结合EC50为3173ng/mL。综合上述结果，嵌合抗体UM05-5L具有非常优良的与CEACAM5结合的亲和力、特异性和选择性。
实施例5：抗体的ADCC活性
使用LOVO细胞为靶细胞，将LOVO细胞按20000个/孔密度接种于96孔细胞培养板，37℃，5％CO 2孵育过夜。第二天，将抗体UM05-5L和阳性对照抗体Erbitux按照20μg/ml配制于培养基中，并按3倍倍比稀释8个浓度。吸尽LOVO细胞上清培养基，将以指定浓度稀释的抗体(阳性对照抗体Erbitux，嵌合抗体UM05-5L或阴性对照无关抗体)按照30μL/孔加入LOVO细胞中。接着，将效应细胞Jurkat/ADC(南京诺艾新生物技术有限公司)按照120000个/30μL/孔铺到LOVO细胞孔中，37℃，5％CO 2孵育16-20h。孵育结束后以Bright-Glo试剂盒(Promega，cat.E2620)检测效应细胞中luciferase表达水 平。该luciferase水平代表了效应细胞的ADCC活化程度。图4a显示了阳性对照Erbitux抗体和阴性对照的ADCC报告基因活性，图4b显示了UM05-5L的ADCC报告基因活性。综合上述结果可知，UM05-5L的ADCC活性非常强，EC50为0.85μg/mL。
实施例6：靶向CEACAM5的CAR-T细胞的构建
我们基于第二代及第三代的CAR-T结构，以抗CEACAM5单链抗体作为CAR-T识别抗体，采用1个或多个41BB，CD28，OX40等共刺激原件进行了不同CEA-CAR的结构设计，并构建相应的慢病毒质粒用于生成可感染细胞并表达相应CAR的慢病毒。该慢病毒可以用于感染自肿瘤病人外周血分离到的T细胞，产生在细胞膜表面表达相应CAR受体的CAR-T细胞。这种CAR-T细胞可以有效识别和杀伤表达CEACAM5的肿瘤细胞。在体外和体内实验中都看到了该细胞免疫疗法具有非常好的安全性和有效性。
将如SEQ ID NO：13所示的单链抗体UM05-5L scFv(以VH-G4S3-VL顺序)与序列CD8α-CD137-CD3ζ-p2A-tEGFR相连接，从而构建嵌合抗原受体CAR，然后将编码所述CAR的核苷酸序列克隆至慢病毒载体(吉凯基因，GV401)中，将载体命名为UM05-5L-CAR。表达框为EF1a启动子-CAR-2A-tEGFR-WPRE。
将UM05-5L-CAR慢病毒载体与包装质粒瞬时转染293T细胞16小时，更换培养基，继续培养24-48小时，收集培养基上清(含慢病毒)，于-80℃保存。
将来源于健康人的PBMC细胞，用CD3/28抗体激活24小时后；按照MOI＝3:1将慢病毒与T细胞混合培养96小时，用PE荧光标记的Cetuximab检测UM05-5L-CAR的表达。结果如图5所示，与对照相比(图5a)，UM05-5L-CAR(图5b)可很好的表达于人T细胞表面。
实施例7：靶向CEACAM5的CAR-T细胞的功能检测
1、细胞因子释放检测
将UM05-5L-CAR-T细胞和表达CEACAM5的肿瘤细胞(例如KATO3和LS174T)混合，并设置培养基对照(即仅使用UM05-5L-CAR-T细胞)和CEACAM5阴性细胞对照(即使用UM05-5L-CAR-T细胞和不表达CEACAM5的细胞，例如RKO细胞)；将两种细胞按照1:1混合培养16小时后，检测上清中IFNγ和IL2的释放。结果如图6所示，UM05-5L-CAR-T可以识别表达CEACAM5的细胞系并释放细胞因子IFNγ和IL-2。
2、肿瘤细胞杀伤检测
将UM05-5L-CAR-T与表达CEACAM5的肿瘤细胞(例如KATO3)按照指定的E:T比例(30:1,10:1,3:1,1:1,0.3:1)混合培养于96孔培养板中。利用Promega LDH检测试剂盒检测培养上清中乳酸脱氢酶(LDH)的释放记录数据。肿瘤细胞杀伤计算结果图7所示，由图中可以看出UM05-5L-CAR-T可以高效的、剂量依赖的杀伤肿瘤细胞。
3、肿瘤细胞刺激的增殖实验
将UM05-5L-CAR-T细胞与表达CEACAM5的肿瘤细胞(例如LS174T)，按照E:T＝1:1混合培养72小时(without IL2 supplement)，通过细胞计数检测其增殖。结果如图8所示，UM05-5L-CAR-T在经过表达CEACAM5的肿瘤细胞刺激时，可高效扩增。
实施例8：LS174T肿瘤模型的体内药效实验
将LS174T细胞(ATCC CL-187)按照1×10 7/只接种于NSG小鼠(百奥赛图)皮下，待肿瘤体积达到200-400mm 3时，经尾静脉分别给予PBS对照、未经修饰的T细胞对照(Mock T)或UM05-5L CAR-T，给与的剂量分别为100μl、1×10 7个细胞、1×10 7tEGFR+个细胞，记录瘤体积变化并分析外周血中IFNγ的释放。结果如图9和图10所示，UM05-5L在小鼠体内识别LS174T肿瘤细胞，并释放大量IFNγ，产生强烈的肿瘤抑制作用。
实施例9：人源化抗体的制备
使用IMGT数据库中的人抗体序列，对UM05-5L抗体进行人源化改造，获得了1种人源化抗体，将其命名为hUM05-3，其具体序列如表6所示。按照实施例2所述，进行抗体的制备。简言之，合成编码SEQ ID NO:16和SEQ ID NO:17的核苷酸序列，分别克隆至表达载体并瞬时表达于HEK293细胞，培养后收集培养液上清，并用Protein A亲和层析法提纯上清中的抗体。纯化后的抗体经PBS溶液透析和冷冻干燥浓缩后，保存于-20℃。
表6.hUM05-3可变区序列
Figure PCTCN2020094045-appb-000005
实施例10：人源化抗体的结合能力
将LS174T细胞(高表达CEACAM5的肿瘤细胞)培养于含10％FBS RPMI1640培养基。将LS174T细胞用TrypLE胰酶消化后，离心，重悬于含2％BSA的DPBS溶液(FACS 缓冲液，4℃)中；然后，按照5×10 5/100μl/孔的浓度，将其加入U型底96孔板中，并加入浓度梯度稀释的抗体，于4℃孵育1小时。孵育后，离心，弃掉上清；每孔加入100μl含有抗人IgG Fc-APC二抗，并于4℃孵育1小时。孵育后，细胞用FACS缓冲液清洗1次，重悬于200μl FACS缓冲液中，于BD C6 plus读取荧光信号值。
实验结果如图11所示。结果显示，人源化抗体hUM05-3能够以剂量依赖的方式结合LS174T细胞。
实施例11：抗体的突变
将人鼠嵌合抗体UM05-5L的VH的第47位氨基酸(即Cys)突变为Trp，突变后的抗体命名为UM05-5L C47W；将人源化抗体hUM05-3的VH的第47位氨基酸(即Trp)突变为Cys，突变后的抗体命名为hUM05-3 W47C。突变后的抗体的可变区序列如表7所示。按照实施例10所述，检测抗体与LS174T细胞的结合能力。实验结果如图12和表8所示。结果显示，人鼠嵌合抗体UM05-5L和人源化抗体hUM05-3在突变前后，均能够结合表达CEACAM5的细胞。
表7.突变抗体的序列
  VH VL
UM05-5L C47W SEQ ID NO:18 SEQ ID NO:8
hUM05-3 W47C SEQ ID NO:19 SEQ ID NO:17
表8.与抗原结合的能力
克隆 EC50μg/ml Emax MFI(平均荧光强度)
UM05-5L 0.81 79739
UM05-5L C47W 2.46 55153
hUM05-3 0.53 52929
hUM05-3 W47C 1.47 34403

Claims (25)

  1. 特异性结合CEACAM5蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:1，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:2，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    和/或，
    (b)包含下述3个互补决定区(CDRs)的轻链可变区(VL)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:4，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:5，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO:6，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，(i)-(vi)中任一项所述的置换为保守置换；
    优选地，(i)-(vi)中任一项所述的CDR根据Kabat编号系统定义；
    优选地，所述抗体或其抗原结合片段包含如下3个重链CDRs：如SEQ ID NO:1或SEQ ID NO:20所示的VH CDR1、如SEQ ID NO:2或SEQ ID NO:21所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:4所示的VL CDR1、如SEQ ID NO:5或SEQ ID NO:22所示的VL CDR2、如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段包含如下3个重链CDRs：如SEQ ID NO:1所示的VH CDR1、如SEQ ID NO:2所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:4所示的VL CDR1、如SEQ ID NO:5所示的VL CDR2、如SEQ ID NO:6所示的VL CDR3；
    优选地，所述抗体或其抗原结合片段包含如下3个重链CDRs：如SEQ ID NO:20所示的VH CDR1、如SEQ ID NO:21所示的VH CDR2、如SEQ ID NO:3所示的VH CDR3；和/或，如下3个轻链CDRs：如SEQ ID NO:4所示的VL CDR1、如SEQ ID NO:22所示的VL CDR2、如SEQ ID NO:6所示的VL CDR3。
  2. 特异性结合CEACAM5蛋白的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (i)如SEQ ID NO:7所示的重链可变区(VH)中含有的CDR1，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    (ii)如SEQ ID NO:7所示的重链可变区(VH)中含有的CDR2，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；和
    (iii)如SEQ ID NO:7所示的重链可变区(VH)中含有的CDR3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；和/或，
    (iv)如SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR1，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    (v)如SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR2，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；和
    (vi)如SEQ ID NO:8所示的轻链可变区(VL)中含有的CDR3，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat、IMGT或Chothia编号系统定义。
  3. 权利要求1或2的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:7所示的序列；
    (ii)与SEQ ID NO:7所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:7所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:8所示的序列；
    (v)与SEQ ID NO:8所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:8所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的重链框架区序列和/或轻链框架区序列；
    优选地，所述抗体或其抗原结合片段包含：具有如SEQ ID NO:7、SEQ ID NO:16、SEQ ID NO:18或SEQ ID NO:19所示的序列的VH和具有如SEQ ID NO:8或SEQ ID NO:17所示的序列的VL；
    优选地，所述抗体或其抗原结合片段包含：
    (1)具有如SEQ ID NO:7所示的序列的VH和具有如SEQ ID NO:8所示的序列的VL；
    (2)具有如SEQ ID NO:18所示的序列的VH和具有如SEQ ID NO:8所示的序列的VL；
    (3)具有如SEQ ID NO:16所示的序列的VH和具有如SEQ ID NO:17所示的序列的VL；或者
    (4)具有如SEQ ID NO:19所示的序列的VH和具有如SEQ ID NO:17所示的序列的VL。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合 片段包含来源于人免疫球蛋白的恒定区或其变体；
    优选地，所述抗体或其抗原结合片段包含：
    (a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；和/或
    (b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加或其任意组合)；
    优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:9所示的重链恒定区(CH)；
    优选地，所述轻链恒定区是κ轻链恒定区；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:11所示的轻链恒定区(CL)。
  5. 权利要求1-4任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、BsFv、dsFv、(dsFv) 2、dsFv-dsFv'、scFv、scFv二聚体、骆驼化单域抗体(camelized single chain domain antibody)、双抗体(diabody)、ds双功能抗体(ds diabody)、纳米抗体、单域抗体(sdAb)、双价域抗体；和/或，所述抗体为鼠源抗体、嵌合抗体、人源化抗体、双特异性抗体或多特异性抗体。
  6. 权利要求1-5任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段带有标记；优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  7. 分离的核酸分子，其编码权利要求1-6任一项所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区；
    优选地，所述核酸分子包含如SEQ ID NO:14或SEQ ID NO:15中所示的核苷酸序列。
  8. 载体，其包含权利要求7所述的核酸分子；优选地，所述载体为克隆载体或表达载体。
  9. 宿主细胞，其包含权利要求7的核酸分子或权利要求8的载体。
  10. 制备权利要求1-6任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求9所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段；
    优选地，所述宿主细胞是哺乳动物细胞；优选地，所述宿主细胞为人、鼠、羊、马、狗或猫的细胞；优选地，所述宿主细胞为中国仓鼠卵巢细胞。
  11. 双特异性或多特异性分子，其包含权利要求1-6任一项所述的抗体或其抗原结合片段；
    优选地，所述双特异性或多特异性分子特异性结合CEACAM5，并且额外地特异性结合一个或多个其他靶标；
    优选地，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
  12. 免疫缀合物，其包含权利要求1-6任一项所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂；
    优选地，所述治疗剂选自细胞毒剂；
    优选地，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合；
    优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
  13. 药物组合物，其包含权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求11所述的双特异性或多特异性分子或者权利要求12所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑 制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述抗体或其抗原结合片段、双特异性或多特异性分子或免疫缀合物与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。
  14. 试剂盒，其含有权利要求1-6任一项所述的抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素；
    优选地，所述试剂盒还包括第二抗体，其特异性识别权利要求1-6任一项所述的抗体或其抗原结合片段；
    优选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  15. 嵌合抗原受体，其包含权利要求1-6任一项所述的抗体或其抗原结合片段的抗原结合结构域；
    优选地，所述抗原结合结构域包含权利要求1-6任一项所述的抗体或其抗原结合片段的重链可变区和轻链可变区；
    优选地，所述抗原结合结构域是scFv；
    优选地，所述抗原结合受体包含权利要求1-6任一项所述的抗体的抗原结合片段；
    优选地，所述抗原结合受体由免疫效应细胞(例如T细胞)所表达。
  16. 分离的核酸分子，其编码权利要求15所述的嵌合抗原受体。
  17. 载体，其包含权利要求16所述的分离的核酸分子；优选地，其用于制备嵌合抗原受体T细胞。
  18. 宿主细胞，其包含权利要求16所述的分离的核酸分子或权利要求17所述的载体；
    优选地，所述宿主细胞是免疫效应细胞(例如，T细胞或NK细胞)；
    优选地，所述宿主细胞是嵌合抗原受体T细胞(CAR-T)。
  19. 一种用于降低CEACAM5在细胞表面的表达水平的方法，其包括将所述细胞与权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求11所述的双特异性或多特异性分子，或权利要求12所述的免疫缀合物，或权利要求13所述的药物组合物，或权利要求15所述的嵌合抗原受体，或权利要求18所述的宿主细胞接触，使得CEACAM5在所述细胞表面的表达水平降低；其中，所述细胞在其表面表达CEACAM5；
    优选地，所述细胞是表达CEACAM5的肿瘤细胞。
  20. 一种抑制表达CEACAM5的肿瘤细胞生长和/或杀伤所述肿瘤细胞的方法，其包括将所述肿瘤细胞与有效量的权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求11所述的双特异性或多特异性分子，或权利要求12所述的免疫缀合物，或权利要求13所述的药物组合物，或权利要求15所述的嵌合抗原受体，或权利要求18所述的宿主细胞接触。
  21. 一种用于在受试者(例如人)中预防和/或治疗肿瘤的方法，所述方法包括向有此需要的受试者施用有效量的权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求11所述的双特异性或多特异性分子，或权利要求12所述的免疫缀合物，或权利要求13所述的药物组合物，或权利要求15所述的嵌合抗原受体，或权利要求18所述的宿主细胞；
    优选地，所述肿瘤表达CEACAM5；
    优选地，所述肿瘤涉及表达CEACAM5的肿瘤细胞；优选地，所述CEACAM5在所述肿瘤细胞表面上表达；
    优选地，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤；
    优选地，所述受试者为哺乳动物，例如人；
    优选地，所述方法还包括施用另外的具有抗肿瘤活性的药物，例如烷化剂、有丝分裂 抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述方法还包括施用另外的抗肿瘤疗法，例如手术、化学治疗、放射治疗、靶向治疗、免疫治疗、激素治疗、基因治疗或姑息治疗。
  22. 权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求11所述的双特异性或多特异性分子，或权利要求12所述的免疫缀合物，或权利要求13所述的药物组合物，或权利要求15所述的嵌合抗原受体，或权利要求16所述的宿主细胞，在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/治疗肿瘤；
    优选地，药物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述肿瘤表达CEACAM5；
    优选地，所述肿瘤涉及表达CEACAM5的肿瘤细胞；优选地，所述CEACAM5在所述肿瘤细胞表面上表达；
    优选地，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
    优选地，所述受试者为哺乳动物，例如人。
  23. 一种检测CEACAM5(例如人CEACAM5)在样品中的存在或其量的方法，其包括以下步骤：
    (1)将所述样品与权利要求1-6任一项所述的抗体或其抗原结合片段接触；
    (2)检测所述抗体或其抗原结合片段与CEACAM5之间复合物的形成或检测所述复合物的量；
    优选地，所述抗体或其抗原结合片段带有可检测的标记；
    优选地，所述CEACAM5是人CEACAM5。
  24. 一种用于检测肿瘤是否能够通过靶向CEACAM5的抗肿瘤疗法来治疗的方法，其包括以下步骤：
    (1)将含有所述肿瘤细胞的样品与权利要求1-6任一项所述的抗体或其抗原结合片段接触；
    (2)检测所述抗体或其抗原结合片段与CEACAM5之间复合物的形成；
    优选地，所述抗体或其抗原结合片段带有可检测的标记；
    优选地，所述CEACAM5是人CEACAM5；
    优选地，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
  25. 权利要求1-6任一项所述的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测肿瘤是否能够通过靶向CEACAM5的抗肿瘤疗法来治疗；
    优选地，所述抗体或其抗原结合片段带有可检测的标记；
    优选地，所述CEACAM5是人CEACAM5；
    优选地，所述肿瘤选自非小细胞肺癌、小细胞肺癌、肾细胞癌、结肠直肠癌、卵巢癌、乳癌、胰脏癌、胃癌、膀胱癌、食管癌、间皮瘤、黑色素瘤、头颈部癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、子宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿(mycosis fungoids)、默克尔细胞癌和其它恶性血液病、如经典型霍奇金淋巴瘤(CHL)、原发性纵膈大B细胞淋巴瘤、T细胞/组织细胞的富B细胞淋巴瘤、EBV阳性和阴性PTLD和EBV 相关弥漫性大B细胞淋巴瘤(DLBCL)、浆母细胞性淋巴瘤、结外NK/T细胞淋巴瘤、鼻咽癌和HHV8相关原发性渗出性淋巴瘤、霍奇金淋巴瘤，中枢神经系统(CNS)肿瘤，例如原发性CNS淋巴瘤，脊轴肿瘤，脑干神经胶质瘤。
PCT/CN2020/094045 2019-06-04 2020-06-03 一种抗ceacam5的单克隆抗体及其制备方法和用途 WO2020244526A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
AU2020289301A AU2020289301A1 (en) 2019-06-04 2020-06-03 CEACAM5-resistant monoclonal antibody and preparation method thereof and use thereof
CN202080040435.2A CN113906052B (zh) 2019-06-04 2020-06-03 一种抗ceacam5的单克隆抗体及其制备方法和用途
CA3142635A CA3142635A1 (en) 2019-06-04 2020-06-03 Anti-ceacam5 monoclonal antibody and preparation method thereof and use thereof
KR1020227000006A KR20220016943A (ko) 2019-06-04 2020-06-03 항 씨이에이씨에이엠5 단클론 항체 및 그의 제조 방법 및 그의 용도
US17/596,183 US20220242968A1 (en) 2019-06-04 2020-06-03 Anti-CEACAM5 monoclonal antibody and preparation method thereof and use thereof
JP2021572365A JP2022536114A (ja) 2019-06-04 2020-06-03 抗ｃｅａｃａｍ５モノクローナル抗体およびその調製方法およびその使用
EP20818729.4A EP3981793A4 (en) 2019-06-04 2020-06-03 CEACAM5-RESISTANT MONOCLONAL ANTIBODY AND METHODS FOR ITS PRODUCTION AND ITS USE
BR112021024544A BR112021024544A2 (pt) 2019-06-04 2020-06-03 Anticorpo monoclonal anti-ceacam5 e método de preparação do mesmo e uso do mesmo

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910481112.6 2019-06-04
CN201910481112 2019-06-04

Publications (1)

Publication Number Publication Date
WO2020244526A1 true WO2020244526A1 (zh) 2020-12-10

Family

ID=73652171

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2020/094053 WO2020244528A1 (zh) 2019-06-04 2020-06-03 一种抗ceacam5的单克隆抗体及其制备方法和用途
PCT/CN2020/094045 WO2020244526A1 (zh) 2019-06-04 2020-06-03 一种抗ceacam5的单克隆抗体及其制备方法和用途

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/094053 WO2020244528A1 (zh) 2019-06-04 2020-06-03 一种抗ceacam5的单克隆抗体及其制备方法和用途

Country Status (10)

Country Link
US (2) US20220242968A1 (zh)
EP (1) EP3981793A4 (zh)
JP (2) JP2022535553A (zh)
KR (1) KR20220016943A (zh)
CN (2) CN113474372B (zh)
AU (1) AU2020289301A1 (zh)
BR (1) BR112021024544A2 (zh)
CA (1) CA3142635A1 (zh)
TW (2) TW202100560A (zh)
WO (2) WO2020244528A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023186112A1 (zh) * 2022-04-02 2023-10-05 普米斯生物技术(珠海)有限公司 靶向ceacam和cd3的多特异性抗体及其用途

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115505043A (zh) * 2021-06-23 2022-12-23 上海吉倍生物技术有限公司 特异性结合糖基化ceacam5的抗体
WO2023078450A1 (en) * 2021-11-05 2023-05-11 Vibrant Pharma Limited Multispecific antibodies and uses thereof

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0404097A2 (de) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispezifische und oligospezifische, mono- und oligovalente Rezeptoren, ihre Herstellung und Verwendung
WO1993011161A1 (en) 1991-11-25 1993-06-10 Enzon, Inc. Multivalent antigen-binding proteins
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO1994025591A1 (en) 1993-04-29 1994-11-10 Unilever N.V. PRODUCTION OF ANTIBODIES OR (FUNCTIONALIZED) FRAGMENTS THEREOF DERIVED FROM HEAVY CHAIN IMMUNOGLOBULINS OF $i(CAMELIDAE)
US6005079A (en) 1992-08-21 1999-12-21 Vrije Universiteit Brussels Immunoglobulins devoid of light chains
CN102482701A (zh) * 2009-09-16 2012-05-30 免疫医疗公司 I类抗-cea抗体及其使用
CN103987729A (zh) * 2011-10-11 2014-08-13 堤乐哈修门医学研究基础建设及服务有限公司 癌胚抗原相关细胞粘附分子(ceacam)的抗体
CN108341876A (zh) * 2017-01-22 2018-07-31 中国人民解放军第四军医大学 抗人ceacam5单克隆抗体及其制备方法和应用
CN109180815A (zh) * 2012-11-20 2019-01-11 赛诺菲 抗ceacam5抗体及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3066470A4 (en) * 2013-11-05 2017-05-31 Immunomedics, Inc. Humanized anti-ceacam5 antibody and uses thereof
TWI608015B (zh) * 2015-01-16 2017-12-11 中央研究院 具有組織標的功能的抗發炎分子
HUE048111T2 (hu) * 2015-03-23 2020-05-28 Bayer Pharma AG Anti-CEACAM6 ellenanyagok és alkalmazásuk
TWI795415B (zh) * 2017-07-07 2023-03-11 日商安斯泰來製藥股份有限公司 新穎的抗人類ＣＥＡＣＡＭ５抗體Ｆａｂ片段

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0404097A2 (de) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispezifische und oligospezifische, mono- und oligovalente Rezeptoren, ihre Herstellung und Verwendung
WO1993011161A1 (en) 1991-11-25 1993-06-10 Enzon, Inc. Multivalent antigen-binding proteins
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
US6005079A (en) 1992-08-21 1999-12-21 Vrije Universiteit Brussels Immunoglobulins devoid of light chains
WO1994025591A1 (en) 1993-04-29 1994-11-10 Unilever N.V. PRODUCTION OF ANTIBODIES OR (FUNCTIONALIZED) FRAGMENTS THEREOF DERIVED FROM HEAVY CHAIN IMMUNOGLOBULINS OF $i(CAMELIDAE)
CN102482701A (zh) * 2009-09-16 2012-05-30 免疫医疗公司 I类抗-cea抗体及其使用
CN103987729A (zh) * 2011-10-11 2014-08-13 堤乐哈修门医学研究基础建设及服务有限公司 癌胚抗原相关细胞粘附分子(ceacam)的抗体
CN109180815A (zh) * 2012-11-20 2019-01-11 赛诺菲 抗ceacam5抗体及其用途
CN108341876A (zh) * 2017-01-22 2018-07-31 中国人民解放军第四军医大学 抗人ceacam5单克隆抗体及其制备方法和应用

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
"Physicians' Desk Reference", November 2002, MEDICAL ECONOMICS COMPANY
AL-LAZIKANI, B.CHOTHIA, C.LESK, AM, J. MOL. BIOL., vol. 273, no. 4, 1997, pages 927
BRENNAN ET AL., SCIENCE, vol. 229, 1985, pages 81
CARTER ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 163 - 167
CHOTHIA, C. ET AL., NATURE, vol. 342, no. 6252, 1989, pages 877 - 83
CHOTHIA, C., J. MOL. BIOL., vol. 186, no. 3, 1985, pages 651 - 63
CHOTHIA, C.LESK, AM, J. MOL. BIOL., vol. 196, 1987, pages 901
FM AUSUBEL ET AL.: "Compiled Molecular Biology Experiment Guide", 1995, JOHN WILEY & SONS, INC.
GRAHAM ET AL., GEN VIROL, vol. 36, 1977, pages 59
GUSS ET AL., EMBO J, vol. 5, 1986, pages 1567 - 1575
HAMERS CASTERMAN C. ET AL., NATURE, vol. 363, no. 6428, 1993, pages 446 - 8
HOLLIGER P. ET AL., PROC NATL. ACAD SCI US A., vol. 90, no. 14, 1993, pages 6444 - 8
HUDSON, CURR. OPIN. IMMUNOL., vol. 11, 1999, pages 548 - 557
HUSTON JS ET AL., PROC NATL ACAD SCI USA, vol. 85, 1988, pages 5879
J. SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH, pages: 647 - 669
KOCH-NOLTE F. ET AL., FASEB J, vol. 21, no. 13, 2007, pages 3490 - 8
LINDMARK ET AL., J. IMMUNOL. METH., vol. 62, 1983, pages 13
LITTLE ET AL., IMMUNOL. TODAY, vol. 21, 2000, pages 364 - 370
MATHER ET AL., ANNALS NY ACAD. SCI., vol. 383, 1982, pages 44 - 68
MATHER JP, BIOL. REPROD., vol. 23, 1980, pages 243 - 252
MORIMOTO ET AL., J. BIOCHEM. BIOPHYS. METHODS, vol. 24, 1992, pages 107 - 117
MUYLDERMANS S., J BIOTECHNOL, vol. 74, no. 4, 2001, pages 277 - 302
NGUYEN VK. ET AL., IMMUNOLOGY, vol. 109, no. 1, 2003, pages 93 - 101
NGUYEN VK. ET AL.: "Heavy-chain antibodies in Camelidae: a case of evolutionary innovation", IMMUNOGENETICS, vol. 54, no. 1, 2002, pages 39 - 47
RIECHMANN L.MUYLDERMANS S., J IMMUNOL METHODS, vol. 231, no. 1-2, 1999, pages 25 - 38
See also references of EP3981793A4
URLAUB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023186112A1 (zh) * 2022-04-02 2023-10-05 普米斯生物技术(珠海)有限公司 靶向ceacam和cd3的多特异性抗体及其用途

Also Published As

Publication number Publication date
CN113906052A (zh) 2022-01-07
TW202100560A (zh) 2021-01-01
US20220242968A1 (en) 2022-08-04
EP3981793A4 (en) 2023-06-07
BR112021024544A2 (pt) 2022-02-08
JP2022535553A (ja) 2022-08-09
KR20220016943A (ko) 2022-02-10
AU2020289301A1 (en) 2022-01-27
US20220235142A1 (en) 2022-07-28
CN113906052B (zh) 2024-02-13
CN113474372B (zh) 2023-08-08
EP3981793A1 (en) 2022-04-13
WO2020244528A1 (zh) 2020-12-10
CN113474372A (zh) 2021-10-01
JP2022536114A (ja) 2022-08-12
CA3142635A1 (en) 2020-12-10
TW202110895A (zh) 2021-03-16

Similar Documents

Publication Publication Date Title
US11643465B2 (en) Anti-PD-1 antibodies
US20210269528A1 (en) Novel anti-pd-l1 antibodies
US10696745B2 (en) Anti-PD-L1 antibodies
CN106243225B (zh) 新型抗-pd-l1抗体
WO2020244526A1 (zh) 一种抗ceacam5的单克隆抗体及其制备方法和用途
JP2019523651A (ja) 抗ｐｓｍａ抗体およびその使用
US20220127366A1 (en) Anti-dectin-1 antibodies and methods of use thereof
CN114728065A (zh) 针对cd3和bcma的抗体和自其制备的双特异性结合蛋白
US20210115138A1 (en) Novel bispecific pd-1/lag-3 antibody molecules
EP4357365A1 (en) Antibody targeting axl protein and antigen binding fragment thereof, and preparation method therefor and application thereof
WO2022116079A1 (zh) 一种抗ceacam5的人源化抗体及其制备方法和用途
WO2021197393A1 (zh) 一种抗人cd47抗体及其抗原结合片段、制备方法和应用
WO2022063272A1 (en) Novel anti-claudin18 antibodies
KR20240039006A (ko) 신규 항-sirpa 항체
CN116120461A (zh) 新型抗药抗体以及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20818729

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021572365

Country of ref document: JP

Kind code of ref document: A

Ref document number: 3142635

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112021024544

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20227000006

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020818729

Country of ref document: EP

Effective date: 20220104

ENP Entry into the national phase

Ref document number: 2020289301

Country of ref document: AU

Date of ref document: 20200603

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112021024544

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20211203