WO2020259550A1 - 抗cea抗体及其应用 - Google Patents

抗cea抗体及其应用 Download PDF

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WO2020259550A1
WO2020259550A1 PCT/CN2020/097984 CN2020097984W WO2020259550A1 WO 2020259550 A1 WO2020259550 A1 WO 2020259550A1 CN 2020097984 W CN2020097984 W CN 2020097984W WO 2020259550 A1 WO2020259550 A1 WO 2020259550A1
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seq
variable region
chain variable
heavy chain
antibody
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PCT/CN2020/097984
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English (en)
French (fr)
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应华
毛浪勇
张玲
葛虎
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to EP20831939.2A priority Critical patent/EP3992211A4/en
Priority to US17/619,558 priority patent/US20230013784A1/en
Priority to JP2021576913A priority patent/JP2022539344A/ja
Priority to CN202080040927.1A priority patent/CN113906053B/zh
Publication of WO2020259550A1 publication Critical patent/WO2020259550A1/zh

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6853—Carcino-embryonic antigens
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure relates to the field of antibody drugs. Specifically, it includes anti-CEA antibody drugs and their applications.
  • Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is one of the first tumor-associated antigens discovered by people. It is a glycoprotein with a molecular weight of about 180kDa.
  • CEA is a member of the immunoglobulin superfamily , And contains 7 domains connected to cell membranes via glycosylphosphatidylinositol (GPI) anchors (Thompson JA, J Clin Lab Anal. 5:344-366, 1991).
  • GPI glycosylphosphatidylinositol
  • CEA was detected in the serum, while the content of CEA in the serum of healthy people or patients with other diseases was extremely low (Thomson, Krupey et al. 1969).
  • the expression of CEA is increased in cancer cells, and the increased CEA promotes cell adhesion, which in turn promotes cell metastasis (Marshall J., Semion Oncol., 30( ⁇ 8): 30-6, 2003).
  • CEA is commonly expressed in epithelial tissues, including cells of the gastrointestinal, respiratory and genitourinary tracts, and cells of the colon, cervix, sweat glands and prostate (Nap et al., Tumour Biol., 9(2-3): 145-53, 1988; Nap et al., Cancer Res., 52(8): 2329-23339, 1992).
  • WO1999043817A1 has disclosed various anti-CEA antibodies.
  • WO2004032962A1 has disclosed various anti-CEA antibodies.
  • WO2005086875A3 has disclosed various anti-CEA antibodies.
  • the present disclosure provides a new anti-CEA antibody.
  • the antibodies of the present disclosure include full-length antibodies and antigen-binding fragments thereof.
  • the anti-CEA antibody of the present disclosure comprises an antibody heavy chain variable region and a light chain variable region, wherein:
  • the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 7, and the HCDR2 of the heavy chain variable region is the same as that shown in SEQ ID NO: 7
  • the HCDR2 of the heavy chain variable region shown in the sequence is the same or has an amino acid difference with it;
  • LCDR1, LCDR2, and LCDR3 of the light chain variable region are the same as LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 8; or
  • the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 9, and the HCDR2 of the heavy chain variable region is the same as that shown in SEQ ID NO:
  • the HCDR2 of the heavy chain variable region shown in the sequence 9 is the same or has one amino acid difference with it;
  • LCDR1, LCDR2, and LCDR3 of the light chain variable region are the same as LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 10; or
  • HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are the same as the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in the sequence of SEQ ID NO: 11;
  • LCDR1, LCDR2, and LCDR3 of the light chain variable region are the same as LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 12; or
  • the HCDR1 and HCDR3 of the heavy chain variable region are the same as the HCDR1 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 13, and the HCDR2 of the heavy chain variable region is the same as that shown in SEQ ID NO:
  • the HCDR2 of the heavy chain variable region shown in the 13 sequence is the same or has one amino acid difference with it;
  • the LCDR1 and LCDR3 of the light chain variable region are the same as the LCDR1 and LCDR3 of the light chain variable region shown in the sequence of SEQ ID NO: 14, and the LCDR2 of the light chain variable region is the same as the sequence of SEQ ID NO: 14
  • the LCDR2 of the light chain variable region shown is the same or has one amino acid difference.
  • the anti-CEA antibody as described in any one of the preceding comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region includes the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively,
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 38 and SEQ ID NO: 17, respectively.
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively;
  • the heavy chain variable region contains the sequences of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively,
  • the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively; or
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 47 and SEQ ID NO: 23, respectively.
  • the light chain variable region comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively; or
  • the heavy chain variable region includes the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, respectively,
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively; or
  • the heavy chain variable region comprises HCDR1 and HCDR3 shown in SEQ ID NO: 33 and SEQ ID NO: 34, and HCDR2 shown in SEQ ID NO: 16 or SEQ ID NO: 38;
  • the light chain variable region includes LCDR1 and LCDR3 shown in SEQ ID NO: 35 and SEQ ID NO: 37, and LCDR2 shown in SEQ ID NO: 36 or SEQ ID NO: 64.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 33, SEQ ID NO: 16 and SEQ ID NO: 34, respectively,
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, respectively; or
  • the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 33, SEQ ID NO: 38 and SEQ ID NO: 34, respectively.
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, respectively; or
  • the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 33, SEQ ID NO: 16 and SEQ ID NO: 34, respectively.
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 35, SEQ ID NO: 64 and SEQ ID NO: 37, respectively; or
  • the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 33, SEQ ID NO: 38 and SEQ ID NO: 34, respectively.
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 35, SEQ ID NO: 64 and SEQ ID NO: 37, respectively.
  • the anti-CEA antibody according to any one of the preceding items, wherein the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.
  • the anti-CEA antibody of any one of the preceding wherein the antibody is a murine antibody or a chimeric antibody, which comprises a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7;
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; or
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 10;
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; or
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 13, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 13;
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 14.
  • any one of the aforementioned (a) to (d) having at least 90% sequence identity is: having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or 100% sequence identity.
  • the anti-CEA antibody of any one of the preceding wherein the antibody is a humanized antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 39, 40, 41 or 42, or is 90% with any one of the amino acid sequences shown in SEQ ID NO: 39, 40, 41 or 42. % Sequence identity; and/or
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 43, 44, 45 or 46, or has 90% sequence identity with any one of the amino acid sequences shown in SEQ ID NO: 43, 44, 45 or 46 Sex
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 48, 49, 50, 51 or 52, or with any one of SEQ ID NO: 48, 49, 50, 51 or 52
  • amino acid sequence has 90% sequence identity; and/or
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 53, 54 or 55, or has 90% sequence identity with any one of the amino acid sequences shown in SEQ ID NO: 53, 54 or 55;
  • variable region of the heavy chain is shown in SEQ ID NO: 56, 57 or 58, or has 90% sequence identity with any one of the amino acid sequences shown in SEQ ID NO: 56, 57 or 58 ;and / or
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 59, 60, 61, 62, or 63, or is combined with any one of the amino acid sequences shown in SEQ ID NO: 59, 60, 61, 62, or 63 90% sequence identity; or
  • the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 65, 66, 67 or 68, or is 90% with any one of the amino acid sequences shown in SEQ ID NO: 65, 66, 67 or 68. % Sequence identity; and/or
  • amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 69, 70, 71, 72, 73, 74, 75 or 76, or with SEQ ID NO: 69, 70, 71, 72, 73, 74 Any one amino acid sequence shown in, 75 or 76 has 90% sequence identity.
  • any of the aforementioned (e) to (h) having at least 90% sequence identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% %, 98%, 99% or 100% sequence identity.
  • the anti-CEA antibody as described above comprises a heavy chain variable region and a light chain variable as shown in Table a-1, Table a-2, Table a-3 or Table a-4 below District combination:
  • Human67V-14 in the table means that the light chain variable region is h67VL4 (SEQ ID NO: 62), and the heavy chain variable region is h67VH3 (SEQ ID NO: 58). Variable region combination.
  • Human103V-32 in the table means that the light chain variable region is h103VL8 (SEQ ID NO: 76), and the heavy chain variable region is h103VH4 (SEQ ID NO: 68). Variable region combination.
  • the humanized anti-CEA antibody as described above comprises a heavy chain variable region and a light chain variable region, wherein: the amino acid sequence of the light chain variable region is as SEQ ID NO: 44, And the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 42; or
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 53, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 52; or
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 62, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 58; or
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 76
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 68.
  • the anti-CEA antibody according to any one of the preceding items, wherein the antibody comprises a framework region derived from a human antibody or a framework region variant thereof, and the framework region variant is in the light chain framework of a human antibody There are 1 to 10 amino acid back mutations in each region and/or heavy chain framework region;
  • the framework region variant comprises an amino acid back mutation selected from the following (i) to (1):
  • the framework region of the light chain variable region contains the selection Back mutation from one or more amino acids in 46P, 47W, 49Y, 70S and 71Y, and/or
  • the framework region of the heavy chain variable region contains one or more amino acid back mutations selected from 38K or 46K;
  • the framework region of the light chain variable region contains the selection Back mutation from one or more amino acids in 2V, 42G, 44V and 71Y, and/or
  • the framework region of the heavy chain variable region contains one or more amino acid back mutations selected from 66K, 67A, 69L, 71V, 73K, 82F, 82AR ;
  • the framework region of the light chain variable region contains the selection Back mutation from one or more amino acids in 3V, 43P and 58V, and/or
  • the framework region of the heavy chain variable region contains selected from 38K, One or more amino acids in 66K and 71V are backmutated;
  • the framework region of the light chain variable region contains one or more amino acid back mutations selected from 4V, 36Y, 43P, 47V, 49E, 70D and 87I, and/or
  • the framework region of the heavy chain variable region contains one or more amino acid back mutations selected from 2I, 38K and 46K;
  • the site of the mutation corresponds to the numbering in the Kabat numbering sequence.
  • "46P” indicates that the amino acid corresponding to position 46 of the Kabat numbering rules is backmutated to "P".
  • the anti-CEA antibody comprises:
  • the heavy chain variable region which includes HCDR1 as shown in SEQ ID NO: 15 and HCDR2 as shown in SEQ ID NO: 16 or SEQ ID NO: 38 and HCDR3 as shown in SEQ ID NO: 17;
  • the framework region of the light chain variable region comprises one or more amino acid back mutations selected from 46P, 47W, 49Y, 70S and 71Y, and/or the framework region of the heavy chain variable region Contains one or more amino acid back mutations selected from 38K or 46K;
  • the heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 21, HCDR2 as shown in SEQ ID NO: 22 or SEQ ID NO: 47, and HCDR3 as shown in SEQ ID NO: 23;
  • the framework region of the light chain variable region contains one or more amino acid back mutations selected from 2V, 42G, 44V and 71Y
  • the framework region of the heavy chain variable region contains One or more amino acid back mutations selected from 48I, 66K, 67A, 69L, 71V, 73K, 82F, 82AR ;
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, respectively;
  • the framework region of the light chain variable region contains one or more amino acid back mutations selected from 3V, 43P and 58V, and/or the framework region of the heavy chain variable region contains a One or more amino acids in 38K, 66K, 71V are backmutated;
  • the light chain variable region which includes LCDR1 as shown in SEQ ID NO: 35, LCDR2 as shown in SEQ ID NO: 36 or SEQ ID NO: 64, and LCDR2 as shown in SEQ ID NO: 37 LCDR3; and
  • the heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 33, HCDR2 as shown in SEQ ID NO: 16 or SEQ ID NO: 38, and HCDR3 as shown in SEQ ID NO: 34;
  • the framework region of the light chain variable region comprises one or more amino acid back mutations selected from 4V, 36Y, 43P, 47V, 49E, 70D and 87I, and/or the heavy chain variable
  • the framework region of the region contains one or more amino acid back mutations selected from 2I, 38K and 46K;
  • the position of the back mutation corresponds to the Kabat numbering sequence number.
  • the anti-CEA antibody according to any one of the preceding, wherein the antibody further comprises an antibody heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, the light chain constant region is selected from human antibody ⁇ and ⁇ chain constant regions and conventional variants thereof; more preferably, the antibody comprises a sequence as shown in SEQ ID NO: 77 The constant region of the heavy chain, and the constant region of the light chain shown in SEQ ID NO: 78 or SEQ ID NO: 79;
  • the antibody comprises:
  • any one of the aforementioned (m) to (p) having at least 85% sequence identity is: having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the anti-CEA antibody of any one of the preceding items wherein the antibody is a full-length antibody or an antigen-binding fragment.
  • the anti-CEA antibody according to any one of the preceding, wherein the anti-CEA antibody comprises:
  • the present disclosure also provides an isolated anti-CEA antibody that competitively binds to human CEA, or human CEA epitopes or fragments, with the anti-CEA antibody described in any one of the preceding items.
  • the aforementioned anti-CEA antibody can bind to human CEA (for example, with high affinity) on the cell membrane surface (membrane-bound or transmembrane), and can also bind to monkey CEA on the cell membrane surface (for example, with high affinity).
  • Binding, and the ratio of the EC50 value of the monkey CEA bound to the cell membrane surface of the anti-CEA antibody to the EC50 value of the human CEA bound to the cell membrane surface is ⁇ 3.5, preferably ⁇ 3.4, ⁇ 3.3, ⁇ 3.2, ⁇ 3.1, ⁇ 3.0, ⁇ 2.9, ⁇ 2.8, ⁇ 2.7, ⁇ 2.6, ⁇ 2.5, ⁇ 2.4, ⁇ 2.3, ⁇ 2.2, ⁇ 2.1, ⁇ 2.0, ⁇ 1.9, ⁇ 1.8, ⁇ 1.7, ⁇ 1.6, ⁇ 1.5, ⁇ 1.0, ⁇ 0.9, ⁇ 0.8 ⁇ 0.7, ⁇ 0.6, ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2 or ⁇ 0.1, more preferably ⁇ 0.7, ⁇ 0.6, ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, the anti-CEA antibody binds to CEA
  • the EC50 (nM) value is determined by the FACS method, and an exemplary example is shown in Test Example 1 of the present disclosure.
  • the aforementioned anti-CEA antibody competes with soluble human CEA (sCEA) to bind to the cell membrane surface human CEA (CEA).
  • the maximum value of the ratio of the fluorescence signal of the anti-CEA antibody bound to the antigen in cells expressing human CEA (such as MKN45) without sCEA and sCEA is less than 2.0, more preferably ⁇ 1.9, ⁇ 1.8, ⁇ 1.7, ⁇ 1.6, ⁇ 1.5, ⁇ 1.4, ⁇ 1.3, ⁇ 1.2, ⁇ 1.1, or ⁇ 1.0, the fluorescence signal value is detected by the FACS method, such as Test Example 2 of the present disclosure.
  • the anti-CEA antibody has a concentration greater than or equal to 10 -7 M (e.g., greater than or equal to 10 -7 M, greater than or equal to 1 ⁇ 10 -6 M, greater than or equal to 1 ⁇ 10 -5 M, Preferably, a KD value greater than or equal to 1 ⁇ 10 -7 M and less than or equal to 5 ⁇ 10 -6 M) is combined with soluble human CEA, and at the same time, it is less than or equal to 10 -9 M (for example, less than or equal to 9 ⁇ 10 -9 M).
  • EC50 value combined with human CEA on the cell membrane surface Or monkey CEA on the cell membrane surface; the EC50 value is determined by the FACS method, such as described in Test Example 1 of the present disclosure; the KD value is determined by the Biacore method, such as Test Example 3 of the present disclosure.
  • the aforementioned anti-CEA antibody has the following characteristics:
  • the anti-CEA antibody competitively binds to the cell membrane surface human CEA; and the anti-CEA antibody is used in cells expressing human CEA (such as MKN45) without sCEA and with sCEA,
  • human CEA such as MKN45
  • the maximum value of the ratio of the fluorescence signal of the anti-CEA antibody and the antigen binding is less than 1.7, and the fluorescence signal value is detected by the FACS method;
  • the anti-CEA antibody binds to human CEA on the cell membrane surface with high affinity, and also binds to the monkey CEA on the cell membrane surface with high affinity, and the anti-CEA antibody binds to the EC50 value of the monkey CEA on the cell membrane surface and the EC50 value of the human CEA on the cell membrane surface
  • the ratio of ⁇ 0.7, the EC50 value is determined by FACS method; and/or
  • the anti-CEA antibody binds to soluble human CEA with a KD value greater than or equal to 1 ⁇ 10 -7 M and less than or equal to 5 ⁇ 10 -6 M, and simultaneously binds with an EC50 value less than or equal to 8 ⁇ 10 -9 M
  • the present disclosure also provides a nucleic acid molecule encoding the anti-CEA antibody described in any one of the preceding items.
  • the present disclosure also provides a host cell, which contains the aforementioned nucleic acid molecule.
  • the present disclosure also provides an antibody-drug conjugate, which is formed by coupling the anti-CEA antibody described in any one of the foregoing with a cytotoxic drug.
  • the present disclosure also provides an antibody drug conjugate, which comprises or consists of the following:
  • the cytotoxic drug is covalently coupled to the anti-CEA antibody.
  • the present disclosure also provides a pharmaceutical composition, which contains a preventive or therapeutically effective amount of the anti-CEA antibody, nucleic acid molecule, or antibody-drug conjugate of any one of the foregoing, and one or more A variety of pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the present disclosure also provides a method for immunodetection or determination of CEA, the method comprising the step of contacting the anti-CEA antibody described in any one of the foregoing with a sample.
  • the present disclosure also provides a kit comprising the anti-CEA antibody described in any one of the foregoing.
  • the present disclosure also provides a method for preventing or treating diseases, the method comprising administering to a subject a preventive or therapeutically effective amount of the anti-CEA antibody, nucleic acid molecule, or antibody drug of any one of the preceding Conjugate or pharmaceutical composition.
  • the present disclosure also provides a method of treating CEA-related diseases, the method comprising administering to a subject a therapeutically effective amount of the anti-CEA antibody, nucleic acid molecule, or antibody drug of any one of the foregoing Conjugate or pharmaceutical composition.
  • the disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, pleomorphic adult Glioblastoma, neuroblastoma, central nervous system cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular tumor, liver Cell cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, Prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myelo
  • the aforementioned preventive or therapeutically effective amount is a unit dose of the composition containing 0.1 mg to 3000 mg (preferably 1 mg to 1000 mg) of the aforementioned anti-CEA antibody, nucleic acid molecule, antibody drug conjugate or drug combination Things.
  • the present disclosure also provides the use of the anti-CEA antibody, nucleic acid molecule, antibody-drug conjugate or pharmaceutical composition as described in any one of the foregoing in the preparation of a medicine for preventing or treating diseases.
  • the present disclosure also provides the use of the anti-CEA antibody, nucleic acid molecule, antibody drug conjugate or pharmaceutical composition as described in any one of the foregoing in the preparation of a medicament for the treatment of diseases or disorders.
  • the aforementioned diseases are preferably diseases related to CEA. In some embodiments, the aforementioned disease is tumor.
  • the aforementioned disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system Cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer Tract cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia , Lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Ke
  • the present disclosure also provides an anti-CEA antibody as described in any one of the foregoing, or the foregoing nucleic acid molecule, or the foregoing antibody-drug conjugate for use in the prevention or treatment of diseases Or a pharmaceutical composition as described above.
  • the aforementioned diseases are preferably diseases related to CEA.
  • the disease is a tumor.
  • the disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system Systemic cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, stomach Intestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, Leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Keukenberg tumor
  • the anti-CEA antibody, nucleic acid molecule, or antibody-drug conjugate according to the present disclosure is the only active ingredient in prevention or treatment.
  • the anti-CEA antibodies, nucleic acid molecules, and antibody drug conjugates according to the present disclosure can be administered in combination with other active ingredients.
  • Figure 1 Endocytosis activity of humanized antibody in MKN45 cells.
  • cytotoxic drug refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction. Cytotoxic drugs include toxins, chemotherapeutic drugs and other compounds that can be used to kill tumor cells.
  • toxin refers to any substance that can produce harmful effects on the growth or proliferation of cells, and can be small molecule toxins and their derivatives from bacteria, fungi, plants or animals, including but not limited to camptothecin derivatives such as Ixatecan, maytansinoid and its derivatives (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (WO 2016/127790 A1, compound 7 ), diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modeccin, ⁇ -sarcin, Aleutitesfordii toxic protein, incense Dianthin toxic protein, Phytolaca americana toxic protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, jatropha curcin, crotonin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictoc
  • chemotherapeutic drugs are chemical compounds that can be used to treat tumors. This definition also includes antihormonal agents that act to modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic therapy. They can be hormones themselves.
  • chemotherapeutic drugs include alkylating agents such as thiotepa; cyclosphamide (CYTOXAN TM ); alkyl sulfonates such as busulfan, improsulfan, and pippo Piposulfan; aziridine such as benaodopa, carboquone, meturedopa and uredopa; aziridine and methyl Melamine (methylamelamine) includes altretamine, triethylenemelamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine; nitrogen mustards Such as chlorambucil, chlorambucil, cholophosphamide, estramustine, ifosfamide, mechlorethamine, chlorambucil hydrochloride; L-phenylalanine nitrogen Melphalan, novembichin, cholesterol mustard, prednimustine, trofosfamide, uracil mustard; nitrosureas such as
  • anti-estrogens include tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxytamoxifen, trioxifene (trioxifene), keoxifene (keoxifene), LY117018, onapristone (onapristone), and toremifene (Fareston); and anti-androgen preparations, such as Flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts of any of the above substances, acids Or derivatives.
  • anti-estrogens include tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxytamoxifen, trioxifene (trioxifene), keoxifene (keoxifene), LY117018, onapristone (onapristone), and toremifene (Fareston);
  • the "antibody” described in the present disclosure is an immunoglobulin.
  • a complete antibody is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain, and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain by the difference of the constant region.
  • Each of the five types of Ig can have ⁇ chain or ⁇ chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies. In addition to full-length antibodies, the antibodies of the present disclosure also include antigen-binding fragments capable of binding antigen.
  • murine antibody in this disclosure is a monoclonal antibody against human CEA prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with the CEA antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine anti-CEA antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine ⁇ , ⁇ chain or a variant thereof, or further comprise murine IgG1, The heavy chain constant region of IgG2, IgG3, or variants thereof.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, and then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed. It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the PD-L1 chimeric antibody further comprises a light chain constant region of a human ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the CEA chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising the human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations (E.g. L234A and/or L235A mutation, and/or S228P mutation) IgG1, IgG2 or IgG4 variants.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the variable region framework of human antibodies, that is, different types of human germline antibodies The antibody produced in the framework sequence. It can overcome the heterogeneous reaction induced by the chimeric antibody carrying a large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), and in Kabat, EA, etc.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further displayed by yeast to undergo affinity maturation mutations to CDRs.
  • the grafting of the CDR may result in the reduced affinity of the produced antibody or its antigen-binding fragment to the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of hypermutation of somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of the humanized antibody.
  • the amino acid residues involved in antigen binding from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable region of animal monoclonal antibodies. Residues in the CDR donor framework that are different from the germline can be considered related.
  • the sequence can be compared with the consensus sequence of a subclass or animal antibody sequence with a high percentage of similarity. Rare framework residues are thought to be the result of hypermutation of somatic cells and thus play an important role in binding.
  • the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of human or murine ⁇ , ⁇ chain or a variant thereof, or further comprise human or murine IgG1 , IgG2, IgG3, IgG4 or variants thereof; preferably comprising the heavy chain constant region of human IgG1, IgG2, or IgG4, or using amino acid mutations (such as L234A and/or L235A mutations, and/or S228P mutations) IgG1, IgG2 or IgG4 variants.
  • the “conventional variants” of the heavy chain constant region of a human antibody and the light chain constant region of a human antibody in the present disclosure refer to the heavy chain that has been disclosed in the prior art and does not change the structure and function of the variable region of the antibody. Variants of the constant region or the light chain constant region. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification of the heavy chain constant region and amino acid substitutions.
  • HumanMAb (HuMAb), “human antibody”, “fully human antibody” and “fully human antibody” can be used interchangeably, and can be antibodies derived from humans or antibodies obtained from a genetically modified organism.
  • the transgenic organism is "engineered” to produce specific human antibodies in response to antigen stimulation and can be produced by any method known in the art.
  • the elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, and the endogenous heavy and light chain loci in these cell lines are targeted Disruption of these cell lines contains targeted destruction of the endogenous heavy and light chain loci.
  • Transgenic organisms can synthesize human antibodies specific to human antigens, and the organisms can be used to produce human antibody-secreting hybridomas.
  • a human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more personal DNA sources.
  • Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display technology, or constructed from B cells activated in vitro, all of which are known in the art.
  • full-length antibody “whole antibody”, “whole antibody” and “whole antibody” are used interchangeably herein and refer to an antibody in a substantially intact form, as distinguished from the antigen-binding fragments defined below.
  • the term specifically refers to antibodies whose light and heavy chains contain constant regions.
  • antibody of the present disclosure includes “full-length antibodies” and antigen-binding fragments thereof.
  • the full-length antibody of the present disclosure includes a full-length antibody formed by a light chain in which a light chain variable region is connected to a light chain constant region and a heavy chain in which a heavy chain variable region is connected to a heavy chain constant region.
  • a light chain in which a light chain variable region is connected to a light chain constant region
  • a heavy chain in which a heavy chain variable region is connected to a heavy chain constant region.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen (eg, CEA). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies.
  • an antigen eg, CEA
  • binding fragment included in the term "antigen-binding fragment" of the antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, including The bivalent fragment of two Fab fragments connected by a disulfide bridge on the hinge region, (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VH and VL domains of one arm of an antibody ; (V) dsFv, a stable antigen-binding fragment formed by VH and VL through interchain disulfide bonds; (vi) diabodies, bispecific antibodies and multispecific antibodies containing fragments such as scFv, dsFv, and Fab.
  • single chain Fv single chain Fv
  • scFv single chain Fv
  • Such single chain antibodies are also included in the term "antigen-binding fragment" of antibodies.
  • antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulin.
  • the antibodies can be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Fab is an antigen-binding fragment that has a molecular weight of about 50,000 and has antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (amino acid residues that cleave the H chain), wherein the N-terminal side of the H chain About half and the entire L chain are joined together by disulfide bonds.
  • protease papain amino acid residues that cleave the H chain
  • F(ab')2 is obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of about 100,000 and has antigen binding activity and contains two Fab regions connected at the hinge position. The antigen-binding fragment.
  • Fab' is an antigen-binding fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of F(ab')2.
  • the Fab' of the present disclosure can be produced by treating the F(ab')2 that specifically recognizes and binds CEA of the present disclosure with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express Fab'.
  • single-chain antibody means to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker Of molecules.
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • a suitable prior art linker consists of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) .
  • linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • Diabodies are antigen-binding fragments in which scFv or Fab are dimerized, and are antigen-binding fragments with bivalent antigen-binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.
  • Bispecific antibodies and multispecific antibodies refer to antibodies that can simultaneously bind to two or more antigens or antigenic determinants, including scFv or Fab fragments that can bind CEA.
  • the diabody of the present disclosure can be produced by the following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes and binds to human CEA, and constructing the DNA encoding scFv so that the amino acid sequence length of the peptide linker is 8 Residues or less, insert the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introduce the expression vector into a prokaryote or eukaryotic to express the diabody.
  • dsFv is obtained by connecting a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected according to a known method (Protein Engineering, 7, 697 (1994)) based on the prediction of the three-dimensional structure of the antibody.
  • the full-length antibody or antigen-binding fragment of the present disclosure can be produced by the following steps: obtaining the cDNA encoding the antibody of the present disclosure that specifically recognizes and binds to human CEA, constructing DNA encoding dsFv, and inserting the DNA into a prokaryotic expression vector Or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or eukaryote to express dsFv.
  • amino acid difference or “amino acid mutation” means that compared with the original protein or polypeptide, the variant protein or polypeptide has amino acid changes or mutations, including one, two, three or one mutation on the basis of the original protein or polypeptide. Insertion, deletion or substitution of more amino acids.
  • antibody framework or "FR region” refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR in the variable domain of the heavy chain (VH) The amino acid residue numbers are 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residue numbers in the light chain variable domain (VL) are 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acids in VL
  • the residue numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDR is defined by the amino acid residues 26-35 (HCDR1) in human VH. ), 50-65 (HCDR2) and 95-102 (HCDR3) and the amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • HCDR1 amino acid residues 26-35
  • HCDR2 amino acid residues 26-65
  • HCDR3 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues
  • the CDR region of an antibody can be determined using the program IMGT/DomainGap Align. Unless otherwise specified, the antibody variable regions and CDR sequences described in the specific embodiments of the present disclosure are applicable "Kabat" numbering rules.
  • epitopes refers to a site on an antigen where an immunoglobulin or antibody specifically binds (for example, a specific site on a CEA molecule).
  • Epitopes include linear epitopes and conformational epitopes.
  • conformational epitopes usually have a unique spatial conformation and include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 Continuous or non-contiguous amino acids. See, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of about less than 10 -7 M, for example, about less than 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less.
  • KD affinity
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.
  • the antibodies of the present disclosure bind CEA with a dissociation equilibrium constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M or 10 -9 M, for example, the affinity of the antibody to the cell surface antigen in the present disclosure
  • KD value was determined by Biacore method.
  • the term “competition” in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding protein or neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay:
  • the antigen-binding protein to be detected for example, an antibody or immunologically functional fragment thereof
  • prevents or inhibits for example, reduces
  • a reference antigen-binding protein for example, a ligand or a reference antibody
  • a common antigen for example, CEA antigen or Its fragment
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, for example, Kirkland et al., 1986, J.Immunol.137:3614-3619
  • solid Phase direct labeling assay solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual (antibody, laboratory manual), Cold Spring Harbor Press); solid-phase direct labeling with I-125 label RIA (see, for example, Morel et al., 1988, Molec.
  • the assay involves the use of purified antigen bound to a solid surface or cell carrying either an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being tested. Usually the tested antigen binding protein is present in excess.
  • the antigen binding proteins identified by the competition assay include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding that binds to adjacent epitopes sufficiently close to the binding epitope of the reference antigen binding protein Proteins, the two epitopes sterically hinder each other from binding. Additional details on the methods used to determine competitive binding are provided in the Examples herein.
  • the competing antigen binding protein is present in excess, it will inhibit (eg reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule can be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • amino acid sequence identity refers to aligning amino acid sequences and introducing gaps when necessary to achieve the maximum percentage of sequence identity, and does not consider any conservative substitutions as part of sequence identity, in the first sequence and in the second sequence The percentage of amino acid residues that are identical.
  • the alignment can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve the maximum alignment over the entire length of the sequence being compared.
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • the vector is a viral vector, in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can integrate into the genome of the host cell after being introduced into the host cell, thereby The host genome replicates together (e.g., non-episomal mammalian vectors).
  • mice can be immunized with human CEA or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the non-human CDR region.
  • Human FR germline sequence can be obtained from ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
  • IMGT ImmunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the family Enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae).
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), 293 cells, and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human CEA. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition containing any one of the combination compounds of the present disclosure, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, in order to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can assess whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • Constant modification or “conservative substitution or substitution” means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in a protein so that they can be frequently Make changes without changing the biological activity of the protein.
  • Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)).
  • substitution of amino acids with similar structure or function is unlikely to disrupt biological activity. Exemplary conservative substitutions are set out below.
  • Effective amount refers to the amount of the drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired preventive or therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, including the biochemistry, tissue, and organization of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease Academic and/or behavioral symptoms.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related disorders of the present disclosure or improving one or more symptoms of the disorder, and reducing the effectiveness of other agents required to treat the disorder
  • the dosage enhances the efficacy of another agent, and/or delays the progression of the patient’s target antigen-related disorder of the present disclosure.
  • Exogenous refers to substances produced outside organisms, cells, or humans according to circumstances.
  • Endogenous refers to substances produced in organisms, cells, or human bodies according to circumstances.
  • “Homology” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100.
  • BLAST algorithm BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol.
  • Polymerase chain reaction or "PCR” as used herein refers to a procedure or technique in which a small amount of a specific portion of nucleic acid, RNA, and/or DNA is amplified as described in, for example, U.S. Patent No. 4,683,195. Generally speaking, it is necessary to obtain sequence information from the end or outside of the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5'terminal nucleotides of the two primers can be identical to the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA.
  • PCR used herein is regarded as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, and the method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Produce specific parts of nucleic acid.
  • isolated refers to a purified state, and in this case means that the designated molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term “isolated” is not intended to mean the complete absence of these materials or the absence of water, buffers or salts unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredients and thus the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments.
  • the carrier can be an anti-adhesive agent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent Agent, emulsifier, buffer, etc.
  • Suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g., olive oil), saline, buffer, buffered saline, and the like Penetrating agents such as sugars, polyols, sorbitol and sodium chloride.
  • the present disclosure includes an agent for treating a disease related to a target antigen (for example, CEA), which includes the anti-CEA antibody of the present disclosure as an active ingredient.
  • a target antigen for example, CEA
  • the CEA-related diseases in the present disclosure are not limited, as long as it is a CEA-related disease.
  • the therapeutic response induced by the molecules of the present disclosure can be achieved by binding to human CEA, and then blocking CEA and its receptor/ligand Binding or killing tumor cells overexpressing CEA. Therefore, when the molecules of the present disclosure are in preparations suitable for therapeutic applications, they are very useful for such subjects who have tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, gastric cancer , Bowel cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.
  • the present disclosure relates to methods for immunodetection or determination of target antigens (for example, CEA), reagents for immunodetection or determination of target antigens (for example, CEA), and cells for immunodetection or determination of target antigens (for example, CEA)
  • target antigens for example, CEA
  • the method and the diagnostic agent for diagnosing diseases related to the target antigen for example, CEA
  • CEA which comprise the antibody or antigen-binding fragment of the present disclosure that specifically recognizes and binds to the target antigen (for example, human CEA) as an active ingredient.
  • the method for detecting or measuring the amount of the target antigen may be any known method.
  • it includes immunodetection or assay methods.
  • the immunoassay or measurement method is a method of detecting or measuring the amount of antibody or antigen using labeled antigen or antibody.
  • immunodetection or measurement methods include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescence immunoassay, western blotting, physicochemical method Wait.
  • the aforementioned CEA-related diseases can be diagnosed by detecting or measuring the level of CEA using the antibodies of the present disclosure.
  • the sample used to detect or measure the target antigen for example, CEA
  • the target antigen for example, CEA
  • the target antigen for example, CEA
  • tissue cells blood, plasma, serum, pancreatic juice , Urine, stool, tissue fluid or culture fluid.
  • the diagnostic agent containing the antibody or antigen-binding fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.
  • the reagents used to perform the antigen-antibody reaction include buffers, salts and the like.
  • the reagents used for detection include reagents commonly used in immunological detection or measurement methods, such as a labeled second antibody that recognizes the monoclonal antibody, its antigen-binding fragment or its conjugate, and a substrate corresponding to the label.
  • Example 1 Preparation of CEA recombinant protein and stably transfected cells
  • the human CEA protein sequences encoding Fc and His tags were cloned into mammalian cell expression vectors, and the recombinant protein was obtained after expression and purification in 293E cells for use in the experiments of subsequent examples.
  • the untagged human CEA gene, human CEACAM1 gene and monkey CEA gene were transfected into CHO cells to form a CHO cell line expressing CEA protein on the cell surface for subsequent antibody screening and identification.
  • the amino acid sequence of related proteins is as follows:
  • Human CEA-his (hCEA-His) amino acid sequence (the soluble human CEA antigen protein used in the test example):
  • monkey CEA (cynoCEA-CHO) expressed on the surface of CHO cells:
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, the Fc-containing recombinant protein and chimeric antibody expression supernatant were purified with a Protein A column, and the hybridoma expression supernatant was purified with a Protein G column.
  • the supernatant is applied to the column at a certain flow rate. Rinse the column with PBS until the A280 reading drops to baseline.
  • the target protein was eluted with 100mM acetic acid, pH 3.0, and neutralized with 1M Tris-HCl, pH 8.0. After the eluted sample is concentrated and replaced with PBS, it is divided into equipment.
  • mice uses hCEA-His protein and cyno-CEA-His protein, or hCEA-CHO cells and cynoCEA-CHO cells for cross-immunization.
  • mice with high antibody titer in the serum and the titer tending to the plateau were selected for fusion of spleen cells, and the splenic lymphocytes and myeloma cells were fused by a PEG-mediated fusion step Sp2/0 cells ( CRL-8287 TM ) was fused to obtain hybridoma cells.
  • the fused hybridoma cells were cultured in MC semi-solid complete medium (RPMI-1640 containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI and 2% methylcellulose at a density of 0.5-1 ⁇ 10 6 cells/ml (Base) resuspended, aliquoted into 35mm cell culture dishes, incubated at 37°C, 5% CO 2 for 7-9 days.
  • the preliminary screening of antibodies was carried out by enzyme-linked immunosorbent assay (ELISA) based on cell surface antigens.
  • ELISA enzyme-linked immunosorbent assay
  • cell fixative Beyotime, Cat#P0098
  • the sealed cell plate can be stored at -20°C or used directly.
  • the screened positive clones are expanded, cryopreserved, and subcloned two to three times until a single cell clone is obtained.
  • the selected hybridoma clones are further prepared and purified by serum-free cell culture method.
  • the obtained hybridoma antibody was detected by flow cytometry for the binding of the antibody to the CEA protein on the cell surface (see Test Example 1 of the present disclosure), and hybridoma cell lines with good binding activity were selected.
  • the binding activity test results of monoclonal hybridoma cell lines mAb47, mAb63, mAb67 and mAb103 are shown in Table 1.
  • the monoclonal hybridoma cell lines mAb47, mAb63, mAb67 and mAb103 were selected to clone the sequence of the monoclonal antibody.
  • the process is as follows: the logarithmic growth phase hybridoma cells are collected, RNA is extracted with Trizol (Invitrogen, Cat#15596-018), and reverse transcribed into cDNA. After PCR amplification using cDNA as a template, it was sent to a sequencing company for sequencing.
  • the amino acid sequence of the antibody corresponding to the obtained DNA sequence is shown in Table 2 below.
  • the antibody CDR sequences in the table are determined according to the Kabat numbering system.
  • the candidate molecules of mAb47, mAb63, mAb67, and mAb103 obtained from the above hybridoma screening can be amplified and sequenced to obtain the variable region coding gene sequence.
  • the first and last primers are designed based on the sequence obtained, and the sequenced gene is used as a template to construct each antibody VH through PCR /VK gene fragment, and then homologous recombination with the expression vector pHr (with signal peptide and hIgG1/hkappa/hlambda constant region gene (CH1-Fc/CL) fragment) to construct a recombinant chimeric antibody full-length expression plasmid VH-CH1- Fc-pHr/VL-CL-pHr to obtain its chimeric antibodies Ch47, Ch63, Ch67 and Ch103.
  • the heavy and light chain variable region germline genes with high homology were selected as templates, and the mouse antibody CDRs were grafted (grafted).
  • the order of formation is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 variable region sequence.
  • the CDR amino acid residues of the antibody in the following example are determined and annotated by the Kabat numbering system.
  • the humanized light chain template of the murine antibody mAb47 is IGKV6-21*01 and IGKJ2*01
  • the humanized heavy chain template is IGHV7. -4-1*02 and IGHJ6*01
  • the humanized back mutation design of the mouse antibody mAb47 is shown in Table 4:
  • the amino acid position numbers in the table are the numbers of the Kabat numbering rules.
  • L46P refers to the numbers corresponding to the Kabat numbering system.
  • the 46th position L is mutated back to P; grafting represents the mouse-derived antibody CDR is implanted into the human germline FR region sequence.
  • the D61S mutation was introduced into the heavy chain h47VH3 (that is, amino acid mutations were made on the antibody HCDR2, so that the antibody HCDR2 sequence was changed from The mutation is: ), the antibody still has good activity.
  • the humanized light chain template of the murine antibody mAb63 is IGKV1-39*01 and IGKJ4*01
  • the humanized heavy chain template is IGHV1.
  • -46*01 and IGHJ1*01 transplant the CDRs of the mouse antibody mAb63 into the corresponding human template for humanization.
  • the humanized back mutation design of the mouse antibody mAb63 is shown in Table 6 below:
  • the amino acid position numbers in the table are the numbers corresponding to the Kabat numbering rules.
  • S 82A R refers to the numbers corresponding to the Kabat numbering system.
  • the 82A position S is mutated back to R; grafting represents mouse-derived antibody CDR implantation into humans Germline FR region sequence.
  • the N54S mutation was introduced into the heavy chain h63VH1 (that is, amino acid mutations were made on the antibody HCDR2, so that the antibody HCDR2 sequence was mutated from DIFPKNGNTDYNRKFKD ( SEQ ID NO: 22) to: ), the antibody still has good activity.
  • the humanized light chain template of the murine antibody mAb67 can select IGKV4-1*01 and IGKJ4*01, IGKV3-15*01 and IGKJ4* 01, or IGKV1-39*01 and IGKJ4*01, humanized heavy chain template select IGHV1-3*01 and IGHJ1*01, or IGHV5-51*01 and IGHJ1*01, and transplant the CDR of the mouse antibody mAb67 respectively
  • Table 8 the humanized back mutation design of the mouse antibody mAb67 is shown in Table 8 below:
  • the amino acid position numbers in the table are the numbers corresponding to the Kabat numbering rules.
  • A43P refers to the numbers corresponding to the Kabat numbering system, and the 43rd position A is mutated back to P; grafting represents mouse-derived antibody CDR implantation into human germline FR District sequence.
  • the humanized light chain template of the murine antibody mAb103 is IGLV4-69*01 and IGLJ2*01
  • the humanized heavy chain template is IGHV7. -4-1*02 and IGHJ1*01
  • the humanized back mutation design of the mouse antibody mAb103 is shown in Table 10 below:
  • the amino acid position numbers in the table are numbers corresponding to the Kabat numbering rules.
  • K49E refers to the numbers corresponding to the Kabat numbering system, and the 49th K is mutated back to E; Grafted represents the mouse-derived antibody CDR implanted into human germline FR District sequence.
  • the D61S mutation was introduced into the heavy chain h103VH1 (that is, amino acid mutations were made on the antibody HCDR2, so that the antibody HCDR2 sequence was changed from The mutation is: ); Introducing the D56E mutation in the light chain h103VL3 (ie, amino acid mutation on the antibody LCDR2, so that the antibody LCDR2 sequence is mutated from LKKDGSHSTGD (SEQ ID NO: 36) to: LKKDGSHSTGE (SEQ ID NO: 64), the antibody still has good activity .
  • the expression vectors for the light chain and heavy chain of the antibody are constructed separately, the humanized antibody light/heavy chains are respectively cross-paired and combined, and the 293E cells are transfected and the culture supernatant is collected and purified to obtain the humanized full-length antibody.
  • the constant region of the humanized antibody heavy chain can be selected from the constant regions of IgG1, IgG2, IgG3, IgG4 and variants thereof; exemplary, the constant region of human heavy chain IgG1 (shown in SEQ ID NO: 77) and the aforementioned human
  • the variable regions of the sourced heavy chain are connected to form the full-length heavy chain of an antibody.
  • the constant region of the humanized antibody light chain can be selected from the constant regions of human kappa, lambda chains or variants thereof; for example, a human light chain constant region kappa chain (shown in SEQ ID NO: 78) or a human light chain
  • the ⁇ chain of the constant chain region (shown in SEQ ID NO: 79) is connected with the variable region of the aforementioned humanized light chain to form the full-length light chain of an antibody.
  • the sequence of the constant region of an exemplary antibody is as follows:
  • the aforementioned humanized antibody heavy chain variable region derived from mAb47 as described in Table 5 is connected to the amino terminal of the human heavy chain IgG1 constant region of SEQ ID NO: 77 to form the full length of the antibody Heavy chain, at the same time, the humanized antibody light chain variable region described in Table 5 is connected to the amino terminal of the human light chain ⁇ constant region shown in SEQ ID NO: 78 to form the antibody full-length light chain, and the following table is obtained
  • Hu47-14 in the table indicates that the light chain variable region of the humanized antibody numbered Hu47-14 is h47VL2, the heavy chain variable region is h47VH4, and the heavy chain constant region sequence is shown in SEQ ID NO: 77 As shown, the light chain constant region sequence is shown in SEQ ID NO: 78.
  • the aforementioned humanized antibody heavy chain variable region derived from mAb63 as described in Table 7 is connected to the amino terminus of the human heavy chain IgG1 constant region of SEQ ID NO: 77 to form the full length of the antibody Heavy chain, at the same time, the humanized antibody light chain variable region described in Table 7 is connected to the amino terminal of the human light chain ⁇ constant region shown in SEQ ID NO: 78 to form the antibody full-length light chain, and the following table is obtained 13 series of mAb63 humanized antibodies:
  • Hu63-13 in the table indicates that the variable region of the light chain of the humanized antibody numbered Hu63-13 is h63VL1, and the variable region of the heavy chain is h63VH5, and its heavy chain constant region sequence is shown in SEQ ID NO: 77 As shown, the light chain constant region sequence is shown in SEQ ID NO: 78.
  • the aforementioned humanized antibody heavy chain variable region derived from mAb67 as described in Table 9 is connected to the amino terminal of the human heavy chain IgG1 constant region with the sequence shown in SEQ ID NO: 77 to form the full length of the antibody Heavy chain, the humanized antibody light chain variable region described in Table 9 is connected with the amino terminal of the human light chain ⁇ constant region shown in SEQ ID NO: 78 to form the antibody full-length light chain, and the following table 14 is obtained The series of mAb67 humanized antibodies shown:
  • Hu67-14 in the table indicates that the variable region of the light chain of the humanized antibody numbered Hu67-14 is h67VL4, and the variable region of the heavy chain is h67VH3, and its heavy chain constant region sequence is shown in SEQ ID NO: 77 As shown, the light chain constant region sequence is shown in SEQ ID NO: 78.
  • the aforementioned humanized antibody heavy chain variable region derived from mAb103 as described in Table 11 is connected to the amino terminal of the human heavy chain IgG1 constant region with the sequence shown in SEQ ID NO: 77 to form the full length of the antibody Heavy chain, the humanized antibody light chain variable region described in Table 11 is connected to the amino terminus of the human light chain ⁇ constant region shown in SEQ ID NO: 79 to form the antibody full-length light chain, and the following table 15 is obtained.
  • Hu103-32 in the table indicates that the variable region of the light chain of the humanized antibody numbered Hu103-32 is h103VL8, and the variable region of the heavy chain is h103VH4, and its heavy chain constant region sequence is shown in SEQ ID NO: 77 As shown, the light chain constant region sequence is shown in SEQ ID NO: 79.
  • ADC molecules targeting CEA SAR-408701 and Rabelizumab-govitecan (also known as Lmab-CL2A-SN38) conjugate.
  • the antibody light/heavy chain sequences are as follows:
  • Test case 1 FACS combined experiment
  • cells expressing CEA on the cell surface were used to detect the binding activity of the antibody by FACS.
  • Cells were collected, 400g, 4 °C 5min centrifugation; prechilled PBS containing 10% FBS at a final concentration of, 400g, 4 °C centrifuge 5min, repeated twice; assigning cells to the 96-well plate, 10 5 cells / well; Per Add 100 ⁇ l of gradient dilution antibody solution to the wells, incubate at 4°C for 60min, centrifuge to remove the supernatant; add 250 ⁇ l of pre-cooled PBS containing a final concentration of 10% FBS to each well to resuspend the cells, centrifuge at 400g, 4°C for 5min, remove the supernatant, Repeat twice; add 50 ⁇ l of 1:200 diluted secondary antibody Alexa Fluor@488 goat anti-human IgG(H+L) (Lifetechologies, Cat#A11013), incuba
  • the experimental results show that the humanized antibodies screened in the present disclosure maintain binding activity similar to that of murine antibodies, and can bind to the human CEA protein on the cell surface; the humanized antibodies screened in the present disclosure can bind to the cell surface monkey CEA protein binding, and the binding activity with monkey CEA protein is better than the positive control antibody.
  • Test Example 2 Competitive experiment with soluble CEA (sCEA)
  • the inventors pre-incubated the antibody with serial dilutions with a certain concentration of sCEA (5 ⁇ g/ml) for 30 minutes, Then collect MKN45 cells (human gastric cancer cells, Nanjing Kebai Biotechnology Co., Ltd., Cat#CBP60488) and distribute the cells to a 96-well plate; add gradient dilutions of antibody and a mixed solution of antibody and sCEA pre-incubated to each well, and incubate at 4°C After 60 minutes, centrifuge to remove the supernatant; add pre-cooled PBS containing a final concentration of 10% FBS to wash the cells, repeat twice; add 50 ⁇ l 1:200 diluted secondary antibody Alexa Fluor@488 goat anti-human IgG(H+L)(Lifetechologies , Cat#A11013), incubate at 4
  • the ratio of the signal without sCEA to the signal with sCEA at each antibody concentration is less than 2, it means that the binding curve of the antibody does not change greatly in the presence of sCEA, and the antibody still preferentially binds to CEA on the cell membrane surface.
  • the experimental results show that the humanized antibodies Hu63-13, Hu47-14, Hu67-14, Hu103-32 screened in the present disclosure have all the fluorescence signal ratios at various antibody concentrations without sCEA and sCEA. Less than 2, for example, the maximum ratio of Hu63-13 is 1.59, while the maximum ratio of the positive control Lmab is 5.18.
  • the antibodies screened in this disclosure are better than the control antibodies.
  • the humanized antibody screened in the present disclosure does not add sCEA and the maximum fluorescence signal ratio of sCEA is less than 2, and is less than the positive control antibody, indicating that the humanized antibody screened in the present disclosure is still in the presence of sCEA It preferentially binds to CEA on the cell membrane surface and is better than the positive control antibody.
  • Test Example 3 Biacore measures the affinity of antibodies to soluble CEA
  • a Biacore (GE, T200) instrument was used to determine the affinity of the humanized antibody to be tested with human and monkey soluble CEA.
  • CM5 biosensing chip
  • GE GE, Cat#BR-1005-30
  • the Biacore instrument To capture a certain amount of antibody to be tested with affinity, then flow a series of soluble CEA antigen (shown in SEQ ID NO: 1) with a concentration gradient on the surface of the chip, and use Biacore to detect the reaction signal in real time to obtain binding and dissociation curves .
  • the biochip is cleaned and regenerated with the regeneration solution configured in the human anti-capture kit.
  • the data obtained in the experiment was fitted with the (1:1) Langmuir model using BIAevaluation version 4.1 software to obtain the affinity value. Since the present disclosure hopes that the CEA binding activity of the selected antibody to the cell membrane surface is stronger than that of soluble CEA, the lower the affinity of the antibody to soluble CEA, the better.
  • the results of the affinity test of humanized antibody and soluble CEA are shown in Table 21 below:
  • the test results show that the affinity of humanized antibodies Hu63-13, Hu47-14 and Hu67-14 to soluble CEA protein is relatively low, which is significantly lower than that of the control antibodies Sanofi and Lmab. This indicates that Hu63-13, Hu47-14 and Hu67-14 are not easily neutralized by soluble CEA in blood in vivo, and a large number of antibodies can bind to cells expressing CEA on the cell membrane surface.
  • Test Example 4 Endocytosis activity of anti-CEA antibody in CEA highly expressing cell MKN45
  • the anti-CEA antibody conjugate in the present disclosure can release toxins to kill the cells after being endocytosed by cells, so the endocytosis activity of CEA antibody in CEA-expressing cells can promote ADC activity.
  • MKN45 cells human gastric cancer cells, Nanjing Kebai Biotechnology Co., Ltd., Cat#CBP60488
  • MKN45 was plated in 96-well plates (Coring, Cat#3795) and cultured overnight.
  • the humanized CEA antibodies Hu63-13, Hu47-14, Hu67-14 and Hu103-3 were pre-incubated with iFL Green Human IgG Labeling Reagent (Invitrogen, Cat#Z25611) for 15 minutes, and the iFL reagent will be humanized Fc binding of antibody, then add antibody and iFL complex to cell culture plate, remove cell culture fluid after 6 hours and 24 hours respectively, add PBS to wash twice, digest and collect cells, use FACS to detect fluorescence signal in cells Intensity, after the antibody is endocytosed by the cell, the iFL bound to the antibody Fc will be brought into the cell. The iFL will be swallowed into the cell and the fluorescent signal can only be detected in an acidic environment.
  • iFL Green Human IgG Labeling Reagent Invitrogen, Cat#Z25611
  • Test Example 5 In vivo pharmacokinetic test
  • the antibodies of the present disclosure all have good pharmacokinetic activity.

Abstract

提供包含重链可变区和轻链可变区的抗CEA抗体、其药物偶联物，以及包含该抗CEA抗体或其药物偶联物的组合物，及其作为药物的用途。

Description

抗CEA抗体及其应用
本公开要求中国专利申请《抗CEA抗体及其应用》的优先权(申请号为201910560329.6；申请日为2019年06月26日)。
技术领域
本公开涉及抗体药物领域。具体地，包括抗CEA抗体药物以及其应用。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
癌胚抗原(CEA，又称为CEACAM-5或CD66e)是最早被人们发现的肿瘤相关抗原之一，它是一种具有约180kDa分子量的糖蛋白，CEA是免疫球蛋白超家族的一名成员，并且含有经由糖基磷脂酰肌醇(GPI)锚与细胞膜连接的7个域(Thompson J.A.,J Clin Lab Anal.5:344-366,1991)。CEA最初由Gold P和Freedman SO在结肠癌组织提取物中发现并报道(Gold and Freedman 1965；Gold and Freedman 1965)，随后报道了利用敏感的放射性免疫分析的方法在结肠癌病人和其他肿瘤病人的血清中检测到CEA，而在健康人或其他疾病患者血清中CEA的含量极低(Thomson，Krupey等人1969)。CEA在癌细胞中表达升高，升高的CEA促进细胞间粘着，进而促进细胞的转移(Marshall J.,Semin Oncol.,30(增刊8):30-6,2003)。CEA常见表达在上皮组织，包括胃肠、呼吸和泌尿生殖道的细胞，及结肠、宫颈、汗腺和前列腺的细胞(Nap等，Tumour Biol.，9(2-3)：145-53，1988；Nap等，Cancer Res.，52(8)：2329-23339，1992)。
目前已见WO1999043817A1、WO2004032962A1、WO2005086875A3、WO2012117002A1等专利公开了多种抗CEA抗体。
发明内容
本公开提供了一种新的抗CEA抗体。本公开的抗体包括全长抗体及其抗原结合片段。
在一些实施方案中，本公开的抗CEA抗体，其包含抗体重链可变区和轻链可变区，其中：
i)所述重链可变区的HCDR1和HCDR3与如SEQ ID NO：7序列所示的重链可变区的HCDR1和HCDR3相同，所述重链可变区的HCDR2与如SEQ ID NO：7序列所示的重链可变区的HCDR2相同或与其具有一个氨基酸差异；
所述轻链可变区的LCDR1、LCDR2和LCDR3与如SEQ ID NO：8序列所示的轻链可变区的LCDR1、LCDR2和LCDR3相同；或
ii)所述重链可变区的HCDR1和HCDR3与如SEQ ID NO：9序列所示的重 链可变区的HCDR1和HCDR3相同，所述重链可变区的HCDR2与如SEQ ID NO：9序列所示的重链可变区的HCDR2相同或与其具有一个氨基酸差异；
所述轻链可变区的LCDR1、LCDR2和LCDR3与如SEQ ID NO：10序列所示的轻链可变区的LCDR1、LCDR2和LCDR3相同；或
iii)所述重链可变区的HCDR1、HCDR2和HCDR3与如SEQ ID NO：11序列所示的重链可变区的HCDR1、HCDR2和HCDR3相同；
所述轻链可变区的LCDR1、LCDR2和LCDR3与如SEQ ID NO：12序列所示的轻链可变区的LCDR1、LCDR2和LCDR3相同；或
iv)所述重链可变区的HCDR1和HCDR3与如SEQ ID NO：13序列所示的重链可变区的HCDR1和HCDR3相同，所述重链可变区的HCDR2与如SEQ ID NO：13序列所示的重链可变区的HCDR2相同或与其具有一个氨基酸差异；
所述轻链可变区的LCDR1和LCDR3与如SEQ ID NO：14序列所示的轻链可变区的LCDR1和LCDR3相同，所述轻链可变区的LCDR2与如SEQ ID NO：14序列所示的轻链可变区的LCDR2相同或与其具有一个氨基酸差异。
在一些实施方案中，如前任一项所述的抗CEA抗体，其包含重链可变区和轻链可变区，其中：
v)所述重链可变区包含序列分别如SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含序列分别如SEQ ID NO：15、SEQ ID NO：38和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3；
vi)所述重链可变区包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含序列分别如SEQ ID NO：21、SEQ ID NO：47和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；或
vii)所述重链可变区包含序列分别如SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32所示的LCDR1、LCDR2和LCDR3；或
viii)所述重链可变区包含序列分别如SEQ ID NO：33和SEQ ID NO：34所示的HCDR1和HCDR3，和序列如SEQ ID NO：16或SEQ ID NO：38所示的HCDR2；
所述轻链可变区包含序列分别如SEQ ID NO：35和SEQ ID NO：37所示的LCDR1和LCDR3，和序列如SEQ ID NO：36或SEQ ID NO：64所示的LCDR2。
在一些实施方案中，所述重链可变区包含序列分别如SEQ ID NO：33、SEQ ID NO：16和SEQ ID NO：34所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：35、SEQ ID NO：36和SEQ ID NO：37所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含序列分别如SEQ ID NO：33、SEQ ID NO：38和SEQ ID NO：34所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：35、SEQ ID NO：36和SEQ ID NO：37所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含序列分别如SEQ ID NO：33、SEQ ID NO：16和SEQ ID NO：34所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：35、SEQ ID NO：64和SEQ ID NO：37所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含序列分别如SEQ ID NO：33、SEQ ID NO：38和SEQ ID NO：34所示的HCDR1、HCDR2和HCDR3，
所述轻链可变区包含序列分别如SEQ ID NO：35、SEQ ID NO：64和SEQ ID NO：37所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如前任一项所述的抗CEA抗体，其中所述抗体是鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，如前任一项所述的抗CEA抗体，其中所述抗体是鼠源抗体或嵌合抗体，其包含重链可变区和轻链可变区，其中：
(a)所述重链可变区的氨基酸序列如SEQ ID NO:7所示，或与SEQ ID NO:7所示的氨基酸序列有至少90％序列同一性；和/或
所述轻链可变区的氨基酸序列如SEQ ID NO:8所示，或与SEQ ID NO:8所示的氨基酸序列有至少90％序列同一性；或
(b)所述重链可变区的氨基酸序列如SEQ ID NO:9所示，或与SEQ ID NO:9所示的氨基酸序列有至少90％序列同一性；和/或
所述轻链可变区的氨基酸序列如SEQ ID NO:10所示，或与SEQ ID NO:10所示的氨基酸序列有至少90％序列同一性；
(c)所述重链可变区的氨基酸序列如SEQ ID NO:11所示，或与SEQ ID NO:11所示的氨基酸序列有至少90％序列同一性；和/或
所述轻链可变区的氨基酸序列如SEQ ID NO:12所示，或与SEQ ID NO:12 所示的氨基酸序列有至少90％序列同一性；或
(d)所述重链可变区的氨基酸序列如SEQ ID NO:13所示，或与SEQ ID NO:13所示的氨基酸序列有至少90％序列同一性；和/或
所述轻链可变区的氨基酸序列如SEQ ID NO:14所示，或与SEQ ID NO:14所示的氨基酸序列有至少90％序列同一性。
在一些实施方案中，前述(a)至(d)任一项所述具有至少90％序列同一性是：具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，如前任一项所述的抗CEA抗体，其中所述抗体是人源化抗体,其包含重链可变区和轻链可变区，其中：
(e)所述重链可变区的氨基酸序列如SEQ ID NO:39、40、41或42所示，或与SEQ ID NO:39、40、41或42所示的任意一条氨基酸序列有90％序列同一性；和/或
所述轻链可变区的氨基酸序列如SEQ ID NO:43、44、45或46所示，或与SEQ ID NO:43、44、45或46所示的任意一条氨基酸序列有90％序列同一性；
(f)所述重链可变区的氨基酸序列如SEQ ID NO:48、49、50、51或52所示，或与SEQ ID NO:48、49、50、51或52所示的任意一条氨基酸序列有90％序列同一性；和/或
所述轻链可变区的氨基酸序列如SEQ ID NO:53、54或55所示，或与SEQ ID NO:53、54或55所示的任意一条氨基酸序列有90％序列同一性；
(g)所述重链可变区的氨基酸序列如SEQ ID NO:56、57或58所示，或与SEQ ID NO:56、57或58所示的任意一条氨基酸序列有90％序列同一性；和/或
所述轻链可变区的氨基酸序列如SEQ ID NO:59、60、61、62或63所示，或与SEQ ID NO:59、60、61、62或63所示的任意一条氨基酸序列有90％序列同一性；或
(h)所述重链可变区的氨基酸序列如SEQ ID NO:65、66、67或68所示，或与SEQ ID NO:65、66、67或68所示的任意一条氨基酸序列有90％序列同一性；和/或
所述轻链可变区的氨基酸序列如SEQ ID NO:69、70、71、72、73、74、75或76所示，或与SEQ ID NO:69、70、71、72、73、74、75或76所示的任意一条氨基酸序列有90％序列同一性。
在一些实施方案中，前述(e)至(h)任一所述具有至少90％序列同一性是具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，如前所述的抗CEA抗体，其包含如下表a-1、表a-2、表a-3或表a-4所示的重链可变区和轻链可变区组合：
表a-1.mAb47来源的人源化抗体轻链可变区和重链可变区组合
Figure PCTCN2020097984-appb-000001
备注：表中例如“Hu47V-14”表示轻链可变区为h47VL2(SEQ ID NO:44)，且重链可变区为h47VH4(SEQ ID NO:42)的轻链可变区和重链可变区组合。
表a-2.mAb63来源的人源化抗体轻链可变区和重链可变区组合
Figure PCTCN2020097984-appb-000002
备注：表中例如“Hu63V-13”表示轻链可变区为h63VL1(SEQ ID NO:53)，且重链可变区为h63VH5(SEQ ID NO:52)的轻链可变区和重链可变区组合。
表a-3.mAb67来源的人源化抗体轻链可变区和重链可变区组合
Figure PCTCN2020097984-appb-000003
备注：表中例如“Hu67V-14”表示轻链可变区为h67VL4(SEQ ID NO:62)，且重链可变区为h67VH3(SEQ ID NO:58)的轻链可变区和重链可变区组合。
表a-4.mAb103来源的人源化抗体轻链可变区和重链可变区组合
Figure PCTCN2020097984-appb-000004
Figure PCTCN2020097984-appb-000005
备注：表中例如“Hu103V-32”表示轻链可变区为h103VL8(SEQ ID NO:76)，且重链可变区为h103VH4(SEQ ID NO:68)的轻链可变区和重链可变区组合。
在一些实施方案中，如前所述的人源化抗CEA抗体，其包含重链可变区和轻链可变区，其中：所述轻链可变区氨基酸序列如SEQ ID NO:44，且所述重链可变区氨基酸序列如SEQ ID NO:42所示；或
所述轻链可变区氨基酸序列如SEQ ID NO:53，且所述重链可变区氨基酸序列如SEQ ID NO:52所示；或
所述轻链可变区氨基酸序列如SEQ ID NO:62，且所述重链可变区氨基酸序列如SEQ ID NO:58所示；或
所述轻链可变区氨基酸序列如SEQ ID NO:76，且所述重链可变区氨基酸序列如SEQ ID NO:68所示。
在一些实施方案中，如前任一项所述的抗CEA抗体，其中所述抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体为在人抗体的轻链框架区和/或重链框架区上分别具有1至10个氨基酸的回复突变；
优选地，所述框架区变体包含选自以下(i)至(l)中所述的氨基酸回复突变：
(i)当轻链可变区包含序列分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3时，轻链可变区的框架区包含选自46P、47W、49Y、70S和71Y中的一个或更多个氨基酸回复突变，和/或
当重链可变区包含序列如SEQ ID NO：15所示的HCDR1、序列如SEQ ID NO：16或SEQ ID NO：38所示的HCDR2、和序列如SEQ ID NO：17所示的HCDR3时，重链可变区的框架区包含选自38K或46K中的一个或更多个氨基酸回复突变；
(j)当轻链可变区包含序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3时，轻链可变区的框架区包含选自2V、42G、44V和71Y中的一个或更多个氨基酸回复突变，和/或
当重链可变区包含序列如SEQ ID NO：21所示的HCDR1、序列如SEQ ID NO：22或SEQ ID NO：47所示的HCDR2、和序列如SEQ ID NO：23所示的HCDR3时，重链可变区的框架区包含选自66K、67A、69L、71V、73K、82F、 82A R中的一个或更多个氨基酸回复突变；
(k)当轻链可变区包含序列分别如SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32所示的LCDR1、LCDR2和LCDR3时，轻链可变区的框架区包含选自3V、43P和58V中的一个或更多个氨基酸回复突变，和/或
当重链可变区包含序列分别如SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29所示的HCDR1、HCDR2和HCDR3时，重链可变区的框架区包含选自38K、66K、71V中的一个或更多个氨基酸回复突变；和
(l)当轻链可变区包含序列如SEQ ID NO：35所示的LCDR1、序列如SEQ ID NO：36或SEQ ID NO：64所示的LCDR2、和序列如SEQ ID NO：37所示的LCDR3时，轻链可变区的框架区包含选自4V、36Y、43P、47V、49E、70D和87I中的一个或更多个氨基酸回复突变，和/或
当重链可变区包含序列如SEQ ID NO：33所示的HCDR1、序列如SEQ ID NO：16或SEQ ID NO：38所示的HCDR2、和序列如SEQ ID NO：34所示的HCDR3时，重链可变区的框架区包含选自2I、38K和46K中的一个或更多个氨基酸回复突变；
其中，所述突变的位点对应于Kabat编号规则的编号。例如“46P”表示将对应于Kabat编号规则第46位的氨基酸回复突变为“P”。
技术人员应当理解，当采用Kabat以外的其他编号规则时，在功能和或结构上具有等同地位的氨基酸残基可能被赋予不同的编号，但仍对应于本公开中所限定的位点。
在一些实施方案中，所述的抗CEA抗体，其包含：
(i-1)轻链可变区，其包含分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3，和
重链可变区，其包含如SEQ ID NO：15所示的HCDR1、如SEQ ID NO：16或SEQ ID NO：38所示的HCDR2、和如SEQ ID NO：17所示的HCDR3；
而且，其中所述轻链可变区的框架区中包含选自46P、47W、49Y、70S和71Y中的一个或更多个氨基酸回复突变，和/或所述重链可变区的框架区中包含选自38K或46K中的一个或更多个氨基酸回复突变；
(j-1)轻链可变区，其包含分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；和
重链可变区，其包含如SEQ ID NO：21所示的HCDR1、如SEQ ID NO：22或SEQ ID NO：47所示的HCDR2、和如SEQ ID NO：23所示的HCDR3；
而且，其中所述轻链可变区的框架区中包含选自2V、42G、44V和71Y中的一个或更多个氨基酸回复突变，和/或所述重链可变区的框架区中包含选自48I、66K、67A、69L、71V、73K、82F、 82A R中的一个或更多个氨基酸回复突变；
(k-1)轻链可变区，其包含分别如SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32所示的LCDR1、LCDR2和LCDR3；和
重链可变区，其包含分别如SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29所示的HCDR1、HCDR2和HCDR3；
而且，其中所述轻链可变区的框架区中包含选自3V、43P和58V中的一个或更多个氨基酸回复突变，和/或所述重链可变区的框架区中包含选自38K、66K、71V中的一个或更多个氨基酸回复突变；和
(l-1)轻链可变区，其包含如SEQ ID NO：35所示的LCDR1、如SEQ ID NO：36或SEQ ID NO：64所示的LCDR2、和如SEQ ID NO：37所示的LCDR3；和
重链可变区，其包含如SEQ ID NO：33所示的HCDR1、如SEQ ID NO：16或SEQ ID NO：38所示的HCDR2、和如SEQ ID NO：34所示的HCDR3；
而且，其中所述轻链可变区的框架区中包含选自4V、36Y、43P、47V、49E、70D和87I中的一个或更多个氨基酸回复突变，和/或所述重链可变区的框架区中包含选自2I、38K和46K中的一个或更多个氨基酸回复突变；
其中，所述回复突变的位点对应于Kabat编号规则编号。
在一些实施方案中，如前任一项所述的抗CEA抗体，其中所述抗体进一步包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗体包含序列如SEQ ID NO:77所示的重链恒定区，和序列如SEQ ID NO:78或SEQ ID NO:79所示的轻链恒定区；
最优选地，所述抗体包含：
(m)序列如SEQ ID NO:80所示或与其具有至少85％序列同一性的重链，和/或序列如SEQ ID NO：81所示或与其具有至少85％序列同一性的轻链；或
(n)序列如SEQ ID NO:82所示或与其具有至少85％序列同一性的重链，和/或序列如SEQ ID NO：83所示或与其具有至少85％序列同一性的轻链；或
(o)序列如SEQ ID NO:84所示或与其具有至少85％序列同一性的重链，和/或序列如SEQ ID NO：85所示或与其具有至少85％序列同一性的轻链；或
(p)序列如SEQ ID NO:86所示或与其具有至少85％序列同一性的重链，和/或序列如SEQ ID NO：87所示或与其具有至少85％序列同一性的轻链。
在一些实施方案中，前述(m)至(p)任一所述具有至少85％序列同一性是：具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性。
在一些实施方案中，如前任一项所述的抗CEA抗体，其中所述抗体为全长抗体或抗原结合片段。
在一些实施方案中，如前任一项所述的抗CEA抗体，其中所述抗CEA抗体包含：
(q)序列如SEQ ID NO:80所示的重链和序列如SEQ ID NO：81所示的轻链；
(r)序列如SEQ ID NO:82所示的重链和序列如SEQ ID NO：83所示的轻链；
(s)序列如SEQ ID NO:84所示的重链和序列如SEQ ID NO：85所示的轻链；或
(t)序列如SEQ ID NO:86所示的重链和序列如SEQ ID NO：87所示的轻链。
在一些实施方案中，本公开还提供一种分离的抗CEA抗体，所述抗体与前面任一项所述的抗CEA抗体竞争性结合人CEA，或人CEA表位或片段。
在一些实施方案中，前述抗CEA抗体，其能与细胞膜表面(膜结合型或跨膜型)的人CEA(例如，以高亲和力)结合，也能与细胞膜表面猴CEA(例如，以高亲和力)结合，且所述抗CEA抗体结合细胞膜表面猴CEA的EC50值与结合细胞膜表面人CEA的EC50值的比值≤3.5，优选≤3.4、≤3.3、≤3.2、≤3.1、≤3.0、≤2.9、≤2.8、≤2.7、≤2.6、≤2.5、≤2.4、≤2.3、≤2.2、≤2.1、≤2.0、≤1.9、≤1.8、≤1.7、≤1.6、≤1.5、≤1.0、≤0.9、≤0.8≤0.7、≤0.6、≤0.5、≤0.4、≤0.3、≤0.2或≤0.1，更优选≤0.7、≤0.6、≤0.5、≤0.4、≤0.3、≤0.2，所述抗CEA抗体与CEA结合的EC50(nM)值通过FACS方法测定，示范性例子如本公开的测试例1所示。
在一些实施方案中，前述抗CEA抗体，相对于可溶性人CEA(sCEA)，其竞争性结合细胞膜表面人CEA(CEA)。在一些实施方案中，所述抗CEA抗体在不加sCEA与加sCEA的表达人CEA的细胞(例如MKN45)中，抗CEA抗体与抗原结合的荧光信号的比值的最大值小于2.0，更优选≤1.9、≤1.8、≤1.7、≤1.6、≤1.5、≤1.4、≤1.3、≤1.2、≤1.1或≤1.0，所述荧光信号值采用FACS方法检测，例如本公开的测试例2。
在一些实施方案中，所述抗CEA抗体以大于或等于10 -7M(例如，大于或等于10 -7M、大于或等于1×10 -6M、大于或等于1×10 -5M，优选大于或等于1×10 -7M且小于或等于5×10 -6M)的KD值结合可溶性人CEA，同时以小于或等于10 -9M(例如，小于或等于9×10 -9M，小于或等于8×10 -9M、小于或等于7×10 -9M、小于或等于6×10 -9M、小于或等于5×10 -9M、小于或等于4×10 -9M、小于或等于3×10 -9M、小于或等于2×10 -9M、小于或等于1×10 -9M、小于或等于1×10 -10M)的EC50值结合细胞膜表面的人CEA或细胞膜表面的猴CEA；所述EC50值采用FACS方法测定，例如本公开的测试例1所述；所述KD值采用Biacore方法测定，例如本公开的测试例3。
在一些实施方案中，前述抗CEA抗体，其具有以下特征：
a.所述抗CEA抗体相对于可溶性人CEA(sCEA)，其竞争性结合细胞膜表面人CEA；且所述抗CEA抗体在不加sCEA与加sCEA的表达人CEA的细胞(例如MKN45)中，抗CEA抗体与抗原结合的荧光信号的比值的最大值小于1.7，所述荧光信号值通过FACS方法检测；
b.所述抗CEA抗体与细胞膜表面的人CEA高亲和力结合，也与细胞膜表面猴CEA高亲和力结合，且所述抗CEA抗体结合细胞膜表面猴CEA的EC50值与结合细胞膜表面人CEA的EC50值的比值≤0.7，所述EC50值通过FACS方法测定； 和/或
c.所述抗CEA抗体以大于或等于1×10 -7M且小于或等于5×10 -6M的KD值结合可溶性人CEA，同时以小于或等于8×10 -9M的EC50值结合细胞膜表面的人CEA和细胞膜表面的猴CEA；所述EC50值采用FACS方法测定，所述KD值采用Biacore方法测定。
在一些实施方案中，本公开还提供一种核酸分子，其编码前面任一项所述的抗CEA抗体。
在一些实施方案中，本公开还提供一种宿主细胞，其包含前面所述的核酸分子。
在一些实施方案中，本公开还提供一种抗体药物偶联物，其由前面任一项所述的抗CEA抗体与细胞毒性药物偶联形成。
在另一些实施方案中，本公开还提供一种抗体药物偶联物，其包含以下或由以下组成：
-前述的抗CEA抗体，和
-细胞毒性药物；
其中，所述细胞毒性药物共价偶联至所述抗CEA抗体。
在一些实施方案中，本公开还提供一种药物组合物，其含有预防或治疗有效量的前面任一项所述的抗CEA抗体，核酸分子、或抗体药物偶联物，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
在一些实施方案中，本公开还提供一种用于免疫检测或测定CEA的方法，所述方法包括使前面任一项所述的抗CEA抗体接触样本的步骤。
在一些实施方案中，本公开还提供一种试剂盒，其包含前面任一项所述的抗CEA抗体。
在一些实施方案中，本公开还提供一种预防或治疗疾病的方法，所述方法包括向受试者施用预防或治疗有效量的前面任一项所述的抗CEA抗体、核酸分子、抗体药物偶联物或药物组合物。
在一些实施方案中，本公开还提供一种治疗与CEA相关的疾病的方法，所述方法包括向受试者施用治疗有效量的前面任一项所述的抗CEA抗体、核酸分子、抗体药物偶联物或药物组合物。
在一些实施方案中，其中前述疾病为肿瘤；在另一些实施方案中，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异 常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；在另一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
在一些实施方案中，前述预防或治疗有效量为单位剂量的组合物中含有0.1mg至3000mg(优选1mg至1000mg)如前所述的抗CEA抗体、核酸分子、抗体药物偶联物或药物组合物。
在一些实施方案中，本公开还提供如前任一所述的抗CEA抗体、核酸分子、抗体药物偶联物或药物组合物在制备用于预防或治疗疾病的药物中的用途。
在一些实施方案中，本公开还提供如前任一所述的抗CEA抗体、核酸分子、抗体药物偶联物或药物组合物在制备用于治疗疾病或病症的药物中的用途。
在一些实施方案中，前述疾病优选为与CEA相干的疾病。在一些实施方案中，其中前述疾病为肿瘤。
在另一些实施方案中，前述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；在另一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
在一些实施方案中，本公开还提供一种用于预防或治疗疾病的如前面任一项所述的抗CEA抗体，或如前所述的核酸分子，或如前所述的抗体药物偶联物或如前所述的药物组合物。在一些实施方案中，前述疾病优选为与CEA相干的疾病。在一些实施方案中，其中所述疾病为肿瘤。在另一些实施方案中，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经 母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；在另一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
在一些实施方案中，根据本公开的抗CEA抗体、核酸分子、抗体药物偶联物，是预防或治疗中的唯一活性成分。
在另一些实施方案中，根据本公开的抗CEA抗体、核酸分子、抗体药物偶联物，能够与其他活性成分组合施用。
附图说明
图1：人源化抗体在MKN45细胞中的内吞活性。
具体实施方式
术语(定义)
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
术语“细胞毒性药物”是指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质，细胞毒性药物包括毒素、化疗药物等能用于肿瘤细胞杀伤的化合物。
术语“毒素”是指能够对细胞的生长或增殖产生有害效果的任何物质，可以是来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括但不限于喜树碱类衍生物如伊沙替康，美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4，auristatin F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790 A1，化合物7)，白喉毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹 (dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。
术语“化疗药物”是可用于治疗肿瘤的化学化合物。该定义还包括起调节、降低、阻断或抑制可促进癌生长的激素效果作用的抗激素剂，且常常是系统或全身治疗的形式。它们自身可以是激素。化疗药物实例包括烷化剂，如噻替哌(thiotepa)；环磷酰胺(cyclosphamide)(CYTOXAN TM)；烷基磺酸脂如白消安(busulfan)，英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶(aziridine)如苯并多巴(benaodopa)，卡波醌(carboquone)，美妥替哌(meturedopa)和尿烷亚胺(uredopa)；氮丙啶和甲基蜜胺(methylamelamine)包括六甲蜜胺(altretamine)，三亚胺嗪(triethylenemelamine)，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)；氮芥(nitrogen mustards)如苯丁酸氮芥，萘氮芥，胆磷酰胺(cholophosphamide)，雌氮芥(estramustine)，异环磷酰胺(ifosfamide)，氮芥(mechlorethamine)，盐酸氧氮芥；左旋苯丙氨酸氮芥(melphalan)，新氮芥(novembichin)，胆甾醇苯乙酸氮芥，松龙苯芥(prednimustine)，曲磷胺(trofosfamide)，尿嘧啶氮芥；亚硝基脲(nitrosureas)如亚硝基脲氮芥(carmustine)，氯脲菌素(chlorozotocin)，福莫司汀(fotemustine)，洛莫司汀(lomustine)，尼莫司汀(nimustine)，雷莫司汀(ranimustine)；抗生素如阿克拉霉素，放线菌素，安曲霉素(authramycin)，重氮丝氨酸，博来霉素，放线菌素C(cactinomycin)，加利车霉素(calicheamicin)，carabicin，洋红霉素(chromomycin)，嗜癌素(carzinophilin)，色霉素，放线菌素D，柔红菌素(daunorubicin)，地托比星(detorubicin)，6-重氮-5-氧-L-正亮氨酸，阿霉素(doxorubicin)，表阿霉素(epirubicin)，依索比星(esorubicin)，伊达比星(idarubicin)，发波霉素(marcellomycin)，丝裂霉素，霉酚酸，诺加霉素(nogalamycin)，橄榄霉素(olivomycin)，培洛霉素(peplomycin)，泊非霉素(potfiromycin)，嘌呤霉素，三铁阿霉素(quelamycin)，罗多比星(rodorubicin)，链黑菌素；链脲霉素(streptozocin)，杀结核菌素，乌苯美司(ubenimex)，净司他丁(zinostatin)，佐柔比星(zorubicin)；抗代谢药如氨甲蝶吟，5-氟尿嘧啶(5-FU)；叶酸类似物如二甲叶酸(denopterin)，氨甲蝶呤，蝶罗呤，三甲曲沙(trimetrexate)；喋吟类似物氟达拉滨(f1udarabine)，6-巯基蝶呤，硫咪蝶呤，硫鸟蝶呤；嘧啶类似物如安西他滨(ancitabine)，阿扎胞苷(azacitidine)，6-氮尿苷，卡莫氟(carmofur)，阿糖胞苷，双脱氧尿苷，去氟氧尿苷(doxitluridine),依诺他滨(enocitabine)，氟尿苷，5-FU；雄激素类如二甲睾酮(calusterone),丙酸甲雄烷酮(dromostanolong propionate)，环硫雄醇(epitiostanol)，美雄氨(mepitiostane)，睾内酯(testolactone)； 抗肾上腺类如氨鲁米特(aminoglutethimide)，米托坦(mitotane)，曲洛司坦(trilostane)；叶酸补充剂如亚叶酸(frolinic acid)；醋葡内脂；醛磷酰胺糖苷(aldophosphamideglycoside)；氨基乙酰丙酸(aminolevulinic acid)；安吖啶(amsacrine)；bestrabucil；比生群(biasntrene)；依达曲沙(edatraxate)；defofamine；秋水仙胺；地吖醌(diaziquone)；依托咪嗪(elfomithine)；依利醋铵(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖(lentinan)；氯尼达明(lonidamine)；米托胍腙(mitoguazone)；米托蒽醌(mitoxantrone)；莫哌达醇(mopidamol)；硝呋旦(nitracrine)；喷司他丁(pintostatin)；phenamet；吡柔比星(pirarubicin)；鬼臼树酸(podophyllinic acid)；2-乙基酰肼；丙卡巴肼(procarbazine)；
Figure PCTCN2020097984-appb-000006
雷佐生(razoxane)；西索菲兰(sizofiran)；锗螺胺(spirogermanium)；细交链孢菌酮酸；三亚胺醌；2，2'，2"-三氯二乙胺(trichlorrotriethylamine)；乌拉坦(urethan)；长春碱酰胺；达卡巴嗪(dacarbazine)；甘露醇氮芥；二溴甘露醇(mitobronitol)；二溴卫矛醇；哌溴烷坑(pipobroman)；gacytosine；阿拉伯糖苷("Ara-C")；环磷酰胺；三胺硫磷(thiotepa)；紫杉烷，如紫杉醇(
Figure PCTCN2020097984-appb-000007
Bristol-Myers Squibb Oncology,Princeton,NJ)和多西他赛(docetaxel)(
Figure PCTCN2020097984-appb-000008
Rhone-Poulenc Rorer,Antony,France)；苯丁酸氮芥；吉西他滨(gemcitabine)；6-硫代鸟嘌呤；巯基嘌呤；氨甲蝶呤；铂类似物如顺铂和卡铂；长春花碱；铂；依托泊甙(etoposide)(VP-16)；异环磷航胶；丝裂霉素C；米托蒽醌；长春新碱；长春瑞宾(vinorelbine)；新霉酰胺(navelbine)；novantrone；替尼泊甙(teniposide)；柔红霉素；氨基蝶呤；xeloda；伊拜磷酸盐(ibandronate)；CPT-11；拓扑异构酶抑制剂RFS2000；二氟甲基鸟氨酸(DMFO)；维甲酸，埃司帕米星(esperamicins)；卡培他滨(capecitabine)；以及上述任何物质的可药用盐，酸或衍生物。此定义还包括能调节或抑制激素对肿瘤的作用的抗激素制剂，如抗雌激素制剂包括他莫昔芬(tamoxifen)，雷洛昔芬(raloxifene)，芳香酶抑制剂4(5)-咪唑，4-羟基他莫昔芬，曲沃昔芬(trioxifene)，酮咯芬(keoxifene)，LY117018，奥那司酮(onapristone)，和托瑞米芬(Fareston)；和抗雄激素制剂，如氟他氨(flutamide)，尼鲁米特(nilutamide)，比卡鲁胺(bicalutamide)，亮丙瑞林(leuprolide)和戈舍瑞林(goserelin)；和上述任何物质的可药用盐，酸或衍生物。
本公开所述的“抗体”是一种免疫球蛋白，通常完整抗体是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有 κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开的抗体包括鼠源抗体、嵌合抗体和人源化抗体。本公开抗体除包括全长抗体外，还包括能结合抗原的抗原结合片段。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的针对人CEA的单克隆抗体。制备时用CEA抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个优选的实施方案中，所述的鼠源抗CEA抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的PD-L1嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的CEA嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变，和/或S228P突变)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由酵母菌展示对CDR进行亲和力成熟突变后的人源化抗体。
由于抗原的接触残基，CDR的移植可由于与抗原接触的构架残基而导致产生 的抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以可能是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查动物单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚类共有序列或具有高相似性百分数的动物抗体序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
在本公开一个的实施方案中，所述的抗体或其抗原结合片段，可进一步包含人源或鼠源κ、λ链或其变体的轻链恒定区，或进一步包含人源或鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区；优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变、和/或S228P突变)的IgG1、IgG2或IgG4变体。
本公开中所述人抗体的重链恒定区和人抗体的轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，S228P突变，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
“人抗体”(HuMAb)、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，可以是源于人的抗体或者是从一种转基因生物体中获得的抗体，该转基因生物体经“改造”以响应于抗原刺激而产生特异性人抗体并且可以通过本领域已知的任何方法产生。在某些技术中，将人重链和轻链基因座的元件引入到源于胚胎干细胞系的生物体的细胞株中，这些细胞系中的内源性重链和轻链基因座被靶向破坏这些细胞系中包含靶向的内源性重链和轻链基因座破坏。转基因生物可以合成对人抗原特异的人抗体，并且该生物可以用于产生人抗体-分泌杂交瘤。人抗体还可以是一种抗体，其中重链和轻链是由源于一个或更多个人DNA来源的核苷酸序列编码的。完全人抗体还可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指轻链和重链包含恒定区的抗体。本公开“抗体”包含“全长抗体”及其抗原结合片段。
在一些实施方案中，本公开的全长抗体包括轻链可变区与轻链恒定区连接的轻链和重链可变区与重链恒定区连接的重链所形成的全长抗体。本领域技术人员可以根据实际需要选择不同的抗体来源的轻链恒定区、重链恒定区，例如人抗体来源的轻链恒定区和重链恒定区。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，CEA)的能力的一个或更多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)dsFv，由VH和VL经链间二硫键形成的稳定的抗原结合片段；(vi)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗原结合片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50，000的分子量并具有抗原结合活性的抗原结合片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100，000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗原结合片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50，000并具有抗原结合活性的抗原结合片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的特异性识别并结合CEA的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等 人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
双抗体是其中scFv或Fab被二聚体化的抗原结合片段，是具有二价抗原结合活性的抗原结合片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
双特异性抗体和多特异性抗体是指能同时结合两个或多个抗原或抗原决定簇的抗体，其中包含能结合CEA的scFv或Fab片段。
本公开的双抗体可以通过以下步骤来生产：获得本公开的特异性识别并结合人CEA的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering，7，697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的全长抗体或抗原结合片段可以通过以下步骤来生产：获得本公开的特异性识别并结合人CEA的抗体的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个、2个、3个或更多个氨基酸的插入、缺失或替换。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、 50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。除非另有说明，本公开具体实施例中所述抗体可变区和CDR序列均适用“Kabat”编号规则。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，CEA分子上的特定部位)。表位包括线性表位和构象表位，例如构象表位通常具有独特的空间构象并包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -8M、10 -9M、10 -10M、10 -11M、10 -12M或更小的亲和力(KD)结合。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合CEA，例如，在本公开中抗体与细胞表面抗原的亲和力采用Biacore法测定KD值。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如CEA抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)； 和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用结合荷有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人CEA或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或更多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站 http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、293细胞和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人CEA特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知 的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下方陈述。
Figure PCTCN2020097984-appb-000009
“有效量”或“有效剂量”指获得任一种或多种有益的或所需的预防或治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本公开靶抗原相关病症的进 展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。
“内源性”指根据情况在生物、细胞或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4，683，195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“分离的”指纯化状态，并且在这种情况下意味着在指定的分子基本上不含其他 生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
此外，本公开包括用于治疗与目标抗原(例如CEA)相关的疾病的药剂，所述药剂包含本公开的抗CEA抗体作为活性成分。
本公开中与CEA相关的疾病没有限制，只要它是与CEA相关的疾病即可，例如利用本公开的分子诱导的治疗反应通过以下方式实现：结合人CEA，然后阻遏CEA与其受体/配体的结合，或杀伤过表达CEA的肿瘤细胞。因此，当本公开的分子处于适于治疗应用的制备物中时，其对这样一些受试者是非常有用的，他们患有肿瘤或癌症，优选黑色素瘤、结肠癌、乳腺癌、肺癌、胃癌、肠癌、肾癌、非小细胞肺癌、膀胱癌等。
此外，本公开涉及用于免疫检测或测定目标抗原(例如CEA)的方法、用于免疫检测或测定目标抗原(例如CEA)的试剂、用于免疫检测或测定表达目标抗原(例如CEA)的细胞的方法和用于诊断与目标抗原(例如CEA)相关的疾病的诊断剂，其包含本公开的特异性识别并结合目标抗原(例如人CEA)的抗体或抗原结合片段作为活性成分。
在本公开中，用于检测或测定目标抗原(例如CEA)的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与CEA相关的疾病可以通过用本公开的抗体检测或测定CEA的水平来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定目标抗原(例如CEA)的样品没有特别限制，只要它具有包含目标抗原(例如CEA)的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的抗体或其抗原结合片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗原结合片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
在以上说明书中提出了本公开一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本公开，但是以下描述优选的方法和材料。通过说明书和权利要求书，本公开的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本公开所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本公开的优选实施方式。这些实施例不应以任何方式理解为限制本公开的范围。
以下结合实施例和测试例进一步描述本公开，但这些实施例和测试例并非限制着本公开的范围。本公开实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件；未注明具体来源的试剂材料，为市场购买获得。
实施例1：CEA重组蛋白和稳定转染的细胞的制备
一、重组CEA抗原及细胞表面表达CEA蛋白的序列
编码带Fc、His标签的人CEA蛋白序列分别克隆到哺乳动物细胞表达载体中，在293E细胞中表达纯化后获得重组蛋白用于后续各实施例的实验中。同时将不带标签的人CEA基因、人CEACAM1基因和猴CEA基因转染到CHO细胞中，形成在细胞表面表达CEA蛋白的CHO细胞株，用于后续抗体的筛选和鉴定。相关蛋白氨基酸序列如下：
1、人CEA-his(hCEA-His)氨基酸序列(测试例中所用的可溶性人CEA抗原蛋白)：
Figure PCTCN2020097984-appb-000010
Figure PCTCN2020097984-appb-000011
2、人CEA-Fc(hCEA-Fc)氨基酸序列：
Figure PCTCN2020097984-appb-000012
3、猴CEA-His(cynoCEA-His)氨基酸序列：
Figure PCTCN2020097984-appb-000013
Figure PCTCN2020097984-appb-000014
4、CHO细胞表面表达人CEA(hCEA-CHO)氨基酸序列：
Figure PCTCN2020097984-appb-000015
5、CHO细胞表面表达猴CEA(cynoCEA-CHO)氨基酸序列：
Figure PCTCN2020097984-appb-000016
6、CHO细胞表面表达人CEACAM1(CEACAM1-CHO)氨基酸序列：
Figure PCTCN2020097984-appb-000017
Figure PCTCN2020097984-appb-000018
二、相关蛋白的纯化
1、带His标签蛋白的纯化
将细胞表达上清样品高速离心去除杂质。用PBS缓冲液(pH 7.4)平衡镍柱，冲洗2-5倍柱体积，将上清样品以一定流速上Ni Sepharose excel柱。用PBS缓冲液冲洗柱子，至A 280读数降至基线，再用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液，最后用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。收集的洗脱液浓缩后用脱盐柱将样品缓冲液换成PBS溶液，以备后续实验使用。
2、含Fc的蛋白、嵌合抗体及杂交瘤抗体的纯化
将细胞表达上清样品高速离心去除杂质，含Fc的重组蛋白、嵌合抗体表达上清用Protein A柱进行纯化，杂交瘤表达上清用Protein G柱进行纯化。上清液以一定流速上柱。用PBS冲洗柱子，至A280读数降至基线。用pH 3.0的100mM乙酸洗脱目的蛋白，用pH 8.0的1M Tris-HCl中和。洗脱样品浓缩换成PBS后分装备用。
实施例2：小鼠抗人CEA单克隆抗体的制备
1、免疫和融合
小鼠的免疫使用hCEA-His蛋白和cyno-CEA-His蛋白，或hCEA-CHO细胞和cynoCEA-CHO细胞进行交叉免疫。蛋白免疫的用量为第一次免疫50μg，之后的免疫用25μg，细胞免疫为每次10 7个细胞，每两周免疫一次。免疫3次后取血测定血清中抗体的效价，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，采用PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2020097984-appb-000019
CRL-8287 TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞以0.5-1×10 6个/ml的密度用MC半固体完全培养基(含20％FBS、1×HAT、1×OPI和2％甲基纤维素的RPMI-1640培养基)重悬，分装于35mm细胞培养皿中，37℃，5％CO 2孵育7-9天。融合后第7-9天，根据细胞克隆大小，挑取单细胞克隆至加有200μl/孔的HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)的96孔细胞培养板中，37℃，5％CO 2培养3天进行检测。
2、杂交瘤细胞筛选
抗体的初步筛选用基于细胞表面抗原的酶联免疫吸附分析法(ELISA)进行。将细胞铺在Elisa板(Corning，Cat#3599)中，37℃培养箱中培养过夜，待细胞完全贴壁，快要长满整个孔时，去上清液，用PBS清洗一次，加入细胞固定液 (Beyotime，Cat#P0098)，室温放置45min，去固定液，洗板机洗板3次，加入5％脱脂奶粉，37℃封闭3h以上。去封闭液，洗板机洗板3次。封闭好的细胞板可以置于-20℃保存或直接使用。使用时加入梯度稀释的杂交瘤细胞培养上清液，37℃孵育1h，洗板机洗3遍，加入100μl 10000倍稀释的羊抗鼠IgG H&L(HRP)二抗(Abcam，Cat#ab205719)，37℃孵育1h，洗板机洗3遍，加入100μl TMB(KPL，Cat#5120-0077)置于37℃显色10min，加入100μl 1M硫酸终止反应，用酶标仪读取450nm的吸光值。测试抗体与细胞表面的CEA结合而不会被可溶性CEA(sCEA)竞争掉时，将抗体与sCEA孵育30min后再加入细胞板中。
将筛选出的阳性克隆进行扩增冻存保种和二到三次亚克隆直至获得单细胞克隆。选择出的杂交瘤克隆用无血清细胞培养法进一步制备和纯化抗体。得到的杂交瘤抗体用流式细胞仪检测抗体与细胞表面CEA蛋白的结合情况(方法见本公开测试例1)，挑选出结合活性好的杂交瘤细胞株。其中，单克隆杂交瘤细胞株mAb47，mAb63、mAb67和mAb103的结合活性检测结果见表1。
表1：鼠源抗体对细胞表面CEA蛋白的结合实验结果
Figure PCTCN2020097984-appb-000020
3、杂交瘤抗体序列测定
选择单克隆杂交瘤细胞株mAb47、mAb63、mAb67和mAb103，克隆单克隆抗体的序列。过程如下：收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat#15596-018)提取RNA，反转录为cDNA。用cDNA为模板进行PCR扩增后送测序公司测序，得到的DNA序列对应的抗体氨基酸序列如下表2所示。
表2：鼠源抗CEA抗体的可变区序列
Figure PCTCN2020097984-appb-000021
Figure PCTCN2020097984-appb-000022
表3：抗CEA抗体的CDR序列
Figure PCTCN2020097984-appb-000023
备注：表中的抗体CDR序列是根据Kabat编号系统确定。
4、人IgG1嵌合抗体制备
将上述杂交瘤筛选所获得的mAb47、mAb63、mAb67和mAb103候选分子经过扩增测序可得到可变区编码基因序列，以测序所得序列设计首尾引物，以测序基因为模板，经过PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG1/hkappa/hlambda恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建重组嵌合抗体全长表达质粒VH-CH1-Fc-pHr/VL-CL-pHr，进而获得其嵌合抗体Ch47、Ch63、Ch67和Ch103。
实施例3：鼠源抗人CEA单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别graft(嫁接)到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。下面范例中抗体的CDR氨基酸残基由Kabat编号系统确定并注释。
1、鼠源抗体mAb47的人源化
挑选同源性高的重轻链可变区种系基因作为模板，例如，鼠源抗体mAb47的人源化轻链模板选IGKV6-21*01和IGKJ2*01，人源化重链模板选IGHV7-4-1*02和IGHJ6*01，将鼠源抗体mAb47的CDR分别移植到相应的人源模板中进行人源化改造，鼠源抗体mAb47的人源化回复突变设计见下表4：
表4：鼠源抗体mAb47的人源化设计
Figure PCTCN2020097984-appb-000024
备注：表中氨基酸位置编号均为Kabat编号规则的编号，例如，L46P，是指对应于Kabat编号系统编号，将第46位L突变回P；嫁接代表鼠源抗体CDR植入人种系FR区序列。
另外，进一步地，在重链h47VH3中引入D61S突变(即在抗体HCDR2上进行氨基酸突变，使抗体HCDR2序列由
Figure PCTCN2020097984-appb-000025
Figure PCTCN2020097984-appb-000026
突变为：
Figure PCTCN2020097984-appb-000027
)，抗体仍具有良好活性。
鼠源抗体mAb47人源化后的具体序列见表5所示：
表5：鼠源抗体mAb47人源化后的可变区序列
Figure PCTCN2020097984-appb-000028
备注：表中下划线为CDR区(根据Kabat编号系统确定)，粗斜体为突变位点。
2、鼠源抗体mAb63的人源化
挑选同源性高的重轻链可变区种系基因作为模板，例如，鼠源抗体mAb63的人源化轻链模板选IGKV1-39*01和IGKJ4*01，人源化重链模板选IGHV1-46*01和IGHJ1*01，将鼠源抗体mAb63的CDR分别移植到相应的人源模板中进行人源化改造，鼠源抗体mAb63的人源化回复突变设计见下表6：
表6：鼠源抗体mAb63的人源化设计
Figure PCTCN2020097984-appb-000029
Figure PCTCN2020097984-appb-000030
备注：表中氨基酸位置编号均为对应于Kabat编号规则的编号，例如，S  82A R，是指对应于Kabat编号系统编号，将第 82A位S突变回R；嫁接代表鼠源抗体CDR植入人种系FR区序列。
另外，进一步地，在重链h63VH1中引入N54S突变(即在抗体HCDR2上进行氨基酸突变，使抗体HCDR2序列由 DIFPKNGNTDYNRKFKD(SEQ ID NO：22)突变为：
Figure PCTCN2020097984-appb-000031
)，抗体仍具有良好活性。
鼠源抗体mAb63人源化及突变后的具体序列见表7所示：
表7：鼠源抗体mAb63人源化后的可变区序列
Figure PCTCN2020097984-appb-000032
Figure PCTCN2020097984-appb-000033
备注：表中下划线为CDR区(对应于Kabat编号系统)，粗斜体为突变位点。
3、鼠源抗体mAb67的人源化
挑选同源性高的重轻链可变区种系基因作为模板，例如，鼠源抗体mAb67的人源化轻链模板选IGKV4-1*01和IGKJ4*01、IGKV3-15*01和IGKJ4*01、或IGKV1-39*01和IGKJ4*01，人源化重链模板选IGHV1-3*01和IGHJ1*01、或IGHV5-51*01和IGHJ1*01，将鼠源抗体mAb67的CDR分别移植到相应的人源模板中进行人源化改造，鼠源抗体mAb67的人源化回复突变设计见下表8：
表8：鼠源抗体mAb67的人源化设计
Figure PCTCN2020097984-appb-000034
备注：表中氨基酸位置编号均为对应于Kabat编号规则的编号，例如，A43P是指对应于Kabat编号系统编号，将第43位A突变回P；嫁接代表鼠源抗体CDR植入人种系FR区序列。
鼠源抗体mAb67人源化后的具体序列见表9所示：
表9：鼠源抗体mAb67人源化后的可变区序列
Figure PCTCN2020097984-appb-000035
Figure PCTCN2020097984-appb-000036
备注：表中下划线为CDR区(对应于Kabat编号系统)，粗斜体为突变位点。
4、鼠源抗体mAb103的人源化
挑选同源性高的重轻链可变区种系基因作为模板，例如，鼠源抗体mAb103的人源化轻链模板选IGLV4-69*01和IGLJ2*01，人源化重链模板选IGHV7-4-1*02和IGHJ1*01，将鼠源抗体mAb103的CDR分别移植到相应的人源模板中进行人源化改造，鼠源抗体mAb103的人源化回复突变设计见下表10：
表10：鼠源抗体mAb103的人源化设计
Figure PCTCN2020097984-appb-000037
Figure PCTCN2020097984-appb-000038
备注：表中氨基酸位置编号均为对应于Kabat编号规则的编号，例如，K49E是指对应于Kabat编号系统编号，将第49位K突变回E；Grafted代表鼠源抗体CDR植入人种系FR区序列。
另外，进一步地，在重链h103VH1中引入D61S突变(即在抗体HCDR2上进行氨基酸突变，使抗体HCDR2序列由
Figure PCTCN2020097984-appb-000039
Figure PCTCN2020097984-appb-000040
突变为：
Figure PCTCN2020097984-appb-000041
)；在轻链h103VL3中引入D56E突变(即在抗体LCDR2上进行氨基酸突变，使抗体LCDR2序列由LKKDGSHSTGD(SEQ ID NO：36)突变为：LKKDGSHSTGE(SEQ ID NO：64)，抗体仍具有良好活性。
鼠源抗体mAb103人源化后的具体序列见表11所示：
表11.鼠源抗体mAb103人源化后的可变区序列
Figure PCTCN2020097984-appb-000042
Figure PCTCN2020097984-appb-000043
备注：表中下划线为CDR区(对应于Kabat编号系统)，粗斜体为突变位点5、人源化抗体的制备
分别构建抗体轻链和重链的表达载体，将人源化的抗体轻/重链分别交叉配对组合，转染293E细胞后收集培养上清纯化即得到人源化的全长抗体。人源化抗体重链恒定区可选自IgG1、IgG2、IgG3、IgG4及其变体的恒定区；示例性的，使用人重链IgG1恒定区(如SEQ ID NO：77所示)与前述人源化重链可变区连接，形成抗体全长重链。人源化抗体轻链恒定区可选自人源κ、λ链或其变体的恒定区；示例性的，使用人轻链恒定区κ链(如SEQ ID NO：78所示)或人轻链恒定区λ链(如SEQ ID NO：79所示)与前述人源化轻链可变区连接，形成抗体全长轻链。
示例性的抗体的恒定区序列如下：
人IgG1重链恒定区：
Figure PCTCN2020097984-appb-000044
Figure PCTCN2020097984-appb-000045
人轻链恒定区κ链：
Figure PCTCN2020097984-appb-000046
人轻链恒定区λ链：
Figure PCTCN2020097984-appb-000047
示例性的，将前述来源自mAb47的如表5中所述的人源化抗体重链可变区与序列如SEQ ID NO：77所示的人重链IgG1恒定区氨基端连接形成抗体全长重链，同时将表5中所述的人源化抗体轻链可变区与序列如SEQ ID NO：78所示的人轻链κ恒定区氨基端连接形成抗体全长轻链，得到如下表12所示的系列mAb47人源化抗体：
表12.mAb47人源化抗体
Figure PCTCN2020097984-appb-000048
备注：表中例如“Hu47-14”表示编号为Hu47-14的人源化抗体的轻链可变区为h47VL2，重链可变区为h47VH4，重链恒定区序列如SEQ ID NO：77所示，轻链恒定区序列如SEQ ID NO：78所示。
示例性的，将前述来源自mAb63的如表7中所述的人源化抗体重链可变区与序列如SEQ ID NO：77所示的人重链IgG1恒定区氨基端连接形成抗体全长重链，同时将表7中所述的人源化抗体轻链可变区与序列如SEQ ID NO：78所示的人轻链κ恒定区氨基端连接形成抗体全长轻链，得到如下表13所示系列mAb63人源化抗体：
表13.mAb63人源化抗体
Figure PCTCN2020097984-appb-000049
Figure PCTCN2020097984-appb-000050
备注：表中例如“Hu63-13”表示编号为Hu63-13的人源化抗体的轻链可变区为h63VL1，重链可变区为h63VH5，其重链恒定区序列如SEQ ID NO：77所示，轻链恒定区序列如SEQ ID NO：78所示。
示例性的，将前述来源自mAb67的如表9中所述的人源化抗体重链可变区与序列如SEQ ID NO：77所示的人重链IgG1恒定区氨基端连接形成抗体全长重链，将表9中所述的人源化抗体轻链可变区与序列如SEQ ID NO：78所示的人轻链κ恒定区氨基端连接形成抗体全长轻链，得到如下表14所示的系列mAb67人源化抗体：
表14.mAb67人源化抗体
Figure PCTCN2020097984-appb-000051
备注：表中例如“Hu67-14”表示编号为Hu67-14的人源化抗体的轻链可变区为h67VL4，重链可变区为h67VH3，其重链恒定区序列如SEQ ID NO：77所示，轻链恒定区序列如SEQ ID NO：78所示。
示例性的，将前述来源自mAb103的如表11中所述的人源化抗体重链可变区与序列如SEQ ID NO：77所示的人重链IgG1恒定区氨基端连接形成抗体全长重链，将表11中所述的人源化抗体轻链可变区与序列如SEQ ID NO：79所示的人轻链λ恒定区氨基端连接形成抗体全长轻链，得到如下表15所示的系列mAb103人源化抗体：
表15.mAb103人源化抗体
Figure PCTCN2020097984-appb-000052
备注：表中例如“Hu103-32”表示编号为Hu103-32的人源化抗体的轻链可变区为h103VL8，重链可变区为h103VH4，其重链恒定区序列如SEQ ID NO：77所示， 轻链恒定区序列如SEQ ID NO：79所示。
示例性的，人源化抗体轻/重链全长序列如下表16所示：
表16.人源化抗体的轻/重链序列
Figure PCTCN2020097984-appb-000053
Figure PCTCN2020097984-appb-000054
备注：表中斜体字为恒定区序列，正体字为可变区序列。
目前已知的2个靶向CEA的ADC分子：SAR-408701和拉贝珠单抗-govitecan(也称Lmab-CL2A-SN38)偶联物，其中的抗体轻/重链序列如下：
SAR-408701中的抗体(简称Sanofi)的重链序列：
Figure PCTCN2020097984-appb-000055
SAR-408701中的抗体(简称Sanofi)的轻链序列
Figure PCTCN2020097984-appb-000056
Lmab-CL2A-SN38中的拉贝珠单抗(labetuzumab，简称Lmab)抗体重链序列
Figure PCTCN2020097984-appb-000057
Figure PCTCN2020097984-appb-000058
Lmab-CL2A-SN38中的拉贝珠单抗(labetuzumab，简称Lmab)轻链序列
Figure PCTCN2020097984-appb-000059
测试例
测试例1：FACS结合实验
为了检测抗体与细胞表面CEA蛋白的结合情况，用在细胞表面表达CEA的细胞通过FACS检测抗体的结合活性。收集细胞，400g，4℃离心5min；加入含有终浓度10％FBS的预冷的PBS，400g，4℃离心5min，重复两次；将细胞分配至96孔板，10 5个细胞/孔；每孔加入100μl梯度稀释的抗体溶液，4℃孵育60min，离心去上清；每孔加入含有终浓度10％FBS的预冷的PBS 250μl重悬细胞，400g，4℃离心5min，去上清液，重复两次；加入50μl 1:200稀释的二抗Alexa Fluor@488羊抗人IgG(H+L)(Lifetechologies，Cat#A11013)，4℃避光孵育45min，离心去上清液；每孔加入含有终浓度10％FBS的预冷的PBS 250μl重悬，400g，4℃离心5min，重复两次；每孔加入100μl预冷的PBS重悬细胞；上流式细胞仪检测(BD，FACSverse)；得到荧光信号值，信号越高表示抗体与细胞表面的蛋白结合活性越强。将检测结果用PRISM分析软件做结合曲线图，拟合得到抗体与细胞表面蛋白人CEA(MKN45人胃癌细胞，南京科佰生物科技有限公司，Cat#CBP60488)、cynoCEA-CHO、CEACAM1-CHO结合活性的EC50值。人源化抗体的结合活性如下表17、18所示：
表17.人源化抗体对不同种属的细胞表面CEA蛋白的结合活性
Figure PCTCN2020097984-appb-000060
Figure PCTCN2020097984-appb-000061
其它人源化抗体对细胞表面CEA蛋白(MKN45、cynoCEA-CHO)的结合活性检测结果见下表18所示：
表18.人源化抗体对细胞表面CEA蛋白的结合活性
Figure PCTCN2020097984-appb-000062
实验结果表明，本公开中筛选到的人源化抗体保持了与鼠源抗体相似的结合活性，均能与细胞表面人CEA蛋白结合；本公开中筛选到的人源化抗体能与细胞表面猴CEA蛋白结合，且与猴CEA蛋白结合活性优于阳性对照抗体。
测试例2：与可溶性CEA(sCEA)竞争实验
为了检测抗体在sCEA(如SEQ ID NO：1所示)存在的情况下是否也优先与细胞膜表面的CEA结合，发明人将梯度稀释的抗体与一定浓度的sCEA(5μg/ml)预先孵育30min，然后收集MKN45细胞(人胃癌细胞，南京科佰生物科技有限公司，Cat#CBP60488)将细胞分配至96孔板；每孔分别加入梯度稀释的抗体和抗体与sCEA预孵育的混合溶液，4℃孵育60min，离心去上清；加入含有终浓度10％FBS的预冷的PBS清洗细胞，重复两次；加入50μl 1:200稀释的二抗Alexa  Fluor@488羊抗人IgG(H+L)(Lifetechologies，Cat#A11013)，4℃避光孵育45min，离心去上清液；加入终浓度10％FBS的预冷的PBS 250μl清洗细胞；重复两次；每孔加入100μl预冷的PBS重悬细胞；上流式细胞仪(FACS)检测(BD，FACSverse)，得到荧光信号值。如果不加sCEA与加sCEA的信号在各个抗体浓度下的比值都小于2，说明抗体在sCEA的存在下结合曲线也没有出现很大的改变，抗体依然优先与细胞膜表面的CEA结合。
实验结果见表19和表20，其中在不加sCEA与加sCEA的情况下，Hu63-13、Hu47-14、Hu67-14、Hu103-32和阳性对照Lmab在抗体不同浓度时的荧光信号比值见下表19所示，不加sCEA与加sCEA的情况下，其它实验抗体的荧光信号最大比值见下表20所示：
表19.人源化抗体不加sCEA与加sCEA的荧光信号比值
Figure PCTCN2020097984-appb-000063
表20.抗体不加sCEA与加sCEA的荧光信号最大比值
Figure PCTCN2020097984-appb-000064
Figure PCTCN2020097984-appb-000065
实验结果表明，不加sCEA与加sCEA的情况下，本公开中筛选到的人源化抗体Hu63-13、Hu47-14、Hu67-14、Hu103-32在各种抗体浓度下的荧光信号比值都小于2，例如Hu63-13最大比值为1.59，而阳性对照Lmab的最大比值达5.18，本公开中筛选到的抗体优于对照抗体。本公开中筛选到的人源化抗体不加sCEA与加sCEA的情况下荧光信号最大比值均小于2，且小于阳性对照抗体，表明本公开中筛选到的人源化抗体在sCEA的存在下依然优先与细胞膜表面的CEA结合，且优于阳性对照抗体。
测试例3：Biacore测定抗体与可溶性CEA的亲和力
用Biacore(GE，T200)仪器测定待测人源化抗体和人、猴可溶性CEA的亲和力。按照人抗捕获试剂盒(GE，Cat#BR-1008-39)说明书中的方法将人抗捕获抗体共价偶联于Biacore仪器的生物传感芯片CM5(GE，Cat#BR-1005-30)上，从而亲和捕获一定量的待测抗体，然后于芯片表面流经一系列浓度梯度的可溶性CEA抗原(如SEQ ID NO：1所示)，利用Biacore实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液将生物芯片洗净再生。实验得到的数据用BIAevaluation version 4.1软件以(1:1)Langmuir模型进行拟合，得出亲和力数值。由于本公开希望筛选到的抗体与细胞膜表面的CEA结合活性强于可溶性CEA，因此抗体与可溶性CEA的亲和力越低越好。人源化抗体与可溶性CEA的亲和力测试结果如下表21所示：
表21.人源化抗体与人可溶性CEA的亲和力
Figure PCTCN2020097984-appb-000066
测试的结果表明，人源化抗体Hu63-13、Hu47-14和Hu67-14与可溶性CEA蛋白的亲和力都比较低，明显低于对照抗体Sanofi和Lmab。这预示了Hu63-13、Hu47-14和Hu67-14在体内不容易被血液中的可溶性CEA中和掉，可以有大量抗体与细胞膜表面表达CEA的细胞结合。
测试例4：抗CEA抗体在CEA高表达细胞MKN45中的内吞活性
本公开中的抗CEA抗体偶联物被细胞内吞后能将毒素释放对细胞起到杀伤作 用，因此CEA抗体在CEA表达的细胞中的内吞活性能促进ADC发挥活性。为了评估人源化抗体在MKN45细胞(人胃癌细胞，南京科佰生物科技有限公司，Cat#CBP60488)中的内吞活性，将MKN45铺在96孔板(Coring，Cat#3795)中过夜培养，次日分别将人源化的CEA抗体Hu63-13、Hu47-14、Hu67-14和Hu103-3与iFL Green Human IgG Labeling Reagent(Invitrogen，Cat#Z25611)预孵育15min，iFL试剂会与人源化抗体的Fc结合，之后将抗体和iFL复合物加入细胞培养板中，分别在6小时和24小时后将细胞培养液去除，加入PBS清洗两遍，消化收集细胞，用FACS检测细胞中的荧光信号强度，抗体被细胞内吞后会将结合在抗体Fc上的iFL带入细胞，iFL被吞入细胞内部在酸性环境下才能检测到荧光信号，因此检测到的信号越强代表抗体的内吞活性越高。各人源化抗体的内吞活性如图1所示：所有人源化抗体都能被MKN45内吞，随着时间的延长，内吞的抗体越多。
测试例5：体内药代动力学测试
用SD大鼠进行体内药代动力学测试。SD大鼠(西普尔-必凯(Sippr-BK)实验动物有限公司)随机分组，每组3只，静脉注射给药，给药剂量为3mg/kg，给药组于给药前及给药后5min，8h，1d，2d，4d，7d，10d，14d，21d，28d采集全血0.3ml，不加抗凝，取血后在4℃放置30min，1000g离心15min，取上层血清置于EP管中，于-80℃保存。用ELISA方法检测血清中的血药浓度，用Winnolin软件计算受试药物的药代动力学参数，检测结果如下表22所示：
表22.分子的体内药代动力学参数
抗体样品 Hu63-13 Hu47-14 Hu67-14 Hu103-32
参数 平均值 平均值 平均值 平均值
t1/2(h) 192.7 188.8 180.6 157.87
t1/2(d) 8.0 7.9 7.5 6.58
Cmax(μg/ml) 67.6 55.2 49.6 66.35
AUC 0-t(μg/ml*h) 4949 3444 4038 3235
AUC 0-∞(μg/ml*h) 5451 3741 4356 3357
Vz(ml/kg) 153.0 218.8 179.9 206.29
CL(ml/天/kg) 13.2 19.3 16.6 21.75
MRT 0-∞(h) 239.0 216.6 237.7 160.14
本公开的抗体均具有良好的药代动力学活性。

Claims (15)

  1. 一种抗CEA抗体，其包含抗体重链可变区和轻链可变区，其中：
    i)所述重链可变区，其HCDR1和HCDR3分别与如SEQ ID NO：7所示的重链可变区包含的HCDR1和HCDR3相同，且其HCDR2与如SEQ ID NO：7所示的重链可变区包含的HCDR2相同或与其具有一个氨基酸差异；和
    所述轻链可变区，其LCDR1、LCDR2和LCDR3分别与如SEQ ID NO：8所示的轻链可变区包含的LCDR1、LCDR2和LCDR3相同；或
    ii)所述重链可变区，其HCDR1和HCDR3分别与如SEQ ID NO：9所示的重链可变区包含的HCDR1和HCDR3相同，且其HCDR2与如SEQ ID NO：9所示的重链可变区包含的HCDR2相同或与其具有一个氨基酸差异；和
    所述轻链可变区，其LCDR1、LCDR2和LCDR3分别与如SEQ ID NO：10所示的轻链可变区包含的LCDR1、LCDR2和LCDR3相同；或
    iii)所述重链可变区，其HCDR1、HCDR2和HCDR3分别与如SEQ ID NO：11所示的重链可变区包含的HCDR1、HCDR2和HCDR3相同；和
    所述轻链可变区，其LCDR1、LCDR2和LCDR3分别与如SEQ ID NO：12所示的轻链可变区包含的LCDR1、LCDR2和LCDR3相同；或
    iv)所述重链可变区，其HCDR1和HCDR3分别与如SEQ ID NO：13所示的重链可变区包含的HCDR1和HCDR3相同，且其HCDR2与如SEQ ID NO：13所示的重链可变区包含的HCDR2相同或与其具有一个氨基酸差异；和
    所述轻链可变区，其LCDR1和LCDR3分别与如SEQ ID NO：14所示的轻链可变区包含的LCDR1和LCDR3相同，且其LCDR2与如SEQ ID NO：14所示的轻链可变区包含的LCDR2相同或与其具有一个氨基酸差异。
  2. 根据权利要求1所述的抗CEA抗体，其包含重链可变区和轻链可变区，其中：
    v)所述重链可变区包含分别如SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3；或
    所述重链可变区包含分别如SEQ ID NO：15、SEQ ID NO：38和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3；或
    vi)所述重链可变区包含分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；或
    所述重链可变区包含分别如SEQ ID NO：21、SEQ ID NO：47和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；或
    vii)所述重链可变区包含分别如SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含分别如SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32所示的LCDR1、LCDR2和LCDR3；或
    viii)所述重链可变区包含分别如SEQ ID NO：33和SEQ ID NO：34所示的HCDR1和HCDR3，和如SEQ ID NO：16或SEQ ID NO：38所示的HCDR2；
    所述轻链可变区包含分别如SEQ ID NO：35和SEQ ID NO：37所示的LCDR1和LCDR3，和如SEQ ID NO：36或SEQ ID NO：64所示的LCDR2；
    优选地，所述重链可变区包含分别如SEQ ID NO：33、SEQ ID NO：38和SEQ ID NO：34所示的HCDR1、HCDR2和HCDR3；和所述轻链可变区包含分别如SEQ ID NO：35、SEQ ID NO：64和SEQ ID NO：37所示的LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1或2所述的抗CEA抗体，其中所述抗CEA抗体是鼠源抗体、嵌合抗体或人源化抗体。
  4. 根据权利要求3所述的抗CEA抗体，其中所述抗CEA抗体是鼠源抗体或嵌合抗体，其包含重链可变区和轻链可变区，其中：
    (a)所述重链可变区，其氨基酸序列如SEQ ID NO:7所示，或与SEQ ID NO:7具有至少90％序列同一性；和/或
    所述轻链可变区，其氨基酸序列如SEQ ID NO:8所示，或与SEQ ID NO:8具有至少90％序列同一性；或
    (b)所述重链可变区，其氨基酸序列如SEQ ID NO:9所示，或与SEQ ID NO:9具有至少90％序列同一性；和/或
    所述轻链可变区，其氨基酸序列如SEQ ID NO:10所示，或与SEQ ID NO:10具有至少90％序列同一性；或
    (c)所述重链可变区，其氨基酸序列如SEQ ID NO:11所示，或与SEQ ID NO:11具有至少90％序列同一性；和/或
    所述轻链可变区，其氨基酸序列如SEQ ID NO:12所示，或与SEQ ID NO:12具有至少90％序列同一性；或
    (d)所述重链可变区，其氨基酸序列如SEQ ID NO:13所示，或与SEQ ID NO:13具有至少90％序列同一性；和/或
    所述轻链可变区，其氨基酸序列如SEQ ID NO:14所示，或与SEQ ID NO:14具有至少90％序列同一性。
  5. 根据权利要求1至3中任一项所述的抗CEA抗体，其中所述抗CEA抗体是人源化抗体,其包含重链可变区和轻链可变区，其中：
    (e)所述重链可变区如SEQ ID NO:39、40、41或42所示，或分别与SEQ ID NO:39、40、41或42具有至少90％序列同一性；和/或
    所述轻链可变区如SEQ ID NO:43、44、45或46所示，或分别与SEQ ID NO:43、44、45或46具有至少90％序列同一性；或
    (f)所述重链可变区如SEQ ID NO:48、49、50、51或52所示，或分别与SEQ ID NO:48、49、50、51或52具有至少90％序列同一性；和/或
    所述轻链可变区如SEQ ID NO:53、54或55所示，或分别与SEQ ID NO:53、54或55具有至少90％同一性；或
    (g)所述重链可变区如SEQ ID NO:56、57或58所示，或分别与SEQ ID NO:56、57或58具有至少90％序列同一性；和/或
    所述轻链可变区如SEQ ID NO:59、60、61、62或63所示，或分别与SEQ ID NO:59、60、61、62或63具有至少90％序列同一性；或
    (h)所述重链可变区如SEQ ID NO:65、66、67或68所示，或分别与SEQ ID NO:65、66、67或68具有至少90％序列同一性；和/或
    所述轻链可变区如SEQ ID NO:69、70、71、72、73、74、75或76所示，或分别与SEQ ID NO:69、70、71、72、73、74、75或76具有至少90％序列同一性；
    优选地，所述轻链可变区如SEQ ID NO:44所示，且所述重链可变区如SEQ ID NO:42所示；或
    优选地，所述轻链可变区如SEQ ID NO:53所示，且所述重链可变区如SEQ ID NO:52所示；或
    优选地，所述轻链可变区如SEQ ID NO:62所示，且所述重链可变区如SEQ ID NO:58所示；或
    优选地，所述轻链可变区如SEQ ID NO:76所示，且所述重链可变区如SEQ ID NO:68所示。
  6. 根据权利要求1、2、3或5所述的抗CEA抗体，其中所述抗CEA抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体在人抗体的轻链框架区和/或重链框架区上分别具有1至10个氨基酸的回复突变；
    优选地，所述抗CEA抗体包含：
    (i)轻链可变区，其包含分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3；和
    重链可变区，其包含如SEQ ID NO：15所示的HCDR1、如SEQ ID NO：16或SEQ ID NO：38所示的HCDR2、和如SEQ ID NO：17所示的HCDR3；
    而且，其中所述轻链可变区的框架区中包含选自46P、47W、49Y、70S和71Y中的一个或更多个氨基酸回复突变，和/或所述重链可变区的框架区中包含选自38K或46K中的一个或更多个氨基酸回复突变；
    (j)轻链可变区，其包含分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；和
    重链可变区，其包含如SEQ ID NO：21所示的HCDR1、如SEQ ID NO：22或SEQ ID NO：47所示的HCDR2、和如SEQ ID NO：23所示的HCDR3；
    而且，其中所述轻链可变区的框架区中包含选自2V、42G、44V和71Y中的一个或更多个氨基酸回复突变，和/或所述重链可变区的框架区中包含选自48I、66K、67A、69L、71V、73K、82F、 82A R中的一个或更多个氨基酸回复突变；
    (k)轻链可变区，其包含分别如SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32所示的LCDR1、LCDR2和LCDR3；和
    重链可变区，其包含分别如SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29所示的HCDR1、HCDR2和HCDR3；
    而且，其中所述轻链可变区的框架区中包含选自3V、43P和58V中的一个或更多个氨基酸回复突变，和/或所述重链可变区的框架区中包含选自38K、66K、71V中的一个或更多个氨基酸回复突变；和
    (l)轻链可变区，其包含如SEQ ID NO：35所示的LCDR1、如SEQ ID NO：36或SEQ ID NO：64所示的LCDR2、和如SEQ ID NO：37所示的LCDR3；和
    重链可变区，其包含如SEQ ID NO：33所示的HCDR1、如SEQ ID NO：16或SEQ ID NO：38所示的HCDR2、和如SEQ ID NO：34所示的HCDR3；
    而且，其中所述轻链可变区的框架区中包含选自4V、36Y、43P、47V、49E、70D和87I中的一个或更多个氨基酸回复突变，和/或
    所述重链可变区的框架区中包含选自2I、38K和46K中的一个或更多个氨基酸回复突变；
    其中，所述回复突变的位点对应于Kabat编号规则编号。
  7. 根据权利要求1至6中任一项所述的抗CEA抗体，所述抗CEA抗体进一步包含重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗体包含如SEQ ID NO:77所示的重链恒定区，和如SEQ ID NO:78或SEQ ID NO:79所示的轻链恒定区；
    最优选地，所述抗CEA抗体包含：
    (m)如SEQ ID NO:80所示或与SEQ ID NO:80具有至少85％序列同一性的重链，和/或
    如SEQ ID NO：81所示或与SEQ ID NO：81具有至少85％序列同一性的轻链；
    (n)如SEQ ID NO:82所示或与SEQ ID NO:82具有至少85％序列同一性的重链，和/或
    如SEQ ID NO：83所示或与SEQ ID NO:83具有至少85％序列同一性的轻链；
    (o)如SEQ ID NO:84所示或与SEQ ID NO:84具有至少85％序列同一性的重链，和/或
    如SEQ ID NO：85所示或与SEQ ID NO:85具有至少85％序列同一性的轻链；或
    (p)如SEQ ID NO:86所示或与SEQ ID NO:86具有至少85％序列同一性的重链，和/或如SEQ ID NO：87所示或与SEQ ID NO:87具有至少85％序列同一性的轻链。
  8. 一种分离的抗CEA抗体，其与权利要求1至7中任一项所述的抗CEA抗体竞争性结合人CEA或人CEA的表位。
  9. 一种核酸分子，其编码权利要求1至8中任一项所述的抗CEA抗体。
  10. 一种宿主细胞，其包含如权利要求9所述的核酸分子。
  11. 一种抗体药物偶联物，其包含以下或由以下组成：
    权利要求1至8中任一项所述的抗CEA抗体，和
    细胞毒性药物；
    其中，所述细胞毒性药物偶联至所述抗CEA抗体。
  12. 一种药物组合物，其含有：
    预防有效量或治疗有效量的根据权利要求1至8中任一项所述的抗CEA抗体，或根据权利要求9所述的核酸分子，或权利要求11所述的抗体药物偶联物，以及
    一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  13. 一种用于免疫检测或测定CEA的方法，所述方法包括使权利要求1至8中任一项所述的抗CEA抗体接触样本的步骤。
  14. 一种试剂盒，其包含根据权利要求1至8中任一项所述的抗CEA抗体。
  15. 一种预防或治疗疾病的方法，所述方法包括：
    向受试者施用预防有效量或治疗有效量的权利要求1至8中任一项所述的抗CEA抗体，或权利要求9所述的核酸分子，或权利要求11所述的抗体药物偶联物，或权利要求12所述的药物组合物，
    其中所述疾病为与CEA相干的疾病，所述疾病优选为肿瘤；
    更优选的，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；
    更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，
    所述肺癌选自：非小细胞肺癌和小细胞肺癌；
    所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
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