WO2023041065A1 - 一种靶向人ceacam5/6的抗体、制备方法和应用 - Google Patents

一种靶向人ceacam5/6的抗体、制备方法和应用 Download PDF

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WO2023041065A1
WO2023041065A1 PCT/CN2022/119437 CN2022119437W WO2023041065A1 WO 2023041065 A1 WO2023041065 A1 WO 2023041065A1 CN 2022119437 W CN2022119437 W CN 2022119437W WO 2023041065 A1 WO2023041065 A1 WO 2023041065A1
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antibody
variable region
ceacam5
heavy chain
chain variable
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PCT/CN2022/119437
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English (en)
French (fr)
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徐伟
莫世甫
赵勇
顾莉蕴
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优迈生物科技(连云港)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the invention belongs to the technical field of biological immunity. Specifically, the present invention provides an antibody targeting human CEACAM5/6, its preparation method and application.
  • CEACAM human carcinoembryonic antigen cell adhesion molecule
  • CEACAM proteins are located outside the cell membrane, among which CEACAM1, CEACAM3 and CEACAM4 are connected to the cell membrane through hydrophobic transmembrane domains; CEACAM5-8 are connected to the cell membrane through glycosylphosphoinositol. These extracellular domains generally serve as adhesion molecules between cells such as epithelial, endothelial, dendritic and leukocytes.
  • CEACAM involves a variety of cellular functions, based on intercellular adhesion function, regulates cell growth and differentiation through signal transduction, and plays an important role in insulin homeostasis, angiogenesis and immune regulation.
  • Members of the CEACAM gene family are involved in a variety of pathophysiological roles, including acting as receptors for microbial pathogens. They play an important role in carcinogenesis, especially in cancer detection, progression and metastasis.
  • Carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5, abbreviated as CEA, also known as CD66e) is a glycoprotein with a molecular weight of about 180 kDa.
  • CEACAM5 contains seven domains linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. The seven domains include a single N-terminal Ig variable domain and six domains (A1-B1-A2-B2-A3-B3) homologous to the Ig constant domains.
  • GPI glycosylphosphatidylinositol
  • CEACAM5 originally classified as a protein expressed only in fetal tissues, has now been identified in several normal adult tissues. Overexpression of CEACAM5 is observed in many types of cancers, for example, CEACAM5 can be detected in the blood of patients with colon cancer, and further studies have established that its overexpression is associated with many malignancies, often with poor prognosis. In prostate and colorectal cancers, overexpression of CEACAM5 was shown to serve as a tumor biomarker.
  • CEACAM5/CEACAM6 was also found to be overexpressed in a variety of malignant tumors, such as breast, pancreas, ovary, colon, lung and gastric adenocarcinoma, and was associated with tumor invasion and metastasis.
  • malignant tumors such as breast, pancreas, ovary, colon, lung and gastric adenocarcinoma
  • CEACAM5 binds to its receptor CEAr, and their interaction leads to the activation and production of pro-inflammatory cytokines, mainly IL-1, IL-6, IL-10 and TNF- ⁇ .
  • pro-inflammatory cytokines mainly IL-1, IL-6, IL-10 and TNF- ⁇ .
  • these cytokines alter the microenvironment of hepatocytes and Kupffer cells, as well as their interactions with hepatic sinusoidal cells. These interactions not only affect tumor cells or other hepatocytes, but also appear to promote the viability of CSCs and other circulating tumor cells in
  • CEACAM5 has become a potentially useful tumor-associated antigen for targeted therapy, but there are still many shortcomings in the existing CEACAM5-targeting drugs in the field. Therefore, there is an urgent need in the art to develop a specific antibody molecule targeting CEACAM5.
  • the object of the present invention is to provide an antibody targeting human CEACAM5/6, a preparation method and an application.
  • a heavy chain variable region of an anti-human CEACAM5/6 antibody is provided, and the heavy chain variable region includes the following three complementarity determining regions:
  • VH-CDR1 shown in SEQ ID NO.1,
  • VH-CDR2 shown in SEQ ID NO.2,
  • any amino acid sequence in the above amino acid sequence also includes optionally adding, deleting, modifying and/or replacing at least one amino acid, and can retain CEACAM5/6 The derived sequence of the binding affinity.
  • amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO.7, 11 or 13.
  • a heavy chain of an anti-human CEACAM5/6 antibody having the heavy chain variable region as described in the first aspect of the present invention.
  • the heavy chain of the antibody further includes a heavy chain constant region.
  • the heavy chain constant region is of human, mouse, camel or rabbit origin, preferably of human origin.
  • the heavy chain constant region is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, preferably, the heavy chain constant region of human IgG1.
  • sequence of the heavy chain of the antibody is shown in SEQ ID NO.9, 14 or 15.
  • a light chain variable region of an anti-human CEACAM5/6 antibody is provided, and the light chain variable region includes the following three CDRs:
  • VL-CDR1 shown in SEQ ID NO.4,
  • VL-CDR2 shown in SEQ ID NO.5, and
  • any amino acid sequence in the above amino acid sequence also includes optionally adding, deleting, modifying and/or replacing at least one amino acid, and can retain CEACAM5/6
  • the derived sequence of the binding affinity
  • sequence of the light chain variable region is shown in SEQ ID NO.8 or 12.
  • a light chain of an anti-human CEACAM5/6 antibody having the light chain variable region as described in the third aspect of the present invention.
  • the light chain of the antibody further includes a light chain constant region.
  • the light chain constant region is of human, mouse, camel or rabbit origin, preferably of human origin.
  • the light chain constant region is selected from the light chain constant region of human ⁇ or ⁇ subtype.
  • the light chain of the antibody is shown in SEQ ID NO.10 or 16.
  • an anti-human CEACAM5/6 antibody is provided, the antibody has:
  • the antibody has: the heavy chain according to the second aspect of the present invention; and/or the light chain according to the fourth aspect of the present invention.
  • the antibody is a humanized antibody.
  • the antibody specifically binds to human CEACAM5 and/or human CEACAM6.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibodies include monospecific, bispecific, trispecific or multispecific antibodies.
  • the light chain of the antibody includes the three light chain CDRs and the light chain framework region used to connect the light chain CDRs; and the heavy chain of the antibody includes the three Heavy chain CDRs and heavy chain framework regions for linking heavy chain CDRs.
  • the heavy chain variable region of the antibody is shown in SEQ ID NO.7, 11, 13; and/or the light chain variable region of the antibody is shown in SEQ ID NO.8, 12 shown.
  • the heavy chain variable region of the antibody is shown in SEQ ID NO.7, and the light chain variable region of the antibody is shown in SEQ ID NO.8.
  • the heavy chain variable region of the antibody is shown in SEQ ID NO.11, and the light chain variable region of the antibody is shown in SEQ ID NO.12.
  • the heavy chain variable region of the antibody is shown in SEQ ID NO.13, and the light chain variable region of the antibody is shown in SEQ ID NO.12.
  • the multispecific antibody includes:
  • CEACAM5/6 antibody or antigen-binding fragment thereof as described in the fifth aspect of the present invention.
  • the other targets are selected from the group consisting of CD47, CD73, CD47, CD3, CTLA4, PD-1, PD-L1, CD28, CD40, OX40, LAG3, DR5, TIM-3, TIGIT , VEGF, VEGFR, Ang2, CD39, CD73, GITR.
  • a multispecific antigen-binding molecule comprising:
  • D1 specifically binds to the target molecule CEACAM5/6 protein
  • D2 specifically binds to the target molecule CD47 protein
  • the D1 is an antibody or an antigen-binding fragment thereof that specifically binds CEACAM5/6 protein;
  • the D2 is an antibody or an antigen-binding fragment thereof that specifically binds to the CD47 protein
  • the D1 has a heavy chain variable region comprising the following three complementarity determining regions CDRs:
  • VH-CDR1 shown in SEQ ID NO.1,
  • VH-CDR2 shown in SEQ ID NO.2,
  • VH-CDR3 shown in SEQ ID NO.3;
  • the light chain variable region comprises the following three complementarity determining region CDRs:
  • VL-CDR1 shown in SEQ ID NO.4,
  • VL-CDR2 shown in SEQ ID NO.5, and
  • VL-CDR3 shown in SEQ ID NO.6;
  • the structure of the antigen-binding fragment is selected from the group consisting of: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv ( scFv) molecules; or (vi) dAb fragments.
  • the heavy chain of the antibody specifically binding to CD47 protein includes: a heavy chain variable region shown in SEQ ID NO.17; and the light chain of the antibody specifically binding to CD47 protein includes : having the light chain variable region shown in SEQ ID NO.18.
  • the heavy chain variable region or the light chain variable region of the anti-human CD47 antibody each contains 1-6 mutations, preferably 1-4 mutations.
  • the mutation of the heavy chain variable region of the anti-human CD47 antibody is selected from the following group:
  • the mutation of the light chain variable region of the anti-human CD47 antibody is selected from the following group:
  • the heavy chain of the antibody specifically binding to the CD47 protein comprises: a heavy chain variable region shown in SEQ ID NO.19; and the light chain of the antibody specifically binding to the CD47 protein comprises : having the light chain variable region shown in SEQ ID NO.20.
  • any amino acid sequence in the above amino acid sequence also includes a derivative sequence optionally undergoing addition, deletion, modification and/or substitution of at least one amino acid and capable of retaining the binding affinity for CD47.
  • the bispecific antigen-binding molecule is a bispecific antibody.
  • the D1 and/or D2 are single-chain antibodies (scFv).
  • D2 is a single-chain antibody (scFv).
  • the D1 and D2 are linked by a linker, and the linker includes an antibody constant region.
  • the D1 is an anti-human CEACAM5/6 antibody.
  • the D2 is an anti-CD47 single chain antibody (scFv).
  • the anti-CEACAM5/6 humanized antibodies include: single-chain antibodies, double-chain antibodies, monoclonal antibodies, chimeric antibodies, murine antibodies, humanized antibodies, and bispecific antibodies .
  • the anti-human CEACAM5/6 antibody comprises the following three CDR heavy chain variable regions:
  • VH-CDR1 shown in SEQ ID NO.1,
  • VH-CDR2 shown in SEQ ID NO.2,
  • VH-CDR3 shown in SEQ ID NO.3;
  • the light chain variable region comprises the following three complementarity determining region CDRs:
  • VL-CDR1 shown in SEQ ID NO.4,
  • VL-CDR2 shown in SEQ ID NO.5, and
  • VL-CDR3 shown in SEQ ID NO.6.
  • the bispecific antibody comprises a heavy chain and/or a light chain selected from Table 4:
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • the recombinant protein also includes protein or polypeptide drugs targeting other tumor targets.
  • the protein or polypeptide drug contains cytokines that regulate immune system functions.
  • the cytokines include wild type or mutant type.
  • the cytokines are selected from the group consisting of IL-1, IL-2, IL-7, IL8, IL-10, IL-12, IL-15, IL-18, IL-21, GM-CSF, IFN ⁇ / ⁇ / ⁇ , OX40-L, 4-1BB-L, CTLA-4, or a combination thereof.
  • a CAR construct is provided, the scFv segment of the antigen-binding domain of the CAR construct specifically binds to human CEACAM5/6, and the scFv segment comprises the The heavy chain variable region of the first aspect and the light chain variable region of the third aspect of the present invention.
  • the immune cells are selected from the group consisting of NK cells and T cells.
  • the immune cells are from humans or non-human mammals (such as mice).
  • an antibody-drug conjugate is provided, and the antibody-drug conjugate contains:
  • an antibody part which is selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, and the heavy chain as described in the third aspect of the present invention
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of detectable labels, drugs, toxins, cytokines, enzymes, or combinations thereof.
  • the antibody part is coupled to the coupling part through a chemical bond or a linker.
  • an active ingredient selected from the group consisting of: the heavy chain variable region as described in the first aspect of the present invention, as described in the second aspect of the present invention
  • the cancer or tumor is selected from the group consisting of colon cancer, rectal cancer, lymphoma, pancreatic cancer, lung cancer, gastric cancer, hepatoma, breast cancer or thyroid cancer, or a combination thereof.
  • a pharmaceutical composition which contains:
  • an active ingredient selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, and the heavy chain as described in the third aspect of the present invention The light chain variable region of the present invention, the light chain as described in the fourth aspect of the present invention, the antibody as described in the fifth aspect of the present invention, the multispecific antigen-binding molecule as described in the sixth aspect of the present invention, the light chain as described in the first aspect of the present invention.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used for treating tumors.
  • the tumor is a tumor that highly expresses human CEACAM5/6.
  • a polynucleotide encoding a polypeptide selected from the group consisting of:
  • the vectors include: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or other vectors.
  • a genetically engineered host cell contains the vector according to the fourteenth aspect of the present invention or the genome is integrated with the vector according to the thirteenth aspect of the present invention said polynucleotide.
  • the host cells are mammalian cells, preferably, cells of human, mouse, sheep, horse, dog or cat, more preferably, cells of Chinese hamster ovary.
  • a method for in vitro non-diagnostic detection (including diagnostic or non-diagnostic) of human CEACAM5/6 protein in a sample comprising the steps of:
  • a method of treating human CEACAM5/6-related diseases comprising:
  • the antibody according to the fifth aspect of the present invention Administer the antibody according to the fifth aspect of the present invention, the multispecific antibody of the antibody, the drug conjugate of the antibody, or the CAR-T cells expressing the antibody, or a combination thereof, to a subject in need.
  • the human CEACAM5/6-related disease is a tumor with high expression of CEACAM5/6, preferably colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatoma, breast cancer and thyroid cancer.
  • the human CEACAM5/6-related disease is a tumor with high expression of CEACAM5/6 and CD47, preferably colorectal cancer.
  • Figure 1 shows the ELISA of UM05-C9 and human CEACAM5 protein.
  • Figure 2 shows the binding of UM05-C9 to CHO-CEACAM5 cells.
  • Figure 3 shows the ELISA of UM05-L9 and bispecific antibody L34 with (A) CEACAM5 protein and (B) CEACAM6 protein from cynomolgus monkeys.
  • Figure 4 shows the results of cellular internalization of UM05-C9.
  • Figure 5 shows different structural combinations of CD47-CEACAM5/6 bispecific antibodies. Among them, green or orange marks the antigen-binding fragments of different antibodies (VH/VL of CD47 antibody or CEACAM5/6 antibody), respectively.
  • Figure 6 shows the detection of the affinity of UM11-L34 molecules to (A) CEACAM5 and (B) CD47 molecules, respectively.
  • Figure 7 shows the affinity detection of UM11-L34 molecules for CEACAM5 and CD47 molecular sequences.
  • the inventors After extensive and in-depth research and extensive screening, the inventors first developed an antibody against CEACAM5 with excellent activity, and unexpectedly found that the antibody also has excellent binding activity to CEACAM6.
  • the present invention provides for the first time an efficient and safe anti-human CD47-CEACAM5/6 bispecific antibody, its preparation method and application.
  • the bispecific antibody of the present invention has better targeting and specific killing effect on tumor cells with high expression of CEACAM5/6 and CD47, and can reduce the binding effect with red blood cells and/or platelets.
  • the anti-CEACAM5/6 and CD47 bispecific antibody of the present invention binds more selectively to CD47 + -CEACAM5/6 + tumor cells, binds weaker to CD47 protein on the surface of red blood cells and platelets, and treats multiple diseases by regulating human immune function. kind of cancer.
  • the present invention has been accomplished on this basis.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked 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 classes, or different types of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ .
  • IgG represents the most important class of immunoglobulins.
  • IgG1 Due to differences in chemical structure and biological function, it can be divided into four subclasses: IgG1, IgG2, IgG3 and IgG4.
  • Light chains are classified as kappa or lambda chains by difference in the constant region.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • Complementary binding determining region is the key sequence structure that determines the specific binding of antibody and antigen.
  • the heavy and light chains each contain three CDRs, named HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, which assign Systems include, for example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al.
  • the residues of each CDR are as follows.
  • the CDRs of an antibody of the invention are bounded by Kabat rules, or by IMGT rules, or by a combination thereof.
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding.
  • a minimal binding unit may be a subsection of a CDR.
  • the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody, as is well known to those skilled in the art. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the rest of the CDR residues defined according to Kabat or IMGT can be replaced by conservative amino acid residues.
  • FR framework region
  • the light and heavy chains of immunoglobulins each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • the light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can thus be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the human antibody FR derivative is substantially identical to a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, or 96%. , 97%, 98%, or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to that of a naturally occurring human antibody .
  • the term "monoclonal antibody” or “mAb” refers to an antibody molecule of a single amino acid composition directed against a particular antigen and should not be construed as requiring that the antibody be produced by any particular method.
  • Monoclonal antibodies can be produced by a single clone of B cells or hybridomas, but can also be recombinant, ie produced by protein engineering.
  • the term "antigen" or “target antigen” refers to a molecule or portion of a molecule capable of being bound by an antibody or antibody-like binding protein. The term further refers to a molecule or portion of a molecule that can be used in an animal to generate antibodies capable of binding to an epitope of the antigen.
  • a target antigen may have one or more epitopes. For each target antigen recognized by an antibody or by an antibody-like binding protein, the antibody-like binding protein is able to compete with intact antibodies recognizing the target antigen.
  • affinity is theoretically defined by the equilibrium association between intact antibody and antigen.
  • the affinity of the bisantibody of the present invention can be evaluated or determined by KD value (dissociation constant) (or other measurement methods), such as bio-layer interferometry (Bio-layer interferometry BLI), determined by Biacore 8K measurement.
  • CEACAM belongs to the immunoglobulin superfamily of adhesion molecules, and its domain is highly glycosylated, usually including 1-2 immunoglobulin variable region-like domains (N domain) and 0-6 immunoglobulin constant region-like domains .
  • CEACAM proteins are located outside the cell membrane, among which CEACAM1, CEACAM3 and CEACAM4 are connected to the cell membrane through hydrophobic transmembrane domains; CEACAM5-8 are connected to the cell membrane through glycosylphosphoinositol. These extracellular domains generally serve as adhesion molecules between cells such as epithelial, endothelial, dendritic and leukocytes.
  • CEACAM5/CEACAM6 was found to be overexpressed in a variety of malignant tumors, such as breast, pancreas, ovary, colon, lung and gastric adenocarcinoma, and was associated with tumor invasiveness and metastasis.
  • anti-CEACAM5/6 antibody and “anti-CEACAM5/CEACAM6 antibody” refer to antibodies that can bind CEACAM5 molecules and CEACAM6 molecules in the present invention with strong specificity, that is, the antibodies have specific cross-reactivity.
  • the present invention provides an anti-CEACAM5/6 antibody (for example, UM05-L9), which not only has strong binding to CEACAM5 molecules, but also has strong binding to CEACAM6 molecules.
  • an anti-CEACAM5/6 antibody for example, UM05-L9
  • the antibody comprises a heavy chain comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain variable region
  • the heavy chain variable region (VH) has a complementarity determining region VH-CDR selected from the group consisting of:
  • VH-CDR1 shown in SEQ ID NO.1,
  • VH-CDR2 shown in SEQ ID NO.2,
  • VH-CDR3 shown in SEQ ID NO.3;
  • the light chain variable region (VL) has a complementarity determining region VL-CDR selected from the group consisting of:
  • VL-CDR1 shown in SEQ ID NO.4,
  • VL-CDR2 shown in SEQ ID NO.5, and
  • VL-CDR3 shown in SEQ ID NO.6;
  • any amino acid sequence in the above amino acid sequence also includes a derivative sequence optionally undergoing addition, deletion, modification and/or substitution of at least one amino acid, and capable of retaining the binding affinity of CEACAM5 and/or CEACAM6.
  • sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology or sequence identity of at least 80%, preferably at least 85%, more preferably Preferably at least 90%, most preferably at least 95% of the amino acid sequence.
  • the number of amino acids added, deleted, modified and/or substituted is preferably no more than 40% of the total amino acid number of the original amino acid sequence, more preferably no more than 35%, more preferably 1-33% , more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted may be 1-7, more preferably 1-5, more preferably 1-3, more preferably For 1-2 pieces.
  • the antibody described herein is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (single chain antibody fragment, scFv), a single domain antibody (single domain antibody) , sdAb) and one or more of single-domain antibodies (Signle-domain antibodies), as well as monoclonal or polyclonal antibodies made from the above-mentioned antibodies.
  • the monoclonal antibody can be developed by various approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is to prepare monoclonal antibody from wild-type or transgenic mice by hybridoma technology.
  • the full-length antibody protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein and the human heavy chain constant region and human light chain constant region constitute a fully human antibody full-length protein.
  • the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.
  • the human light chain constant region is selected from the light chain constant region of human kappa or lambda subtype; further preferably, the human heavy chain constant region is the heavy chain constant region of human IgG1, and the human light chain constant region for the ⁇ chain.
  • the antibody (anti-CEACAM5/6 antibody) in the present invention can be a full-length protein (such as IgG1, IgG2a, IgG2b or IgG2c), or a protein fragment (such as Fab, F( ab'), sdAb, scFv fragment).
  • a full-length protein such as IgG1, IgG2a, IgG2b or IgG2c
  • a protein fragment such as Fab, F( ab'), sdAb, scFv fragment.
  • antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking antibody hypervariable regions (e.g., independent complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3 - CDR3-FR4 peptide.
  • CDRs independent complementarity determining regions
  • the CEACAM5/6 antibody or antigen-binding fragment thereof of the present invention is selected from the group consisting of camel-derived single domain antibody, scFv, scFv dimer, BsFv, dsFv, dsFv2, dsFv-dsFv', Fv fragment , Fab, Fab', F(ab') 2 , ds diabody, nanobody, domain antibody or bivalent domain antibody.
  • the antibody (anti-CEACAM5/6 antibody) in the present invention can be a wild-type protein, or a mutant protein that has undergone a specific mutation to achieve a specific effect, for example, the effector function of the antibody is eliminated by mutation.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully human derivatized antibodies.
  • the antibody derivatives of the present invention can be single-chain antibodies, and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other known antibody derivatives in the field, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
  • the single-chain antibody is a conventional single-chain antibody in the field, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15-20 amino acids.
  • the animal is preferably a mammal, such as a mouse.
  • the antibodies of the present invention may be chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies targeting CEACAM5/6 (eg, human CEACAM5/6).
  • the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.7, 11 or 13.
  • the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.8 or 12.
  • the heavy chain variable region (VH) amino acid sequence of the antibody targeting CEACAM5/6, and/or the light chain variable region (VL) amino acid sequence is shown in Table 2 below:
  • UM05-L9 is a human-mouse chimeric antibody
  • UM05-C9 is a humanized antibody
  • UM05-C9' is another version of a humanized antibody.
  • the VH-CDR and VL-CDR of the three are the same.
  • CD47 is a transmembrane glycoprotein of 45-50kD and belongs to the immunoglobulin (Ig) superfamily. Studies have found that the highly expressed CD47 on tumor cells binds to the ligand SIRP ⁇ on macrophages, which makes SIRP ⁇ tyrosine phosphorylated, thereby sending out inhibitory regulatory signals and inhibiting the phagocytosis of macrophages. Correspondingly, blocking this pathway can release the inhibitory effect of macrophages on tumor cell phagocytosis, improve the body's immune response to tumor cells, and provide a new way for tumor immunotherapy.
  • Ig immunoglobulin
  • CD47-SIRP ⁇ In tumor therapy targeting the CD47-SIRP ⁇ axis, the main mechanism is the activation of macrophages, thereby enhancing the phagocytosis of tumor cells by macrophages.
  • blocking CD47 may further recruit macrophages into tumor tissue, as well as cytokines and chemokines that recruit additional immune cells to tumor tissue, such as monocyte chemoattractant protein 3 (MCP-3), which The secretion of factors contributes to the efficacy of CD47 blockade therapy.
  • MCP-3 monocyte chemoattractant protein 3
  • therapies targeting CD47-SIRP ⁇ may also alter the polarization state of macrophages in tumors. Macrophages can be divided into M1 (type I) and M2 (type II) according to their phenotype and functional activity.
  • M1 macrophages can produce a large number of pro-inflammatory cytokines and mediate resistance to intracellular parasites and Inhibit tumor growth; M2 macrophages produce less pro-inflammatory molecules, participate in tissue damage repair, angiogenesis and promote tumor growth.
  • other immune cells can also respond to CD47-blocking therapy. SIRP ⁇ is highly expressed on myeloid immune cells, so it may be a key regulator of the myeloid lineage.
  • CD47 regulates antigen uptake by SIRP ⁇ + dendritic cells, and using a syngeneic immunocompetent tumor model, the therapeutic effect of CD47 blockade was found to be dendritic cell dependent.
  • Therapies targeting the CD47-SIRP ⁇ axis can promote tumor adaptive immune responses by stimulating antigen presentation by macrophages or dendritic cells.
  • CD47 is widely expressed in a variety of cells, especially in neonatal erythrocytes. Therefore, therapeutic antibodies targeting CD47 may cause anemia. On the other hand, the concentration of free anti-human CD47 antibody in the body will also be greatly reduced due to the adsorption of anti-human CD47 antibody by red blood cells.
  • Preferred anti-human CD47 antibodies in the present invention include anti-human CD47 antibodies or antigen-binding fragments thereof described in Chinese patent 202010240238.7, which is incorporated herein by reference (as used herein, represented by the code UMO3-C4, its heavy chain variable region As SEQ ID NO.17; light chain variable region as shown in SEQ ID NO.18).
  • the IgG4 subtype of the CD47 antibody is represented by the code UM03-C4 in this patent.
  • a derivative antibody human CD47 antibody obtained by mutating its VH and VL regions (as used herein, represented by the code UMO3-C4') is also included.
  • the mutated anti-human CD47 antibodies involved include the following mutations or a combination of the following mutations:
  • Heavy chain H-I37V, H-P45L, H-I48V, H-R94K, H-T52R, H-T52S, H-T52L, H-T52I, H-T52H, H-T52A, H-T52N, H-T52Q ;
  • Light chains L-L4M, L-I46L, L-V58I, L-T93N.
  • anti-human CD47 antibody or its antigen-binding domain different combinations of anti-human CD47 antibody or its antigen-binding domain and anti-human CEACAM5/6 antibody or its antigen-binding domain are used to obtain the bispecific antibody described in the present invention.
  • a bispecific antibody (Bispecific Antibody, bsAb) is a non-natural antibody that can target two different antigens or proteins simultaneously, block two different signaling pathways, and stimulate a specific immune response.
  • the role of bifunctionality and bifunctionality in tumor immunotherapy is becoming more and more important, and it has become a research hotspot in antibody engineering treatment of tumors in the world today.
  • bispecific antibodies mainly mediate the killing of immune cells on tumors in tumor immunotherapy; combining dual targets, blocking dual signaling pathways, and exerting unique or overlapping functions can effectively prevent drug resistance; Strong specificity, targeting, and reduced off-target toxicity; effective reduction in treatment costs and other advantages (excerpted from the antibody circle), so the use of bispecific antibody drugs can reduce the chance of tumor cell escape, clear tumor cells, and improve curative effect.
  • Bispecific antibodies can be produced by means of double hybridoma cells, chemical coupling, and recombinant genes, among which the recombinant gene technology is flexible in terms of binding sites and yields. According to incomplete statistics, there are currently more than 60 types of bispecific antibodies. According to their characteristics and structural differences, the structure of bispecific antibodies mainly includes bispecific antibodies containing Fc fragments (IgG-like bispecific antibodies with Fc Mediated effector function) and bispecific antibody without Fc fragment (non-IgG-like bispecific antibody, which acts through antigen binding, has the advantages of small molecular weight and low immunogenicity).
  • the present invention also includes antibodies that simultaneously bind anti-human CEACAM5/6 antibodies and other targets.
  • the other targets are selected from the group consisting of CD47, CD73, CD47, CD3, CTLA4, PD-1, PD-L1, CD28, CD40, OX40, LAG3, DR5, TIM-3, TIGIT, VEGF , VEGFR, Ang2, CD39, CD73, GITR, or a combination thereof.
  • the antibody that binds to other targets in the bispecific antibody of the present invention is an anti-human CD47 antibody.
  • bispecific antibody As used herein, the terms “bispecific antibody”, “bifunctional antibody”, “antibody of the present invention”, “double antibody of the present invention”, “double antibody”, “bifunctional fusion antibody”, “anti-human CEACAM5/6 and CD47 double “Specific antibody” and “anti-CEACAM5/6 and CD47 antibody” are used interchangeably and refer to bispecific antibodies that simultaneously bind CEACAM5/6 and CD47.
  • linker refers to one or more segments inserted into an immunoglobulin domain that provide sufficient mobility for the domains of the light and heavy chains to fold into an interchangeable dual variable region immunoglobulin. amino acid residues.
  • the linker is a flexible linker, preferably a peptide linker.
  • suitable peptide linkers include monoglycine (Gly), or serine (Ser) residues, and the identity and sequence of amino acid residues in the peptide linker can vary with the type of secondary structural elements that need to be achieved in the peptide linker.
  • the present invention provides a bispecific antibody constructed based on the aforementioned anti-human CEACAM5/6 antibody and anti-human CD47 antibody or an antigen-binding fragment thereof.
  • These bispecific antibodies connect anti-human CEACAM5/6 antibodies and anti-human CD47 antibodies or their antigen-binding fragments through linkers to form multivalent antibodies with symmetrical or asymmetrical structures, which can simultaneously bind CEACAM5/6 and CD47 protein effect.
  • the linker sequences that can be selected and used here include:
  • the linker is (GGGGS)n, wherein n is an integer between 0 and 5; or SSSSKAPPPSLPSPSRLPGPSDTPILPQ, or GGGSSSSKAPPPPSLPSPSRLPGPSDTPILPQGGG; more preferably, the linker is GGGSSSSKAPPPPSLPSPSRLPGPSDTPILPQGGG.
  • the preferred linker sequence is as shown in 119-152 positions in SEQ ID NO.64 or SEQ ID NO.66.
  • the bispecific antibody constructed from the anti-human CEACAM5/6 antibody and anti-human CD47 antibody or antigen-binding fragment thereof described in the present invention may further comprise a human heavy chain constant region and/or a human light chain constant region; more preferably , the human heavy chain constant region is selected from the heavy chain constant region of human IgG1, IgG2 or IgG4, and the human light chain constant region is selected from the light chain constant region of human kappa or lambda chain; further preferably, the The human heavy chain constant region is the heavy chain constant region of human IgG1, and the human light chain constant region is the ⁇ chain; further preferably, the human heavy chain constant region is the heavy chain constant region of human IgG1.
  • Mutations are generally known to enhance or weaken the biological functions mediated by the constant region of IgG1 antibodies, including ADCC, ADCP (Antibody-dependent cellular phagocytosis), CDC and FcRn binding activities. These mutations include, but are not limited to, those listed in the literature (Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life. Front. Immunol., 2019, 10:1296):
  • the inventors designed anti-human CEACAM5/6-CD47 bispecific antibodies with different structural forms.
  • the sequence of the anti-human CEACAM5/6-CD47 bispecific antibody is shown in Table 4.
  • the anti-human CEACAM5/6-CD47 antibody of the present invention has a stronger binding selectivity to the CD47 protein on the surface of CEACAM5/6 + -CD47 + double positive tumor cells, a stronger killing effect, and a stronger binding effect on the surface of red blood cells and platelets
  • the CD47 protein binds weaker and has no agglutinating effect on red blood cells.
  • the bispecific antibody of the present invention also includes its conservative variant, which means that compared with the amino acid sequence of the bispecific antibody of the present invention, there are at most 10, preferably at most 8, and more preferably at most 5 , preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • Lys(K) Arg Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • a polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.
  • a polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.
  • Nucleic acids (and combinations of nucleic acids) of the invention can be used to produce recombinant antibodies of the invention in a suitable expression system.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.
  • biomolecules nucleic acid, protein, etc.
  • the biomolecules involved in the present invention include biomolecules in an isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • the present invention also provides host cells comprising the vector of the present invention, preferably, the host cells are mammalian cells, more preferably human, mouse, sheep, horse, dog or cat cells, further preferably Chinese Hamster ovary cells.
  • the host cells are mammalian cells, more preferably human, mouse, sheep, horse, dog or cat cells, further preferably Chinese Hamster ovary cells.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with CaCl2 using procedures well known in the art.
  • Another way is to use MgCl2. Transformation can also be performed by electroporation, if desired.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional media according to the host cells used.
  • the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
  • the expression level of the bispecific antibody can reach 3.9g/L, the purity is above 97%, and the lactic acid can be well metabolized during the culture process.
  • the recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the BsAb of the present invention can be used alone, or can be combined or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, or any combination of these substances.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biological toxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Tumor therapeutic agents (eg, cisplatin) or any form of antitumor drugs, etc.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned anti-human CEACAM5/6 antibody or its active fragment or its fusion protein of the present invention, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • the prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.
  • routes including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.
  • the pharmaceutical composition of the present invention can be directly used for binding CEACAM5/6, and thus can be used for treating tumors.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the anti-human CEACAM5/6 antibody of the present invention and a pharmaceutically acceptable carrier or excipients.
  • a pharmaceutically acceptable carrier or excipients include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the anti-human CEACAM5/6 antibodies of the present invention can also
  • the anti-human CEACAM5/6 antibody of the present invention can be used alone, and the optimal target response can be obtained by adjusting the dosage regimen. For example, a single administration, or multiple administrations over time, or the dosage may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • a safe and effective amount of the anti-human CEACAM5/6 antibody of the present invention is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g/kg body weight, and in most cases no more than about 50 ⁇ g/kg body weight. mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the present invention also provides the use of the antibody, bispecific antibody, antibody conjugate ADC, recombinant protein, and/or immune cell of the present invention, such as for preparing diagnostic preparations or preparing medicines. Further, the present invention also provides the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of medicaments or cells used for cell therapy, the medicaments or cells are used to prevent and/or treat the benefit by enhancing the immune response disease.
  • the medicament is a medicament for preventing and/or treating diseases related to abnormal expression or function of CEACAM5/6 (CEACAM5 protein or CEACAM6 protein).
  • the disorder is cancer
  • the disorder is a tumor with high expression of CEACAM5 or CEACAM6, preferably colorectal cancer.
  • the human CEACAM5/6-related disease is a tumor with simultaneous high expression of CEACAM5/6 and CD47, preferably colorectal cancer.
  • the anti-human CEACAM5/6 antibody of the present invention has cross-reactivity for simultaneously binding CEACAM5 and CEACAM6;
  • the anti-human CEACAM5/6 and CD47 bispecific antibody of the present invention has better targeting and specific killing effect on tumor cells with high expression of CEACAM5/6 and CD47, and can reduce the interaction with red blood cells and/or platelets binding effect.
  • the parental sequence of the humanized CD47 monoclonal antibody is quoted from Chinese patent 202010240238.7, its heavy chain variable region sequence is shown in SEQ ID NO:17; its light chain variable region sequence is shown in SEQ ID NO:18.
  • the IgG4 subtype of the humanized CD47 monoclonal antibody is represented by the code number UM03-C4 in this patent.
  • the anti-human CD47 antibody also includes a derivative antibody human CD47 antibody obtained by mutating its VH and VL regions (as used herein, represented by the code UMO3-C4', and its heavy chain variable region sequence is as follows: Shown in SEQ ID NO:19; Its light chain variable region sequence is shown in SEQ ID NO:20).
  • the mutated anti-human CD47 antibodies involved include the following mutations or a combination of the following mutations:
  • Heavy chain H-I37V, H-P45L, H-I48V, H-R94K, H-P101D, H-T64K, H-T52R, H-T52S, H-T52L, H-T52I, H-T52H, H-T52A , H-T52G, H-T52N, H-T52Q;
  • Light chains L-L4M, L-I46L, L-V58I, L-T93N.
  • the mutations of M18 are all in the FR region, and M19, M20 and M21 are one or two mutations with CDR added on the basis of M18.
  • Example 2 Obtaining of humanized CEACAM5/6 monoclonal antibody
  • the present inventor constructed a CHO cell line overexpressing human CEACAM5 protein, and immunized mice with it. Splenocytes from immunized mice were fused with SP2/0-AG14 cells for hybridoma cell fusion, and an appropriate amount of fused cells were spread on a 96-well plate. On the 10th day after fusion, the supernatant of each well was taken, and the binding activity of the mouse antibody secreted by the hybridoma cells to human CEACAM5 was detected by ELISA (see Example 3 for the method), and a series of hybridoma cells with higher activity were obtained.
  • the hybridoma cells with excellent activity were selected, and among them, unexpectedly, an antibody strain also had excellent binding activity to CEACAM6.
  • the cDNA sequence of the heavy chain variable region and the cDNA sequence of the light chain variable region corresponding to the secreted antibody were obtained by sequencing, and the amino acid sequence of the heavy chain variable region encoded by it was shown in SEQ ID NO.7; the encoded light chain variable region The amino acid sequence is shown in SEQ ID NO.8.
  • the heavy chain variable region and the light chain variable region of the mouse antibody were linked to the constant region of the human IgG1 heavy chain and the constant region of the ⁇ chain, respectively, to obtain the human-mouse chimeric antibody UM05-L9, and its heavy chain sequence is shown in SEQ Shown in ID NO.9, the light chain sequence is shown in SEQ ID NO.10.
  • the human CEACAM5 protein solution with a concentration of 1 ⁇ g/mL was coated on a 96-well high-affinity plate at 100 ⁇ L/well, and shaken overnight at 4°C. The next day, wash 3 times with 300 ⁇ L PBST (Tween20: 0.5 ⁇ ), then block with 100 ⁇ L/well of 5% BSA/PBS for 1 hour, and shake at room temperature. Wash 3 times with 300 ⁇ L PBST. Prepare serial dilutions of antibody samples in PBS. Add 100 ⁇ L/well to a 96-well plate and shake at room temperature for 1 hour. Wash 3 times with 300 ⁇ L PBST.
  • FACS Fluorescence Activated Cell Sorting
  • the test results are shown in Figure 2.
  • the EC50 of UM05-C9 binding to CHO-CEACAM5 is 636.9 ng/mL.
  • Antigen-antibody binding affinity was detected by SPR technology (Surface Plasmon Resonance). Briefly, the chimeric antibody UM05-L9 at a concentration of 4 ⁇ g/mL was incubated with protein A sensor chip (GE, Cat#29127556) for 30s for antibody capture. In the antigen binding stage, the mobile phase of serially diluted human CEACAM5 protein was combined with the UM05-L9 antibody captured on the sensor chip for 120s. During the dissociation stage, the elution was continued for 360s with HBS-EP buffer. The binding of CEACAM5 protein to the antibody on the sensor chip was quantitatively detected with Biacore T20x0 (GE Healthcare). The test results are shown in Table 8, the affinity of UM05-L9 is 0.68nM.
  • UM05-L9 was used to detect its binding to cynomolgus monkey CEACAM5 and CEACAM6 proteins by ELISA method.
  • the detection method refers to the description in 3.1.
  • the results are shown in Figure 3.
  • CEACAM5/6 antibody of the present invention can bind to the CEACAM5/6 protein of cynomolgus monkey provides the convenience of animal model research for the drug development of related antibodies.
  • the tumor tissue-specific analysis of UM05-C9 was carried out by Sichuan Xiapaixen Medical Technology Co., Ltd. using the multi-organ tumor tissue chip of Shanghai Xinchao Biotechnology Co., Ltd. (Cat No: HOrgC120PG04, Lot No: XT19-008).
  • Example 5 Endocytic properties of CEACAM5/6 antibody UM05-C9
  • the cDNA sequence encoding the heavy chain or light chain of the antibody is linked to the sequence encoding the signal peptide, and cloned into the mammalian cell expression vector pcDNA3.4 respectively.
  • the heavy chain expression plasmid and the light chain expression plasmid were transfected into HEK293 cells with Lipofectamine 2000 transfection reagent (Invitrogen) at a molar ratio of 2:1, and cultured at 37°C and 5% carbon dioxide for 7 days. The culture supernatant was collected, and the antibody in the supernatant was purified by Protein A affinity chromatography. The quality of the purified antibody can be analyzed by classical protein analysis methods such as SDS-PAGE electrophoresis and molecular sieve chromatography. The purified antibody was dialyzed with PBS solution, concentrated by freeze-drying, and stored at -20°C.
  • the human CD47 protein solution with a concentration of 1 ⁇ g/mL was coated with 100 ⁇ L/well of a 96-well high-affinity plate, and shaken overnight at 4°C. The next day, wash 3 times with 300 ⁇ L PBST (Tween20: 0.5 ⁇ ), then block with 100 ⁇ L/well of 5% BSA/PBS for 1 hour, and shake at room temperature. Wash 3 times with 300 ⁇ L PBST. Prepare serial dilutions of antibody samples in PBS. Add 100 ⁇ L/well to a 96-well plate and shake at room temperature for 1 hour. Wash 3 times with 300 ⁇ L PBST.
  • Table 12 shows representative data on the binding of antibodies involved in the present invention to CD47 protein.
  • the method for detecting the binding of the bispecific antibody to the human CEACAM5 protein was the same as that described in Example 5.1, except that the coating protein was changed to 1 ⁇ g/mL human CEACAM5 protein solution.
  • the data are shown in Table 12.
  • Example 9 Binding of bispecific antibody to CD47 + -CEACAM5 + human tumor cell A549
  • Lung cancer cell A549 is a human tumor cell that expresses both CD47 and CEACAM5.
  • the inventors of the present application tested antibody binding using flow cytometry on A549 cells. Use PBS to prepare a concentration gradient solution of the antibody to be detected, and prepare a final concentration of 2 ⁇ working solution.
  • Example 10 Binding of bispecific antibody to human erythrocytes and agglutination of human erythrocytes
  • erythrocytes were separated from the peripheral blood of volunteers, and suspended into a 1% erythrocyte suspension with physiological saline.
  • the concentration gradient solution of the antibody was prepared in PBS, and the final concentration of 2 ⁇ working solution was prepared.
  • Example 11 Bispecific antibody inhibits the binding of CD47 and SIRP ⁇ protein on A549 cells
  • Lung cancer cell A549 is a human tumor cell that expresses both CD47 and CEACAM5.
  • the inventors of the present application used flow cytometry on A549 cells to detect that the antibody inhibited the binding of CD47 and SIRP ⁇ protein on A549 cells.
  • Use PBS to prepare a concentration gradient solution of the antibody to be detected, and prepare a final concentration of 2 ⁇ working solution.
  • Antibody code Inhibition (with reference to UM03-C4) UM03-C4 +++ L8 +++ L10 +++ L11 + L12 +++ L13 ++ L14 +++ L15 +++ L16 +++ L17 ++ L18 + L23 + L25 +++ L26 +++ L27 ++ L28 ++ L29 + L30 + L31 + L32 + L33 + L34 ++ L35 + L36 + L37 + L38 + L39 + L40 +++ L41 +++
  • the number of "+” represents the ability of the antibody to block the binding of CD47 and SIRP ⁇ on A549 cells.
  • the inventors of the present application tested the ADCC killing effect of some antibodies on A549 cells and human erythrocytes.
  • Collect A549 cells resuspend the culture medium and count, dilute to 1.5 ⁇ 10 5 /ml cell suspension, plate A549 cells at 50ul/well one day in advance, discard the culture medium the next day, and add the prepared antibody gradient dilution to the well plate
  • erythrocyte killing effect For the detection of erythrocyte killing effect, firstly, separate erythrocytes from the peripheral blood of volunteers, wash them twice with culture medium, count them, and make 1 ⁇ 10 7 /mL cell suspension, take 50ul/well cell suspension and 2 ⁇ antibody Mix 50ul of the gradient dilution solution and incubate in a cell incubator at 37°C for 1 hour; after washing twice with PBS, add the effector cells Jurkat-NFAT-CD16A, count the cells and dilute to a cell suspension of 3 ⁇ 10 6 /ml, add 30ul/ml Add the wells into the well plate, continue to incubate for 4 hours in a 37°C cell culture incubator, then add 30ul/well One-Glo, wait for 3min, and then use a microplate reader to detect the bioluminescence intensity. The detection results are shown in Table 16 below.
  • the inventors of the present application tested the ADCP effect of some antibodies on A549 cells and human erythrocytes.
  • Collect A549 cells resuspend the medium and count, dilute to 1 ⁇ 10 5 /ml cell suspension, plate A549 cells at 50ul/well one day in advance, discard the medium the next day, and add the prepared antibody gradient dilution to the well plate
  • the results are shown in Table 17.
  • the affinity of L34 molecule to human CEACAM5 and human CD47 was detected by SPR technology (Surface Plasmon Resonance). Briefly, the antibody solution with a concentration of 4 ⁇ g/mL was incubated with protein A sensor chip (GE, Cat#29127556) for 30s for antibody capture. In the antigen binding stage, human CEACAM5 protein and human CD47 protein were used as the mobile phase to bind to the antibody captured on the sensor chip for 120s. During the dissociation stage, the elution was continued for 360s with HBS-EP buffer. The combination of antigen and antibody on the sensor chip was quantitatively detected by Biacore 8K. The results are shown in Figure 6 and Table 18.
  • the epitope competition between the two antigens and the double antibody was also detected by SPR technology, and the method was the same as described above.
  • the antibody solution was first incubated with protein A sensor chip for 30s for antibody capture.
  • CEACAM5 protein and CD47 protein were used as the mobile phase to bind to the antibody captured on the sensor chip for 120s.
  • the combination of antigen and antibody on the sensor chip was quantitatively detected by Biacore 8K. The result is shown in Figure 7.
  • A549 cells are CD47 + -CEACAM + tumor cells, and the A549 cells were subcutaneously inoculated into female NOD-Scid mice at an amount of 6 ⁇ 10 6 cells/mouse. After the tumor grew to about 70 mm 3 , the antibody solution prepared in normal saline was given intravenously. Six mice in each group were administered once every 3 days for a total of 3 weeks.
  • CEACAM5 As a tumor marker, immunological assays to measure elevated CEACAM5 in the blood of cancer patients have been used clinically for cancer prognosis and control, and more importantly, CEACAM5 has become a target for Potentially useful tumor-associated antigens for therapeutics. There are 2 main approaches that have been reported to use CEACAM5-targeted immunotherapy for cancer.
  • CEACAM5 antibodies to elicit lytic activity of immune cells, specifically through antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), to eliminate CEACAM5-expressing tumor cells;
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • CEACAM5 antibodies or antibody fragments are conjugated to effector molecules such as drugs, toxins, radioactive nucleotides, immunomodulators or cytokines to specifically target tumor cells expressing CEACAM5, thereby exerting the therapeutic effect of effector molecules.
  • effector molecules such as drugs, toxins, radioactive nucleotides, immunomodulators or cytokines to specifically target tumor cells expressing CEACAM5, thereby exerting the therapeutic effect of effector molecules.
  • effector molecules such as drugs, toxins, radioactive nucleotides, immunomodulators or cytokines
  • the current study suggests that therapeutic approaches targeting CEACAM5 will help suppress the metastatic process of tumors. Therefore, the anti-CEACAM5/6 antibody with affinity to CEACAM5 and CEACAM6 provided by the present invention has important application value.
  • the anti-human CEACAM5/6 and CD47 bispecific antibody of the present invention has better targeting and specific killing effect on tumor cells with high expression of CEACAM5/6 and CD47, and can also reduce the concentration of red blood cells and/or platelets. the combined effect.

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Abstract

提供了一种靶向人癌胚抗原CEACAM5/6的单克隆抗体，还提供了能同时靶向人癌胚抗原CEACAM5/6和人CD47的双特异性抗体及其制备方法和应用。

Description

一种靶向人CEACAM5/6的抗体、制备方法和应用 技术领域
本发明属于生物免疫技术领域。具体地，本发明提供了一种靶向人CEACAM5/6的抗体、制备方法和应用。
背景技术
人癌胚抗原细胞粘附分子(CEACAM)基因家族早在上世纪60年代就被发现，包括位于19号染色体(q13.1-13.3之间)的35个基因/伪基因，其中21个编码蛋白质。CEACAM属于免疫球蛋白超家族黏附分子，其结构域高度糖基化，通常包括1-2个免疫球蛋白可变区样结构域(N domain)和0-6个免疫球蛋白恒定区样结构域。CEACAM蛋白位于细胞膜外，其中CEACAM1、CEACAM3和CEACAM4通过疏水跨膜结构域与细胞膜连接；CEACAM5-8则通过糖基磷酰肌醇连接到细胞膜上。这些细胞外域通常作为细胞(例如上皮、内皮、树突状和白细胞)间的粘附分子。
CEACAM涉及多种细胞功能，以细胞间的黏附功能为基础，通过信号转导调节细胞生长和分化，并在胰岛素稳态、血管生成及免疫调节中发挥重要作用。CEACAM基因家族成员参与各种各样的病理生理作用，包括作为微生物病原体的受体。它们在致癌中起重要作用，特别是在癌症检测、进展和转移中。癌胚抗原细胞粘附分子5(CEACAM5，简称为CEA，又称为CD66e)是具有约180kDa分子量的糖蛋白。CEACAM5含有经由糖基磷脂酰肌醇(GPI)锚与细胞膜连接的7个结构域。7个结构域包括单一N端Ig可变域和与Ig恒定域同源的6个结构域(A1-B1-A2-B2-A3-B3)。CEACAM5最初分类为仅在胎儿组织中表达的蛋白质，现在已经在几种正常成年组织中被鉴定出来。CEACAM5的过量表达在许多类型的癌症中观察到，例如，在结肠癌患者的血液中可以检测到CEACAM5，并且进一步研究确定了它的过度表达与许多恶性肿瘤，通常是不良预后相关。在前列腺癌和结直肠癌中，CEACAM5的过度表达被证明可以作为肿瘤生物标志物。
此外，在多种恶性肿瘤，如乳房、胰腺、卵巢、结肠、肺和胃腺肿瘤中CEACAM5/CEACAM6亦被发现呈过度表达，并且与肿瘤的侵袭性和转移有关。在肝转移的起始过程中，CEACAM5与其受体CEAr结合，它们的相互作用导致促炎细胞因子的活化和产生，主要是IL-1、IL-6、IL-10和TNF-α。总之，这些细胞因子改变肝细胞和Kupffer细胞的微环境，以及它们与肝窦细胞的相互作用。这些相互作用不仅影响肿瘤细胞或其他肝细胞，似乎也促进了脑脊液中CSC和其他循环肿瘤细胞的活力。
综上，CEACAM5已成为用于靶向治疗的潜在有用的肿瘤相关抗原，而本领域当前已有的靶向CEACAM5的药物仍有诸多不足。因此，本领域迫切需要开发一种靶向CEACAM5的特异性抗体分子。
发明内容
本发明的目的在于提供一种靶向人CEACAM5/6的抗体、制备方法和应用。
在本发明的第一方面，提供了一种抗人CEACAM5/6抗体的重链可变区，所述的重链可变区包括以下三个互补决定区：
SEQ ID NO.1所示的VH-CDR1，
SEQ ID NO.2所示的VH-CDR2，和
SEQ ID NO.3所示的VH-CDR3；其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CEACAM5/6的结合亲和力的衍生序列。
在另一优选例中，所述重链可变区的氨基酸序列选自下组：SEQ ID NO.7、11或13。
在本发明的第二方面，提供了一种抗人CEACAM5/6抗体的重链，所述的重链具有如本发明的第一方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源、骆驼源或兔源的，较佳地为人源的。
在另一优选例中，所述的重链恒定区选自人IgG1、IgG2、IgG3或IgG4的重链恒定区，较佳地，为人IgG1的重链恒定区。
在另一优选例中，所述的抗体的重链的序列如SEQ ID NO.9、14或15所示。
在本发明的第三方面，提供了一种抗人CEACAM5/6抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.4所示的VL-CDR1，
SEQ ID NO.5所示的VL-CDR2，和
SEQ ID NO.6所示的VL-CDR3；其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CEACAM5/6的结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区的序列如SEQ ID NO.8或12所示。
在本发明的第四方面，提供了一种抗人CEACAM5/6抗体的轻链，所述的轻链具有如本发明的第三方面所述的轻链可变区。
在另一优选例中，所述抗体的轻链还包括轻链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源、骆驼源或兔源的，较佳地为人源的。
在另一优选例中，所述的轻链恒定区选自人κ或λ亚型的轻链恒定区。
在另一优选例中，所述的抗体的轻链如SEQ ID NO.10或16所示。
在本发明的第五方面，提供了一种抗人CEACAM5/6抗体，所述抗体具有：
(1)如本发明的第一方面所述的重链可变区；和/或
(2)如本发明的第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明的第二方面所述的重链；和/或如本发明的第四方面所述的轻链。
在另一优选例中，所述抗体为人源化抗体。
在另一优选例中，所述抗体特异性结合人CEACAM5和/或人CEACAM6。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体包括单特异性、双特异性、三特异性或多特异性抗体。
在另一优选例中，所述的抗体的轻链包括所述的三个轻链CDR以及用于连接轻链CDR的轻链框架区；和所述的抗体的重链包括所述的三个重链CDR以及用于连接重链CDR的重链框架区。
在另一优选例中，所述的抗体的重链可变区如SEQ ID NO.7、11、13所示；和/或所述的抗体的轻链可变区如SEQ ID NO.8、12所示。
在另一优选例中，所述的抗体的重链可变区如SEQ ID NO.7所示，并且所述抗体的轻链可变区如SEQ ID NO.8所示。
在另一优选例中，所述的抗体的重链可变区如SEQ ID NO.11所示，并且所述抗体的轻链可变区如SEQ ID NO.12所示。
在另一优选例中，所述的抗体的重链可变区如SEQ ID NO.13所示，并且所述抗体的轻链可变区如SEQ ID NO.12所示。
在另一优选例中，所述的多特异性抗体，包括：
(1)如本发明的第五方面中所述的CEACAM5/6抗体或其抗原结合片段；
(2)结合其他靶点的抗体或其抗原结合片段。
在另一优选例中，所述其他靶点选自下组：CD47、CD73、CD47、CD3、CTLA4、PD-1、PD-L1、CD28、CD40、OX40、LAG3、DR5、TIM-3、TIGIT、VEGF、VEGFR、Ang2、CD39、CD73、GITR。
在本发明的第六方面，提供了一种多特异性抗原结合分子，包含：
第一抗原结合域D1；和
第二抗原结合域D2；
其中，D1特异性结合靶分子CEACAM5/6蛋白；
D2特异性结合靶分子CD47蛋白；
所述D1为特异性结合CEACAM5/6蛋白的抗体或其抗原结合片段；
所述D2为特异性结合CD47蛋白的抗体或其抗原结合片段；
其中，所述D1具有包含以下三个互补决定区CDR的重链可变区：
SEQ ID NO.1所示的VH-CDR1，
SEQ ID NO.2所示的VH-CDR2，和
SEQ ID NO.3所示的VH-CDR3；以及
包含以下三个互补决定区CDR的轻链可变区：
SEQ ID NO.4所示的VL-CDR1，
SEQ ID NO.5所示的VL-CDR2，和
SEQ ID NO.6所示的VL-CDR3；
其中，所述抗原结合片段的结构选自下组：(i)Fab片段；(ii)F(ab') 2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；或(vi)dAb片段。
在另一优选例中，所述特异性结合CD47蛋白的抗体的重链包括：具有SEQ ID NO.17所示的重链可变区；和所述特异性结合CD47蛋白的抗体的轻链包括：具有SEQ ID NO.18所示的轻链可变区。
在另一优选例中，所述抗人CD47抗体的重链可变区或轻链可变区各自包含1-6个突变，较佳地，1-4个突变。
在另一优选例中，所述抗人CD47抗体的重链可变区的突变选自下组：
H-I37V、H-P45L、H-I48V、H-T52S、H-T52L、H-T52I、H-T52H、H-T52A、H-T52G、H-T52N、H-T52Q、H-T64K、H-R94K、H-P101D、或其组合。
在另一优选例中，所述抗人CD47抗体的轻链可变区的突变选自下组：
L-L4M、L-I46L、L-V58I、L-T93N、或其组合。
在另一优选例中，所述特异性结合CD47蛋白的抗体的重链包括：具有SEQ ID NO.19所示的重链可变区；和所述特异性结合CD47蛋白的抗体的轻链包括：具有SEQ ID NO.20所示的轻链可变区。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CD47的结合亲和力的衍生序列。
在另一优选例中，所述的双特异性抗原结合分子是双特异性抗体。
在另一优选例中，所述的D1和/或D2为单链抗体(scFv)。
在另一优选例中，所述的双特异性抗原结合分子中，仅D2为单链抗体(scFv)。
在另一优选例中，所述的D1和D2通过接头连接，所述接头包括抗体恒定区。
在另一优选例中，所述的D1为抗人CEACAM5/6抗体。
在另一优选例中，所述的D2为抗CD47单链抗体(scFv)。
在另一优选例中，所述的抗CEACAM5/6人源化抗体包括：单链抗体、双链抗体、单克隆抗体、嵌合抗体、鼠源抗体、人源化抗体、和双特异性抗体。
在另一优选例中，所述的抗人CEACAM5/6抗体包含以下三个互补决定区CDR的重链可变区：
SEQ ID NO.1所示的VH-CDR1，
SEQ ID NO.2所示的VH-CDR2，和
SEQ ID NO.3所示的VH-CDR3；以及
包含以下三个互补决定区CDR的轻链可变区：
SEQ ID NO.4所示的VL-CDR1，
SEQ ID NO.5所示的VL-CDR2，和
SEQ ID NO.6所示的VL-CDR3。
在另一优选例中，所述的双特异性抗体包含选自表4所示的重链和/或轻链：
表4.
Figure PCTCN2022119437-appb-000001
Figure PCTCN2022119437-appb-000002
在本发明的第七方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、如本发明第五方面所述的抗体或如本发明第六方面所述的多特异性抗原结合分子；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在另一优选例中，所述的重组蛋白还包括靶向其他肿瘤靶点的蛋白或多肽药物。
在另一优选例中，所述蛋白或多肽药物包含具有调节免疫系统功能的细胞因子。
在另一优选例中，所述细胞因子包括野生型或突变型。
在另一优选例中，所述细胞因子选自下组：IL-1、IL-2、IL-7、IL8、IL-10、IL-12、IL-15、IL-18、IL-21、GM-CSF、IFNα/β/γ，OX40-L、4-1BB-L、CTLA-4、或其组合。
在本发明的第八方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合结构域的scFv区段特异性结合于人CEACAM5/6，并且所述scFv区段包含如本发明的第一方面所述的重链可变区和如本发明的第三方面所述的轻链可变区。
在本发明的第九方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明的第八方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、如本发明第五方面所述的抗体、或如本发明的第六方面所述的多特异性抗原结合分子、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
在本发明的第十一方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、如本发明第五方面所述的抗体、如本发明的第六方面所述的多特异性抗原结合分子、如本发明的第七方面所述的重组蛋白、如本发明的第八方面所述的CAR构建物、如本发明的第九方面所述的重组的免疫细胞、如本发明的第十方面所述的抗体药物偶联物、或其组合，所述活性成分用于
(a)制备检测试剂或试剂盒；
(b)制备预防和/或治疗人CEACAM5/6相关疾病的药物或制剂；和/或
(c)制备预防和/或治疗人CEACAM5/6相关的癌症或肿瘤的药物或制剂。
在另一优选例中，所述癌症或肿瘤选自下组：结肠癌、直肠癌、淋巴瘤、胰腺癌、肺癌、胃癌、肝细胞瘤、乳腺癌或甲状腺癌、或其组合。
在本发明的第十二方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、如本发明第五方面所述的抗体、如本发明第六方面所述的多特异性抗原结合分子、如本发明第七方面所述的重组蛋白、如本发明第八方面所述的CAR构建物、如本发明第九方面所述的重组的免疫细胞、如本发明第十方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物用于治疗肿瘤。
在另一优选例中，所述肿瘤为高表达人CEACAM5/6的肿瘤。
在本发明的第十三方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、如本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的多特异性抗原结合分子；
(3)如本发明第七方面所述的重组蛋白；或
(4)如本发明第八方面所述的CAR构建物。
在本发明的第十四方面，提供了一种载体，所述的载体含有如本发明的第十三方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第十五方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明的第十四方面所述的载体或基因组中整合有如本发明的第十三方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为哺乳动物细胞，较佳地，为人、鼠、羊、马、狗或猫的细胞，更佳地，为中国仓鼠卵巢细胞。
在本发明的第十六方面，提供了一种体外非诊断的检测(包括诊断性或非诊断性)样品中人CEACAM5/6蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明的第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CEACAM5/6蛋白。
在本发明的第十七方面，提供了一种治疗人CEACAM5/6相关疾病的方法，所述方法包括：
给需要的对象施用如本发明的第五方面所述的抗体、所述抗体的多特异性抗体、所述抗体的药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在另一优选例中，所述人CEACAM5/6相关疾病为CEACAM5/6高表达的肿瘤，优选为结直肠癌、胰腺癌、肺癌、胃癌、肝细胞瘤、乳腺癌和甲状腺癌。
在另一优选例中，所述人CEACAM5/6相关疾病为CEACAM5/6和CD47高表达的肿瘤，优选为结直肠癌。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了UM05-C9与人CEACAM5蛋白的ELISA。
图2显示了UM05-C9与CHO-CEACAM5细胞的结合。
图3显示了UM05-L9和双特异性抗体L34与食蟹猴(A)CEACAM5蛋白和(B)CEACAM6蛋白的ELISA。
图4显示了UM05-C9的细胞内化结果。
图5显示了CD47-CEACAM5/6双特异性抗体的不同结构组合。其中，绿色或橙色分别标志不同抗体的抗原结合片段(CD47抗体或CEACAM5/6抗体的VH/VL)。
图6显示了UM11-L34分子对(A)CEACAM5和(B)CD47分子分别进行的亲和力检测。
图7显示了UM11-L34分子对CEACAM5和CD47分子顺序进行的亲和力检测。
具体实施方式
本发明人经过广泛而深入地研究，通过大量筛选，首次开发了一种活性优异的抗CEACAM5的抗体，并且意外发现，所述的抗体竟然对CEACAM6也具有优异的结合活性。此外，本发明还首次提供了一种高效、安全的抗人CD47-CEACAM5/6的双特异性抗体、及其制备方法和用途。本发明的双特异性抗体对CEACAM5/6和CD47高表达的肿瘤细胞有更好的靶向和特异性杀伤作用，同时可以降低与红细胞和/或血小板的结合作用。具体地，本发明的抗CEACAM5/6和CD47双特异性抗体与CD47 +-CEACAM5/6 +肿瘤细胞结合选择性更强，与红细胞和血小板表面CD47蛋白结合更弱，通过调节人免疫功能治疗多种癌症。在此基础上完成了本发明。
术语
如本文所用，术语“抗体”指免疫球蛋白，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不同种型，即IgM、IgD、IgG、IgA和IgE，对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。IgG代表免疫球蛋白中最重要的一类，由于化学结构和生物功能差异，它又可以分为4个子类：IgG1、IgG2、IgG3和IgG4。轻链通过恒定区的不同分为κ或λ链。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
互补结合决定区(complement determine region，CDR)是决定抗体与抗原特异性结合的关键序列结构。重链和轻链各包含了3个CDR，分别命名为HCDR1、HCDR2和HCDR3，以及LCDR1、LCDR2和LCDR3。在一个给定的抗体的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等 人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际Immuno GeneTics database(IMGT)，以及基于loop结构位置的Chothia定义。
例如，根据不同的CDR确定方案，每一个CDR的残基如下所述。
表1 CDR定义方案
Figure PCTCN2022119437-appb-000003
注1：不同文献中的标注略有不同，特别是Chothia标注方案。
注2：除了contact标注方案使用Chothia或者Martin编号方案外，其他的标注方案都能与各种编号方案兼容。
注3：Chothia CDR-H1环的末端在使用Kabat编号惯例时根据环的长度在H32和H34之间变化。(这是因为Kabat编号方案将插入放置在H35A和H35B处)如果H35A和H35B都不存在时，那么CDR-H1结束在32位；如果只有H35A存在时，那么CDR-H1结束在33位；如果H35A和H35B同时存在，那么CDR-H1结束在34位。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
在一个实施方案中，本发明抗体的CDR通过Kabat规则确定边界，或通过IMGT规则确定，或通过其组合确定规则。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat、Chothia、AbM、Contact和IMGT方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员熟知的，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或IMGT定义的其余CDR残基可以被保守氨基酸残基替代。
如本文所用，术语“框架区”(FR)指插入CDR间的氨基酸序列，即指在单一物种 中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语″人框架区″是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
如本文所用，术语“单克隆抗体”或“mAb”指针对特定抗原的具有单一氨基酸组成的抗体分子，且不应理解为需要通过任何特定方法产生该抗体。单克隆抗体可由B细胞或杂交瘤的单一克隆产生，但还可为重组的，即通过蛋白工程产生。
如本文所用，术语“抗原”或“靶抗原”指能够由抗体或抗体样结合蛋白所结合的分子或分子的部分。该术语进一步指能够用于动物以产生能够与该抗原的表位结合的抗体的分子或分子的部分。靶抗原可具有一个或多个表位。对于每种由抗体或由抗体样结合蛋白识别的靶抗原，抗体样结合蛋白能够与识别靶抗原的完整抗体竞争。
如本文所用，术语“亲和力”理论上通过完整抗体和抗原间的平衡缔合来定义。本发明双抗的亲和力可以通过KD值(解离常数)(或其它测定方式)进行评估或测定，例如生物膜层干涉技术(Bio-layer interferometry BLI)，使用Biacore 8K测量确定。
抗CEACAM5/6抗体
CEACAM属于免疫球蛋白超家族黏附分子，其结构域高度糖基化，通常包括1-2个免疫球蛋白可变区样结构域(N domain)和0-6个免疫球蛋白恒定区样结构域。CEACAM蛋白位于细胞膜外，其中CEACAM1、CEACAM3和CEACAM4通过疏水跨膜结构域与细胞膜连接；CEACAM5-8则通过糖基磷酰肌醇连接到细胞膜上。这些细胞外域通常作为细胞(例如上皮、内皮、树突状和白细胞)间的粘附分子。在多种恶性肿瘤，如乳房、胰腺、卵巢、结肠、肺和胃腺肿瘤中CEACAM5/CEACAM6被发现呈过度表达，并且与肿瘤的侵袭性和转移有关。
如本文所用，“抗CEACAM5/6抗体”、“抗CEACAM5/CEACAM6抗体”是指本发明中能够与CEACAM5分子和CEACAM6分子有较强特异性结合的抗体，即所述抗体具有针对CEACAM5和CEACAM6分子的交叉反应性。
本发明提供了一种抗CEACAM5/6抗体(例如，UM05-L9)，其与CEACAM5分子有强结合外，亦与CEACAM6分子有较强结合。
优选地，所述抗体包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
所述的重链可变区(VH)具有选自下组的互补决定区VH-CDR：
SEQ ID NO.1所示的VH-CDR1，
SEQ ID NO.2所示的VH-CDR2，和
SEQ ID NO.3所示的VH-CDR3；
所述的轻链可变区(VL)具有选自下组的互补决定区VL-CDR：
SEQ ID NO.4所示的VL-CDR1，
SEQ ID NO.5所示的VL-CDR2，和
SEQ ID NO.6所示的VL-CDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留CEACAM5和/或CEACAM6结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
较佳地，本文所述抗体为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。所述人源轻链恒定区选自人κ或λ亚型的轻链恒定区；进一步优选地，所述人源重链恒定区为人IgG1的重链恒定区，所述人源轻链恒定区为κ链。
本发明中的抗体(抗CEACAM5/6的抗体)可以是全长蛋白(如IgG1,IgG2a,IgG2b或者IgG2c)，也可以是包含抗原抗体结合域或抗原结合片段的蛋白片段(例如Fab，F(ab'),sdAb,scFv片段)。
如本文所用，抗原结合片段的非限制性例子包括：(i)Fab片段；(ii)F(ab') 2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段；和(vii)由模拟抗体高变区的氨基酸残基组成的最小识别单位(例如，独立的互补性决定区(CDR)如CDR3肽) 或受约束的FR3-CDR3-FR4肽。在另一优选例，本发明的CEACAM5/6的抗体或其抗原结合片段选自下组：驼源单域抗体、scFv、scFv二聚体、BsFv、dsFv、dsFv2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab') 2、ds双功能抗体、纳米抗体、域抗体或双价域抗体。
本发明中的抗体(抗CEACAM5/6的抗体)可以是野生型蛋白，也可以是经过特定突变已达到某种特定效果的突变型蛋白，例如利用突变消除抗体的效应子功能。
本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向CEACAM5/6(例如人CEACAM5/6)的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO.7、11或13所示的氨基酸序列。
在另一优选例中，所述抗体的轻链可变区含有SEQ ID NO.8或12所示的氨基酸序列。
在另一优选例中，所述靶向CEACAM5/6的抗体的重链可变区(VH)氨基酸序列，和/或轻链可变区(VL)氨基酸序列如下表2所示：
表2.抗体编号及VH、VL的序列编号(SEQ ID NO.)
抗体编号 VH序列编号 VL序列编号
UM05-L9 7 8
UM05-C9 11 12
UM05-C9’ 13 12
其中，UM05-L9是人-鼠嵌合抗体，UM05-C9是人源化抗体，UM05-C9’是另一个版本的人源化抗体，三者的VH-CDR以及VL-CDR相同。
CD47及其抗体
CD47是45～50kD的跨膜糖蛋白，属于免疫球蛋白(Ig)超家族的成员。研究发现，肿瘤细胞上高表达的CD47与巨噬细胞上的配体SIRPα结合，使得SIRPα酪氨酸磷酸化，从而发出抑制性调节信号，抑制巨噬细胞的吞噬作用。对应的，阻断该通路可以解除巨噬细胞对肿瘤细胞吞噬的抑制作用，提高机体对肿瘤细胞的免疫反应，为肿瘤的免疫治疗提供一条新途径。
在以CD47-SIRPα轴为靶标的肿瘤治疗中，最主要的机制是巨噬细胞的激活，从而增强巨噬细胞对肿瘤细胞的吞噬作用。其次，阻断CD47可能进一步将巨噬细胞募集到肿瘤组织中，以及向肿瘤组织募集额外的免疫细胞的细胞因子和趋化因子，如单 核细胞趋化蛋白3(MCP-3)，这些细胞因子的分泌有助于CD47阻断疗法的功效。再次，靶向CD47-SIRPα的疗法也可能改变肿瘤中巨噬细胞的极化状态。巨噬细胞按其表型和功能活性又可以分为M1(Ⅰ型)和M2(Ⅱ型)两种，M1巨噬细胞能产生大量的促炎细胞因子，并介导抵抗细胞内寄生虫和抑制肿瘤生长；M2巨噬细胞则产生较少的促炎分子，参与组织损伤修复、血管生成和促进肿瘤生长。研究发现，阻断CD47-SIRPα后可显著增加小鼠微环境中的M1巨噬细胞肿，而小鼠M2巨噬细胞则没有显著的增加。最后，其它免疫细胞也可以响应CD47阻断疗法。SIRPα在骨髓免疫细胞上高度表达，因此它可能是骨髓谱系的关键调节剂。在小鼠中，CD47通过SIRPα+树突状细胞来调节抗原摄取，使用同源免疫活性肿瘤模型发现，CD47阻断的治疗效果取决于树突状细胞。通过刺激巨噬细胞或树突细胞的抗原呈递，靶向CD47-SIRPα轴的疗法可以促进肿瘤的适应性免疫应答。
CD47广泛表达于多种细胞，特别是在新生红细胞上高表达。因此靶向CD47的治疗性抗体有可能会引起贫血。另一方面，体内游离抗人CD47抗体的浓度也会由于红细胞对抗人CD47抗体的吸附作用而大大降低。
因此，开发新的治疗性抗人CD47抗体，使得其与肿瘤细胞表面CD47蛋白的结合选择性更强，与红细胞表面CD47蛋白结合更弱，成为新型治疗性抗人CD47抗体的目标之一。
本发明中优选的抗人CD47抗体包括中国专利202010240238.7中描述的抗人CD47抗体或其抗原结合片段，其通过引用并入本文(如本文所用，以代号UM03-C4表示，其重链可变区如SEQ ID NO.17；轻链可变区如SEQ ID NO.18所示)。该CD47抗体的IgG4亚型在本专利中以代号UM03-C4表示。此外，在该抗人CD47抗体基础上，还包括对其VH、VL区进行突变而得的衍生抗体人CD47抗体(如本文所用，以代号UM03-C4’表示)。所涉及的突变抗人CD47抗体包括如下突变或者下述突变的组合：
重链：H-I37V、H-P45L、H-I48V、H-R94K、H-T52R、H-T52S、H-T52L、H-T52I、H-T52H、H-T52A、H-T52N、H-T52Q；
轻链：L-L4M、L-I46L、L-V58I、L-T93N。
优选地，将抗人CD47抗体或其抗原结合域与抗人CEACAM5/6的抗体或其抗原结合域进行不同组合从而获得本发明中所述的双特异性抗体。
双特异性抗体
双特异性抗体(Bispecific Antibody，bsAb)是一种非天然抗体，它能同时靶向两种不同的抗原或蛋白，阻断两种不同的信号通路，激发具有特异性的免疫反应，其特异性和双功能性在肿瘤免疫治疗中的作用越来越重要，已成为当今世界抗体工程治疗肿瘤方面的研究热点。研究表明，双特异性抗体在肿瘤免疫治疗方面主要有介导免疫细胞对肿瘤的杀伤；结合双靶点，阻断双信号通路，发挥独特的或重叠的功能，可以有效防止耐药性；具有强特异性、靶向性和降低脱靶毒性；有效降低治疗成本等优势(摘自抗体圈)，因此采用双特异性抗体药物可以降低肿瘤细胞逃逸几率，清除肿瘤细胞， 提高疗效。
双特异性抗体可通过双杂交瘤细胞，化学偶联，重组基因等手段制备，其中重组基因技术在结合位点以及产量等方面灵活性强。据不完全统计，目前已有60多种双特异性抗体，根据其特点以及结构差异性，双特异性抗体结构主要有含Fc片段的双特异性抗体(IgG-like双特异性抗体，具有Fc介导的效应功能)和不含Fc片段的双特异性抗体(non-IgG-like双特异性抗体，通过抗原结合力发挥作用，具有分子量小、免疫原性低等优势)两种结构。
本发明还包括同时结合抗人CEACAM5/6抗体和其他靶点的抗体。较佳地，所述的其他靶点选自下组：CD47、CD73、CD47、CD3、CTLA4、PD-1、PD-L1、CD28、CD40、OX40、LAG3、DR5、TIM-3、TIGIT、VEGF、VEGFR、Ang2、CD39、CD73、GITR、或其组合。
在另一优选例中，本发明的双特异性抗体中结合其他靶点的抗体为抗人CD47抗体。
如本文所用，术语“双特异性抗体”、“双功能抗体”“本发明抗体”、“本发明双抗”“双抗”、“双功能融合抗体”、“抗人CEACAM5/6和CD47双特异性抗体”、“抗CEACAM5/6和CD47抗体”可互换使用，是指同时结合CEACAM5/6和CD47的双特异性抗体。
如本文所用，术语“接头(linker)”是指插入免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。
如本文所用，所述接头为柔性接头，优选为肽接头。合适的肽接头实例包括单甘氨酸(Gly)、或丝氨酸(Ser)残基，肽接头中氨基酸残基的标识和序列可随着肽接头中需要实现的次级结构要素的类型而变化。
本发明中提供了一种基于前述抗人CEACAM5/6抗体和抗人CD47抗体或其抗原结合片段而构建的双特异性抗体。这些双特异性抗体通过接头(linker)把抗人CEACAM5/6抗体和抗人CD47抗体或其抗原结合片段连接起来，组成对称或者不对称结构的多价抗体，达到可同时结合CEACAM5/6和CD47蛋白的效果。这里可以选取使用的接头序列包括：
(G)n、(GGGGS)n、(GGGSG)n、(GGSGG)n、
GGGGSGSAGSAAGSGEFGGGGSGGG(SEQ ID NO.65)、
SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO.66)、
KESGSVSSEQLAQFRSLD(SEQ ID NO.67)、
EGKSSGSGSESKST(SEQ ID NO.68)、
GSAGSAAGSGEF(SEQ ID NO.69)等。优选地，所述接头为(GGGGS)n，其中n为0-5之间的整数；或SSSSKAPPPSLPSPSRLPGPSDTPILPQ，或GGGSSSSKAPPPSLPSPSRLPGPSDTPILPQGGG；更优选地，接头为GGGSSSSKAPPPSLPSPSRLPGPSDTPILPQGGG。
在本发明的一个具体实施例中，优选的接头序列如SEQ ID NO.64中的119-152位或SEQ ID NO.66所示。
本发明所描述的抗人CEACAM5/6抗体和抗人CD47抗体或其抗原结合片段而构建的双特异性抗体可以还包含人源重链恒定区和/或人源轻链恒定区；更优选地，所述人源重链恒定区选自人IgG1、IgG2或IgG4的重链恒定区，所述人源轻链恒定区选自人κ或λ链的轻链恒定区；进一步优选地，所述人源重链恒定区为人IgG1的重链恒定区，所述人源轻链恒定区为κ链；进一步优选地，所述人源重链恒定区为人IgG1的重链恒定区可以携带本领域技术人员所通常知晓得突变，以增强或减弱IgG1抗体恒定区介导的生物学功能，包括ADCC、ADCP(Antibody-dependent cellular phagocytosis)、CDC和FcRn结合活性等。这些突变包括但不限于如文献(Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life.Front.Immunol.,2019,10:1296)中所列的突变：
表3 IgG1重链恒定区突变及其功能
Figure PCTCN2022119437-appb-000004
Figure PCTCN2022119437-appb-000005
根据上述技术，本发明人设计了不同结构形式的抗人CEACAM5/6-CD47双特异性抗体。优选地，组成的抗人CEACAM5/6-CD47双特异性抗体的序列如表4所示。
表4.抗人CEACAM5/6-CD47双特异性抗体的序列
Figure PCTCN2022119437-appb-000006
Figure PCTCN2022119437-appb-000007
与现有技术相比，本发明的抗人CEACAM5/6-CD47抗体与CEACAM5/6 +-CD47 +双阳性肿瘤细胞表面CD47蛋白的结合选择性更强，杀伤作用更强，与红细胞和血小板表面CD47蛋白结合更弱，并且对红细胞没有凝集作用。
在本发明中，本发明的双特异性抗体还包括其保守性变异体，指与本发明双抗的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
编码核酸和表达载体
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明的核酸(以及核酸组合)可用于在合适的表达系统产生本发明的重组抗体。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是 高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
较佳地，本发明还提供了包含本发明的载体的宿主细胞，优选地，所述宿主细胞是哺乳动物细胞，更优选为人、鼠、羊、马、狗或猫的细胞，进一步优选为中国仓鼠卵巢细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在早期培养条件中，双特异性抗体的表达量可达3.9g/L，纯度均在97％以上，且在培养过程中可以很好地代谢乳酸。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的双抗可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.肿瘤治疗剂(例如，顺铂)或任何形式的抗肿瘤药物等。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有本发明上述的抗人CEACAM5/6抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治 疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射(如腹膜内)、颅内注射、或腔内注射。
本发明的药物组合物可直接用于结合CEACAM5/6，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明的抗人CEACAM5/6抗体以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的抗人CEACAM5/6抗体还可与其他治疗剂一起使用。
在本发明中，可单独使用本发明的抗人CEACAM5/6抗体，通过调整给药方案以获得最佳目的反应。例如，单次给药，或在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
使用药物组合物时，是将安全有效量的本发明的抗人CEACAM5/6抗体施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
应用
本发明还提供了本发明抗体、双特异性抗体、抗体偶联物ADC、重组蛋白、和/或免疫细胞的用途，例如用于制备诊断制剂或制备药物。进一步地，本发明还提供了包含本发明的抗体或其抗原结合片段在制备药物或用于细胞疗法的细胞中的用途，所述药物或细胞用于预防和/或治疗通过增强免疫应答而受益的病症。
较佳地，所述的药物是用于预防和/或治疗与CEACAM5/6(CEACAM5蛋白或CEACAM6蛋白)表达或功能异常相关的疾病的药物。
在本发明的一个实施方案中，其中所述病症是癌症，优选地，所述病症是CEACAM5或CEACAM6高表达的肿瘤，优选为结直肠癌。
在另一优选例中，所述人CEACAM5/6相关疾病为CEACAM5/6和CD47同时高表达的肿瘤，优选为结直肠癌。
本发明的主要优点包括
(1)本发明的抗人CEACAM5/6抗体具有同时结合CEACAM5以及CEACAM6的交叉反应性；
(2)本发明的抗人CEACAM5/6和CD47双特异性抗体对CEACAM5/6和CD47高表达的肿瘤细胞有更好的靶向和特异性杀伤作用，同时可以降低与红细胞和/或血小板的结合作用。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明的抗体序列如表5所示：
表5.本发明的抗体序列
Figure PCTCN2022119437-appb-000008
Figure PCTCN2022119437-appb-000009
Figure PCTCN2022119437-appb-000010
Figure PCTCN2022119437-appb-000011
Figure PCTCN2022119437-appb-000012
Figure PCTCN2022119437-appb-000013
Figure PCTCN2022119437-appb-000014
Figure PCTCN2022119437-appb-000015
Figure PCTCN2022119437-appb-000016
Figure PCTCN2022119437-appb-000017
Figure PCTCN2022119437-appb-000018
实施例1：人源化CD47单克隆抗体的突变
人源化CD47单克隆抗体的母本序列引自中国专利202010240238.7，其重链可变区序列如SEQ ID NO:17所示；其轻链可变区序列如SEQ ID NO:18所示。该人源化CD47单克隆抗体的IgG4亚型在本专利中以UM03-C4代号表示。
UM03-C4重链VH：
Figure PCTCN2022119437-appb-000019
UM03-C4轻链VL：
Figure PCTCN2022119437-appb-000020
其中，CD47抗体的重链和轻链中以Kabat定义的CDR序列用下划线标出。
此外，在该抗人CD47抗体基础上，还包括对其VH、VL区进行突变而得的衍生抗体人CD47抗体(如本文所用，以代号UM03-C4’表示，其重链可变区序列如SEQ ID NO:19所示；其轻链可变区序列如SEQ ID NO:20所示)。所涉及的突变抗人CD47抗体包括如下突变或者下述突变的组合：
重链：H-I37V、H-P45L、H-I48V、H-R94K、H-P101D、H-T64K、H-T52R、H-T52S、H-T52L、H-T52I、H-T52H、H-T52A、H-T52G、H-T52N、H-T52Q；
轻链：L-L4M、L-I46L、L-V58I、L-T93N。
其中，M18的突变都是在FR区，M19、M20和M21是在M18基础上添加了CDR的1个或两个突变。
部分突变对CD47单抗的活性影响如下表6所列：
表6 CD47单抗突变及其功能影响
Figure PCTCN2022119437-appb-000021
注：位置编号均根据Kabat规则。
实施例2：人源化CEACAM5/6单克隆抗体的获得
2.1小鼠抗人CEACAM5/6单克隆抗体的获得
本发明人构建了过表达人CEACAM5蛋白的CHO细胞株，以之免疫小鼠。取免疫小 鼠的脾细胞与SP2/0-AG14细胞进行杂交瘤细胞融合，并取适量融合后的细胞铺至96孔板。融合后第10天取各孔上清，以ELISA法检测杂交瘤细胞分泌的小鼠抗体与人CEACAM5的结合活性(方法参见实施例3)，得到了一系列具有较高活性的杂交瘤细胞。
选择其中活性优异的杂交瘤细胞，其中，出乎意料的是一株抗体居然对CEACAM6也具有优异的结合活性。
经测序获得其分泌抗体对应的重链可变区cDNA序列和轻链可变区cDNA序列，其编码的重链可变区氨基酸序列如SEQ ID NO.7所示；编码的轻链可变区氨基酸序列如SEQ ID NO.8所示。将小鼠抗体的重链可变区和轻链可变区分别连接至人IgG1重链的恒定区和κ链的恒定区，得到人-鼠嵌合抗体UM05-L9，其重链序列如SEQ ID NO.9所示，轻链序列如SEQ ID NO.10所示。
2.2小鼠抗人CEACAM5/6抗体的人源化
对实施例2中得到的小鼠抗体UM05-L9的重链可变区和轻链可变区序列进行分析，得到其重链互补决定区CDR如下表所示：
表7小鼠CEACAM5/6单抗UM05-L9的CDR区界定
  Kabat定义 IMGT定义
H-CDR1 DYAMH GYTFTDYA
H-CDR2 VISTYSGHTNYNQKFKG ISTYSGHT
H-CDR3 GSTTAHYYTMDF VRGSTTAHYYTMDF
L-CDR1 GASENIYGTLN ENIYGT
L-CDR2 GATNLAD GAT
L-CDR3 QNVLSIPYT QNVLSIPYT
同时参考Kabat定义和IMGT定义的CDR。搜索人germline抗体序列数据库(IGMT和IgBlast)，分别获得与小鼠抗体重/轻链可变区同源性较高的人germline抗体序列，并将其框架区与上述小鼠抗体的CDR进行组合，即所谓CDR grafting，同时对框架区的部分氨基酸进行了回复突变，最终获得了人源化抗体UM05-C9和UM05-C9’，其重链可变区序列分别如SEQ ID NO.11和SEQ ID NO.13所示；轻链可变区序列则两者相同，如SEQ ID NO.12所示。
实施例3：抗体UM05-C9/UM05-L9与CEACAM家族蛋白的结合
3.1抗体与人CEACAM5蛋白的结合
将浓度为1μg/mL的人CEACAM5蛋白溶液以100μL/well包被96孔高亲和力板，4℃，振荡过夜。第二天先以300μL PBST(Tween20：0.5‰)洗涤3次，之后用100μL/well的5％BSA/PBS封闭1小时，室温振荡。300μL PBST洗涤3次。用PBS配制抗体样品的梯度稀释溶液。以100μL/孔加入96孔板，室温振荡1小时。300μL PBST洗涤3次。配制二抗羊抗人(goat anti-human)IgG HRP溶液，以100μL/孔加入96孔板，室温振荡30min。300μL PBST洗涤4次。加入100μL/孔TMB，显色20min。加入100μL/孔0.6N H 2SO 4，终止显色，检测OD 450nm。
经检测，结果如图1所示，人源化抗体UM05-C9与人CEACAM5结合的EC50为0.399nM。
3.2抗体与CHO-CEACAM5细胞的结合
用FACS检测抗体与过表达人CEACAM5的CHO细胞的结合情况。简单来说，首先收集细胞，PBS洗涤一遍后计数，稀释成2×10 6/ml细胞悬液；分别取10μl抗体工作液加入100μl细胞悬液中，4℃避光孵育30min；PBS洗涤2次后，加入对应的荧光标记二抗Goat-anti-Human IgG(H+L)(Invitrogen)，4℃避光孵育30min，PBS洗涤2次后，以400μl FACS buffer悬起，以流式细胞仪检测抗体与细胞的结合情况。
检测结果如图2所示，UM05-C9与CHO-CEACAM5结合的EC50为636.9ng/mL。
3.3UM05-L9的亲和力检测
抗原抗体结合的亲和力用SPR技术(Surface Plasmon Resonance)进行检测。简单来说，将4μg/mL浓度的嵌合抗体UM05-L9与protein A sensor chip(GE,Cat#29127556)孵育30s进行抗体捕获。在抗原结合阶段，以梯度稀释的人CEACAM5蛋白为流动相与sensor chip上捕获的UM05-L9抗体进行120s的结合。在解离阶段，以HBS-EP缓冲液持续洗脱360s。CEACAM5蛋白在sensor chip上与抗体结合的情况以Biacore T20x0(GE Healthcare)进行定量检测。检测结果如表8所示，UM05-L9的亲和力为0.68nM。
表8.UM05-L9与人CEACAM5的亲和力和动力学数据
受体 配体 ka(1/Ms) kd(1/s) KD(M)
CEACAM5 UM05-L9 6.54×10 4 4.45×10 -4 6.80×10 -9
3.4 UM05-L9抗体与人CEACAM分子家族其它分子的结合
进一步使用UM05-L9检测了其与CEACAM分子家族其成员的结合情况。使用了SW620-CEACAM1、SW620-CEACAM3、CHO-CEACAM6、CHO-CEACAM7、CHO-CEACAM8等稳转细胞株。检测方法如3.2中所述。结果如下表9所示。
结果显示，UM05-L9分子除与CEACAM5分子有强结合外，亦与CEACAM6分子有较强结合。而与其它被检测的分子，包括CEACAM1、3、7、8在内，都基本没有结合。由于CEACAM5和CEACAM6都是肿瘤细胞特异性高表达的分子，这提示UM05-L9(推测也包括UM05-C9分子)具有较好的肿瘤靶向性。
表9.UM05-L9与人CEACAM分子家族其它分子的结合
  UM05-L9 倍数(以LOVO细胞结合为基准)
LOVO 25653.4 1
SW620-CEACAM1 823.1 0.032
SW620-CEACAM3 646.7 0.025
  UM05-L9 倍数(以LOVO细胞结合为基准)
LOVO 9398.2 1
CHO-CEACAM6 4035.1 0.429
CHO-CEACAM7 375.8 0.040
CHO-CEACAM8 386.1 0.041
3.5 UM05-L9与食蟹猴CEACAM5和CEACAM6分子的结合
进一步使用UM05-L9以ELISA法检测了其与食蟹猴CEACAM5和CEACAM6蛋白的结合情况。检测方法参考3.1中所述。结果图3所示。
结果显示UM05-L9与食蟹猴的CEACAM5和CEACAM6分子都有较强结合，其结合 EC50分别为0.6784nM和0.4629nM。
本发明的CEACAM5/6抗体可以与食蟹猴CEACAM5/6蛋白结合的特性，为将相关抗体进行药物开发提供了动物模型研究的便利。
实施例4:UM05-C9的肿瘤组织特异性
UM05-C9的肿瘤组织特异性分析由四川夏派森医药科技有限公司采用上海芯超生物科技有限公司的多器官肿瘤组织芯片进行(Cat No：HOrgC120PG04，Lot No：XT19-008)。
对免疫组化所得的结果进行图像扫描及半定量分析显示，UM05-C9检测到结直肠癌肿瘤组织中有强染色，在部分肺癌和胰腺癌中亦有较强染色，而在对应的癌旁组织中都呈阴性或低表达(表10)。
表10 UM05-C9对多器官肿瘤组织芯片染色结果(部分)
Figure PCTCN2022119437-appb-000022
Figure PCTCN2022119437-appb-000023
实施例5：CEACAM5/6抗体UM05-C9的细胞内吞性质
实验步骤：A549用F12K+10％FBS+1％P/S培养基培养，按照5万/孔，200μl/孔接96孔板中。用SiteClick TM Antibody Azido Modification Kit(Invitrogen,Cat#S20026)和Click-iT TM pHrodo TM iFL Red sDIBO Alkyne for Antibody Labeling(Invitrogen,Cat#C20034)试剂盒按说明书标记抗体，并将标记好的抗体按照1：20、1：40、1：80、1：160、1：320、1：640用完全培养基稀释，至浓度为45、22.5、11.25、5.62、2.8、1.4μg/ml。将前一天接板的细胞去上清，然后将上述配制的抗体按照100μl/孔，加入到细胞中，在孵箱中继续孵育16h。用胰酶消化细胞，收集细胞，流式细胞法检测抗体内吞情况(Ex＝560nM，Em＝585nM)。抗体被细胞内吞后会有荧光被激发，而未被内吞的抗体不会有荧光被激发。
结果如图4所示。表明CEACAM5/6抗体结合到A549细胞后会发生内化(internalization)。
实施例6：CD47-CEACAM5/6双特异性抗体的设计
将抗人CD47抗体或其抗原结合域与抗人CEACAM5/6的抗体或其抗原结合域进行不同组合，可以组成双特异性抗体。进行了如图5所示的几种双抗结构的尝试。
由此得到的双抗组合如表11所示。
表11.CEACAM5/6-CD47双特异性抗体序列及类型
Figure PCTCN2022119437-appb-000024
Figure PCTCN2022119437-appb-000025
实施例7：双特异性抗体的制备
将编码抗体重链或轻链的cDNA序列连接在编码信号肽的序列之后，分别克隆到哺乳动物细胞表达载体pcDNA3.4上。将重链表达质粒和轻链表达质粒按2：1的摩尔比用Lipofectamine 2000转染试剂(Invitrogen)转染入HEK293细胞，并在37℃、5％二氧化碳条件下培养7天。收集培养液上清，并用Protein A亲和层析法提纯上清中的抗体。纯化后抗体的质量可以用SDS-PAGE电泳和分子筛层析等经典的蛋白分析方法分析。纯化后的抗体经PBS溶液透析和冷冻干燥浓缩后，保存于-20℃。
实施例8：双特异性抗体与CD47蛋白或CEACAM家族蛋白的结合
8.1双特异性抗体与CD47蛋白的结合
将浓度为1μg/mL的人CD47蛋白溶液以100μL/孔包被96孔高亲和力板，4℃，振荡过夜。第二天先以300μL PBST(Tween20：0.5‰)洗涤3次，之后用100μL/孔的5％BSA/PBS封闭1小时，室温振荡。300μL PBST洗涤3次。用PBS配制抗体样品的梯度稀释溶液。以100μL/孔加入96孔板，室温振荡1小时。300μL PBST洗涤3次。配制二抗羊抗鼠(goat anti-mouse)IgG HRP或羊抗人(goat anti-human)IgG HRP溶液，以100μL/孔加入96孔板，室温振荡1小时。300μLPBST洗涤4次。加入100μL/孔TMB，显色20min。加入100μL/孔0.6N H 2SO 4，终止显色，检测OD 450nm。
本发明涉及的抗体与CD47蛋白结合代表性数据如表12所示。
8.2双特异性抗体与CEACAM5蛋白的结合
双特异性抗体与人CEACAM5蛋白结合的检测方法同实施例5.1所描述，差别在于将包被蛋白改为1μg/mL的人CEACAM5蛋白溶液。数据如表12所示。
表12双抗与靶点结合活性
Figure PCTCN2022119437-appb-000026
Figure PCTCN2022119437-appb-000027
实施例9：双特异性抗体与CD47 +-CEACAM5 +的人肿瘤细胞A549的结合
肺癌细胞A549(ATCC)是同时表达CD47和CEACAM5的人源肿瘤细胞。本申请发明人在A549细胞上使用流式细胞术检测了抗体的结合。用PBS配制待检测抗体的浓度梯度溶液，配制成终浓度的2×工作液。收集A549细胞，PBS洗涤一遍后计数，稀释成4×10 6/ml细胞悬液；取50μL抗体工作液加入50μL细胞悬液中，4℃避光孵育60min；PBS洗两遍后加入对应的荧光标记二抗(anti-huIgG 633)，4℃避光孵育30min，PBS洗涤两次后，以400μL FACS buffer悬起，以流式细胞仪检测抗体与细胞的结合情况。数据如表13所示。
表13双抗体与人肿瘤细胞A549的结合EC50(nM)
抗体代号 EC50(nM)
UM03-C4 2.284
UM05-L9 20.69
L5* >100
L7* >100
L8 4.52
L9* >100
L10 21.59
L11 12.85
L12 10.48
L13 16.27
L14* ～100
L15* ～100
L16* ～100
L17 7.477
L18 7.336
L20* >100
L21* >100
L22* >100
L23 32.39
L25 79.58
L26 7.857
L27 24.82
L28 23.74
L29 13.24
L30 13.45
L31 7.912
L32 10.29
L33 9.172
L34 16.83
L35 13.48
L36 7.935
L37 7.463
L38 8.148
L39 9.050
L40 NA
L41 NA
*表示这些分子在检测浓度范围内，检测信号没有达到平台，无法准确计算EC50。“NA”代表未做检测。
实施例10：双特异性抗体与人红细胞的结合及对人红细胞的凝集作用
本发明人检测了本发明所设计的部分双特异性抗体以及阳性对照抗体Hu5F9(美国专利US 9017675B2及文献PLoS ONE 2015,10(9):e0137345)与人红细胞的结合以及对人红细胞的凝集作用。首先从志愿者外周血中分离红细胞，用生理盐水悬成1％浓度红细胞悬液。用PBS配制抗体的浓度梯度溶液，配制成终浓度的2×工作液。取50μL抗体工作液加入50μL红细胞悬液中，4℃避光孵育60min；PBS洗两遍后加入对应的荧光标记二抗，4℃避光孵育30min，PBS洗涤两次后，以400μL FACS buffer悬起，以流式细胞仪检测抗体与红细胞的结合情况。对于红细胞凝集检测，红细胞用生理盐水悬成2％浓度悬液，同样取50μL抗体工作液加入50μL红细胞悬液中，置于96孔圆底板，室温静置2h后观察拍照。数据如表14所示。
表14双特异性抗体与人红细胞的结合和凝集作用
抗体代号 结合(以Hu5F9 凝集(以Hu5F9
  为参照) 1 为参照) 2
Hu5F9 +++ +++
L5 + -
L7 + ++
L8 ++ ++
L9 + -
L10 +++ -
L11 ++ -
L12 +++ -
L13 +++ -
L14 ++ -
L15 +++ -
L16 +++ -
L17 +++ -
L18 + ++
L20 ++ -
L21 ++ -
L22 ++ -
L23 - -
L25 ++ +
L26 +++ -
L27 - ++
L28 - +
L29 - -
L30 - -
L31 - -
L32 - -
L33 - -
L34 - -
L35 - -
L36 NA -
L37 NA -
L38 - -
L39 - -
L40 ++++ ++
L41 - -
注1：“+”个数代表与人红细胞的结合强弱。“-”代表在最高浓度为100nM的抗体浓度检测范围内，未检测到抗体与红细胞的明显结合。“NA”代表未做检测。
注2：“+”个数代表抗体引起人红细胞凝聚的能力。“-”代表在最高浓度为200μg/mL的抗体浓度检测范围内，抗体未引起明显的红细胞凝集。“NA”代表未做检测。
实施例11：双特异性抗体抑制A549细胞上CD47与SIRPα蛋白的结合
肺癌细胞A549(ATCC)是同时表达CD47和CEACAM5的人源肿瘤细胞。本申请发明人在A549细胞上使用流式细胞术检测了抗体抑制A549细胞上CD47与SIRPα蛋白的结合。用PBS配制待检测抗体的浓度梯度溶液，配制成终浓度的2×工作液。收集A549细胞，PBS洗涤一遍后计数，稀释成4×10 6/ml细胞悬液；取50μL抗体工作液加入50μL 细胞悬液中，4℃避光孵育60min；PBS洗两遍后加入human-SIRPa-mouse-Fc tag蛋白(SIA-H52A8)，4℃避光孵育30min；PBS洗两遍后加入羊抗鼠IgG1-Fc特异性检测二抗(115-095-071)，4℃避光孵育30min；PBS洗两遍后以400μL FACS buffer悬起，以流式细胞仪检测标记二抗的结合情况，得到抗体抑制A549细胞上CD47与SIRPα蛋白的结合的活性，结果如表15所示。
表15双特异性抗体阻断A549细胞上CD47-SIRPα结合的活性
抗体代号 抑制(以UM03-C4为参考)
UM03-C4 +++
L8 +++
L10 +++
L11 +
L12 +++
L13 ++
L14 +++
L15 +++
L16 +++
L17 ++
L18 +
L23 +
L25 +++
L26 +++
L27 ++
L28 ++
L29 +
L30 +
L31 +
L32 +
L33 +
L34 ++
L35 +
L36 +
L37 +
L38 +
L39 +
L40 +++
L41 +++
注：“+”个数代表抗体阻断A549细胞上CD47与SIRPα结合的能力。
实施例12：双特异性抗体的ADCC
本申请发明人在A549细胞和人红细胞上检测了部分抗体对两个细胞的ADCC杀伤作用。收集A549细胞，培养基重悬计数，稀释成1.5×10 5/ml的细胞悬液，A549细胞以50ul/孔提前一天铺板，次日弃去培养基将配置好的抗体梯度稀释液加入孔板中，37℃细胞培养箱孵育1小时；PBS洗两遍后，加入效应细胞Jurkat-NFAT-luc/CD16A，细胞计数稀释成 3×10 6/ml的细胞悬液，以30ul/孔加入孔板中，继续放入37℃细胞培养箱孵育4小时，后加入30ul/孔One-Glo，等待3min后用酶标仪检测生物发光强度。对红细胞杀伤作用的检测，首先从志愿者外周血中分离红细胞，以培养基洗2遍之后计数，配置成1×10 7/mL细胞悬液，取50ul/well细胞悬液与2×的抗体梯度稀释液50ul混合后置于37℃细胞培养箱孵育1小时；PBS洗两遍后，加入效应细胞Jurkat-NFAT-CD16A，细胞计数稀释成3×10 6/ml的细胞悬液，以30ul/孔加入孔板中，继续放入37℃细胞培养箱孵育4小时，后加入30ul/孔One-Glo，等待3min后用酶标仪检测生物发光强度。检测出结果如下表16所示。
表16双特异性抗体对A549及RBC细胞的ADCC作用
Figure PCTCN2022119437-appb-000028
注：“+”个数定性的代表抗体介导的对靶细胞的ADCC活性。
“NA”代表未检测。
实施例13双特异性抗体的ADCP效应
本申请发明人在A549细胞和人红细胞上检测了部分抗体对两个细胞的ADCP作用。收集A549细胞，培养基重悬计数，稀释成1×10 5/ml的细胞悬液，A549细胞以50ul/孔提前一天铺板，次日弃去培养基将配置好的抗体梯度稀释液加入孔板中，37℃细胞培养箱孵育1小时；PBS洗两遍后，加入效应细胞Jurkat-NFAT-luc/CD32A，细胞计数稀释成3×10 6/ml的细胞悬液，以30ul/孔加入孔板中，继续放入37℃细胞培养箱孵育4小时，后加入30ul/ 孔One-Glo，等待3min后用酶标仪检测生物发光强度。结果如表17所示。
表17双特异性抗体对A549及RBC细胞的ADCP作用
Figure PCTCN2022119437-appb-000029
注：“+”个数定性的代表抗体介导的对靶细胞的ADCP活性。
“NA”代表未检测。
实施例14：抗原抗体亲和力检测
用SPR技术(Surface Plasmon Resonance)分别检测了L34分子与人CEACAM5和人CD47的亲和力。简单来说，将4μg/mL浓度的抗体溶液与protein A sensor chip(GE,Cat#29127556)孵育30s进行抗体捕获。在抗原结合阶段，分别以人CEACAM5蛋白和人CD47蛋白为流动相与sensor chip上捕获的抗体进行120s的结合。在解离阶段，以HBS-EP缓冲液持续洗脱360s。抗原在sensor chip上与抗体结合的情况以Biacore 8K进行定量检测。结果如图6和表18所示。
表18 L34分子与人CEACAM5和人CD47蛋白的亲和力
受体 ka(1/Ms) kd(1/s) KD(M)
CD47 6.86×10 5 4.51×10 -2 6.57×10 -8
CEACAM5 2.33×10 4 1.73×10 -4 7.43×10 -9
上述亲和力检测结果显示，以L34分子为代表的双抗分子对人CD47靶点的亲和力显著弱化，而保持了与CEACAM5的亲和力。
在上述亲和力检测的基础上，用SPR技术还检测了两个抗原与双抗结合的表位竞争情况，方法同前所述。在本实验中，先将抗体溶液与protein A sensor chip孵育30s进行抗体捕获。在抗原结合阶段，先后以CEACAM5蛋白和CD47蛋白为流动相与sensor chip上捕获的抗体进行120s的结合。抗原在sensor chip上与抗体结合的情况以Biacore 8K进行定量检测。结果如图7所示。
实施例15：小鼠肿瘤药效模型
A549细胞(ATCC，人非小细胞肺癌细胞)为CD47 +-CEACAM +的肿瘤细胞，按6×10 6细胞/只的量将A549细胞接种于雌性NOD-Scid小鼠皮下。待肿瘤长至约70mm 3后，静脉给予配制在生理盐水中的抗体溶液。每组6只小鼠，每3天给药一次，共给药3周。
实施例16：食蟹猴的药物代谢和毒理
对食蟹猴(n＝2)静脉注射抗体溶液，剂量为20mg/kg。在给药前后0、0.25、4、8、24、48、72、96、144、192、240和336小时采血，ELISA法检测血药浓度，并做药时曲线图。同时计算药代动力学参数。
讨论
除了已知的CEACAM5被用作肿瘤标记，测量癌症患者血液中升高的CEACAM5的免疫学测定法已在临床上用于癌症的预后和控制之外，更重要的是，CEACAM5已成为用于靶向治疗的潜在有用的肿瘤相关抗原。已经报道的使用CEACAM5靶向免疫治疗癌症主要有2种主要方法。一种方法使用抗CEACAM5抗体引发免疫细胞的溶解活性，特别是通过抗体依赖性细胞毒性(ADCC)或补体依赖性细胞毒性(CDC)，以消除表达CEACAM5的肿瘤细胞；另一种方法是通过抗CEACAM5抗体或抗体片段与例如药物、毒素、放射性核苷酸、免疫调节剂或细胞因子等效应分子缀合，特异性靶向表达CEACAM5的肿瘤细胞，从而发挥效应分子的治疗作用。鉴于CEACAM5更多地过量表达于一些诸如结肠直肠癌、胰腺癌、肺癌、胃癌、肝细胞瘤、乳腺癌和甲状腺癌等实体肿瘤中，因此当前的研究集中在抗CEACAM5抗体的抗原识别能力。
总之，当前的研究表明，靶向CEACAM5的治疗方法将有助于抑制肿瘤的转移过程。因此，本发明提供的对CEACAM5和CEACAM6具有亲和力的抗CEACAM5/6抗体，具有重要的应用价值。此外，本发明中的抗人CEACAM5/6和CD47双特异性抗体对CEACAM5/6和CD47高表达的肿瘤细胞具有更好的靶向和特异性杀伤作用外，还可以降低与红细胞和/或血小板的结合作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种抗人CEACAM5/6抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区：
    SEQ ID NO.1所示的VH-CDR1，
    SEQ ID NO.2所示的VH-CDR2，和
    SEQ ID NO.3所示的VH-CDR3；其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CEACAM5/6的结合亲和力的衍生序列。
  2. 一种抗人CEACAM5/6抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗人CEACAM5/6抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.4所示的VL-CDR1，
    SEQ ID NO.5所示的VL-CDR2，和
    SEQ ID NO.6所示的VL-CDR3；其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CEACAM5/6的结合亲和力的衍生序列。
  4. 一种抗人CEACAM5/6抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗人CEACAM5/6抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链。
  6. 一种多特异性抗原结合分子，其特征在于，包含：
    第一抗原结合域D1；和
    第二抗原结合域D2；
    其中，D1特异性结合靶分子CEACAM5/6蛋白；
    D2特异性结合靶分子CD47蛋白；
    所述D1为特异性结合CEACAM5/6蛋白的抗体或其抗原结合片段；
    所述D2为特异性结合CD47蛋白的抗体或其抗原结合片段；
    其中，所述D1具有包含以下三个互补决定区CDR的重链可变区：
    SEQ ID NO.1所示的VH-CDR1，
    SEQ ID NO.2所示的VH-CDR2，和
    SEQ ID NO.3所示的VH-CDR3；以及
    包含以下三个互补决定区CDR的轻链可变区：
    SEQ ID NO.4所示的VL-CDR1，
    SEQ ID NO.5所示的VL-CDR2，和
    SEQ ID NO.6所示的VL-CDR3；
    其中，所述抗原结合片段的结构选自下组：(i)Fab片段；(ii)F(ab') 2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；或(vi)dAb片段。
  7. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体或如权利要求6所述的多特异性抗原结合分子；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  8. 一种CAR构建物，其特征在于，所述的CAR构建物的抗原结合结构域的scFv区段特异性结合于人CEACAM5/6，并且所述scFv区段包含如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区。
  9. 一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如权利要求8所述的CAR构建物。
  10. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体、或如权利要求6所述的多特异性抗原结合分子、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、酶、或其组合。
  11. 一种活性成分的用途，其特征在于，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体、或如权利要求6所述的多特异性抗原结合分子、如权利要求7所述的重组蛋白、如权利要求8所述的CAR构建物、如权利要求9所述的重组的免疫细胞、如权利要求10所述的抗体药物偶联物、或其组合，所述活性成分用于
    (a)制备检测试剂或试剂盒；
    (b)制备预防和/或治疗人CEACAM5/6相关疾病的药物或制剂；和/或
    (c)制备预防和/或治疗人CEACAM5/6相关的癌症或肿瘤的药物或制剂。
  12. 如权利要求11所述的用途，其特征在于，所述癌症或肿瘤选自下组：结肠癌、直肠癌、淋巴瘤、胰腺癌、肺癌、胃癌、肝细胞瘤、乳腺癌或甲状腺癌、或其组合。
  13. 一种药物组合物，所述的药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5 所述的抗体、或如权利要求6所述的多特异性抗原结合分子、如权利要求7所述的重组蛋白、如权利要求8所述的CAR构建物、如权利要求9所述的重组的免疫细胞、如权利要求10所述的抗体药物偶联物、或其组合；以及
    (ii)药学上可接受的载体。
  14. 一种多核苷酸，其特征在于，所述的多核苷酸编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体；或
    (2)如权利要求6所述的多特异性抗原结合分子；
    (3)如权利要求7所述的重组蛋白；或
    (4)如权利要求8所述的CAR构建物。
  15. 一种载体，其特征在于，所述的载体含有如权利要求13所述的多核苷酸。
  16. 一种体外的检测(包括诊断性或非诊断性)样品中人CEACAM5/6蛋白的方法，其特征在于，所述方法包括步骤：
    (1)在体外，将所述样品与如权利要求5所述的抗体接触；
    (2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CEACAM5/6蛋白。
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CN101607985A (zh) * 2008-12-24 2009-12-23 中国科学院生物物理研究所 抗人cea的单克隆抗体，包含其的组合物，及其用途
CN102482701A (zh) * 2009-09-16 2012-05-30 免疫医疗公司 I类抗-cea抗体及其使用
CN103403029A (zh) * 2011-03-02 2013-11-20 罗切格利卡特公司 抗cea抗体
CN108659131A (zh) * 2018-05-28 2018-10-16 长春力太生物技术有限公司 一种抗ceacam-5的单域抗体及其应用
CN112566938A (zh) * 2018-06-03 2021-03-26 拉姆卡普生物测试有限公司 针对ceacam5和cd47的双特异性抗体

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US20050147614A1 (en) * 2002-07-01 2005-07-07 Begent Richard J.H. Antibodies against tumor surface antigens
CN101607985A (zh) * 2008-12-24 2009-12-23 中国科学院生物物理研究所 抗人cea的单克隆抗体，包含其的组合物，及其用途
CN102482701A (zh) * 2009-09-16 2012-05-30 免疫医疗公司 I类抗-cea抗体及其使用
CN103403029A (zh) * 2011-03-02 2013-11-20 罗切格利卡特公司 抗cea抗体
CN108659131A (zh) * 2018-05-28 2018-10-16 长春力太生物技术有限公司 一种抗ceacam-5的单域抗体及其应用
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