WO2023125349A1 - 抗gucy2c抗体及其应用 - Google Patents

抗gucy2c抗体及其应用 Download PDF

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WO2023125349A1
WO2023125349A1 PCT/CN2022/141755 CN2022141755W WO2023125349A1 WO 2023125349 A1 WO2023125349 A1 WO 2023125349A1 CN 2022141755 W CN2022141755 W CN 2022141755W WO 2023125349 A1 WO2023125349 A1 WO 2023125349A1
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antibody
amino acid
antigen
acid sequence
seq
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PCT/CN2022/141755
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English (en)
French (fr)
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成广存
付雅媛
曹卓晓
唐任宏
任晋生
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山东先声生物制药有限公司
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Priority to CN202280074760.XA priority Critical patent/CN118215685A/zh
Publication of WO2023125349A1 publication Critical patent/WO2023125349A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present application relates to the field of antibodies, in particular, to anti-GUCY2C antibodies.
  • colorectal cancer patients are microsatellite-stable, with low tumor mutational burden and reduced immune infiltrate density, often accompanied by KRAS or BRAF oncogene mutations, resulting in colorectal cancer patients lacking response to currently approved immunotherapy, failing to show Significant survival benefit.
  • Guanylate cyclase C (GUCY2C or GCC) is a target widely expressed in colorectal cancer and other gastrointestinal tumors.
  • GUCY2C In normal tissues, GUCY2C plays an important role in maintaining intestinal fluid, electrolyte balance and cell proliferation.
  • the intracellular enzyme catalytic domain can bind GTP, catalyze the conversion of GTP to cGMP, and generate second messengers to affect downstream signaling pathways.
  • GUCY2C is expressed only in mucosal cells of the small intestine, large intestine, and rectum, and is expressed in all primary and metastatic colorectal tumors. Therefore, GUCY2C becomes an attractive target, and the development of drugs targeting GUCY2C has important clinical significance for the treatment of colorectal cancer.
  • the application provides an antibody or antigen-binding fragment thereof that specifically binds GUCY2C, wherein the antibody or antigen-binding fragment thereof comprises a VH with HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises SEQ ID NO.17 , 20, 22, 30, 33 or 35, or an amino acid sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequence;
  • the HCDR2 comprises The amino acid sequence shown in SEQ ID NO.18, 21, 23, 31, 34, 36, 52 or 53, or having 1, 2, 3 or more amino acid insertions, deletions and/or compared with said amino acid sequence and
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.19, 24, 32 or 37, or has 1, 2, 3 or more amino acid insertions, deletions and / or substituted amino acid sequences,
  • the antibody or its antigen-binding fragment comprises a VL with LCDR1, LCDR2 and LCDR3, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.25, 28, 38 or 41, or the same as the amino acid sequence Compared with an amino acid sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions;
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO.26, 29 or 39, or compared with the amino acid sequence Amino acid sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions;
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO.27 or 40, or has 1 compared with said amino acid sequence , an amino acid sequence of 2, 3 or more amino acid insertions, deletions and/or substitutions.
  • the present application provides a multispecific molecule comprising any Nanobody or antigen-binding fragment thereof described above.
  • the application provides a chimeric antigen receptor (CAR), which at least comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain Comprising any one of the above Nanobodies or antigen-binding fragments thereof.
  • CAR chimeric antigen receptor
  • the present application provides an immune effector cell, which expresses the above-mentioned chimeric antigen receptor, or comprises a nucleic acid fragment encoding the chimeric antigen receptor.
  • the present application provides a method for preparing any one of the above-mentioned Nanobodies or antigen-binding fragments thereof or multispecific molecules, which comprises culturing said host cells, and isolating the Nanobodies or nanobodies expressed by said cells. Antigen-binding fragments, or isolated multispecific molecules expressed by said cells.
  • any one of the above-mentioned Nanobodies or antigen-binding fragments thereof disclosed in the present application multispecific molecules, immune effector cells, nucleic acid fragments, vectors, host cells, and products prepared by any of the above-mentioned methods are also provided. or the use of the pharmaceutical composition in the preparation of drugs for the prevention and/or treatment of tumors; the tumors may be selected from colorectal cancer, gastric cancer, small intestine cancer, esophageal cancer, pancreatic cancer, lung cancer, soft tissue sarcoma and neuroendocrine tumors.
  • the present application provides a method for preventing and/or treating tumors, comprising administering to a patient in need thereof an effective amount of any of the aforementioned Nanobodies or antigen-binding fragments thereof, multispecific molecules, immune effectors Cells, nucleic acid fragments, vectors, host cells, products prepared by any of the above methods; or pharmaceutical compositions; wherein the tumor is selected from colorectal cancer, gastric cancer, small intestine cancer, esophageal cancer, pancreatic cancer, lung cancer, soft tissue Sarcomas and neuroendocrine tumors.
  • the present application also provides any of the above Nanobodies or antigen-binding fragments thereof, multispecific molecules, immune effector cells, nucleic acid fragments, vectors, host cells, and products prepared by any of the above methods; or A pharmaceutical composition for preventing and/or treating tumors; wherein the tumors are selected from colorectal cancer, gastric cancer, small intestine cancer, esophageal cancer, pancreatic cancer, lung cancer, soft tissue sarcoma and neuroendocrine tumors.
  • the present application provides a kit comprising any of the above-mentioned Nanobodies or antigen-binding fragments thereof, multispecific antibodies, immune effector cells, nucleic acid fragments, vectors, host cells, any of the above-mentioned methods Preparation of the obtained product, or pharmaceutical composition.
  • the present application provides a method for detecting the expression of GUCY2C, wherein, under the condition that a complex can be formed between any of the above-mentioned Nanobodies or their antigen-binding fragments and GUCY2C, the sample to be detected and any of the above-mentioned A Nanobody or antigen-binding fragment thereof is contacted.
  • the present application provides a method for inhibiting the proliferation or migration of GUCY2C-expressing cells in vitro, wherein, under the condition that a complex can be formed between any of the above Nanobodies or antigen-binding fragments thereof and GUCY2C, the The cells are contacted with any of the Nanobodies or antigen-binding fragments thereof described above.
  • the anti-GUCY2C antibody or antigen-binding fragment thereof provided in this application can specifically bind GUCY2C at the protein and cell levels, and has good affinity with human and monkey GUCY2C proteins, providing an excellent choice for the development of drugs targeting GUCY2C.
  • Figure 1 shows the detection of expression levels of 293T-hGUCY2C, CHO-cynoGUCY2C, CHO-hGUCY2C and 293T-mGUCY2C cell lines by flow cytometric analysis.
  • Fig. 2A is an ELISA detection of the binding reaction between mouse anti-GCC001 and human GUCY2C-his protein.
  • Fig. 3A is ELISA detection of the binding reaction between mouse anti-GCC001 and monkey GUCY2C-his protein.
  • Fig. 3B is ELISA detection of the binding reaction between mouse anti-GCC003 and monkey GUCY2C-his protein.
  • Fig. 4A is ELISA detection of the binding reaction between mouse anti-GCC001 and mouse GUCY2C-his protein.
  • Fig. 4B is ELISA detection of the binding reaction between mouse anti-GCC003 and mouse GUCY2C-his protein.
  • Figure 5A is the FACS detection of the binding reaction between mouse anti-GCC001 and 293T-hGUCY2C cells.
  • Figure 5B is the FACS detection of the binding reaction between mouse anti-GCC003 and 293T-hGUCY2C cells.
  • Fig. 6A is the FACS detection of the binding reaction between mouse anti-GCC001 and human tumor cell HT-55.
  • Fig. 6B is the FACS detection of the binding reaction between mouse anti-GCC003 and human tumor cell HT-55.
  • 7A-7B are ELISA detection of the binding of chimeric antibody to human GUCY2C-his protein.
  • 8A-8B are ELISA detection of the binding of chimeric antibody to monkey GUCY2C-his protein.
  • 9A-9B are ELISA detection of the binding of chimeric antibody to mouse GUCY2C-his protein.
  • Figures 10A-10B are FACS detection of chimeric antibody binding to 293T-hGUCY2C cells
  • 11A-11B are FACS detection of the binding of chimeric antibody to human tumor cell HT-55.
  • Figure 12 shows the epitope grouping among antibodies.
  • 13A-13B are ELISA method to detect the binding of h001 humanized antibody to human GUCY2C-his protein.
  • Fig. 14 is an ELISA method to detect the binding of h003 humanized antibody to human GUCY2C-his protein.
  • 15A-15B are ELISA method to detect the binding of h001 humanized antibody to mouse GUCY2C-his protein.
  • Fig. 16 is an ELISA method to detect the binding of h003 humanized antibody to mouse GUCY2C-his protein.
  • 17A-17B are ELISA method to detect the binding of h001 humanized antibody to monkey GUCY2C-his protein.
  • Figure 18 shows the detection of the binding of h003 humanized antibody to monkey GUCY2C-his protein by ELISA method.
  • 19A-19B are FACS detection of the binding of h001 humanized antibody to 293T-hGUCY2C cells.
  • Figure 20 is the FACS detection of the binding of h003 humanized antibody to 293T-hGUCY2C cells.
  • 21A-21B are FACS detection of the binding of h001 humanized antibody to tumor cell HT-55.
  • 22A-22B are FACS detection of the binding of h001 humanized antibody to CHO-cynoGUCY2C cells.
  • Figure 23 is the FACS detection of the binding of h003 humanized antibody to CHO-cynoGUCY2C cells.
  • 24A-24B are FACS detection of the binding of h001 humanized antibody to 293T-mGUCY2C cells.
  • Figure 25 is the FACS detection of the binding of h003 humanized antibody to 293T-mGUCY2C cells.
  • compositions including A and B should be understood as the following technical scheme: a composition composed of A and B, and a composition containing other components in addition to A and B, all fall into Into the scope of the aforementioned "a composition”.
  • GUI2c refers to mammalian guanylate cyclase C (GUCY2C), preferably human GUCY2C protein.
  • GUI2C may be used interchangeably with "STAR", “GUC2C", “GCC” or "ST receptor”.
  • the nucleotide sequence of human GUCY2C is disclosed as GenBank accession number NM_004963, and the amino acid sequence of human GUCY2C is disclosed as GenBank accession number NP_004954.
  • Naturally occurring allelic variants have an amino acid sequence that is at least 95%, 97%, or 99% identical to the protein described in GenBank Accession No. NP.Sub.-004954.
  • GUCY2C protein is a transmembrane cell surface receptor protein that plays an important role in maintaining intestinal fluid, electrolyte homeostasis and cell proliferation.
  • KD equilibrium dissociation constant
  • high affinity generally refers to having about 1 ⁇ 10 -6 M or lower, 1 ⁇ 10 -7 M or lower, about 1 ⁇ 10 -8 M or lower, about 1 ⁇ 10 -9 M or A KD of lower or about 1 ⁇ 10 ⁇ 10 M or lower.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg, Biacore) or equilibrium dialysis. For example, refer to the method for obtaining the KD value shown in Example 10 herein.
  • antigen binding molecule is used herein in the broadest sense to refer to a molecule that specifically binds an antigen.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimic refers to an organic compound or binding domain that can specifically bind to an antigen, but has nothing to do with the structure of an antibody.
  • antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical "four-chain antibody”, which belongs to the immunoglobulins composed of two heavy chains (HC) and two light chains (LC); In the N-terminal to C-terminal direction, it consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • CH1 domain a heavy chain constant region
  • HR hinge region
  • CH2 domain a heavy chain constant region CH2 domain
  • CH3 domain heavy chain constant region
  • the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain
  • the light chain is
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • IgA2 Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • an “antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , proto-ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).
  • Antibody herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies , conjugated antibody, chimeric antibody, humanized antibody or fully human antibody.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variants (such as containing naturally occurring mutations or arising during the manufacture of a formulation, such variants typically appear as In addition to being present in small amounts), the individual antibodies comprising the population are identical and/or bind the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), in monoclonal antibody preparations each monoclonal antibody is directed against a single determinant on the antigen.
  • monoclonal antibodies can be produced by a variety of techniques including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology and the use of transgenic animals containing all or part of the human immunoglobulin loci methods and other methods known in the art.
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.
  • multispecific herein refers to having at least two antigen binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen. combined.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • full-length antibody intact antibody
  • intact antibody intact antibody
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein, and do not possess the full structure of an intact antibody, but only include partial or partial variants of an intact antibody that possess the ability to bind Antigen capacity.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody and single domain antibody.
  • Papain digestion of intact antibodies yields two identical antigen-binding fragments, termed "Fab” fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is a Fab' fragment in which the cysteine residue of the constant domain bears a free thiol group. Pepsin treatment yields an F(ab')2 fragment with two antigen-combining sites (two Fab fragments) and part of the Fc region.
  • Fd refers to an antibody consisting of VH and CH1 domains.
  • Fv refers to an antibody fragment consisting of a single-armed VL and VH domain.
  • the Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a variable region (such as the Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although perhaps with a lower affinity than the full binding site.
  • scFv single-chain variable fragment
  • linker see, e.g., Bird et al., Science 242:423- 426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers that can be used in the present application are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al.
  • diabody herein, whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thus forcing the domains to The complementary domains of the other chain pair and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2 : 1121-1123 (1994)).
  • naked antibody refers to an antibody that is not conjugated to a therapeutic agent or tracer
  • conjugated antibody refers to an antibody conjugated to a therapeutic agent or tracer
  • Chimeric antibody herein refers to an antibody whose light chain and/or heavy chain are partly derived from an antibody (which may be derived from a specific species or belong to a specific class or subclass of antibodies). class), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the Binding activity to target antigen (U.S.P 4,816,567 by Cabilly et al; Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the antibody's heavy and light chain variable regions are derived from a primary antibody (e.g., a murine antibody), and the antibody's heavy and light chains are The light chain constant region is from a second antibody (eg, a human antibody).
  • a primary antibody e.g., a murine antibody
  • the light chain constant region is from a second antibody (eg, a human antibody).
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.
  • Fully human antibody refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region herein refers to the region in the heavy or light chain of an antibody that is involved in making the antibody bind to an antigen
  • “heavy chain variable region” is used interchangeably with “VH” and “HCVR”
  • “light chain variable region”” can be used interchangeably with “VL” and “LCVR”.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., p.91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). It can form a precise complementarity with the antigen epitope, so it is also called complementarity determining region.
  • the CDR of the variable region of the heavy chain can be abbreviated as HCDR
  • the CDR of the variable region of the light chain can be abbreviated as LCDR.
  • framework region or “FR region” are used interchangeably and refer to those amino acid residues in an antibody heavy chain variable region or light chain variable region other than the CDRs.
  • CDRs For a further description of CDRs, refer to Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest” (1991); Chothia et al. , J.Mol.Biol.196:901-917 (1987); Al-Lazikani B. et al., J.Mol.Biol., 273:927-948 (1997); MacCallum et al., J.Mol.Biol.262: 732-745 (1996); Abhinandan and Martin, Mol.
  • CDR herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health , Bethesda, Md., 1991).
  • IMGT numbering system herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol.27:55 -77, 2003.
  • IMGT ImMunoGeneTics information system
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • heavy chain constant region herein refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences.
  • the "heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • Heavy chain constant region includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • light chain constant region refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the carboxy-terminal part of the antibody obtained by papain hydrolysis of the whole antibody, which typically includes the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, thus the Fc region can comprise or excluding Lys447.
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • identity may be calculated by aligning said sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (for example, may be optimal alignment to introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between two sequences will vary with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or The PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5 or 6, determining the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.ncbi.nlm.nih.gov.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen-binding domain, such as an antibody The variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs are able to redirect T cells and other immune effector cells to a target of choice, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen-binding domain.
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules which are suitable as vectors for direct expression of the antibodies of the present application in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).
  • An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology, such as the progression of cancer, in the subject being treated.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject refers to an organism receiving treatment for a particular disease or condition as described herein.
  • subjects and patients include mammals, such as humans, primate (eg, monkeys) or non-primate mammals, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • the term "about” refers to all values within plus or minus 10% of the indicated numerical value. For example about 10 may refer to all values within the range of 9-11.
  • the present application provides an anti-GUCY2C antibody, a nucleic acid encoding the same, a method for preparing the antibody, a pharmaceutical composition containing the antibody, and related uses of the pharmaceutical composition for treating tumors.
  • the application provides an antibody or antigen-binding fragment thereof that specifically binds GUCY2C, wherein the antibody or antigen-binding fragment thereof comprises a VH with HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises SEQ ID NO.17 , 20, 22, 30, 33 or 35, or an amino acid sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the amino acid sequence;
  • the HCDR2 comprises The amino acid sequence shown in SEQ ID NO.18, 21, 23, 31, 34, 36, 52 or 53, or having 1, 2, 3 or more amino acid insertions, deletions and/or compared with said amino acid sequence and
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO.19, 24, 32 or 37, or has 1, 2, 3 or more amino acid insertions, deletions and / or substituted amino acid sequences,
  • the antibody or its antigen-binding fragment comprises a VL with LCDR1, LCDR2 and LCDR3, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO.25, 28, 38 or 41, or the same as the amino acid sequence Compared with an amino acid sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions;
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO.26, 29 or 39, or compared with the amino acid sequence Amino acid sequence having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions;
  • said LCDR3 comprises the amino acid sequence shown in SEQ ID NO.27 or 40, or has 1 compared with said amino acid sequence , an amino acid sequence of 2, 3 or more amino acid insertions, deletions and/or substitutions.
  • the substitutions are conservative amino acid substitutions.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in any one of groups (1)-(4):
  • the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO.17, 20 or 22; the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO.18, 21, 23, 52 or 53 , and the HCDR3 comprises the amino acid sequence shown in any sequence of SEQ ID NO.19 or 24; and/or, the LCDR1 comprises the amino acid sequence shown in any sequence of SEQ ID NO.25 or 28, and the LCDR2 comprises The amino acid sequence shown in any sequence of SEQ ID NO:26 or 29, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.27;
  • the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO.30, 33 or 35; the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO.31, 34 or 36, and the HCDR3 comprises the amino acid sequence shown in any sequence of SEQ ID NO.32 or 37; And/or, the LCDR1 comprises the amino acid sequence shown in any sequence of SEQ ID NO.38 or 41, and the LCDR2 comprises SEQ ID NO: 39 or the amino acid sequence shown in any sequence of 29, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO.40;
  • HCDR1, HCDR2 and HCDR3 derived from the heavy chain variable region shown in any one of SEQ ID NO.13, 15, 42, 47-51, 54, 57-60; and/or, derived from SEQ ID LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in any one of NO.14, 16, 43, 44, 45, 46, 55 and 56;
  • the heavy chain variable region comprises the amino acid sequence shown in any sequence of SEQ ID NO.13, 42, 47-51, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.
  • the heavy chain variable region comprises any sequence shown in SEQ ID NO.15, 54, 57-60 and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO.16, 55 or 56; or (3) the heavy chain variable region and/or the light chain
  • the variable region comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence shown in group (1) or (2) , 98%, 99% or 100% identity sequence; or, having at most 20, 19, 18, 17, 16 amino acid sequences compared with the amino acid sequence shown in group (1) or (2) , 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutated sequence; all The mutation can be selected from insertion
  • the heavy chain variable region and/or the light chain variable region are selected from VH and/or VL shown in Table 3 or Table 7; preferably, the heavy chain variable region and the The light chain variable regions were paired as shown in Table 4 or Table 8.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region may be selected from IgG, such as IgGl, IgG2, IgG3 or IgG4.
  • the IgG may be selected from human IgG, such as human IgG4.
  • the heavy chain constant region may be selected from an Fc region, a CH3 region or an entire heavy chain constant region.
  • the heavy chain constant region is a human Fc region.
  • the light chain constant region may be selected from a kappa chain or a lambda chain, preferably a kappa chain.
  • the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO.8, and/or the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO.10.
  • the human Fc region comprises the amino acid sequence shown in SEQ ID NO.12.
  • the antibody or antigen-binding fragment thereof specifically binds to human GUCY2C protein, preferably, its KD for binding to human GUCY2C protein is better than 1.00E-8M.
  • the antibody or antigen-binding fragment thereof is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.
  • the antibody or antigen-binding fragment thereof may be selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific molecules (e.g., bispecific antibodies), monovalent Antibody, multivalent antibody, intact antibody, fragment of intact antibody, naked antibody, conjugated antibody, chimeric antibody, humanized antibody, fully human antibody, Fab, Fab', Fab'-SH, F(ab')2 , Fd, Fv, scFv, diabody or single domain antibody.
  • monoclonal antibodies polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific molecules (e.g., bispecific antibodies), monovalent Antibody, multivalent antibody, intact antibody, fragment of intact antibody, naked antibody, conjugated antibody, chimeric antibody, humanized antibody, fully human antibody, Fab, Fab', Fab'-SH, F(ab')2 , Fd, Fv, scFv, diabody or single domain antibody.
  • the antibody or antigen-binding fragment thereof may also be coupled with a therapeutic agent or tracer; preferably, the therapeutic agent is selected from the group consisting of drugs, toxins, radioisotopes, chemotherapeutics or immunomodulators.
  • the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the present application provides a multispecific molecule comprising any of the above-mentioned antibodies or antigen-binding fragments thereof; preferably, the multispecific molecule further comprises an antigen specifically binding to GUCY2C or binding to an antigen other than GUCY2C An antibody or an antigen-binding fragment thereof that differs from any of the above-mentioned antibodies or antigen-binding fragments of the GUCY2C epitope.
  • the antigens other than GUCY2C are selected from the group consisting of: (1) tumor-specific antigen (TSA) or tumor-associated antigen (TAA); (2) immune checkpoint; (3) recruitment and/or Targets for activating immune cells.
  • TSA tumor-specific antigen
  • TAA tumor-associated antigen
  • immune checkpoint immune checkpoint
  • the application provides a chimeric antigen receptor (CAR), which at least comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain Comprising any one of the above-mentioned antibodies or antigen-binding fragments thereof.
  • CAR chimeric antigen receptor
  • the present application provides an immune effector cell, which expresses the above-mentioned chimeric antigen receptor, or comprises a nucleic acid fragment encoding the chimeric antigen receptor.
  • the immune effector cells are selected from T cells, NK cells (natural killer cell), NKT cells (natural killer T cell), DNT cells (double negative T cell), monocytes, macrophages, dendritic cells or mast cells, said T cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells.
  • the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
  • the present application provides an isolated nucleic acid fragment encoding any of the antibodies or antigen-binding fragments thereof, or the multispecific molecule, or the chimeric antigen receptor described above.
  • the present application provides a vector comprising the nucleic acid fragment.
  • the application provides a host cell comprising the vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (such as Escherichia coli), fungi (such as yeast), insect cells or mammalian cells (eg CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (such as Escherichia coli), fungi (such as yeast), insect cells or mammalian cells (eg CHO cell line or 293T cell line).
  • the present application provides a method for preparing any of the above-mentioned antibodies or antigen-binding fragments thereof or the multispecific molecule, which comprises culturing the cells, and isolating the antibodies or antigens thereof expressed by the cells Fragments are combined, or multispecific molecules expressed by said cells are isolated.
  • the present application provides a method for preparing the immune effector cells, which includes introducing the nucleic acid fragment encoding the CAR into the immune effector cells, and optionally, also including promoting the immune effector cells to express The CAR.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising any of the above-mentioned antibodies or antigen-binding fragments thereof, multispecific molecules, immune effector cells, nucleic acid fragments, vectors, host cells, or any of the above-mentioned Process for the preparation of the obtained product.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.
  • the pharmaceutically acceptable carrier is a carrier that does not weaken the viability and function of immune cells, and does not affect the specific binding of the antibody or its antigen-binding fragment to the antigen, including but not limited to cell culture medium, buffer, physiological saline and balanced salt solution wait.
  • buffers include isotonic phosphates, acetates, citrates, borates, carbonates, and the like.
  • the pharmaceutically acceptable carrier is phosphate buffer containing 1% serum.
  • any of the above-mentioned antibodies or antigen-binding fragments thereof disclosed in the present application multispecific molecules, immune effector cells, nucleic acid fragments, vectors, host cells, and products prepared by any of the above-mentioned methods are also provided, Or the application of the pharmaceutical composition in the preparation of medicines for preventing and/or treating tumors.
  • Said tumor may be selected from colorectal cancer, gastric cancer, small intestinal cancer, esophageal cancer, pancreatic cancer.
  • the present application provides a method for preventing and/or treating tumors, comprising administering an effective amount of any of the above-mentioned antibodies or antigen-binding fragments thereof, multispecific molecules, and immune effector cells to patients in need thereof , a nucleic acid fragment, a vector, a host cell, a product prepared by any of the above methods, or a pharmaceutical composition.
  • Said tumor may be selected from colorectal cancer, gastric cancer, small intestinal cancer, esophageal cancer, pancreatic cancer.
  • the present application also provides any of the above-mentioned antibodies or antigen-binding fragments thereof, multispecific molecules, immune effector cells, nucleic acid fragments, vectors, host cells, products prepared by any of the above-mentioned methods, or drugs
  • the composition is used for preventing and/or treating tumors.
  • Said tumor may be selected from colorectal cancer, gastric cancer, small intestinal cancer, esophageal cancer, pancreatic cancer.
  • the present application provides a kit comprising any of the above-mentioned antibodies or antigen-binding fragments thereof, multispecific molecules, immune effector cells, nucleic acid fragments, vectors, host cells, prepared by any of the above-mentioned methods The obtained product, or pharmaceutical composition.
  • GUCY2C recombinant protein Human GUCY2C protein (UniProt No.: P25092) was used as template sequence to design tagged fusion protein, cloned into pTT5 vector (Youbao Biology, VT2202), and GUCY2C plasmid was constructed and grown in Expi 293F cells (Gibco, A14527 ) was expressed transiently to obtain the antigen and protein for detection in this example.
  • the preparation method of cynomolgus monkey and mouse GUCY2C recombinant protein is the same as that of human recombinant protein.
  • the cynomolgus monkey GUCY2C sequence comes from Uniprot: A0A2K5TZ15, the mouse GUCY2C sequence comes from Uniprot: Q3UWA6, and the specific sequence information of the recombinant protein is as follows:
  • Cyno GUCY2C ECD his-tagged cynomolgus monkey GUCY2C protein extracellular domain fusion protein
  • Mouse GUCY2C ECD his-tagged mouse GUCY2C protein extracellular domain fusion protein (SEQ ID NO: 3):
  • control antibodies used in this example are all from published patents.
  • the PF1608 antibody was derived from the published international patent No. WO2019224716A2
  • the 5F9 antibody was derived from the published international patent No. WO2017136693A1.
  • the PF1608 and 5F9 control antibodies were recombinantly expressed using human IgG1+ ⁇ subtype.
  • the expression and purification process of the control antibody was as follows: the antibody sequence gene was synthesized and cloned into the expression vector pTT5, then transiently transfected into Expi293F cells (purchased from Gibco, A14527), cultured on a shaker at 37°C for 7 days, and the supernatant of the cells was collected for protein A
  • Expi293F cells purchased from Gibco, A14527
  • the resulting control antibodies were named PF1608-hIgG1 and 5F9-hIgG1.
  • the specific sequence information of the antibody is shown in Table 1.
  • step 1.1 After constructing and expressing the relevant recombinant protein according to step 1.1, purify it as follows: centrifuge the cell expression supernatant sample at high speed to remove impurities, equilibrate the nickel column with 20mM PBS+500mM NaCl solution, and wash 2-5 times the column volume.
  • the collected eluted products containing the target protein can be further purified by gel chromatography Superdex200 (GE) after concentration, and the mobile phase is PBS to remove aggregates and miscellaneous protein peaks, and collect the eluted peaks of the target product.
  • the obtained protein was identified as correct by electrophoresis, peptide map, and LC-MS, and then sorted for use.
  • the proteins purified by this protocol include human GUCY2C-His, monkey GUCY2C-His and mouse GUCY2C-His.
  • the cell culture supernatant expressing the antibody was collected by high-speed centrifugation.
  • Protein A affinity column was washed with 0.1M NaOH for 3-5 times of column volume, and then washed with pure water for 3-5 times of column volume.
  • 1 ⁇ PBS (pH7.4) buffer system as the equilibration buffer to equilibrate the chromatography column for 3-5 times of column volume.
  • the cell supernatant was loaded and combined at a low flow rate, and the flow rate was controlled so that the retention time was about 1 min or longer.
  • the chromatography column was washed with 1 ⁇ PBS (pH7.4) for 3-5 times the column volume until the UV absorption dropped back to the baseline.
  • solution replacement can be performed by methods well known to those skilled in the art, such as using ultrafiltration tubes for ultrafiltration concentration and solution replacement to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required A buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove aggregate components in the eluted product to improve sample purity.
  • Embodiment 2 stable transfection cell line construction
  • 293T cells were transfected with plasmids ( 3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015), and selectively cultured in DMEM medium containing 10 ⁇ g/ml puromycin for 2 weeks, with human GUCY2C antibody (5F9, self-produced) and anti-human IgG (H+ L) Antibody (Jackson, product number: 109-605-088) was used to sort positive monoclonal cells on a flow cytometer FACS AriaII (purchased from BD Biosciences) to a 96-well plate, and placed at 37 ° C, 5% (v/ v) CO 2 culture, select some monoclonal wells for expansion after about 2 weeks.
  • plasmids 3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015
  • human GUCY2C antibody 5F9, self-produced
  • anti-human IgG (H+ L) Antibody Jackson, product number: 109-605-088
  • the amplified clones were screened by flow cytometry. Cell lines with better growth, higher fluorescence intensity, and monoclonality were selected to continue to be expanded and cultured and frozen in liquid nitrogen. The obtained cell line was named: 293T-hGUCY2C, and the expression status detected by FACS is shown in A in FIG. 1 .
  • the nucleotide sequence encoding the full-length amino acid sequence of cynomolgus monkey GUCY2C (UniProt: A0A2K5TZ15) was cloned into pcDNA5-FRT vector (Invitrogen TM , V601020) and a plasmid was prepared. After plasmid transfection, the FlpinCHO cell line was selectively cultured in F12 medium containing 800 ⁇ g/ml hygromycin for 2 weeks, and the resulting cell line was named: CHO-cyno GUCY2C.
  • the GUCY2C positive control 5F9 antibody was used to detect the expression by FACS. The situation is shown in B in Figure 1.
  • the nucleotide sequence encoding the full-length amino acid sequence of human GUCY2C (UniProt: P25092) was cloned into the pcDNA5-FRT vector and a plasmid was prepared. After plasmid transfection of the FlpinCHO cell line, it was selectively cultured in F12 medium containing 600 ⁇ g/ml hygromycin for 2 weeks. The resulting cell line was named: CHO-hGUCY2C, and the positive control 5F9 antibody of GUCY2C was used to detect the expression by FACS. As shown in C in Figure 1.
  • the nucleotide sequence encoding the full-length amino acid sequence of mouse GUCY2C (Uniprot: Q3UWA6) was cloned into pcDNA3.1 vector and a plasmid was prepared. After plasmid transfection, the 293T cell line was selectively cultured in DMEM medium containing 10 ⁇ g/ml puromycin for 2 weeks, and the resulting cell line was named: 293T-mGUCY2C.
  • the GUCY2C positive control PF1608 antibody was used to detect the expression by FACS. As shown in D in Figure 1.
  • the monoclonal antibody of this example was produced by immunizing mice.
  • the experimental mice were 6-8 weeks old, female SPF Balb/C mice or SJL mice (purchased from Charles River).
  • the immunogen is the human GUCY2C-his protein prepared in Example 1 or the CHO-hGUCY2C stably transfected cell line prepared in Example 2.
  • the immunogen was emulsified with TiterMax (purchased from Sigma, T2684-1M), and then injected 0.1 mL subcutaneously (SC) and intraperitoneally (IP), respectively, that is, each mouse was injected with 50 ⁇ g of immunogen; Originally, 0.1 mL of Imject Alum (purchased from Thermo) was injected subcutaneously and intraperitoneally, that is, each mouse was injected with 25 ⁇ g of immunogen.
  • TiterMax purchased from Sigma, T2684-1M
  • IP intraperitoneally
  • 0.1 mL of TiterMax was emulsified with normal saline and injected intraperitoneally with 0.1 mL of cell suspension, that is, 1 ⁇ 10 7 cells were injected into each mouse after 15 minutes; 10 7 cell suspensions.
  • the frequency of immunization was once a week, and blood was collected on the 3rd, 19th, 47th, and 61st days.
  • the combination with human GUCY2C protein His tag, coating concentration 4 ⁇ g/mL was detected by ELISA method, and the presence and antibody titer of human GUCY2C antibody in the immunized animal serum were tested.
  • Spleen and lymph nodes were aseptically collected, ground and filtered with a 40 ⁇ m cell strainer (purchased from BD Falcon), and ACK Lysing Buffer (purchased from Gibco) was added to lyse the erythrocytes doped in the splenocytes to obtain a spleen cell suspension, which was washed with DMEM (purchased from Gibco) The cells were washed once by centrifugation at 1500 rpm from Gibco) basal medium.
  • DMEM purchased from Gibco
  • mice myeloma cells SP2/0 purchased from ATCC
  • Cytofusion Medium C purchased from BTX
  • BTX ECM2001+electrofusion method Refer to ECM2001+ELECTROFUSION PROTOCOL
  • the fused cells were diluted into DMEM medium containing 20% fetal bovine serum (purchased from ExCell Bio) and 1 ⁇ HAT (purchased from Sigma).
  • splenocytes were added to 96-well cell culture plates at 2 ⁇ 10 4 per well, and placed in a 37°C, 5% CO 2 incubator.
  • the supernatant on the cell fusion plate was screened by ELISA, and the positive clones detected by ELISA were amplified into 24-well plates for expanded culture. After culturing for 3 days, the culture fluid in the 24-well plate was taken, and the binding activity to human GUCY2C protein was determined by ELISA and FACS.
  • the optimal clones were selected, and the optimal clones were expanded and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO 2 , and frozen in liquid nitrogen.
  • the hybridoma cells of this example were obtained.
  • the supernatant of the hybridoma cells was taken, and the combination with human GUCY2C protein was detected by protein ELISA method, and positive clones were selected; the next day, the positive clones were detected by protein and cell ELISA methods and 293T-hGUCY2C cells, human GUCY2C cells Binding of -his protein, monkey GUCY2C-his protein and mouse GUCY2C-his protein.
  • the clones with higher positive signal in the primary screening were selected for subcloning, and the hybridoma antibodies after subcloning were detected again by the same method.
  • the hybridoma clone after strain identification was expanded and cultured, the supernatant was collected and centrifuged, and the antibody was purified according to the purification method described in Example 1.3.2.
  • two antibodies specifically binding to GUCY2C, GCC001 and GCC003, were obtained through screening.
  • Enzyme-linked immunosorbent assay was used to detect the binding of mouse monoclonal antibody to human, monkey and mouse GUCY2C-his proteins.
  • Human, monkey and mouse GUCY2C-his proteins were diluted with PBS to a final concentration of 2 ⁇ g/mL, and then added to 96-well ELISA plates at 50 ⁇ l per well. Seal with a plastic film and incubate at 4°C overnight, wash the plate twice with PBST the next day, add blocking solution [PBS+2% (w/w) BSA] to block at room temperature for 2 hours. Pour off the blocking solution, add 100nM as the initial concentration, 3-fold serial dilution of the antibody to be tested, and 50 ⁇ l of positive and negative control antibodies per well. After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST.
  • the purified GCC001 and The GCC003 antibody binds efficiently to human, monkey and mouse GUCY2C proteins.
  • the positive control antibody 5F9 recognized human and monkey GUCY2C, but not mouse GUCY2C.
  • the negative control antibody anti-hel-hIgG1 (purchased from Baiying, catalog number: B117901) had no binding activity.
  • FACS buffer PBS+2% fetal bovine serum
  • the binding of the antibody to the endogenous tumor cell HT55 is shown in Figure 6.
  • the GCC001 antibody can specifically bind to HT55 cells, while the GCC003 antibody has a weaker binding ability to HT55 cells, which is consistent with the trend of the PF1608 control antibody. Subsequent epitope competition analysis found that the difference in binding ability was due to different epitopes.
  • the hybridoma cells in the logarithmic growth phase were collected, and the cells were frozen with Trizol (Invitrogen, Cat No. 15596-018) according to the kit instructions, and NGS sequencing was performed.
  • the sequences of the murine anti-human GUCY2C antibodies GCC001 and GCC003 were obtained by sequencing, and the amino acid sequences of the heavy chain variable region (HCVR) and light chain variable region (LCVR) are as follows:
  • the CDR region of the above-mentioned GUCY2C monoclonal antibody was analyzed, wherein the CDR region was determined and annotated using the Kabat numbering system, the Chothia numbering system and the IMGT numbering system, and the specific results are shown in Table 2.
  • VH/VL gene fragments of each antibody were constructed by PCR.
  • the resulting VH/VL gene fragments were subjected to homologous recombination with the expression vector pTT5 to construct the full-length expression plasmid pTT5-VH-(G4S) 3 -VL-Fc of the recombinant chimeric antibody to form GCC001 ScFv-hFc and GCC003 ScFv-hFc chimeric antibodies .
  • the amino acid sequence of the human Fc region is shown in Table 1.
  • the plasmid was prepared, it was transfected into Expi293F cells, cultured on a shaker at 37°C for 7 days, the supernatant was collected, centrifuged, and the antibody was purified according to the purification method described in Example 1.3.2.
  • ELISA was used to detect the binding of chimeric antibody to human, monkey and mouse GUCY2C-his proteins.
  • the GUCY2C chimeric antibody was grouped by competition ELISA. Referring to the method of Example 3.5.1, 2 ⁇ g/mL chimeric antibody was used to coat the ELISA plate, human GUCY2C-his protein was serially diluted starting from 30 ⁇ g/mL, and EC80 was calculated as the antigen concentration in the competitive ELISA.
  • GCC001 ScFv-hFc is similar to the positive control 5F9-hIgG1 binding epitope
  • the antibody, GCC003 ScFv-hFc is an antibody with a similar binding epitope to the positive control PF1608-hIgG1.
  • the binding activity of antibodies similar to the binding epitope of PF1608 at the cellular level was significantly weaker than that of antibodies similar to the binding epitope of 5F9.
  • the heavy chain and mouse antibody with high homology were selected.
  • the light chain variable region germline gene is used as a template, and the CDRs of the mouse antibody are transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Back mutations and/or hotspot mutations were performed as needed.
  • the CDR sequences of this embodiment are divided according to the Kabat numbering system.
  • the humanized light chain templates of murine antibody GCC001 are IGKV3-15*01, IGKV4-1*01 and IGKJ2*01, the humanized heavy chain templates are IGHV1-46*01 and IGHJ6*01, and the murine antibody GCC001
  • the CDRs of the GCC001 were transplanted into the corresponding human templates, that is, the humanized antibody h001 of GCC001 was obtained, and its variable region sequence is as follows:
  • the key amino acids in the FR region sequence of the GCC001 humanized antibody were reverse-mutated to ensure the original affinity.
  • computer simulation was used according to the antibody structure Amino acid mutations were performed on NG to eliminate the risk of molecular modification.
  • the specific mutation design is shown in Table 3 (reverse mutations are in natural numbering order).
  • Graft means the mouse antibody CDR is implanted into the human germline FR region sequence; Graft+G72R means the 72nd G of Graft is mutated to R, and so on.
  • h001H1L1 means GCC001 humanized antibody h001 has the light chain variable region as described in L1 and the heavy chain variable region as described in H1, and so on.
  • amino acid sequences of the humanized heavy chain and light chain variable regions are shown in Table 5:
  • the humanized light chain templates of murine antibody GCC003 are IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-46*01 and IGHJ6*01.
  • the humanized antibody h003 of GCC003 was obtained.
  • the variable region sequence of h003 is as follows:
  • Graft means the mouse antibody CDR is implanted into the human germline FR region sequence; G72R means the 72nd G of Graft is mutated back to R, and so on.
  • h003H1L1 means GCC003 humanized antibody h003 has the light chain variable region as described in L1 and the heavy chain variable region as described in H1, and so on.
  • amino acid sequences of the heavy chain and light chain variable regions after humanization are shown in Table 9 below, wherein the sequences of the CDR regions are consistent with those before humanization.
  • the h003 humanized antibody was expressed in the scFv-hFc combination format, and the h001 humanized antibody was expressed in the human IgG1 format (see Table 1 for the Fc sequence, heavy chain constant region and light chain constant region sequence).
  • Expi293F cells were transiently transfected, and the supernatant was collected by centrifugation 7 days later, and the antibody was purified according to the purification method described in Example 1.3.2.
  • both h001 and h003 humanized antibodies can effectively bind to human GUCY2C-his protein, maintaining the binding ability of the chimeric antibody.
  • both h001 and h003 humanized antibodies can effectively bind to the mouse GUCY2C-his protein, maintaining the binding ability of the chimeric antibody.
  • both h001 and h003 humanized antibodies can effectively bind to the monkey GUCY2C-his protein, maintaining the binding ability of the chimeric antibody.
  • the h001 humanized antibody can specifically bind to the endogenous tumor cell HT55, maintaining the binding ability of the chimeric antibody.
  • both h001 and h003 humanized antibodies can efficiently bind to CHO-cynoGUCY2C cells.
  • the BIAcore 8K instrument was used to detect the binding strength of the antibody to the antigen using the Protein A capture method.
  • protein A was immobilized on the CM4 chip (purchased from GE, BR-1005-34) by amino coupling method, and was mixed with HBS-EP+pH7. 4 is the mobile phase.
  • NHS and EDC activate the chip for about 600 seconds, dilute Protein A to 50 ⁇ g/mL with 10 mM sodium acetate pH4.5, inject for 600 seconds, and finally block the remaining activation sites with ethanolamine. Then, the affinity of the antibody to the antigen is determined by a multi-cycle kinetic method.
  • the protein A chip is used to capture the antibody to be tested, and then a single concentration of the antigen protein is injected to record the binding and dissociation process of the antibody and the antigen protein.
  • the chip regeneration was completed with Glycine pH1.5, where the mobile phase was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20), the flow rate was 30 ⁇ L/min, the regeneration time was 30s, and the detection temperature was 25°C; , according to the 1:1binding model, analyze the data, and fit the antibody-antigen binding kinetic parameters, including the binding rate constant Ka, the dissociation rate constant Kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax.
  • binding rate (Ka), dissociation rate (Kd) and binding affinity (KD) of chimeric antibody and humanized antibody to human GUCY2C protein are shown in Table 10.

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Abstract

一种能够特异性地结合GUCY2C的抗体或其抗原结合片段，所述抗体或其抗原结合片段能够以高亲和力与GUCY2C特异性结合，可作为药物治疗胃肠道恶性肿瘤。

Description

抗GUCY2C抗体及其应用
相关专利申请的交叉引用
本申请要求于2021年12月27日向中国国家知识产权局提交的，专利申请号为202111609922.9，发明名称为《抗GUCY2C抗体及其应用》的中国专利申请的优先权。上述在先申请的全文通过引用的方式并入本申请中。
技术领域
本申请涉及抗体领域，具体而言，涉及抗GUCY2C抗体。
背景技术
胃肠道恶性肿瘤包括结直肠癌(CRC)、胃癌和食管癌。结直肠癌又名大肠癌，是消化系统常见的恶性肿瘤。目前，CRC的治疗手段主要为手术、放化疗，早期阶段的CRC患者可以实行手术或/和化疗，但对于晚期患者以及转移性结直肠癌患者，临床上缺乏有效的治疗手段。近年来，以PD1/L1为代表的免疫检查点抑制剂在多种肿瘤治疗中取得了长足发展，但对于结直肠癌来说却仍然充满挑战。大多数结直肠癌患者属于微卫星稳定型，肿瘤突变负荷低以及免疫浸润密度降低，且经常伴有KRAS或BRAF致癌基因突变，导致结直肠癌患者对目前批准的免疫治疗缺乏反应，未能显示出显著的生存获益。
鸟苷环化酶C(GUCY2C或GCC)是广泛表达于结直肠癌和其他胃肠道肿瘤的靶点。在正常组织中，GUCY2C在维持肠液、电解质平衡和细胞增殖中发挥重要功能，胞内酶催化区能够结合GTP，催化GTP向cGMP转化，产生第二信使影响下游信号通路。GUCY2C仅在小肠、大肠和直肠的黏膜细胞处表达，同时在所有原发性和转移性结肠直肠肿瘤中都有表达。因此GUCY2C成为一个有吸引力的靶标，靶向GUCY2C的药物开发对结直肠癌的治疗有重要的临床意义。
发明内容
本申请提供了如下实施方案。
在一方面，本申请提供了一种特异性结合GUCY2C的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含具有HCDR1，HCDR2和HCDR3的VH，所述HCDR1包含SEQ ID NO.17、20、22、30、33或35所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；所述HCDR2包含SEQ ID NO.18、21、23、31、34、36、52或53所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；并且所述HCDR3包含SEQ ID NO.19、24、32或37所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列，
和/或，所述抗体或其抗原结合片段包含具有LCDR1，LCDR2和LCDR3的VL，所述LCDR1包含SEQ ID NO.25、28、38或41所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；所述LCDR2包含SEQ ID  NO.26、29或39所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；并且所述LCDR3包含SEQ ID NO.27或40所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列。
在另一方面，本申请提供了一种多特异性分子，其包含上述任一种纳米抗体或其抗原结合片段。
在另一方面，本申请提供了一种嵌合抗原受体(CAR)，其至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含上述任一种纳米抗体或其抗原结合片段。
在另一方面，本申请提供了一种免疫效应细胞，其表达上述嵌合抗原受体，或包含编码所述嵌合抗原受体的核酸片段。
在另一方面，本申请提供了一种分离的核酸片段，其编码上述任一种纳米抗体或其抗原结合片段，或所述多特异性分子，或所述嵌合抗原受体。
在另一方面，本申请提供了一种载体(vector)，其包含所述核酸片段。
在另一方面，本申请提供了一种宿主细胞，其包含所述载体。
在另一方面，本申请提供了一种制备上述任一种纳米抗体或其抗原结合片段或多特异性分子的方法，其包括培养所述宿主细胞，以及分离所述细胞表达的纳米抗体或其抗原结合片段，或分离所述细胞表达的多特异性分子。
在另一方面，本申请提供了一种制备所述免疫效应细胞的方法，其包括将编码所述CAR的核酸片段导入所述免疫效应细胞。
在另一方面，本申请提供了一种药物组合物，其包含上述任一种纳米抗体或其抗原结合片段，多特异性抗体，免疫效应细胞，核酸片段，载体，宿主细胞，或上述任一项方法制备获得的产品。
在另一方面，还提供了本申请公开的上述任一种纳米抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品；或药物组合物在制备预防和/或治疗肿瘤的药物中的用途；所述肿瘤可以选自结直肠癌、胃癌、小肠癌、食道癌、胰脏癌、肺癌、软组织肉瘤和神经内分泌肿瘤。
在另一方面，本申请提供了一种预防和/或治疗肿瘤的方法，包括向有此需要的患者施用有效量的上述任一种纳米抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品；或药物组合物；其中所述肿瘤选自结直肠癌、胃癌、小肠癌、食道癌、胰脏癌、肺癌、软组织肉瘤和神经内分泌肿瘤。
在另一方面，本申请还提供了上述任一种纳米抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品；或药物组合物，用于预防和/或治疗肿瘤；其中所述肿瘤选自结直肠癌、胃癌、小肠癌、食道癌、胰脏癌、肺癌、软组织肉瘤和神经内分泌肿瘤。
在另一方面，本申请提供了一种试剂盒，其包含上述任一种纳米抗体或其抗原结合片段， 多特异性抗体，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品，或药物组合物。
在另一方面，本申请提供了一种检测GUCY2C表达的方法，其中，在上述任一种纳米抗体或其抗原结合片段与GUCY2C之间能够形成复合物的条件下，使待检测样品与上述任一种纳米抗体或其抗原结合片段接触。
在另一方面，本申请提供了一种体外抑制表达GUCY2C的细胞增殖或迁移的方法，其中，在上述任一种纳米抗体或其抗原结合片段与GUCY2C之间能够形成复合物的条件下，使所述细胞与上述任一种纳米抗体或其抗原结合片段接触。
本申请提供的抗GUCY2C抗体或其抗原结合片段在蛋白和细胞水平均能特异性结合GUCY2C，与人和猴GUCY2C蛋白具备良好的亲和力，对于开发靶向GUCY2C的药物提供了一项优异的选择。
附图说明
图1为流式细胞分析方法检测293T-hGUCY2C、CHO-cynoGUCY2C、CHO-hGUCY2C和293T-mGUCY2C细胞系表达水平。
图2A为ELISA检测鼠抗GCC001与人GUCY2C-his蛋白的结合反应。
图2B为ELISA检测鼠抗GCC003与人GUCY2C-his蛋白的结合反应。
图3A为ELISA检测鼠抗GCC001与猴GUCY2C-his蛋白的结合反应。
图3B为ELISA检测鼠抗GCC003与猴GUCY2C-his蛋白的结合反应。
图4A为ELISA检测鼠抗GCC001与鼠GUCY2C-his蛋白的结合反应。
图4B为ELISA检测鼠抗GCC003与鼠GUCY2C-his蛋白的结合反应。
图5A为FACS检测鼠抗GCC001与293T-hGUCY2C细胞的结合反应。
图5B为FACS检测鼠抗GCC003与293T-hGUCY2C细胞的结合反应。
图6A为FACS检测鼠抗GCC001与人肿瘤细胞HT-55的结合反应。
图6B为FACS检测鼠抗GCC003与人肿瘤细胞HT-55的结合反应。
图7A-7B为ELISA检测嵌合抗体与人GUCY2C-his蛋白的结合。
图8A-8B为ELISA检测嵌合抗体与猴GUCY2C-his蛋白的结合。
图9A-9B为ELISA检测嵌合抗体与鼠GUCY2C-his蛋白的结合。
图10A-10B为FACS检测嵌合抗体与293T-hGUCY2C细胞的结合
图11A-11B为FACS检测嵌合抗体与人肿瘤细胞HT-55的结合。
图12为抗体之间的表位分组情况。
图13A-13B为ELISA方法检测h001人源化抗体与人GUCY2C-his蛋白的结合。
图14为ELISA方法检测h003人源化抗体与人GUCY2C-his蛋白的结合。
图15A-15B为ELISA方法检测h001人源化抗体与鼠GUCY2C-his蛋白的结合。
图16为ELISA方法检测h003人源化抗体与鼠GUCY2C-his蛋白的结合。
图17A-17B为ELISA方法检测h001人源化抗体与猴GUCY2C-his蛋白的结合。
图18为ELISA方法检测h003人源化抗体与猴GUCY2C-his蛋白的结合。
图19A-19B为FACS检测h001人源化抗体与293T-hGUCY2C细胞的结合。
图20为FACS检测h003人源化抗体与293T-hGUCY2C细胞的结合。
图21A-21B为FACS检测h001人源化抗体与肿瘤细胞HT-55的结合。
图22A-22B为FACS检测h001人源化抗体与CHO-cynoGUCY2C细胞的结合。
图23为FACS检测h003人源化抗体与CHO-cynoGUCY2C细胞的结合。
图24A-24B为FACS检测h001人源化抗体与293T-mGUCY2C细胞的结合。
图25为FACS检测h003人源化抗体与293T-mGUCY2C细胞的结合。
发明的详细描述
术语定义和说明
除非本申请另外定义，与本申请相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的组合物，均落入前述“一种组合物”的范围内。
本文术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文术语“GUCY2c”是指哺乳动物鸟苷酸环化酶C(GUCY2C)，优选人GUCY2C蛋白。术语“GUCY2C”可以与“STAR”、“GUC2C”、“GCC”或“ST受体”互换使用。人GUCY2C的核苷酸序列公开为GenBank登录号NM_004963，人GUCY2C的氨基酸序列公开为GenBank登录号NP_004954。通常，自然发生的等位基因变体具有与GenBank登录号NP.Sub.-004954中描述的蛋白质至少95％、97％或99％相同的氨基酸序列。GUCY2C蛋白是一种跨膜细胞表面受体蛋白，在维持肠液、电解质稳态和细胞增殖等方面起着重要作用。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约1×10 -6M或更低、1×10 -7M或更低、约1×10 -8M或更低、约1×10 -9M或更低或约1×10 -10M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定，示例性地，可参见本文实施例10所示KD值获得方法。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer等，2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque等，Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于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，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文“抗体”包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体或全人抗体。
本文术语“单克隆抗体”是指从基本上同质的抗体群体获得的抗体，即，除了可能的变异体(例如含有天然存在的突变或在制剂的生产过程中产生，此类变体通常以少量存在)之外，包含所述群体的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单一决定簇。本文修饰语“单克隆”不应解释为需要通过任何特定方法产生所述抗体或抗原结合分子。举例来说，单克隆抗体可通过多种技术制得，包括(但不限于)杂交瘤技术、重组DNA方法、噬菌体库展示技术和利用含有全部或部分人免疫球蛋白基因座的转殖基因动物的方法和其它本领域已知的方法。
本文术语“单特异性”是指表示具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’) 2、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此,本文术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段,和重链的VH域和第一恒定域(CH1)的抗体片段。Fab’片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同,包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫醇基团的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’)2片段。
本文术语“Fd”是指由VH和CH1结构域组成的抗体。本文术语“Fv”是指由单臂VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
本文术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如，Bird等,Science 242:423-426(1988)；Huston等,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本申请的其他接头由Alfthan等(1995)，Protein Eng.8:725-731，Choi等(2001)，Eur.J.Immunol.31:94-106，Hu等(1996)，Cancer Res.56:3055-3061，Kipriyanov等(1999)，J.Mol.Biol.293:41-56和Roovers等(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。
本文术语“双抗体(diabody)”，其VH和VL结构域在单个多肽链上表达，但使用太短 的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等，Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等,Structure 2:1121-1123(1994))。
本文术语“裸抗体”是指不与治疗剂或示踪剂缀合的抗体；术语“缀合抗体”是指与治疗剂或示踪剂缀合的抗体。
本文术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(Cabilly等的U.S.P 4,816,567；Morrison等，Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt等，Kuby Immunology,6 th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等,J.Biol.Chem.,252:6609-6616(1977)；Kabat等,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等，J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等,J.Mol.Biol.,273:927-948(1997)；MacCallum等,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定 义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat等，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc等，Dev.Comparat.Immunol.27:55-77,2003。
本文术语“Chothia编号系统”通常是指由Chothia等提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等(1989)Nature 342:878-883)。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白酶水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据Kabat编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本申请所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等，(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本申请核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本申请蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等，(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖 (即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本申请的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP 2 101 823 B1)。本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本申请所述的特定疾病或病症的治疗的生物体。对象和患者的实例包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。 当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
如本文所用，术语“约”是指指定数值正负10％范围内的所有值。例如约10可以指9-11的范围内的所有值。
具体实施方式
本申请提供抗GUCY2C抗体，编码其的核酸，抗体制备方法，含有所述抗体的药物组合物，以及药物组合物用于治疗肿瘤的相关用途。
在一方面，本申请提供了一种特异性结合GUCY2C的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含具有HCDR1，HCDR2和HCDR3的VH，所述HCDR1包含SEQ ID NO.17、20、22、30、33或35所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；所述HCDR2包含SEQ ID NO.18、21、23、31、34、36、52或53所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；并且所述HCDR3包含SEQ ID NO.19、24、32或37所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列，
和/或，所述抗体或其抗原结合片段包含具有LCDR1，LCDR2和LCDR3的VL，所述LCDR1包含SEQ ID NO.25、28、38或41所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；所述LCDR2包含SEQ ID NO.26、29或39所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；并且所述LCDR3包含SEQ ID NO.27或40所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列。
在优选的实施方案中，所述替换为保守氨基酸的替换。
在一些实施方案中，所述抗体或其抗原结合片段包含(1)-(4)组任一组所示的氨基酸序列：
(1)所述HCDR1包含SEQ ID NO.17、20或22任一序列所示的氨基酸序列；所述HCDR2包含SEQ ID NO.18、21、23、52或53任一序列所示的氨基酸序列，并且所述HCDR3包含SEQ ID NO.19或24任一序列所示的氨基酸序列；和/或，所述LCDR1包含SEQ ID NO.25或28任一序列所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26或29任一序列所示的氨基酸序列，并且所述LCDR3包含SEQ ID NO.27所示的氨基酸序列；
(2)所述HCDR1包含SEQ ID NO.30、33或35任一序列所示的氨基酸序列；所述HCDR2包含SEQ ID NO.31、34或36任一序列所示的氨基酸序列，并且所述HCDR3包含SEQ ID NO.32或37任一序列所示的氨基酸序列；和/或，所述LCDR1包含SEQ ID NO.38或41任一序列所示的氨基酸序列，所述LCDR2包含SEQ ID NO:39或29任一序列所示的氨基酸序列，并且所述LCDR3包含SEQ ID NO.40所示的氨基酸序列；
(3)来源于SEQ ID NO.13、15、42、47-51、54、57-60任一序列所示的重链可变区的HCDR1，HCDR2和HCDR3；和/或，来源于SEQ ID NO.14、16、43、44、45、46、55、56任一序列所示的轻链可变区的LCDR1，LCDR2和LCDR3；
(4)与(1)-(3)组中的每个CDR相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合，优选地，所述替换为保守氨基酸的替换。
在一些实施方案中，所述抗体或其抗原结合片段包括：具有SEQ ID NO.13、15、42、47-51、54、57-60任一项所示的重链可变区；或，具有与SEQ ID NO.13、15、42、47-51、54、57-60任一序列所示的氨基酸序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；或，具有与SEQ ID NO.13、15、42、47-51、54、57-60任一序列所示的氨基酸序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；
和/或，具有SEQ ID NO.14、16、43、44、45、46、55、56任一项所示的轻链可变区，或，具有与SEQ ID NO.14、16、43、44、45、46、55、56任一序列所示的氨基酸序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；或，具有与SEQ ID NO.14、16、43、44、45、46、55、56任一序列所示的氨基酸序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在优选的实施方案中，(1)所述重链可变区包含如SEQ ID NO.13、42、47-51任一序列所示的氨基酸序列，并且所述轻链可变区包含如SEQ ID NO.14、43、44、45或46任一序列所示的氨基酸序列；或者(2)所述重链可变区包含如SEQ ID NO.15、54、57-60任一序列所示的氨基酸序列，并且所述轻链可变区包含如SEQ ID NO.16、55或56任一序列所示的氨基酸序列；或者(3)所述重链可变区和/或所述轻链可变区包含与第(1)或(2)组所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；或，具有与第(1)或(2)组所示的氨基酸序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些实施方案中，所述重链可变区和/所述轻链可变区选自表3或表7所示的VH和/或VL；优选地，所述重链可变区和所述轻链可变区如表4或表8所示的进行配对。
在一些实施方案中，所述抗体或其抗原结合片段还包含重链恒定区和/或轻链恒定区。
在优选的实施方案中，所述重链恒定区可以选自IgG，例如IgG1、IgG2、IgG3或IgG4。所述IgG可选自人IgG，例如人IgG4。可选地，所述重链恒定区可选自Fc区、CH3区或完整重链恒定区。可选地，所述重链恒定区为人Fc区。可选地，所述轻链恒定区可选自κ链或λ链，优选为κ链。
在更优选的实施方案中，所述重链恒定区可以包含SEQ ID NO.8所示的氨基酸序列，和/或所述轻链恒定区可以包含SEQ ID NO.10所示的氨基酸序列。可选地，所述人Fc区包含SEQ ID NO.12所示的氨基酸序列。
在一些实施方案中，所述抗体或其抗原结合片段特异性结合人GUCY2C蛋白，优选地，其与人GUCY2C蛋白结合的KD优于1.00E-8M。
在一些实施方案中，所述抗体或其抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
在一些实施方案中，所述抗体或其抗原结合片段可以选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性分子(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体、全人抗体、Fab、Fab’、Fab’-SH、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
在一些实施方案中，所述抗体或其抗原结合片段还可以偶联有治疗剂或示踪剂；优选地，所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂。优选地，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
在另一方面，本申请提供了一种多特异性分子，其包含上述任一种抗体或其抗原结合片段；优选地，所述多特异性分子进一步包含特异性结合GUCY2C以外的抗原或结合与上述任一项抗体或其抗原结合片段不同的GUCY2C表位的抗体或其抗原结合片段。
在优选的实施方案中，所述GUCY2C以外的抗原选自下组：(1)肿瘤特异性抗原(TSA)或肿瘤相关抗原(TAA)；(2)免疫检查点；(3)募集和/或激活免疫细胞的靶点。
在另一方面，本申请提供了一种嵌合抗原受体(CAR)，其至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含上述任一项抗体或其抗原结合片段。
在另一方面，本申请提供了一种免疫效应细胞，其表达上述嵌合抗原受体，或包含编码所述嵌合抗原受体的核酸片段。优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞。优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
在另一方面，本申请提供了一种分离的核酸片段，其编码上述任一种抗体或其抗原结合片段，或所述多特异性分子，或所述嵌合抗原受体。
在另一方面，本申请提供了一种载体(vector)，其包含所述核酸片段。
在另一方面，本申请提供了一种宿主细胞，其包含所述载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(例如大肠杆菌)、真菌(例如酵母)、昆虫细胞或哺乳动物细胞(例如CHO细胞系或293T细胞系)。
在另一方面，本申请提供了一种制备上述任一种抗体或其抗原结合片段或所述多特异性分子的方法，其包括培养所述细胞，以及分离所述细胞表达的抗体或其抗原结合片段，或分离所述细胞表达的多特异性分子。
在另一方面，本申请提供了一种制备所述免疫效应细胞的方法，其包括将编码所述CAR的核酸片段导入所述免疫效应细胞，可选地，还包括启动所述免疫效应细胞表达所述CAR。
在另一方面，本申请提供了一种药物组合物，其包含上述任一种抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，或上述任一项方法制备获得的产品。可选地，所述药物组合物还包含药学上可接受的载体(carrier)、稀释剂或助剂。
所述药学上可接受的载体为不减弱免疫细胞活力以及功能、不影响抗体或其抗原结合片段与抗原特异性结合的载体，包括但不限于细胞培养基、缓冲液、生理盐水和平衡盐溶液等。缓冲液的实例包括等渗磷酸盐、醋酸盐、柠檬酸盐、硼酸盐以及碳酸盐等。在具体的实施方式中，所述药学上可接受的载体为含1％血清的磷酸盐缓冲液。
在另一方面，还提供了本申请公开的上述任一种抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品，或药物组合物在制备预防和/或治疗肿瘤的药物中的用途。所述肿瘤可以选自结肠直肠癌、胃癌、小肠癌、食道癌、胰脏癌。
在另一方面，本申请提供了一种预防和/或治疗肿瘤的方法，包含向有此需要的患者施用有效量的上述任一种抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品，或药物组合物。所述肿瘤可以选自结肠直肠癌、胃癌、小肠癌、食道癌、胰脏癌。
在另一方面，本申请还提供了上述任一种抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品，或药物组合物，用于预防和/或治疗肿瘤。所述肿瘤可以选自结肠直肠癌、胃癌、小肠癌、食道癌、胰脏癌。
在另一方面，本申请提供了一种试剂盒，其包含上述任一种抗体或其抗原结合片段，多特异性分子，免疫效应细胞，核酸片段，载体，宿主细胞，上述任一项方法制备获得的产品，或药物组合物。
在另一方面，本申请提供了一种体外检测GUCY2C表达的方法，其中，在上述任一种抗体或其抗原结合片段与GUCY2C之间能够形成复合物的条件下，使待检测样品与所述抗体或其抗原结合片段接触。
在另一方面，本申请提供了一种体外抑制表达GUCY2C细胞增殖或迁移的方法，其中，在上述任一种抗体或其抗原结合片段与GUCY2C之间能够形成复合物的条件下，使所述细胞与所述抗体或其抗原结合片段接触。
实施例
下面结合具体实施例来进一步描述本申请，本申请的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本申请的实施例仅是范例性的，并不对本申请的范围构成任何限制。本领域技术人员应 该理解的是，在不偏离本申请的精神和范围下可以对本申请的技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本申请的保护范围内。
实施例1、重组蛋白和对照抗体的制备及其纯化方法
1.1重组蛋白的设计和表达
构建GUCY2C重组蛋白：以人GUCY2C蛋白(UniProt号：P25092)为模板序列，设计带标签融合蛋白，克隆到pTT5载体(优宝生物，VT2202)，构建GUCY2C质粒，并在Expi 293F细胞(Gibco，A14527)瞬转表达，获得本实施例中的抗原及检测用蛋白。食蟹猴和小鼠GUCY2C重组蛋白的制备方法同人重组蛋白制备方法。食蟹猴GUCY2C序列来自于Uniprot号：A0A2K5TZ15，小鼠GUCY2C序列来自Uniprot：Q3UWA6，重组蛋白的具体序列信息如下所示：
人GUCY2C ECD(his标签人GUCY2C蛋白胞外区融合蛋白)(SEQ ID NO:1)：
Figure PCTCN2022141755-appb-000001
Cyno GUCY2C ECD(his标签食蟹猴GUCY2C蛋白胞外区融合蛋白)(SEQ ID NO:2)：
Figure PCTCN2022141755-appb-000002
小鼠GUCY2C ECD(his标签小鼠GUCY2C蛋白胞外区融合蛋白)(SEQ ID NO:3)：
Figure PCTCN2022141755-appb-000003
1.2对照抗体的设计和表达
本实施例采用的对照抗体均来自于已公开发表的专利。PF1608抗体来源于已公开发表的国际专利第WO2019224716A2号，5F9抗体来源于已公开发表的国际专利第WO2017136693A1号，如无特别说明，PF1608和5F9对照抗体采用人IgG1+κ亚型进行重组表达。
对照抗体的表达和纯化过程如下：将抗体序列基因合成后克隆至表达载体pTT5上，然后瞬时转染Expi293F细胞(购自Gibco，A14527)，37℃摇床培养7天后收集细胞上清进行protein A抗体纯化，纯化过程参见“1.3.2 Protein A亲和层析纯化对照抗体”。所得对照抗体命名为PF1608-hIgG1和5F9-hIgG1。抗体具体序列信息见表1。
表1.对照抗体序列表
Figure PCTCN2022141755-appb-000004
Figure PCTCN2022141755-appb-000005
1.3重组蛋白的纯化以及对照抗体的纯化
1.3.1镍柱纯化重组蛋白
根据步骤1.1构建和表达相关重组蛋白后，按以下方法进行纯化：将细胞表达上清样品高速离心去除杂质，用20mM PBS+500mM NaCl溶液平衡镍柱，冲洗2-5倍柱体积。将培养上清液上样到Ni亲和层析柱(购自GE Healthcare)，同时用紫外检测仪监测紫外吸收值(A280nm)的变化，用平衡液冲洗柱子，至A280读数降至基线，后分别用含有10mM，20mM，40mM，90mM，250mM，500mM咪唑的平衡液梯度洗脱，收集各洗脱峰，根据SDS-PAGE胶图确定目的蛋白所在组分。收集的含有目的蛋白洗脱产物浓缩后可用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS，去除聚体及杂蛋白峰，收集目的产物洗脱峰。所得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。通过此方案纯化得到蛋白包括人GUCY2C-His、猴GUCY2C-His和鼠GUCY2C-His。
1.3.2 Protein A亲和层析纯化对照抗体
首先高速离心收取表达抗体的细胞培养上清。Protein A亲和柱利用0.1M NaOH洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积。利用1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用50mM柠檬酸/柠檬酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
实施例2、稳转细胞株构建
编码人源GUCY2C全长氨基酸序列(UniProt：P25092)的核苷酸序列被克隆到pcDNA3.1载体(优宝生物，VT1001)并制备质粒。对293T细胞进行质粒转染(
Figure PCTCN2022141755-appb-000006
3000 Transfection Kit，购自Invitrogen，货号：L3000-015)后，在含10μg/ml puromycin的DMEM培养基中选择性培养2周，用人源GUCY2C抗体(5F9，自产)和抗人IgG(H+L)抗体(Jackson，货号：109-605-088)在流式细胞仪FACS AriaII(购自BD Biosciences)上分选阳性单克隆细胞到96孔板，并置于37℃，5％(v/v)CO 2培养，大约2周后选择部分单克隆孔进行扩增。对扩增后的克隆经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存。所得细胞株命名为：293T-hGUCY2C，FACS检测表达情况如图1中A所示。
编码食蟹猴GUCY2C全长氨基酸序列(UniProt：A0A2K5TZ15)的核苷酸序列被克隆到pcDNA5-FRT载体(Invitrogen TM，V601020)并制备质粒。对FlpinCHO细胞系进行质粒转染后，在含800μg/ml hygromycin的F12培养基中选择性培养2周，所得细胞株命名为：CHO-cyno GUCY2C，用GUCY2C阳性对照5F9抗体经FACS进行检测，表达情况如图1中B所示。
编码人GUCY2C全长氨基酸序列(UniProt：P25092)的核苷酸序列被克隆到pcDNA5-FRT载体并制备质粒。对FlpinCHO细胞系进行质粒转染后，在含600μg/ml hygromycin的F12培养基中选择性培养2周，所得细胞株命名为：CHO-hGUCY2C，用GUCY2C阳性对照5F9抗体经FACS进行检测，表达情况如图1中C所示。
编码鼠GUCY2C全长氨基酸序列(Uniprot：Q3UWA6)的核苷酸序列被克隆到pcDNA3.1载体并制备质粒。对293T细胞系进行质粒转染后，在含10μg/ml puromycin的DMEM培养基中选择性培养2周，所得细胞株命名为：293T-mGUCY2C，用GUCY2C阳性对照PF1608抗体经FACS进行检测，表达情况如图1中D所示。
实施例3、针对鸟苷酸环化酶C蛋白的杂交瘤抗体制备
3.1动物免疫
本实施例的单克隆抗体通过免疫小鼠产生。实验用小鼠为6～8周龄、雌性的SPF级Balb/C小鼠或SJL小鼠(购自Charles River)。免疫原为实施例1制备的人GUCY2C-his蛋白或实施例2制备的CHO-hGUCY2C稳转细胞株。蛋白组初次免疫时，免疫原用TiterMax(购自Sigma，T2684-1M)乳化后皮下(SC)与腹腔(IP)分别注射0.1mL，即每只小鼠注射50μg免疫原；加强免疫时，免疫原用Imject Alum(购自Thermo)皮下与腹腔分别注射0.1mL，即每只小鼠注射25μg免疫原。细胞组初次免疫时，TiterMax与生理盐水乳化后腹腔注射0.1mL，15min后腹腔注射0.1mL细胞悬液，即每只小鼠注射1×10 7细胞；加强免疫时，腹腔注射细胞量为1×10 7的细胞悬液。免疫频次每周一次，于第3，19，47，61天取血。通过ELISA方法检测与人GUCY2C蛋白(His标签，包被浓度4μg/mL)的结合，测试经免疫接种的动物血清中识别人GUCY2C抗体的存在和抗体滴度。依据血清滴度，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，皮下、腹腔注射50μg/只的生理盐水配制的抗原溶液。
3.2细胞融合
无菌取脾脏和淋巴结，研磨并用40μm细胞滤网(购自BD Falcon)过滤，加入ACK Lysing Buffer(购自Gibco)，裂解脾细胞中掺杂的红细胞，获得脾细胞悬液，用DMEM(购自Gibco)基础培养基1500rpm离心清洗细胞1次。然后按照活细胞数目2:1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC)混合，用Cytofusion Medium C(购自BTX)清洗2次，采用BTX ECM2001+电融合方法(参照ECM2001+ELECTROFUSION PROTOCOL)进行细胞融合。融合后的细胞稀释到含20％胎牛血清(购自ExCell Bio)、1×HAT(购自Sigma)的DMEM培养基中。然后脾细胞按2×10 4每孔加入到96孔细胞培养板中，放入37℃、5％CO 2培养箱中。10天后用ELISA筛选细胞融合板上清，将ELISA检测的阳性克隆扩增到24孔板进行扩大培养。培养3天后取24孔板中的培养液，用ELISA、FACS确定对人GUCY2C蛋白的结合活性。
根据24孔板筛选结果，选择符合条件的杂交瘤细胞用Medium D(购自STEMCELL)在6孔板进行亚克隆。亚克隆后7天挑取单克隆于10％FBS、1×HT(购自Sigma)的DMEM培养基中，在37℃，5％CO 2条件下培养2天，挑选阳性单克隆扩增到24孔板继续培养，2天后用ELISA、FACS确定抗原结合阳性筛选标准。根据24孔板样品检测结果，挑选出最优的克隆，并于含10％FBS的DMEM培养基中在37℃，5％CO 2条件下将最优的克隆进行扩大培养，液氮冻存即得本实施例的杂交瘤细胞。
3.3杂交瘤抗体筛选
融合7～10天后，取杂交瘤细胞上清，通过蛋白ELISA方法检测与人GUCY2C蛋白的结合，挑选出阳性克隆；第二天通过蛋白和细胞ELISA方法检测阳性克隆与293T-hGUCY2C细胞、人GUCY2C-his蛋白、猴GUCY2C-his蛋白和鼠GUCY2C-his蛋白的结合。选择初筛阳性信号较高的克隆进行亚克隆，亚克隆后的杂交瘤抗体运用同样的方法再次进行检测。
3.4鼠源单克隆抗体生产
将定株后的杂交瘤克隆进行扩大培养，收取上清离心后并按实施例1.3.2所述纯化方法纯化抗体。本实施例共筛选获得2株特异性结合GUCY2C的抗体，GCC001和GCC003。
3.5鼠单克隆抗体鉴定
3.5.1酶联免疫吸附实验(ELISA)检测鼠单克隆抗体与人、猴和鼠GUCY2C-his蛋白的结合。
人、猴和鼠GUCY2C-his蛋白用PBS稀释到终浓度2μg/mL，然后以50μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用PBST洗板2次，加入封闭液[PBS+2％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入100nM为起始浓度，3倍梯度稀释的待测抗体，阳性及阴性对照抗体50μl每孔。37℃孵育1小时后，用PBST洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Merck，货号：AP113P)，37℃孵育1小时后，用PBST洗板5次。加入TMB底物50μl每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μl每孔。用酶标仪(PowerWave HT+Biostack3,购自biotek)读取OD 450nm数值，鼠单克隆抗体与人、猴和鼠GUCY2C-his蛋白结合活性结果如图2-4所示，纯化后的GCC001和GCC003抗体与人、猴和鼠GUCY2C蛋白均有效结合。阳性对照抗体5F9可识别人和猴GUCY2C，但不识别鼠GUCY2C。阴性对照抗体anti-hel-hIgG1(购自百英，货号：B117901)没有结合 活性。
3.5.2流式细胞实验(FACS)检测抗体与人GUCY2C细胞的结合。
将所需细胞扩大培养至对数生长期，消化收集细胞并吹打细胞至单细胞悬液。细胞计数后，离心，将细胞沉淀用FACS缓冲液(PBS+2％胎牛血清)重悬至2×10 6细胞每毫升，按每孔100μl加入到96孔FACS反应板中，离心，弃掉上清，加入待测抗体样品(100nM为起始浓度，5倍梯度稀释)每孔50μl，与细胞混匀，4度孵育1小时。用PBS缓冲液离心洗涤3次，每孔加入50μl Alexa
Figure PCTCN2022141755-appb-000007
647 AffiniPure Goat Anti-Human IgG,Fcγfragment specific标记的二抗(购自Jackson，货号：109-605-098)，4度孵育1小时。用PBS缓冲液离心洗涤3次，100μl PBS重悬后用FACS检测和分析结果。通过软件(FlowJo)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism8)分析，进行数据拟合，分析结果如图5所示，GCC001和GCC003可特异性结合人293T-hGUCY2C细胞。抗体与内源肿瘤细胞HT55的结合如图6所示，GCC001抗体可特异结合HT55细胞，GCC003抗体与HT55细胞结合能力较弱，与PF1608对照抗体趋势一致。后续表位竞争分析发现这种结合能力的差异是由于表位不同所导致的。
实施例4杂交瘤抗体序列测定
收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书冻存细胞，并进行NGS测序。经测序得到鼠源抗人GUCY2C抗体GCC001和GCC003序列，其重链可变区(HCVR)和轻链可变区(LCVR)氨基酸序列如下所示：
GCC001 HCVR：
Figure PCTCN2022141755-appb-000008
GCC001 LCVR：
Figure PCTCN2022141755-appb-000009
GCC003 HCVR：
Figure PCTCN2022141755-appb-000010
GCC003 LCVR：
Figure PCTCN2022141755-appb-000011
分析上述GUCY2C单克隆抗体的CDR区，其中，所述CDR区采用Kabat编号系统、Chothia编号系统和IMGT编号系统进行确定和注释，具体结果如表2所示。
表2抗体CDR序列及编号
Figure PCTCN2022141755-appb-000012
Figure PCTCN2022141755-appb-000013
实施例5 scFv嵌合抗体构建和表达纯化
根据实施例4获得的GCC001和GCC003抗体可变区基因的测序结果，设计引物，PCR搭建各抗体VH/VL基因片段。所得VH/VL基因片段与表达载体pTT5进行同源重组，构建重组嵌合抗体全长表达质粒pTT5-VH-(G4S) 3-VL-Fc，形成GCC001 ScFv-hFc和GCC003 ScFv-hFc嵌合抗体。其中人Fc区的氨基酸序列参见表1。质粒制备完成后转染入Expi293F细胞，37℃摇床培养7天后，收取上清，离心后并按实施例1.3.2所述纯化方法纯化抗体。
实施例6 scFv嵌合抗体鉴定
6.1 ELISA检测嵌合抗体与人、猴和鼠GUCY2C-his蛋白的结合。
按照实施例3.5.1的方法进行ELISA检测与数据分析。嵌合抗体与人、猴和鼠GUCY2C-his蛋白结合活性结果如图7-9所示，本申请中的嵌合抗体GCC001 ScFv-hFc和GCC003 ScFv-hFc与人、猴和鼠GUCY2C-his蛋白均能有效结合。
6.2 FACS检测嵌合抗体与GUCY2C在细胞水平的结合。
按照实施例3.5.2的方法进行FACS检测与数据分析。分析结果如图10A-10B所示，嵌合抗体GCC003 ScFv-hFc和GCC001 ScFv-hFc可特异性结合过表达细胞293T-hGUCY2C。图11A-11B结果显示嵌合抗体GCC003 ScFv-hFc和GCC001 ScFv-hFc可特异性结合内源肿瘤细胞HT55。
实施例7抗体表位竞争实验
为了鉴定抗体对抗原的结合位点，采用竞争ELISA的方法对GUCY2C嵌合抗体进行分组。参照实施例3.5.1的方法使用2μg/mL嵌合抗体包被ELISA板，人GUCY2C-his蛋白从30μg/mL开始进行梯度稀释，计算出EC80作为竞争性ELISA中的抗原浓度。用PBS稀释嵌 合抗体至2μg/mL，以50μL/孔包被96孔高吸附酶标板，4℃过夜包被后用250μL封闭液(含有2％(w/w)BSA的PBS)进行室温两小时封闭，加入40μg/mL待检测的抗体后，再加入每个待检测抗体对应的EC80浓度的人GUCY2C-his蛋白，孵育2小时，用PBS洗5次后加入HRP标记的anti-His二抗(购自Genescript，货号：A00612)，孵育1小时，洗板5次。加入TMB底物50μL每孔，室温孵育10分钟后，加入终止液(1.0M HCl)50μL每孔。用酶标仪(Insight，购自PerkinElmer)读取OD450nm数值，利用公式计算出抗体相互之间的竞争率，结果如图12所示，GCC001 ScFv-hFc是与阳性对照5F9-hIgG1结合表位相似的抗体，GCC003 ScFv-hFc是与阳性对照PF1608-hIgG1结合表位相似的抗体。与PF1608结合抗原表位相近的抗体在细胞水平的结合活性显著弱于与5F9结合表位相近的抗体。
实施例8 GUCY2C抗体人源化设计
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库和MOE(Molecular Operating Environment)软件，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，进行回复突变和/或热点突变。本实施例CDR序列按Kabat编号系统划分。
8.1 GCC001抗体人源化
8.1.1 GCC001构架选择
鼠源抗体GCC001的人源化轻链模板为IGKV3-15*01、IGKV4-1*01和IGKJ2*01，人源化重链模板为IGHV1-46*01和IGHJ6*01，将鼠源抗体GCC001的CDR分别移植到对应的人源模板中，即获得GCC001的人源化抗体h001，其可变区序列如下所示：
h001 HCVR(VH-CDR graft，IGHV1-46*01)：
Figure PCTCN2022141755-appb-000014
h001 LCVR1(VL-CDR graft，IGKV3-15*01)：
Figure PCTCN2022141755-appb-000015
h001 LCVR2(VL-CDR graft，IGKV4-1*01)：
Figure PCTCN2022141755-appb-000016
8.1.2 GCC001人源化抗体的回复突变及热点突变设计
根据需要，将GCC001人源化抗体的FR区序列中关键氨基酸进行回复突变，以保证原有的亲和力，同时鉴于GCC001重链CDR2区存在高风险易修饰位点NG，故根据抗体结构采用计算机模拟的方式对NG进行氨基酸突变以消除分子修饰风险，具体突变设计见表3(回复突变以自然编号顺序)。
表3 GCC001的人源化抗体回复突变及热点突变设计
Figure PCTCN2022141755-appb-000017
Figure PCTCN2022141755-appb-000018
注：Graft代表将鼠抗体CDR植入人种系FR区序列；Graft+G72R表示将Graft第72位G突变为R，其它依此类推。
8.1.3 GCC001人源化抗体
对上述表3的GCC001的人源化抗体回复突变及热点突变设计进行组合，最终获得多种GCC001人源化抗体(详见表4)。
表4 GCC001人源化抗体对应氨基酸序列
Figure PCTCN2022141755-appb-000019
注：h001H1L1表示GCC001人源化抗体h001具有如L1所述轻链可变区和如H1所述重链可变区，其它依此类推。
人源化后的重链、轻链可变区氨基酸序列如表5所示：
表5 GCC001人源化抗体回复突变的可变区氨基酸序列
Figure PCTCN2022141755-appb-000020
Figure PCTCN2022141755-appb-000021
根据Kabat编号系统，上述GCC001人源化抗体重链、轻链可变区CDR序列分析结果如表6所示。
表6 GCC001人源化抗体重轻链可变区CDR序列的Kabat分析结果
Figure PCTCN2022141755-appb-000022
8.2 GCC003抗体人源化
8.2.1 GCC003构架选择
鼠源抗体GCC003的人源化轻链模板为IGKV4-1*01和IGKJ4*01，人源化重链模板为IGHV1-46*01和IGHJ6*01，将鼠源抗体GCC003的CDR分别移植到其人源模板中，即获得GCC003的人源化抗体h003。h003可变区序列如下所示：
h003 HCVR(VH-CDR graft,IGHV1-46*01)：
Figure PCTCN2022141755-appb-000023
h003 LCVR1(VL-CDR graft 1,IGKV4-1*01)：
Figure PCTCN2022141755-appb-000024
8.2.2 GCC003的人源化抗体的回复突变设计
根据需要，将GCC003的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，具体回复突变设计见表7(回复突变以自然编号顺 序)。
表7 GCC003的人源化抗体回复突变设计
Figure PCTCN2022141755-appb-000025
注：Graft代表将鼠抗体CDR植入人种系FR区序列；G72R表示将Graft第72位G突变回R，其它依此类推。
8.2.3 GCC003人源化抗体
对上述表7的GCC003人源化抗体回复突变设计进行组合，最终获得多种GCC003人源化抗体，具体组合参见表8。
表8 GCC003人源化抗体可变区对应氨基酸序列
Figure PCTCN2022141755-appb-000026
注：h003H1L1表示GCC003人源化抗体h003具有如L1所述轻链可变区和如H1所述重链可变区，其它依此类推。
人源化后的重链、轻链可变区氨基酸序列如下表9所示，其中CDR区域序列与人源化之前的序列一致。
表9 GCC003人源化抗体回复突变的重链和轻链氨基酸序列
Figure PCTCN2022141755-appb-000027
Figure PCTCN2022141755-appb-000028
8.3 GUCY2C人源化全长抗体的构建和表达纯化
设计PCR引物搭建各人源化抗体VH/VL基因片段，再与载体进行同源重组，构建人源化抗体全长表达载体。其中，h003人源化抗体以scFv-hFc组合型式表达，h001人源化抗体以人IgG1型式表达(Fc序列、重链恒定区和轻链恒定区序列见表1)。质粒构建完成后瞬时转染Expi293F细胞，7天后离心收集上清，并按实施例1.3.2所述纯化方法纯化抗体。
实施例9人源化抗体鉴定
9.1 ELISA检测人源化抗体与人、猴和鼠GUCY2C蛋白的结合
按照实施例3.5.1的方法进行ELISA检测与数据分析。
如图13A、13B、14所示，h001和h003人源化抗体与人GUCY2C-his蛋白均能够有效结合，维持了嵌合抗体的结合能力。
如图15A、15B、16所示，h001和h003人源化抗体与鼠GUCY2C-his蛋白均能够有效结合，维持了嵌合抗体的结合能力。
如图17A、17B、18所示，h001和h003人源化抗体与猴GUCY2C-his蛋白均能够有效结合，维持了嵌合抗体的结合能力。
9.2流式细胞实验(FACS)检测人源化抗体与GUCY2C在细胞水平的结合
按照实施例3.5.2的方法进行FACS检测与数据分析。
分析结果如图19和图20所示，本申请中的h001人源化抗体和h003人源化抗体均可特异性结合293T-hGUCY2C重组细胞，维持了嵌合抗体的结合能力。
如图21所示，h001人源化抗体可特异性结合内源肿瘤细胞HT55，维持了嵌合抗体的结合能力。
如图22和23所示，h001和h003人源化抗体均可与CHO-cynoGUCY2C细胞有效结合。
如图24-25所示，本申请的人源化抗体均能够与293T-mGUCY2C细胞有较好的结合活性，维持了嵌合抗体的结合能力。其中，h003和PF1608抗体结合表位相似，二者与人、猴和鼠GUCY2C在细胞水平的结合活性均较弱。
实施例10、抗体的亲和力测定
应用BIAcore 8K仪器，采用Protein A捕获法检测抗体与抗原的结合强度。首先，应用氨基偶联法将Protein A固定到CM4芯片(购自GE，BR-1005-34)上，根据Amine Coupling Kit试剂盒(购自GE，BR100633)的指导，以HBS-EP+pH7.4为流动相，将NHS和EDC混合后，活化芯片约600秒，用10mM乙酸钠pH4.5将Protein A稀释至50μg/mL，注射600s， 最后用乙醇胺对剩余的活化位点进行封闭。然后，采用多循环动力学法测定抗体与抗原的亲和力，在每一个循环中，首先用Protein A芯片捕获待测抗体，然后注入单一浓度的抗原蛋白，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5完成芯片再生，其中流动相为HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)，流速30μL/min，再生时间为30s，检测温度为25℃；最后，根据1:1binding模型，对数据进行分析，拟合抗体抗原结合动力学参数，包括结合速率常数Ka、解离速率常数Kd、平衡解离常数KD、最大结合信号Rmax。
嵌合抗体及人源化抗体与人GUCY2C蛋白的结合速率(Ka)、解离速率(Kd)及结合亲和力(KD)如表10所示。
表10 SPR(biacore)检测嵌合和人源化抗体与人GUCY2C蛋白的亲和力
重组抗体名称 Ka(1/Ms) Kd(1/s) KD(M)
5F9-hIgG1 1.48E+05 5.68E-04 3.84E-09
PF1608-hIgG1 1.71E+05 2.46E-04 1.44E-09
GCC003 ScFv-hFc 2.85E+05 1.27E-03 4.48E-09
h003H1L1-hFc 3.29E+05 1.60E-03 4.86E-09
h003H2L1-hFc 3.20E+05 1.16E-03 3.64E-09
h003H3L1-hFc 2.99E+05 1.13E-03 3.79E-09
h003H4L1-hFc 3.01E+05 1.28E-03 4.27E-09
GCC001 ScFv-hFc 2.46E+04 9.25E-05 3.76E-09
h001H1L1-hIgG1 5.13E+04 8.56E-05 1.67E-09
h001H2L1-hIgG1 5.37E+04 7.68E-05 1.43E-09
h001H1L2-hIgG1 4.80E+04 7.60E-05 1.58E-09
h001H3L1-hIgG1 5.37E+04 5.74E-05 1.07E-09
h001H2L2-hIgG1 4.83E+04 8.72E-05 1.81E-09
h001H3L2-hIgG1 5.09E+04 8.19E-05 1.61E-09
h001H1bL2-hIgG1 5.35E+04 1.02E-04 1.91E-09
h001H1dL2-hIgG1 4.85E+04 9.14E-05 1.88E-09

Claims (24)

  1. 一种特异性结合GUCY2C的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含具有HCDR1，HCDR2和HCDR3的VH，所述HCDR1包含SEQ ID NO.17、20、22、30、33或35所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；所述HCDR2包含SEQ ID NO.18、21、23、31、34、36、52或53所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；并且所述HCDR3包含SEQ ID NO.19、24、32或37所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列，
    和/或，所述抗体或其抗原结合片段包含具有LCDR1，LCDR2和LCDR3的VL，所述LCDR1包含SEQ ID NO.25、28、38或41所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；所述LCDR2包含SEQ ID NO.26、29或39所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列；并且所述LCDR3包含SEQ ID NO.27或40所示的氨基酸序列，或者与所述氨基酸序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的氨基酸序列，优选地，所述替换为保守氨基酸的替换。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含下述(1)-(4)组任一组所示的氨基酸序列：
    (1)所述HCDR1包含SEQ ID NO.17、20或22任一序列所示的氨基酸序列；所述HCDR2包含SEQ ID NO.18、21、23、52或53任一序列所示的氨基酸序列，并且所述HCDR3包含SEQ ID NO.19或24任一序列所示的氨基酸序列；和/或，所述LCDR1包含SEQ ID NO.25或28任一序列所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26或29任一序列所示的氨基酸序列，并且所述LCDR3包含SEQ ID NO.27所示的氨基酸序列；
    (2)所述HCDR1包含SEQ ID NO.30、33或35任一序列所示的氨基酸序列；所述HCDR2包含SEQ ID NO.31、34或36任一序列所示的氨基酸序列，并且所述HCDR3包含SEQ ID NO.32或37任一序列所示的氨基酸序列；和/或，所述LCDR1包含SEQ ID NO.38或41任一序列所示的氨基酸序列，所述LCDR2包含SEQ ID NO:39或29任一序列所示的氨基酸序列，并且所述LCDR3包含SEQ ID NO.40所示的氨基酸序列；
    (3)来源于SEQ ID NO.13、15、42、47-51、54、57-60任一序列所示的重链可变区的HCDR1，HCDR2和HCDR3；和/或，来源于SEQ ID NO.14、16、43、44、45、46、55、56任一序列所示的轻链可变区的LCDR1，LCDR2和LCDR3；
    (4)与(1)-(3)组中的每个CDR相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合，优选地，所述替换为保守氨基酸的替换。
  3. 根据权利要1或2所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包括：具有SEQ ID NO.13、15、42、47-51、54、57-60任一序列所示的重链可变区；或，具有与SEQ ID NO.13、15、42、47-51、54、57-60任一序列所示的氨基酸序列相比具有至少80％、 85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或，具有与SEQ ID NO.13、15、42、47-51、54、57-60任一序列所示的氨基酸序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列；所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；
    和/或，具有SEQ ID NO.14、16、43、44、45、46、55、56任一序列所示的轻链可变区，或，具有与SEQ ID NO.14、16、43、44、45、46、55、56任一序列所示的氨基酸序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的序列；或，具有与SEQ ID NO.14、16、43、44、45、46、55、56任一序列所示的氨基酸序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换；
    优选地，(1)所述重链可变区包含如SEQ ID NO.13、42、47-51任一序列所示的氨基酸序列，并且所述轻链可变区包含如SEQ ID NO.14、43、44、45或46任一序列所示的氨基酸序列；
    优选地，(2)所述重链可变区包含如SEQ ID NO.15、54、57-60任一序列所示的氨基酸序列，并且所述轻链可变区包含如SEQ ID NO.16、55或56任一序列所示的氨基酸序列；
    优选地，(3)所述重链可变区和/或所述轻链可变区包含与第(1)或(2)组所示的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或，具有与第(1)或(2)组所示的氨基酸序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列；所述突变选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  4. 根据权利要求3所述的抗体或其抗原结合片段，其中所述重链可变区和/或所述轻链可变区选自表3或表7所示的VH和/或VL；优选地，所述重链可变区和所述轻链可变区如表4或表8配对。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段还包含重链恒定区和/或轻链恒定区；
    优选地，所述重链恒定区选自IgG，例如IgG1、IgG2、IgG3或IgG4，所述IgG选自人IgG，例如人IgG4；可选地，所述重链恒定区选自Fc区、CH3区或完整重链恒定区，可选地，所述重链恒定区为人Fc区；所述轻链恒定区选自κ链或λ链，优选为κ链；
    更优选地，所述重链恒定区包含SEQ ID NO.8所示的氨基酸序列，所述轻链恒定区包含SEQ ID NO.10所示的氨基酸序列，所述人Fc区包含SEQ ID NO.12所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段特异性结合人GUCY2C蛋白；优选地，其与人GUCY2C蛋白结合的KD优于1.00E-8M。
  7. 根据权利要求1-6中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段为：(1)嵌合抗体或其片段；(2)人源化抗体或其片段；或(3)全人抗体或其片段。
  8. 根据权利要求1-7中任一项所述的抗体或其抗原结合片段，其选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性分子(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体、全人抗体、Fab、Fab’、Fab’-SH、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
  9. 根据权利要求1-8中任一项所述的抗体或其抗原结合片段，其还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂，优选地，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
  10. 一种多特异性分子，其包含权利要求1-9中任一项所述的抗体或其抗原结合片段；优选地，所述多特异性分子进一步包含特异性结合GUCY2C以外的抗原或结合与权利要求1-9任一项抗体或其抗原结合片段不同的GUCY2C表位的抗体或其抗原结合片段；
    优选地，所述GUCY2C以外的抗原选自下组：(1)肿瘤特异性抗原(TSA)或肿瘤相关抗原(TAA)；(2)免疫检查点；(3)募集和/或激活免疫细胞的靶点。
  11. 一种嵌合抗原受体(CAR)，其至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求1-9中任一项所述的抗体或其抗原结合片段。
  12. 一种免疫效应细胞，其表达权利要求11所述的嵌合抗原受体，或包含编码权利要求11所述的嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、DNT细胞、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
  13. 一种分离的核酸片段，其编码权利要求1-9中任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，或权利要求11所述的嵌合抗原受体。
  14. 一种载体，其包含权利要求13所述的核酸片段。
  15. 一种宿主细胞，其包含权利要求14所述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(例如大肠杆菌)、真菌(例如酵母)、昆虫细胞或哺乳动物细胞(例如CHO细胞系或293T细胞系)。
  16. 一种制备权利要求1-9中任一项所述的抗体或其抗原结合片段或权利要求10所述的多特异性分子的方法，其包括培养权利要求15所述的宿主细胞，以及分离所述细胞表达的抗体或其抗原结合片段，或分离所述细胞表达的多特异性分子。
  17. 一种制备权利要求12所述的免疫效应细胞的方法，其包括将编码权利要求11所述的CAR的核酸片段导入免疫效应细胞，可选地，还包括启动免疫效应细胞表达权利要求11所述的CAR。
  18. 一种药物组合物，其包含权利要求1-9中任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，或权利要求12所述的免疫效应细胞，或权利要求13所述的核酸片段，或权利要求14所述的载体，或权利要求15所述的宿主细胞，或权利要求16-17 中任一项所述的方法制备获得的产品；可选地，所述药物组合物还包含药学上可接受的载体、稀释剂或助剂。
  19. 权利要求1-9中任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，或权利要求12所述的免疫效应细胞，或权利要求13所述的核酸片段，或权利要求14所述的载体，或权利要求15所述的宿主细胞，或权利要求16-17中任一项所述的方法制备获得的产品；或权利要求18所述的药物组合物在制备预防和/或治疗肿瘤的药物中的用途；所述肿瘤选自结肠直肠癌、胃癌、小肠癌、食道癌、胰脏癌。
  20. 一种预防和/或治疗肿瘤的方法，包含向有此需要的患者施用有效量的权利要求1-9中任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，或权利要求12所述的免疫效应细胞，或权利要求13所述的核酸片段，或权利要求14所述的载体，或权利要求15所述的宿主细胞，或权利要求16-17中任一项所述的方法制备获得的产品；或权利要求18所述的药物组合物；其中所述肿瘤选自结肠直肠癌、胃癌、小肠癌、食道癌、胰脏癌。
  21. 权利要求1-9中任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，或权利要求12所述的免疫效应细胞，或权利要求13所述的核酸片段，或权利要求14所述的载体，或权利要求15所述的宿主细胞，或权利要求16-17中任一项所述的方法制备获得的产品；或权利要求18所述的药物组合物，用于预防和/或治疗肿瘤；其中所述肿瘤选自结肠直肠癌、胃癌、小肠癌、食道癌、胰脏癌。
  22. 一种试剂盒，其包含权利要求1-9中任一项所述的抗体或其抗原结合片段，或权利要求10所述的多特异性分子，或权利要求12所述的免疫效应细胞，或权利要求13所述的核酸片段，或权利要求14所述的载体，或权利要求15所述的宿主细胞，或权利要求16-17中任一项所述的方法制备获得的产品，或权利要求18所述的药物组合物。
  23. 一种体外检测GUCY2C表达的方法，其中，在权利要求1-9中任一项所述的抗体或其抗原结合片段与GUCY2C之间能够形成复合物的条件下，使待检测样品与权利要求1-9中任一项所述的抗体或其抗原结合片段接触。
  24. 一种体外抑制表达GUCY2C的细胞增殖或迁移的方法，其中，在权利要求1-9中任一项所述的抗体或其抗原结合片段与GUCY2C之间能够形成复合物的条件下，使所述细胞与权利要求1-9中任一项所述的抗体或其抗原结合片段接触。
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