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WO2025061149A1 - 抗gcc抗体、嵌合抗原受体及其用途 - Google Patents

抗gcc抗体、嵌合抗原受体及其用途 Download PDF

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Publication number
WO2025061149A1
WO2025061149A1 PCT/CN2024/120038 CN2024120038W WO2025061149A1 WO 2025061149 A1 WO2025061149 A1 WO 2025061149A1 CN 2024120038 W CN2024120038 W CN 2024120038W WO 2025061149 A1 WO2025061149 A1 WO 2025061149A1
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seq
amino acid
acid sequence
variable region
chain variable
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French (fr)
Inventor
徐庞连
庄秋传
魏敏
丁凯强
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Nanjing Legend Biotechnology Co Ltd
Legend Biotech Ireland Ltd
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Nanjing Legend Biotechnology Co Ltd
Legend Biotech Ireland Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present disclosure belongs to the field of biomedicine and relates to an anti-GCC antibody, a chimeric antigen receptor comprising the anti-GCC antibody and uses thereof.
  • Guanylate cyclase C (GUCY2C or GCC for short) is a membrane-bound guanylate cyclase that belongs to a single transmembrane protein receptor, including an extracellular domain, a transmembrane domain, and an intracellular domain.
  • This enzyme is an intestinal receptor for bacterial heat-stable enterotoxins, so GUCY2C is also called a heat-stable enterotoxin receptor.
  • Guanylin and uroguanylin as well as artificial GCC agonists can bind to and activate GCC.
  • the activation of GCC can stimulate the production of the intracellular second messenger cGMP, which plays a role in regulating water and electrolyte balance, maintaining intestinal barrier function, and exerting anti-inflammatory activity.
  • GCC In normal human tissues, GCC is expressed at the top of intestinal epithelial cells. Loss of function caused by GCC mutations will lead to reduced fluid and ion secretion, thus causing constipation; enhancement of function caused by GCC mutations will increase fluid and ion secretion, thus causing secretory diarrhea.
  • the GCC-cGMP signaling axis also plays an important role in the pathogenesis of diseases such as intestinal tumors and inflammatory bowel disease: the inactivation of GCC-cGMP signaling caused by significant downregulation of guanylin and uroguanylin is closely related to tumorigenesis. Upregulation of GCC-cGMP signaling activates the STAT1 signaling pathway, leading to inflammatory bowel disease, etc. GCC is specifically and highly expressed in most gastrointestinal tumor tissues, making it an ideal target for targeted therapy of gastrointestinal tumors.
  • Chimeric antigen receptor T (CAR-T) cell immunotherapy is a new type of antigen-targeted precision treatment technology. After T cells are transfected and express specific CAR genes, they can recognize and bind to specific antigens on the surface of tumor cells, directly activate T cells without relying on MHC co-stimulatory signals, and then attack tumor cells.
  • CAR-T cell therapy has potential advantages over other T cell redirection therapy strategies. Other treatment strategies usually require long-term treatment until the disease progresses, while CAR-T cells can be expanded in vivo and only a single infusion is needed to achieve long-term disease remission. CAR-T cell therapy is becoming a potential treatment option for cancer treatment.
  • the present disclosure provides a new anti-guanylate cyclase C (GCC) antibody that can recognize human and mouse GCC antigen targets, has strong cell membrane localization and strong specificity, and can be used for companion diagnostic antibody development in the future.
  • the rabbit-derived antibody targeting GCC provided in this application can not only be used to develop a chimeric antigen receptor (CAR) targeting GCC, but also can be used for immunohistochemical analysis.
  • CAR chimeric antigen receptor
  • an anti-GCC antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementary determining regions HCDR1 to HCDR3, and the light chain variable region comprises light chain complementary determining regions LCDR1 to LCDR3, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:37;
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:38;
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:39;
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:40; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO:41.
  • the anti-GCC antibody or antigen-binding fragment thereof wherein:
  • the HCDR1 to HCDR3 of the heavy chain variable region and the LCDR1 to LCDR3 of the light chain variable region are determined according to the Kabat numbering system, the AbM numbering system, the IMGT numbering system, the Chothia numbering system or the Contact numbering system.
  • the anti-GCC antibody or antigen-binding fragment thereof wherein:
  • amino acid sequence of HCDR1 is shown in SEQ ID NO: 1
  • amino acid sequence of HCDR2 is shown in SEQ ID NO: 6
  • amino acid sequence of HCDR3 is shown in SEQ ID NO: 11
  • amino acid sequence of LCDR1 is shown in SEQ ID NO: 16
  • amino acid sequence of LCDR2 is shown in SEQ ID NO: 21
  • amino acid sequence of LCDR3 is shown in SEQ ID NO: 26;
  • the amino acid sequence of HCDR1 is shown in SEQ ID NO: 2
  • the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7
  • the amino acid sequence of HCDR3 is shown in SEQ ID NO: 12
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 17
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 22
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 27;
  • amino acid sequence of HCDR1 is shown in SEQ ID NO: 3
  • amino acid sequence of HCDR2 is shown in SEQ ID NO: 8
  • amino acid sequence of HCDR3 is shown in SEQ ID NO: 13
  • amino acid sequence of LCDR1 is shown in SEQ ID NO: 18
  • amino acid sequence of LCDR2 is shown in SEQ ID NO: 23
  • amino acid sequence of LCDR3 is shown in SEQ ID NO: 28;
  • HCDR1 is as shown in SEQ ID NO:4
  • amino acid sequence of HCDR2 is as shown in SEQ ID NO:9
  • amino acid sequence of HCDR3 is as shown in SEQ ID NO:14
  • amino acid sequence of LCDR1 is as shown in SEQ ID NO:19
  • amino acid sequence of LCDR2 is as shown in SEQ ID NO:24
  • amino acid sequence of LCDR3 is as shown in SEQ ID NO:29;
  • HCDR1 is shown in SEQ ID NO: 5
  • amino acid sequence of HCDR2 is shown in SEQ ID NO: 10
  • amino acid sequence of HCDR3 is shown in SEQ ID NO: 15
  • amino acid sequence of LCDR1 is shown in SEQ ID NO: 20
  • amino acid sequence of LCDR2 is shown in SEQ ID NO: 25
  • amino acid sequence of LCDR3 is shown in SEQ ID NO: 30.
  • the anti-GCC antibody or antigen-binding fragment thereof wherein:
  • the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:31
  • the light chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:37;
  • the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:32
  • the light chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:38.
  • the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:33
  • the light chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:39;
  • the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:34
  • the light chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:40; or
  • the heavy chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO:41.
  • the anti-GCC antibody or antigen-binding fragment thereof wherein:
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37;
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 39;
  • the anti-GCC antibody or its antigen-binding fragment wherein the anti-GCC antibody includes a non-CDR region, and the non-CDR region is from human, mouse or rabbit; in some specific embodiments, the non-CDR region includes a framework region, a heavy chain constant region and/or a light chain constant region.
  • the anti-GCC antibody or its antigen-binding fragment wherein the antigen-binding fragment is Fab, Fab', F(ab')2, single-chain antibody (scFv) or disulfide-stabilized variable fragment (dsFv).
  • the anti-GCC antibody or its antigen-binding fragment comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO:43 to SEQ ID NO:47; or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:57 and SEQ ID NO:58; in some specific embodiments, the amino acid sequence of the anti-GCC antibody or its antigen-binding fragment is as shown in any one of SEQ ID NO:43 to SEQ ID NO:47.
  • Another aspect of the present disclosure relates to an isolated nucleic acid encoding the anti-GCC antibody or antigen-binding fragment thereof according to any one of the present disclosure.
  • Yet another aspect of the present disclosure relates to a vector comprising the isolated nucleic acid of the present disclosure.
  • Yet another aspect of the present disclosure relates to a host cell comprising the isolated nucleic acid of the present disclosure, or comprising the vector of the present disclosure.
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor wherein the anti-GCC antibody or antigen-binding fragment is a single-chain antibody (scFv).
  • the chimeric antigen receptor further comprises a signal peptide located at the N-terminus of the polypeptide
  • the signal peptide is derived from CD8 ⁇ , CD28 or CD4 molecules;
  • the signal peptide comprises an amino acid sequence as shown in SEQ ID NO:61, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:61.
  • the chimeric antigen receptor further comprises a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain;
  • the hinge region is derived from IgG4Fc CH2CH3, CD28 or CD8 ⁇ ;
  • the hinge region comprises an amino acid sequence as shown in SEQ ID NO:62, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:62.
  • the chimeric antigen receptor wherein the transmembrane domain is derived from one or more transmembrane regions of CD8 ⁇ , CD4, CD28, CD137, CD80, CD86, CD152 and PD1 molecules;
  • the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO:63, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:63.
  • the chimeric antigen receptor wherein the intracellular signaling domain comprises a co-stimulatory signaling domain
  • the costimulatory signaling domain is derived from the following costimulatory molecules: ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof;
  • the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and/or the cytoplasmic domain of CD137;
  • the co-stimulatory signaling domain comprises an amino acid sequence as shown in SEQ ID NO:64, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:64.
  • the chimeric antigen receptor wherein the intracellular signaling domain comprises a primary intracellular signaling domain
  • the primary intracellular signaling domain is derived from CD3 ⁇ ;
  • the primary intracellular signaling domain comprises an amino acid sequence as shown in SEQ ID NO:65, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:65.
  • the amino acid sequence of the chimeric antigen receptor is as shown in any one of SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:71 to SEQ ID NO:75.
  • Yet another aspect of the present disclosure relates to an isolated nucleic acid encoding the chimeric antigen receptor described in any one of the present disclosure.
  • Yet another aspect of the present disclosure relates to a vector comprising the isolated nucleic acid of the present disclosure.
  • Another aspect of the present disclosure relates to a cell comprising the chimeric antigen receptor described in any one of the present disclosure, comprising the isolated nucleic acid of the present disclosure, or comprising the vector of the present disclosure;
  • the cell is an immune cell.
  • the cells wherein the cells are selected from: T cells, NK cells, peripheral blood mononuclear cells (PBMC), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells and combinations thereof.
  • PBMC peripheral blood mononuclear cells
  • Yet another aspect of the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-GCC antibody or antigen-binding fragment thereof described in any one of the present disclosure, or comprising the chimeric antigen receptor described in any one of the present disclosure, or comprising the cell described in any one of the present disclosure;
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
  • Another aspect of the present disclosure relates to use of the anti-GCC antibody or antigen-binding fragment thereof described in any one of the present disclosure or the chimeric antigen receptor described in any one of the present disclosure in immunohistochemical detection or in the preparation of a detection kit.
  • Another aspect of the present disclosure relates to use of the anti-GCC antibody or antigen-binding fragment thereof described in any one of the present disclosure, the chimeric antigen receptor described in any one of the present disclosure, or the cell described in any one of the present disclosure in the preparation of a medicament for treating or preventing a GCC-related disease or condition;
  • the GCC-related disease or condition is a tumor
  • the GCC-related disease or condition is a digestive tract tumor
  • the GCC-related disease or condition is one or more selected from gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, esophageal junction cancer, small intestine cancer, pancreatic cancer and liver cancer.
  • the GCC-related disease or condition is a tumor
  • the GCC-related disease or condition is a digestive tract tumor
  • the GCC-related disease or condition is one or more selected from gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, esophageal junction cancer, small intestine cancer, pancreatic cancer and liver cancer.
  • Another aspect of the present disclosure relates to a method for treating or preventing a GCC-related disease or disorder, comprising the step of administering to a subject in need thereof an effective amount of the anti-GCC antibody or antigen-binding fragment thereof described in any one of the present disclosure, the chimeric antigen receptor described in any one of the present disclosure, or the cell described in any one of the present disclosure;
  • the GCC-related disease or condition is a tumor
  • the GCC-related disease or condition is a digestive tract tumor
  • the GCC-related disease or condition is one or more selected from gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, esophageal junction cancer, small intestine cancer, pancreatic cancer and liver cancer.
  • EC50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can induce 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a "heavy” (H) chain.
  • Antibody light chains can be classified as ⁇ and ⁇ light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ or ⁇ , and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain CL.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability (called complementary determining regions, CDRs), interspersed with more conservative regions called framework regions (FRs).
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR
  • FR refers to those variable region residues that flank the CDRs. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
  • CDR refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the VH ⁇ -fold framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the VL ⁇ -fold framework of an antibody.
  • the positions of CDRs and FRs can be determined using a variety of definition methods well known in the art, for example, the Kabat numbering system, the AbM numbering system, the IMGT numbering system, the Chothia numbering system, or the Contact numbering system.
  • the Kabat numbering system is an 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).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the IMGT numbering system is based on the numbering system of the international ImMunoGeneTics information (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the contact numbering system is based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-745).
  • CDRs are determined based on amino acid sequence and position in the light chain or heavy chain. Since the "position" of CDRs within the immunoglobulin variable region domain is conserved between species, CDR and framework region amino acid residues are easily identified by comparing the structural features divided using the known numbering system with the variable region domain amino acid sequence. The amino acid residues from each of these hypervariable regions or CDRs are illustrated in Table 1 below.
  • CDR complementary metal-oxide-semiconductor
  • HCDR1 complementarity determining region 2 of an antibody or region thereof
  • a scheme for identifying a particular CDR is specified, such as by IMGT, Kabat, AbM, CDR defined by Chothia or Contact method.
  • the specific amino acid sequence of CDR is given.
  • CDR region can also be determined by a combination of various numbering systems, such as a combination of Kabat and Chothia numbering systems, a combination of Kabat and AbM numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, for example, the term "CDR1 as described in a specific VH or VL” includes any CDR1 as defined by the above-mentioned exemplary CDR numbering system, but is not limited thereto.
  • a variable region e.g., VH or VL
  • those skilled in the art will understand that the CDRs in this region can be defined by different numbering systems or their combinations.
  • antibody is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • the antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
  • Monoclonal antibodies have high specificity for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, which usually recognize different epitopes on an antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but it can also be obtained by recombinant DNA technology (see, for example, US Patent 4,816,567).
  • humanized antibody refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • the antigen-binding fragments of antibodies are diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448 and Poljak RJ et al., Structure 1994; 2: 1121-1123).
  • single chain antibody refers to a molecule comprising an antibody heavy chain variable region ( VH ) and an antibody light chain variable region ( VL ) connected by a linker.
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • the VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242: 423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85: 5879-5883).
  • Such scFv molecules may have the general structure: NH2- VL -linked fragment- VH -COOH or NH2- VH -linked fragment- VL -COOH.
  • Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 69) amino acid sequences or variants thereof.
  • GGGGS linker having an amino acid sequence
  • SEQ ID NO: 70 linker having an amino acid sequence
  • Other linkers useful in the present disclosure are described by Alfthan et al, Protein Eng. 1995; 8: 725-731, Choi et al, Eur. J.
  • the term “isolated” or “isolated” refers to that obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated.
  • isolated or “isolated” does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • plasmids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • bacteriophages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses include adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain a replication initiation site.
  • the term "host cell” refers to cells that can be used to introduce a vector, including but not limited to The invention is limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen means that the antibody binds to the antigen with an affinity ( KD ) of less than about 10-5 M, such as less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less.
  • KD refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
  • an antibody binds to an antigen (e.g., a GCC protein) with a dissociation equilibrium constant ( KD ) of less than about 10-5 M, such as less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M, or 10-10 M or less.
  • KD can be determined using methods known to those skilled in the art, such as using a Fortebio molecular interaction instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • immunohistochemistry refers to an immunocytochemical study conducted at the tissue level.
  • immunohistochemical staining is a type of immunostaining, which is to combine fluorescent or colorable chemicals on antibodies, and use the immunological principle that the modified antibodies have specific binding to antigens in biological tissues to selectively identify antigens in tissue sections or smear cells, and detect whether there is a target antigen in cells or tissues; this method can not only be used to detect the expression of antigens, but also to observe the location of antigen expression, and can improve the sensitivity and specificity of pathological diagnosis.
  • the artificially prepared and modified specific antibodies are labeled with colorants, such as enzymes, metal ions, isotopes, etc.; and the target antigens of the cells or tissues to be detected can be any substance that can bind to antibodies, that is, antigenic substances, including proteins, polypeptides, nucleic acids, polysaccharides, pathogens, etc. Both of them locate, quantify and characterize the antigens of tissues or cells through immunological antigen-antibody reactions and histochemical color reactions, combined with tissue cell morphological analysis.
  • colorants such as enzymes, metal ions, isotopes, etc.
  • the target antigens of the cells or tissues to be detected can be any substance that can bind to antibodies, that is, antigenic substances, including proteins, polypeptides, nucleic acids, polysaccharides, pathogens, etc. Both of them locate, quantify and characterize the antigens of tissues or cells through immunological antigen-antibody reactions and histochemical color reactions, combined with tissue cell morphological analysis.
  • chimeric antigen receptor refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune cells such as T cells. Some CARs are also called “artificial T cell receptors,””chimeric T cell receptors,” or “chimeric immune receptors.” In some embodiments, CARs comprise an extracellular antigen binding domain, a transmembrane domain, and T cells and/or their The term “CAR-T cell” refers to a T cell that expresses CAR.
  • the subject is a mammal, such as a non-primate or a primate (e.g., a human).
  • the subject can be a mammal, such as a human, diagnosed with a disease or condition.
  • the subject can be a mammal, such as a human, who is at risk of developing a disease or condition.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., a tumor
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.
  • the anti-GCC antibody or antigen-binding fragment thereof disclosed herein has good specificity.
  • the anti-GCC antibody or antigen-binding fragment thereof disclosed herein has a high affinity.
  • the anti-GCC antibody or antigen-binding fragment thereof disclosed in the present invention has high sensitivity in immunohistochemical analysis and has good application prospects in tumor detection and preparation of detection kits.
  • the anti-GCC antibody or antigen-binding fragment thereof, the chimeric antigen receptor or the chimeric antigen receptor T cell of the present disclosure have good potential for treating or preventing tumors.
  • Figures 1A and 1B show the binding curves of anti-GCC antibody 22G5-Rabbit IgG and target proteins human GCC-his protein ( Figure 1A) and mouse GCC-his protein ( Figure 1B) detected by ELISA.
  • PF0098-Rabbit IgG was used as a control, and the binding of 22G5-Rabbit IgG to GCC target protein was concentration-dependent, and its EC 50 was similar to that of PF0098-Rabbit IgG antibody.
  • Figures 2A to 2C show the binding curves of the anti-GCC antibody 22G5-Rabbit IgG and target cells detected by flow cytometry.
  • PF0098-Rabbit IgG was used as a control.
  • the binding of 22G5-Rabbit IgG to target cells 293T-human GCC.Luc ( Figure 2A) and 293T-mouse GCC.Luc ( Figure 2B) was concentration-dependent, and there was no nonspecific binding to 293T cells ( Figure 2C), and the specificity was better than PF0098-Rabbit IgG. Therefore, 22G5-Rabbit IgG is a new type of anti-GCC antibody with high specificity.
  • Figure 3 shows the results of immunohistochemical detection of tissue sections of human intestinal cancer cell SW948 subcutaneous tumor, human intestinal cancer cell LS1034 subcutaneous tumor, mouse small intestine and mouse spleen using different anti-GCC antibodies at the same concentration (0.5 ⁇ g/mL).
  • the isotype control antibody ab172730 did not stain on the four tissue sections.
  • the PF0098-Rabbit IgG antibody had strong GCC-specific staining in SW948 subcutaneous tumor and LS1034 subcutaneous tumor sections, and produced non-specific staining in mouse spleen.
  • the 22G5-Rabbit IgG antibody had strong GCC-specific staining in SW948 subcutaneous tumor, LS1034 subcutaneous tumor and mouse small intestine sections, and its cell membrane staining was stronger than that of the PF0098-Rabbit IgG antibody, and there was no non-specific staining in the four tissues.
  • the staining of anti-GCC antibody ab122404 in SW948 subcutaneous tumor and LS1034 subcutaneous tumor was concentrated in the cytoplasm, and no specific staining of GCC on the cell membrane surface was shown. In addition, non-specific staining was produced in the mouse small intestine and mouse spleen. Therefore, in the application of immunohistochemical staining, 22G5-Rabbit IgG antibody has excellent specificity and sensitivity.
  • Figures 4A to 4C show the cell killing results of the constructed CAR-T cells against the target cells CT26-mouse GCC.Luc overexpressing mouse GCC at effector-target ratios of 5:1 ( Figure 4A), 2.5:1 ( Figure 4B) and 1.25:1 ( Figure 4C).
  • PF0098 CAR-T was used as a positive control, and all CAR-Ts were able to effectively kill mouse GCC-positive target cells.
  • Figures 5A to 5C show the cell killing results of the constructed CAR-T cells against the target cells LS1034.Luc that naturally express human GCC at effector-target ratios of 5:1 ( Figure 5A), 2.5:1 ( Figure 5B), and 1.25:1 ( Figure 5C).
  • PF0098 CAR-T was used as a control, and 14C2 CAR-T that recognized the mouse target could not effectively kill the target cells.
  • the remaining CAR-T cells had strong cytotoxicity against human GCC-positive target cells. This shows that rabbit-derived scFv is also suitable for the antigen binding domain of CAR, and enables CAR-T to play a highly efficient recognition and killing function.
  • Figures 6A to 6C show the cytotoxicity of the constructed CAR-T cells against 293T.Luc cells without GCC expression at effector-target ratios of 5:1 (Figure 6A), 2.5:1 ( Figure 6B), and 1.25:1 ( Figure 6C). None of them could kill 293T.Luc, indicating that the constructed CAR-T cells can only specifically recognize GCC-positive cells.
  • Figures 7A to 7D show the cytokine secretion levels of the constructed CAR-T cells after co-incubation with target cells CT26-mouse GCC.Luc ( Figure 7A), LS1034.Luc ( Figure 7B), and 293T.Luc ( Figure 7C) for 24 hours. After incubation with target cells CT26-mouse GCC.Luc or LS1034.Luc, the IFN ⁇ secreted by CAR-T cells was upregulated.
  • Figure 7D shows the background IFN ⁇ levels secreted by UnT and each group of CAR-T cells without co-culture with target cells.
  • Genscript Biotech Co., Ltd. Through the service order of Genscript Biotech Co., Ltd. (Genscript, Cat. NO. C5825HF280), New Zealand rabbits were immunized with GCC protein to obtain antibodies that bind to GCC with high affinity.
  • the specific screening method and steps are described as follows.
  • the following method was used to construct Dubca-human GCC.Luc and Dubca-mouse GCC.Luc overexpression cell lines.
  • the human GCC coding sequence (NM_004963.3) and the mouse GCC coding sequence (NM_001127318.1) were synthesized and inserted into the lentiviral expression vector pLVX-Luc-puro (Clontech, Cat. No. 632164), respectively, and then transfected into Lenti-X 293T host cells together with the auxiliary packaging plasmids psPAX2 and pMD.2G, and the lentivirus was obtained by concentration and purification.
  • the two lentiviruses obtained were transduced into the Dubca cell line ( CRL2276 TM ), and finally Dubca-human GCC.Luc and Dubca-mouse GCC.Luc were obtained after three rounds of puromycin resistance screening.
  • the expression of human GCC target was detected by incubating Dubca-human GCC.Luc cells with 5F9-hIgGFc (Genscript, Cat. NO. C0224DK260-1) antibody. 2.0 ⁇ 10 5 Dubca-human GCC.Luc and Dubca cells were incubated with 1:100 diluted 5F9-hIgGFc antibody at 4°C for 0.5 hours; washed 3 times with DPBS, and then resuspended with 1:100 fluorescent secondary antibody (PE anti-human IgG Fc Antibody, Biolegend, Cat. No. 409304), incubated at 4°C in the dark for 0.5 hours; washed 3 times with DPBS, and then loaded on the flow cytometer for reading.
  • 5F9-hIgGFc Genscript, Cat. NO. C0224DK260-1
  • 2.0 ⁇ 10 5 Dubca-human GCC.Luc and Dubca cells were incubated with 1:100 diluted 5F9-hIgGFc antibody at 4°C for
  • PF0098 is an anti-GCC antibody disclosed in patent application WO2019224716A2 (VH as shown in SEQ ID NO:36, VL as shown in SEQ ID NO:42), which can simultaneously recognize human GCC, monkey GCC and mouse GCC, and can also perform immunohistochemical detection. This antibody will also be cited as a standard control in the subsequent examples.
  • CT26 cell line ATCC, CRL-2638TM
  • HEK293T cell line Clontech, Cat. No. 632180
  • overexpressed mouse GCC NM_001127318.1
  • human GCC NM_004963.3
  • CT26-mouse GCC.Luc 293T-mouse GCC.Luc
  • 293T-human GCC.Luc 293T-human GCC.Luc cells.
  • HEK293T cell line Clontech, Cat. No.
  • colon cancer cell line LS1034 (ATCC, CRL-2158TM) that naturally overexpresses human GCC were also constructed to express firefly luciferase, i.e., LS1034.Luc cells, and used for subsequent in vitro screening.
  • Anti-GCC rabbit monoclonal antibodies were produced through the MonoRab TM platform service order of GenScript Biotech Co., Ltd.
  • the His-tagged mouse GUCY2C/Guanylyl cyclase C protein (ACRO GUC-M52H3, referred to as mouse GCC-his protein) was modified with ImmunoPlus TM technology and immunized two New Zealand white rabbits using traditional immunization methods, with an interval of two weeks.
  • 293T-mouse GCC cells were used for immunization in the third round.
  • the FACS detection method is as follows: digest and collect the target cells, divide them into 96-well pointed bottom plates, 2 ⁇ 10 5 cells per well, centrifuge at 400g for 3 minutes, discard the supernatant, add 100 ⁇ L of B cell clone supernatant and incubate at 4°C for 1 hour, wash 3 times with 4°C pre-cooled DPBS buffer, and add 1:100 diluted Alexa 488 goat anti-rabbit IgG (H+L) (Invitrogen, Cat.NO.A11034) was incubated at 4°C for 30 minutes and washed again with 4°C pre-cooled DPBS for 3 times.
  • the positive B cell clones were isolated by MonoRab TM platform. Reagent (Ambion, Cat.No.: 15596-026) standard technical process to extract RNA, according to PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara, Cat.No.: 6110A) instruction manual for cDNA reverse transcription, amplification of the antibody light chain and heavy chain fragments, the second generation sequencing method to obtain the antibody light and heavy chain sequence.
  • Table 3 and Table 4 respectively show the antibody heavy chain variable region (VH) and light chain variable region (VL) sequences divided according to the Kabat partition rule, and the corresponding complementary determining region (CDR) sequence.
  • the antibody heavy chain variable region (VH) and light chain variable region (VL) are connected by a linker, and the linker can be composed of a repeated GGGGS (SEQ ID NO: 69) amino acid sequence or a variant thereof (e.g., SEQ ID NOs: 67-68).
  • Rabbit IgG molecules are divided into IgG-k and IgG- ⁇ according to the light chain. Generally, most antibodies obtained from New Zealand white rabbits are IgG-k type, and only ⁇ 1% are IgG- ⁇ type.
  • the molecular weight of natural rabbit IgG-k is about 150KD, containing two identical light chains and two identical heavy chains.
  • the light chain is composed of the N-terminal variable domain VL and the constant domain CL (e.g., SEQ ID NO: 56)
  • the heavy chain is composed of the N-terminal variable domain VH and three constant domains (CH1, CH2 and CH3, e.g., SEQ ID NO: 55), of which VL and VH contain three complementary determining regions CDR1, CDR2 and CDR3.
  • the light chain and the heavy chain, as well as the two heavy chains, are connected in the form of disulfide bonds.
  • the heavy chain variable region and light chain variable region sequences selected above were fused with rabbit IgG (IgG1k), such as 22G5 single-chain antibody sequence or PF0098 single-chain antibody sequence, to obtain 22G5 VH-Rabbit IgG CH1-CH2-CH3 (SEQ ID NO: 57), 22G5 VL-Rabbit IgG CL (SEQ ID NO: 58) or PF0098 VH-Rabbit IgG CH1-CH2-CH3 (SEQ ID NO: 59) and PF0098 VL-Rabbit IgG CL (SEQ ID NO: 60).
  • IgG1k rabbit IgG
  • the coding sequence of the fusion sequence was inserted into the pcDNA3.4 vector to construct an expression plasmid, and then the 22G5-Rabbit IgG antibody and PF0098-Rabbit IgG antibody were expressed in HD CHO-S cell culture, and the antibody was further purified.
  • the concentration of the obtained antibodies was determined using Nano Drop A280nm, and the purity was evaluated by SDS-PAGE.
  • the purification information of 22G5-Rabbit IgG and PF0098-Rabbit IgG antibodies is shown in Table 5. This shows that the 22G5 antibody can be efficiently recombinantly expressed.
  • the binding ability of the purified antibody and the target protein was detected by enzyme-linked immunosorbent assay.
  • the two target proteins, human GCC-his and mouse GCC-his, were diluted with coating solution and coated on 96-well Costar ELISA plates at a concentration of 1 ⁇ g/mL, 100 ⁇ L per well, and placed at 4°C overnight.
  • Flow cytometry was used to detect the binding ability of the purified antibodies to target cells.
  • 293T-human GCC.Luc and 293T-mouse GCC.Luc cells were used as target cells, and 293T cells were used as negative controls.
  • the cells were plated in a 96-well plate at a density of 0.2M per well, incubated with serially diluted antibodies at 4°C for 60 minutes, washed three times with DPBS, and the supernatant was discarded.
  • Alexa Fluor 500 was used with a 1:100 dilution. Resuspend the cell pellet with 488 goat anti-rabbit IgG (H+L) (Invitrogen, Cat.NO.A11034) and incubate at 4°C in the dark for 30 minutes.
  • the flow cytometer was loaded and read. As shown in Figures 2A to 2C, the binding of 22G5-Rabbit IgG and PF0098-Rabbit IgG antibodies to target cells is strongly concentration-dependent, and the EC 50 is shown in Table 7. In addition, 22G5-Rabbit IgG did not bind to 293T cells, but PF0098-Rabbit IgG antibody showed strong non-specific binding to 293T cells at high concentrations. In summary, 22G5-Rabbit IgG has a high specificity for anti-GCC antigen.
  • the paraffin tissue sections used for detection were dewaxed and then soaked in 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol and pure water. Then, the sections were microwave-heated for antigen repair using an improved sodium citrate antigen repair solution (BEYOTIME#P0083). After the repair system cooled, the sections were washed with PBS and then blocked with 10% hydrogen peroxide (Sinopharm Chemical Reagent Co., Ltd. #10011218) and 5% goat serum (BEYOTIME#C0265) in sequence.
  • the anti-GCC antibody was diluted to a working concentration of 0.5 ⁇ g/mL using antibody diluent (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
  • the experimental results are shown in Figure 3.
  • the isotype control antibody ab172730 (Abcam, Recombinant Rabbit IgG, monoclonal [EPR25A] -Isotype Control) showed no staining on the four tissue sections.
  • the PF0098-Rabbit IgG antibody had strong GCC-specific staining in the SW948 subcutaneous tumor and LS1034 subcutaneous tumor sections, and produced non-specific staining in the mouse spleen.
  • the 22G5-Rabbit IgG antibody had strong specific staining for the SW948 subcutaneous tumor, LS1034 subcutaneous tumor, and mouse small intestine tissue sections expressing GCC.
  • the backbone structure sequence encoding the chimeric antigen receptor (CAR) was synthesized using gene synthesis technology, that is, the coding structures from N-terminus to C-terminus were: CD8 ⁇ hinge region (SEQ ID NO: 62), CD8 ⁇ transmembrane domain (SEQ ID NO: 63), CD137 co-stimulatory signal domain (SEQ ID NO: 64) and CD3 ⁇ intracellular signal domain (SEQ ID NO: 65) nucleotide molecules and cloned into a lentiviral vector (pLSINK-BBzBB), and the resulting backbone vector was named "pLSINK-CE11A16".
  • the multiple cloning site (MCS) in the vector allows the nucleic acid sequence containing the Kozak sequence (SEQ ID NO: 66) to be operably linked to the CD8 ⁇ signal peptide (SEQ ID NO: 61) of the CAR backbone sequence and inserted upstream of the CAR backbone vector.
  • the synthesized nucleic acid sequences encoding CD8 ⁇ signal peptide and anti-GCC single-chain antibody were cloned into the lentiviral vector pLSINK-CE11A16 through EcoRI (5′-GAATTC-3′) and SpeI (5′-ACTAGT-3′) restriction sites.
  • the constructed chimeric antigen receptors (CARs) were 14C2 CAR, 21E1 CAR, 22G5 CAR, 25E2 CAR, 48D5 CAR and PF0098 CAR (e.g., SEQ ID NOs: 49-53 and SEQ ID NO: 54).
  • a mixture of lentiviral packaging plasmids containing pMDLg.pRRE (Addgene #12251), pRSV-REV (Addgene #12253), and pMD2.G (Addgene #12259) was added to the vector expressing the CAR construct.
  • PKI polyetherimide
  • the mixture was then added to HEK293 cells for transfection. Afterwards, the cells were incubated overnight at 37°C in a cell culture incubator containing 5% CO 2. The supernatant was collected and centrifuged at 4°C, 3000g for 15 minutes.
  • the supernatant was added with 20% PEG 8000 prepared in advance at a ratio of 4:1 and carefully mixed by inversion.
  • the virus was precipitated at 4°C overnight. Subsequently, the supernatant was centrifuged at 4°C, 3000g for 30 minutes, the supernatant was carefully discarded, and the virus pellet was carefully resuspended with pre-cooled DPBS and stored at -80°C for use.
  • the virus titer was determined by titration of transduction of the SUP-T1 (human T lymphoblastoid) cell line.
  • Human T cells were purified from commercial PBMCs using the Miltenyi Pan T Cell Isolation Kit (Cat#130-096-535) according to the manufacturer's protocol as described below. First, determine the cell number and centrifuge the cell suspension at 300g for 10 minutes. Then discard the supernatant completely and resuspend the cell pellet in 40 ⁇ L MACS buffer (DPBS supplemented with 8 ⁇ M EDTA and 0.5% FBS) per 10 7 total cells. Add 10 ⁇ L Pan T Cell Biotin-Antibody Cocktail per 10 7 total cells, mix thoroughly and incubate in a refrigerator (2-8°C) for about 5 minutes. Then add 30 ⁇ L MACS buffer per 10 7 cells. Add 20 ⁇ L Pan T Cell MicroBead Cocktail per 10 7 cells.
  • MACS buffer DPBS supplemented with 8 ⁇ M EDTA and 0.5% FBS
  • the prepared T cells were activated for 48 hours using human T Cell TransAct TM (Miltenyi #130-111-160), in which anti-CD3/CD28 MACSiBead beads were added at a ratio of 1:2 to T cells.
  • human T Cell TransAct TM Miltenyi #130-111-160
  • anti-CD3/CD28 MACSiBead beads were added at a ratio of 1:2 to T cells.
  • the activated T cells were transduced with lentivirus at a certain multiplicity of infection, and R10 medium containing 300 IU/mL IL-2 was added 72 hours later.
  • the transduced cells were transferred to a new cell culture plate and expanded at 37°C with 5 % CO2.
  • CAR expression levels were evaluated by flow cytometry using directly labeled antibodies.
  • 5 ⁇ 10 5 T cells were collected from each group and antibodies were prepared using pre-cooled DPBS buffer, in which PF0098 CAR-T and untransduced CAR T cells (UnT) were incubated with 1:100 diluted Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen, Cat.NO.A11001) was incubated at 4°C for 30 minutes. The remaining CAR-T cells and untransduced UnT cells were incubated with 1:100 diluted Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen, Cat. NO.
  • the CAR-T cells prepared in Example 4 were subjected to a cytotoxicity assay.
  • the above-mentioned CAR-T cells were co-incubated with the LS1034.Luc, CT26-mouse GCC.Luc and 293T.Luc cells prepared in Example 1 at an effector cell to target cell ratio (E/T) of 5:1, 2.5:1 and 1.25:1 for 24 hours, respectively.
  • E/T effector cell to target cell ratio
  • PF0098 CAR-T was a positive control.
  • One-glo luminescent luciferase assay reagent Promega #E6120
  • the remaining luciferase activity is directly related to the number of remaining target cells in the well. Cytotoxicity is calculated by the following formula:
  • Cytotoxicity % 100% x (1 - (RLU sample - RLU min ) / (RLU unT - RLU min )).
  • RLU sample refers to the luciferase activity measured in the wells of CAR-T cells transduced with anti-GCC CAR.
  • RLU min refers to the luciferase activity measured in the wells to which Triton X-100 was added at a final concentration of 1% at the beginning of the cytotoxicity assay, and
  • RLU unT refers to the luciferase activity measured in the wells of T cells not transduced with CAR.
  • GCC CAR-T cells prepared in Example 4
  • target cells LS1034.Luc, CT26-mouse GCC.Luc and 293T.Luc cells prepared in Example 1
  • GCC CAR-T cells were mixed with target cells at an E/T ratio of 2.5:1 (2000 Target cells/well) were co-incubated in a 384-well plate for 24 hours.
  • the IFN- ⁇ concentration produced in the culture supernatant after co-incubation of CAR-T with target cells and in the supernatant of CAR-T cells themselves was measured by HTRF kit (Cisbio, Cat#62HIFNGPEG).
  • the HTRF reagent needs to be stable at room temperature for 30 minutes.
  • the co-culture supernatant was transferred to a 384-well assay plate (Greiner Bio-One, #784075) 16 ⁇ L/well, and then 4 ⁇ L/well premixed HTRF reagent prepared according to the kit operation manual was added.
  • the plate was sealed with a sealing film and incubated overnight at room temperature.
  • HTRF values were read on Tecan Spark 10M.
  • the IFN- ⁇ concentration was calculated by referring to the signal obtained from the standard curve provided by the kit.
  • the concentrations of IFN ⁇ secreted by the five CAR-T cells were detected to range from 1488.3 pg/mL to 2692.5 pg/mL (co-incubated with CT26-mGCC.Luc target cells) and 1173.1 pg/mL to 1939.0 pg/mL (co-incubated with LS1034.Luc target cells, except 14C2 CAR-T), which were upregulated relative to the UnT control (detection values were 600.4 pg/mL and 910.8 pg/mL).

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Abstract

本公开属于生物医药领域，涉及一种抗鸟苷酸环化酶C(GCC)抗体或抗原结合片段、包含该抗GCC抗体或抗原结合片段的嵌合抗原受体(CAR)及用途。所披露的抗GCC抗体或抗原结合片段可以用于GCC相关疾病或病症的伴随诊断，如免疫组化分析；所披露的GCC CAR构建体转导的免疫细胞可用于GCC相关疾病或病症的细胞免疫疗法。

Description

抗GCC抗体、嵌合抗原受体及其用途
相关申请的交叉引用
本申请是以PCT申请号为PCT/CN2023/120440，申请日为2023年9月21日的申请为基础，并主张其优先权，该PCT申请的全部内容在此作为整体引入本申请中。
技术领域
本公开属于生物医药领域，涉及一种抗GCC抗体、包含该抗GCC抗体的嵌合抗原受体及用途。
背景技术
鸟苷酸环化酶C(guanylyl cyclase C，简称GUCY2C或GCC)是一种膜结合型的鸟苷酸环化酶，属于单次跨膜蛋白受体，包括细胞外结构域、跨膜结构域及胞内结构域。该酶是细菌热稳定肠毒素的肠道受体，所以GUCY2C又称为热稳定肠毒素受体。鸟苷素和尿鸟苷素以及人工GCC激动剂均可结合并激活GCC。GCC的激活可刺激胞内第二信使cGMP的产生，起到调节水和电解质平衡、维持肠道屏障功能和发挥抗炎活性等作用。
在正常人体组织中，GCC表达于肠上皮细胞顶端。GCC突变导致的功能丧失，会导致液体和离子分泌减少，从而引起便秘；GCC突变导致的功能增强，会使液体和离子分泌增加，从而引起分泌性腹泻。GCC-cGMP信号轴在肠道肿瘤和炎症性肠病等疾病的发病过程中也具有重要作用：由鸟苷素和尿鸟苷素显著下调导致的GCC-cGMP信号传导失活与肿瘤发生密切相关。GCC-cGMP信号传导上调会激活STAT1信号通路，导致炎症性肠病等。GCC在大多数消化道肿瘤组织中呈特异性高表达，因此是消化道肿瘤靶向治疗的理想靶标。
嵌合抗原受体T(CAR-T)细胞免疫疗法是一种新型的抗原靶向性的精准治疗技术，T细胞转染和表达特异性CAR基因后，能识别并结合肿瘤细胞表面特定抗原，不依赖于MHC共刺激信号而直接激活T细胞，进而攻击肿瘤细胞。CAR-T细胞疗法具有优于其他T细胞重定向治疗策略的潜在优势。其他治疗策略通常需要长期治疗直到疾病进展，而CAR-T细胞可以在体内扩增，只需要单次输注，即可获得长期的疾病缓解。CAR-T细胞疗法正在成为癌症治疗中有潜力的治疗方案。
目前尚需要开发新的抗GCC抗体。
发明内容
经过深入的研究和创造性的劳动，本公开提供了一种新的抗鸟苷酸环化酶C(GCC)抗体，能识别人和鼠GCC抗原靶点，细胞膜定位强，特异性强，未来可以用于伴随诊断抗体开发。本申请提供的靶向GCC的兔源抗体，不但能用于开发靶向GCC的嵌合抗原受体(CAR)，而且能够用于免疫组化分析。由此提供了下述发明：
本公开的一个方面涉及一种抗GCC抗体或其抗原结合片段，包含重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1至HCDR3，所述轻链可变区包含轻链互补决定区LCDR1至LCDR3，其中：
(1)所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:37所示的氨基酸序列中的LCDR1、LCDR2和LCDR3；
(2)所述重链可变区包含如SEQ ID NO:32所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:38所示的氨基酸序列中的LCDR1、LCDR2和LCDR3；
(3)所述重链可变区包含如SEQ ID NO:33所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:39所示的氨基酸序列中的LCDR1、LCDR2和LCDR3；
(4)所述重链可变区包含如SEQ ID NO:34所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:40所示的氨基酸序列中的LCDR1、LCDR2和LCDR3；或者
(5)所述重链可变区包含如SEQ ID NO:35所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:41所示的氨基酸序列中的LCDR1、LCDR2和LCDR3。
在本公开的一些实施方式中，所述的抗GCC抗体或其抗原结合片段，其中，
所述重链可变区的HCDR1至HCDR3以及所述轻链可变区的LCDR1至LCDR3按照Kabat编号系统、AbM编号系统、IMGT编号系统、Chothia编号系统或Contact编号系统确定。
在本公开的一些实施方式中，所述的抗GCC抗体或其抗原结合片段，其中：
(1)HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:6所示、HCDR3的氨基酸序列如SEQ ID NO:11所示、LCDR1的氨基酸序列如SEQ ID NO:16所示、LCDR2的氨基酸序列如SEQ ID NO:21所示和LCDR3的氨基酸序列如SEQ ID NO:26所示；
(2)HCDR1的氨基酸序列如SEQ ID NO:2所示、HCDR2的氨基酸序列如SEQ ID NO:7所示、HCDR3的氨基酸序列如SEQ ID NO:12所示、LCDR1的氨基酸序列如SEQ ID NO:17所示、LCDR2的氨基酸序列如SEQ ID NO:22所示和LCDR3的氨基酸序列如SEQ ID NO:27所示；
(3)HCDR1的氨基酸序列如SEQ ID NO:3所示、HCDR2的氨基酸序列如SEQ ID NO:8所示、HCDR3的氨基酸序列如SEQ ID NO:13所示、LCDR1的氨基酸序列如SEQ ID NO:18所示、LCDR2的氨基酸序列如SEQ ID NO:23所示和LCDR3的氨基酸序列如SEQ ID NO:28所示；
(4)HCDR1的氨基酸序列如SEQ ID NO:4所示、HCDR2的氨基酸序列如SEQ ID NO:9所示、HCDR3的氨基酸序列如SEQ ID NO:14所示、LCDR1的氨基酸序列如SEQ ID NO:19所示、LCDR2的氨基酸序列如SEQ ID NO:24所示和LCDR3的氨基酸序列如SEQ ID NO:29所示；或者
(5)HCDR1的氨基酸序列如SEQ ID NO:5所示、HCDR2的氨基酸序列如SEQ ID NO:10所示、HCDR3的氨基酸序列如SEQ ID NO:15所示、LCDR1的氨基酸序列如SEQ ID NO:20所示、LCDR2的氨基酸序列如SEQ ID NO:25所示和LCDR3的氨基酸序列如SEQ ID NO:30所示。
在本公开的一些实施方式中，所述的抗GCC抗体或其抗原结合片段，其中，
(1)所述重链可变区包含与SEQ ID NO:31所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:37所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列；
(2)所述重链可变区包含与SEQ ID NO:32所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:38所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性 的氨基酸序列；
(3)所述重链可变区包含与SEQ ID NO:33所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:39所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列；
(4)所述重链可变区包含与SEQ ID NO:34所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:40所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列；或者
(5)所述重链可变区包含与SEQ ID NO:35所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:41所示氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列。
在本公开的一些实施方式中，所述的抗GCC抗体或其抗原结合片段，其中，
(1)所述重链可变区的氨基酸序列如SEQ ID NO:31所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:37所示；
(2)所述重链可变区的氨基酸序列如SEQ ID NO:32所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:38所示；
(3)所述重链可变区的氨基酸序列如SEQ ID NO:33所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:39所示；
(4)所述重链可变区的氨基酸序列如SEQ ID NO:34所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:40所示；或者
(5)所述重链可变区的氨基酸序列如SEQ ID NO:35所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:41所示。
在本公开的一些实施方式中，所述的抗GCC抗体或其抗原结合片段，其中，所述抗GCC抗体包括非CDR区，且所述非CDR区来自人、小鼠或兔；在一些具体实施方式中，所述非CDR区包括框架区、重链恒定区和/或轻链恒定区。
在本公开的一些实施方式中，所述的抗GCC抗体或其抗原结合片段，其中，所述抗原结合片段是Fab、Fab'、F(ab')2、单链抗体(scFv)或二硫键稳定的可变片段(dsFv)。
在本公开的一些实施方式中，所述的抗GCC抗体或其抗原结合片段，其包含与SEQ ID NO:43至SEQ ID NO:47中的任一序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列；或者包含与SEQ ID NO:57和SEQ ID NO:58具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列；在一些具体实施方式中，所述抗GCC抗体或其抗原结合片段的氨基酸序列如SEQ ID NO:43至SEQ ID NO:47中的任一序列所示。
本公开的另一方面涉及一种分离的核酸，其编码本公开中任一项所述的抗GCC抗体或其抗原结合片段。
本公开的再一方面涉及一种载体，其包含本公开的分离的核酸。
本公开的再一方面涉及一种宿主细胞，其包含本公开的分离的核酸，或者包含本公开的载体。
本公开的再一方面涉及一种嵌合抗原受体(CAR)，其包含多肽，该多肽包含：
(1)细胞外抗原结合结构域，该细胞外抗原结合结构域包含如本公开中任一项所述的抗GCC抗体或抗原结合片段；
(2)跨膜结构域；以及
(3)细胞内信号传导结构域。
在本公开的一些实施方式中，所述的嵌合抗原受体，其中该抗GCC抗体或抗原结合片段是单链抗体(scFv)。
在本公开的一些实施方式中，所述的嵌合抗原受体，其进一步包含位于该多肽的N-末端的信号肽；
在一些具体实施方式中，所述信号肽来源于CD8α、CD28或CD4分子；
在一些具体实施方式中，所述信号肽包含如SEQ ID NO:61所示的氨基酸序列，或者与SEQ ID NO:61具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列。
在本公开的一些实施方式中，所述的嵌合抗原受体，其进一步包含位于所述细胞外抗原结合结构域的C-末端和所述跨膜结构域的N-末端之间的铰链结构域；
在一些具体实施方式中，所述铰链区来源于IgG4Fc CH2CH3、CD28或CD8α；
在一些具体实施方式中，所述铰链区包含如SEQ ID NO:62所示的氨基酸序列，或者与SEQ ID NO:62具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列。
在本公开的一些实施方式中，所述的嵌合抗原受体，其中所述跨膜结构域来源于CD8α、CD4、CD28、CD137、CD80、CD86、CD152和PD1分子跨膜区的一种或多种；
在一些具体实施方式中，所述跨膜结构域包含如SEQ ID NO:63所示的氨基酸序列，或者与SEQ ID NO:63具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列。
在本公开的一些实施方式中，所述的嵌合抗原受体，其中所述细胞内信号传导结构域包含共刺激信号传导结构域；
在一些具体实施方式中，所述共刺激信号传导结构域源自以下共刺激分子：CD27、CD28、CD137、OX40、CD30、CD40、CD3、HVEM、ICOS、Myd88、LFA-1、ICOS、CD2、CD7、LIGHT、NKG2C、B7-H3、CD83的配体以及它们的组合；
在一些具体实施方式中，所述共刺激信号传导结构域包含CD28的胞质域和/或CD137的胞质域；
在一些具体实施方式中，所述共刺激信号传导结构域包含如SEQ ID NO:64所示的氨基酸序列，或者与SEQ ID NO:64具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列。
在本公开的一些实施方式中，所述的嵌合抗原受体，其中所述细胞内信号传导结构域包含初级细胞内信号传导结构域；
在一些具体实施方式中，所述初级细胞内信号传导结构域源自CD3ζ；
在一些具体实施方式中，所述初级细胞内信号传导结构域包含如SEQ ID NO:65所示的氨基酸序列，或者与SEQ ID NO:65具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列。
在本公开的一些实施方式中，所述的嵌合抗原受体，其氨基酸序列如SEQ ID NO:49至SEQ ID NO:53或者SEQ ID NO:71至SEQ ID NO：75中任一序列所示。
本公开的再一方面涉及一种分离的核酸，其编码本公开中任一项所述的嵌合抗原受体。
本公开的再一方面涉及一种载体，其包含本公开的分离的核酸。
本公开的再一方面涉及一种细胞，其包含本公开中任一项所述的嵌合抗原受体， 包含本公开的分离的核酸，或者包含本公开的载体；
在一些具体实施方式中，所述细胞为免疫细胞。
在本公开的一些实施方式中，所述的细胞，其中，所述细胞选自：T细胞、NK细胞、外周血单核细胞(PBMC)、造血干细胞、多能干细胞、胚胎干细胞以及它们的组合。
本公开的再一方面涉及一种药物组合物，其包含本公开中任一项所述的抗GCC抗体或其抗原结合片段，或者包含本公开中任一项所述的嵌合抗原受体，或者包含本公开中任一项所述的细胞；
在一些具体实施方式中，所述药物组合物还包含一种或多种药学上可接受的辅料。
本公开的再一方面涉及本公开中任一项所述的抗GCC抗体或其抗原结合片段或者本公开中任一项所述的嵌合抗原受体在免疫组化检测或制备检测试剂盒中的用途。
本公开的再一方面涉及本公开中任一项所述的抗GCC抗体或其抗原结合片段、本公开中任一项所述的嵌合抗原受体或者本公开中任一项所述的细胞在制备治疗或预防GCC相关疾病或病症的药物中的用途；
在一些具体实施方式中，所述GCC相关疾病或病症为肿瘤；
在一些具体实施方式中，所述GCC相关疾病或病症为消化道肿瘤；
在一些具体实施方式中，所述GCC相关疾病或病症为选自胃肠道癌、结直肠癌、胃癌、食道癌、食管胃结合部癌、小肠癌、胰腺癌和肝癌中的一种或多种。
根据本公开中任一项所述的抗GCC抗体或其抗原结合片段、本公开中任一项所述的嵌合抗原受体或者本公开中任一项所述的细胞，其用于治疗或预防GCC相关疾病或病症；
在一些具体实施方式中，所述GCC相关疾病或病症为肿瘤；
在一些具体实施方式中，所述GCC相关疾病或病症为消化道肿瘤；
在一些具体实施方式中，所述GCC相关疾病或病症为选自胃肠道癌、结直肠癌、胃癌、食道癌、食管胃结合部癌、小肠癌、胰腺癌和肝癌中的一种或多种。
本公开的再一方面涉及一种治疗或预防GCC相关疾病或病症的方法，包括给予有需求的受试者以有效量的本公开中任一项所述的抗GCC抗体或其抗原结合片段、本公开中任一项所述的嵌合抗原受体或者本公开中任一项所述的细胞的步骤；
在一些具体实施方式中，所述GCC相关疾病或病症为肿瘤；
在一些具体实施方式中，所述GCC相关疾病或病症为消化道肿瘤；
在一些具体实施方式中，所述GCC相关疾病或病症为选自胃肠道癌、结直肠癌、胃癌、食道癌、食管胃结合部癌、小肠癌、胰腺癌和肝癌中的一种或多种。
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
如本文中所使用的，术语EC50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区，CDR)，其间散布有较保守的称为框架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。
术语“框架”或“FR”是指那些位于CDR两侧的可变区残基。FR残基是除了高变区残基或CDR残基之外的那些可变结构域残基。
术语“高变区”、“互补决定区”和“CDR”可互换使用。“CDR”是指免疫球蛋白(Ig或抗体)VHβ-折叠框架的非框架区内的三个高变区(H1、H2或H3)之一，或抗体VLβ-折叠框架的非框架区内的三个高变区(L1、L2或L3)之一。CDR和FR的位置可以使用本领域熟知的多种定义方法确定，例如，Kabat编号系统、AbM编号系统、IMGT编号系统、Chothia编号系统或Contact编号系统。Kabat编号系统由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat etal.,Sequences of Proteins of Immunological Interest,5th Ed.Public HealthService,National Institutes of  Health,Bethesda,Md.,1991)。AbM编号系统使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program forModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。IMGT编号系统基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置。(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。Contact编号系统基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-745)。在本文中，根据氨基酸序列和在轻链或重链中的位置来确定CDR。由于免疫球蛋白可变区结构域内CDR的“位置”在物种之间是保守的，通过使用已知的编号系统划分的结构特征与可变区结构域氨基酸序列比对，很容易鉴定CDR和框架区氨基酸残基。来自这些高变区或CDR中的每一者的氨基酸残基在下表1中举例说明。
表1.根据各种编号系统的示例性CDR
CDR的边界可能根据使用的编号方法而变化。因此，除非另有说明，否则术语给定抗体或其区域，例如可变区的“CDR”和“互补决定区”，以及抗体或其区域的单个CDR(例如，HCDR1、HCDR2)，应理解为涵盖如上文所述的任何已知方案所定义的互补决定区。在一些情况下，指定用于鉴定特定CDR的方案，例如由IMGT、Kabat、AbM、 Chothia或Contact方法定义的CDR。在其它情况下，给出CDR的特定氨基酸序列。应当注意，CDR区也可以通过各种编号系统的组合，例如Kabat和Chothia编号系统的组合、Kabat和AbM编号系统的组合或Kabat和IMGT编号系统的组合来确定。因此，例如“如特定VH或VL中阐述的CDR1”的术语包括如由上述示例性CDR编号系统定义的任何CDR1，但不限于此。一旦给出可变区(例如，VH或VL)的氨基酸序列，本领域技术人员将理解该区域内的CDR可以由不同的编号系统或它们的组合来定义。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517):495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522 525；Reichmann et al.,Nature,1988；332:323 329；Presta,Curr.Op.Struct.Biol.1992；2:593-596；和Clark,Immunol.Today 2000；21:397 402。在一些情况下，抗体的抗原结合片段是双抗体(Diabodies)，其中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.et al.,Proc.Natl.Acad.Sci.USA 1993；90:6444-6448和Poljak R.J.et al.,Structure 1994；2:1121-1123)。
如本文中所使用的，术语“单链抗体(single chain fragment variable，ScFv)”是指，包含通过连接体连接的抗体重链可变区(VH)和抗体轻链可变区(VL)的分子。其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird et al,Science 1988；242:423-426和Huston et al,Proc.Natl.Acad.Sci.USA 1988；85:5879-5883)。此类scFv分子可具有一般结构：NH2-VL-连接片段-VH-COOH或NH2-VH-连接片段-VL-COOH。合适的现有技术接头由重复的GGGGS(SEQ ID NO:69)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(SEQ ID NO:70)的连接子，但也可使用其变体(Holliger et al，Proc.Natl.Acad.Sci.USA 1993；90:6444-6448)。可用于本公开的其他接头由Alfthan et al，Protein Eng.1995；8:725-731，Choi et al，Eur.J.Immunol.2001；31:94-106，Hu et al，Cancer Res.1996；56:3055-3061，Kipriyanov et al，J.Mol.Biol.1999；293:41-56和Roovers et al,Cancer Immunology,Immunotherapy,2001,50(1):51-59.描述。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不 限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，GS细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原(例如，GCC蛋白)。可以使用本领域技术人员知悉的方法测定KD，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用。并且在本公开中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所用，术语“免疫组织化学”或“免疫组化”，是在组织水平下，进行的免疫细胞化学研究。“免疫组织化学染色”属于一种免疫染色，是在抗体上结合荧光或可呈色的化学物质，利用此经修饰后的抗体与生物组织中的抗原具有特异性结合的免疫学原理，选择性地识别组织切片或涂片细胞中的抗原，检测细胞或组织中是否有目标抗原的存在；此方式不仅可以用来检测抗原的表达量，也可以观察抗原表达的位置，并可提高病理诊断的灵敏度和特异度。免疫组织化学染色中，经人为制备修饰后的特异性抗体带有显色剂标记，如：酶、金属离子、同位素等；而待检测的细胞或组织的目标抗原，则可为任何能让抗体结合的物质，也就是具有抗原性的物质，包括蛋白质、多肽、核酸、多糖、病原体等。两者通过免疫学的抗原抗体反应和组织化学的呈色反应，再结合组织细胞形态学分析，对组织或细胞的抗原进行定位、定性及定量。
如本文中所用，“嵌合抗原受体”或“CAR”是指基因工程受体，其可用于将一种或多种抗原特异性移植到免疫细胞如T细胞上。一些CAR也被称为“人工T细胞受体”、“嵌合T细胞受体”或“嵌合免疫受体”。在一些实施方案中，CAR包含对一种或多种抗原(例如肿瘤抗原)具有特异性的细胞外抗原结合结构域、跨膜结构域和T细胞和/或其 它受体的细胞内信号传导结构域。“CAR-T细胞”是指表达CAR的T细胞。
术语“受试者”和“患者”在本文中可互换使用。如本文中所用，在某些实施方案中，受试者是哺乳动物，例如非灵长类动物或灵长类动物(例如人)。受试者可以是被诊断患有疾病或病症的哺乳动物，例如人。受试者可以是处于发展疾病或病症的风险中的哺乳动物，例如人。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
有益效果
本公开取得了如下技术效果中的一项或者几项：
(1)本公开的抗GCC抗体或其抗原结合片段具有良好的特异性。
(2)本公开的抗GCC抗体或其抗原结合片段具有较高的亲和力。
(3)本公开的抗GCC抗体或其抗原结合片段在免疫组化分析中具有较高的灵敏度，在肿瘤检测及制备检测试剂盒中具有良好的应用前景。
(4)本公开的抗GCC抗体或其抗原结合片段、本公开的嵌合抗原受体或者本公开的嵌合抗原受体T细胞具有良好的治疗或预防肿瘤的潜力。
附图说明
图1A至图1B显示通过酶联免疫吸附实验检测抗GCC抗体22G5-Rabbit IgG与靶蛋白人GCC-his蛋白(图1A)及鼠GCC-his蛋白(图1B)的结合曲线。PF0098-Rabbit IgG为对照，22G5-Rabbit IgG和GCC靶蛋白的结合呈现浓度依赖性，其EC50和PF0098-Rabbit IgG抗体相近。
图2A至图2C显示通过流式细胞术检测抗GCC抗体22G5-Rabbit IgG与靶细胞的结合曲线。PF0098-Rabbit IgG为对照。22G5-Rabbit IgG和靶细胞293T-human GCC.Luc(图2A)及293T-mouse GCC.Luc(图2B)的结合呈现浓度依赖性，且对293T细胞(图2C)没有非特异性结合，特异性优于PF0098-Rabbit IgG。因此，22G5-Rabbit IgG是一种新型的、特异性高的抗GCC抗体。
图3显示使用不同抗GCC抗体在相同浓度下(0.5μg/mL)对人肠癌细胞SW948皮下瘤、人肠癌细胞LS1034皮下瘤、小鼠小肠及小鼠脾脏的组织切片进行免疫组织化学检测的实验结果。同型对照抗体ab172730在四种组织切片上均无染色。PF0098-Rabbit IgG抗体在SW948皮下瘤、LS1034皮下瘤切片中有较强的GCC特异性染色，且在小鼠脾脏中产生了非特异性染色。22G5-Rabbit IgG抗体在SW948皮下瘤、LS1034皮下瘤及小鼠小肠切片中均有较强的GCC特异性染色，其细胞膜染色强于PF0098-Rabbit IgG抗体，在四种组织中均无非特异性染色。抗GCC抗体ab122404在SW948皮下瘤、LS1034皮下瘤中的染色集中在细胞质，未显示出GCC在细胞膜表面的特异性染色，且在小鼠小肠及小鼠脾脏中产生了非特异性染色。因此，在免疫组织化学染色的应用上，22G5-Rabbit IgG抗体具有优越的特异性和敏感性。
图4A至图4C显示构建的CAR-T细胞对过表达鼠GCC的靶细胞CT26-mouse GCC.Luc分别在效靶比为5:1(图4A)、2.5:1(图4B)及1.25:1(图4C)时的细胞杀伤结果。PF0098 CAR-T作为阳性对照，所有CAR-T均能有效杀伤鼠GCC阳性的靶细胞。
图5A至图5C显示构建的CAR-T细胞对天然表达人GCC的靶细胞LS1034.Luc分别在效靶比为5:1(图5A)、2.5:1(图5B)及1.25:1(图5C)时的细胞杀伤结果。PF0098 CAR-T为对照，识别小鼠靶点的14C2 CAR-T不能有效杀伤靶细胞，其余CAR-T细胞对人GCC阳性的靶细胞具有较强的细胞毒性。这表明，兔源scFv也适用于CAR的抗原结合域，并使得CAR-T发挥高效识别和杀伤的功能。
图6A至图6C显示构建的CAR-T细胞对无GCC表达的293T.Luc细胞分别在效靶比为5:1(图6A)、2.5:1(图6B)及1.25:1(图6C)时的细胞毒性。所有CAR-T 均不能杀伤293T.Luc，表明所构建的CAR-T细胞只能特异性的识别GCC阳性的细胞。
图7A至7D显示构建的CAR-T细胞分别与靶细胞CT26-mouse GCC.Luc(图7A)、LS1034.Luc(图7B)、及293T.Luc(图7C)共孵育24小时后的细胞因子分泌水平。与靶细胞CT26-mouse GCC.Luc或LS1034.Luc孵育后，CAR-T细胞分泌的IFNγ有所上调。图7D表示UnT及各组CAR-T细胞在没有和靶细胞共培养的条件下，所分泌的本底IFNγ水平。
本公开涉及的部分序列如下。
SEQ ID NO:1 14C2重链可变区CDR1
SEQ ID NO:2 21E1重链可变区CDR1
SEQ ID NO:3 22G5重链可变区CDR1
SEQ ID NO:4 25E2重链可变区CDR1
SEQ ID NO:5 48D5重链可变区CDR1
SEQ ID NO:6 14C2重链可变区CDR2
SEQ ID NO:7 21E1重链可变区CDR2
SEQ ID NO:8 22G5重链可变区CDR2
SEQ ID NO:9 25E2重链可变区CDR2
SEQ ID NO:10 48D5重链可变区CDR2
SEQ ID NO:11 14C2重链可变区CDR3
SEQ ID NO:12 21E1重链可变区CDR3
SEQ ID NO:13 22G5重链可变区CDR3
SEQ ID NO:14 25E2重链可变区CDR3
SEQ ID NO:15 48D5重链可变区CDR3
SEQ ID NO:16 14C2轻链可变区CDR1
SEQ ID NO:17 21E1轻链可变区CDR1
SEQ ID NO:18 22G5轻链可变区CDR1
SEQ ID NO:19 25E2轻链可变区CDR1
SEQ ID NO:20 48D5轻链可变区CDR1
SEQ ID NO:21 14C2轻链可变区CDR2
SEQ ID NO:22 21E1轻链可变区CDR2
SEQ ID NO:23 22G5轻链可变区CDR2
SEQ ID NO:24 25E2轻链可变区CDR2
SEQ ID NO:25 48D5轻链可变区CDR2
SEQ ID NO:26 14C2轻链可变区CDR3
SEQ ID NO:27 21E1轻链可变区CDR3
SEQ ID NO:28 22G5轻链可变区CDR3
SEQ ID NO:29 25E2轻链可变区CDR3
SEQ ID NO:30 48D5轻链可变区CDR3
SEQ ID NO:31 14C2重链可变区
SEQ ID NO:32 21E1重链可变区
SEQ ID NO:33 22G5重链可变区
SEQ ID NO:34 25E2重链可变区
SEQ ID NO:35 48D5重链可变区
SEQ ID NO:36PF0098重链可变区
SEQ ID NO:37 14C2轻链可变区
SEQ ID NO:38 21E1轻链可变区
SEQ ID NO:39 22G5轻链可变区
SEQ ID NO:40 25E2轻链可变区
SEQ ID NO:41 48D5轻链可变区
SEQ ID NO:42PF0098轻链可变区
SEQ ID NO:43 14C2单链抗体
SEQ ID NO:44 21E1单链抗体

SEQ ID NO:45 22G5单链抗体
SEQ ID NO:46 25E2单链抗体
SEQ ID NO:47 48D5单链抗体
SEQ ID NO:48PF0098单链抗体
SEQ ID NO:49 14C2 CAR

SEQ ID NO:50 21E1 CAR
SEQ ID NO:51 22G5 CAR
SEQ ID NO:52 25E2 CAR

SEQ ID NO:53 48D5 CAR
SEQ ID NO:54 PF0098 CAR
SEQ ID NO:55 Rabbit IgG CH1-CH2-CH3

SEQ ID NO:56 Rabbit IgG CL
SEQ ID NO:57 22G5 VH-Rabbit IgG CH1-CH2-CH3
SEQ ID NO:58 22G5 VL-Rabbit IgG CL
SEQ ID NO:59 PF0098 VH-Rabbit IgG CH1-CH2-CH3

SEQ ID NO:60 PF0098 VL-Rabbit IgG CL
SEQ ID NO:61 CD8α信号肽
SEQ ID NO:62 CD8α铰链区
SEQ ID NO:63 CD8α跨膜结构域
SEQ ID NO:64 CD137共刺激信号结构域
SEQ ID NO:65 CD3ζ细胞内信号结构域
SEQ ID NO:66 Kozak序列
SEQ ID NO:67 连接子1
SEQ ID NO:68连接子2
SEQ ID NO:69连接子重复单元
SEQ ID NO:70连接子
SEQ ID NO:71 14C2 CAR-无信号肽
SEQ ID NO:72 21E1 CAR-无信号肽
SEQ ID NO:73 22G5 CAR-无信号肽
SEQ ID NO:74 25E2 CAR-无信号肽
SEQ ID NO:75 48D5 CAR-无信号肽
具体实施方式
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：抗GCC兔单克隆抗体的筛选
通过金斯瑞生物科技有限公司的服务订单(Genscript，Cat.NO.C5825HF280)，使用GCC蛋白免疫新西兰兔子，获得高亲和力结合GCC的抗体。具体筛选方法及步骤如下所述。
1.靶细胞构建
使用如下方法构建Dubca-human GCC.Luc和Dubca-mouse GCC.Luc过表达细胞系。合成人GCC编码序列(NM_004963.3)和鼠GCC编码序列(NM_001127318.1)并分别插入慢病毒表达载体pLVX-Luc-puro(Clontech,Cat.No.632164)中，随后同辅助包装质粒psPAX2、pMD.2G一起转染Lenti-X 293T宿主细胞，通过浓缩纯化获得慢病毒。将获得的两种慢病毒分别转导Dubca细胞系(CRL2276TM)，经过3轮嘌呤霉素抗性的筛选最终获得了Dubca-human GCC.Luc和Dubca-mouse GCC.Luc。
通过使用5F9-hIgGFc(Genscript，Cat.NO.C0224DK260-1)抗体与Dubca-human GCC.Luc细胞孵育检测人GCC靶点的表达量。取Dubca-human GCC.Luc和Dubca细胞各2.0×105与1：100稀释的5F9-hIgGFc抗体4℃条件下孵育0.5小时；使用DPBS洗涤3次，随后使用1：100配制的荧光二抗(PE anti-human IgG Fc Antibody,Biolegend,Cat.No.409304)重悬细胞，4℃避光孵育0.5小时；使用DPBS洗涤3次后流式细胞仪上样读取。通过使用表达纯化的PF0098-Rabbit IgG抗体与Dubca-mouse GCC.Luc细胞孵育检测鼠GCC靶点的表达量，检测方法同上。PF0098是在专利申请WO2019224716A2中披露的一种抗GCC抗体(VH如SEQ ID NO:36所示，VL如SEQ ID NO:42所示)，它能够同时识别人GCC，猴子GCC以及鼠GCC，并同时可进行免疫组织化学检测。实施例后续也将引用此抗体作为标准对照。
使用上述方法在CT26细胞系(ATCC,CRL-2638TM)和HEK293T细胞系 (Clontech,Cat.No.632180)过表达鼠GCC(NM_001127318.1)或人GCC(NM_004963.3)，构建得到了CT26-mouse GCC.Luc、293T-mouse GCC.Luc及293T-human GCC.Luc细胞。HEK293T细胞系(Clontech,Cat.No.632180)以及天然高表达人GCC的结肠癌细胞系LS1034(ATCC,CRL-2158TM)也被构建表达萤火虫荧光素酶，即LS1034.Luc细胞，并用于后续的体外筛选。
2.动物免疫和滴度检测
通过金斯瑞生物科技有限公司的MonoRabTM平台服务订单生产抗GCC兔单克隆抗体。将His标签标记的鼠GUCY2C/Guanylyl cyclase C蛋白(ACRO GUC-M52H3，简称鼠GCC-his蛋白)，经过ImmunoPlusTM技术修饰后，利用传统免疫方式免疫两只新西兰白兔，免疫间隔两周一次，为了增加免疫效果，在第三轮使用了293T-mouse GCC细胞免疫。四轮免疫结束后第7天，采血并针对两种靶蛋白鼠GCC-his和His标签标记的人GUCY2C/Guanylyl cyclase C蛋白(ACRO GUC-H52H5，简称人GCC-his蛋白)进行滴度检测。
3.阳性克隆筛选
免疫结束后，采集兔子30mL新鲜血用于分离PBMC，通过B细胞克隆的挑选，将抗原阳性的B细胞铺于50块96孔细胞板培养。使用间接ELISA检测细胞上清与鼠GCC-his和人GCC-his靶蛋白的结合特性，选择具有特异性交叉结合的克隆上清对Dubca-mouse GCC.Luc和Dubca细胞进行FACS结合的验证，阳性克隆被选择扩大到24孔板中继续培养。扩大培养后的上清使用如上方式再次进行ELISA和FACS的检测确认。FACS检测方式如下：消化并收集靶细胞，分装入96孔尖底板中，每孔2×105个细胞，400g离心3分钟后，弃去上清，加入100μL B细胞克隆上清液4℃共孵育1小时，使用4℃预冷的DPBS buffer洗涤3次，加入1：100稀释的Alexa488山羊抗兔IgG(H+L)(Invitrogen，Cat.NO.A11034)在4℃孵育30分钟，再次使用4℃预冷的DPBS洗涤3次。将上述孵育后的细胞在Attune NxT流式细胞仪(Thermofisher)上读取。结果展示在表2中，克隆14C2只与靶细胞Dubca-mouse GCC.Luc结合，其余4个克隆可与Dubca-mouse GCC.Luc和Dubca-human GCC.Luc两种靶细胞特异性结合。因此，所筛选到4个抗体克隆与小鼠和人GCC蛋白有种属交叉性。
表2.B细胞克隆上清与靶细胞结合的结果

通过MonoRabTM平台分离阳性B细胞克隆。将检测确认后选定的阳性B细胞克隆使用试剂(Ambion,Cat.No.:15596-026)标准技术流程提取RNA，根据PrimeScriptTM 1st Strand cDNA Synthesis Kit(Takara,Cat.No.:6110A)指导手册进行cDNA反转录，扩增出抗体的轻链和重链片段，二代测序法得到抗体的轻重链序列。表3和表4分别展示根据Kabat分区规则划分的抗体重链可变区(VH)和轻链可变区(VL)序列，以及相应的互补决定区(CDR)序列。抗体重链可变区(VH)和轻链可变区(VL)之间通过连接子连接，连接子可以由重复的GGGGS(SEQ ID NO:69)氨基酸序列或其变体组成(例如SEQ ID NOs:67-68)。
表3.抗体重链可变区及CDR氨基酸序列
表4.抗体轻链可变区及CDR氨基酸序列

实施例2：候选抗体的制备、纯化和鉴定
兔IgG分子根据轻链的不同划分为IgG-k和IgG-λ，一般情况下由新西兰白兔获得的抗体大部分为IgG-k型，只有<1％为是IgG-λ型。天然的兔IgG-k分子量约150KD，包含两条相同的轻链和两条相同的重链。轻链由N端可变结构域VL和恒定结构域CL(例如SEQ ID NO:56)组成，重链由N端可变结构域VH和三个恒定结构域(CH1，CH2和CH3，例如SEQ ID NO:55)组成，其中VL以及VH包含三个互补决定区CDR1、CDR2和CDR3。轻链与重链，以及两条重链之间以二硫键形式连接。
将上述筛选后的重链可变区和轻链可变区序列分别与兔IgG(IgG1k)融合，例如22G5单链抗体序列或PF0098单链抗体序列，经融合后分别获得22G5 VH-Rabbit IgG CH1-CH2-CH3(SEQ ID NO:57)、22G5 VL-Rabbit IgG CL(SEQ ID NO:58)或PF0098VH-Rabbit IgG CH1-CH2-CH3(SEQ ID NO:59)及PF0098 VL-Rabbit IgG CL(SEQ ID NO:60)。将融合序列的编码序列插入到pcDNA3.4载体中以构建表达质粒，随后在HD CHO-S细胞培养物中表达22G5-Rabbit IgG抗体和PF0098-Rabbit IgG抗体，并进一步进行抗体纯化。获得的抗体使用Nano Drop A280nm测定浓度，并采用SDS-PAGE评估纯度。表5中展示22G5-Rabbit IgG和PF0098-Rabbit IgG抗体的纯化信息。这表明，22G5抗体能高效地重组表达。
表5.抗体纯化信息
利用酶联免疫吸附实验检测纯化抗体和靶蛋白的结合能力。分别将两种靶蛋白即人GCC-his和鼠GCC-his使用包被液稀释，以1μg/mL浓度，每孔体系100μL包被于96孔Costar酶标板，置4℃过夜。第二天，经过0.05％ PBST洗液洗涤2次后使用5％MPBS室温封闭2小时；封闭结束，经洗涤后加入梯度稀释的抗体溶液室温孵育1.5小时；孵育结束洗液洗涤3次后加入1：10000稀释的酶标二抗Mouse Anti-Rabbit IgG Antibody(M205)[HRP],mAb(Genscript,Cat#A01827)，室温结合45分钟。洗液洗涤4次，加入TMB显色10分钟，随后使用1M HCl终止显色反应，并使用酶标仪在OD450nm处读数分析。结合曲线见图1A和图1B，EC50见表6。结果显示，22G5-Rabbit IgG和 PF0098-Rabbit IgG两种抗体均具有较强的结合GCC蛋白的能力。
表6.抗体与靶蛋白结合EC50
利用流式细胞术检测纯化抗体与靶细胞的结合能力。利用293T-human GCC.Luc和293T-mouse GCC.Luc细胞作为靶细胞，293T细胞作为阴性对照。将细胞以0.2M每孔的密度铺于96孔板中，与连续稀释后的抗体在4℃下孵育60分钟后DPBS洗涤三次，弃上清。使用1：100稀释的Alexa488山羊抗兔IgG(H+L)(Invitrogen，Cat.NO.A11034)重悬细胞沉淀，在4℃避光孵育30分钟。DPBS再次洗涤三次后流式细胞仪上样读取。如图2A至图2C所示，22G5-Rabbit IgG和PF0098-Rabbit IgG抗体与靶细胞的结合具有较强的浓度依赖，EC50见表7。除此之外，22G5-Rabbit IgG与293T细胞没有结合，但PF0098-Rabbit IgG抗体在高浓度下和293T细胞出现较强的非特异结合。综上，22G5-Rabbit IgG具有较高的抗GCC抗原的特异性。
表7.抗体与靶细胞结合EC50
实施例3：候选抗体的免疫组织化学检测
将用于检测的石蜡组织切片进行脱蜡处理，然后依次进行100％乙醇、95％乙醇、80％乙醇、70％乙醇及纯水浸泡处理。接着使用改进型柠檬酸钠抗原修复液(BEYOTIME#P0083)对切片进行微波加热抗原修复。待修复体系冷却后，将切片用PBS进行清洗，然后依次使用10％过氧化氢(国药集团化学试剂有限公司#10011218)及5％山羊血清(BEYOTIME#C0265)进行封闭处理。将抗GCC抗体用抗体稀释液(北京中杉金桥生物技术有限公司#ZLI-9030)稀释到工作浓度0.5μg/mL，甩去切片上的山羊血清，将抗体滴加到切片组织上，4℃孵育过夜。抗体孵育完成后用PBS清洗切片三次，将即用型快捷免疫组化检测试剂盒(福州迈新生物技术开发有限公司#KIT-5005)的羊抗兔抗体滴加到切片组织上，室温避光孵育15分钟。抗体孵育完成后用PBS清洗切片三次，之后使用DAB显色液(VECTOR LABORATORIES#sk-4105)进行显色。显色完成后使用苏木素染液(珠海贝索生物#BA4097)对切片进行染色。然后依次进行70％乙醇、80％乙醇、95％乙醇、100％乙醇及二甲苯浸泡处理，处理完 成后在切片组织上滴加中性树脂(国药集团化学试剂有限公司#10004160)并进行封片。待切片干燥后镜下观察并拍照。
实验结果见图3，同型对照抗体ab172730(Abcam，Recombinant Rabbit IgG,monoclonal[EPR25A]-Isotype Control)在四种组织切片上均无染色。PF0098-Rabbit IgG抗体在SW948皮下瘤、LS1034皮下瘤切片中有较强的GCC特异性染色，且在小鼠脾脏中产生了非特异性染色。在相同实验条件下并且抗体浓度均为0.5μg/mL，22G5-Rabbit IgG抗体对表达GCC的SW948皮下瘤、LS1034皮下瘤及小鼠小肠组织切片，均具有较强的特异性染色，其细胞膜染色强于高于PF0098-Rabbit IgG抗体，而且对不表达GCC的小鼠脾脏组织切片，未见非特异染色。此外，抗GCC抗体ab122404(Abcam)在SW948皮下瘤、LS1034皮下瘤中的染色集中在细胞质，未显示出细胞膜表面的特异性染色，且在小鼠小肠及小鼠脾脏中产生了非特异性染色。因此，22G5-Rabbit IgG抗体对GCC阳性细胞膜的染色强度优于对照抗体，特异性也优于对照抗体。22G5-Rabbit IgG抗体是一种新型的抗GCC抗体，适用于人类及小鼠组织切片的免疫组织化学检测。
实施例4：CAR-T细胞的构建
利用基因合成技术合成编码嵌合抗原受体(CAR)的骨架结构序列，即从N端到C端的编码结构分别为：CD8α铰链区(SEQ ID NO:62)、CD8α跨膜结构域(SEQ ID NO:63)、CD137共刺激信号结构域(SEQ ID NO:64)和CD3ζ细胞内信号结构域(SEQ ID NO:65)的核苷酸分子并克隆到慢病毒载体(pLSINK-BBzBB)中，由此产生的骨架载体被命名为“pLSINK-CE11A16”。载体中的多克隆位点(MCS)允许将包含Kozak序列(SEQ ID NO:66)的核酸序列可操作地连接到CAR骨架序列的CD8α信号肽(SEQ ID NO：61)并插入到CAR骨架载体上游。通过本领域已知的分子克隆技术，将合成的编码CD8α信号肽和抗GCC单链抗体(例如22G5 scFv,SEQ ID NO:45)的核酸序列通过EcoRI(5′-GAATTC-3′)和SpeI(5′-ACTAGT-3′)限制性位点克隆到慢病毒载体pLSINK-CE11A16中。所构建的嵌合抗原受体(CAR)分别为14C2 CAR、21E1 CAR、22G5 CAR、25E2 CAR、48D5 CAR及PF0098 CAR(例如SEQ ID NOs:49-53及SEQ ID NO:54)。
将含有pMDLg.pRRE(Addgene#12251)、pRSV-REV(Addgene#12253)和pMD2.G(Addgene#12259)的慢病毒辅助包装质粒混合物与表达CAR构建体的载体 以预先优化的比例与聚醚酰亚胺(PEI)混合，然后在25℃下孵育5分钟。然后将混合物加入HEK293细胞中转染。之后，将细胞在37℃下并含有5％ CO2的细胞培养箱中孵育过夜。收集上清，在4℃，3000g条件下离心15分钟，上清液中以4:1加入提前配制的20％ PEG 8000并小心上下颠倒混匀，4℃过夜沉淀病毒。随后4℃，3000g条件下离心30分钟，小心地弃去上清液，使用预冷的DPBS小心重悬病毒沉淀，储存在-80℃备用。通过SUP-T1(人类T淋巴母细胞)细胞系转导的滴定法测定病毒滴度。
使用Miltenyi Pan T细胞分离试剂盒(Cat#130-096-535)按照如下所述制造商的方案，从商业化的PBMC中纯化人T细胞。首先确定细胞数，并将细胞悬浮液以300g离心10分钟。然后完全弃去上清液，将细胞沉淀以每107个总细胞重悬于40μLMACS缓冲液(DPBS添加8μM EDTA以及0.5％ FBS)中。每107个总细胞加入10μL Pan T Cell Biotin-Antibody Cocktail，充分混匀后放入冰箱(2～8℃)孵育5分钟左右。然后每107个细胞加入30μL MACS缓冲液。每107个细胞加入20μL Pan T Cell MicroBead Cocktail。将细胞悬浮液混合物充分混合并在冰箱(2～8℃)中再孵育10分钟。磁分离至少需要500μL体系。对于磁分离，将LS柱置于合适的MACS分离器的磁场中。该柱通过用3mL缓冲液冲洗进行预处理。然后将细胞悬浮液上样到柱子上，收集含有未标记细胞的流出液，流出液代表富集的T细胞组分。通过用3mL缓冲液清洗柱子并收集通过的未标记细胞来收集剩余的T细胞。将两次收集的T细胞组分相结合，离心并重悬于含有300IU/mL IL-2的TexMACS GMP Medium&1L(Miltenyi,Cat#170-076-309)中。
随后，将制备的T细胞使用human T Cell TransActTM(Miltenyi#130-111-160)激活48小时，其中添加anti-CD3/CD28 MACSiBead珠与T细胞的比例为1:2。
将慢病毒以一定感染复数转导激活的T细胞，72小时后补加含有300IU/mL IL-2的R10培养基，并将转导的细胞转移到新细胞培养板中，在37℃具有5％ CO2的条件下进行细胞扩增。
使用直标抗体通过流式细胞术评估CAR表达水平。转导后第6天，每组收集5×105个T细胞，使用预冷的DPBS buffer配制抗体，其中PF0098 CAR-T和未转导CAR的T细胞(UnT)与1：100稀释的Alexa488山羊抗鼠IgG(H+L)(Invitrogen，Cat.NO.A11001)在4℃孵育30分钟。其余CAR-T和未转导的UnT与1：100稀释的Alexa488山羊抗兔IgG(H+L)(Invitrogen，Cat.NO.A11034)在4℃孵育30分钟，使用4℃预冷的DPBS洗涤3次。将上述孵育后的细胞在Attune NxT流式细胞 仪(Thermofisher)上读取。结果展示在表8中，CAR表达范围为43.94％至75.10％，PF0098 CAR-T为对照，UnT代表未转导CAR的T细胞。
表8.转导T细胞后CAR的表达水平
实施例5：CAR-T细胞的功能评估
将实施例4中制备的CAR-T细胞进行细胞毒性测定。上述CAR-T细胞与实施例1中制备的LS1034.Luc、CT26-mouse GCC.Luc和293T.Luc细胞分别以效应细胞与靶细胞比(E/T)为5：1、2.5：1及1.25：1共孵育24小时。其中，PF0098 CAR-T为阳性对照。为了测定CAR-T细胞对靶细胞的细胞毒性，One-glo发光荧光素酶测定试剂(Promega#E6120)被添加到共培养的细胞中以检测孔中剩余的荧光素酶活性值。剩余的荧光素酶活性与孔中剩余靶细胞的数量直接相关。细胞毒性由下式计算：
细胞毒性％＝100％×(1-(RLUsample-RLUmin)/(RLUunT-RLUmin))。
RLUsample是指在抗GCC CAR转导的CAR-T细胞孔中测量的荧光素酶活性值。RLUmin是指在细胞毒性测定开始时以终浓度为1％加入Triton X-100的孔中测定的荧光素酶活性值，RLUunT是指在未转导CAR的T细胞孔中测定的荧光素酶活性值。
如图4A至图4C所示，所有CAR-T细胞对CT26-mouse GCC.Luc细胞表现出较强的细胞毒性。如图5A至图5C所示，由于14C2不结合人GCC蛋白，所以14C2 CAR-T细胞对天然表达人GCC的LS1034.Luc细胞无杀伤作用。25E2 CAR-T细胞只在5：1的高E/T比情况下对靶细胞展现出细胞毒性。其余3种CAR-T对靶细胞LS1034.Luc细胞均能够保持较强的细胞毒性。如图6A至图6C所示，这些CAR-T对293T.Luc在较高E/T比下依然没有细胞毒性，证明这5种CAR-T细胞只识别GCC阳性的靶细胞。
除了对靶细胞的细胞毒性外，对GCC CAR-T细胞(实施例4制备)与靶细胞(实施例1中制备的LS1034.Luc、CT26-mouse GCC.Luc和293T.Luc细胞)共孵育后产生IFN-γ的能力也进行了分析。将GCC CAR-T细胞以2.5：1的E/T比与靶细胞混合(2000 个靶细胞/孔)，在384孔板中共孵育24小时。通过HTRF试剂盒(Cisbio，Cat#62HIFNGPEG)测量CAR-T与靶细胞共孵育后的培养上清液以及CAR-T细胞自身上清液中产生的IFN-γ浓度。在测定前，HTRF试剂需要室温稳定30分钟。将共培养上清液转移到384孔测定板(Greiner Bio-One，#784075)16μL/孔，然后加入根据试剂盒操作手册制备的4μL/孔预混合HTRF试剂。将板用封口膜密封并在室温下孵育过夜。在Tecan Spark 10M上读取HTRF值。IFN-γ浓度通过参考试剂盒提供的标准曲线获得的信号来计算。
如图7A至图7D所示，检测到5种CAR-T分泌的IFNγ的浓度范围为1488.3pg/mL-2692.5pg/mL(与CT26-mGCC.Luc靶细胞共孵育)和1173.1pg/mL-1939.0pg/mL(与LS1034.Luc靶细胞共孵育，除14C2 CAR-T外)，相对于UnT对照(检测值为600.4pg/mL和910.8pg/mL)有所上调。
尽管本公开的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本公开的保护范围之内。本公开的全部范围由所附权利要求及其任何等同物给出。

Claims (28)

  1. 抗GCC抗体或其抗原结合片段，包含重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1至HCDR3，所述轻链可变区包含轻链互补决定区LCDR1至LCDR3，其中：
    (1)所述重链可变区包含如SEQ ID NO:31所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:37所示的氨基酸序列中的LCDR1、LCDR2和LCDR3；
    (2)所述重链可变区包含如SEQ ID NO:32所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:38所示的氨基酸序列中的LCDR1、LCDR2和LCDR3；
    (3)所述重链可变区包含如SEQ ID NO:33所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:39所示的氨基酸序列中的LCDR1、LCDR2和LCDR3；
    (4)所述重链可变区包含如SEQ ID NO:34所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:40所示的氨基酸序列中的LCDR1、LCDR2和LCDR3；或者
    (5)所述重链可变区包含如SEQ ID NO:35所示的氨基酸序列中的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含如SEQ ID NO:41所示的氨基酸序列中的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的抗GCC抗体或其抗原结合片段，其中，
    所述重链可变区的HCDR1至HCDR3以及所述轻链可变区的LCDR1至LCDR3按照Kabat编号系统、AbM编号系统、IMGT编号系统、Chothia编号系统或contact编号系统确定。
  3. 抗GCC抗体或其抗原结合片段，包含重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1至HCDR3，所述轻链可变区包含轻链互补决定区LCDR1至LCDR3，其中：
    (1)HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:6所示、HCDR3的氨基酸序列如SEQ ID NO:11所示、LCDR1的氨基酸序列如 SEQ ID NO:16所示、LCDR2的氨基酸序列如SEQ ID NO:21所示和LCDR3的氨基酸序列如SEQ ID NO:26所示；
    (2)HCDR1的氨基酸序列如SEQ ID NO:2所示、HCDR2的氨基酸序列如SEQ ID NO:7所示、HCDR3的氨基酸序列如SEQ ID NO:12所示、LCDR1的氨基酸序列如SEQ ID NO:17所示、LCDR2的氨基酸序列如SEQ ID NO:22所示和LCDR3的氨基酸序列如SEQ ID NO:27所示；
    (3)HCDR1的氨基酸序列如SEQ ID NO:3所示、HCDR2的氨基酸序列如SEQ ID NO:8所示、HCDR3的氨基酸序列如SEQ ID NO:13所示、LCDR1的氨基酸序列如SEQ ID NO:18所示、LCDR2的氨基酸序列如SEQ ID NO:23所示和LCDR3的氨基酸序列如SEQ ID NO:28所示；
    (4)HCDR1的氨基酸序列如SEQ ID NO:4所示、HCDR2的氨基酸序列如SEQ ID NO:9所示、HCDR3的氨基酸序列如SEQ ID NO:14所示、LCDR1的氨基酸序列如SEQ ID NO:19所示、LCDR2的氨基酸序列如SEQ ID NO:24所示和LCDR3的氨基酸序列如SEQ ID NO:29所示；或者
    (5)HCDR1的氨基酸序列如SEQ ID NO:5所示、HCDR2的氨基酸序列如SEQ ID NO:10所示、HCDR3的氨基酸序列如SEQ ID NO:15所示、LCDR1的氨基酸序列如SEQ ID NO:20所示、LCDR2的氨基酸序列如SEQ ID NO:25所示和LCDR3的氨基酸序列如SEQ ID NO:30所示。
  4. 根据权利要求1至3中任一权利要求所述的抗GCC抗体或其抗原结合片段，其中，
    (1)所述重链可变区包含与SEQ ID NO:31所示氨基酸序列具有至少80％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:37所示氨基酸序列具有至少80％同一性的氨基酸序列；
    (2)所述重链可变区包含与SEQ ID NO:32所示氨基酸序列具有至少80％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:38所示氨基酸序列具有至少80％同一性的氨基酸序列；
    (3)所述重链可变区包含与SEQ ID NO:33所示氨基酸序列具有至少80％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:39所示氨基酸序列具有至少80％同一性的氨基酸序列；
    (4)所述重链可变区包含与SEQ ID NO:34所示氨基酸序列具有至少80％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:40所示氨基酸序列具有至少80％同一性的氨基酸序列；或者
    (5)所述重链可变区包含与SEQ ID NO:35所示氨基酸序列具有至少80％同一性的氨基酸序列，并且所述轻链可变区包含与SEQ ID NO:41所示氨基酸序列具有至少80％同一性的氨基酸序列。
  5. 根据权利要求1至4中任一权利要求所述的抗GCC抗体或其抗原结合片段，其中，
    (1)所述重链可变区的氨基酸序列如SEQ ID NO:31所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:37所示；
    (2)所述重链可变区的氨基酸序列如SEQ ID NO:32所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:38所示；
    (3)所述重链可变区的氨基酸序列如SEQ ID NO:33所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:39所示；
    (4)所述重链可变区的氨基酸序列如SEQ ID NO:34所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:40所示；或者
    (5)所述重链可变区的氨基酸序列如SEQ ID NO:35所示，并且所述轻链可变区的氨基酸序列如SEQ ID NO:41所示。
  6. 根据权利要求1至5中任一权利要求所述的抗GCC抗体或其抗原结合片段，其中，所述抗GCC抗体包括非CDR区，且所述非CDR区来自人、小鼠或兔；
    优选地，所述非CDR区包括框架区、重链恒定区和/或轻链恒定区。
  7. 根据权利要求1至6中任一权利要求所述的抗GCC抗体或其抗原结合片段，其中，所述抗原结合片段是Fab、Fab'、F(ab')2、单链抗体或二硫键稳定的可变片段。
  8. 根据权利要求1至7中任一权利要求所述的抗GCC抗体或其抗原结合片段，其包含与SEQ ID NO:43至SEQ ID NO:47中的任一序列具有至少80％同一性的氨基酸序列；或者包含与SEQ ID NO:57和SEQ ID NO:58具有至少80％同一性的氨基酸 序列；
    优选地，所述抗GCC抗体或其抗原结合片段的氨基酸序列如SEQ ID NO:43至SEQ ID NO:47中的任一序列所示。
  9. 一种分离的核酸，其编码权利要求1至8中任一权利要求所述的抗GCC抗体或其抗原结合片段。
  10. 一种载体，其包含权利要求9所述的分离的核酸。
  11. 一种宿主细胞，其包含权利要求9所述的分离的核酸，或者包含权利要求10所述的载体。
  12. 一种嵌合抗原受体，其包含多肽，该多肽包含：
    (1)细胞外抗原结合结构域，该细胞外抗原结合结构域包含如权利要求1至8中任一权利要求所述的抗GCC抗体或抗原结合片段；
    (2)跨膜结构域；以及
    (3)细胞内信号传导结构域。
  13. 根据权利要求12所述的嵌合抗原受体，其中该抗GCC抗体或抗原结合片段是单链抗体。
  14. 根据权利要求12至13中任一权利要求所述的嵌合抗原受体，其进一步包含位于该多肽的N-末端的信号肽；
    优选地，所述信号肽来源于CD8α、CD28或CD4分子；
    优选地，所述信号肽包含如SEQ ID NO:61所示的氨基酸序列，或者与SEQ ID NO:61具有至少85％同一性的氨基酸序列。
  15. 根据权利要求12至14任一权利要求所述的嵌合抗原受体，其进一步包含位于所述细胞外抗原结合结构域的C-末端和所述跨膜结构域的N-末端之间的铰链结构域；
    优选地，所述铰链区来源于IgG4Fc CH2CH3、CD28或CD8α；
    优选地，所述铰链区包含如SEQ ID NO:62所示的氨基酸序列，或者与SEQ ID NO:62具有至少85％同一性的氨基酸序列。
  16. 根据权利要求12至15任一权利要求所述的嵌合抗原受体，其中所述跨膜结构域来源于CD8α、CD4、CD28、CD137、CD80、CD86、CD152和PD1分子跨膜区的一种或多种；
    优选地，所述跨膜结构域包含如SEQ ID NO:63所示的氨基酸序列，或者与SEQ ID NO:63具有至少85％同一性的氨基酸序列。
  17. 根据权利要求12至16任一权利要求所述的嵌合抗原受体，其中所述细胞内信号传导结构域包含共刺激信号传导结构域；
    优选地，所述共刺激信号传导结构域源自以下共刺激分子：CD27、CD28、CD137、OX40、CD30、CD40、CD3、HVEM、ICOS、Myd88、LFA-1、ICOS、CD2、CD7、LIGHT、NKG2C、B7-H3、CD83的配体以及它们的组合；
    优选地，所述共刺激信号传导结构域包含CD28的胞质域和/或CD137的胞质域；
    优选地，所述共刺激信号传导结构域包含如SEQ ID NO:64所示的氨基酸序列，或者与SEQ ID NO:64具有至少85％同一性的氨基酸序列。
  18. 根据权利要求12至17中任一权利要求所述的嵌合抗原受体，其中所述细胞内信号传导结构域包含初级细胞内信号传导结构域；
    优选地，所述初级细胞内信号传导结构域源自CD3ζ；
    优选地，所述初级细胞内信号传导结构域包含如SEQ ID NO:65所示的氨基酸序列，或者与SEQ ID NO:65具有至少85％同一性的氨基酸序列。
  19. 根据权利要求12至18中任一权利要求所述的嵌合抗原受体，其氨基酸序列如SEQ ID NO:49至SEQ ID NO:53或者SEQ ID NO:71至SEQ ID NO：75中任一序列所示。
  20. 一种分离的核酸，其编码权利要求12至19中任一权利要求所述的嵌合抗原受体。
  21. 一种载体，其包含权利要求20所述的分离的核酸。
  22. 一种细胞，其包含权利要求12至19中任一权利要求所述的嵌合抗原受体，包含权利要求20所述的分离的核酸，或者包含权利要求21所述的载体；
    优选地，所述细胞为免疫细胞。
  23. 根据权利要求22所述的细胞，其中，所述细胞选自：T细胞、NK细胞、外周血单核细胞、造血干细胞、多能干细胞、胚胎干细胞以及它们的组合。
  24. 一种药物组合物，其包含权利要求1至8中任一权利要求所述的抗GCC抗体或其抗原结合片段，或者包含权利要求12至19中任一权利要求所述的嵌合抗原受体，或者包含权利要求22或23所述的细胞；
    优选地，所述药物组合物还包含一种或多种药学上可接受的辅料。
  25. 权利要求1至8中任一权利要求所述的抗GCC抗体或其抗原结合片段或者权利要求12至19中任一权利要求所述的嵌合抗原受体在免疫组化检测或制备检测试剂盒中的用途。
  26. 权利要求1至8中任一权利要求所述的抗GCC抗体或其抗原结合片段、权利要求12至19中任一权利要求所述的嵌合抗原受体或者权利要求22或23所述的细胞在制备治疗或预防GCC相关疾病或病症的药物中的用途；
    优选地，所述GCC相关疾病或病症为肿瘤；
    优选地，所述GCC相关疾病或病症为消化道肿瘤；
    优选地，所述GCC相关疾病或病症为选自胃肠道癌、结直肠癌、胃癌、食道癌、食管胃结合部癌、小肠癌、胰腺癌和肝癌中的一种或多种。
  27. 根据权利要求1至8中任一权利要求所述的抗GCC抗体或其抗原结合片段、权利要求12至19中任一权利要求所述的嵌合抗原受体或者权利要求22或23所述的细胞，其用于治疗或预防GCC相关疾病或病症；
    优选地，所述GCC相关疾病或病症为肿瘤；
    优选地，所述GCC相关疾病或病症为消化道肿瘤；
    优选地，所述GCC相关疾病或病症为选自胃肠道癌、结直肠癌、胃癌、食道癌、食管胃结合部癌、小肠癌、胰腺癌和肝癌中的一种或多种。
  28. 一种治疗或预防GCC相关疾病或病症的方法，包括给予有需求的受试者以有效量的权利要求1至8中任一权利要求所述的抗GCC抗体或其抗原结合片段、权利要求12至19中任一权利要求所述的嵌合抗原受体或者权利要求22或23所述的细胞的步骤；
    优选地，所述GCC相关疾病或病症为肿瘤；
    优选地，所述GCC相关疾病或病症为消化道肿瘤；
    优选地，所述GCC相关疾病或病症为选自胃肠道癌、结直肠癌、胃癌、食道癌、食管胃结合部癌、小肠癌、胰腺癌和肝癌中的一种或多种。
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