Anti-GCC antibody, chimeric antigen receptor, and use

Abstract

The present disclosure relates to the field of biological medicines, and in particular to an anti-guanylyl cyclase C (GCC) antibody or an antigen binding fragment, a chimeric antigen receptor (CAR) containing the anti-GCC antibody or the antigen binding fragment, and a use. The disclosed anti-GCC antibody or antigen-binding fragment can be used for companion diagnostics of GCC-related diseases or conditions, e.g., immunohistochemical analysis. The disclosed GCC CAR construct-transduced immune cells can be used for cellular immunotherapy of GCC-related diseases or conditions.

Description

Anti-GCC antibodies, chimeric antigen receptors and their uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on and claims priority to PCT application number PCT / CN2023 / 120440, filed on September 21, 2023. The entire contents of the PCT application are hereby incorporated into this application as a whole. Technical Field

[0003] 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. Background Art

[0004] Guanylyl cyclase C (GUCY2C or GCC) is a membrane-bound guanylate cyclase and a single-pass transmembrane protein receptor consisting of an extracellular domain, a transmembrane domain, and an intracellular domain. This enzyme is the intestinal receptor for heat-stable bacterial enterotoxins, hence GUCY2C is also known as the heat-stable enterotoxin receptor. Guanylin and uroguanylin, as well as artificial GCC agonists, can bind to and activate GCC. GCC activation stimulates 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.

[0005] In normal human tissues, GCC is expressed at the apical end of intestinal epithelial cells. Loss of function caused by GCC mutations leads to decreased fluid and ion secretion, causing constipation; enhancement of function caused by GCC mutations leads to increased fluid and ion secretion, 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: inactivation of GCC-cGMP signaling due to 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.

[0006] Chimeric antigen receptor T (CAR-T) cell immunotherapy is a novel, antigen-targeted precision medicine 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 activating T cells and attacking tumor cells without relying on MHC co-stimulatory signals. CAR-T cell therapy has potential advantages over other T cell redirection therapy strategies. While other treatment strategies typically require long-term treatment until disease progression, CAR-T cells can expand in vivo and only require a single infusion to achieve long-term disease remission. CAR-T cell therapy is becoming a promising treatment option for cancer.

[0007] There is currently a need to develop new anti-GCC antibodies.

[0008] Summary of the Invention

[0009] After in-depth research and creative work, 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 high 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 chimeric antigen receptors (CARs) targeting GCC, but also can be used for immunohistochemical analysis. The following invention is thus provided:

[0010] One aspect of the present disclosure relates to an anti-GCC antibody or 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 complementarity determining regions HCDR1 to HCDR3, and the light chain variable region comprises light chain complementarity determining regions LCDR1 to LCDR3, wherein:

[0011] (1) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 37;

[0012] (2) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 32, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 38;

[0013] (3) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 33, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 39;

[0014] (4) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 34, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 40; or

[0015] (5) 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.

[0016] In some embodiments of the present disclosure, the anti-GCC antibody or antigen-binding fragment thereof, wherein,

[0017] 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.

[0018] In some embodiments of the present disclosure, the anti-GCC antibody or antigen-binding fragment thereof, wherein:

[0019] (1) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 16, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 26;

[0020] (2) 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, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 27;

[0021] (3) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 18, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 23, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 28;

[0022] (4) the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 9, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 14, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 19, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 24, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 29; or

[0023] (5) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 15, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 20, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 30.

[0024] In some embodiments of the present disclosure, the anti-GCC antibody or antigen-binding fragment thereof, wherein,

[0025] (1) 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, 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 of SEQ ID NO:37;

[0026] (2) 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, 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 of SEQ ID NO:38;

[0027] (3) 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, 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 of SEQ ID NO:39;

[0028] (4) 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, 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 of SEQ ID NO:40; or

[0029] (5) 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: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 of SEQ ID NO:41.

[0030] In some embodiments of the present disclosure, the anti-GCC antibody or antigen-binding fragment thereof, wherein,

[0031] (1) the 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;

[0032] (2) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38;

[0033] (3) the 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;

[0034] (4) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40; or

[0035] (5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 41.

[0036] In some embodiments of the present disclosure, the anti-GCC antibody or antigen-binding fragment thereof, wherein the anti-GCC antibody includes a non-CDR region, and the non-CDR region is derived 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.

[0037] In some embodiments of the present disclosure, the anti-GCC antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, single-chain antibody (scFv) or disulfide-stabilized variable fragment (dsFv).

[0038] In some embodiments of the present disclosure, the anti-GCC antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof is as shown in any one of SEQ ID NO: 43 to SEQ ID NO: 47.

[0039] 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.

[0040] Yet another aspect of the present disclosure relates to a vector comprising the isolated nucleic acid of the present disclosure.

[0041] 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.

[0042] Yet another aspect of the present disclosure relates to a chimeric antigen receptor (CAR), comprising a polypeptide comprising:

[0043] (1) an extracellular antigen-binding domain comprising an anti-GCC antibody or antigen-binding fragment as described in any one of the present disclosure;

[0044] (2) transmembrane domain; and

[0045] (3) Intracellular signal transduction domain.

[0046] In some embodiments of the present disclosure, the chimeric antigen receptor, wherein the anti-GCC antibody or antigen-binding fragment is a single-chain antibody (scFv).

[0047] In some embodiments of the present disclosure, the chimeric antigen receptor further comprises a signal peptide located at the N-terminus of the polypeptide;

[0048] In some embodiments, the signal peptide is derived from CD8α, CD28 or CD4 molecules;

[0049] In some embodiments, the signal peptide comprises the 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.

[0050] In some embodiments of the present disclosure, 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;

[0051] In some embodiments, the hinge region is derived from IgG4Fc CH2CH3, CD28 or CD8α;

[0052] In some embodiments, the hinge region comprises the amino acid sequence 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.

[0053] In some embodiments of the present disclosure, the chimeric antigen receptor, wherein the transmembrane domain is derived from one or more transmembrane regions of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1 molecules;

[0054] In some embodiments, 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.

[0055] In some embodiments of the present disclosure, the chimeric antigen receptor, wherein the intracellular signaling domain comprises a costimulatory signaling domain;

[0056] In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule comprising a ligand of CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof;

[0057] In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137;

[0058] In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of 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.

[0059] In some embodiments of the present disclosure, the chimeric antigen receptor, wherein the intracellular signaling domain comprises a primary intracellular signaling domain;

[0060] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ;

[0061] In some specific embodiments, the primary intracellular signaling domain comprises the amino acid sequence as set forth 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.

[0062] In some embodiments of the present disclosure, 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.

[0063] Yet another aspect of the present disclosure relates to an isolated nucleic acid encoding the chimeric antigen receptor of any one of the present disclosure.

[0064] Yet another aspect of the present disclosure relates to a vector comprising the isolated nucleic acid of the present disclosure.

[0065] Yet another aspect of the present disclosure relates to a cell comprising the chimeric antigen receptor according to any one of the present disclosure, the isolated nucleic acid according to the present disclosure, or the vector according to the present disclosure;

[0066] In some embodiments, the cell is an immune cell.

[0067] In some embodiments of the present disclosure, the cells are selected from the group consisting of: T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0068] Yet another aspect of the present disclosure relates to a pharmaceutical composition comprising the anti-GCC antibody or antigen-binding fragment thereof according to any one of the present disclosure, or the chimeric antigen receptor according to any one of the present disclosure, or the cell according to any one of the present disclosure;

[0069] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0070] 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.

[0071] Another aspect of the present disclosure relates to use of the anti-GCC antibody or antigen-binding fragment thereof, the chimeric antigen receptor, 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.

[0072] In some embodiments, the GCC-related disease or disorder is a tumor;

[0073] In some embodiments, the GCC-related disease or condition is a digestive tract tumor;

[0074] In some embodiments, the GCC-related disease or condition is one or more selected from gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, esophagogastric junction cancer, small intestine cancer, pancreatic cancer and liver cancer.

[0075] The anti-GCC antibody or antigen-binding fragment thereof according to any one of the present disclosure, the chimeric antigen receptor according to any one of the present disclosure, or the cell according to any one of the present disclosure, for use in treating or preventing a GCC-related disease or disorder;

[0076] In some embodiments, the GCC-related disease or disorder is a tumor;

[0077] In some embodiments, the GCC-related disease or condition is a digestive tract tumor;

[0078] In some embodiments, the GCC-related disease or condition is one or more selected from gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, esophagogastric junction cancer, small intestine cancer, pancreatic cancer and liver cancer.

[0079] Yet another aspect of the present disclosure relates to a method for treating or preventing a GCC-related disease or condition, 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;

[0080] In some embodiments, the GCC-related disease or disorder is a tumor;

[0081] In some embodiments, the GCC-related disease or condition is a digestive tract tumor;

[0082] In some embodiments, the GCC-related disease or condition is one or more selected from gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, esophagogastric junction cancer, small intestine cancer, pancreatic cancer and liver cancer.

[0083] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0084] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0085] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, 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 mediates the binding of the immunoglobulin 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 be further subdivided into regions of high variability (called complementarity determining regions, CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0086] The term "framework" or "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.

[0087] The terms "hypervariable region", "complementarity determining region" and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the VH β-sheet 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 β-sheet framework of an antibody. The positions of CDRs and FRs can be determined using a variety of definitions 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, for example, 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; "AbM™, 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 system (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 takes into account the location 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). Herein, CDRs are identified by amino acid sequence and position in the light or heavy chain.Since the "position" of CDRs within the variable region of an immunoglobulin is conserved between species, it is easy to identify CDR and framework amino acid residues by comparing the structural features divided using a known numbering system with the variable region domain amino acid sequence. The amino acid residues from each of these hypervariable regions or CDRs are exemplified in Table 1 below.

[0088] Table 1. Exemplary CDRs according to various numbering systems

[0089] The boundary of CDR may change according to the numbering method used.Therefore, unless otherwise indicated, otherwise term given antibody or its zone, such as " CDR " and " complementary determining region " of variable region, and single CDR (such as, HCDR1, HCDR2) of antibody or its zone, should be interpreted as containing the complementary determining region defined by any known scheme as described above.In some cases, be specified for identifying the scheme of specific CDR, such as by IMGT, Kabat, AbM, the CDR defined by Chothia or Contact method.In other cases, provide the specific amino acid sequence of CDR.It should be noted that CDR district also can be determined by the combination of such as the combination of Kabat and Chothia numbering system, the combination of Kabat and AbM numbering system or the combination of Kabat and IMGT numbering system.Therefore, such as the term of " CDR1 as set forth in specific VH or VL " includes any CDR1 as defined by above-mentioned exemplary CDR numbering system, but is not limited thereto.Once provide the amino acid sequence of variable region (such as, VH or VL), those skilled in the art will understand that the CDR in this zone can be defined by different numbering systems or their combination.

[0090] The term "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 of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0091] As used herein, 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 possible spontaneous natural mutations. 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, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology 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 using recombinant DNA technology (see, for example, US Patent 4,816,567).

[0092] As used herein, the term "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 can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the recipient antibody can also be replaced with amino acid residues of the corresponding non-human antibody, or with amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, 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; and Clark, Immunol. Today 2000; 21: 397 402. In some cases, the antigen-binding fragment of an antibody is a diabodies, in which V H and V L The domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on 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).

[0093] As used herein, the term "single chain antibody (ScFv)" refers to a single chain antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ) molecule. Where V L and V HThe domains are paired to form monovalent molecules via linkers that enable their production 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-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L -COOH. Suitable prior art linkers consist of repeated GGGGS (SEQ ID NO: 69) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 70) may be used, but variants thereof may also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448). Other linkers useful in the present disclosure are described by 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 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0094] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "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.

[0095] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. 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 virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (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. Additionally, a vector may contain an origin of replication.

[0096] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not 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.

[0097] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity of M or less (K D ) binds to the antigen.

[0098] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind with a dissociation equilibrium constant of less than about 10 -5 M, for example, less than about 10 -6M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binds to an antigen (e.g., GCC protein). K can be determined using methods known to those skilled in the art. D , for example, using the Fortebio molecular interaction instrument.

[0099] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. In addition, in the present disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0100] As used herein, the term "immunohistochemistry" or "immunohistochemistry" refers to immunocytochemical studies performed at the tissue level. "Immunohistochemical staining" is a type of immunostaining that involves conjugating fluorescent or colorable chemicals to antibodies. Using the immunological principle that these modified antibodies specifically bind to antigens in biological tissues, they selectively identify antigens in cells in tissue sections or smears, detecting the presence of target antigens in cells or tissues. This method can be used not only to detect the expression level of antigens, but also to observe the location of antigen expression, thereby improving the sensitivity and specificity of pathological diagnosis. In immunohistochemical staining, artificially prepared and modified specific antibodies are labeled with a colorimetric agent, such as an enzyme, metal ion, or isotope. The target antigen in the cells or tissues to be detected can be any substance that can bind to the antibody, that is, an antigenic substance, including proteins, peptides, nucleic acids, polysaccharides, and pathogens. Through the immunological antigen-antibody reaction and histochemical color reaction, combined with tissue and cell morphological analysis, the antigens in tissues or cells can be located, identified, and quantified.

[0101] As used herein, "chimeric antigen receptor" or "CAR" 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 referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, CAR comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain of T cells and / or other receptors that are specific for one or more antigens (e.g., tumor antigens). "CAR-T cells" refer to T cells that express CAR.

[0102] The terms "subject" and "patient" are used interchangeably herein. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., a human). A subject can be a mammal, such as a human, diagnosed with a disease or condition. A subject can be a mammal, such as a human, who is at risk of developing a disease or condition.

[0103] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, 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, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0104] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of 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 amount effective 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 sex, the mode of administration of the drug, and other treatments administered simultaneously, etc. Beneficial effects

[0105] The present disclosure achieves one or more of the following technical effects:

[0106] (1) The anti-GCC antibodies or antigen-binding fragments thereof disclosed herein have good specificity.

[0107] (2) The anti-GCC antibodies or antigen-binding fragments thereof disclosed herein have high affinity.

[0108] (3) The anti-GCC antibody or antigen-binding fragment thereof disclosed herein has high sensitivity in immunohistochemical analysis and has good application prospects in tumor detection and preparation of detection kits.

[0109] (4) The anti-GCC antibody or antigen-binding fragment thereof, the chimeric antigen receptor or chimeric antigen receptor T cell disclosed herein have good potential for treating or preventing tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figures 1A and 1B show the binding curves of the anti-GCC antibody 22G5-Rabbit IgG to the target proteins human GCC-his protein (Figure 1A) and mouse GCC-his protein (Figure 1B) detected by enzyme-linked immunosorbent assay. PF0098-Rabbit IgG was used as a control. The binding of 22G5-Rabbit IgG to the GCC target protein was concentration-dependent, with an EC of 50 Similar to PF0098-Rabbit IgG antibody.

[0111] Figures 2A to 2C show flow cytometry binding curves of the anti-GCC antibody 22G5-Rabbit IgG to target cells. PF0098-Rabbit IgG served as a control. 22G5-Rabbit IgG exhibited concentration-dependent binding to target cells, 293T-human GCC.Luc (Figure 2A) and 293T-mouse GCC.Luc (Figure 2B), with no nonspecific binding to 293T cells (Figure 2C), demonstrating superior specificity compared to PF0098-Rabbit IgG. Therefore, 22G5-Rabbit IgG is a novel, highly specific anti-GCC antibody.

[0112] Figure 3 shows the results of immunohistochemistry using different anti-GCC antibodies at the same concentration (0.5 μg / mL) on tissue sections of human intestinal cancer cell SW948 subcutaneous tumors, human intestinal cancer cell LS1034 subcutaneous tumors, mouse small intestine, and mouse spleen. The isotype control antibody ab172730 showed no staining on any of the four tissue sections. The PF0098-Rabbit IgG antibody showed strong GCC-specific staining in SW948 and LS1034 subcutaneous tumor sections, and nonspecific staining in the mouse spleen. The 22G5-Rabbit IgG antibody showed strong GCC-specific staining in SW948 and LS1034 subcutaneous tumor sections, as well as mouse small intestine sections. Its cell membrane staining was stronger than that of the PF0098-Rabbit IgG antibody, and there was no nonspecific staining in any of the four tissue sections. Anti-GCC antibody ab122404 showed concentrated staining in the cytoplasm of SW948 and LS1034 subcutaneous tumors, lacking specific staining of GCC on the cell membrane surface. Furthermore, nonspecific staining was observed in the mouse small intestine and spleen. Therefore, the 22G5-Rabbit IgG antibody exhibits superior specificity and sensitivity in immunohistochemical staining.

[0113] Figures 4A to 4C show the cell-killing results of the constructed CAR-T cells against CT26-mouse GCC.Luc target cells 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 served as a positive control. All CAR-T cells were able to effectively kill mouse GCC-positive target cells.

[0114] 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 served as a control. 14C2 CAR-T that recognizes mouse targets was unable to effectively kill target cells. The remaining CAR-T cells showed strong cytotoxicity against human GCC-positive target cells. This indicates that rabbit-derived scFv is also suitable for the antigen binding domain of CAR and enables CAR-T to exert its efficient recognition and killing functions.

[0115] Figures 6A to 6C show the cytotoxicity of the constructed CAR-T cells against 293T.Luc cells lacking GCC expression at effector-target ratios of 5:1 (Figure 6A), 2.5:1 (Figure 6B), and 1.25:1 (Figure 6C). All CAR-T cells were unable to kill 293T.Luc cells, indicating that the constructed CAR-T cells specifically recognize only GCC-positive cells.

[0116] Figures 7A to 7D show the cytokine secretion levels of the constructed CAR-T cells after 24 hours of co-incubation with target cells CT26-mouse GCC.Luc (Figure 7A), LS1034.Luc (Figure 7B), and 293T.Luc (Figure 7C). IFNγ secretion by CAR-T cells was upregulated after incubation with target cells CT26-mouse GCC.Luc or LS1034.Luc. Figure 7D shows the background IFNγ secretion levels of UnT and each group of CAR-T cells without co-culture with target cells.

[0117] Some of the sequences involved in this disclosure are as follows.

[0118] SEQ ID NO: 1 14C2 heavy chain variable region CDR1

[0119] SEQ ID NO:2 21E1 heavy chain variable region CDR1

[0120] SEQ ID NO:3 22G5 heavy chain variable region CDR1

[0121] SEQ ID NO:4 25E2 heavy chain variable region CDR1

[0122] SEQ ID NO:5 48D5 heavy chain variable region CDR1

[0123] SEQ ID NO:6 14C2 heavy chain variable region CDR2

[0124] SEQ ID NO:7 21E1 heavy chain variable region CDR2

[0125] SEQ ID NO:8 22G5 heavy chain variable region CDR2

[0126] SEQ ID NO:9 25E2 heavy chain variable region CDR2

[0127] SEQ ID NO: 10 48D5 heavy chain variable region CDR2

[0128] SEQ ID NO:11 14C2 heavy chain variable region CDR3

[0129] SEQ ID NO: 12 21E1 heavy chain variable region CDR3

[0130] SEQ ID NO:13 22G5 heavy chain variable region CDR3

[0131] SEQ ID NO:14 25E2 heavy chain variable region CDR3

[0132] SEQ ID NO:15 48D5 heavy chain variable region CDR3

[0133] SEQ ID NO:16 14C2 light chain variable region CDR1

[0134] SEQ ID NO: 17 21E1 light chain variable region CDR1

[0135] SEQ ID NO:18 22G5 light chain variable region CDR1

[0136] SEQ ID NO: 19 25E2 light chain variable region CDR1

[0137] SEQ ID NO:20 48D5 light chain variable region CDR1

[0138] SEQ ID NO:21 14C2 light chain variable region CDR2

[0139] SEQ ID NO:22 21E1 light chain variable region CDR2

[0140] SEQ ID NO:23 22G5 light chain variable region CDR2

[0141] SEQ ID NO:24 25E2 light chain variable region CDR2

[0142] SEQ ID NO:25 48D5 light chain variable region CDR2

[0143] SEQ ID NO:26 14C2 light chain variable region CDR3

[0144] SEQ ID NO:27 21E1 light chain variable region CDR3

[0145] SEQ ID NO:28 22G5 light chain variable region CDR3

[0146] SEQ ID NO:29 25E2 light chain variable region CDR3

[0147] SEQ ID NO:30 48D5 light chain variable region CDR3

[0148] SEQ ID NO:31 14C2 heavy chain variable region

[0149] SEQ ID NO:32 21E1 heavy chain variable region

[0150] SEQ ID NO:33 22G5 heavy chain variable region

[0151] SEQ ID NO:34 25E2 heavy chain variable region

[0152] SEQ ID NO:35 48D5 heavy chain variable region

[0153] SEQ ID NO:36PF0098 heavy chain variable region

[0154] SEQ ID NO:37 14C2 light chain variable region

[0155] SEQ ID NO:38 21E1 light chain variable region

[0156] SEQ ID NO:39 22G5 light chain variable region

[0157] SEQ ID NO:40 25E2 light chain variable region

[0158] SEQ ID NO:41 48D5 light chain variable region

[0159] SEQ ID NO:42PF0098 light chain variable region

[0160] SEQ ID NO:43 14C2 single-chain antibody

[0161] SEQ ID NO:44 21E1 single-chain antibody

[0162] SEQ ID NO:45 22G5 single-chain antibody

[0163] SEQ ID NO:46 25E2 single-chain antibody

[0164] SEQ ID NO:47 48D5 single-chain antibody

[0165] SEQ ID NO:48PF0098 single-chain antibody

[0166] SEQ ID NO:49 14C2 CAR

[0167] SEQ ID NO:50 21E1 CAR

[0168] SEQ ID NO:51 22G5 CAR

[0169] SEQ ID NO:52 25E2 CAR

[0170] SEQ ID NO:53 48D5 CAR

[0171] SEQ ID NO:54 PF0098 CAR

[0172] SEQ ID NO:55 Rabbit IgG CH1-CH2-CH3

[0173] SEQ ID NO:56 Rabbit IgG CL

[0174] SEQ ID NO:57 22G5 VH-Rabbit IgG CH1-CH2-CH3

[0175] SEQ ID NO:58 22G5 VL-Rabbit IgG CL

[0176] SEQ ID NO:59 PF0098 VH-Rabbit IgG CH1-CH2-CH3

[0177] SEQ ID NO:60 PF0098 VL-Rabbit IgG CL

[0178] SEQ ID NO:61 CD8α signal peptide

[0179] SEQ ID NO:62 CD8α hinge region

[0180] SEQ ID NO:63 CD8α transmembrane domain

[0181] SEQ ID NO:64 CD137 co-stimulatory signaling domain

[0182] SEQ ID NO:65 CD3ζ intracellular signaling domain

[0183] SEQ ID NO:66 Kozak sequence

[0184] SEQ ID NO:67 Linker 1

[0185] SEQ ID NO:68 Linker 2

[0186] SEQ ID NO:69 Linker repeat unit

[0187] SEQ ID NO: 70 Linker

[0188] SEQ ID NO:71 14C2 CAR-no signal peptide

[0189] SEQ ID NO:72 21E1 CAR-no signal peptide

[0190] SEQ ID NO:73 22G5 CAR-no signal peptide

[0191] SEQ ID NO:74 25E2 CAR-no signal peptide

[0192] SEQ ID NO:75 48D5 CAR-no signal peptide DETAILED DESCRIPTION

[0193] The embodiments of the present disclosure will be described in detail below with reference to the examples. However, those skilled in the art will appreciate that the following examples are intended only to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0194] Example 1: Screening of anti-GCC rabbit monoclonal antibodies

[0195] Through a service order from Genscript Biotech (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 below.

[0196] 1. Target Cell Construction

[0197] The following method was used to construct Dubca-human GCC.Luc and Dubca-mouse GCC.Luc overexpressing 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. Subsequently, the vectors were transfected into Lenti-X 293T host cells together with the auxiliary packaging plasmids psPAX2 and pMD.2G. The lentiviruses were concentrated and purified to obtain the lentiviruses. The two lentiviruses were transduced into the Dubca cell line ( CRL2276 TM ), and after three rounds of puromycin resistance screening, Dubca-human GCC.Luc and Dubca-mouse GCC.Luc were finally obtained.

[0198] The expression level of human GCC target was detected by incubating Dubca-human GCC.Luc cells with 5F9-hIgGFc (Genscript, Cat. NO. C0224DK260-1). 2.0×10 Dubca-human GCC.Luc and Dubca cells were taken. 5 Incubate with 5F9-hIgGFc antibody diluted 1:100 at 4°C for 0.5 hours; wash three times with DPBS, then resuspend the cells with a fluorescent secondary antibody (PE anti-human IgG Fc Antibody, Biolegend, Cat. No. 409304) prepared at 1:100 and incubate at 4°C in the dark for 0.5 hours; wash three times with DPBS and load the sample on a flow cytometer for reading. The expression level of the mouse GCC target was detected by incubating the purified PF0098-Rabbit IgG antibody with Dubca-mouse GCC.Luc cells, using the same detection method as above. PF0098 is an anti-GCC antibody disclosed in patent application WO2019224716A2 (VH is shown in SEQ ID NO:36, VL is shown in SEQ ID NO:42). It can simultaneously recognize human GCC, monkey GCC, and mouse GCC, and can also be detected by immunohistochemistry. This antibody will also be cited as a standard control in the subsequent examples.

[0199] Using the above method, mouse GCC (NM_001127318.1) or human GCC (NM_004963.3) was overexpressed in the CT26 cell line (ATCC, CRL-2638™) and HEK293T cell line (Clontech, Cat. No. 632180), resulting in the construction of CT26-mouse GCC.Luc, 293T-mouse GCC.Luc, and 293T-human GCC.Luc cells. The HEK293T cell line (Clontech, Cat. No. 632180) and the LS1034 colon cancer cell line (ATCC, CRL-2158™), which naturally overexpresses human GCC, were also constructed to express firefly luciferase, i.e., LS1034.Luc cells, and used for subsequent in vitro screening.

[0200] 2. Animal Immunization and Titer Testing

[0201] MonoRab from GenScript Biotech TM Platform service orders produce anti-GCC rabbit monoclonal antibodies. His-tagged mouse GUCY2C / Guanylyl cyclase C protein (ACRO GUC-M52H3, referred to as mouse GCC-his protein) was immunoprecipitated with ImmunoPlus TM After technical modifications, two New Zealand white rabbits were immunized using traditional methods, with immunizations repeated every two weeks. To enhance the efficacy of the immunization, 293T-mouse GCC cells were used in the third round of immunization. Seven days after the completion of the four rounds of immunization, blood was collected and titered for two target proteins: mouse GCC-his and His-tagged human GUCY2C / Guanylyl cyclase C protein (ACRO GUC-H52H5, referred to as human GCC-his protein).

[0202] 3. Positive clone screening

[0203] After the immunization, 30 mL of fresh blood was collected from the rabbit to isolate PBMC. After the B cell clones were selected, the antigen-positive B cells were plated on 50 96-well cell plates for culture. Indirect ELISA was used to detect the binding characteristics of the cell supernatant to the mouse GCC-his and human GCC-his target proteins. The clone supernatant with specific cross-binding was selected to verify the FACS binding of Dubca-mouse GCC.Luc and Dubca cells. The positive clones were selected and expanded to 24-well plates for continued culture. The supernatant after the expanded culture was again confirmed by ELISA and FACS detection as above. The FACS detection method is as follows: digest and collect the target cells, and distribute them into 96-well pointed bottom plates, with 2×105 After centrifugation at 400 g for 3 minutes, the supernatant was discarded and 100 μL of B cell clone supernatant was added and incubated at 4°C for 1 hour. The supernatant was washed three times with 4°C pre-cooled DPBS buffer and 1:100 diluted Alexa Incubate with 488 goat anti-rabbit IgG (H+L) (Invitrogen, Cat. No. A11034) at 4°C for 30 minutes and wash three times with pre-chilled 4°C DPBS. After incubation, cells were analyzed on an Attune NxT flow cytometer (Thermofisher). The results are shown in Table 2. Clone 14C2 bound exclusively to the target Dubca-mouse GCC.Luc cells, while the remaining four clones specifically bound to both Dubca-mouse GCC.Luc and Dubca-human GCC.Luc target cells. Therefore, the four antibody clones screened showed cross-species affinity for both mouse and human GCC proteins.

[0204] Table 2. Results of B cell clone supernatant binding to target cells

[0205] By MonoRab TM The positive B cell clones selected after the test were used RNA was extracted using standard procedures using Ambion reagents (Cat. No.: 15596-026) and analyzed using PrimeScript TM cDNA reverse transcription was performed using the 1st Strand cDNA Synthesis Kit (Takara, Cat. No.: 6110A) instruction manual to amplify the antibody light and heavy chain fragments. The light and heavy chain sequences of the antibody were obtained by next-generation sequencing. Tables 3 and 4 show the antibody heavy chain variable region (VH) and light chain variable region (VL) sequences, respectively, divided according to the Kabat partitioning rules, as well as the corresponding complementary determining region (CDR) sequences. The antibody heavy chain variable region (VH) and light chain variable region (VL) are connected by a linker, which 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).

[0206] Table 3. Antibody heavy chain variable region and CDR amino acid sequences

[0207] Table 4. Antibody light chain variable region and CDR amino acid sequences

[0208] Example 2: Preparation, purification and identification of candidate antibodies

[0209] Rabbit IgG molecules are divided into IgG-k and IgG-λ according to the difference in light chain. Generally, most antibodies obtained from New Zealand white rabbits are of IgG-k type, and only <1% are of IgG-λ type. The natural rabbit IgG-k has a molecular weight of about 150KD and contains two identical light chains and two identical heavy chains. The light chain consists of the N-terminal variable domain VL and the constant domain CL (for example, SEQ ID NO: 56), and the heavy chain consists of the N-terminal variable domain VH and three constant domains (CH1, CH2 and CH3, for example, 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.

[0210] The selected heavy and light chain variable region sequences were fused to rabbit IgG (IgG1k), such as the 22G5 single-chain antibody sequence or the 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), respectively. The coding sequences of the fusion sequences were inserted into the pcDNA3.4 vector to construct expression plasmids. The 22G5-Rabbit IgG antibody and PF0098-Rabbit IgG antibody were then expressed in HD CHO-S cell culture and further purified. The concentration of the obtained antibodies was determined using a NanoDrop A280 nm analyzer, and the purity was assessed using 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.

[0211] Table 5. Antibody purification information

[0212] The binding ability of the purified antibodies to the target proteins was tested using an enzyme-linked immunosorbent assay (ELISA). Human GCC-his and mouse GCC-his were diluted in coating buffer at a concentration of 1 μg / mL, and 100 μL per well was coated onto a 96-well Costar microtiter plate. The plates were incubated at 4°C overnight. The next day, the plates were washed twice with 0.05% PBST and blocked with 5% MPBS for 2 hours at room temperature. Following blocking, the plates were washed and incubated with serially diluted antibody solutions for 1.5 hours at room temperature. Following incubation, the plates were washed three times with the ELISA solution, and a 1:10,000 dilution of the enzyme-linked secondary antibody, Mouse Anti-Rabbit IgG Antibody (M205) [HRP], mAb (Genscript, Cat# A01827), was added and allowed to bind for 45 minutes at room temperature. The plates were washed four times with the ELISA solution, and TMB was added for 10 minutes. The color reaction was then stopped with 1 M HCl and analyzed using a microplate reader at OD 450 nm. Binding curves are shown in Figures 1A and 1B. EC 50 See Table 6. The results showed that both 22G5-Rabbit IgG and PF0098-Rabbit IgG antibodies had strong ability to bind to GCC protein.

[0213] Table 6. EC of antibody binding to target protein 50

[0214] 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 diluted 1:100. 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. Wash three times with DPBS and then load the sample onto the flow cytometer for reading. 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, with EC 50 See Table 7. In addition, 22G5-Rabbit IgG did not bind to 293T cells, but PF0098-Rabbit IgG showed strong nonspecific binding to 293T cells at high concentrations. In summary, 22G5-Rabbit IgG has high specificity against GCC antigens.

[0215] Table 7. Antibody binding EC to target cells50

[0216] Example 3: Immunohistochemical detection of candidate antibodies

[0217] The paraffin tissue sections used for testing were dewaxed and then soaked in 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, and pure water. The sections were then microwave-heated for antigen retrieval using an improved sodium citrate antigen retrieval solution (BEYOTIME #P0083). After the retrieval 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). Anti-GCC antibody was diluted to a working concentration of 0.5 μg / mL using antibody diluent (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. #ZLI-9030). The goat serum on the sections was shaken off, and the antibody was added dropwise to the tissue sections and incubated overnight at 4°C. After the antibody incubation is completed, the sections are washed three times with PBS, and the goat anti-rabbit antibody of the ready-to-use rapid immunohistochemistry detection kit (Fuzhou Maixin Biotechnology Development Co., Ltd. #KIT-5005) is added to the slice tissue and incubated in the dark at room temperature for 15 minutes. After the antibody incubation is completed, the sections are washed three times with PBS, and then DAB color development solution (VECTOR LABORATORIES#sk-4105) is used for color development. After the color development is completed, the sections are stained with hematoxylin stain (Zhuhai Besuo Biological #BA4097). Then, 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol and xylene are soaked in sequence. After the treatment is completed, neutral resin (Sinopharm Chemical Reagent Co., Ltd. #10004160) is added to the slice tissue and the slice is sealed. After the sections are dried, they are observed under a microscope and photographed.

[0218] The experimental results are shown in Figure 3. The isotype control antibody ab172730 (Abcam, Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control) did not stain on any of the four tissue sections. The PF0098-Rabbit IgG antibody had strong GCC-specific staining in SW948 and LS1034 subcutaneous tumor sections, and produced nonspecific staining in mouse spleen. Under the same experimental conditions and at an antibody concentration of 0.5 μg / mL, the 22G5-Rabbit IgG antibody had strong specific staining for GCC-expressing SW948 and LS1034 subcutaneous tumors, as well as mouse small intestinal tissue sections. Its cell membrane staining was stronger than that of the PF0098-Rabbit IgG antibody, and no nonspecific staining was observed in mouse spleen tissue sections that did not express GCC. Furthermore, the anti-GCC antibody ab122404 (Abcam) showed concentrated staining in the cytoplasm of SW948 and LS1034 subcutaneous tumors, lacking specific staining of the cell membrane surface. Furthermore, it produced nonspecific staining in the mouse small intestine and spleen. Therefore, the 22G5-Rabbit IgG antibody exhibited superior staining intensity and specificity on GCC-positive cell membranes compared to the control antibody. The 22G5-Rabbit IgG antibody is a novel anti-GCC antibody suitable for immunohistochemical detection in human and mouse tissue sections.

[0219] Example 4: Construction of CAR-T cells

[0220] Gene synthesis technology was used to synthesize the backbone structure sequence encoding the chimeric antigen receptor (CAR), that is, the coding structures from N-terminus to C-terminus were: CD8α hinge region (SEQ ID NO: 62), CD8α transmembrane domain (SEQ ID NO: 63), CD137 co-stimulatory signaling domain (SEQ ID NO: 64) and CD3ζ intracellular signaling domain (SEQ ID NO: 65) nucleotide molecules and cloned into the lentiviral vector (pLSINK-BBzBB). 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 CD8α signal peptide (SEQ ID NO: 61) of the CAR backbone sequence and inserted upstream of the CAR backbone vector. By molecular cloning techniques known in the art, the synthesized nucleic acid sequences encoding CD8α signal peptide and anti-GCC single-chain antibody (e.g., 22G5 scFv, SEQ ID NO: 45) were cloned into the lentiviral vector pLSINK-CE11A16 through EcoRI (5′-GAATTC-3′) and SpeI (5′-ACTAGT-3′) restriction sites. The chimeric antigen receptors (CARs) constructed 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).

[0221] A mixture of lentiviral auxiliary packaging plasmids containing pMDLg.pRRE (Addgene#12251), pRSV-REV (Addgene#12253), and pMD2.G (Addgene#12259) was mixed with a vector expressing a CAR construct in a pre-optimized ratio with polyetherimide (PEI) and then incubated at 25°C for 5 minutes. 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% CO2. The supernatant was collected and centrifuged at 4°C, 3000g for 15 minutes. 20% PEG 8000 prepared in advance was added to the supernatant 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 in pre-chilled DPBS and stored at -80°C for later use. Virus titers were determined by titration using the SUP-T1 (human T lymphoblastoid) cell line.

[0222] 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. The cell number was first determined and the cell suspension was centrifuged at 300 g for 10 minutes. The supernatant was then completely discarded and the cell pellet was plated at 10 [mu]l. 7 Resuspend 100 μL of total cells in 40 μL MACS buffer (DPBS supplemented with 8 μM EDTA and 0.5% FBS). 7 Add 10 μL Pan T Cell Biotin-Antibody Cocktail to each total cell, mix thoroughly and place in refrigerator (2-8°C) for about 5 minutes. 7 Add 30 μL MACS buffer to each 10 cells. 7 Add 20 μL Pan T Cell MicroBead Cocktail to each cell. Mix the cell suspension mixture thoroughly and incubate in the refrigerator (2-8°C) for another 10 minutes. Magnetic separation requires at least a 500 μL system. For magnetic separation, place the LS column in the magnetic field of a suitable MACS separator. The column is pretreated by flushing with 3 mL of buffer. The cell suspension is then loaded onto the column and the effluent containing unlabeled cells is collected, representing the enriched T cell component. The remaining T cells are collected by washing the column with 3 mL of buffer and collecting the unlabeled cells that pass through. The T cell components collected twice are combined, centrifuged and resuspended in TexMACS GMP Medium & 1L (Miltenyi, Cat#170-076-309) containing 300 IU / mL IL-2.

[0223] Subsequently, the prepared T cells were transfected with human T Cell TransAct TM The cells were activated for 48 hours with anti-CD3 / CD28 MACSiBeads (Miltenyi #130-111-160) at a ratio of 1:2.

[0224] Activated T cells were transduced with lentivirus at a certain multiplicity of infection. 72 hours later, R10 medium containing 300 IU / mL IL-2 was added, and the transduced cells were transferred to a new cell culture plate and expanded at 37°C with 5% CO2.

[0225] CAR expression levels were assessed by flow cytometry using directly labeled antibodies. On day 6 after transduction, 5 × 10 5T cells were prepared using pre-cooled DPBS buffer, in which PF0098 CAR-T and untransduced CAR T cells (UnT) were diluted with Alexa Fluor 500 Antibody at a dilution of 1:100. 488 goat anti-mouse IgG (H+L) (Invitrogen, Cat. NO. A11001) was incubated at 4°C for 30 minutes. The remaining CAR-T and untransduced UnT were incubated with 1:100 diluted Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen, Cat. NO. A11034) was incubated at 4°C for 30 minutes and washed three times with 4°C pre-cooled DPBS. The cells after the above incubation were read on an Attune NxT flow cytometer (Thermofisher). The results are shown in Table 8. The CAR expression range is 43.94% to 75.10%. PF0098 CAR-T is the control, and UnT represents T cells that are not transduced with CAR.

[0226] Table 8. CAR expression levels after transduction of T cells

[0227] Example 5: Functional evaluation of CAR-T cells

[0228] 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. Among them, PF0098 CAR-T was a positive control. In order to determine the cytotoxicity of CAR-T cells against target cells, One-glo luminescent luciferase assay reagent (Promega #E6120) was added to the co-cultured cells to detect the remaining luciferase activity value in the well. The remaining luciferase activity is directly related to the number of target cells remaining in the well. Cytotoxicity is calculated by the following formula:

[0229] Cytotoxicity % = 100% × (1-(RLU sample -RLU min ) / (RLU unT -RLU min )).

[0230] RLU sample Refers to the luciferase activity value measured in the anti-GCC CAR-T cell wells. RLU min Refers to the luciferase activity value measured in the wells to which Triton X-100 was added at a final concentration of 1% at the beginning of the cytotoxicity assay, RLUunT Refers to the luciferase activity value measured in wells with non-CAR-transduced T cells.

[0231] As shown in Figures 4A to 4C, all CAR-T cells exhibited strong cytotoxicity against CT26-mouse GCC.Luc cells. As shown in Figures 5A to 5C, because 14C2 does not bind to the human GCC protein, 14C2 CAR-T cells had no killing effect on LS1034.Luc cells, which naturally express human GCC. 25E2 CAR-T cells exhibited cytotoxicity against target cells only at a high E / T ratio of 5:1. The other three CAR-T cells all maintained strong cytotoxicity against the target LS1034.Luc cells. As shown in Figures 6A to 6C, these CAR-T cells showed no cytotoxicity against 293T.Luc cells even at higher E / T ratios, demonstrating that these five CAR-T cells only recognize GCC-positive target cells.

[0232] In addition to the cytotoxicity against target cells, the ability of GCC CAR-T cells (prepared in Example 4) to produce IFN-γ after co-incubation with target cells (LS1034.Luc, CT26-mouse GCC.Luc, and 293T.Luc cells prepared in Example 1) was also analyzed. GCC CAR-T cells were mixed with target cells at an E / T ratio of 2.5:1 (2000 target cells / well) and co-incubated in 384-well plates for 24 hours. The IFN-γ concentration produced in the culture supernatant after co-incubation of CAR-T with target cells and in the supernatant of the CAR-T cells themselves was measured using an HTRF kit (Cisbio, Cat#62HIFNGPEG). Prior to measurement, the HTRF reagent was allowed to stabilize at room temperature for 30 minutes. The co-culture supernatant was transferred to a 384-well assay plate (Greiner Bio-One, #784075) at 16 μL / well, and then 4 μL / well of pre-mixed HTRF reagent prepared according to the kit manual was added. The plate was sealed with sealing film and incubated overnight at room temperature. HTRF values ​​were read on a Tecan Spark 10M. IFN-γ concentrations were calculated by referring to the signal obtained from the standard curve provided with the kit.

[0233] As shown in Figures 7A to 7D , the concentrations of IFNγ secreted by the five CAR-Ts 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 ​​of 600.4 pg / mL and 910.8 pg / mL).

[0234] Although the specific embodiments of the present disclosure have been described in detail, it will be understood by those skilled in the art that various modifications and replacements may be made to those details based on all the teachings disclosed, and such changes are within the scope of protection of the present disclosure. The full scope of the present disclosure is given by the appended claims and any equivalents thereof.

Claims

1. 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: (1) 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; (2) 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; (3) 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; (4) 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 (5) 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.

2. The anti-GCC antibody or antigen-binding fragment thereof according to claim 1, 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.

3. 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: (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, and the amino acid sequence of LCDR1 is shown in The amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 26; (2) 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, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:27; (3) the amino acid sequence of HCDR1 is shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is shown in SEQ ID NO:8, the amino acid sequence of HCDR3 is shown in SEQ ID NO:13, the amino acid sequence of LCDR1 is shown in SEQ ID NO:18, the amino acid sequence of LCDR2 is shown in SEQ ID NO:23, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:28; (4) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:14, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:19, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:24, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:29; or (5) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 15, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 20, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 25 and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

30.

4. The anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein (1) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:31, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:37; (2) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:32, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:38; (3) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:33, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:39; (4) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:34, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:40; or (5) The heavy chain variable region comprises an amino acid sequence that is at least 80% 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% identical to the amino acid sequence shown in SEQ ID NO:

41.

5. The anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein (1) the 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; (2) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38; (3) the 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; (4) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40; or (5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:35, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

41.

6. The anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein The anti-GCC antibody comprises a non-CDR region, and the non-CDR region is derived from human, mouse or rabbit; Preferably, the non-CDR regions include framework regions, heavy chain constant regions and / or light chain constant regions.

7. The anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The antigen binding fragment is Fab, Fab', F(ab')2, single chain antibody or disulfide bond stabilized variable fragment.

8. The anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 43 to SEQ ID NO: 47; or comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 57 and SEQ ID NO: 58; Preferably, the amino acid sequence of the anti-GCC antibody or antigen-binding fragment thereof is as shown in any one of SEQ ID NO:43 to SEQ ID NO:

47.

9. An isolated nucleic acid encoding the anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising the isolated nucleic acid of claim 9.

11. A host cell comprising the isolated nucleic acid of claim 9, or comprising the vector of claim 10.

12. A chimeric antigen receptor comprising a polypeptide comprising: (1) an extracellular antigen-binding domain, which comprises the anti-GCC antibody or antigen-binding fragment according to any one of claims 1 to 8; (2) transmembrane domain; and (3) Intracellular signal transduction domain.

13. The chimeric antigen receptor of claim 12, wherein the anti-GCC antibody or antigen-binding fragment is a single chain antibody.

14. The chimeric antigen receptor according to any one of claims 12 to 13, further comprising a signal peptide located at the N-terminus of the polypeptide; Preferably, the signal peptide is derived from CD8α, CD28 or CD4 molecule; Preferably, the signal peptide comprises the amino acid sequence as shown in SEQ ID NO:61, or an amino acid sequence having at least 85% identity with SEQ ID NO:

61.

15. The chimeric antigen receptor according to any one of claims 12 to 14, further comprising a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain; Preferably, the hinge region is derived from IgG4Fc CH2CH3, CD28 or CD8α; Preferably, the hinge region comprises the amino acid sequence as shown in SEQ ID NO:62, or an amino acid sequence that is at least 85% identical to SEQ ID NO:

62.

16. The chimeric antigen receptor according to any one of claims 12 to 15, wherein the transmembrane domain is derived from one or more transmembrane regions of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1 molecules; Preferably, the transmembrane domain comprises the amino acid sequence as shown in SEQ ID NO:63, or an amino acid sequence having at least 85% identity with SEQ ID NO:

63.

17. The chimeric antigen receptor of any one of claims 12 to 16, wherein the intracellular signaling domain comprises a co-stimulatory signaling domain; Preferably, 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; Preferably, the co-stimulatory signaling domain comprises the cytoplasmic domain of CD28 and / or the cytoplasmic domain of CD137; Preferably, the costimulatory signaling domain comprises the amino acid sequence as shown in SEQ ID NO:64, or an amino acid sequence that is at least 85% identical to SEQ ID NO:

64.

18. The chimeric antigen receptor of any one of claims 12 to 17, wherein the intracellular signaling domain comprises a primary intracellular signaling domain; Preferably, the primary intracellular signaling domain is derived from CD3ζ; Preferably, the primary intracellular signaling domain comprises the amino acid sequence as shown in SEQ ID NO:65, or an amino acid sequence having at least 85% identity with SEQ ID NO:

65.

19. The chimeric antigen receptor according to any one of claims 12 to 18, wherein the amino acid sequence thereof is shown in any one of SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:71 to SEQ ID NO:

75.

20. An isolated nucleic acid encoding the chimeric antigen receptor of any one of claims 12 to 19.

21. A vector comprising the isolated nucleic acid of claim 20.

22. A cell comprising the chimeric antigen receptor of any one of claims 12 to 19, comprising the isolated nucleic acid of claim 20, or comprising the vector of claim 21; Preferably, the cells are immune cells.

23. The cell according to claim 22, wherein The cells are selected from the group consisting of: T cells, NK cells, peripheral blood mononuclear cells, hematopoietic stem cells, pluripotent stem cells, embryonic stem cells and combinations thereof.

24. A pharmaceutical composition comprising the anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the chimeric antigen receptor according to any one of claims 12 to 19, or the cell according to claim 22 or 23; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

25. Use of the anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the chimeric antigen receptor according to any one of claims 12 to 19 in immunohistochemical detection or in the preparation of a detection kit.

26. Use of the anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the chimeric antigen receptor according to any one of claims 12 to 19, or the cell according to claim 22 or 23 in the preparation of a medicament for treating or preventing a GCC-related disease or condition; Preferably, the GCC-related disease or disorder is a tumor; Preferably, the GCC-related disease or condition is a digestive tract tumor; Preferably, 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.

27. The anti-GCC antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the chimeric antigen receptor according to any one of claims 12 to 19, or the cell according to claim 22 or 23, for use in treating or preventing a GCC-related disease or disorder; Preferably, the GCC-related disease or disorder is a tumor; Preferably, the GCC-related disease or condition is a digestive tract tumor; Preferably, 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.

28. 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 according to any one of claims 1 to 8, the chimeric antigen receptor according to any one of claims 12 to 19, or the cell according to claim 22 or 23; Preferably, the GCC-related disease or disorder is a tumor; Preferably, the GCC-related disease or condition is a digestive tract tumor; Preferably, 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.

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