Anti-GUCY2C humanized antibody and chimeric antigen receptor and application thereof

Abstract

The invention relates to the field of antibodies, and particularly discloses an anti-GUCY2C humanized antibody as well as a chimeric antigen receptor and application thereof. The anti-GUCY2C humanized antibody disclosed by the invention can be specifically combined with human GUCY2C, and a CAR-T constructed by the anti-GUCY2C humanized antibody has a good in-vitro killing function.

Description

[0001] Priority declaration

[0002] This application claims priority to the Chinese invention patent application with application number 202311208651.5, filing date September 18, 2023, and invention name “An anti-GUCY2C humanized antibody, its chimeric antigen receptor and application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of antibodies, and in particular, to anti-GUCY2C humanized antibodies, chimeric antigen receptors thereof, and applications thereof. Background Art

[0004] Guanylate cyclase C (GUCY2C) is a transmembrane receptor that is mainly expressed on the top of intestinal glands and villus cells. GUCY2C is expressed in various gastrointestinal malignancies, including more than 95% of colorectal cancer and more than 50% of gastric or gastroesophageal junction cancer. GUCY2C is highly expressed in most colon cancers of all stages, including liver metastases. Moderately differentiated and well-differentiated gastrointestinal tumors are often associated with higher membrane expression of GUCY2C.

[0005] GUCY2C is largely restricted in expression at the top of the tight junctions of the intestinal epithelium in normal tissues. However, tumors destroy the normal tight junction structure, exposing GUCY2C more thoroughly. Therefore, compared with normal tissues, it only binds to GUCY2C antibodies and has no effect on normal tissues.

[0006] Chimeric antigen receptor T cell immunotherapy, referred to as CAR-T, is a method of modifying the patient's T cells in vitro to enable the patient's T cells to recognize tumor cells, and then infusing them back into the patient's body for treatment after in vitro expansion and culture. At present, CAR-T targeting CD19 has achieved great results in the treatment of B-cell blood tumors. GUCY2C has good target specificity, and CAR-T cells targeting GUCY2C can be developed to treat, for example, colorectal cancer. Summary of the invention

[0007] The present disclosure provides an anti-human GUCY2C humanized antibody, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 1, 3, 5 or 7, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 2, 4, 6 or 8; optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.

[0008] In some embodiments, the anti-human GUCY2C humanized antibody described above, the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 1, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 2. Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system; or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.

[0009] In some embodiments, the anti-human GUCY2C humanized antibody described above, the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 3, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 4. Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system; or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.

[0010] In some embodiments, the anti-human GUCY2C humanized antibody described above, the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 5, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 6. Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system; or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.

[0011] In some embodiments, the anti-human GUCY2C humanized antibody described above, the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 7, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 8. Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system; or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.

[0012] In some embodiments, the anti-human GUCY2C humanized antibody described in any of the preceding items, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody respectively include the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region of the antibody respectively include the amino acid sequences shown in SEQ ID NO:12, amino acid AAS and SEQ ID NO:14.

[0013] In some embodiments, the anti-human GUCY2C humanized antibody of any of the preceding items, the heavy chain variable region of the antibody comprises an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 5, 3 or 7, and the light chain variable region comprises an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 6, 4 or 8;

[0014] In some embodiments, in any of the above anti-human GUCY2C humanized antibodies, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:6; the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:4; or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:8.

[0015] In some embodiments, the anti-human GUCY2C humanized antibody described in any of the above items comprises a heavy chain variable region as shown in SEQ ID NO:5 and a light chain variable region as shown in SEQ ID NO:6.

[0016] In some embodiments, the anti-human GUCY2C humanized antibody described in any of the preceding items comprises a constant region;

[0017] In some embodiments, in any of the above anti-human GUCY2C humanized antibodies, the constant region includes a heavy chain constant region and / or a light chain constant region;

[0018] In some embodiments, in any of the above anti-human GUCY2C humanized antibodies, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from the κ type or λ type light chain constant region;

[0019] In some embodiments, in any of the above anti-human GUCY2C humanized antibodies, the species origin of the constant region is human.

[0020] In some embodiments, the anti-human GUCY2C humanized antibody described in any of the above items is an antigen-binding fragment selected from any one of scFv, F(ab')2, Fab', Fab, scFab, dsFv and Fv. In some embodiments, the anti-human GUCY2C humanized antibody described in any of the above items is scFv (single-chain antibody). In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO: 20, 19 or 21.

[0021] In some embodiments, the present disclosure provides a humanized antibody that competitively binds to human GUCY2C with any of the foregoing anti-human GUCY2C humanized antibodies, or an anti-human GUCY2C humanized antibody that binds to the same epitope as any of the foregoing anti-human GUCY2C humanized antibodies.

[0022] In some embodiments, the anti-human GUCY2C humanized antibody described in any of the above items, wherein the antibody has at least one of the following characteristics:

[0023] A. capable of binding to human GUCY2C; optionally, it can bind to human GUCY2C (ECD) -His antigen with an EC50 value of ≤ 1 nM, wherein the EC50 value is determined by ELISA method;

[0024] B. capable of binding to human GUCY2C; optionally, it can bind to human GUCY2C (ECD) -His antigen with a KD value of ≤ 0.05 nM, wherein the KD value is determined by the Fortebio method;

[0025] C. Capable of binding to human GUCY2C; optionally, it can bind to cells expressing human GUCY2C with an EC50 of ≤21 nM, wherein the EC50 value is determined by flow cytometry.

[0026] In addition, the present disclosure provides a chimeric antigen receptor, which comprises the anti-human GUCY2C humanized antibody described in any of the above items;

[0027] In some embodiments, in any of the above chimeric antigen receptors, the anti-human GUCY2C humanized antibody is a scFv;

[0028] In some embodiments, in the chimeric antigen receptor of any of the preceding items, the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:6; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO:3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:4; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:8.

[0029] In some embodiments, the chimeric antigen receptor described in any of the preceding items includes a transmembrane domain; optionally, the transmembrane domain is a transmembrane domain selected from the following proteins: α / β or ζ chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154; optionally, the transmembrane domain is human CD8a hinge-transmembrane region; optionally, the human CD8a hinge-transmembrane region has the amino acid sequence shown in SEQ ID NO:15.

[0030] In some embodiments, the chimeric antigen receptor described in any of the above items comprises an intracellular signaling domain; optionally, the intracellular signaling domain is at least one functional signaling domain of a protein selected from the following: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules, activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, 4-1BB, B7-H3, CDS, ICAM-1, ICOS, GITR, BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NK p44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA 6. VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD2 9. ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, RANKL, CD226, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD22 9, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83 ligands; optionally, the intracellular signaling domain includes the co-stimulatory molecule 4-1BB intracellular signaling domain and / or the human CD3 molecule signaling domain CD3Zeta; optionally, the 4-1BB intracellular signaling domain has the amino acid sequence shown in SEQ ID NO:16, and the human CD3 molecule signaling domain CD3Zeta has the amino acid sequence shown in SEQ ID NO:17.

[0031] In some embodiments, the chimeric antigen receptor described in any of the above items includes a signal peptide; optionally, the signal peptide is a human CD8a signal peptide; optionally, the human CD8a signal peptide has an amino acid sequence as shown in SEQ ID NO:18.

[0032] In some embodiments, the chimeric antigen receptor described in any of the above items comprises a polypeptide: [anti-human GUCY2C humanized antibody]-[transmembrane domain]-[intracellular signaling domain]; optionally,

[0033] A) the anti-human GUCY2C humanized antibody is the anti-human GUCY2C humanized antibody according to any one of claims 1 to 6; optionally, the anti-human GUCY2C humanized antibody is a scFv, and optionally, the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8;

[0034] B) the transmembrane domain is a human CD8a hinge-transmembrane region; optionally, the human CD8a hinge-transmembrane region has an amino acid sequence as shown in SEQ ID NO: 15; and / or

[0035] C) the intracellular signaling domain is the intracellular region of the 4-1BB immune co-stimulatory molecule and / or the intracellular region of the human CD3 molecule CD3Zeta; optionally, the 4-1BB intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 16, and the human CD3 molecule signaling domain CD3Zeta has the amino acid sequence shown in SEQ ID NO: 17.

[0036] In addition, the present disclosure provides a nucleic acid encoding the anti-human GUCY2C humanized antibody described in any of the foregoing items or encoding the chimeric antigen receptor described in any of the foregoing items.

[0037] In addition, the present disclosure provides a vector comprising any of the above nucleic acids.

[0038] In addition, the present disclosure provides a cell comprising any of the nucleic acids or vectors described above; optionally, the cell is an engineered immune effector cell; optionally, the immune effector cell is selected from T cells, NK cells, NKT cells, macrophages and CIK cells; optionally, the immune effector cell is a T cell; optionally, the vector comprises a nucleotide sequence: [a nucleotide sequence encoding a signal peptide] - [a nucleotide sequence encoding an anti-human GUCY2C humanized antibody] - [a nucleotide sequence encoding a transmembrane domain] - [a nucleotide sequence encoding an intracellular signaling domain]; optionally,

[0039] A) the nucleotide sequence encoding the signal peptide is a nucleotide sequence encoding a human CD8a signal peptide; optionally, the human CD8a signal peptide has an amino acid sequence as shown in SEQ ID NO: 18;

[0040] B) the nucleotide sequence encoding the anti-human GUCY2C humanized antibody, wherein the anti-human GUCY2C humanized antibody is a scFv; optionally, the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8;

[0041] C) the nucleotide sequence encoding the transmembrane domain is a nucleotide sequence encoding the hinge-transmembrane region of human CD8a; optionally, the hinge-transmembrane region of human CD8a has an amino acid sequence as shown in SEQ ID NO: 15;

[0042] D) the nucleotide sequence encoding the intracellular signaling domain, wherein the intracellular signaling domain is the intracellular region of the 4-1BB immune co-stimulatory molecule and / or the intracellular region of the human CD3 molecule CD3Zeta; optionally, the 4-1BB intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 16; the human CD3 molecule signaling domain CD3Zeta has the amino acid sequence shown in SEQ ID NO: 17.

[0043] In addition, the present disclosure provides an engineered immune effector cell, which expresses a chimeric antigen receptor, wherein the chimeric antigen receptor is any of the chimeric antigen receptors described above; optionally, the immune effector cell is selected from T cells, NK cells, NKT cells, macrophages and CIK cells; optionally, the immune effector cell is a T cell; optionally, the chimeric antigen receptor comprises: [anti-human GUCY2C humanized antibody]-[transmembrane domain]-[intracellular signaling domain]; optionally,

[0044] A) the anti-human GUCY2C humanized antibody is a scFv, and optionally, the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8;

[0045] B) the transmembrane domain is a human CD8a hinge-transmembrane region; optionally, the human CD8a hinge-transmembrane region has an amino acid sequence as shown in SEQ ID NO: 15; and / or

[0046] C) the intracellular signaling domain is the intracellular region of the 4-1BB immune co-stimulatory molecule and / or the intracellular region of the human CD3 molecule CD3Zeta; optionally, the 4-1BB intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 16, and the human CD3 molecule signaling domain CD3Zeta has the amino acid sequence shown in SEQ ID NO: 17.

[0047] In addition, the present disclosure provides a pharmaceutical composition, comprising: the anti-human GUCY2C humanized antibody described in any of the foregoing items, the chimeric antigen receptor described in any of the foregoing items, the foregoing nucleic acid, the foregoing vector or the foregoing cell, and optionally, further comprising one or more pharmaceutically acceptable carriers, diluents or excipients.

[0048] In addition, the present disclosure provides the use of any of the foregoing anti-human GUCY2C humanized antibodies, chimeric antigen receptors, nucleic acids, vectors, cells or pharmaceutical compositions in the preparation of drugs for treating or diagnosing diseases related to GUCY2C expression; optionally, the GUCY2C expression-related disease is a tumor; optionally, the tumor is selected from at least one of colorectal tumors, large intestine tumors, gastric tumors, esophageal tumors, gastroesophageal tumors and pancreatic tumors.

[0049] In addition, the present disclosure provides an anti-human GUCY2C humanized antibody, chimeric antigen receptor, nucleic acid, vector, cell or pharmaceutical composition as described in any of the above for use as a drug. In some embodiments, the drug treats or diagnoses a GUCY2C expression-related disease. Optionally, the GUCY2C expression-related disease is a tumor; optionally, the tumor is selected from at least one of a colorectal tumor, a large intestine tumor, a gastric tumor, an esophageal tumor, a gastroesophageal tumor and a pancreatic tumor.

[0050] In addition, the present disclosure provides a method for diagnosing / treating a disease, the method comprising administering a diagnostic / therapeutic effective amount of an anti-human GUCY2C humanized antibody, chimeric antigen receptor, nucleic acid, vector, cell or pharmaceutical composition as described above to an individual in need. Optionally, the GUCY2C expression-related disease is a tumor; optionally, the tumor is selected from at least one of a colorectal tumor, a large intestine tumor, a gastric tumor, an esophageal tumor, a gastroesophageal tumor and a pancreatic tumor.

[0051] In addition, the present disclosure provides a method for detecting or measuring human GUCY2C, comprising using the anti-human GUCY2C humanized antibody as described in any of the preceding items.

[0052] In addition, the present disclosure provides a method for preparing the anti-human GUCY2C humanized antibody as described in any of the preceding items, comprising culturing the cell as described in any of the preceding items.

[0053] The present disclosure has the following beneficial effects: the anti-human GUCY2C humanized antibody of the present disclosure can specifically bind to human GUCY2C; in some embodiments, the anti-human GUCY2C humanized antibody, chimeric antigen receptor, nucleic acid, vector, cell or pharmaceutical composition has an anti-tumor effect in vitro / in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0055] Figure 1 This is the result of the binding test (ELISA) of the anti-human GUCY2C humanized antibody to human GUCY2C;

[0056] Figure 2 This is the result of the binding test (FCM) between the anti-human GUCY2C humanized antibody and LS1034;

[0057] Figure 3 This is the result of affinity test of anti-human GUCY2C humanized antibody (Fortebio);

[0058] Figure 4 Structural diagram of the CAR vector;

[0059] Figure 5 This is a graph showing the results of the cell positivity rate detection of CAR-T cells;

[0060] Figure 6 This is the result of CAR-T in vitro killing function test. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure is clearly and completely described below, but the description and embodiments should not be interpreted as limiting the scope of the present disclosure. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by ordinary technicians in the field to which the present disclosure belongs.

[0062] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0063] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs or amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine; common natural amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0064] In the present disclosure, the term "antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, chimeric antibodies, full-length antibodies or antigen-binding fragments thereof (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

[0065] In the present disclosure, the term "natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains. From N to C-terminus, each heavy chain has a heavy chain variable region (VH, also known as a variable heavy domain), followed by a heavy chain constant region, which includes three constant domains (CH1, CH2, and CH3); from N to C-terminus, each light chain has a light chain variable region (VL, also known as a variable light domain), followed by a light chain constant region (CL, also known as a light chain constant domain).

[0066] The term "full length antibody" or "intact antibody" refers to an antibody that contains a structure substantially similar to a native antibody structure, or the heavy chain contains an Fc region.

[0067] In the present disclosure, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, which is the region within the antibody variable domain that mainly contributes to specific binding to the antigen. In a specific embodiment of the present disclosure, "CDRs" refers to more than two highly variable regions in the heavy and light chains of the antibody.

[0068] In the present disclosure, the heavy chain complementarity determining region is represented by HCDR, and the heavy chain variable region contains three CDR regions: HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, and the light chain variable region contains three CDR regions: LCDR1, LCDR2 and LCDR3. The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering rules (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering rules, "ABM" numbering rules, "contact" numbering rules (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering rules (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc. In some embodiments of the present disclosure, the CDR is determined according to the IMGT coding rules. In some embodiments, the anti-human GUCY2C humanized antibody disclosed herein, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody have the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region of the antibody have the amino acid sequences shown in SEQ ID NO:12, amino acid AAS and SEQ ID NO:14, respectively.

[0069] In the present disclosure, "framework region" or "FR" region refers to the region other than CDR in the heavy chain variable region and light chain variable region of an antibody. The heavy chain framework region can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3 and FR4), wherein the heavy chain framework region can be further subdivided into adjacent regions separated by HCDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by LCDR, including LFR1, LFR2, LFR3 and LFR4 framework regions. The heavy chain variable region is connected by the following numbered CDRs and FRs (arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus)): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is connected by the following numbered CDRs and FRs (arranged from the amino terminus to the carboxyl terminus): LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. In an optional embodiment, the antibody disclosed herein includes a heavy chain framework region in the heavy chain variable region shown in any one of SEQ ID NOs: 1, 3, 5 or 7, and a light chain framework region in the light chain variable region shown in any one of SEQ ID NOs: 2, 4, 6 or 8. In an optional embodiment, the framework region amino acid sequence of the antibody has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above-mentioned framework region.

[0070] The term "identity" or "sequence identity" refers to the degree (i.e., percentage) to which the amino acids / nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned (introducing gaps, if necessary, to obtain the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity). To determine the percentage of sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, for example, using well-known computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0071] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular species and the remainder of the heavy chain and / or light chain is derived from another species.

[0072] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in the human body. For example, it can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the framework region of the heavy chain variable region of a human antibody, the framework region of the light chain variable region, and the human antibody constant region).

[0073] The term "antigen binding fragment" is a portion of a complete antibody that specifically binds to an antigen to which the complete antibody binds. It will be readily understood by those skilled in the art based on the disclosure that the antigen binding fragment can be prepared by methods known in the art, for example, by enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds, or by recombinant genetics techniques or by an automatic peptide synthesizer (such as the automatic peptide synthesizer of Applied BioSystems).

[0074] In an optional embodiment, the antigen binding fragment is any one of F(ab')2, Fab', Fab, scFab, dsFv, Fv and scFv. "Fab" is a monovalent fragment composed of VL, VH, CL and CH1 domains. "Fv" is composed of VH and VL; "dsFv" is a disulfide-stabilized Fv fragment. "Fab'" contains VL, CL as well as VH and CH1, and also contains a region between the CH1 and CH2 domains so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule. "F(ab')2" is a bivalent fragment containing two Fab' fragments connected by a disulfide bond at the hinge region. "Single-chain Fab fragment" or "scFab" is a polypeptide consisting of VH, CH1, VL, CL and a linker, wherein the antibody domain and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. "scFv" is a fusion protein comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are connected by a peptide linker, can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, scFv herein can have VL and VH variable regions in any order, for example, from the N-terminus to the C-terminus of the scFv polypeptide, can include: a) VL-linker-VH or b) VH-linker-VL.

[0075] In the present disclosure, the term "linker", "connecting peptide", "connecting polypeptide", "Linker" or "linker" refers to a connecting unit that connects two polypeptide fragments, which usually has a certain flexibility, and the use of the linker will not cause the loss of the original function of the protein domain. The linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. In some embodiments, the linker is selected from (GxS)y linkers, wherein x is selected from integers selected from 1-5 and y is selected from integers selected from 0-6. In some embodiments, the linker is selected from (GxS)y linkers, wherein x is selected from integers selected from 1-5 and y is selected from integers selected from 1-6.

[0076] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody. An epitope can be formed by a continuous string of amino acids (linear epitope) or contain non-continuous amino acids (conformational epitope), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen by protein properties) and become spatially close. The difference between a conformational epitope and a linear epitope is that the binding of the antibody to the conformational epitope is lost in the presence of a denaturing solvent. A conformational epitope generally contains at least 3, at least 4, at least 5, at least 6, at least 7 or more amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using methods known in the art, including but not limited to alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking, etc.

[0077] An antibody that binds to the same epitope as a reference antibody or an antibody that competes for binding with a reference antibody means an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, or an antibody whose binding to the antigen is blocked by the reference antibody by 50% or more. For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to the antigen under saturating conditions, and then the ability of the test antibody to bind to the antigen is assessed after removing excess reference antibody; if the test antibody is able to bind to the antigen after saturation binding of the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody; and if the test antibody is unable to bind to the antigen after saturation binding of the reference antibody, then the test antibody can bind to the same epitope as the reference antibody. To confirm whether the test antibody binds to the same epitope, routine experiments (e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art) can be used. In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if an excess (in a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amount) of one antibody inhibits antigen binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0078] The terms "specifically bind" and "specific binding" refer to an antibody that binds to an antigen or an epitope within the antigen with a higher affinity than it does to other antigens or epitopes. -7 M or less (e.g., about 1×10 -8 M or smaller, about 1×10 -9 M or smaller, about 1×10 -10 M or smaller, about 1×10 -11 M or smaller, or about 1×10 -12 The KD of an antibody is determined by measuring the equilibrium dissociation constant (KD) of the antibody with a molecular weight of 4 M or less. Surface plasmon resonance measurements.

[0079] The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, in conjunction with "affinity" refers to internal binding affinity, which reflects 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art. The term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to a dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd / ka) and is expressed as a molar concentration (M). The KD value of an antibody can be measured using methods well known in the art. For example, a biosensor system such as a system measuring surface plasmon resonance (e.g., Biacore) is used, or the affinity in a solution is measured by a solution equilibrium titration method (SET).

[0080] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an Fc region of amino acid sequence mutation). Examples of antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0081] The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequence of the antibody molecules included in the group is identical, except that a small amount of natural mutations may exist. In contrast, polyclonal antibody preparations are usually included in a variety of different antibodies with different amino acid sequences in their variable domains, which are usually specific for different epitopes. "Monoclonal" represents the characteristics of the antibody obtained from a substantially homogeneous antibody population, and should not be construed as requiring the production of antibodies by any ad hoc method. In some embodiments, antibody provided by the invention is a monoclonal antibody.

[0082] The term "antigen" refers to a molecule that can be bound by an antigen binding protein (eg, an antibody) selectively binding to an antigen. An antigen may have one or more epitopes that can interact with different antigen binding proteins (eg, antibodies).

[0083] The term "anti-human GUCY2C antibody" or "antibody that binds to human GUCY2C" refers to an antibody that can bind to human GUCY2C with sufficient affinity. In certain embodiments, the antibody that binds to human GUCY2C has a binding affinity of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10 -8M or smaller, e.g. 10 -8 M to 10 -12 M, for example 10 -9 M to 10 -10 In certain embodiments, the anti-human GUCY2C antibody binds to a GUCY2C epitope that is conserved in GUCY2C from different species (eg, monkey, human).

[0084] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which cells typically contain the nucleic acid molecules, but the nucleic acid molecules are present outside the chromosome or at a chromosomal position different from its natural chromosomal position. The isolated nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or a separate vector, and such one or more nucleic acid molecules present in one or more positions in a host cell. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly indicated. Specifically, as described in detail below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues.

[0085] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0086] The term "vector" is a construct capable of transporting another polynucleotide connected thereto. An expression vector is a vector suitable for transforming a host cell and containing a nucleic acid for guiding or controlling the expression of a heterologous coding region connected thereto, and the vector may include, but is not limited to, sequences that affect or control transcription, translation, and RNA splicing of the coding region connected thereto when introns are present. In an optional embodiment, the vector is a "plasmid", which is a circular double-stranded DNA loop, to which additional DNA segments may be connected. In an optional embodiment, the vector is a viral vector, such as a lentiviral retrovirus adenovirus, an adeno-associated virus vector (AAV or AAV2), wherein additional DNA segments may be connected to the viral genome. In an optional embodiment, the vector is capable of autonomous replication in a host cell into which they are introduced. In an optional embodiment, the vector may be integrated into the genome of the host cell after being introduced into the host cell and replicated together with the host genome.

[0087] The term "host cell" is a cell or a progeny cell thereof into which an exogenous nucleic acid has been introduced. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, without regard to the number of passages, including mutant progeny having the same function or biological activity as screened or selected in the initial transformed cell. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Exemplary host cells are as follows: NSO, SP2 cells, Chinese hamster ovary (CHO) cells, HeLa cells, 3T3 cells and HEK-293 cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, Pichia pastoris, Pichia finlandica, Candida albicans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei.

[0088] Herein, "CAR" is a chimeric antigen receptor, which is an artificially modified receptor, for example, it is a receptor that can anchor specific molecules (such as antibodies) that recognize tumor cell surface antigens or viral antigens on immune cells (such as T cells), so that immune cells recognize tumor antigens or viral antigens and kill tumor cells or viruses. Generally, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane region, and an intracellular signaling domain in sequence. The chimeric antigen receptor of the present disclosure can be constructed using transmembrane regions and intracellular signaling domains known in the art for constructing CAR. In an optional embodiment, the CAR is a chimeric antigen receptor, and its main components may also include a transmembrane domain and an intracellular domain. In an optional embodiment, the CAR may also include a costimulatory molecule. "Costimulatory molecule" refers to a molecule that is present on the surface of an antigen presenting cell and can bind to a costimulatory molecule receptor on a Th cell to produce a co-stimulatory signal. The proliferation of T lymphocytes requires not only the binding of antigens, but also the acceptance of costimulatory molecule signals. The costimulatory signal is transmitted to T cells mainly by binding of costimulatory molecules CD80 and CD86 expressed on the surface of antigen presenting cells to CD28 molecules on the surface of T cells. B cells can receive costimulatory signals through general pathogen components such as LPS, or through complement components, or through activated antigen-specific Th cell surface CD40L. Costimulatory molecules known in the art for constructing CAR can be used, including but not limited to CD27, CD28, 4-1BB, OX40, ICOS, B7-H3 and / or any combination thereof.

[0089] The term "pharmaceutically acceptable carrier" refers to an inactive substance used to deliver drugs such as antibodies in a formulation. Pharmaceutically acceptable carriers can be antiadhesives, binders, coatings, disintegrants, fillers, diluents, preservatives, sweeteners, absorption delaying agents, wetting agents, emulsifiers, buffers, etc.

[0090] The term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids, such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy specimens, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.

[0091] The terms "administering" or "administering," as applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.

[0092] The term "effective amount" is an amount sufficient to reduce the severity or frequency of symptoms, or to eliminate these symptoms or potential causes, or to prevent the occurrence of symptoms or their potential causes, or to improve or ameliorate the amount of damage caused by or associated with a disease state. In some embodiments, an effective amount is a therapeutically effective amount or a preventive effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, a state or symptom particularly associated with the disease state, or to otherwise hinder, delay or reverse the disease state or the progress of other undesirable symptoms associated with the disease in any way. A "preventive effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms.

[0093] On the basis of the amino acid sequence of the antibody disclosed in the present disclosure, those skilled in the art can prepare the antibody by genetic engineering technology or other technology (chemical synthesis, recombinant expression), for example, the antibody can be separated and purified from the culture product of the recombinant cell capable of recombinantly expressing the antibody as described in any of the above items, which is easy for those skilled in the art to achieve. Based on this, no matter what technology is used to prepare the antibody of the present disclosure, it belongs to the protection scope of the present disclosure. In order to make the purpose, technical scheme and advantages of the embodiments of the present disclosure clearer, the technical scheme in the embodiments of the present disclosure will be clearly and completely described below. If the specific conditions are not indicated in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques used or considered herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art.This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0094] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0095] Example 1. Preparation of GUCY2C monoclonal antibody

[0096] 1. Anti-human GUCY2C mouse antibody

[0097] For the preparation of anti-human GUCY2C mouse antibodies, please refer to the Chinese patent application (application number: 202211138356.2; invention name: Anti-human guanylate cyclase C antibody, kit and application thereof), the entire text of which is incorporated into the present disclosure by reference.

[0098] GUCY2C ECD-His tag protein (the extracellular segment of human GUCY2C protein is connected to a His tag (6 consecutive histidines) at the C-terminus) was prepared as an immunogen, and Balb / c mice were immunized multiple times. Immunization was performed once a week for a total of 5-7 times. Starting from the fourth immunization, mouse serum was collected weekly to detect the serum titer of anti-human GUCY2C antibodies. The mouse serum was diluted in multiples and incubated with K562 (human chronic myeloid leukemia cells) cell line (K562-hGUCY2C) and K562 cell line overexpressing human GUCY2C, and stained with PE-labeled goat anti-mouse Fc antibody (Biolegend), and the serum titer was detected by flow cytometry. At the same time, the GUCY2C ECD-His tag protein was coated in a 96-well plate, and the serum diluted in multiples was added for incubation, followed by the addition of HRP-labeled goat anti-mouse Fc antibody, and the serum titer was detected by ELISA. According to the results of flow cytometry and ELISA, the mice with the highest titer were determined, the mice were euthanized, spleen cells were collected, and fused with SP20 cell lines. The fused cells were plated into 96-well plates, and after the cells were confluent, the binding of cell supernatants to K562-hGUCY2C was detected by flow cytometry and ELISA. Cells with strong supernatant binding to K562-hGUCY2C were selected for monoclonalization. After the monoclonal cells were confluent, the binding of antibodies in the cell supernatants to K562-hGUCY2C and K562 cell lines was detected by flow cytometry.

[0099] The monoclonal cells in the supernatant with strong binding activity to K562-hGUCY2C and not binding to the K562 cell line were selected for expansion culture. The supernatant was collected one week later and the antibodies in the supernatant were purified. A mouse monoclonal antibody targeting human GUCY2C (clone number 1D1) was obtained, and its variable region sequence is:

[0100] The amino acid sequence of the heavy chain variable region of the mouse monoclonal antibody 1D1 is SEQ ID NO: 1

[0101] EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGINPNNGNTNYNQKFKGKATLTVD KSSSTAYMELRSLTSEDSAVYYCGRSPFVHYYDYYAMDYWGQGTSVTVSS

[0102] The amino acid sequence of the light chain variable region of the mouse monoclonal antibody 1D1 is SEQ ID NO: 2

[0103] DIVLTQSPASLAVSLGQRATISCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFS LNIHPMEEDDTAMYFCQQSKEVPFTFGSGTNLEIK

[0104] 2. Anti-human GUCY2C humanized antibody

[0105] The mouse monoclonal antibody targeting human GUCY2C (clone number 1D1) was humanized and designed to replace the mouse FR with the human FR. After pairing, humanized antibodies R2092, R2093 and R2097 were obtained, and the amino acid sequences of their variable regions are as follows:

[0106] The VH of humanized antibody R2092 is SEQ ID NO: 3

[0107] QVQLVQSGAEVKKPGSSVKVSCKTSGYTFTEYTMHWVRQAPGQSLEWMGGINPNNGNTNYNQKFQGRVTIT ADKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSS;

[0108] The VL of humanized antibody R2092 is SEQ ID NO: 4

[0109] DIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFT LTINPVEADDTANYYCQQSKEVPFTFGQGTKLEIK;

[0110] The VH of humanized antibody R2097 is SEQ ID NO: 5

[0111] QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWMGGINPNNGNTIYNQKFQGRVTMT VDKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSS;

[0112] The VL of humanized antibody R2097 is SEQ ID NO: 6

[0113] DIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFT LTINPVEADDTAMYFCQQSKEVPFTFGQGTKLEIK;

[0114] The VH of humanized antibody R2093 is SEQ ID NO: 7

[0115] QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWMGGINPNNGNTIYNQKFQGRVTMT VDKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSS;

[0116] The VL of humanized antibody R2093 is SEQ ID NO: 8

[0117] DIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFT LTINPVEADDTANYYCQQSKEVPFTFGQGTKLEIK.

[0118] Table 1. CDRs sequences of antibodies

[0119]

[0120] Note: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the antibodies described in Table 1 above are CDRs of 1D1 and its humanized antibodies confirmed according to the IMGT coding system.

[0121] Example 2: Humanized Antibody Binding EC50 Detection (ELISA)

[0122] The binding of humanized antibodies and human GUCY2C (ECD)-His antigen (the C-terminus of the extracellular segment of the human GUCY2C protein connected to a His tag (6 consecutive histidines)) was detected by the ELISA method. First, 96-well plates were coated with 2 μg / mL, 100 μL per well, and incubated at 4°C overnight. The next day, the supernatant was discarded and the plate was washed 3 times. Then, 1% BSA was used to block for 1 hour, and then the plate was washed three times. The humanized antibody was diluted to 30 μg / mL, and then 3-fold gradient dilution was made to 11 wells, added to the 96-well plate, incubated at room temperature for 2 hours, washed 4 times, and then anti-human Fc-HRP was added. Incubated for 30 minutes, washed 5 times, 100 μL of substrate was added to each well for 15 minutes, and the OD450 value was detected on the machine after termination. The graph was plotted and the EC50 value was counted using graphpad software. The experimental results are shown in Figure 1 , the experimental results show that the humanized antibody constructed by the present disclosure can specifically bind to human GUCY2C.

[0123] Example 3: Humanized Antibody Binding EC50 Detection (FCM)

[0124] The binding of anti-GUCY2C humanized antibody to human GUCY2C on the membrane of LS1034 tumor cells (ATCC / CRL-2158) was detected by flow cytometry. The cells were dissociated with cell dissociation buffer (Sigma) and kept on ice.

[0125] After blocking with PBS + 3% BSA, cells were stained with primary antibody for 1 hour on ice, then washed with cold PBS, and 10 μg / mL of secondary antibody (Biolegend, 410708) was added on ice for one hour. Cells were washed again with cold PBS and resuspended in PBS, and GUCY2C binding was analyzed on Cytoflex. The experimental results are shown in Figure 2 ,The experimental results show that the humanized antibody disclosed in the present invention has good binding activity with the GUCY2C-expressing cell line LS1034.

[0126] Example 4: Fortebio detects the affinity of anti-human GUCY2C humanized antibody

[0127] Fortebio was used to detect the binding affinity of the anti-GUCY2CY humanized antibody to the human GUCY2C (ECD)-His antigen (the C-terminus of the extracellular segment of the human GUCY2C protein is connected to a His tag (6 consecutive histidines)). The anti-GUCY2C humanized antibody was diluted to 5 μg / mL in calcium- and magnesium-free phosphate buffered saline (PBS) and captured on an anti-human Fc biosensor (ForteBio). Human GUCY2C (ECD)-His was diluted to 5 μg / mL and KD was tested on the machine. The experimental results are shown in Table 2 and Figure 3 , the affinity of R2092 is 3.10E-11M, the affinity of R2093 is 3.63E-11M, and the affinity of R2097 is 3.91E-11M.

[0128] Table 2. Affinity test results of anti-human GUCY2C humanized antibodies

[0129]

[0130] Example 5: Construction of CAR lentiviral vector of humanized antibody

[0131] Gene synthesize the following structural nucleotide sequence, construct the nucleotide fragment into the lentiviral vector according to the restriction site of the lentiviral vector, design primers, and verify the correctness of the vector construction through sequencing results. The CAR structure of the humanized antibody is shown in Figure 4 ,in:

[0132] Human CD8a hinge-transmembrane region amino acid sequence (SEQ ID NO: 15):

[0133] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0134] 4-1BB intracellular signaling domain amino acid sequence (SEQ ID NO: 16):

[0135] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0136] Human CD3 molecule signaling domain CD3 Zeta amino acid sequence (SEQ ID NO: 17):

[0137] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0138] Human CD8a signal peptide amino acid sequence (SEQ ID NO: 18): MALPVTALLLPLALLLHAARP

[0139] Amino acid sequence of R2092 scFv (SEQ ID NO: 19):

[0140] QVQLVQSGAEVKKPGSSVKVSCKTSGYTFTEYTMHWVRQAPGQSLEWMGGINPNNGNTNYNQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLTINPVEADDTANYYCQQSKEVPFTFGQGTKLEIK

[0141] Amino acid sequence of R2097scFv (SEQ ID NO:20):

[0142] QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWMGGINPNNGNTIYNQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLTINPVEADDTAMYFCQQSKEVPFTFGQGTKLEIK

[0143] Amino acid sequence of R2093scFv (SEQ ID NO:21):

[0144] QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWMGGINPNNGNTIYNQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLTINPVEADDTANYYCQQSKEVPFTFGQGTKLEIK

[0145] Nucleotide sequence of CAR-GUCY2C (R2092) (SEQ ID NO:22):

[0146]

[0147] CAR-GUCY2C (R2097) nucleotide sequence (SEQ ID NO: 23):

[0148]

[0149] CAR-GUCY2C (R2093) nucleotide sequence (SEQ ID NO: 13):

[0150]

[0151] Example 6: Packaging and concentrating lentivirus

[0152] 293t according to 5x10 6 The cells were inoculated at a density of cells / 10 cm culture dish, and the state of the cells was observed the next day. The third-generation lentiviral packaging vector and the target gene vector (the vector of Example 5) were co-transfected into 293T by PEI transfection method. The medium was changed 6 hours after transfection at a density of 15 mL / 150 mm 2 DMEM medium containing 10% fetal bovine serum was added to the culture dish. Virus supernatant was collected 48 hours and 72 hours after transfection, and centrifuged at 2000rpm and 4℃ for 10min to remove cell debris. Impurities were then filtered with a 0.45-micron filter. The filtered virus suspension was concentrated with 25000rpm and 4℃ for 2 hours to concentrate the lentivirus. The concentrated virus was resuspended in an appropriate amount of culture medium and stored at -80℃.

[0153] Example 7: Production of CAR-T and control cells

[0154] 20 mL of blood was drawn, and PBMC was separated by Ficall gradient centrifugation. T cells were separated using the Stemcell T cell negative selection kit (Cat. No. 19051). The separated T cells were resuspended in medium supplemented with 5% human AB serum and 300 units / mL IL-2X-VIVO 15 to 1×10 6 cells / mL, wash the beads with X-VIVO 15 containing 1% FBS, add pre-washed magnetic beads (Cat#40203D, 10mL, Life technology) at a ratio of magnetic beads: T cells = 2:1, and resuspend T cells in fresh culture medium to 3-5×10 cells / mL after 2-3 days. 6 cells / mL, add lentivirus (lentivirus of Example 6) at an MOI of 10, and add 8 μg / mL Polybrene at the same time. After 4-6 hours, add culture medium to dilute the cells to 1×10 6 cells / mL, and fresh culture medium was replaced the next day to maintain the cell concentration at 0.2-0.3×10 6 PBMC / mL, and then the culture medium was replaced every 2-3 days. The cell positive rate was analyzed by flow cytometry 72 hours after virus infection. Figure 5The experimental results showed that the positive rate of CART-R2092 (CAR-GUCY2C (R2092) nucleotide) cells was 88.22%, the positive rate of CART-R2093 (CAR-GUCY2C (R2093) nucleotide) cells was 81.84%, and the positive rate of CART-R2097 (CAR-GUCY2C (R2097) nucleotide) cells was 81.05%.

[0155] Example 8: Evaluation of CAR-T cell killing function in vitro

[0156] The xCELLigence real-time, cell-mediated cytotoxicity system (Acea Biosciences Inc.) was used to assess CAR-T cell-mediated cytotoxicity, with 1.0E+04 T84 cells in 150 μL growth medium in each well of an E-Plate 16 (Acea Biosciences) and cultured overnight in a 37°C incubator, with electrical impedance quantified every 15 minutes using the RTCADP Analyzer system and RTCA software version 2.0 (Acea Biosciences Inc.). Approximately 24 hours later, 50 μL of CAR-T cells (E:T ratio of 5:1) or 50 μL of culture medium was added as a negative, and cell-mediated killing was quantified over the next 24 hours, with electrical impedance read every 15 minutes. The experimental results are shown in the attached Figure 6 The experimental results show that the CAR-T cells prepared by using the CAR constructed with the humanized antibody disclosed in the present invention have a very good growth inhibitory effect on T84 cells.

[0157] The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. An anti-human GUCY2C humanized antibody, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 1, 3, 5 or 7, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 2, 4, 6 or 8; Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system; Optionally, the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID NO:5, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID NO:6; Optionally, HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the antibody include the amino acid sequences shown as SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively, and LCDR1, LCDR2 and LCDR3 of the light chain variable region of the antibody include the amino acid sequences shown as SEQ ID NO:12, amino acid AAS and SEQ ID NO:14, respectively.

2. The anti-human GUCY2C humanized antibody according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 5, 3 or 7, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 6, 4 or 8; Alternatively, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6; or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4; or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; Optionally, the antibody comprises a heavy chain variable region as shown in SEQ ID NO:5 and a light chain variable region as shown in SEQ ID NO:

6.

3. The anti-human GUCY2C humanized antibody according to claim 1 or 2, wherein the antibody comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; the light chain constant region is selected from the κ type or λ type light chain constant region; Optionally, the species origin of the constant region is human; Optionally, the anti-human GUCY2C humanized antibody is a full-length antibody.

4. The anti-human GUCY2C humanized antibody according to any one of claims 1 to 3, wherein the antibody is an antigen-binding fragment selected from any one of scFv, F(ab')2, Fab', Fab, scFab, dsFv and Fv; Optionally, the anti-human GUCY2C humanized antibody is a scFv; Optionally, the scFv has the amino acid sequence shown in SEQ ID NO: 20, 19 or 21.

5. A humanized antibody that competitively binds to human GUCY2C with the antibody of any one of claims 1 to 4, or an anti-human GUCY2C humanized antibody that binds to the same epitope as the antibody of any one of claims 1 to 4.

6. The anti-human GUCY2C humanized antibody according to any one of claims 1 to 5, wherein the antibody has at least one of the following properties: A. can bind to human GUCY2C; optionally, it can bind to human GUCY2C (ECD) -His antigen with an EC50 value of ≤ 1 nM, wherein, The EC50 value was determined by ELISA method; B. capable of binding to human GUCY2C; optionally, it can bind to human GUCY2C (ECD) -His antigen with a KD value of ≤ 0.05 nM, wherein the KD value is determined by the Fortebio method; C. Capable of binding to human GUCY2C; optionally, it can bind to cells expressing human GUCY2C with an EC50 of ≤21 nM, wherein the EC50 value is determined by flow cytometry.

7. A chimeric antigen receptor comprising the anti-human GUCY2C humanized antibody according to any one of claims 1 to 6; Optionally, the anti-human GUCY2C humanized antibody is a scFv; Optionally, the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6; the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; Optionally, the scFv has the amino acid sequence shown in SEQ ID NO: 20, 19 or 21.

8. The chimeric antigen receptor of claim 7, comprising a transmembrane domain; Optionally, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the α / β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154; Optionally, the transmembrane domain is a human CD8a hinge-transmembrane region; Optionally, the human CD8a hinge-transmembrane region has the amino acid sequence shown in SEQ ID NO:

15.

9. The chimeric antigen receptor according to claim 7 or 8, comprising an intracellular signaling domain; Optionally, the intracellular signaling domain is at least one functional signaling domain of a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules, activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, 4-1BB, B7-H3, CDS, ICAM-1, ICOS, GITR, BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D , ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, R ANKL, CD226, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and CD83 ligands; Optionally, the intracellular signaling domain includes a co-stimulatory molecule 4-1BB intracellular signaling domain and / or a human CD3 molecule signaling domain CD3Zeta; Optionally, the 4-1BB intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO:16, and the human CD3 molecule signaling domain CD3Zeta has an amino acid sequence as shown in SEQ ID NO:

17.

10. The chimeric antigen receptor according to any one of claims 7 to 9, comprising a signal peptide; Optionally, the signal peptide is a human CD8a signal peptide; Optionally, the human CD8a signal peptide has an amino acid sequence as shown in SEQ ID NO:

18.

11. The chimeric antigen receptor according to any one of claims 7 to 9, comprising a polypeptide as follows: [anti-human GUCY2C humanized antibody]-[transmembrane domain]-[intracellular signaling domain]; Optionally, A) the anti-human GUCY2C humanized antibody is the anti-human GUCY2C humanized antibody according to any one of claims 1 to 6; optionally, the anti-human GUCY2C humanized antibody is a scFv; optionally, the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; optionally, the scFv has the amino acid sequence shown in SEQ ID NO: 20, 19 or 21; B) the transmembrane domain is a human CD8a hinge-transmembrane region; optionally, the human CD8a hinge-transmembrane region has an amino acid sequence as shown in SEQ ID NO: 15; and / or C) the intracellular signaling domain is the intracellular region of the 4-1BB immune co-stimulatory molecule and / or the intracellular region of the human CD3 molecule CD3Zeta; optionally, the 4-1BB intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 16, and the human CD3 molecule signaling domain CD3Zeta has the amino acid sequence shown in SEQ ID NO:

17.

12. A nucleic acid encoding the anti-human GUCY2C humanized antibody according to any one of claims 1 to 6 or encoding the chimeric antigen receptor according to any one of claims 7 to 11; Optionally, the nucleic acid has the nucleotide sequence shown in SEQ ID NO:23, 22 or 13. A vector comprising the nucleic acid according to claim 12.

14. A cell comprising the nucleic acid of claim 12 or the vector of claim 13; Optionally, the cell is an engineered immune effector cell; Optionally, the immune effector cells are selected from T cells, NK cells, NKT cells, macrophages and CIK cells; Optionally, the immune effector cells are T cells; Optionally, the vector comprises the following nucleotide sequence: [nucleotide sequence encoding a signal peptide]-[nucleotide sequence encoding an anti-human GUCY2C humanized antibody]-[nucleotide sequence encoding a transmembrane domain]-[nucleotide sequence encoding an intracellular signaling domain]; Optionally, A) the nucleotide sequence encoding the signal peptide is a nucleotide sequence encoding a human CD8a signal peptide; optionally, the human CD8a signal peptide has an amino acid sequence as shown in SEQ ID NO: 18; B) the nucleotide sequence encoding the anti-human GUCY2C humanized antibody, the anti-human GUCY2C humanized antibody is a scFv; optionally, the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; optionally, the scFv has the amino acid sequence shown in SEQ ID NO: 20, 19 or 21; C) the nucleotide sequence encoding the transmembrane domain is a nucleotide sequence encoding the hinge-transmembrane region of human CD8a; optionally, the hinge-transmembrane region of human CD8a has an amino acid sequence as shown in SEQ ID NO: 15; D) the nucleotide sequence encoding the intracellular signaling domain, wherein the intracellular signaling domain is the intracellular region of the 4-1BB immune co-stimulatory molecule and / or the intracellular region of the human CD3 molecule CD3Zeta; optionally, the 4-1BB intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 16; the human CD3 molecule signaling domain CD3Zeta has the amino acid sequence shown in SEQ ID NO: 17; Optionally, the vector comprises the nucleotide sequence shown in SEQ ID NO:23, 22 or 13.

15. An engineered immune effector cell, the immune effector cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor is the chimeric antigen receptor described in any one of claims 7 to 11; Optionally, the immune effector cells are selected from T cells, NK cells, NKT cells, macrophages and CIK cells; Optionally, the immune effector cells are T cells; Optionally, the chimeric antigen receptor comprises: [anti-human GUCY2C humanized antibody]-[transmembrane domain]-[intracellular signaling domain]; Optionally, A) The anti-human GUCY2C humanized antibody is a scFv, and optionally, the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4; or the heavy chain variable region of the scFv comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8; optionally, the scFv has the amino acid sequence shown in SEQ ID NO: 20, 19 or 21 B) the transmembrane domain is a human CD8a hinge-transmembrane region; optionally, the human CD8a hinge-transmembrane region has an amino acid sequence as shown in SEQ ID NO: 15; and / or C) the intracellular signaling domain is the intracellular region of the 4-1BB immune co-stimulatory molecule and / or the intracellular region of the human CD3 molecule CD3Zeta; optionally, the 4-1BB intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 16, and the human CD3 molecule signaling domain CD3Zeta has the amino acid sequence shown in SEQ ID NO:

17.

16. A pharmaceutical composition comprising: the anti-human GUCY2C humanized antibody according to any one of claims 1 to 6, the chimeric antigen receptor according to any one of claims 7 to 11, the nucleic acid according to claim 12, the vector according to claim 13, or the cell according to claim 14 or 15; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

17. Use of the anti-human GUCY2C humanized antibody according to any one of claims 1 to 6, the chimeric antigen receptor according to any one of claims 7 to 11, the nucleic acid according to claim 12, the vector according to claim 13, the cell according to claim 14 or 15, or the pharmaceutical composition according to claim 16 in the preparation of a drug for treating or diagnosing a disease related to GUCY2C expression; Optionally, the GUCY2C expression-related disease is a tumor; Optionally, the tumor is selected from at least one of a colorectal tumor, a large intestine tumor, a gastric tumor, an esophageal tumor, a gastroesophageal tumor and a pancreatic tumor.

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