CN119638845A

CN119638845A - Anti-GUCY 2C humanized antibody, chimeric antigen receptor thereof and application thereof

Info

Publication number: CN119638845A
Application number: CN202411274268.4A
Authority: CN
Inventors: 通莉莉; 王先进; 彭亮; 蒋飞; 阎丽君; 余祥
Original assignee: Shenzhen Wenbo Investment Co ltd; Shenzhen Fapon Biological Therapy Co ltd; Guangdong Fapon Biopharma Inc
Current assignee: Shenzhen Wenbo Investment Co ltd; Shenzhen Fapon Biological Therapy Co ltd; Guangdong Fapon Biopharma Inc
Priority date: 2023-09-18
Filing date: 2024-09-10
Publication date: 2025-03-18

Abstract

The present disclosure relates to the field of antibodies, and in particular, discloses anti-GUCY2C humanized antibodies and chimeric antigen receptors and applications thereof. The anti-GUCY2C humanized antibodies disclosed in the present disclosure can specifically bind to human GUCY2C, and the CAR-T constructed therefrom has good in vitro killing function.

Description

Anti-GUCY 2C humanized antibody, chimeric antigen receptor thereof and application thereof

Priority statement

The application claims priority to a chinese patent application with application number 202311208651.5, application day 2023, 9 and 18, entitled "an anti-GUCY 2C humanized antibody and chimeric antigen receptor and use thereof", the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to the field of antibodies, in particular, to anti-GUCY 2C humanized antibodies, chimeric antigen receptors thereof, and uses thereof.

Background

Guanylate cyclase C (guanylyl cyclase C, abbreviation: GUCY 2C) is a transmembrane receptor expressed predominantly on the intestinal glands and on top of villous cells. GUCY2C is expressed in a variety of gastrointestinal malignancies, including more than 95% of large bowel cancers and more than 50% of stomach or gastroesophageal junction cancers, with GUCY2C being highly expressed in most colon cancers at all stages including liver metastases, moderately differentiated and highly differentiated gastrointestinal tumors often being associated with higher membrane expression of GUCY 2C.

GUCY2C is limited and expressed at the top end of intestinal epithelial tight connection of normal tissues to a great extent, and the tumor breaks the normal tight connection structure to expose the GUCY2C more thoroughly, so that compared with the normal tissues, only GUCY2C antibody is combined, and the effect on the normal tissues is avoided.

Chimeric antigen receptor T cell immunotherapy, abbreviated as CAR-T, is a method for treating a patient by modifying T cells of the patient in vitro, so that the T cells of the patient have the capability of recognizing tumor cells, and reinjecting the T cells into the patient for treatment after in vitro expansion culture. Currently, CD 19-targeted CAR-T has achieved tremendous results in the treatment of B-cell hematological tumors. GUCY2C targets have good specificity, and CAR-T cells targeting GUCY2C can be developed to treat colorectal cancer and the like.

Disclosure of Invention

The present disclosure provides 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 comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 1, 3, 5, or 7, and the light chain variable region of the antibody comprises LCDR1, LCDR2, and LCDR3 of 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.

In some embodiments, the anti-human GUCY2C humanized antibody described above, the heavy chain variable region of which comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 1, and the light chain variable region of which comprises LCDR1, LCDR2, and LCDR3 of 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.

In some embodiments, the anti-human GUCY2C humanized antibody described above, the heavy chain variable region of which comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO. 3, and the light chain variable region of which comprises 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.

In some embodiments, the anti-human GUCY2C humanized antibody described above, the heavy chain variable region of which comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 5, and the light chain variable region of which comprises LCDR1, LCDR2, and LCDR3 of 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.

In some embodiments, the anti-human GUCY2C humanized antibody described above, the heavy chain variable region of which comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO. 7, and the light chain variable region of which comprises 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.

In some embodiments, the anti-human GUCY2C humanized antibody of any preceding claim, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the antibody comprise the amino acid sequences set forth 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 comprise the amino acid sequences set forth in SEQ ID NO:12, amino acids AAS, and SEQ ID NO:14, respectively.

In some embodiments, the heavy chain variable region of the anti-human GUCY2C humanized antibody of any one of the preceding claims comprises an amino acid sequence having 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%) identity to SEQ ID No. 5, 3, or 7, and the light chain variable region comprises an amino acid sequence having 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%) identity to SEQ ID No. 6, 4, or 8;

In some embodiments, the anti-human GUCY2C humanized antibody of any preceding claim, 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.

In some embodiments, the anti-human GUCY2C humanized antibody of any preceding claim, said antibody comprising a heavy chain variable region as set forth in SEQ ID NO. 5 and a light chain variable region as set forth in SEQ ID NO. 6.

In some embodiments, the anti-human GUCY2C humanized antibody of any one of the preceding claims, said antibody comprising a constant region;

in some embodiments, the anti-human GUCY2C humanized antibody of any one of the preceding claims, the constant regions comprising a heavy chain constant region and/or a light chain constant region;

In some embodiments, the anti-human GUCY2C humanized antibody of any preceding claim, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD;

In some embodiments, the anti-human GUCY2C humanized antibody of any one of the preceding claims, the constant region is of human species origin.

In some embodiments, the anti-human GUCY2C humanized antibody of any one of the preceding claims, which 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 of any one of the preceding claims is a scFv (single chain antibody). In some embodiments, the scFv has the amino acid sequence shown in SEQ ID NO. 20, 19 or 21.

In some embodiments, the disclosure provides humanized antibodies that competitively bind to human GUCY2C with the anti-human GUCY2C humanized antibodies of any of the preceding claims, or anti-human GUCY2C humanized antibodies that bind to the same epitope as the anti-human GUCY2C humanized antibodies of any of the preceding claims.

In some embodiments, the anti-human GUCY2C humanized antibody of any one of the preceding claims, wherein the antibody has at least one of the following properties:

A. Can bind to human GUCY2C, alternatively, it can bind to human GUCY2C (ECD) -His antigen with an EC50 value of less than or equal to 1nM, wherein the EC50 value is determined by ELISA method;

B. Can bind to human GUCY2C, alternatively, it can bind to human GUCY2C (ECD) -His antigen with a KD value of less than or equal to 0.05nM, wherein said KD value is determined by the Fortebio method;

C. can bind to human GUCY2C, alternatively, it can bind to cells expressing human GUCY2C with an EC50 of 21nM or less, wherein the EC50 value is determined by flow cytometry.

In addition, the present disclosure provides a chimeric antigen receptor comprising an anti-human GUCY2C humanized antibody of any one of the preceding claims;

in some embodiments, the chimeric antigen receptor of any one of the preceding claims, the anti-human GUCY2C humanized antibody is a scFv;

In some embodiments, the chimeric antigen receptor of any one of the preceding claims, 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.

In some embodiments, the chimeric antigen receptor of any of the preceding claims comprises a transmembrane domain, optionally the transmembrane domain is a transmembrane domain selected from the group consisting of the alpha/beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, 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 an amino acid sequence as set forth in SEQ ID NO 15.

In some embodiments, the chimeric antigen receptor of any of the preceding claims comprises an intracellular signaling domain, optionally the intracellular signaling domain is at least one functional signaling domain selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a lymphocyte activation signaling molecule, an activated NK cell receptor, BTLA, a Toll ligand receptor 、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、RANKL、CD226、SLAMF4、CD84、CD96、CEACAM1、CRTAM、CD229、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a, and a CD83 ligand, optionally the intracellular signaling domain comprises 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.

In some embodiments, the chimeric antigen receptor of any one of the preceding claims, 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 set forth in SEQ ID No. 18.

In some embodiments, the chimeric antigen receptor of any one of the preceding claims, comprising a polypeptide of [ anti-human GUCY2C humanized antibody ] - [ transmembrane domain ] - [ intracellular signaling domain ] -, alternatively,

A) The anti-human GUCY2C humanized antibody is the anti-human GUCY2C humanized antibody of any one of claims 1 to 6, optionally the anti-human GUCY2C humanized antibody is an 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;

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 signal domain is a 4-1BB immune co-stimulatory molecule intracellular region and/or a human CD3 molecule intracellular region CD3Zeta, optionally, the 4-1BB intracellular signal domain has an amino acid sequence as shown in SEQ ID NO. 16, and the human CD3 molecule signal domain CD3Zeta has an amino acid sequence as shown in SEQ ID NO. 17.

In addition, the present disclosure provides a nucleic acid encoding the anti-human GUCY2C humanized antibody of any one of the preceding claims or encoding the chimeric antigen receptor of any one of the preceding claims.

In addition, the present disclosure provides a vector comprising the nucleic acid of any one of the preceding claims.

In addition, the present disclosure provides a cell comprising the nucleic acid or vector of any one of the preceding claims, optionally the cell is an engineered immune effector cell, optionally the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage and a CIK cell, optionally the immune effector cell is a T cell, optionally the vector comprises a nucleotide sequence of [ 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 signal domain ],

A) The nucleotide sequence of the coded signal peptide is a nucleotide sequence of coded human CD8a signal peptide, and optionally, the human CD8a signal peptide has an amino acid sequence shown as SEQ ID NO. 18;

b) The nucleotide sequence encoding an anti-human GUCY2C humanized antibody is an 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;

C) The nucleotide sequence of the coding transmembrane domain is a nucleotide sequence of a human CD8a hinge-transmembrane region, and optionally, the human CD8a hinge-transmembrane region has an amino acid sequence shown as SEQ ID NO. 15;

D) The nucleotide sequence of the encoding intracellular signal domain, wherein the intracellular signal domain is a 4-1BB immune co-stimulatory molecule intracellular region and/or a human CD3 molecule intracellular region CD3Zeta, optionally the 4-1BB intracellular signal domain has an amino acid sequence as shown in SEQ ID NO. 16, and the human CD3 molecule signal domain CD3Zeta has an amino acid sequence as shown in SEQ ID NO. 17.

In addition, the present disclosure provides an engineered immune effector cell that expresses a chimeric antigen receptor, wherein the chimeric antigen receptor is a chimeric antigen receptor of any one of the preceding claims, optionally the immune effector cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, and a CIK cell, 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,

A) The anti-human GUCY2C humanized antibody is an 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;

In addition, the present disclosure provides a pharmaceutical composition comprising an anti-human GUCY2C humanized antibody according to any preceding claim, a chimeric antigen receptor according to any preceding claim, a nucleic acid according to any preceding claim, a vector according to any preceding claim or a cell according to any preceding claim, optionally further comprising one or more pharmaceutically acceptable carriers, diluents or excipients.

In addition, the present disclosure provides the use of an anti-human GUCY2C humanized antibody, chimeric antigen receptor, nucleic acid, vector, cell or pharmaceutical composition of any preceding claim in the manufacture of a medicament for treating or diagnosing a disease associated with GUCY2C expression, optionally the disease associated with GUCY2C expression is a tumor, optionally the tumor is selected from at least one of colorectal tumor, large intestine tumor, gastric tumor, esophageal tumor, gastroesophageal tumor and pancreatic tumor.

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 one of the preceding claims for use as a medicament. In some embodiments, the medicament treats or diagnoses a disease associated with the expression of GUCY 2C. Optionally, the GUCY2C expression related disease is tumor, and optionally, the tumor is at least one selected from colorectal tumor, large intestine tumor, stomach tumor, esophageal tumor, gastroesophageal tumor and pancreatic tumor.

In addition, the present disclosure provides a method of diagnosing/treating a disease comprising administering to an individual in need thereof a diagnostically/therapeutically effective amount of an anti-human GUCY2C humanized antibody, chimeric antigen receptor, nucleic acid, vector, cell or pharmaceutical composition of any of the foregoing. Optionally, the GUCY2C expression related disease is tumor, and optionally, the tumor is at least one selected from colorectal tumor, large intestine tumor, stomach tumor, esophageal tumor, gastroesophageal tumor and pancreatic tumor.

In addition, the present disclosure provides a method of detecting or assaying human GUCY2C, the method comprising using an anti-human GUCY2C humanized antibody as described in any one of the preceding claims.

In addition, the present disclosure provides a method of making an anti-human GUCY2C humanized antibody of any one of the preceding claims, comprising culturing a cell of any one of the preceding claims.

The disclosure has the beneficial effect that the anti-human GUCY2C humanized antibody disclosed by the disclosure can specifically bind to human GUCY2C, and 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.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the results of the binding assay (ELISA) of anti-human GUCY2C humanized antibodies to human GUCY 2C;

FIG. 2 is a graph of the results of anti-human GUCY2C humanized antibodies binding to LS1034 detection (FCM);

FIG. 3 is a graph showing the results of affinity detection (Fortebio) of humanized anti-human GUCY2C antibodies;

FIG. 4 is a block diagram of CAR vector construction;

FIG. 5 is a graph of the results of cell positive rate detection of CAR-T cells;

FIG. 6 is a graph of the results of detection of the in vitro killing function of CAR-T.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions in the embodiments of the present disclosure are clearly and completely described below, but the description and the embodiments should not be construed as limiting the scope of the present disclosure. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

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).

The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs or amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids such as hydroxyproline, gamma-carboxyglutamic acid 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 as a naturally occurring amino acid (i.e., an alpha carbon to which hydrogen, carboxyl, amino, and R groups are bound), e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to naturally occurring amino acids.

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

In the present disclosure, the term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, a natural IgG antibody is an iso-tetralin of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains. From N to C-terminal, each heavy chain has one heavy chain variable region (VH, also called variable heavy domain) followed by a heavy chain constant region comprising three constant domains (CH 1, CH2 and CH 3), and from N to C-terminal, each light chain has one light chain variable region (VL, also called variable light domain) followed by one light chain constant region (CL, also called light chain constant domain).

The term "full length antibody" or "whole antibody" refers to an antibody comprising a structure substantially similar to the structure of a natural antibody, or a heavy chain comprising an Fc region.

In the present disclosure, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, which are regions within the antibody variable domains that contribute primarily to specific binding to an antigen. In particular embodiments of the present disclosure, "CDRs" refers to more than 2 highly variable regions in the heavy and light chains of the antibody.

In the present disclosure, the heavy chain complementarity determining region is denoted by HCDR, the 3 CDR regions comprising HCDR1, HCDR2 and HCDR3 in the heavy chain variable region, the light chain complementarity determining region is denoted by LCDR, and the 3 CDR regions comprising LCDR1, LCDR2 and LCDR3 in the light chain variable region. The amino acid sequence boundaries of the CDRs can be determined by various well-known schemes, such as "Kabat" numbering convention (see Kabat et al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR.protein Sequence and Structure Analysis of Antibody Variable Domains [ J ]. 2001), and ImMunoGenTics (IMGT) numbering convention (Lefranc, M.P. et al, dev.Comp.Immunol.,27,55-77 (2003); front Immunol.2018Oct 16; 9:2278), and the like. In some embodiments of the present disclosure, the CDRs are determined according to IMGT encoding rules. In some embodiments, the anti-human GUCY2C humanized antibody of the disclosure has the heavy chain variable regions of HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and the light chain variable regions of the antibody has the LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:12, amino acids AAS, and SEQ ID NO:14, respectively.

In the present disclosure, "framework region" or "FR" region refers to regions of the antibody heavy and light chain variable regions other than CDRs. The heavy chain framework regions can be further subdivided into contiguous regions (FR 1, FR2, FR3, and FR 4) separated by CDRs, wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by HCDR comprising HFR1, HFR2, HFR3, and HFR4 framework regions, and the light chain framework regions can be further subdivided into contiguous regions separated by LCDR comprising LFR1, LFR2, LFR3, and LFR4 framework regions. The heavy chain variable region is obtained by aligning and linking HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with FR (from amino terminus to carboxyl terminus) and the light chain variable region is obtained by aligning and linking LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with FR (from amino terminus to carboxyl terminus). In alternative embodiments, the antibodies of the present disclosure include a heavy chain framework region in a heavy chain variable region as set forth in any one of SEQ ID NOs 1, 3, 5 or 7, and a light chain framework region in a light chain variable region as set forth in any one of SEQ ID NOs 2,4,6 or 8. In alternative embodiments, the antibody has a framework region amino acid sequence that is 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 the framework region described above.

The term "identity" or "sequence identity" refers to the degree to which the amino acids/nucleic acids of two sequences are identical at equivalent positions (i.e., percent) when the two sequences are optimally aligned (with gaps introduced as necessary to obtain the maximum percent sequence identity, and without any conservative substitutions being considered as part of the sequence identity). To determine percent sequence identity, the alignment may be accomplished 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-2 or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains are derived from a particular species and the remainder of the heavy and/or light chains are derived from other species.

The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be accomplished by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterparts (i.e., the framework regions of the heavy chain variable region, the framework regions of the light chain variable region, and the human antibody constant region of a human antibody).

The term "antigen binding fragment" is a portion of an intact antibody that specifically binds to an antigen to which the intact antibody binds. It will be readily appreciated by those skilled in the art from the disclosure herein that antigen binding fragments may be obtained by methods known in the art, for example, enzymatic digestion methods (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds, and may also be obtained by recombinant genetic techniques or by synthesis using an automated peptide synthesizer (e.g., an automated peptide synthesizer of Applied BioSystems).

In alternative embodiments, the antigen binding fragment is any one of F (ab ') ₂, fab', fab, scFab, dsFv, fv, and scFv. "Fab" is a monovalent fragment consisting of the VL, VH, CL and CH1 domains. "Fv" is composed of VH and VL, and "dsFv" is a disulfide stabilized Fv fragment. "Fab '" contains VL, CL and 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 the F (ab ') 2 molecule. "F (ab ') 2" comprises a bivalent fragment of two Fab' fragments linked at the hinge region by a disulfide bond. A "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 sequences in the N-terminal to C-terminal direction a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, C) VH-CL-linker-VL-CH 1 or d) VL-CH 1-linker-VH-CL. An "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 linked by a peptide linker, are capable of being expressed as a single chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified herein, an scFv may have VL and VH variable regions in any order, e.g., the N-to C-terminus of an scFv polypeptide may comprise a) a VL-linker-VH or b) a VH-linker-VL.

The terms "Linker", "connecting peptide", "connecting polypeptide", "Linker" or "Linker" in this disclosure refer to a connecting unit that connects two polypeptide fragments, typically with some flexibility, without loss of function of the original protein domain by the use of a Linker. The linker may be a peptide linker comprising one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. In some embodiments, the linker is a linker selected from (GxS) y, wherein x is selected from an integer from 1 to 5, y is selected from an integer from 0 to 6, in some embodiments, the linker is a linker selected from (GxS) y, wherein x is selected from an integer from 1 to 5, and y is selected from an integer from 1 to 6.

The term "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody. Epitopes can be formed by continuous amino acid strings (linear epitopes) or comprise discontinuous amino acids (conformational epitopes), for example becoming spatially close due to folding of the antigen (i.e. tertiary folding of the antigen by the nature of the protein). Conformational epitopes differ from linear epitopes in that the binding of the antibody to the conformational epitope is lost in the presence of denaturing solvents. Conformational epitopes typically comprise 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 a particular epitope (i.e., those that bind the same epitope) can be performed using methods well 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, among others.

An antibody that binds to the same epitope as the reference antibody or that competes for binding with the reference antibody means an antibody that blocks the binding of the reference antibody to its antigen by 50% or more, or an antibody whose binding to its antigen is blocked by 50% or more by the reference antibody in a competition assay. For example, in order to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to an antigen under saturated conditions, and then the ability of the test antibody to bind to the antigen is evaluated after removing excess reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody if the test antibody is able to bind to the antigen after saturation binding of the reference antibody, and that the test antibody can bind to the same epitope as the reference antibody if the test antibody is unable to bind to the antigen after saturation binding of the reference antibody. To confirm whether the antibodies to be tested bind to the same epitope, they can be detected using routine experimentation (e.g., peptide mutation and binding assays using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if one excess (in 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amounts) of the antibody inhibits antigen binding of the other 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).

The terms "specifically bind", "specifically bind" and "specifically bind" refer to antibodies that bind to an antigen or epitope within the antigen with higher affinity than other antigens or epitopes. Typically, an antibody binds an antigen or epitope within an antigen with an equilibrium dissociation constant (KD) of about 1 x 10 ^-7 M or less (e.g., about 1 x 10 ^-8 M or less, about 1 x 10 ^-9 M or less, about 1 x 10 ^-10 M or less, about 1 x 10 ^-11 M or less, or about 1 x 10 ^-12 M or less). Antibody KD can be measured using conventional procedures, e.g., bySurface plasmon resonance measurement.

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 ligand (e.g., an antigen). As used herein, unless otherwise indicated, binding "affinity" refers to an internal binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art. The term "kassoc" or "ka" refers to the rate of association of a particular antibody-antigen interaction, and the term "kdis" or "kd" refers to the rate of dissociation of a particular antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is obtained from the ratio of KD to ka (i.e., KD/ka) and is expressed as molar concentration (M). The KD values of antibodies can be determined using methods well known in the art. For example, surface plasmon resonance (e.g., biacore) is measured using a biosensing system such as a system, or affinity in solution is measured by Solution Equilibrium Titration (SET).

The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody (either the Fc region of a native sequence or the Fc region of a mutation in an amino acid sequence). 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, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules comprised in the population are identical, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically include a plurality of different antibodies having different amino acid sequences in their variable domains, which are typically specific for different epitopes. "monoclonal" refers to the characteristics of the antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. In some embodiments, the antibodies provided herein are monoclonal antibodies.

The term "antigen" refers to a molecule capable of being bound by an antigen binding protein (e.g., an antibody) selective binding agent. An antigen may have one or more epitopes that are capable of interacting with different antigen binding proteins (e.g., antibodies).

The term "anti-human GUCY2C antibody" or "binding to human GUCY2C antibody" refers to an antibody that is capable of binding human GUCY2C with sufficient affinity. In certain embodiments, antibodies that bind to human GUCY2C have a dissociation constant (KD) of < about 1 μm, < about 100nM, < about 10nM, < about 1nM, < about 0.1nM, < about 0.01nM, or < about 0.001nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-12 M, e.g., 10 ^-9 M to 10 ^-10 M). In certain embodiments, the anti-human GUCY2C antibodies bind to GUCY2C epitopes that are conserved in GUCY2C from different species (e.g., monkey, human).

The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An 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 separate vectors, and such one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues, as described in detail below.

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 are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "vector" is a construct capable of transporting another polynucleotide to which it is linked. Expression vectors are vectors suitable for transformation of host cells and contain nucleic acids that direct or control expression of a heterologous coding region to which they are linked, and may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, RNA splicing of the coding region to which they are linked. In alternative embodiments, the vector is a "plasmid" that is a circular double stranded DNA loop in which additional DNA segments may be ligated. In alternative embodiments, the vector is a viral vector, such as a lentiviral retroviral adenovirus, an adeno-associated viral vector (AAV or AAV 2), wherein additional DNA segments may be ligated into the viral genome. In alternative embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced. In alternative embodiments, the vector may be integrated into the genome of the host cell and replicated together with the host genome after introduction into the host cell.

The term "host cell" is a cell into which exogenous nucleic acid has been introduced or a progeny cell thereof. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages, including mutant progeny that have the same function or biological activity as selected or selected in the original transformed cells. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell line plant cells, and fungal cells. Exemplary host cells are 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 finland (PICHIA FINLANDICA), candida albicans (Candida albicans), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), trichoderma reesei (Trichoderma reesei).

Herein "CAR" is a chimeric antigen receptor, an engineered receptor, e.g., a receptor capable of anchoring a specific molecule (e.g., an antibody) that recognizes a tumor cell surface antigen or a viral antigen to an immune cell (e.g., a T cell), causing the immune cell to recognize the tumor antigen or viral antigen and kill the tumor cell or virus. Typically, a chimeric antigen receptor comprises, in sequence, an antigen binding domain, a transmembrane region, and an intracellular signaling domain. Chimeric antigen receptors of the present disclosure can be constructed using transmembrane regions and intracellular signaling domains known in the art for constructing CARs. In alternative embodiments, the CAR is a chimeric antigen receptor, the major components of which may also include a transmembrane domain and an intracellular domain. In alternative embodiments, the CAR may further comprise a co-stimulatory molecule. "costimulatory molecule" refers to a molecule that is present on the surface of antigen presenting cells and that binds to a costimulatory molecule receptor on Th cells, producing a costimulatory signal. Proliferation of T lymphocytes requires not only antigen binding but also receiving a co-stimulatory molecular signal. The co-stimulatory signal is transmitted to the T cell primarily by binding of co-stimulatory molecules CD80, CD86 expressed on the surface of the antigen presenting cell to the CD28 molecule on the surface of the T cell. B cells receive costimulatory signals through common pathogen components such as LPS, or through complement components, or through activated antigen-specific CD40L on Th cell surfaces. Costimulatory molecules known in the art for constructing CARs may be employed, including but not limited to CD27, CD28, 4-1BB, OX40, ICOS, B7-H3, and/or any combination thereof.

The term "pharmaceutically acceptable carrier" refers to an inactive substance in a formulation used to deliver a drug such as an antibody. The pharmaceutically acceptable carrier may be an anti-adherent, an adhesive, a coating, a disintegrant, a filler, a b diluent, a preservative, a sweetener, an absorption delaying agent, a wetting agent, an emulsifier, a buffer, etc.

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 the body of a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cyst fluid, tears, fecal matter, sputum, mucous secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneal cavity and other body cavity fluids, 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 fluid, and the like, tissue biopsy samples, fine needle punctures, surgically excised tissues, organ cultures, or cell cultures.

The term "administering" or "administering," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

The term "effective amount" is an amount sufficient to reduce the severity or frequency of symptoms, or to eliminate such symptoms or underlying etiology, or to prevent the appearance of symptoms or underlying etiology thereof, or to ameliorate or improve the damage caused by or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or condition, particularly a state or condition associated with the disease state, or otherwise hinder, delay or reverse the progression of the disease state or other undesirable condition associated with the disease in any way. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, 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 related symptoms.

Based on the disclosure of the amino acid sequence of the antibody, a person skilled in the art may use genetic engineering techniques or other techniques (chemical synthesis, recombinant expression) to prepare the antibody, e.g. by separating and purifying the antibody from a culture product of recombinant cells capable of recombinantly expressing the antibody according to any of the above, which is easy to achieve for a person skilled in the art, and on the basis of this, any technique is used to prepare the antibody of the disclosure, which falls within the scope of protection of the disclosure. In order to make the objects, technical solutions and advantages of the embodiments of the present disclosure more apparent, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be 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 ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).

The features and capabilities of the present disclosure are described in further detail below in connection with the examples.

EXAMPLE 1 preparation of GUCY2C monoclonal antibody

1. Anti-human GUCY2C murine antibody

Anti-human GUCY2C murine antibodies can be prepared as described in Chinese patent application (application number: 202211138356.2; title: anti-human guanylate cyclase C antibodies, kit and use thereof), the disclosure of which is incorporated by reference in its entirety.

GUCY2C ECD-His tag protein (the extracellular segment of human GUCY2C protein is C-terminally connected with His tag (6 continuous histidines)) is prepared as immunogen, and Balb/C mice are immunized for multiple times. Immunization was performed once a week for a total of 5-7 times. From the fourth immunization, mouse serum was taken weekly for detection of serum titers of anti-human GUCY2C antibodies. Mice were serum-fold diluted and incubated with K562 (human chronic myelogenous leukemia cell) cell lines (K562-hGUCY C) and K562 cell lines over-expressing human GUCY2C, stained with PE-labeled goat anti-mouse Fc antibody (bioleged), and serum titers detected by flow cytometry. Meanwhile, GUCY2C ECD-His tag protein is coated into a 96-well plate, diluted serum is added for incubation, then HRP-labeled goat anti-mouse Fc antibody is added, and serum titer is detected by ELISA. The mice with the highest titers were determined by flow cytometry and ELISA results, euthanized, splenocytes were collected and fused with the SP20 cell line. And paving the fused cells into a 96-well plate, detecting the binding condition of cell supernatant and K562-hGUCY C by flow cytometry and ELISA after the cells grow fully, selecting cells with strong binding between the supernatant and K562-hGUCY C for monoclonal, and detecting the binding condition of antibodies in the cell supernatant and K562-hGUCY C and K562 cell lines by flow cytometry after the monoclonal cells grow fully.

Monoclonal cells that bind strongly to K562-hGUCY C and that do not bind to the K562 cell line were selected for expanded culture. After one week, the supernatant was collected and the antibodies in the supernatant were purified. A murine monoclonal antibody targeting human GUCY2C (clone No. 1D 1) was obtained, whose variable region sequence:

The heavy chain variable region amino acid sequence of the murine monoclonal antibody 1D1 SEQ ID NO.1

EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGINPNNGNTNYNQKFKGKATLTVD KSSSTAYMELRSLTSEDSAVYYCGRSPFVHYYDYYAMDYWGQGTSVTVSS

The amino acid sequence of the light chain variable region of the murine monoclonal antibody 1D1 SEQ ID NO. 2

DIVLTQSPASLAVSLGQRATISCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFS LNIHPMEEDDTAMYFCQQSKEVPFTFGSGTNLEIK

2. Humanized antibodies against human GUCY2C

The humanized antibodies R2092, R2093 and R2097 are obtained by changing the murine FR into the humanized FR through humanized design transformation of the murine monoclonal antibody (clone number 1D 1) targeting human GUCY2C, and pairing, and the amino acid sequences of the variable regions are as follows:

the VH of the humanized antibody R2092 is SEQ ID NO 3

QVQLVQSGAEVKKPGSSVKVSCKTSGYTFTEYTMHWVRQAPGQSLEWMGGINPNNGNTNYNQKFQGRVTIT ADKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSS;

VL of humanized antibody R2092 is SEQ ID NO. 4

DIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFT LTINPVEADDTANYYCQQSKEVPFTFGQGTKLEIK;

The VH of the humanized antibody R2097 is SEQ ID NO. 5

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWMGGINPNNGNTIYNQKFQGRVTMT VDKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSS;

VL of humanized antibody R2097 is SEQ ID NO. 6

DIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFT LTINPVEADDTAMYFCQQSKEVPFTFGQGTKLEIK;

The VH of the humanized antibody R2093 is SEQ ID NO 7

VL of humanized antibody R2093 is SEQ ID NO. 8

DIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFT LTINPVEADDTANYYCQQSKEVPFTFGQGTKLEIK.

TABLE 1 CDRs sequences of antibodies

Note that HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the antibodies described in table 1 above are CDRs of 1D1 and humanized antibodies thereof as confirmed by the IMGT coding system.

Example two humanized antibody binding EC50 detection (ELISA)

The binding of the humanized antibody to human GUCY2C (ECD) -His antigen (His tag (6 consecutive histidines) attached to the C-terminus of the extracellular portion of human GUCY2C protein) was detected by ELISA method by first coating 96-well plates with 2. Mu.g/mL, 100. Mu.L each empty, 4℃overnight, washing the plates 3 times after the next day, blocking 1 hour with 1% BSA, then washing the plates three times, diluting the humanized antibody to 30. Mu.g/mL, then 3-fold gradient to 11 wells, adding 96-well plates, incubating at room temperature for 2 hours, washing the plates 4 times, then adding anti-human Fc-HRP, incubating for 30 minutes, washing the plates 5 times, adding 100. Mu.L of substrate each empty, reacting for 15min, detecting OD450 values on machine after termination, mapping with graphpad software, and counting EC50 values. The experimental results are shown in fig. 1, and the experimental results show that the humanized antibody constructed by the present disclosure can specifically bind to human GUCY 2C.

Example III humanized antibody binding EC50 detection (FCM)

Binding of anti-GUCY 2C humanized antibodies to human GUCY2C on LS1034 tumor cell (ATCC/CRL-2158) membranes was detected by flow cytometry. Cells were dissociated with cell dissociation buffer (Sigma) and on ice

Blocking with PBS+3% BSA, staining cells with primary antibody for 1 hour on ice, then washing with cold PBS, adding 10. Mu.g/mL secondary antibody (Biolegend, 410708) on ice for one hour, washing cells again with cold PBS and re-suspending in PBS, and analyzing GUCY2C binding on Cytoflex. The experimental results are shown in fig. 2, and the experimental results show that the humanized antibody has good binding activity with the GUCY2C expression cell line LS 1034.

Example IV Fortebio detection of affinity of anti-human GUCY2C humanized antibody

Binding affinity of anti-GUCY 2CY 2 humanized antibodies to human GUCY2C (ECD) -His antigen (the extracellular segment C-terminal end of human GUCY2C protein was linked to His tag (6 consecutive histidines)) was detected using Fortebio, diluted to 5 μg/mL in Phosphate Buffered Saline (PBS) without calcium and magnesium, and captured on an anti-human Fc biosensor (Fortebio), diluted to 5 μg/mL for human GUCY2C (ECD) -His, and performed KD detection on-line. The results are shown in Table 2 below and in FIG. 3, R2092 has an affinity of 3.10E-11M, R2093 has an affinity of 3.63E-11M, and R2097 has an affinity of 3.91E-11M.

TABLE 2 affinity assay results for humanized anti-human GUCY2C antibodies

Example five construction of humanized antibody CAR lentiviral vector

The following structural nucleotide sequences are synthesized, nucleotide fragments are constructed on a lentiviral vector according to the enzyme cutting sites of the lentiviral vector, primers are designed, the correctness of vector construction is verified through sequencing results, and the CAR structure of the humanized antibody is shown in figure 4, wherein:

human CD8a hinge-transmembrane region amino acid sequence (SEQ ID NO: 15):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

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

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

human CD3 molecule signaling domain CD3Zeta amino acid sequence (SEQ ID NO: 17):

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

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

Amino acid sequence of R2092scFv (SEQ ID NO: 19):

QVQLVQSGAEVKKPGSSVKVSCKTSGYTFTEYTMHWVRQAPGQSLEWMGGINPNNGNTNYNQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLTINPVEADDTANYYCQQSKEVPFTFGQGTKLEIK

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

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWMGGINPNNGNTIYNQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLTINPVEADDTAMYFCQQSKEVPFTFGQGTKLEIK

amino acid sequence of R2093scFv (SEQ ID NO: 21):

QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYTMHWVRQAPGKSLEWMGGINPNNGNTIYNQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCGRSPFVHYYDYYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSPGQRATITCRASESVYNSGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTLTINPVEADDTANYYCQQSKEVPFTFGQGTKLEIK

CAR-GUCY2C (R2092) nucleotide sequence (SEQ ID NO: 22):

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAAGTGCAACTGGTGCAGTCAGGAGCCGAGGTGAAGAAGCCCGGATCATCCGTGAAGGTGTCTTGTAAGACCTCTGGCTACACTTTTACCGAATACACTATGCACTGGGTGCGCCAGGCTCCCGGACAGTCCCTCGAATGGATGGGCGGGATTAACCCTAATAACGGGAATACAAATTACAATCAGAAATTCCAGGGTAGGGTTACCATAACAGCTGATAAGTCCACTAGTACAGCCTACATGGAGCTCTCCAGCCTGCGCTCTGAGGATACTGCCGTGTACTACTGCGGCAGGAGCCCATTTGTTCACTACTACGATTACTATGCAATGGACTATTGGGGGCAAGGTACTCTCGTGACCGTGAGCTCTGGTGGAGGCGGTAGCGGAGGAGGCGGCTCCGGTGGCGGCGGTTCTGACATCGTCCTCACCCAGTCACCAGCTTCACTGGCTGTGTCACCAGGGCAAAGGGCAACCATCACTTGTAGAGCTAGTGAGAGTGTCTACAACAGTGGGATCTCCTTCATGAACTGGTTCCAACAGAAGCCTGGTCAGCCTCCCAAACTCCTGATATACGCCGCCTCTAACAAGGGCACCGGCGTCCCTGCTCGCTTTTCTGGTTCCGGGAGTGGCACTGATTTCACACTGACAATAAACCCAGTTGAGGCTGATGACACTGCCAACTATTACTGCCAGCAGAGTAAGGAGGTCCCATTCACATTTGGCCAGGGTACAAAGCTGGAGATAAAGACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAA

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

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAGCTGGTCCAAAGTGGAGCAGAAGTTAAGAAACCCGGCGCTTCTGTCAAAGTGTCTTGCAAAACTTCAGGCTATACATTCACTGAGTACACAATGCACTGGGTGCGCCAAGCACCAGGCAAAAGTCTGGAATGGATGGGTGGCATTAACCCTAATAACGGCAACACAATCTATAATCAGAAGTTTCAGGGACGCGTGACCATGACAGTGGACAAATCTACATCCACAGCCTACATGGAACTGTCCTCCCTCAGATCCGAGGATACAGCCGTCTACTATTGCGGTAGAAGCCCTTTTGTGCATTACTATGACTACTATGCCATGGACTACTGGGGTCAAGGCACACTGGTGACCGTGAGTAGCGGAGGAGGAGGATCTGGAGGAGGAGGCTCTGGAGGTGGTGGATCAGACATTGTCCTGACCCAGAGTCCTGCCTCACTCGCCGTCAGCCCAGGACAGAGGGCAACAATCACCTGTCGGGCCAGTGAGAGCGTGTACAATAGCGGAATCTCCTTCATGAATTGGTTTCAGCAGAAACCAGGACAACCTCCTAAACTGCTGATCTACGCCGCCTCCAACAAAGGCACTGGAGTGCCAGCAAGGTTCAGCGGATCAGGCTCCGGCACTGATTTCACACTGACCATTAATCCAGTCGAGGCCGATGACACTGCAATGTACTTCTGCCAGCAGAGCAAGGAAGTTCCATTCACATTCGGCCAGGGCACCAAACTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGC CGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAA

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

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAGCTGGTCCAAAGTGGAGCAGAAGTTAAGAAACCCGGCGCTTCTGTCAAAGTGTCTTGCAAAACTTCAGGCTATACATTCACTGAGTACACAATGCACTGGGTGCGCCAAGCACCAGGCAAAAGTCTGGAATGGATGGGTGGCATTAACCCTAATAACGGCAACACAATCTATAATCAGAAGTTTCAGGGACGCGTGACCATGACAGTGGACAAATCTACATCCACAGCCTACATGGAACTGTCCTCCCTCAGATCCGAGGATACAGCCGTCTACTATTGCGGTAGAAGCCCTTTTGTGCATTACTATGACTACTATGCCATGGACTACTGGGGTCAAGGCACACTGGTGACCGTGAGTAGCGGAGGAGGAGGATCTGGAGGAGGAGGCTCTGGAGGTGGTGGATCAGACATTGTCCTGACCCAGAGTCCTGCCTCACTCGCCGTCAGCCCAGGACAGAGGGCAACAATCACCTGTCGGGCCAGTGAGAGCGTGTACAATAGCGGAATCTCCTTCATGAATTGGTTTCAGCAGAAACCAGGACAACCTCCTAAACTGCTGATCTACGCCGCCTCCAACAAAGGCACTGGAGTGCCAGCAAGGTTCAGCGGATCAGGCTCCGGCACTGATTTCACACTGACCATTAATCCAGTCGAGGCCGATGACACTGCAAATTACTACTGCCAGCAGAGCAAGGAAGTTCCATTCACATTCGGCCAGGGCACCAAACTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAA

EXAMPLE six packaging and concentrating lentiviruses

293T was inoculated at a density of 5X10 ⁶ cells/10cm dish, the state of cells was observed the next day, 3-generation lentiviral packaging vector and the objective gene vector (vector of example 5) were co-transferred to 293t by PEI transfection method, the medium was changed after 6 hours of transfection, DMEM medium containing 10% fetal bovine serum was added to 15mL/150mm ² dish, virus supernatant was collected 48 hours and 72 hours after transfection, centrifugation was performed at 2000rpm for 10min, cell debris was removed, then 0.45 μm filter was used to filter impurities, the filtered virus suspension was concentrated lentivirus at 25000rpm for 4 ℃ for 2 hours, the concentrated virus was resuspended in an appropriate amount of medium, and stored at-80 ℃.

Example seven production of CAR-T and control cells

PBMC were isolated by 20mL of blood, ficall gradient centrifugation, T cells were isolated using Stemcell company T cell negative selection kit (cat# 19051), T cells were resuspended to 1X 10 ⁶ cells/mL using medium supplemented with 5% human AB serum and 300 units/mL IL-2X-VIVO 15, beads were washed with 1% FBS X-VIVO 15, pre-washed beads (Cat# 40203D,10mL,Life technology) were added at a ratio of magnetic beads to T cells=2:1, T cells were resuspended to 3-5X 10 ⁶ cells/mL using fresh medium after 2-3days, lentiviral (lentiviral of example 6) was added at a value of MOI=10, polybrene was added at the same time after 8. Mu.g/mL, 4-6hours were added, the cells were diluted to 1X 10 ⁶ cells/mL, fresh medium was daily maintained at a concentration of 0.2-0.3X ⁶/mL, and the virus infection rate was analyzed for 72 days after 2-3days of positive cell infection. The experimental results are shown in fig. 5, and the experimental results show that the cell positive rate of CART-R2092 (CAR-GUCY 2C (R2092) nucleotide) is 88.22%, the cell positive rate of CART-R2093 (CAR-GUCY 2C (R2093) nucleotide) is 81.84%, and the cell positive rate of CART-R2097 (CAR-GUCY 2C (R2097) nucleotide) is 81.05%.

Example eight CAR-T in vitro killing function assessment

Xcelligent real-time, cell-mediated cytotoxicity system (ace Biosciences inc.) was used to assess CAR-T cell-mediated cytotoxicity, 1.0e+04T 84 cells were grown in 150 μl of growth medium in each well of E-Plate 16 (ace Biosciences) and incubated overnight in a 37 ℃ incubator, and impedance and RTCA software version 2.0 (ace Biosciences inc.) were quantified every 15 minutes using the RTCADP Analyzer system. After about 24 hours, 50. Mu.L of CAR-T cells (E: T ratio 5:1) or 50. Mu.L of medium were added as negative and cell-mediated killing was quantified over the next 24 hours, with electrical impedance read every 15 minutes. The experimental result is shown in figure 6, and the experimental result shows that the CAR-T cell prepared by adopting the CAR constructed by the humanized antibody disclosed by the disclosure has a very good effect of inhibiting the growth of T84 cells.

The foregoing description of the preferred embodiments of the present disclosure is provided only and not intended to limit the disclosure so that various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure should 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 comprises HCDR1, HCDR2 and HCDR3 of SEQ ID No. 1, 3,5 or 7, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 of SEQ ID No. 2,4, 6 or 8;

Optionally, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT numbering system, or by a Kabat numbering system, or by a Chothia numbering system, or by a Contact numbering system, or by an AbM numbering system;

Alternatively, the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 of SEQ ID No. 5, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 of SEQ ID No. 6;

Alternatively, HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the antibody comprise 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 comprise 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 of claim 1, the heavy chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID No. 5, 3 or 7, and the light chain variable region comprising an amino acid sequence having at least 80% identity 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;

alternatively, 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 of claim 1 or 2, comprising constant regions;

Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;

Alternatively, the heavy chain constant region is selected from the group consisting of a heavy chain constant region of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD;

Optionally, the constant region is of human species origin;

alternatively, the anti-human GUCY2C humanized antibody is a full-length antibody.

4. The humanized anti-human GUCY2C antibody of any one of claims 1-3, which is an antigen binding fragment selected from any one of scFv, F (ab ') ₂, fab', fab, scFab, dsFv, and Fv;

alternatively, the anti-human GUCY2C humanized antibody is scFv;

alternatively, the scFv has the amino acid sequence shown in SEQ ID NO. 20, 19 or 21.

5. A humanized antibody that competes for binding 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 of any one of claims 1-5, wherein the antibody has at least one of the following properties:

7. A chimeric antigen receptor comprising the anti-human GUCY2C humanized antibody of any one of claims 1 to 6;

alternatively, the anti-human GUCY2C humanized antibody is an scFv;

Alternatively, 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;

8. The chimeric antigen receptor of claim 7, comprising a transmembrane domain;

Alternatively, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha/beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, 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;

alternatively, the human CD8a hinge-transmembrane region has the amino acid sequence shown in SEQ ID NO. 15.

9. The chimeric antigen receptor of claim 7 or 8, comprising an intracellular signaling domain;

Alternatively, 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 receptor 、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、RANKL、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 comprises a co-stimulatory molecule 4-1BB intracellular signaling domain and/or a human CD3 molecule signaling domain CD3Zeta;

Alternatively, the 4-1BB intracellular signaling domain has the amino acid sequence shown as SEQ ID NO. 16, and the human CD3 molecule signaling domain CD3Zeta has the amino acid sequence shown as SEQ ID NO. 17.

10. The chimeric antigen receptor according to any one of claims 7 to 9, comprising a signal peptide;

alternatively, the signal peptide is a human CD8a signal peptide;

alternatively, the human CD8a signal peptide has the amino acid sequence shown as SEQ ID NO. 18.

11. The chimeric antigen receptor according to any one of claims 7 to 9, comprising a polypeptide of [ anti-human GUCY2C humanized antibody ] - [ transmembrane domain ] - [ intracellular signaling domain ];

alternatively, the process may be carried out in a single-stage,

A) The anti-human GUCY2C humanized antibody is the anti-human GUCY2C humanized antibody of any one of claims 1 to 6, optionally the anti-human GUCY2C humanized antibody is an 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 by SEQ ID NO:20, 19 or 21;

12. A nucleic acid encoding the anti-human GUCY2C humanized antibody of any one of claims 1 to 6 or encoding the chimeric antigen receptor of any one of claims 7 to 11;

Alternatively, the nucleic acid has the nucleotide sequence shown as SEQ ID NO. 23, 22 or 13.

13. A vector comprising the nucleic acid of 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;

alternatively, the immune effector cell is selected from the group consisting of T cells, NK cells, NKT cells, macrophages, and CIK cells;

Optionally, the immune effector cell is a T cell;

Optionally, the vector comprises the nucleotide sequence of [ 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 signal domain ];

alternatively, the process may be carried out in a single-stage,

B) The nucleotide sequence encoding an anti-human GUCY2C humanized antibody is an 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 as SEQ ID NO. 20, 19 or 21;

d) The nucleotide sequence of the encoding intracellular signal domain, wherein the intracellular signal domain is a 4-1BB immune co-stimulatory molecule intracellular region and/or a human CD3 molecule intracellular region CD3Zeta, optionally the 4-1BB intracellular signal transduction domain has an amino acid sequence as shown in SEQ ID NO. 16, the human CD3 molecule signal transduction domain CD3Zeta has an amino acid sequence as shown in SEQ ID NO. 17;

Alternatively, the vector comprises the nucleotide sequence shown as SEQ ID NO. 23, 22 or 13.

15. An engineered immune effector cell that expresses a chimeric antigen receptor, wherein the chimeric antigen receptor is the chimeric antigen receptor of any one of claims 7 to 11;

Optionally, the immune effector cell is a T cell;

Alternatively, the chimeric antigen receptor comprises [ anti-human GUCY2C humanized antibody ] - [ transmembrane domain ] - [ intracellular signaling domain ];

alternatively, the process may be carried out in a single-stage,

A) The humanized anti-human GUCY2C antibody is an 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

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

17. Use of an anti-human GUCY2C humanized antibody according to any one of claims 1 to 6, a chimeric antigen receptor according to any one of claims 7 to 11, a nucleic acid according to claim 12, a vector according to claim 13, a cell according to claim 14 or 15, or a pharmaceutical composition according to claim 16 for the preparation of a medicament for the treatment or diagnosis of a disease associated with GUCY2C expression;

alternatively, the disease associated with GUCY2C expression is a tumor;

Optionally, the tumor is selected from at least one of colorectal tumor, large intestine tumor, stomach tumor, esophageal tumor, gastroesophageal tumor, and pancreatic tumor.