GUCY2C polypeptide and application thereof

Abstract

The invention relates to the technical field of biology, and discloses a GUCY2C polypeptide and application thereof. The GUCY2C polypeptide disclosed by the invention can be used for preparing an antibody.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and specifically, to a GUCY2C polypeptide and its applications. Background Art

[0002] Guanylyl cyclase C (GUCY2C or GCC for short) belongs to the receptor guanylyl cyclase family. The human guanylyl cyclase C protein mainly consists of five parts: ① the extracellular N-terminal receptor-binding region, which contains 40% of its own protein and can bind specific ligands; ② the hydrophobic transmembrane region, which transmits extracellular information into the cell; ③ the cytoplasmic region, that is, the kinase homology region, which transfers signals from the ligand-bound receptor region to the catalytic region; ④ the catalytic region; ⑤ the carboxyl terminus. The endogenous ligand of GUCY2C is uroguanylin, and when GUCY2C is activated by the ligand, it can initiate a series of downstream signal transmissions, regulate the homeostasis of body fluids and electrolytes, and maintain the integrity of the intestinal barrier.

[0003] In normal human body tissues, GUCY2C is mainly expressed in intestinal epithelial cells. In recent years, studies have found that GUCY2C is stably expressed in primary colorectal cancer cells, while in metastatic colorectal cancer cells, GUCY2C is abnormally highly expressed and is considered a specific marker molecule for metastatic colorectal cancer. Other data also show that GUCY2C is expressed in pancreatic cancer, gastric cancer, and esophageal cancer, and GUCY2C has become a new tumor treatment target. Summary of the Invention

[0004] The present disclosure constructs a new GUCY2C polypeptide.

[0005] In some embodiments, the present disclosure provides a polypeptide, which includes a first domain, a second domain, and a linker, wherein the first domain and the second domain are human GUCY2C polypeptide fragments; the N-terminus of the linker is connected to the C-terminus of the first domain, and the C-terminus of the linker is connected to the N-terminus of the second domain.

[0006] In some embodiments, for the polypeptide as described in any one of the above, the first domain includes the amino acid residues at positions 24 to 161 of SEQ ID NO.1, and the second domain includes the amino acid residues at positions 300 to 376 of SEQ ID NO.1; or the first domain includes the amino acid residues at positions 162 to 299 of SEQ ID NO.1, and the second domain includes the amino acid residues at positions 377 to 430 of SEQ ID NO.1.

[0007] In some embodiments, for the polypeptide as described in any one of the above, wherein,

[0008] A. The amino acid sequence of the first domain has 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%, 99% or 100%) sequence identity with SEQ ID NO.2, and / or the amino acid sequence of the second domain has 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%, 99% or 100%) sequence identity with SEQ ID NO.3; or

[0009] B. The amino acid sequence of the first domain has 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%, 99% or 100%) sequence identity with SEQ ID NO.4, and / or the amino acid sequence of the second domain has 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%, 99% or 100%) sequence identity with SEQ ID NO.5.

[0010] In some embodiments, the polypeptide as described in any one of the above, wherein,

[0011] a. The amino acid sequence of the first domain is as shown in SEQ ID NO.2, and the amino acid sequence of the second domain is as shown in SEQ ID NO.3; or

[0012] b. The amino acid sequence of the first domain is as shown in SEQ ID NO.4, and the amino acid sequence of the second domain is as shown in SEQ ID NO.5.

[0013] In some embodiments, the polypeptide as described in any one of the above, wherein the linker is a flexible peptide linker;

[0014] In some embodiments, the polypeptide as described in any one of the above, wherein the linker is selected from (G x S y ) Z linkers, wherein x and y are independently selected from integers of 0 - 5, and y is selected from integers of 1 - 6;

[0015] In some embodiments, the polypeptide as described in any one of the above, wherein the linker is the linker shown in SEQ ID NO: 6 or 7.

[0016] In some embodiments, the polypeptide as described in any one of the above, wherein the polypeptide is represented by the following formula (I):

[0017] (I): X-L1-Y,

[0018] wherein X is a first GUCY2C polypeptide fragment, Y is a second GUCY2C polypeptide fragment, and L1 is a linker;

[0019] In some embodiments, the polypeptide as described in any one of the above, wherein,

[0020] A. The amino acid sequence of the first GUCY2C polypeptide fragment has 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%, 99% or 100%) sequence identity with SEQ ID NO.2, and / or the amino acid sequence of the second GUCY2C polypeptide fragment has 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%, 99% or 100%) sequence identity with SEQ ID NO.3; or

[0021] B. The amino acid sequence of the first GUCY2C polypeptide fragment has 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%, 99% or 100%) sequence identity with SEQ ID NO.4, and / or the amino acid sequence of the second GUCY2C polypeptide fragment has 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%, 99% or 100%) sequence identity with SEQ ID NO.5;

[0022] In some embodiments, for the polypeptide as described in any of the above, the first GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.2, and the second GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.3; or the first GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.4, and the second GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.5;

[0023] In some embodiments, for the polypeptide as described in any of the above, the linker is a flexible peptide linker;

[0024] In some embodiments, for the polypeptide as described in any of the above, wherein the linker is selected from (G x S y ) Z linkers, wherein x and y are independently selected from integers of 0-5, and y is selected from integers of 1-6;

[0025] In some embodiments, for the polypeptide as described in any of the above, the linker is the linker as shown in SEQ ID NO:6 or 7.

[0026] In some embodiments, the present disclosure provides a polypeptide having an amino acid sequence with 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%, 99% or 100%) sequence identity to SEQ ID NO.8 or 9;

[0027] In some embodiments, for the polypeptide as described in any of the above, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.8 or SEQ ID NO.9.

[0028] In some embodiments, for the polypeptide as described in any of the above, wherein the N-terminus of the polypeptide comprises a signal peptide;

[0029] In some embodiments, for the polypeptide as described in any of the above, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.10.

[0030] In some embodiments, for the polypeptide as described in any of the above, wherein the C-terminus of the polypeptide comprises a protein tag;

[0031] In some embodiments, for the polypeptide as described in any of the above, the amino acid sequence of the protein tag is as shown in SEQ ID NO.11;

[0032] In some embodiments, for the polypeptide as described in any one of the above, the amino acid sequence of the polypeptide has 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%, 99% or 100%) sequence identity with SEQ ID NO.12 or 13;

[0033] In some embodiments, for the polypeptide as described in any one of the above, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.12 or SEQ ID NO.13.

[0034] In some embodiments, the application of the polypeptide as described in any one of the above in the preparation of an anti-human GUCY2C antibody;

[0035] In some embodiments, the application of the polypeptide as described in any one of the above as an immunogen for the preparation of an anti-human GUCY2C antibody;

[0036] In some embodiments, the polypeptide as described in any one of the above is used to screen for an anti-human GUCY2C antibody, and the anti-human GUCY2C antibody specifically binds to the polypeptide described in any one of the preceding items;

[0037] In some embodiments, for the polypeptide as described in any one of the above, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.8 or SEQ ID NO.12 is stronger than the binding activity to the polypeptide shown in SEQ ID NO.9 or SEQ ID NO.13. In some embodiments, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.8 is more than 1-fold (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20 or a greater multiple) of the binding activity to the polypeptide shown in SEQ ID NO.9; In some embodiments, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.8 is more than 9-fold of the binding activity to the polypeptide shown in SEQ ID NO.9; In some embodiments, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.8 or 9 is detected by the ELISA method.

[0038] In some embodiments, for the polypeptide as described in any one of the above, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.9 or SEQ ID NO.13 is stronger than that to the polypeptide shown in SEQ ID NO.8 or SEQ ID NO.12. In some embodiments, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.9 is more than 1-fold (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20 or greater fold) of that to the polypeptide shown in SEQ ID NO.8; in some embodiments, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.9 is more than 9-fold of that to the polypeptide shown in SEQ ID NO.8; in some embodiments, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.8 or 9 is detected by ELISA method.

[0039] In some embodiments, the present disclosure provides a method for screening an anti-human GUCY2C antibody, the method comprising the step of detecting the binding activity of the anti-human GUCY2C antibody to the polypeptide as described in any one of the above.

[0040] In some embodiments, for the method for screening an anti-human GUCY2C antibody as described in any one of the above, the screening method comprises the steps:

[0041] A) Prepare the anti-human GUCY2C antibody to be tested;

[0042] B) Detect the binding activity of the anti-human GUCY2C antibody obtained in step A) to the polypeptide shown in SEQ ID NO.8 respectively;

[0043] C) Detect the binding activity of the anti-human GUCY2C antibody obtained in step A) to the polypeptide shown in SEQ ID NO.9 respectively;

[0044] D) Compare the binding activities of the anti-human GUCY2C antibodies in steps B) and C) to the polypeptide, and select the anti-human GUCY2C antibody with stronger binding activity to the polypeptide shown in SEQ ID NO.8 than to the polypeptide shown in SEQ ID NO.9; in some embodiments, select the anti-human GUCY2C antibody with binding activity to the polypeptide shown in SEQ ID NO.8 being more than 1-fold (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20 or greater fold) of that to the polypeptide shown in SEQ ID NO.9; in some embodiments, select the anti-human GUCY2C antibody with binding activity to the polypeptide shown in SEQ ID NO.8 being more than 9-fold of that to the polypeptide shown in SEQ ID NO.9.

[0045] In some embodiments, for the method for screening anti-human GUCY2C antibodies as described in any one of the above, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.8 or 9 is detected by an ELISA method. In some embodiments, the method for detecting the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.8 or 9 is as described in Example 3 of the present disclosure. In some embodiments, the binding activity of the anti-human GUCY2C antibody to the polypeptides shown in SEQ ID NO.8 and 9 is determined by comparing their OD450 readings.

[0046] In some embodiments, the present disclosure provides an anti-human GUCY2C antibody that binds to the polypeptide as described in any one of the above;

[0047] In some embodiments, for the antibody as described in any one of the above, the affinity of the anti-human GUCY2C antibody for the polypeptide shown in SEQ ID NO.8 is higher than its affinity for the polypeptide shown in SEQ ID NO.9; in some embodiments, the affinity of the anti-human GUCY2C antibody for the polypeptide shown in SEQ ID NO.8 is at least 50% higher than its affinity for the polypeptide shown in SEQ ID NO.9 (for example, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 500%, 1000%, 10000% or more higher). In some embodiments, the anti-human GUCY2C antibody is not R0591, R0826, F021-3, F021-14, F021-15, F021-18, F021-36, F021-43, and F021-70;

[0048] In some embodiments, the present disclosure provides an isolated nucleic acid encoding the polypeptide or anti-human GUCY2C antibody as described in any one of the above.

[0049] In some embodiments, the present disclosure provides a host cell containing the nucleic acid as described in any one of the above.

[0050] In some embodiments, the present disclosure provides a polypeptide for detecting an anti-human GUCY2C antibody, and the polypeptide is the polypeptide as described in any one of the above; in some embodiments, the polypeptide is the polypeptide shown in SEQ ID NO.8 or SEQ ID NO.12.

[0051] In some embodiments, the present disclosure provides a kit comprising the polypeptide as described in any one of the above; in some embodiments, the polypeptide is the polypeptide shown in SEQ ID NO.8 or SEQ ID NO.12.

[0052] The present disclosure also provides a method for detecting or assaying anti-human GUCY2C antibodies, the method comprising the step of detecting or assaying anti-human GUCY2C antibodies using the polypeptide described in any one of the preceding items.

[0053] The present disclosure also provides a method for preparing the polypeptide or anti-human GUCY2C antibody described in any one of the preceding items, which comprises culturing the cells described in any one of the preceding items, and then separating and purifying to obtain the polypeptide or anti-human GUCY2C antibody.

[0054] The present disclosure newly constructs a GUCY2C polypeptide; in some embodiments, the polypeptide can be used for immunization, antibody screening or antibody function analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0056] Figure 1 It is a 3D structure diagram of the GUCY2C antigen polypeptide; wherein Figure 1 A is the 3D structure of the GUCY2C antigen protein predicted by AlphaFold, Figure 1 B is the structure of the S24-M161-GGGG-S300-D376 protein predicted by AlphaFold (see the yellow structure in Attachment Figure 1 B), Figure 1 C is the protein structure of S162-L299-GGGGG-S377-Q430 predicted by AlphaFold (see the purple structure in Attachment Figure 1 C);

[0057] Figure 2 It is the SDS-Page electrophoresis result of the GUCY2C polypeptide;

[0058] Figure 3 It is the reverse column chromatography result diagram of the N-terminal domain (R2230);

[0059] Figure 4 It is the reverse column chromatography result diagram of the C-terminal domain (R2231);

[0060] Figure 5 It is a schematic diagram of the CAR structure constructed with anti-human GUCY2C antibodies;

[0061] Figure 6 It is a flow cytometry data diagram for detecting the CAR positive rate of CAR-T cells;

[0062] Figure 7 It is for the evaluation of the in vitro anti-tumor activity of CAR-T cells. Specific implementation manners

[0063] To make the objectives, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. However, the description and embodiments should not be construed as limiting the scope of the present disclosure. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase. Unless clearly defined in the present disclosure, the technical terms and scientific terms used in the present disclosure have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure pertains.

[0064] In the present disclosure, "a / an", "one", "the", "above-mentioned", "aforementioned" include plural references, unless the context clearly indicates otherwise. For example, "an antibody" means one antibody or more than one antibody. Additionally, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. The meaning of the term "plural" is at least two, such as 2, 3, etc., unless otherwise clearly and specifically stated otherwise.

[0065] Unless the context clearly indicates otherwise, in the description and claims of the present disclosure, words such as "comprise" or "have" should be understood in the sense of "include", rather than in an exclusive or exhaustive sense; that is, in the sense of "including but not limited to". For example, "comprising A" means "including A, but not limited to A".

[0066] Unless the context clearly indicates otherwise, in the description and claims of the present disclosure, "optional", "optionally", "alternative" or "alternatively" means that the subsequent described event or circumstance may but does not necessarily occur, that is, including the cases where the event or circumstance occurs or does not occur.

[0067] "About" means within an acceptable error range of a specific value as determined by those of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular measurement, result or embodiment, unless otherwise clearly stated elsewhere in the embodiment or the description, "about" means within one standard deviation or up to a range of 5% according to the convention in the art.

[0068] The term "and / or" means each or a combination of two specific features, including or not including the other specific feature. For example, "A and / or B" means any of the following cases: A and B, A or B, A (alone) and B (alone); "A, B and / or C" means any of the following cases: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0069] The three-letter and one-letter codes of amino acids used in the present disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0070] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids are, for example: 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 having the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Amino acid analogs usually have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds having a structure different from the general chemical structure of amino acids, but acting in a manner similar to naturally occurring amino acids.

[0071] The term "antibody" in the present disclosure is used in the broadest sense and encompasses various antibody structures, including but not limited to: monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetra-specific antibodies), murine antibodies, chimeric antibodies, humanized antibodies, human antibodies, full-length antibodies, antigen-binding fragments (also referred to as antigen-binding portions, or antibody fragments), etc., as long as they exhibit the desired antigen-binding activity.

[0072] "Natural antibody" refers to immunoglobulin molecules that exist naturally. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus of an IgG antibody, each heavy chain has a heavy chain variable region (VH, also known as variable heavy domain or heavy chain variable domain), followed by a heavy chain constant region (CH, also known as constant heavy domain), and the heavy chain constant region includes three constant domains (CH1, CH2, and CH3); similarly, from the N-terminus to the C-terminus, each light chain has a light chain variable region (VL, also known as variable light domain or light chain variable domain), followed by a light chain constant region (CL, also known as constant light domain). Antibodies can be classified into five isotypes, IgA, IgD, IgE, IgG, and IgM, according to whether they contain heavy chains of α, δ, ε, γ, and μ, and isotype antibodies can be further divided into different subclasses. For example, the IgG isotype includes four subtypes, IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), and IgG4 (γ4 heavy chain), and the IgA isotype is divided into two subtypes, IgA1 (α1 heavy chain) and IgA2 (α2 heavy chain), etc.

[0073] The terms "full-length antibody", "intact antibody", and "whole antibody" in the present disclosure are used interchangeably and refer to an antibody having a structure substantially similar to that of a natural antibody, or an antibody containing the heavy chain of the Fc region.

[0074] An "isolated" antibody is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody can be purified to a purity greater than 90% or 99%, and can be purified and assayed by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0075] The term "variable region" or "variable domain" refers to the domain in the heavy or light chain of an antibody that is involved in antibody binding to an antigen. The heavy chain variable region (VH) and light chain variable region (VL) of a natural antibody, each variable region contains 4 conserved framework regions (FRs) and 3 hypervariable regions (also known as complementarity determining regions, HVRs or CDRs).

[0076] The terms "complementary determining region", "hypervariable region" or "CDR" refer to the major regions within the variable domain that contribute to antigen binding; "framework" or "FR" refers to the variable domain residues of the variable region other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2 and HCDR3; VL contains three CDR regions: LCDR1, LCDR2 and LCDR3. Each VH and VL is typically composed of three CDRs and four FRs arranged in the following order (from the amino terminus to the carboxyl terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid sequence boundaries of the CDRs can be determined by methods well known to those skilled in the art, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001) and the ImMunoGenTics (IMGT) numbering convention (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between the various numbering systems is well known to those skilled in the art.

[0077] The term "light chain" includes the light chain variable region (VL) and the light chain constant region (CL). VL is at the amino terminus of the light chain, and CL is at the carboxyamino terminus of the light chain. The light chain can be a κ chain or a λ chain, etc.

[0078] The term "heavy chain" includes the heavy chain variable region (VH) and the heavy chain constant region (CH). The heavy chain variable region is at the amino terminus of the heavy chain, and the heavy chain constant region is at the carboxy terminus of the heavy chain. The heavy chain constant region of an IgG antibody includes three constant region domains: CH1, CH2 and CH3. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgD, IgM and IgE, etc.

[0079] The terms "constant region" or "constant domain" refer to the carboxyl-terminal portions of the light and heavy chains that do not directly participate in antibody-antigen binding and have a relatively conserved amino acid sequence compared to VH and VL.

[0080] The term "antigen-binding fragment" refers to a portion of an intact antibody that, unlike the intact antibody, is capable of specifically binding the antigen to which the intact antibody binds. Some examples of antigen-binding fragments include, but are not limited to: Fv, Fab, Fab’, Fab’-SH, F(ab′)2, dsFv, (dsFv)2, single-chain Fab (scFab), single-chain antibodies (e.g., scFv), diabodies, linear antibodies, and multispecific antibodies formed from antigen-binding fragments. Among them, "Fab" is a monovalent fragment composed of the VL, VH, CL, and CH1 domains; "Fv" is composed of VH and VL; "Fab’" is a Fab fragment that includes a portion of the hinge region; "F(ab’)2" is a divalent fragment that includes two Fab’ fragments linked by a disulfide bond in the hinge region; "scFab" is a polypeptide composed of VH, CH1, VL, CL, and a linker, where the antibody domains and the linker have one of the following sequences 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 that includes a light-chain variable region and a heavy-chain variable region, where the light-chain variable region and the heavy-chain variable region are linked by a peptide linker and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, in this article, the scFv can have the VL and VH variable regions in any one of the sequences. For example, from the N-terminal to the C-terminal of the scFv polypeptide, it can include: a) VL-linker-VH or b) VH-linker-VL; "dsFv" is a disulfide bond-stabilized Fv fragment; (dsFv)2 is a dimerized dsFv; "Fab'-SH" is a Fab' fragment in which the cysteine residue in the hinge region carries a free thiol group.

[0081] The term "Fc region" refers to the C-terminal region of an antibody heavy chain, including the native antibody Fc region and Fc region variants. In some embodiments, the Fc region of a human IgG heavy chain extends from the amino acid residue at position Cys226 to its carboxyl terminus. In some embodiments, the Fc region of a human IgG heavy chain extends from Pro230 to its carboxyl terminus. The C-terminus of the Fc region can also be, for example, a deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or a deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). In some embodiments, a composition of intact antibodies can include a population of antibodies lacking the K447 residue and / or the G446+K447 residues. In some embodiments, a composition of intact antibodies can include a population of antibodies without a deletion of the K447 residue and / or the G446+K447 residues. In some embodiments, a composition of intact antibodies has a population of antibodies that is a mixture of antibodies with and without the K447 residue and / or the G446+K447 residues. The Fc regions for the antibodies described in this disclosure include the Fc regions of human IgG1, IgG2 (IgG2a, IgG2b), IgG3, and IgG4. Unless otherwise specified in this disclosure, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0082] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from one particular species and the remaining portion of the heavy chain and / or light chain is derived from other species.

[0083] 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, this can be achieved by retaining the CDR regions of the non-human antibody and replacing the remainder of the antibody with their human counterparts (i.e., the framework regions of the heavy chain variable region of a human antibody, the framework regions of the light chain variable region of a human antibody, and the human antibody constant regions).

[0084] The terms "human antibody", "human-derived antibody", "fully human antibody", "completely human antibody" can be used interchangeably and refer to an antibody in which the variable region and constant region sequences are human sequences. Human antibodies include antibodies having human-derived genes but with altered residues such as cysteine or glycosylation sites. Human antibodies can be antibodies recombinantly produced in non-human cells. Human antibodies include antibodies obtained from transgenic mice with human immunoglobulin heavy and light chain loci.

[0085] 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). Unless otherwise specified, as used in this disclosure, "affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its ligand Y is typically expressed as the dissociation constant (KD). Affinity can be measured by conventional methods known in the art. The terms "kassoc" or "ka" refer to the association rate of a particular antibody-antigen interaction, and the terms "kdis" or "kd" refer to the dissociation rate 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 typically expressed in molar concentration (M). The KD value of an antibody can be determined using methods well known in the art. Methods for determining the KD of an antibody include using a biosensing system such as a surface plasmon resonance system, or measuring the affinity in solution by solution equilibrium titration (SET).

[0086] The term "affinity matured" antibody refers to an antibody that has one or more amino acid residue changes in one or more CDRs of the antibody, resulting in an improvement in the affinity of the antibody for the antigen compared to the parental antibody. In some embodiments, the affinity matured antibody will have an affinity for the target antigen in the nanomolar or even picomolar range. Affinity matured antibodies can be generated by methods known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. The following references describe random mutagenesis of CDR and / or framework residues: Barbas et al., PNAS, 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995) and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0087] The term "monoclonal antibody" refers to a substantially homogeneous population of antibodies, i.e., the amino acid sequences of the antibody molecules contained in the population are the same (except for possible minor natural mutations). In contrast, polyclonal antibodies usually contain different antibodies with variable domains having different amino acid sequences, which are usually specific for different epitopes and / or different antigens. "Monoclonal" indicates an antibody obtained from a substantially homogeneous population of antibodies. Monoclonal antibodies can be produced by methods well known in the art. For example, they can be prepared by the hybridoma method (Kohler et al., (1975) Nature 256:495), by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), by isolation from phage antibody libraries (Clackson et al., (1991) Nature 352:624-628 and Marks et al., (1991) J. Mol. Biol. 222:581-597), and monoclonal antibodies can also be obtained using transgenic animals containing all or part of the human immunoglobulin locus (see Presta (2005) J. Allergy Clin. Immunol. 116:731).

[0088] The term "antigen" refers to a protein that can selectively bind to an antigen-binding protein (such as an antibody). An antigen (such as human GUCY2C) can have one or more epitopes that interact with different antigen-binding proteins (such as antibodies).

[0089] The term "epitope" refers to the area or region on an antigen that can specifically bind to an antibody. Epitopes are usually composed of antigenic determinants, which are specific chemical groups with a certain structure. Epitopes can be formed by contiguous amino acid residues (linear epitopes) or by non - contiguous amino acid residues (conformational epitopes), such as non - contiguous amino acid residues that are spatially close due to the folding of the antigen. In some embodiments, an epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8 - 10 amino acid residues in a unique spatial conformation. Epitopes can be determined by any method well - known in the art, such as conventional immunoassays, antibody competitive binding assays, or X - ray crystallography or related structure determination methods (e.g., nuclear magnetic resonance spectroscopy). Antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be obtained using methods well - known in the art, including but not limited to, for example, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443 - 463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487 - 496), and cross - blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0090] An antibody that "binds the same epitope as a reference antibody" or "competitively binds with a reference antibody" refers to an antibody that blocks 50% or more of the binding of the reference antibody to an antigen in a competitive assay, or an antibody whose binding to the antigen is blocked by the reference antibody by 50% or more. To determine whether a test antibody binds the same epitope as a reference antibody, it can be detected under saturation conditions whether the reference antibody is allowed to bind the antigen. For example, after removing the excess reference antibody, the ability of the test antibody to bind the antigen is evaluated; if the test antibody can bind the antigen after the saturation binding of the reference antibody, then the test antibody binds a different epitope from the reference antibody; however, if the test antibody cannot bind the antigen after the saturation binding of the reference antibody, then the test antibody may bind the same epitope as the reference antibody. To confirm whether a test antibody binds the same epitope or is only hindered from binding due to steric reasons, conventional experiments (such as peptide mutagenesis and detection using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative method known in the art) can be used. If in two settings (i.e., the two antibodies are used as saturation antibodies respectively), only the saturation antibody can bind the antigen, then it can be concluded that the test antibody and the reference antibody competitively bind the antigen. In some embodiments, in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 50 (1990) 1495 - 1502), if an excess (such as 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amount) of one antibody inhibits the binding of another antibody to the antigen by at least 50%, at least 75%, at least 90%, or even 99% or more, the two antibodies are considered to bind the same or overlapping epitopes.

[0091] The terms "specifically bind" or "binds specifically" refer to non-random binding between two molecules. For example, an antibody binds to the antigen or epitope corresponding to that antibody with a higher affinity than to other antigens or epitopes. Generally, an antibody binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1×10 -7 M or less (such as about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -12 M or less). In some embodiments, the KD of the antibody binding to the antigen is 10%, 1%, or less of the KD of the antibody binding to non-specific antigens (such as BSA, casein). KD values can be measured using methods well known in the art, such as by Surface plasmon resonance assay measurement. Antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity with other related antigens, for example, with corresponding antigens from other species such as humans or monkeys, e.g., cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp), or common marmoset (Callithrix jacchus) (marmoset).

[0092] It has been found through research in the present disclosure that CART constructed with antibodies binding to the juxtamembrane region (near the C-terminus) of GUCY2C has better anti-tumor effects than CART constructed with antibodies binding to the juxtamembrane region (near the N-terminus) of GUCY2C. In order to obtain antibodies that bind to the juxtamembrane region (near the N-terminus) of GUCY2C, the inventors conducted extensive research and constructed a brand-new GUCY2C polypeptide, which can be used as an immunogen to prepare anti-human GUCY2C antibodies that bind to the juxtamembrane region of GUCY2C by immunizing animals; it can also be used as an antigen polypeptide to screen anti-human GUCY2C antibodies that bind to the juxtamembrane region of GUCY2C.

[0093] In some embodiments, the present disclosure constructs a polypeptide, which is a GUCY2C construct and includes a first domain, a second domain, and a linker, wherein the first domain and the second domain are human GUCY2C polypeptide fragments; the N-terminus of the linker is connected to the C-terminus of the first domain, and the C-terminus of the linker is connected to the N-terminus of the second domain.

[0094] In some embodiments, for the polypeptide as described in any of the above, which is a GUCY2C construct, the first domain includes amino acid residues 24 to 161 of SEQ ID NO.1, and the second domain includes amino acid residues 300 to 376 of SEQ ID NO.1; or the first domain includes amino acid residues 162 to 299 of SEQ ID NO.1, and the second domain includes amino acid residues 377 to 430 of SEQ ID NO.1.

[0095] In some embodiments, the polypeptide as described in any one of the above is a GUCY2C construct, wherein: A. the amino acid sequence of the first domain has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.2, and / or the amino acid sequence of the second domain has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.3; or B. the amino acid sequence of the first domain has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.4, and / or the amino acid sequence of the second domain has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.5. In some embodiments, the polypeptide as described in any one of the above is a GUCY2C construct, wherein: a. the amino acid sequence of the first domain is as shown in SEQ ID NO.2, and the amino acid sequence of the second domain is as shown in SEQ ID NO.3; or b. the amino acid sequence of the first domain is as shown in SEQ ID NO.4, and the amino acid sequence of the second domain is as shown in SEQ ID NO.5.

[0096] In some embodiments, the polypeptide as described in any one of the above is a GUCY2C construct, wherein the linker is a flexible peptide linker; in some embodiments, the polypeptide as described in any one of the above, wherein the linker is selected from (G x S y ) Z linker, wherein x and y independently are selected from integers of 0 - 5, and y is selected from integers of 1 - 6; in some embodiments, the polypeptide as described in any one of the above, wherein the linker is the linker as shown in SEQ ID NO:6 or 7.

[0097] In some embodiments, the polypeptide as described in any of the above is a GUCY2C construct, wherein the polypeptide is represented by the following formula (I): (I): X-L1-Y, where X is a first GUCY2C polypeptide fragment, Y is a second GUCY2C polypeptide fragment, and L1 is a linker; in some embodiments, the polypeptide as described in any of the above, wherein A. the amino acid sequence of the first GUCY2C polypeptide fragment has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.2, and / or the amino acid sequence of the second GUCY2C polypeptide fragment has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.3; or B. the amino acid sequence of the first GUCY2C polypeptide fragment has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.4, and / or the amino acid sequence of the second GUCY2C polypeptide fragment has at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO.5; in some embodiments, the polypeptide as described in any of the above is a GUCY2C construct, the first GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.2, and the second GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.3; or the first GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.4, and the second GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.5; in some embodiments, the polypeptide as described in any of the above, the linker is a flexible peptide linker; in some embodiments, the polypeptide as described in any of the above, wherein the linker is selected from (G x S y ) ZLinker, wherein x and y are independently selected from integers of 0-5, and y is selected from integers of 1-6; In some embodiments, for the polypeptide as described in any one of the above, the linker is the linker shown in SEQ ID NO: 6 or 7. In some embodiments, the present disclosure provides a polypeptide having an amino acid sequence with 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%, 99% or 100%) sequence identity to SEQ ID NO.8 or 9; In some embodiments, for the polypeptide as described in any one of the above, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.8 or SEQ ID NO.9. In some embodiments, for the polypeptide as described in any one of the above, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.8.

[0098] In some embodiments, for the polypeptide as described in any one of the above, it is a GUCY2C construct, wherein the N-terminus of the polypeptide includes a signal peptide; In some embodiments, for the polypeptide as described in any one of the above, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.10.

[0099] In some embodiments, for the polypeptide as described in any one of the above, it is a GUCY2C construct, wherein the C-terminus of the polypeptide includes a protein tag; In some embodiments, for the polypeptide as described in any one of the above, the amino acid sequence of the protein tag is as shown in SEQ ID NO.11; In some embodiments, for the polypeptide as described in any one of the above, the amino acid sequence of the polypeptide has 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%, 99% or 100%) sequence identity to SEQ ID NO.12 or 13; In some embodiments, for the polypeptide as described in any one of the above, it is a GUCY2C construct, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.12 or SEQ ID NO.13. In some embodiments, for the polypeptide as described in any one of the above, it is a GUCY2C construct, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.12.

[0100] The terms "anti-human GUCY2C antibody" and "antibody that binds to human GUCY2C" refer to antibodies that can bind to the human GUCY2C antigen with sufficient affinity. In one example, the binding of the anti-human GUCY2C antibody to the non-human GUCY2C protein is less than about 10%, 1% or less of the binding to human GUCY2C, and the binding can be determined by Measured by surface plasmon resonance assay. In certain embodiments, the antibody that binds to GUCY2C has the following dissociation constant (KD): < 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 or less. In certain embodiments, the anti-human GUCY2C antibody binds to a GUCY2C epitope conserved in GUCY2C from different species. In certain embodiments, the anti-human GUCY2C antibody binds to the polypeptide near the membrane terminus of human GUCY2C; in certain embodiments, the anti-human GUCY2C antibody binds to the polypeptide far from the membrane terminus of human GUCY2C; in certain embodiments, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO. 8 or SEQ ID NO. 12 is stronger than the binding activity to the polypeptide shown in SEQ ID NO. 9 or SEQ ID NO. 13. In certain embodiments, the anti-human GUCY2C antibody specifically binds to the polypeptide shown in SEQ ID NO. 8 or SEQ ID NO. 12. In certain embodiments, the anti-human GUCY2C antibody prepared or screened from the novel GUCY2C polypeptide of the present disclosure (such as the polypeptide shown in SEQ ID NO. 8 or SEQ ID NO. 12) can be used to construct CART cells, prepare pharmaceutical compositions, and be used for treating tumors.

[0101] The binding of the anti-human GUCY2C antibody provided by the present disclosure to human GUCY2C can also be represented by the "half maximal effective concentration" (EC50) value. Generally, the smaller the EC50, the better the affinity, indicating that the antibody can bind to the antigen at a lower concentration. The EC50 value can be determined by binding detection methods known in the art, such as direct or indirect binding detection methods (such as enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting technology, and other binding detection methods).

[0102] The terms "linking peptide", "linking polypeptide", "linker", "Linker" or "adapter" refer to a linking unit that links two polypeptide fragments, usually having 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 the (GxS y )wGzSt linker, wherein x, y, w, z, t are independently selected from integers from 0 to 6 (when it is 0, it means that the corresponding amino acid residue does not exist, for example, the amino acid residue of (G4S1)0G5S0 is: GGGGG). In some embodiments, the linker is selected from the Gx linker, wherein x is selected from integers from 1 to 10. In some embodiments, the linker is the polypeptide shown in SEQ ID NO: 6 or 7.

[0103] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in the present disclosure and refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and their polymers. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, and nucleic acids can be synthetic, naturally occurring, and non-naturally occurring, such as non-naturally occurring nucleic acids having binding properties similar to reference nucleic acids and metabolized in a manner similar to reference nucleotides. Including but not limited to, phosphorothioates, phosphoroamidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and modified nucleic acids. An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain such nucleic acid molecules, but the nucleic acid molecules are present extrachromosomally or at a chromosomal location different from their natural chromosomal location. A "nucleic acid encoding an anti-human GUCY2C antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including one or more nucleic acid molecules in a single vector or separate vectors, and one or more nucleic acid molecules present at one or more locations in a host cell. In some embodiments, the nucleic acid sequence also includes conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the specifically recited sequences. For example, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0104] The terms "polypeptide" and "protein" are used interchangeably herein and refer to polymers 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 acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0105] The terms "identity" and "sequence identity" refer to the degree (percentage) to which the amino acids (amino acid sequence identity) or nucleic acids (nucleic acid sequence identity) of two sequences are the same at equivalent positions when the two sequences are optimally aligned. To obtain the optimal alignment of two sequences, gaps may be introduced if necessary to obtain the maximum percentage of sequence identity. The percentage of amino acid sequence identity can be determined in a variety of ways well known in the art, such as by using software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA, which are well known in the art. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve the maximum alignment of the entire length of the comparison sequences.

[0106] The term "conservative modified variant" or "conservative substitution" of an amino acid refers to the replacement of an amino acid in a sequence with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, backbone conformation, and rigidity, etc.), but without changing the biological activity of the protein. Those skilled in the art know that, generally, single amino acid substitutions in non-essential regions of a polypeptide usually do not change the biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). The term "conservative modified variant" or "conservative substitution" of a nucleic acid refers to those nucleic acids that encode the same or substantially the same amino acid sequence, and also includes nucleic acids of sequences that are substantially the same in cases where the nucleic acid does not encode an amino acid sequence. Due to the degeneracy of the genetic code, any given protein can be encoded by multiple functionally identical nucleic acids. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where a codon specifies alanine, the codon can be changed to any of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", and they are one type of conservative modified variation. Each nucleic acid sequence encoding a polypeptide in the present disclosure includes every possible silent variation of the nucleic acid. Each codon in a nucleic acid (except for AUG - usually the only codon for methionine; and TGG - usually the only codon for tryptophan) can be modified to produce a functionally identical molecule.

[0107] The term "vector" is a vehicle that can transport a genetic element (such as nucleic acid) linked thereto. A vector can be used to transform, transduce, or transfect a host cell so that the genetic element it carries can be expressed in the host cell. Exemplarily, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses, etc. In some embodiments, the vector is a "plasmid", which is a circular double-stranded DNA loop to which additional DNA segments can be linked. In some embodiments, the vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), and the DNA segment can be linked to the viral genome. A vector can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector can also contain an origin of replication. A vector can also include components that assist its entry into cells, including but not limited to, viral particles, liposomes, or protein coats. A vector can be an expression vector. In some embodiments, the vector (such as an expression vector) contains a nucleic acid sequence encoding an antibody of the present disclosure, a promoter (such as, SV40, CMV, EF-1α), and can also contain at least one selection marker.

[0108] The term "expression vector" or "expression construct" is a vector that is suitable for transforming a host cell and contains a nucleic acid sequence that directs and / or controls (together with the host cell) the expression of one or more coding regions operably linked thereto. Expression vectors can include, but are not limited to: sequences that affect or control transcription, translation, and RNA splicing of coding regions operably linked thereto in the presence of introns.

[0109] The term "operably linked" refers to a linkage relationship in which the components referred to by the term allow them to perform their inherent functions under suitable conditions. For example, a control sequence "operably linked" to a protein-coding sequence refers to a sequence that is linked thereto and enables the expression of the protein-coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0110] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical, nucleic acid content-wise, to the parental cell, but may contain mutations. Mutant progeny having the same function or biological activity as those screened or selected in the original transformed cell are included herein. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells, including, but not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, the genus Pichia, Saccharomyces cerevisiae, the genus Saccharomyces, Hansenula polymorpha, the genus Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, the genus Fusarium, Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. The genus Pichia, any species of the genus Saccharomyces, Hansenula polymorpha, any species of the genus Kluyveromyces, Candida albicans, any species of the genus Aspergillus, Trichoderma reesei, Chrysosporium lucknowense, any species of the genus Fusarium, Yarrowia lipolytica, and Neurospora crassa.

[0111] The term "pharmaceutical composition" refers to a mixture containing one or more drugs (such as the antibodies described in the present disclosure) and other components, such as physiological / pharmaceutically acceptable carriers and excipients.

[0112] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is different from the active ingredient and non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives, etc.

[0113] The terms "subject" or "individual" include humans and non-human animals. Non-human animals include all vertebrates (such as mammals and non-mammals), such as non-human primates (such as cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably in the present disclosure. Unless otherwise clearly defined in the present disclosure, the term "cyno" or "cynomolgus" refers to Macaca fascicularis. In certain embodiments, the subject is a human.

[0114] "Administer" or "administering", when 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.

[0115] The term "sample" refers to a collection of fluid, cells, or tissue similar to those isolated from a subject, as well as fluids, cells, or tissues present in the subject. Exemplary samples are biological fluids, such as blood, serum, and serous fluid, plasma, lymph fluid, urine, saliva, cyst fluid, tears, excreta, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, abdominal, and other body cavity fluids, fluids collected by bronchoalveolar 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 samples, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.

[0116] The term "treatment" refers to a clinical intervention that attempts to alter the natural course of the individual being treated and can be implemented for prevention or during the course of a clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the formation of a disease or slow the progression of a disease.

[0117] The term "administer" or "administering", when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to the contact of an exogenous agent with the animal, human, subject, cell, tissue, organ or biological fluid.

[0118] The term "effective amount" generally refers to an amount sufficient to reduce the severity or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent the appearance of symptoms and / or their underlying causes, or improve an impairment (such as a pulmonary disease) caused by and associated with a disease state. In some embodiments, the effective amount may be a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a condition or symptom associated with the disease state, or otherwise prevent, impede, delay or reverse the disease state or the progression of any other undesirable symptom 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. A complete therapeutic or prophylactic effect may not occur after administration of a single dose and may occur after administration of a series of doses. Thus, the therapeutically effective amount or prophylactically effective amount may be administered in one or more administrations. The "therapeutically effective amount" and "prophylactically effective amount" can vary depending on a variety of factors, such as the disease state, age, sex and weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual.

[0119] The terms "cancer" and "tumor" refer to diseases in mammals characterized by unregulated cell growth. Tumors include benign tumors and malignant tumors, and early stage tumors refer to non-invasive or metastatic cancers, or cancers classified as stage 0, I, or II cancer. In some embodiments, the tumor is a tumor expressing GUCY2C. Examples of cancers include, but are not limited to, lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and hematological cancers.

[0120] In some embodiments, the present disclosure prepares or detects the antibodies of the present disclosure using conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology well-known in the art. Related techniques have been disclosed in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by M.J. Gait, 1984); "Animal Cell Culture" (edited by R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (edited by D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (edited by J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); and "Current Protocols in Immunology" (edited by J.E. Coligan et al., 1991), which are hereby incorporated by reference.

[0121] The features and properties of the present disclosure will be further described in detail below in conjunction with examples.

[0122] Example 1. Construction of GUCY2C Polypeptide

[0123] The inventors comprehensively studied the structure of the human GUCY2C antigen protein (the 3D structure is shown in the appendix Figure 1(A), two polypeptides of the extracellular region of human GUCY2C, S162-L299 and S377-Q430, were linked by a linker peptide GGGGG to construct the C-terminal domain of the GUCY2C antigen (S162-L299-GGGGG-S377-Q430). The constructed C-terminal domain was expressed as an independent protein. The structure of the S162-L299-GGGGG-S377-Q430 protein constructed by AlphaFold analysis (see attached Figure 1 C purple structure) was superimposed with the structure of the GUCY2C antigen protein, and it was found that the predicted structure of the C-terminal domain of the GUCY2C antigen constructed in the present disclosure was well superimposed with the predicted structure of the GUCY2C antigen protein, and did not have an obvious impact on the spatial structure. In addition, in the present disclosure, two polypeptides of the extracellular region of human GUCY2C, S24-M161 and S300-D376, were linked by a linker peptide GGGG to construct the N-terminal domain of the GUCY2C antigen (S24-M161-GGGG-S300-D376). The constructed C-terminal domain was expressed as an independent protein. The structure of the GUCY2C antigen N-terminal domain (S24-M161-GGGG-S300-D376) protein was analyzed by AlphaFold (see attached Figure 1 B yellow structure) was superimposed with the GUCY2C antigen structure, and it was found that the predicted structure of the GUCY2C antigen N-terminal domain in the present disclosure was well superimposed with the predicted structure of the GUCY2C antigen protein, and did not have an obvious impact on the spatial structure.

[0124] Amino acid sequence of the extracellular region of human GUCY2C (SEQ ID NO.1):

[0125] MKTLLLDLALWSLLFQPGWLSFSSQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLMSPARKLMYFLVNFWKTNDLPFKTYSWSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQDKEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVAEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTLSPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVDSTMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQ;

[0126] Amino acid sequence of the polypeptide at positions S162 - L299 in the extracellular region of human GUCY2C (SEQ ID NO.2):

[0127] SPARKLMYFLVNFWKTNDLPFKTYSWSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQDKEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVAEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTL;

[0128] Amino acid sequence of the polypeptide at positions S377 - Q430 in the extracellular region of human GUCY2C (SEQ ID NO.3):

[0129] STMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQ;

[0130] Amino acid sequence of the polypeptide at positions S24 - M161 in the extracellular region of human GUCY2C (SEQ ID NO.4):

[0131] SQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLM;

[0132] Amino acid sequence (SEQ ID NO.5) of the polypeptide at positions S300 - D376 in the extracellular region of human GUCY2C:

[0133] SPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVD;

[0134] Amino acid sequence of Linker polypeptide 1 (SEQ ID NO.6): GGGGG

[0135] Amino acid sequence of Linker polypeptide 2 (SEQ ID NO.7): GGGG

[0136] Amino acid sequence of the C - terminal domain of the constructed GUCY2C antigen (SEQ ID NO.8): SPARKLMYFLVNFWKTNDLPFKTYSWSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQDKEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVAEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTL GGGGG STMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQ;

[0137] Amino acid sequence of the N - terminal domain of the constructed GUCY2C antigen (SEQ ID NO.9): SQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLM GGGG SPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVD;

[0138] Amino acid sequence of the signal peptide (SEQ ID NO.10): MGWSCIILFLVATATGVHS;

[0139] Amino acid sequence of the protein tag (SEQ ID NO.11): HHHHHH;

[0140] Amino acid sequence of the C-terminal domain of the GUCY2C antigen containing the signal peptide and the protein tag (SEQ ID NO.12): MGWSCIILFLVATATGVHSSPARKLMYFLVNFWKTNDLPFKTYSWSTSYVYKNGTETEDCFWYLNALEASVSYFSHELGFKVVLRQDKEFQDILMDHNRKSNVIIMCGGPEFLYKLKGDRAVAEDIVIILVDLFNDQYFEDNVTAPDYMKNVLVLTLGGGGGSTMVLLYTSVDTKKYKVLLTYDTHVNKTYPVDMSPTFTWKNSKLPNDITGRGPQHHHHHH

[0141] Amino acid sequence of the N-terminal domain of the GUCY2C antigen containing the signal peptide and the protein tag (SEQ ID NO.13): MGWSCIILFLVATATGVHSSQVSQNCHNGSYEISVLMMGNSAFAEPLKNLEDAVNEGLEIVRGRLQNAGLNVTVNATFMYSDGLIHNSGDCRSSTCEGLDLLRKISNAQRMGCVLIGPSCTYSTFQMYLDTELSYPMISAGSFGLSCDYKETLTRLMGGGGSPGNSLLNSSFSRNLSPTKRDFALAYLNGILLFGHMLKIFLENGENITTPKFAHAFRNLTFEGYDGPVTLDDWGDVDHHHHHH

[0142] Example 2. Preparation and detection of the newly constructed GUCY2C polypeptide

[0143] A signal peptide was added to the N-terminus of the constructed C-terminal domain / N-terminal domain of the GUCY2C antigen to ensure that the protein could be secreted extracellularly, and a 6XHis tag was added to the C-terminus for Ni column purification. The C-terminal domain of the GUCY2C antigen (SEQ ID NO.12) containing the signal peptide and the protein tag and the N-terminal structure of the GUCY2C antigen (SEQ ID NO.13) containing the signal peptide and the protein tag were constructed.

[0144] The synthetic open reading frame was inserted into the pcDNA3.4 expression vector. After correct sequencing, the plasmid was extracted and transfected into Expi293 cells using PEI for expression. After 7 days of culture, the supernatant was collected by centrifugation. The target molecule was purified using an NI-TED affinity chromatography column. The packing material was equilibrated with 1xPBS. After sample loading, impurities were washed with 1XPBS + 10 mM imidazole, and the target molecule was eluted with 1XPBS + 250 mM imidazole. Then, the solution was replaced with 1XPBS for binding force detection. The SDS-Page electrophoresis results are shown in the appendix Figure 2 , and the results showed that the purity of the N-terminal domain of the GUCY2C antigen constructed in this disclosure was low, while the purity of the C-terminal domain of the GUCY2C antigen was good.

[0145] After removing the N-glycosylation modification of the target molecule using PNG, liquid chromatography-mass spectrometry was used for detection. The reverse column chromatography results of the N-terminal domain of the GUCY2C antigen (R2230 in the figure) are shown in the appendix Figure 3 , and the molecular weight of the molecule with a retention time of 11.84 min was the same as that of the target molecule (theoretical molecular weight: 24701.8 Da, detected molecular weight: 24698.7 Da), confirming that the target molecule was purified. Due to the large amount of impurities and the presence of multiple glycosylation sites on the target molecule, which could cause smearing of the electrophoresis bands, clear bands could not be obtained by electrophoresis.

[0146] The same method was used to detect the C-terminal domain of the GUCY2C antigen (R2231 in the figure). The reverse column chromatography results are shown in the appendix Figure 4 , and the molecular weight of the molecule with a retention time of 11.00 min was the same as that of the target molecule (theoretical molecular weight: 23495.8 Da, detected molecular weight: 23494.3 Da), and the target molecule was also obtained.

[0147] Example 3. Screening of anti-human GUCY2C antibodies using the constructed GUCY2C polypeptide

[0148] 1. Preparation of anti-human GUCY2C antibodies

[0149] The anti-human GUCY2C antibodies F021-3, F021-14, F021-15, F021-18, F021-36, and F021-43 in this disclosure were prepared according to the preparation of F021-3, F021-14, F021-15, F021-18, F021-36, and F021-43 antibodies described in the international patent application (Application No.: PCT / CN2023 / 119458); the anti-human GUCY2C antibody F021-70 was prepared according to the preparation of the 2A1 antibody described in the Chinese patent application (Application No.: 202310004900.2).

[0150] 2. Screening of anti-human GUCY2C antibodies that bind to the juxtamembrane region

[0151] The binding of the anti-human GUCY2C antibody to be tested with the C-terminal domain / N-terminal domain of the GUCY2C antigen constructed in the present disclosure was detected by ELISA, and the antibodies that bind to the C-terminal domain of the GUCY2C antigen were screened out. First, the GUCY2C antigen polypeptides: R2230 (human GUCY2C antigen N-terminal domain, SEQ ID NO.9), R2231 (human GUCY2C antigen C-terminal domain, SEQ ID NO.8) antigen and R0596 (human GUCY2C extracellular domain, SEQ ID NO.1) were respectively added to a 96-well enzyme-linked immunosorbent assay (ELISA) plate and coated on the 96-well plate at a concentration of 2 μg / mL, 100 μL per well, at 37 °C for 3 h. After incubation, the supernatant was discarded and the plate was washed 3 times, then blocked with 1% bovine serum albumin (BSA) for 1 h, and then the plate was washed three times. The antibody was diluted to 2 μg / mL and added to the 96-well plate, incubated at room temperature for 1 h, the plate was washed 4 times, then anti-human Fc-HRP was added and incubated for 30 min, and the plate was washed 5 times. 100 μL of the substrate was added to each well and reacted for 15 min. After termination, the OD450 value was detected by a microplate reader. In this experiment, the negative control was an IgG protein (Isotype) unrelated to GUCY2C, and the positive control antibodies were R0591 (9H3 antibody described in US Patent Application (Publication No. US20200010566A1)) and R0826 (5F9 antibody described in US Patent (Publication No.: US8785600B2)). The experimental results are shown in Table 1:

[0152] Table 1. Table of the binding of antibodies to GUCY2C polypeptides

[0153]

[0154] Note: The data in the table represent the OD450 readings

[0155] The experimental results showed that the anti-human GUCY2C antibodies prepared in the present disclosure could all bind to the full-length polypeptide of the extracellular domain of human GUCY2C; however, there were differences in the binding activities of the anti-human GUCY2C antibodies to the C-terminal domain (SEQ ID NO.8) and the N-terminal domain (SEQ ID NO.9) of the human GUCY2C antigen. Among them, R0591, R0826, F021-43, and F021-70 had the highest binding activity to the N-terminal domain of the human GUCY2C antigen, and the OD450 reading of their binding to the N-terminal domain of the human GUCY2C antigen was approximately 10 times that of their binding to the C-terminal domain of the human GUCY2C antigen; F021-15 bound to both the C-terminal domain and the N-terminal domain of the human GUCY2C antigen, but the OD450 reading of its binding to the N-terminal domain of the human GUCY2C antigen was nearly 3 times that of its binding to the C-terminal domain of the human GUCY2C antigen; F021-18 weakly bound to both the C-terminal domain and the N-terminal domain of the human GUCY2C antigen, and the OD450 reading of its binding to the C-terminal domain of the human GUCY2C antigen was similar to that of its binding to the N-terminal domain of the human GUCY2C antigen; while F021-3, F021-14, and F021-36 preferred to bind to the C-terminal domain of GUCY2CN, and the OD450 readings of their binding to the C-terminal domain of the human GUCY2C antigen were 1.8, 2.4, and 9.9 times that of their binding to the N-terminal domain of the human GUCY2C antigen, respectively. Finally, the anti-human GUCY2C antibody F021-36 with a binding activity to the C-terminal domain of the human GUCY2C antigen more than 9 times that of its binding to the N-terminal domain of the human GUCY2C antigen was screened out.

[0156] Example 4. Preparation and detection of anti-human GUCY2C antibody CAR-T cells

[0157] 1. Construction of lentiviral vector

[0158] Genes were synthesized to encode the following CAR structure (see Appendix Figure 5 ), and according to the restriction enzyme sites of the lentiviral vector, the nucleotide fragments were constructed onto the lentiviral vector. Primers were designed, and the correctness of the vector construction was verified by the sequencing results. Antibody and its corresponding CART numbers: R0591 (CART-R1191), R0826 (CART-R1192), F021-3 (CART-R1434), F021-14 (CART-R1091), F021-15 (CART-R1435), F021-18 (CART-R1436), F021-36 (CART-R1437), F021-43 (CART-R1093), F021-70 (CART-R1092).

[0159] 2. Package and concentrate lentivirus

[0160] Inoculate 293t at a density of 5x10 6 cells / 10 cm culture dish. Observe the cell state the next day. Co-transfect the third-generation lentiviral packaging vector into 293t by the PEI transfection method. Change the medium 6 hours after transfection. Add DMEM medium containing 10% fetal bovine serum according to 15 ml / 150 mm 2 culture dish. Collect the virus supernatant 48 hours and 72 hours after transfection. Centrifuge at 2000 rpm at 4°C for 10 min to remove cell debris. Then filter the impurities through a 0.45-μm filter. Concentrate the lentivirus in the filtered virus suspension at 25000 rpm at 4°C for 2 hours. Resuspend the concentrated virus in an appropriate amount of medium and store it at -80°C.

[0161] 3. Produce CAR-T and control cells

[0162] Draw 20 mL of blood. Isolate PBMC by Ficoll gradient centrifugation. Isolate T cells using the Stemcell T cell negative selection kit (product number: 19051). Resuspend the isolated T cells in X-VIVO 15 medium supplemented with 5% human AB serum and 300 units / mL IL-2 to 1×10 6 cells / mL. Wash the beads with X-VIVO 15 containing 1% FBS. Add the pre-washed beads (Cat#40203D, 10ML, Life technology) at a ratio of magnetic beads: T cells = 2:1. After 2 - 3 days, resuspend the T cells in fresh medium to 3 - 5×10 6 cells / mL. Add lentivirus at an MOI of 10, and at the same time add 8 μg / ML of Polybrene. After 4 - 6 hours, supplement the medium to dilute the cells to 1×10 6 cells / mL. Replace the fresh medium the next day to maintain the cell concentration at 0.2 - 0.3×10 6 PBMC / mL. Then replace the medium every 2 - 3 days. Analyze the positive rate of cells by flow cytometry 72 hours after virus infection. The experimental results are shown in Table 2, and the flow cytometry results are shown in Figure 6 .

[0163] Table 2. Detection results of CAR positive rate of CAR-T cells

[0164]

[0165] Example 5. Evaluation of in vitro killing function of CAR-T

[0166] The xCELLigence real-time, cell-mediated cytotoxicity system (Acea Biosciences Inc.) was used to evaluate CAR-T cell-mediated cytotoxicity. 1.0E+04 LS1034 cells were placed in 150 μL of growth medium in each well of an E-Plate 16 (Acea Biosciences) and cultured overnight in a 37°C incubator. Impedance was quantified every 15 minutes using the RTCA DP Analyzer system and RTCA software version 2.0 (Acea Biosciences Inc.). Approximately 24 hours later, 50 μL of CAR-T (CART-R1191, CART-R1091, CART-R1435, CART-R1436, CART-R1437, CART-R1092) cells (E:T ratio of 5:1) or 50 μL of medium were added as a negative control, and cell-mediated killing was quantified over the next 24 hours with impedance readings taken every 15 minutes. The experimental results are shown in Appendix Figure 7 , and the experimental results showed that CAR-T cells prepared with CARs constructed using humanized antibodies had a very good effect on inhibiting the growth of T84 cells; among them, the CART constructed with the anti-human GUCY2C antibody F021-36 that binds to the C-terminal domain (near the membrane end) of the GUCY2CN antigen had the most significant tumor-suppressing effect.

[0167] The above are only the preferred embodiments of the present disclosure and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure shall be included within the protection scope of the present disclosure.

Claims

1. A polypeptide, which comprises a first domain, a second domain and a linker, wherein the first domain and the second domain are human GUCY2C polypeptide fragments; the N-terminus of the linker is connected to the C-terminus of the first domain, and the C-terminus of the linker is connected to the N-terminus of the second domain.

2. The polypeptide according to claim 1, wherein, the first domain comprises the amino acid residues at positions 162 to 299 of SEQ ID NO.1, and the second domain comprises the amino acid residues at positions 377 to 430 of SEQ ID NO.1; or the first domain comprises the amino acid residues at positions 24 to 161 of SEQ ID NO.1, and the second domain comprises the amino acid residues at positions 300 to 376 of SEQ ID NO.

1.

3. The polypeptide according to claim 1 or 2, wherein, A. the amino acid sequence of the first domain has at least 80% sequence identity with SEQ ID NO.2, and / or the amino acid sequence of the second domain has at least 80% sequence identity with SEQ ID NO.3; or B. the amino acid sequence of the first domain has at least 80% sequence identity with SEQ ID NO.4, and / or the amino acid sequence of the second domain has at least 80% sequence identity with SEQ ID NO.5; Optionally, wherein, a. the amino acid sequence of the first domain is as shown in SEQ ID NO.2, and the amino acid sequence of the second domain is as shown in SEQ ID NO.3; or b. the amino acid sequence of the first domain is as shown in SEQ ID NO.4, and the amino acid sequence of the second domain is as shown in SEQ ID NO.

5.

4. The polypeptide according to any one of claims 1 to 3, wherein, the polypeptide is shown as the following formula (I): (I): X-L1-Y, wherein, X is a first GUCY2C polypeptide fragment, Y is a second GUCY2C polypeptide fragment, and L1 is a linker; Optionally, A. the amino acid sequence of the first GUCY2C polypeptide fragment has at least 80% sequence identity with SEQ ID NO.2, and / or the amino acid sequence of the second GUCY2C polypeptide fragment has at least 80% sequence identity with SEQ ID NO.3; or B. the amino acid sequence of the first GUCY2C polypeptide fragment has at least 80% sequence identity with SEQ ID NO.4, and / or the amino acid sequence of the second GUCY2C polypeptide fragment has at least 80% sequence identity with SEQ ID NO.5; Optionally, the first GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.2, and the second GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.3; or the first GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.4, and the second GUCY2C polypeptide fragment comprises the amino acid sequence of SEQ ID NO.5; Optionally, the linker is a flexible peptide linker; optionally, the linker is selected from (G x S) y linkers, where x is an integer selected from 1 to 5 and y is an integer selected from 1 to 6; optionally, the linker is the linker shown in SEQ ID NO: 6 or 7; Optionally, the amino acid sequence of the polypeptide has at least 80% sequence identity with SEQ ID NO.8 or 9; optionally, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.8 or SEQ ID NO.

9. Optionally, the N-terminus of the polypeptide includes a signal peptide; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.10; Optionally, the C-terminus of the polypeptide includes a protein tag; optionally, the amino acid sequence of the protein tag is as shown in SEQ ID NO.11; Optionally, the amino acid sequence of the polypeptide has at least 80% sequence identity with SEQ ID NO.12 or 13; optionally, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.12 or SEQ ID NO.

13.

5. Use of the polypeptide according to any one of claims 1 to 4 in the preparation of an anti-human GUCY2C antibody; Optionally, the polypeptide is used as an immunogen for the preparation of an anti-human GUCY2C antibody; Optionally, the polypeptide is used for screening an anti-human GUCY2C antibody, and the anti-human GUCY2C antibody specifically binds to the polypeptide; Optionally, the binding activity of the anti-human GUCY2C antibody to the polypeptide shown in SEQ ID NO.8 or SEQ ID NO.12 is stronger than the binding activity to the polypeptide shown in SEQ ID NO.9 or SEQ ID NO.13; optionally, an anti-human GUCY2C antibody with a binding activity to the polypeptide shown in SEQ ID NO.8 more than 1-fold that of the binding activity to the polypeptide shown in SEQ ID NO.9 is selected; optionally, an anti-human GUCY2C antibody with a binding activity to the polypeptide shown in SEQ ID NO.8 more than 9-fold that of the binding activity to the polypeptide shown in SEQ ID NO.9 is selected; optionally, the binding activity of the anti-human GUCY2C antibody to the polypeptide is detected by ELISA.

6. A method for screening an anti-human GUCY2C antibody, the method comprising the step of detecting the binding activity of the anti-human GUCY2C antibody to the polypeptide according to any one of claims 1-4; Optionally, the screening method comprises the steps: A) Prepare the anti-human GUCY2C antibody to be tested; B) Detect the binding activity of the anti-human GUCY2C antibody obtained in step A) to the polypeptide shown in SEQ ID NO.8; C) Detect the binding activity of the anti-human GUCY2C antibody obtained in step A) to the polypeptide shown in SEQ ID NO.9; D) Compare the binding activities of the antibodies and polypeptides detected in steps B) and C), and select an anti-human GUCY2C antibody with a stronger binding activity to the polypeptide shown in SEQ ID NO.8 than to the polypeptide shown in SEQ ID NO.9; Optionally, select an anti-human GUCY2C antibody with a binding activity to the polypeptide shown in SEQ ID NO.8 that is more than 1-fold that to the polypeptide shown in SEQ ID NO.9; Optionally, select an anti-human GUCY2C antibody with a binding activity to the polypeptide shown in SEQ ID NO.8 that is more than 9-fold that to the polypeptide shown in SEQ ID NO.

9. Optionally, the binding activity of the anti-human GUCY2C antibody and the polypeptide in steps B) and C) is detected by ELISA.

7. An anti-human GUCY2C antibody that binds to the polypeptide according to any one of claims 1 to 4. Optionally, the affinity of the anti-human GUCY2C antibody for the polypeptide shown in SEQ ID NO.8 is higher than that for the polypeptide shown in SEQ ID NO.9; Optionally, the affinity of the anti-human GUCY2C antibody for the polypeptide shown in SEQ ID NO.8 is at least 50% higher than that for the polypeptide shown in SEQ ID NO.

8.

8. An isolated nucleic acid encoding the polypeptide according to any one of claims 1 to 4 or the anti-human GUCY2C antibody according to claim 7.

9. A polypeptide for detecting an anti-human GUCY2C antibody, which is the polypeptide according to any one of claims 1 to 4; Optionally, the polypeptide is the polypeptide shown in SEQ ID NO.8 or SEQ ID NO.12; or the polypeptide is the polypeptide shown in SEQ ID NO.9 or SEQ ID NO.

13.

10. A kit comprising the polypeptide according to any one of claims 1 to 4; Optionally, the polypeptide is the polypeptide shown in SEQ ID NO.8 or SEQ ID NO.12; or the polypeptide is the polypeptide shown in SEQ ID NO.9 or SEQ ID NO.13.

Images