CN116284408A - Antibody binding to human MUC17, preparation method and application thereof - Google Patents

Abstract

The present invention relates to novel antibodies or antigen-binding fragments thereof that specifically bind to human MUC17 and compositions containing the antibodies or antigen-binding fragments thereof. The invention also relates to polynucleotides encoding the antibodies or antigen binding fragments thereof and host cells comprising the same, as well as related uses. The invention furthermore relates to therapeutic and diagnostic uses of these antibodies or antigen binding fragments thereof.

Description

Antibodies binding to human MUC17, preparation methods and uses thereof

Technical Field

The present invention belongs to the field of tumor treatment and biotechnology, and relates to an antibody binding to human MUC17, a preparation method and use thereof.

Background Art

Mucin17 (MUC17) is a transmembrane protein expressed on the apical membrane of normal gastrointestinal mucosal epithelial cells. It belongs to the mucin family. Its primary structure includes a signal peptide, a large extracellular domain consisting of a central region of 61 tandem repeats, two epidermal growth factor (EGF) domains, aggregin (SEA) and a cytoplasmic tail with 80 amino acids. MUC17 is abnormally expressed in some cancers. For example, MUC17 mRNA is expressed in a pancreatic cell line and three colon cancer cell lines (Gun et al. 2002); immunohistochemical studies confirmed the expression of MUC17 protein in pancreatic cancer (Moniaux et al. 2006); MUC17 is overexpressed on the membrane of gastric cancer and gastroesophageal junction cancer cells; however, in colon cancer, MUC17 protein expression is low (Senapati et al., J. Clin. Pathol. 2010). Nevertheless, the expression pattern of MUC17 makes it a potential target for the treatment of different forms of malignant tumors.

Therefore, there is an urgent need to develop novel drugs targeting human MUC17 and to provide more and more effective treatment options for patients with diseases with abnormal MUC17 expression.

Summary of the invention

The purpose of the present invention is to provide an antibody or an antigen-binding fragment thereof that binds to human MUC17; to provide a polynucleotide molecule encoding the antibody or the antigen-binding fragment thereof that binds to human MUC17; to provide an expression vector comprising the polynucleotide molecule; to provide a host cell comprising the expression vector; to provide a method for preparing the antibody or the antigen-binding fragment thereof that binds to human MUC17; to provide a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof that binds to human MUC17; and to provide the use of the antibody or the antigen-binding fragment thereof that binds to human MUC17 in the preparation of drugs.

In a first aspect of the present invention, an antibody or antigen-binding fragment thereof that binds to human MUC17 is provided, wherein the antibody or antigen-binding fragment thereof that binds to human MUC17 comprises:

(a) heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of the HCDR1 is selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82 and 90, the amino acid sequence of the HCDR2 is selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83 and 91, and the amino acid sequence of the HCDR3 is selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84 and 92; and

(b) light chain complementary determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is selected from the group consisting of SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86 and 94, the amino acid sequence of LCDR2 is selected from the group consisting of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87 and 95, and the amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NO: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88 and 96.

In another preferred embodiment, the antibody or antigen-binding fragment thereof that binds to human MUC17 is characterized by:

(1) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 2, 3 and 4, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 6, 7 and 8, respectively;

(2) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 10, 11 and 12, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 14, 15 and 16, respectively;

(3) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 18, 19 and 20, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 22, 23 and 24, respectively;

(4) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 26, 27 and 28, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 30, 31 and 32, respectively;

(5) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 34, 35 and 36, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 38, 39 and 40, respectively;

(6) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 42, 43 and 44, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 46, 47 and 48, respectively;

(7) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 50, 51 and 52, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 54, 55 and 56, respectively;

(8) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 58, 59 and 60, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 62, 63 and 64, respectively;

(9) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 66, 67 and 68, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 70, 71 and 72, respectively;

(10) the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 74, 75 and 76, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 78, 79 and 80, respectively;

(11) the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 82, 83 and 84, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 86, 87 and 88, respectively; or

(12) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 90, 91 and 92, respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs: 94, 95 and 96, respectively.

In another preferred embodiment, the antibody that binds to human MUC17 is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.

In another preferred embodiment, the antibody that binds to human MUC17 is a single-chain antibody or a double-chain antibody.

In another preferred embodiment, the antibody that binds to human MUC17 is a murine antibody, a chimeric antibody or a humanized antibody.

In another preferred embodiment, the antibody that binds to human MUC17 is a humanized antibody.

In another preferred embodiment, the antigen-binding fragment that binds to human MUC17 includes a Fab fragment, a F(ab’)2 fragment, a Fab’ fragment, a Fv fragment or a single-chain Fv (scFv) fragment.

In another preferred embodiment, the antigen-binding fragment that binds to human MUC17 comprises a heavy chain variable region (VH) and a light chain variable region (VL),

(1) the amino acid sequence of VH is selected from the group consisting of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 99 and 103; and

(2) The amino acid sequence of VL is selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 100 and 104.

In another preferred embodiment, the antibody or antigen-binding fragment thereof that binds to human MUC17 is characterized by:

(1) the amino acid sequence of the VH is shown in SEQ ID NO: 1, and the amino acid sequence of the VL is shown in SEQ ID NO: 5;

(2) the amino acid sequence of the VH is shown in SEQ ID NO: 9, and the amino acid sequence of the VL is shown in SEQ ID NO: 13;

(3) the amino acid sequence of the VH is shown in SEQ ID NO: 17, and the amino acid sequence of the VL is shown in SEQ ID NO: 21;

(4) the amino acid sequence of the VH is as shown in SEQ ID NO: 25, and the VL comprises the amino acid sequence as shown in SEQ ID NO: 29;

(5) the amino acid sequence of the VH is shown in SEQ ID NO: 33, and the amino acid sequence of the VL is shown in SEQ ID NO: 37;

(6) the amino acid sequence of the VH is shown in SEQ ID NO: 41, and the amino acid sequence of the VL is shown in SEQ ID NO: 45;

(7) the amino acid sequence of the VH is shown in SEQ ID NO: 49, and the amino acid sequence of the VL is shown in SEQ ID NO: 53;

(8) the amino acid sequence of the VH is shown in SEQ ID NO: 57, and the amino acid sequence of the VL is shown in SEQ ID NO: 61;

(9) the amino acid sequence of the VH is shown in SEQ ID NO: 65, and the amino acid sequence of the VL is shown in SEQ ID NO: 69;

(10) the amino acid sequence of the VH is shown in SEQ ID NO: 73, and the amino acid sequence of the VL is shown in SEQ ID NO: 77;

(11) the amino acid sequence of the VH is shown in SEQ ID NO: 81, and the amino acid sequence of the VL is shown in SEQ ID NO: 85;

(12) The amino acid sequence of the VH is shown in SEQ ID NO: 89, and the amino acid sequence of the VL is shown in SEQ ID NO: 93;.

(13) the amino acid sequence of the VH is shown in SEQ ID NO: 99, and the amino acid sequence of the VL is shown in SEQ ID NO: 100; or

(14) The amino acid sequence of the VH is shown in SEQ ID NO: 103, and the amino acid sequence of the VL is shown in SEQ ID NO: 104.

In another preferred embodiment, the antibody or antigen-binding fragment thereof that binds to human MUC17 comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region is selected from κ or λ light chain constant region; preferably, the heavy chain constant region is IgG1 heavy chain constant region, and the light chain constant region is κ light chain constant region; preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 97, and the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 98.

In another preferred embodiment, the antibody or antigen-binding fragment thereof that binds to human MUC17 comprises a heavy chain and a light chain selected from:

(1) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 101, and the amino acid sequence of the light chain is shown in SEQ ID NO: 102; and

(2) The amino acid sequence of the heavy chain is shown in SEQ ID NO: 105, and the amino acid sequence of the light chain is shown in SEQ ID NO: 106.

In the second aspect of the present invention, an isolated polynucleotide molecule is provided, wherein the polynucleotide molecule encodes the antibody or antigen-binding fragment thereof that binds to human MUC17 as described in the first aspect of the present invention.

In the third aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide molecule as described in the second aspect of the present invention.

In another preferred embodiment, the expression vector is a virus or a plasmid, preferably a phage or a phagemid.

In another preferred embodiment, the expression vector is selected from the following group: pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, preferably pTT5.

In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector according to the third aspect of the present invention.

In another preferred embodiment, the host cell is selected from the following group: COS, CHO, 293F, 293E, NS0, sf9, sf21, DH5α, BL21 (DE3) or TG1, more preferably E. coli TG1, BL21 (DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies).

In another preferred embodiment, the host cell is a eukaryotic cell, preferably a CHO cell, a 293F cell or a 293E cell.

In a fifth aspect of the present invention, a method for preparing an antibody or an antigen-binding fragment thereof that binds to human MUC17 as described in the first aspect of the present invention is provided, the method comprising the following steps:

(a) culturing the host cell according to the fourth aspect of the present invention under expression conditions to express an antibody or an antigen-binding fragment thereof that binds to human MUC17;

(b) isolating and purifying the antibody or antigen-binding fragment thereof that binds to human MUC17 described in step (a).

In the sixth aspect of the present invention, a pharmaceutical composition is provided, comprising an effective amount of an antibody or an antigen-binding fragment thereof that binds to human MUC17 as described in the first aspect of the present invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.

In the seventh aspect of the present invention, there is provided a use of an antibody or antigen-binding fragment thereof that binds to human MUC17 as described in the first aspect of the present invention, or a pharmaceutical composition as described in the sixth aspect of the present invention in the preparation of a drug for cancer or tumor.

In another preferred embodiment, the cancer or tumor is selected from: pancreatic cancer, colorectal cancer, colon cancer, rectal cancer, colorectal cancer, small intestine cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, gastroesophageal cancer or a combination thereof.

In an eighth aspect of the present invention, an immunoconjugate is provided, comprising:

(a) an antibody or antigen-binding fragment thereof that binds to human MUC17 as described in the first aspect of the present invention; and

(b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

In another preferred embodiment, the conjugate portion is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.).

In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate (ADC).

In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating cancer or tumor.

In the ninth aspect of the present invention, a method for treating cancer or tumor is provided, the method comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof that binds to human MUC17 as described in the first aspect of the present invention, a pharmaceutical composition as described in the sixth aspect of the present invention, or an immunoconjugate as described in the eighth aspect of the present invention.

In another preferred embodiment, the method further comprises administering the drug in combination with other anti-tumor drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows ELISA detection of the binding of hybridoma antibodies to MUC17 antigen.

FIG. 2 shows the binding of hybridoma antibodies to LS174T cells detected by FACS.

FIG3 shows the ELISA detection of the binding of chimeric antibodies to the MUC17 antigen.

4A-4B show the binding of chimeric antibodies to LS174T cells detected by FACS.

FIG5 shows the ELISA detection of the binding of humanized antibodies to MUC17 antigen.

FIG. 6A shows the binding of humanized antibody hu-27A3 to LS174T cells detected by FACS; FIG. 6B shows the binding of humanized antibody hu-19E11 to LS174T cells detected by FACS.

DETAILED DESCRIPTION

After extensive and in-depth research, from antigen immunization, hybridoma screening, antibody expression and purification to biological activity identification, the inventors screened and obtained a new mouse monoclonal antibody that can specifically bind to human MUC17, and further constructed and obtained its chimeric antibody and humanized antibody. The antibody of the present invention has high affinity and specificity for human MUC17, can be used to prepare drugs for treating cancer or tumors, and has good clinical application prospects.

the term

In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

In the present invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" are heterotetrameric proteins with the same structural characteristics, which are composed of two identical light chains (L) and two identical heavy chains (H), each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes (isotypes) is different, and each heavy chain and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end. The light chain constant region includes a domain CL. The constant region of the light chain is paired with the CH1 domain of the heavy chain constant region, and the variable region of the light chain is paired with the variable region of the heavy chain. The constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes. The light chain constant region includes κ (Kappa) or λ (Lambda). The heavy chain and light chain of the antibody are covalently linked together by a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of the antibody are covalently linked together by an inter-polypeptide disulfide bond formed between the hinge region.

In the present invention, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a class of substantially homogeneous populations, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies for different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by culturing hybridoma cells and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring any special method to produce the antibody.

In the present invention, the term "mouse antibody" refers to an antibody derived from rats or mice. The murine antibodies of the present invention are obtained by immunizing mice with human MUC17 as an antigen and screening hybridoma cells. More preferably, the murine antibodies of the present invention include 8H2, 19E11, 23F6, 27A3, 42H4, 44H4, 11C8, 15D10, 45D1, 50A5, 51E12 and 56E8.

In the present invention, the term "chimeric antibody" refers to an antibody comprising a heavy chain variable region and a light chain variable region sequence derived from one species and a constant region sequence derived from another species, such as an antibody having a mouse heavy chain variable region and a light chain variable region connected to a human constant region. Preferably, the heavy chain of the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region sequence of a mouse antibody with the IgG1 constant region of a human, and the light chain is obtained by splicing the light chain variable region of a mouse antibody with the Kappa chain of a human. More preferably, the chimeric antibodies of the present invention include ch-8H2, ch-19E11, ch-23F6, ch-27A3, ch-42H4, ch-44H4, ch-11C8, ch-15D10, ch-45D1, ch-50A5, ch-51E12 and ch-56E8.

In the present invention, the term "humanized antibody" refers to an antibody whose CDR is derived from a non-human species (preferably mouse), and the remaining part of the antibody molecule (including the framework region and the constant region) is derived from a human antibody. In addition, the framework region residues can be changed to maintain binding affinity. Preferably, the humanized antibody of the present invention is recombined by the CDR region of the mouse antibody 27A3 or 19E11 and the non-CDR region derived from a human antibody, and the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important influence on the conformation of the VL and VH of 27A3 or 19E11 are back mutated to obtain. More preferably, the humanized antibodies of the present invention include hu-27A3 and hu-19E11.

In the present invention, the term "antigen-binding fragment" refers to a fragment of an antibody that can specifically bind to human MUC17. Non-limiting examples of the antigen-binding fragment of the present invention include Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, single-chain antibodies (scFv), single-domain antibodies (sdAb), etc. The Fab fragment is a fragment of an antibody that is digested with papain and cleaved into two identical Fab segments and an Fc segment. The Fab segment consists of the VH and CH1 of the heavy chain of the antibody and the VL and CL domains of the light chain. The F(ab')2 fragment is a fragment produced by digesting an antibody with pepsin. The F(ab')2 fragment has two antigen-binding Fab' parts linked together by a disulfide bond. The Fv fragment is composed of a dimer in which the heavy chain variable region and the light chain variable region of the antibody are tightly non-covalently associated. A single-chain antibody (scFv) is an antibody formed by connecting the heavy chain variable region and the light chain variable region of the antibody through a short peptide linker of 15 to 20 amino acids. Single-domain antibodies (sdAbs), also known as nanobodies or heavy-chain antibodies, are composed only of heavy chains, and their antigen-binding region is only a single domain connected to the Fc region through a hinge region.

In the present invention, the term "variable" means that some parts of the variable region in the antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The more conservative part of the variable region is called the framework region (FR). The variable region of the natural heavy chain and light chain each contains four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC).

As used herein, the term "framework region (FR)" refers to the amino acid sequence inserted between CDRs, i.e., refers to those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved between different immunoglobulins in a single species. The light chain and heavy chain of an immunoglobulin each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain can therefore be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially identical to the naturally occurring human antibody FR, i.e., the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

Knowing the amino acid sequence of the CDRs, one skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.

As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody.

In the present invention, the terms "anti", "binding", "specific binding" refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. Typically, an antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about ^10-7 M, for example, less than about ^10-8 M, ^10-9 M, ^10-10 M, ^10-11 M or less. In the present invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, the binding affinity of an antibody to an antigen is measured in a BIACORE instrument using surface plasmon resonance (SPR) or the relative affinity of an antibody to an antigen is measured using ELISA.

In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitope of the present invention is a region of an antigen that is bound by an antibody.

Encoding nucleic acid and expression vector

Another aspect of the present invention provides a polynucleotide molecule, which encodes the above-mentioned antibody or antigen-binding fragment thereof that binds to human MUC17. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

The preparation method of the polynucleotide molecule described in the present invention is a conventional preparation method in the art, and preferably includes the following preparation method: obtaining a polynucleotide molecule encoding the above-mentioned antibody or its antigen-binding fragment through gene cloning technology such as PCR method, or obtaining a polynucleotide molecule encoding the above-mentioned antibody or its antigen-binding fragment through artificial full sequence synthesis method.

Those skilled in the art will appreciate that the nucleotide sequence encoding the amino acid sequence of the above-mentioned antibody or antigen-binding fragment thereof may be appropriately introduced with substitution, deletion, alteration, insertion or addition to provide a homologue of a polynucleotide. The homologue of the polynucleotide of the present invention may be prepared by replacing, deleting or adding one or more bases of the gene encoding the antibody or antigen-binding fragment thereof within the range of maintaining the antibody activity.

Another aspect of the present invention provides an expression vector, which contains the above-mentioned polynucleotide molecule.

The expression vector is a conventional expression vector in the art, which refers to an expression vector containing appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences and other appropriate sequences. The expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid. For more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, 1989. For many known techniques and protocols for nucleic acid manipulation, please refer to Current Protocols in Molecular Biology, Second Edition, compiled by Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.4, pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, and more preferably pTT5.

The present invention further provides a host cell, wherein the host cell contains the above expression vector.

The host cell described in the present invention is any conventional host cell in the art, as long as the above-mentioned recombinant expression vector can be stably replicated by itself and the nucleotide carried can be effectively expressed. The host cell includes prokaryotic expression cells and eukaryotic expression cells, and the host cell preferably includes: COS, CHO (Chinese Hamster Ovary), 293E, 293F, NS0, sf9, sf21, DH5α, BL21 (DE3) or TG1, and more preferably E.coli TG1, BL21 (DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies). The aforementioned expression vector is transformed into a host cell to obtain a preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

As a preferred embodiment, the host cell is a eukaryotic cell, preferably a CHO cell, a 293F cell or a 293E cell.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

Another aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof that binds to human MUC17, the preparation method comprising the following steps:

(a) culturing the host cell described above under expression conditions to express an antibody or an antigen-binding fragment thereof that binds to human MUC17;

(b) isolating and purifying the antibody or antigen-binding fragment thereof that binds to human MUC17 described in step (a).

The host cell culture method and the antibody separation and purification method described in the present invention are conventional methods in the art. For specific operation methods, please refer to the corresponding cell culture technology manual and antibody separation and purification technology manual. The preparation method of the antibody or antigen-binding fragment thereof that binds to human MUC17 disclosed in the present invention comprises: culturing the above-mentioned host cell under expression conditions to express the antibody or antigen-binding fragment thereof that binds to human MUC17; isolating and purifying the antibody or antigen-binding fragment thereof that binds to human MUC17. Using the above method, the recombinant protein can be purified into a substantially uniform substance.

The antibody or antigen-binding fragment thereof binding to human MUC17 disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, the antibody or antigen-binding fragment thereof binding to human MUC17 bound to the affinity column can be eluted by conventional methods such as high salt buffer, pH change, etc. The inventors of the present invention conducted a detection experiment on the obtained antibody or antigen-binding fragment thereof binding to human MUC17, and the experimental results showed that the anti-MUC17 antibody can bind well to the target cells and has a high affinity.

Pharmaceutical composition

Another aspect of the present invention provides a composition comprising the above-mentioned antibody or antigen-binding fragment thereof that binds to human MUC17, and one or more pharmaceutically acceptable carriers, diluents or excipients. Preferably, the composition is a pharmaceutical composition.

The anti-MUC17 antibody or antigen-binding fragment thereof provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert the therapeutic effect more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the antibody of the present invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to coagulation, deamination or oxidation). Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-8, preferably about 5-7 or 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, or intracavitary injection. Generally, for liquid preparations, they can usually be stored at 2°C-8°C for at least one year, and for lyophilized preparations, they can be kept stable at 30°C for at least six months. The antibody preparation can be a suspension, aqueous injection, freeze-dried preparation, etc. commonly used in the pharmaceutical field.

In the present invention, the term "pharmaceutical composition" refers to the antibody or antigen-binding fragment thereof that binds to human MUC17 of the present invention, and a pharmaceutically acceptable carrier that together constitute a pharmaceutical preparation composition so as to more stably exert the therapeutic effect. These preparations can ensure the conformational integrity of the amino acid core sequence of the antibody disclosed in the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to aggregation, deamination or oxidation).

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 milligrams/kg body weight per day. In addition, the antibodies of the present invention can also be used with other therapeutic agents.

When using a pharmaceutical composition, a safe and effective amount of the antibody or its immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 milligrams/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 milligrams/kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

application

Another aspect of the present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof that binds to human MUC17, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing, diagnosing, or treating cancer or tumors.

The drugs for treating cancer or tumors referred to in the present invention refer to drugs that have the function of inhibiting and/or treating tumors, which may include delaying the development of tumor-related symptoms and/or reducing the severity of these symptoms, further including alleviating existing tumor-associated symptoms and preventing the occurrence of other symptoms, and also including reducing or preventing tumor metastasis, etc.

The tumors targeted by the drug of the present invention preferably include but are not limited to: pancreatic cancer, colorectal cancer, colon cancer, rectal cancer, colorectal cancer, small intestine cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, gastroesophageal cancer or a combination thereof.

When the anti-MUC17 antibody and the composition thereof of the present invention are administered to animals including humans, the dosage varies depending on the patient's age and weight, the characteristics and severity of the disease, and the route of administration. The total dosage may be determined by reference to the results of animal experiments and various circumstances. The anti-MUC17 antibody and the composition thereof of the present invention may also be administered in combination with other anti-tumor drugs to achieve the purpose of more effectively treating tumors.

The anti-MUC17 antibodies or antigen-binding fragments thereof, compositions or conjugates thereof of the present invention can also be used to detect the presence or level of MUC17 in a sample.

Beneficial Effects

The present invention provides novel antibodies and antigen-binding fragments thereof that bind to human MUC17, which have high affinity and specificity for human MUC17 and are expected to become better potential therapeutic drugs for patients with diseases with abnormal MUC17 expression.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

The experimental materials and sources used in the following examples and the preparation methods of the reagents are specifically described as follows.

Experimental reagents and materials:

Mouse myeloma cells SP2/0: purchased from ATCC, catalog number CRL-1581;

Balb/c mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd.

LS174T cells: ATCC, catalog number CL-188 ^TM ;

Sheep anti-mouse secondary antibody: purchased from Bio-Long Immunotechnology Co., Ltd., catalog number BF03001-1ML;

Donkey anti-mouse PE fluorescent secondary antibody: purchased from Jackson, catalog number 715-116-150;

Goat anti-human PE fluorescent secondary antibody: purchased from Jackson, catalog number 109-115-098;

96-well ELISA plate: purchased from costar, catalog number 9018;

HRP-labeled goat anti-human Fc antibody: purchased from Sigma, catalog number A0170;

PBS buffer: Shanghai Yuanpei Biotechnology Co., Ltd., catalog number B320KJ;

Bovine serum albumin (BSA): purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number A600332-0100;

PBST: PBS + 0.05% Tween 20;

TMB: purchased from BD, catalog number 555214;

RPMI 1640 Medium: purchased from Gibco, catalog number 61870127;

FBS: purchased from Gibco, catalog number 10099;

Penicillin-streptomycin: purchased from Gibco, catalog number 15140122;

Fetal bovine serum: purchased from Gibco, catalog number 10091-148;

Polyethylene glycol solution (PEG): purchased from Sigma, product number P7181;

Hybridoma-SFM: purchased from life technologies, catalog number 12045-076;

HAT: purchased from Gibco, catalog number 21060017;

The experimental instruments used in the following examples are described as follows:

PCR instrument: purchased from BioRad, product number C1000 Touch Thermal Cycler;

Beckman Coulter CytoFLEX flow cytometer: purchased from Beckman;

SpectraMax M5 microplate reader: purchased from Molecular Devices.

Example 1. Antigen immunization of animals and preparation and screening of hybridoma cells

1. Antigen Immunization of Mice

Balb/c mice were routinely immunized with human MUC17-His protein expressed in mammalian cells 293E (according to the sequence Q685J3 provided by UniProtKB, the amino acid aa4131-4390 segment was taken, a signal peptide sequence was added to its N-terminus, and a His tag was added to the C-terminus, and the two restriction sites of EcoRI and HindIII were respectively constructed into the expression vector, transfected into HEK-293E cells for expression and purified, and the purity after expression and purification was >95%). On the first day, the soluble human MUC17-His protein was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple points into Balb/c mice (human MUC17-His 100 μg/mouse/0.5 mL); on the 14th day, the soluble human MUC17-His protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously into Balb/c mice (human MUC17-His 50 μg/mouse/0.5 mL); on the 28th day, the soluble human MUC17-His protein was emulsified with Freund's incomplete adjuvant and injected subcutaneously into Balb/c mice (human MUC17-His 50 μg/mouse/0.5 mL); three weeks later, the soluble human MUC17-His protein, 50 μg/mouse/0.2 mL, was stimulated by intraperitoneal injection, and 3-4 days later, the spleens of the mice were taken for fusion experiments.

2. Preparation and Screening of Hybridoma Cells

3-4 days after the last immunization of mice, mouse spleen cells were PEG-fused with mouse myeloma cells SP2/0 using conventional hybridoma technology protocols. The fused cells were evenly suspended in complete culture medium, which was a culture medium composed of RPMI1640-GLUMAX with 1% Penicillin-streptomycin, 20% FBS (fetal bovine serum), and 1×HAT. The fused cells were cultured in 60 96-well culture plates at 3×10 ⁴ cells/200μL/well. After 7-12 days, the supernatant was harvested and the hybridoma wells with positive human MUC17 binding activity were screened by indirect enzyme-linked immunosorbent assay (ELISA).

Among them, the method of screening the hybridoma wells with positive human MUC17 binding activity by ELISA is as follows: MUC17-Fc (according to the sequence Q685J3 provided by UniProtKB, the amino acid aa4131-4390 sequence is taken, the signal peptide sequence is added to its N-terminus, and the Fc tag is added to the C-terminus, and the expression vector is constructed through the two restriction sites of EcoRI and HindIII, and HEK-293E cells are transfected for expression and purified) is diluted to 1μg/mL with PBS buffer, 100μL/well is added to the plate, and cultured at 4℃ overnight. The next day, the supernatant is discarded, 5% skim milk powder is added to block at 37℃ for 2 hours, and the plate is washed 3 times with PBST for standby use. The collected hybridoma supernatant is added to the blocked plate in sequence, 100μL/well, and placed at 37℃ for 1 hour. Wash the plate 3 times with PBST, add HRP-labeled goat anti-mouse IgG secondary antibody, and place at 37°C for 30 minutes; after washing the plate 3 times with PBST, pat dry the residual droplets on absorbent paper as much as possible, add 100 μL of TMB to each well, and place it at room temperature (20±5°C) away from light for 5 minutes; add 50 μL of 2M H ₂ SO ₄ stop solution to each well to terminate the substrate reaction, read the OD value at 450nm on an enzyme reader, and analyze the binding ability of the test antibody to the target antigen MUC17. A total of 12 hybridoma cell lines were obtained through screening. The 12 hybridoma cell lines obtained by amplification and screening in serum-containing complete medium were centrifuged and replaced with serum-free medium Hybridoma-SFM medium to a cell density of 1-2×10 ⁷ /mL. The cells were cultured at 8% CO ₂ and 37°C for 1 week, and the culture supernatant was obtained by centrifugation. The supernatant was purified by Protein G affinity chromatography to obtain anti-human MUC17 monoclonal antibody proteins, which were named 8H2, 19E11, 23F6, 27A3, 42H4, 44H4, 11C8, 15D10, 45D1, 50A5, 51E12 and 56E8, respectively.

Example 2. Binding ability of mouse antibodies to human MUC17-Fc protein

ELISA was used to determine the binding ability of mouse antibodies to human MUC17-Fc protein. The specific method is as follows:

The MUC17-Fc protein was diluted to 1 μg/mL with coating solution (50 mM carbonate coating buffer, pH 9.6) and coated at 4°C overnight; then blocked with 5% skim milk powder and incubated at 37°C for 2 hours; after washing the plate 3 times with PBST, the anti-human MUC17 antibody protein prepared in Example 1 was diluted 3 times from 1 μg/mL in 11 gradients with 1% BSA buffer, and 100 μL/well was added to the pre-coated MUC17-Fc plate and incubated at 37°C for 1 hour; the plate was washed 3 times with PBST, and HRP-labeled goat anti-mouse IgG secondary antibody was added and placed at 37°C for 30 minutes; after washing the plate 3 times with PBST, the residual droplets were patted dry on absorbent paper as much as possible, 100 μL of TMB was added to each well, and the plate was placed at room temperature (20±5°C) away from light for 5 minutes, and 50 μL of 2M H ₂ SO _4. Stop the substrate reaction with the stop solution, read the OD value at 450nm on the microplate reader, and analyze the binding ability of the test antibody to the target antigen human MUC17-Fc. GraphPad Prism9 was used for data analysis, plotting and calculating EC _50. The results are shown in Figure 1. The data are shown in Table 1. The clone with the strongest affinity is 27A3; among them, 42H4 and 11C8 have poor affinity.

Table 1. ELISA detection of the binding of hybridoma antibodies to human MUC17 antigen

Sample 42H4 27A3 8H2 56E8 50A5 11C8 Top 2.605 2.419 2.737 1.97 2.644 2.253 HillSlope 0.9669 1.281 0.8767 1.143 0.9144 0.748 _EC50 (ng/mL) 237 20.2 55.56 27.57 81.34 108 Sample 23F6 44H4 15D10 51E12 45D1 19E11 Top 2.883 2.696 2.787 2.883 2.865 2.528 HillSlope 0.957 1.081 1.369 1.212 1.063 1.181 _EC50 (ng/mL) 62.33 67.69 31.71 37.15 35.96 37.55

Example 3. Binding ability of mouse antibodies to colorectal cancer cell LS174T

Flow cytometry (FACS) was used to detect the binding activity of mouse antibodies to colorectal cancer LS174T cells. The specific method is as follows:

LS174T cells were collected, centrifuged to remove the cell culture medium, and washed twice with PBS buffer; counted and diluted to 2×10 ⁶ cells/mL with 1% BSA FACS buffer, and cells were plated into 96-well round-bottom plates for use, 100 μL/well; the anti-human MUC17 antibody protein prepared in Example 1 was diluted 3 times from 1 μg/mL with 1% BSA buffer in 11 gradients and added to the above-mentioned cell round-bottom plates, and incubated at 4°C for 1 hour; after centrifugation, the supernatant was discarded, washed 3 times with 1% BSA FACS buffer, and 100 μL of donkey anti-mouse PE fluorescent secondary antibody or goat anti-human PE fluorescent secondary antibody was added to each well at a ratio of 1:500 (see the instructions for fluorescent secondary antibody for details), incubated at 4°C for 1 hour; washed 3 times with 1% BSA FACS buffer, and then resuspended with 1% BSA FACS buffer, 200 μL/well, and the samples were measured and analyzed using BD FACSCelesta ^TM . The obtained data were fitted and analyzed using GraphPad Prism9, and the graphs were drawn and EC ₅₀ was calculated. The results of the antibody binding to cells are shown in FIG2 , and the data are shown in Table 2.

As shown in Table 2, the hybridoma antibody 27A3 has the strongest affinity, followed by 19E11 and 51E12.

Table 2. FACS detection of the binding of hybridoma antibodies to LS174T cells

Sample 42H4 27A3 8H2 56E8 50A5 11C8 Top 725.8 734.9 444.6 296.2 500.4 468.9 HillSlope 1.335 1.422 1.276 1.448 1.166 0.9413 _EC50 (ng/mL) 468.3 6.562 164.3 110.3 541.6 869.6 Sample 23F6 44H4 15D10 51E12 45D1 19E11 Top 459.5 574 382.1 878.4 456.7 560.8 HillSlope 1.219 1.407 0.959 1.279 1.079 1.457 _EC50 (ng/mL) 395.4 48.8 3426 47.21 273.2 23.98

Example 4. Preparation and activity identification of chimeric antibodies against human MUC17

1. Preparation of Chimeric Antibodies

The antibody genes of the 12 hybridoma cells prepared in Example 1 were obtained.

In this example, the heavy chain variable region and light chain variable region of the hybridoma were obtained by molecular biology related methods, and chimeric antibodies were further constructed.

RNA of hybridoma cells was extracted by Trizol and reverse transcribed by mRNA to obtain cDNA. Then, cDNA was used as a template to perform PCR with degenerate primers for the heavy chain and light chain of mouse antibody (Antibody Engineering Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combined primers is from page 323). The obtained PCR products were sequenced and analyzed by Kabat database to determine that the obtained sequences were the variable region sequences of mouse antibody. The sequence information is shown in Table 6.

The obtained heavy chain variable region sequence was spliced with the human IgG1 constant region (amino acid sequence as shown in SEQ ID NO: 97), and the light chain variable region sequence was spliced with the human kappa chain constant region (amino acid sequence as shown in SEQ ID NO: 98), and the heavy chain and light chain of the chimeric antibody were respectively constructed into the pTT5 expression vector, transfected into HEK-293E cells, and the chimeric antibodies were obtained by Protein A affinity purification, sterile filtered through a 0.22 μM filter membrane, and stored at 4° C. for use, and were named ch-8H2, ch-19E11, ch-23F6, ch-27A3, ch-42H4, ch-44H4, ch-11C8, ch-15D10, ch-45D1, ch-50A5, ch-51E12 and ch-56E8, respectively.

2. Determination of the affinity of chimeric antibodies for the target antigen human MUC17

The affinity of the chimeric antibody to human MUC17 was determined by ELISA (refer to Example 2 for the experimental method, the secondary antibody was HRP-goat anti-human Fc). The obtained data were fitted and analyzed using GraphPad Prism9, and the graph was plotted and EC ₅₀ was calculated.

The results are shown in Figure 3 and the data are shown in Table 3. The results show that the chimeric antibody has a binding affinity to human MUC17 comparable to that of the mouse antibody. From the ELISA results, it can be seen that except for the low high platform of the chimeric antibody ch-56E8 and the fact that ch-42H4 did not detect binding to the antigen MUC17 under the experimental conditions, the affinity of the other clones is acceptable.

Table 3. ELISA detection of affinity of chimeric antibodies to MUC17

Sample ch-8H2 ch-19E11 ch-23F6 ch-27A3 ch-42H4 ch-44H4 Top 2.53 2.328 2.106 2.31 7.583 2.26 HillSlope 1.368 1.335 1.304 1.347 1.622 1.386 _EC50 (nM) 0.1164 0.1404 0.1203 0.07855 2646 0.1247 Sample ch-11C8 ch-15D10 ch-45D1 ch-50A5 ch-51E12 ch-56E8 Top 1.984 2.465 2.516 2.004 2.315 1.697 HillSlope 1.248 1.362 1.261 1.232 1.37 1.148 _EC50 (nM) 0.1553 0.1172 0.1233 0.1747 0.2178 0.1207

3. Determination of the binding affinity of chimeric antibodies to colorectal cancer LS174T cells

The binding affinity of the chimeric antibody to colorectal cancer LS174T cells was determined by FACS (refer to Example 3 for the experimental method). The obtained data were fitted and analyzed using GraphPad Prism9, and the graph was plotted and EC ₅₀ was calculated.

The results are shown in Figures 4A-4B. The data are shown in Tables 4A-4B. As can be seen from the data, ch-27A3, ch-19E11 and ch-51E12 have a strong affinity for binding to LS174T cells.

Table 4A. FACS detection of the binding of chimeric antibodies to LS174T cells

Sample ch-8H2 ch-23F6 ch-27A3 ch-42H4 Top 7034 8046 7961 5975 HillSlope 0.9588 1.041 0.7482 0.8707 _EC50 (nM) 1.762 4.866 0.04347 10.32

Table 4B. FACS detection of the binding of chimeric antibodies to LS174T cells

Example 5. Preparation of humanized antibodies against human MUC17

The mouse antibodies 27A3 and 19E11 with stronger affinity were selected for antibody humanization.

The amino acid sequences of the light chain variable region and the heavy chain variable region were analyzed, and the three antigen complementary determining regions (CDRs) and four framework regions (FRs) of the murine antibodies 27A3 and 19E11 were determined according to the Kabat rule. The heavy chain variable region sequence of 27A3 is shown in SEQ ID NO: 25, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 26, 27 and 28, respectively; the light chain variable region sequence of 27A3 is shown in SEQ ID NO: 29, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 30, 31 and 32, respectively. The heavy chain variable region sequence of 19E11 is shown in SEQ ID NO: 9, and HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs: 10, 11 and 12, respectively; the light chain variable region sequence of 19E11 is shown in SEQ ID NO: 13, and the LCDR1, LCDR2 and LCDR3 amino acid sequences are shown in SEQ ID NOs: 14, 15 and 16, respectively.

The humanized template that best matches the non-CDR region of 27A3 was selected from the Germline database. The CDR region of 27A3 was then transplanted onto the selected humanized template to replace the CDR region of the humanized template. At the same time, based on the three-dimensional structure of the antibody, the buried residues, the residues that directly interact with the CDR region, and the residues that have an important influence on the conformation of VL and VH of 27A3 were back-mutated to obtain the heavy chain variable region (SEQ ID NO: 99) and light chain variable region (SEQ ID NO: 100) of humanized 27A3. The back-mutated variable region sequence was recombined with human IgG1 (SEQ ID NO: 97) and Kappa chain constant region (SEQ ID NO: 98) to obtain the full-length heavy chain (SEQ ID NO: 101) and full-length light chain (SEQ ID NO: 102) of the 27A3 humanized antibody (hu-27A3). The heavy chain and light chain gene sequences of 27A3 were linked to the pTT5 expression vector, transfected into HEK-293E cells, and purified by Protein A affinity to obtain humanized antibodies. After sterile filtration through a 0.22 μM filter membrane, the antibody was stored at 4°C for later use.

Similarly, a humanized template that best matches the non-CDR region of 19E11 was selected from the Germline database. The CDR region of 19E11 was then transplanted onto the selected humanized template to replace the CDR region of the humanized template. At the same time, based on the three-dimensional structure of the antibody, the buried residues, residues that directly interact with the CDR region, and residues that have an important influence on the conformation of VL and VH of 19E11 were back-mutated to obtain the heavy chain variable region (SEQ ID NO: 103) and light chain variable region (SEQ ID NO: 104) of humanized 19E11. The back-mutated variable region sequence was recombined with human IgG1 (SEQ ID NO: 97) and Kappa chain constant region (SEQ ID NO: 98) to obtain the full-length heavy chain (SEQ ID NO: 105) and full-length light chain (SEQ ID NO: 106) of the 19E11 humanized antibody (hu-19E11). The heavy chain and light chain gene sequences of 19E11 were linked to the pTT5 expression vector, transfected into HEK-293E cells, and purified by Protein A affinity to obtain humanized antibodies. After sterile filtration through a 0.22 μM filter membrane, the antibody was stored at 4°C for later use.

Example 6. Affinity identification of humanized antibodies against human MUC17

1. ELISA to detect the affinity of humanized antibodies to the target antigen MUC17

The affinity of the humanized antibody to MUC17 was determined by ELISA (refer to Example 2 for the experimental method). The obtained data were fitted and analyzed using GraphPad Prism9, and the graph was plotted and EC ₅₀ was calculated.

The results are shown in Figure 5 and Table 5. The results showed that the humanized 27A3 and 19E11 well maintained the affinity of the original mouse antibodies.

Table 5. ELISA detection of affinity of hu-27A3 and hu-19E11 humanized antibodies to MUC17

Sample ch-27A3 hu-27A3 ch-19E11 hu-19E11 _EC50 (nM) 0.07855 0.07659 0.1404 0.1311

2. Determination of the binding affinity of humanized antibodies to colorectal cancer cells LS174T

The binding affinity of the humanized antibody to colorectal cancer cell LS174T was determined by FACS (refer to Example 3 for the experimental method). The obtained data were fitted and analyzed using GraphPad Prism9, and the graph was plotted and EC ₅₀ was calculated.

The results are shown in Figures 6A-6B. The EC ₅₀ of hu-27A3 is 0.03nM, and the EC ₅₀ of hu-19E11 is 0.085nM, and the affinity of hu-27A3 is higher than that of hu-19E11. The results show that the humanized 27A3 and 19E11 have well maintained the affinity of the original mouse antibodies.

In summary, the present invention immunizes mice with MUC17 protein, fuses mouse spleen cells with myeloma cells, and performs affinity screening of anti-MUC17 hybridoma antibodies. The antibody gene sequences of 12 hybridomas were obtained by retrieving and sequencing the genes of the hybridomas. Chimeric antibodies were constructed and affinity compared. According to the affinity and sequencing comparison results, 27A3 and 19E11 were selected for humanization. The affinity test results showed that the humanized 27A3 and 19E11 well maintained the affinity of the original mouse antibodies.

The high-affinity anti-MUC17 humanized antibody obtained by the present invention can be used for screening anti-tumor drugs.

Table 6. Antibody sequence list

Sequence Listing

<110> Dansheng Pharmaceutical Technology (Shanghai) Co., Ltd.

<120> Antibodies that bind to human MUC17, preparation methods and uses thereof

<160> 106

<170> SIPOSequenceListing 1.0

<210> 1

<211> 116

<212> PRT

<213> Artificial Sequence

<400> 1

Glu Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Asn Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ser Val Asn Trp Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210> 2

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 2

Gly Phe Ala Phe Ser Asn Tyr Asp Met Ser

1 5 10

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 3

Thr Ile Ser Gly Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Asn Val Lys

1 5 10 15

Gly

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 4

Val Asn Trp Gly Phe Pro Tyr

1 5

<210> 5

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 5

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ile Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Arg Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Ile Leu Leu Ile Tyr Trp Thr Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Gly Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Thr Ser Pro Trp Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile

100 105 110

Lys

<210> 6

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 6

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Arg Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 7

Trp Thr Ser Thr Arg Glu Ser

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 8

Gln Gln Tyr Tyr Thr Ser Pro Trp Thr

1 5

<210> 9

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 9

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Lys Gln Ser Asn Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Ile Ser Ser Ser Thr Ala His

65 70 75 80

Met Glu Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Val Thr Thr Val Gly Tyr Pro Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 10

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 10

Gly Phe Ser Phe Thr Gly Tyr Asn Met Asn

1 5 10

<210> 11

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 11

Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 12

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 12

Ser Val Thr Thr Val Gly Tyr Pro Met Asp Tyr

1 5 10

<210> 13

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 13

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly

1 5 10 15

Gln Lys Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Tyr Asn His Lys Asn Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Ser Phe Ala Ser Ala Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys His Gln

85 90 95

His Tyr Thr Ser Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 14

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 14

Lys Ser Ser Gln Ser Leu Leu Asn Ser Tyr Asn His Lys Asn Cys Leu

1 5 10 15

Ala

<210> 15

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 15

Phe Ala Ser Ala Arg Glu Ser

1 5

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 16

His Gln His Tyr Thr Ser Pro Leu Thr

1 5

<210> 17

<211> 117

<212> PRT

<213> Artificial Sequence

<400> 17

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Gly Ile Trp Ala Gly Gly Asn Thr Gln Ser His Ser Ala Leu Met

50 55 60

Ser Arg Val Ser Ile Ser Lys Asp Ile Ser Lys Arg Gln Val Phe Leu

65 70 75 80

Lys Met Asp Arg Leu Gln Thr Asp Asp Ser Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Phe Tyr Gly Asn Phe Asp Val Trp Gly Ala Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 18

Gly Phe Ser Leu Thr Ala Tyr Gly Val His

1 5 10

<210> 19

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 19

Gly Ile Trp Ala Gly Gly Asn Thr Gln Ser His Ser Ala Leu Met Ser

1 5 10 15

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 20

Ala Leu Phe Tyr Gly Asn Phe Asp Val

1 5

<210> 21

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 21

Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Thr Ser

20 25 30

Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Glu Ala Leu Ile

35 40 45

Phe Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Asn Leu Glu Met Arg

100 105

<210> 22

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 22

Lys Ala Ser Gln Ser Val Asp Thr Ser Val Ala

1 5 10

<210> 23

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 23

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210> 24

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 24

Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

1 5

<210> 25

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 25

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ile Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ser His Pro Ser Asp Ser Glu Ser Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Pro Tyr Tyr Gly Ser Ser Ala Trp Phe Ala Asp Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 26

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 26

Gly Tyr Ser Phe Thr Ile Tyr Trp Met His

1 5 10

<210> 27

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 27

Met Ser His Pro Ser Asp Ser Glu Ser Arg Leu Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 28

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 28

Pro Tyr Tyr Gly Ser Ser Ala Trp Phe Ala Asp

1 5 10

<210> 29

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 29

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly

1 5 10 15

Gln Lys Val Thr Met Ser Cys Lys Ser Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln

85 90 95

His Tyr Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Arg

<210> 30

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 30

Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 31

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 31

Phe Ala Ser Thr Arg Glu Ser

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 32

Gln Gln His Tyr Ser Phe Pro Leu Thr

1 5

<210> 33

<211> 123

<212> PRT

<213> Artificial Sequence

<400> 33

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Ala Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Ser Asn

20 25 30

Tyr Ile Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Lys Pro Thr Asn Gly Val Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Thr Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Phe Ser Leu Leu Arg Leu Gln Gly Tyr Ala Met Asp Asp

100 105 110

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 34

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 34

Gly Tyr Thr Phe Asn Ser Asn Tyr Ile Tyr

1 5 10

<210> 35

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 35

Glu Ile Lys Pro Thr Asn Gly Val Thr Asn Phe Asn Glu Lys Phe Lys

1 5 10 15

Thr

<210> 36

<211> 14

<212> PRT

<213> Artificial Sequence

<400> 36

Gly Phe Ser Leu Leu Arg Leu Gln Gly Tyr Ala Met Asp Asp

1 5 10

<210> 37

<211> 111

<212> PRT

<213> Artificial Sequence

<400> 37

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Thr Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Ser Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Ser Ile Thr

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Phe Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 38

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 38

Arg Ala Ser Glu Ser Val Glu Thr Tyr Gly Asn Ser Phe Met His

1 5 10 15

<210> 39

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 39

Arg Ala Ser Ser Leu Glu Ser

1 5

<210> 40

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 40

Gln Gln Ser Asn Glu Asp Pro Tyr Thr

1 5

<210> 41

<211> 121

<212> PRT

<213> Artificial Sequence

<400> 41

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Phe Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Gly Pro Arg Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ala Ser Asn Ala Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Gly Thr Ser Tyr Pro Asn Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 42

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 42

Gly Phe Asn Ile Lys Asp Thr Tyr Ile His

1 5 10

<210> 43

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 43

Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Gly Pro Arg Phe Gln

1 5 10 15

Gly

<210> 44

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 44

Asn Tyr Gly Thr Ser Tyr Pro Asn Ala Met Asp Tyr

1 5 10

<210> 45

<211> 111

<212> PRT

<213> Artificial Sequence

<400> 45

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Thr Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Ser Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Thr

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Phe Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 46

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 46

Arg Ala Ser Glu Ser Val Glu Thr Tyr Gly Asn Ser Phe Met His

1 5 10 15

<210> 47

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 47

Arg Ala Ser Ser Leu Glu Ser

1 5

<210> 48

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 48

Gln Gln Ser Asn Glu Asp Pro Tyr Thr

1 5

<210> 49

<211> 117

<212> PRT

<213> Artificial Sequence

<400> 49

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Gly Ile Trp Ala Gly Gly Ser Thr Gln Ser Asn Ser Ala Leu Met

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Arg Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Phe Tyr Gly Asn Phe Asp Val Trp Gly Ala Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 50

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 50

Gly Phe Ser Leu Thr Ala Tyr Gly Val His

1 5 10

<210> 51

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 51

Gly Ile Trp Ala Gly Gly Ser Thr Gln Ser Asn Ser Ala Leu Met Ser

1 5 10 15

<210> 52

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 52

Ala Leu Phe Tyr Gly Asn Phe Asp Val

1 5

<210> 53

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 53

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Thr Ser

20 25 30

Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Glu Ala Leu Ile

35 40 45

Phe Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys

100 105

<210> 54

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 54

Lys Ala Ser Gln Ser Val Asp Thr Ser Val Ala

1 5 10

<210> 55

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 55

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210> 56

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 56

Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

1 5

<210> 57

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 57

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asp Pro Ser Asp Ser Tyr Thr Arg Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Gly Ile Gly Asn Ser Phe Ile Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 58

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 58

Gly Tyr Thr Phe Thr Thr Tyr Trp Met His

1 5 10

<210> 59

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 59

Val Ile Asp Pro Ser Asp Ser Tyr Thr Arg Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 60

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 60

Ser Gly Ile Gly Asn Ser Phe Ile Met Asp Tyr

1 5 10

<210> 61

<211> 106

<212> PRT

<213> Artificial Sequence

<400> 61

Gln Val Val Leu Thr Gln Ser Pro Ala Ile Met Pro Ala Phe Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Ser Cys Ser Ala Ser Ser Ser Val Ile Tyr Met

20 25 30

Phe Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Thr Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Val Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 62

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 62

Ser Ala Ser Ser Ser Val Ile Tyr Met Phe

1 5 10

<210> 63

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 63

Arg Thr Ser Asn Leu Ala Ser

1 5

<210> 64

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 64

Gln Gln Tyr His Ser Tyr Pro Leu Thr

1 5

<210> 65

<211> 116

<212> PRT

<213> Artificial Sequence

<400> 65

Glu Met Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Asp Met Ser Trp Ile Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Gln Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Ser Leu Asn Trp Gly Phe Asp His Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 66

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 66

Gly Phe Thr Phe Ser Thr Tyr Asp Met Ser

1 5 10

<210> 67

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 67

Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

Gly

<210> 68

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 68

Leu Asn Trp Gly Phe Asp His

1 5

<210> 69

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 69

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Gln Lys Val Thr Met Ser Cys Lys Ser Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ala Arg Phe Ile Gly Asp Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln

85 90 95

His Tyr Asn Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 70

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 70

Lys Ser Ser Gln Ser Leu Leu Asn Ser Asn Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 71

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 71

Phe Ala Ser Thr Arg Glu Ser

1 5

<210> 72

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 72

Gln Gln His Tyr Asn Pro Pro Trp Thr

1 5

<210> 73

<211> 117

<212> PRT

<213> Artificial Sequence

<400> 73

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Gly Ile Trp Ala Gly Gly Asn Thr Gln Ser His Ser Ala Leu Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Ile Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Arg Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Phe Tyr Gly Asn Phe Asp Val Trp Gly Ala Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 74

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 74

Gly Phe Ser Leu Thr Ala Tyr Gly Val His

1 5 10

<210> 75

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 75

Gly Ile Trp Ala Gly Gly Asn Thr Gln Ser His Ser Ala Leu Met Ser

1 5 10 15

<210> 76

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 76

Ala Leu Phe Tyr Gly Asn Phe Asp Val

1 5

<210> 77

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 77

Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Thr Ser

20 25 30

Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Glu Ala Leu Ile

35 40 45

Phe Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys

100 105

<210> 78

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 78

Lys Ala Ser Gln Ser Val Asp Thr Ser Val Ala

1 5 10

<210> 79

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 79

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210> 80

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 80

Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr

1 5

<210> 81

<211> 119

<212> PRT

<213> Artificial Sequence

<400> 81

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Asn Ala Phe Ser Leu Ser

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Phe

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Asn Tyr Tyr Gly Ser Ser Ser Trp Phe Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 82

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 82

Gly Asn Ala Phe Ser Leu Ser Trp Met Asn

1 5 10

<210> 83

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 83

Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Lys Phe Lys

1 5 10 15

Gly

<210> 84

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 84

Tyr Tyr Gly Ser Ser Ser Trp Phe Val Tyr

1 5 10

<210> 85

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 85

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Tyr Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Thr Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 86

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 86

Arg Ser Ser Gln Ser Leu Val His Ser Tyr Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 87

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 87

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 88

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 88

Ser Gln Ser Thr His Phe Pro Trp Thr

1 5

<210> 89

<211> 117

<212> PRT

<213> Artificial Sequence

<400> 89

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Gly Ile Trp Ala Gly Gly Ser Thr Lys Ser Asn Ser Ala Leu Met

50 55 60

Ser Ser Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Phe Tyr Gly Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 90

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 90

Gly Phe Ser Leu Thr Ala Tyr Gly Val His

1 5 10

<210> 91

<211> 16

<212> PRT

<213> Artificial Sequence

<400> 91

Gly Ile Trp Ala Gly Gly Ser Thr Lys Ser Asn Ser Ala Leu Met Ser

1 5 10 15

<210> 92

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 92

Ala Leu Phe Tyr Gly Tyr Phe Asp Val

1 5

<210> 93

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 93

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Ser Val Asp Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Glu Ala Leu Ile

35 40 45

Phe Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Val Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys

100 105

<210> 94

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 94

Lys Ala Ser Gln Ser Val Asp Thr Asn Val Ala

1 5 10

<210> 95

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 95

Ser Ala Ser Tyr Arg Tyr Ser

1 5

<210> 96

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 96

Gln Gln Tyr Asn Val Tyr Pro Tyr Thr

1 5

<210> 97

<211> 330

<212> PRT

<213> Artificial Sequence

<400> 97

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 98

<211> 107

<212> PRT

<213> Artificial Sequence

<400> 98

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 99

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 99

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ile Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ser His Pro Ser Asp Ser Glu Ser Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Pro Tyr Tyr Gly Ser Ser Ala Trp Phe Ala Asp Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 100

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 100

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

His Tyr Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Arg

<210> 101

<211> 450

<212> PRT

<213> Artificial Sequence

<400> 101

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ile Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ser His Pro Ser Asp Ser Glu Ser Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Pro Tyr Tyr Gly Ser Ser Ala Trp Phe Ala Asp Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 102

<211> 220

<212> PRT

<213> Artificial Sequence

<400> 102

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

His Tyr Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Arg Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 103

<211> 120

<212> PRT

<213> Artificial Sequence

<400> 103

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Ile Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Val Thr Thr Val Gly Tyr Pro Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 104

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 104

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Tyr Asn His Lys Asn Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Ser Phe Ala Ser Ala Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys His Gln

85 90 95

His Tyr Thr Ser Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 105

<211> 450

<212> PRT

<213> Artificial Sequence

<400> 105

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Ile Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Val Thr Thr Val Gly Tyr Pro Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 106

<211> 220

<212> PRT

<213> Artificial Sequence

<400> 106

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Tyr Asn His Lys Asn Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Ser Phe Ala Ser Ala Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys His Gln

85 90 95

His Tyr Thr Ser Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

Claims (18)

1. An antibody or an antigen-binding fragment thereof that binds to human MUC17, characterized in that it comprises: (a) Heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of said HCDR1 is selected from the group consisting of SEQ ID NO: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82 and 90 The group, the amino acid sequence of the HCDR2 is selected from the group consisting of SEQ ID NO: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83 and 91, the amino acid sequence of the HCDR3 is selected from the group consisting of SEQ ID NO: NO: Groups consisting of 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84 and 92; and (b) Light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, the amino acid sequence of said LCDR1 is selected from the group consisting of SEQ ID NO: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86 and 94 The group, the amino acid sequence of the LCDR2 is selected from the group consisting of SEQ ID NO: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87 and 95, the amino acid sequence of the LCDR3 is selected from the group consisting of SEQ ID NO: Groups consisting of 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88 and 96. 2. The antibody or antigen-binding fragment thereof that binds to human MUC17 according to claim 1, wherein: (1) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 2, 3 and 4 respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 6, 7 and 8 respectively Show; (2) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 10, 11 and 12 respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 14, 15 and 16 respectively Show; (3) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 18, 19 and 20 respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 22, 23 and 24 respectively Show; (4) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 26, 27 and 28, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 30, 31 and 32 Show; (5) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 34, 35 and 36, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 38, 39 and 40 Show; (6) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 42, 43 and 44 respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 46, 47 and 48 respectively Show; (7) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 50, 51 and 52 respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 54, 55 and 56 respectively Show; (8) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 58, 59 and 60, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 62, 63 and 64 Show; (9) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 66, 67 and 68 respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 70, 71 and 72 respectively Show; (10) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 74, 75 and 76, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 78, 79 and 80 Show; (11) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 82, 83 and 84 respectively, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 86, 87 and 88 respectively show; or (12) The amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 90, 91 and 92, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 94, 95 and 96 Show. 3. The antibody or antigen-binding fragment thereof that binds to human MUC17 according to claim 1 or 2, wherein the antibody is a monoclonal antibody or a polyclonal antibody; preferably, the antibody is a monoclonal antibody. 4. The antibody or antigen-binding fragment thereof that binds to human MUC17 according to claim 1 or 2, wherein the antibody is a murine antibody, a chimeric antibody or a humanized antibody. 5. The antibody or antigen-binding fragment thereof in conjunction with human MUC17 according to claim 1 or 2, wherein the antigen-binding fragment comprises a Fab fragment, F(ab')2 fragment, Fab' fragment, Fv fragment or Single-chain Fv (scFv) fragments. 6. The antibody or antigen-binding fragment thereof that binds to human MUC17 according to claim 1 or 2, wherein: The antibody or antigen-binding fragment thereof that binds to human MUC17 includes a heavy chain variable region (VH) and a light chain variable region (VL), (1) the amino acid sequence of the VH is selected from the group consisting of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 99 and 103; and (2) The amino acid sequence of the VL is selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 100 and 104. 7. The antibody or antigen-binding fragment thereof binding to human MUC17 as claimed in claim 6, characterized in that, (1) The amino acid sequence of the VH is shown in SEQ ID NO: 1, and the amino acid sequence of the VL is shown in SEQ ID NO: 5; (2) The amino acid sequence of the VH is shown in SEQ ID NO: 9, and the amino acid sequence of the VL is shown in SEQ ID NO: 13; (3) The amino acid sequence of the VH is shown in SEQ ID NO: 17, and the amino acid sequence of the VL is shown in SEQ ID NO: 21; (4) The amino acid sequence of the VH is shown in SEQ ID NO: 25, and the VL includes the amino acid sequence shown in SEQ ID NO: 29; (5) The amino acid sequence of the VH is shown in SEQ ID NO: 33, and the amino acid sequence of the VL is shown in SEQ ID NO: 37; (6) The amino acid sequence of the VH is shown in SEQ ID NO: 41, and the amino acid sequence of the VL is shown in SEQ ID NO: 45; (7) The amino acid sequence of the VH is shown in SEQ ID NO: 49, and the amino acid sequence of the VL is shown in SEQ ID NO: 53; (8) The amino acid sequence of the VH is shown in SEQ ID NO: 57, and the amino acid sequence of the VL is shown in SEQ ID NO: 61; (9) The amino acid sequence of the VH is shown in SEQ ID NO: 65, and the amino acid sequence of the VL is shown in SEQ ID NO: 69; (10) The amino acid sequence of the VH is shown in SEQ ID NO: 73, and the amino acid sequence of the VL is shown in SEQ ID NO: 77; (11) The amino acid sequence of the VH is shown in SEQ ID NO: 81, and the amino acid sequence of the VL is shown in SEQ ID NO: 85; (12) The amino acid sequence of the VH is shown in SEQ ID NO: 89, and the amino acid sequence of the VL is shown in SEQ ID NO: 93; (13) The amino acid sequence of the VH is shown in SEQ ID NO: 99, and the amino acid sequence of the VL is shown in SEQ ID NO: 100; or (14) The amino acid sequence of the VH is shown in SEQ ID NO: 103, and the amino acid sequence of the VL is shown in SEQ ID NO: 104. 8. The antibody or antigen-binding fragment thereof that binds to human MUC17 as claimed in claim 6 or 7, wherein the antibody or antigen-binding fragment thereof that binds to human MUC17 comprises a heavy chain constant region and a light chain constant region, so The heavy chain constant region is selected from IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, and the light chain constant region is selected from kappa or lambda light chain constant region; preferably, the heavy chain constant region is an IgG1 heavy chain constant region region, the light chain constant region is a kappa light chain constant region; preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 97, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 98. 9. The antibody or antigen-binding fragment thereof that binds to human MUC17 according to claim 1, wherein: The antibody or antigen-binding fragment thereof that binds to human MUC17 includes a heavy chain and a light chain, selected from: (1) The amino acid sequence of the heavy chain is shown in SEQ ID NO: 101, and the amino acid sequence of the light chain is shown in SEQ ID NO: 102; and (2) The amino acid sequence of the heavy chain is shown in SEQ ID NO:105, and the amino acid sequence of the light chain is shown in SEQ ID NO:106. 10. An isolated polynucleotide molecule, characterized in that the polynucleotide molecule encodes the antibody or antigen-binding fragment thereof that binds to human MUC17 according to any one of claims 1-9. 11. An expression vector, characterized in that the expression vector contains the polynucleotide molecule according to claim 10. 12. A host cell, characterized in that the host cell contains the expression vector according to claim 11. 13. A method for preparing an antibody or an antigen-binding fragment thereof that binds to human MUC17 according to any one of claims 1-9, wherein the preparation method comprises the following steps: (a) under expression conditions, culturing the host cell as claimed in claim 12, thereby expressing the antibody or antigen-binding fragment thereof in conjunction with human MUC17; (b) isolating and purifying the antibody or antigen-binding fragment thereof that binds to human MUC17 described in step (a). 14. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises an effective amount of the antibody or antigen-binding fragment thereof binding to human MUC17 according to any one of claims 1-9; optionally, the The pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent or excipient. 15. The pharmaceutical composition of claim 14, further comprising one or more additional therapeutic agents. 16. Use of the antibody or antigen-binding fragment thereof that binds to human MUC17 according to any one of claims 1-9, or the pharmaceutical composition according to claim 14 or 15 in the preparation of medicines for cancer or tumors. 17. The use according to claim 16, wherein the cancer or tumor is selected from: pancreatic cancer, large intestine cancer, colon cancer, rectal cancer, colorectal cancer, small intestine cancer, gastrointestinal cancer, gastric cancer, esophageal cancer , gastroesophageal cancer, or a combination thereof. 18. An immunoconjugate, characterized in that, the immunoconjugate comprises: (a) an antibody or an antigen-binding fragment thereof binding to human MUC17 according to any one of claims 1-9; and (b) A conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.

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