CN112898430B

CN112898430B - Binding protein of CA242, application thereof, detection method and kit

Info

Publication number: CN112898430B
Application number: CN201911229292.5A
Authority: CN
Inventors: 孟媛; 钟冬梅; 娄文娟; 覃婷; 王晨
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2022-11-04
Anticipated expiration: 2039-12-04
Also published as: CN112898430A

Abstract

The invention discloses a binding protein of CA242, and an application, a detection method and a kit thereof, relating to the technical field of antibodies, wherein the binding protein disclosed by the invention comprises an antigen binding domain, and the antigen binding domain comprises at least one of the following complementarity determining regions: CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3. The binding protein provided by the invention can be specifically bound with CA242, has good binding activity and affinity, strong specificity and high sensitivity, and can be used for specific detection of CA242 protein in the fields of scientific research, detection, tumor diagnosis and the like.

Description

Binding protein of CA242, application thereof, detection method and kit

Technical Field

The invention relates to the technical field of antibodies, and particularly relates to a binding protein of CA242, and an application, a detection method and a kit thereof.

Background

Tumor markers (Tumor markers) are chemical species that reflect the presence of tumors. They are not existed in normal adult tissue but only in embryonic tissue, or their content in tumor tissue is greatly greater than that in normal tissue, and their quantity can indicate the property of tumor, so that it can know the tissue generation, cell differentiation and cell function of tumor to help diagnosis, classification, prognosis and treatment guidance of tumor. CA242 is a sialylated mucin-type carbohydrate antigen, which has little or no content in normal tissues of human body, and can be increased in the content of tumor tissues and serum when malignant tumors occur, particularly in pancreatic cancer and colorectal cancer, so that the CA242 can be used as a new tumor marker.

In the 80s of the 20 th century, experts obtained glycoprotein antigens (CA) capable of recognizing tumor-specific macromolecules using hybridoma technology, and developed monoclonal antibody recognition systems. CA is a tumor cell-associated antigen. The commonly used CA series are: CA125 (ovarian cancer associated antigen); CA19-9 (pancreatic, intestinal cancer associated antigen); CA15-3 (breast cancer associated antigen). After obtaining a series of antibodies by Limdholm et al immunized mice with human colorectal cancer cell COLD205 in 1983, a series of antigens corresponding to these antibodies, including CA19-9, CA50, CA242, etc., were successively discovered. The series of antigens appear on the surface of homomucin, and antigenic determinants are all of sugar chain structures but have different tumor specificities, so the antigens serve as different tumor markers.

The monitoring system formed by CA19-9 and CA50 has been widely used for diagnosing digestive tract malignant tumors, especially colorectal cancer. CA242 is a sialylated glycosphingolipid antigen, almost always expressed with CA50, but both recognized by different monoclonal antibodies. Is clinically used for diagnosing digestive tract malignant tumors, particularly pancreatic cancer and colorectal cancer. CA50, CA19-9 are susceptible to liver function and cholestasis, with false positives being frequent in benign obstructive jaundice and liver parenchymal lesions. Compared with CA19-9 and CA50, the sensitivity and specificity of the CA242 of the new generation are higher in pancreatic cancer, gallbladder cancer and digestive tract cancer. CA242 is mainly present in malignant cells of the pancreas and colon. There was significant expression in malignant tissues of pancreatic and colon cancers, whereas CA242 appeared much lower in benign tumors than CA19-9 and CA50. CA242, a novel tumor marker, has the advantage of higher specificity, namely, the increase is obvious in malignant tumors and is not generally increased in benign diseases. Therefore, clinical diagnosis of malignant tumors by CA242 is more specific than CA19-9 and CA50, and CA242 is a more valuable indicator than CA50 and CA19-9 in the diagnosis of pancreatic and colorectal cancers.

Foreign studies have shown that CA242 is generally significantly elevated in the serum of patients with pancreatic cancer and advanced colorectal cancer. For colorectal cancer, CA242 has a higher sensitivity relative to CA19-9 and CA50. Many factors affect the content of CA242 in serum, and besides the antigen itself, the degree of malignancy, the rate and extent of diffusion, and the metabolism and excretion of the antigen of a tumor all affect the serum level. The principle of detecting the serum CA242 by using a high-affinity CA242 monoclonal antibody is based on the fact that C242 can be specifically combined with antigen CA242 on the surface of mucin, and the detection is carried out by using an immune serological method after the labeling.

In the diagnosis of colorectal cancer, CEA (carcinoembryonic antigen) is currently the most widely used tumor marker, but its sensitivity in the early stage of colorectal cancer (Dukes stage a, B) is very low. However, the combined use of CEA and CA242 makes diagnosis of colon cancer more sensitive than the use of either alone. When colorectal cancer is diagnosed, the increase of CA242 is earlier than the increase of CEA and the appearance of clinical relapse symptoms, so the combination of CA242 and CEA has better diagnostic value. In the postoperative detection, CA242 is better than CEA and becomes a good supplementary diagnostic index of CEA.

CA242 is the first tumor-associated antigen to appear in pancreatic cancer patients. In diagnosing pancreatic cancer, most reports suggest that it has sensitivity comparable to or lower than that of CA50 and CA19-9, but has higher specificity, i.e., serum CA242 is only slightly elevated in benign hepatobiliary disease and pancreatitis, and thus its false positive rate is significantly lower than that of CA19-9 and CA50.

In order to improve the level of tumor diagnosis and the therapeutic effect, the methods for detecting the CA242 antigen in clinical practice at present mainly include radioimmunoassay, enzyme-linked immunosorbent assay, time-resolved fluoroimmunoassay, chemiluminescence, and the like from the detection point of view. In the past, the human CA242 antigen determination kit represented by the radioimmunoassay has serious defects of poor sensitivity and anti-interference capability due to the limitation of methodology, and has basically exited the market; the prior application of the technology is enzyme-linked immunosorbent assay, time-resolved fluoroimmunoassay and chemiluminescence, wherein the chemiluminescence technology is developed in the 80 th century and is a new technology developed after the enzyme-linked immunosorbent assay and the radioimmunoassay.

The different methods have their own advantages and disadvantages, but all require specific monoclonal antibodies directed against CA242. At present, the monoclonal antibody for detecting CA242 in China has some defects in sensitivity, specificity and affinity and has a larger space for improvement, so that the monoclonal antibody for detecting CA242 still has a strong demand in the field.

Disclosure of Invention

The invention aims to provide a binding protein of CA242, and application, a detection method and a kit thereof. The binding protein provided by the invention can be specifically bound with CA242, has good binding activity and affinity, strong specificity and high sensitivity, and can be used for specific detection of CA242 protein in the fields of scientific research, detection, tumor diagnosis and the like.

Noun definitions

The term "binding protein" broadly refers to all proteins/protein fragments, in particular antibodies or functional fragments of antibodies, comprising CDR regions. The term "antibody" includes polyclonal and monoclonal antibodies, and "antibody functional fragments" include antigen-compound-binding fragments of these antibodies, including Fab, F (ab') 2, fd, fv, scFv, diabodies, and minimal recognition units, as well as single chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isoforms (antibodies). The term "antibody" is used interchangeably with "immunoglobulin".

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions, called "complementarity determining regions" or "CDRs". The extent of framework regions and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of Health and Human Services (U.S. department of Health and Human Services), (1983), and Chothia. The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, "framework region" or "FR" region means the region of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR 1, FR2, FR3 and FR 4) separated by CDRs.

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

As used herein, the term "purified" or "isolated" in relation to a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include the naturally-produced polypeptide contained in a cell lysate, the polypeptide in purified or partially purified form, recombinant polypeptides, the polypeptide expressed or secreted by a cell, and the polypeptide in a heterologous host cell or culture. In connection with a nucleic acid, the terms "isolated" or "purified" mean that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Exemplary embodiments of the invention:

in a first aspect, embodiments of the present invention provide a binding protein for detecting CA242, the binding protein comprising an antigen binding domain; the antigen binding domain comprises at least one of the following complementarity determining regions, or a similar complementarity determining region having at least 80% sequence identity with the sequence of at least one of the complementarity determining regions:

a complementarity determining region CDR-VH1 having an amino acid sequence of D-X1-T-F-X2-Y-G-M-X3, wherein: x1 is F or Y, X2 is T or S, X3 is S, N or H;

a complementarity determining region CDR-VH2 having an amino acid sequence of W-X1-D-T-X2-T-G-E-P-X3-Y-A-X4-D-F-K-G, wherein: x1 is I, V or L, X2 is T, N or Y, X3 is S or T, X4 is D or E;

a complementarity determining region CDR-VH3 having an amino acid sequence a-R-X1-P-Y-X2-W-Y-F-D-X3, wherein: x1 is G or A, X2 is GG, D or N, X3 is I, V or L;

a complementarity determining region CDR-VL1 having the amino acid sequence R-S-S-X1-S-X2-H-S-X3-G-N-T-Y-X4-Y, wherein: x1 is R or K, X2 is II, IL, LL or LI, X3 is D or N, X4 is I or L;

a complementarity determining region CDR-VL2 having the amino acid sequence X1-M-S-N-X2-X3-S, wherein: x1 is R or K, X2 is I, V or L, X3 is I, V or L;

a complementarity determining region CDR-VL3 having the amino acid sequence X1-Q-H-X2-E-X3-P-F-T, wherein: x1 is I or L, X2 is I, V or L, and X3 is F or Y.

The binding protein provided by the embodiment of the invention contains an antigen binding domain, the antigen binding domain comprises at least one of the complementarity determining regions, the amino acid sequence of the complementarity determining region is discovered and disclosed for the first time, the binding protein is a new sequence, the binding protein can be endowed with the capability of specifically binding the CA242 antigen, and has better binding activity and affinity which are respectively equal to or higher than those of products in the current market.

In alternative embodiments, in the complementarity determining region CDR-VH1, X1 is Y; in the CDR-VH2, X3 is T; in the CDR-VH3, X1 is G; in the complementarity determining region CDR-VL1, X1 is K; in the complementarity determining region CDR-VL2, X1 is R; in the CDR-VL3, X3 is Y.

In alternative embodiments, in the complementarity determining region CDR-VH1, X2 is T.

In alternative embodiments, in the complementarity determining region CDR-VH1, X2 is S.

In alternative embodiments, in the complementarity determining region CDR-VH1, X3 is S.

In alternative embodiments, in the complementarity determining region CDR-VH1, X3 is N.

In alternative embodiments, in the complementarity determining region CDR-VH1, X3 is H.

In alternative embodiments, in the complementarity determining region CDR-VH2, X1 is I.

In alternative embodiments, in the complementarity determining region CDR-VH2, X1 is V.

In alternative embodiments, in the complementarity determining region CDR-VH2, X1 is L.

In alternative embodiments, in the complementarity determining region CDR-VH2, X2 is T.

In an alternative embodiment, in the complementarity determining region CDR-VH2, X2 is N.

In alternative embodiments, in the complementarity determining region CDR-VH2, X2 is Y.

In alternative embodiments, in the complementarity determining region CDR-VH2, X4 is D.

In alternative embodiments, in the complementarity determining region CDR-VH2, X4 is E.

In an alternative embodiment, in the complementarity determining region CDR-VH3, X2 is GG.

In alternative embodiments, in the complementarity determining region CDR-VH3, X2 is D.

In alternative embodiments, in the complementarity determining region CDR-VH3, X2 is N.

In alternative embodiments, in the complementarity determining region CDR-VH3, X3 is I.

In alternative embodiments, in the complementarity determining region CDR-VH3, X3 is V.

In alternative embodiments, in the complementarity determining region CDR-VH3, X3 is L.

In alternative embodiments, in the complementarity determining region CDR-VL1, X2 is II.

In alternative embodiments, in the complementarity determining region CDR-VL1, X2 is IL.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X2 is LL.

In alternative embodiments, in the complementarity determining region CDR-VL1, X2 is LI.

In alternative embodiments, in the complementarity determining region CDR-VL1, X3 is D.

In alternative embodiments, in the complementarity determining region CDR-VL1, X3 is N.

In alternative embodiments, in the complementarity determining region CDR-VL1, X4 is I.

In an alternative embodiment, in the complementarity determining region CDR-VL1, X4 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL2, X2 is I.

In alternative embodiments, in the complementarity determining region CDR-VL2, X2 is V.

In alternative embodiments, in the complementarity determining region CDR-VL2, X2 is L.

In alternative embodiments, in the complementarity determining region CDR-VL2, X3 is I.

In alternative embodiments, in the complementarity determining region CDR-VL2, X3 is V.

In alternative embodiments, in the complementarity determining region CDR-VL2, X3 is L.

In alternative embodiments, in the complementarity determining region CDR-VL3, X1 is I.

In alternative embodiments, in the complementarity determining region CDR-VL3, X1 is L.

In an alternative embodiment, in the complementarity determining region CDR-VL3, X2 is I.

In alternative embodiments, in the complementarity determining region CDR-VL3, X2 is V.

In alternative embodiments, in the complementarity determining region CDR-VL3, X2 is L.

In alternative embodiments, the similar complementarity determining regions have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequences of the complementarity determining regions described above.

In alternative embodiments, the antigen binding domain has a K with the CA242 protein _D ≤9.5×10 ^-7 Affinity of mol/L.

In alternative embodiments, the antigen binding domain has a K with the CA242 protein _D ≤9×10 ^-7 mol/L、8×10 ^-7 mol/L、7×10 ^-7 mol/L、6×10 ^-7 mol/L、5×10 ^-7 mol/L、4×10 ^-7 mol/L、3×10 ^-7 mol/L、2×10 ^-7 mol/L、1×10 ^-7 mol/L、9×10 ^-8 mol/L、8×10 ^-8 mol/L、7×10 ^-8 mol/L、6×10 ^-8 mol/L、5×10 ^- ⁸ mol/L、4×10 ^-8 mol/L、3×10 ^-8 mol/L、2×10 ^-8 mol/L、1×10 ^-8 mol/L or 9X 10 ^-9 Affinity in mol/L.

In an alternative embodiment, the antigen binding domain has a 9.47 x 10 to CA242 protein ^-9 mol/L≤K _D ≤9.5×10 ^-7 Affinity of mol/L.

K _D The detection of (2) is carried out with reference to the method in the examples of the present invention.

In an alternative embodiment, the mutation site (i.e., xn site, n =1,2,3 or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 1-65:

in alternative embodiments, in the complementarity determining region CDR-VH1, X1 is F; in the CDR-VH2, X3 is S; in the CDR-VH3, X1 is A; in the complementarity determining region CDR-VL1, X1 is R; in the complementarity determining region CDR-VL2, X1 is K; in the CDR-VL3, X3 is F.

In alternative embodiments, the mutation site (i.e., xn site, n =1,2,3 or 4) in each of the complementarity determining regions described above is selected from any one of the following combinations of mutations 66-78:

in alternative embodiments, the binding protein includes at least 3 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain, or3 complementarity determining regions of a light chain); alternatively, the binding protein comprises at least 6 complementarity determining regions (e.g., 3 complementarity determining regions of a heavy chain and 3 complementarity determining regions of a light chain);

in alternative embodiments, the binding protein is a whole antibody comprising a variable region and a constant region.

In alternative embodiments, the binding protein is a functional fragment of an antibody, such as any one of a nanobody, F (ab ') 2, fab', fab, fv, scFv, diabody, and antibody minimal recognition unit;

functional fragments of the above antibodies typically have the same binding specificity as the antibody from which they are derived. As will be readily understood by those skilled in the art based on the teachings of the present invention, functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reduction to cleave disulfide bonds.

Functional fragments of the above antibodies can also be obtained by recombinant genetic techniques also known to those skilled in the art or synthesized by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In alternative embodiments, the binding protein comprises light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4, as set forth in sequence in SEQ ID NOS 1-4, and/or heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4, as set forth in sequence in SEQ ID NOS 5-8.

In addition, based on the disclosure of the present invention, the species source of the heavy chain or light chain framework region of the binding protein may be human, so as to constitute a humanized antibody.

In alternative embodiments, the binding protein further comprises an antibody constant region.

In alternative embodiments, the antibody constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE and IgD.

In alternative embodiments, the species of the antibody constant region is from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, or human.

In alternative embodiments, the antibody constant region is derived from a mouse.

In alternative embodiments, the light chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 9 and the heavy chain constant region sequence of the antibody constant region is set forth in SEQ ID NO. 10.

The sequences of SEQ ID NOS: 1-10 are shown in the following table:

in a second aspect, the embodiments of the present invention provide a use of a binding protein according to any one of the preceding embodiments in the preparation of a diagnostic reagent or kit for a tumor, wherein the marker for the tumor comprises CA242.

In alternative embodiments, the tumor is selected from any one of colorectal cancer, pancreatic cancer, gallbladder cancer, and digestive tract cancer.

In a third aspect, the embodiments of the present invention provide a tumor diagnostic reagent or kit, where the tumor marker includes CA242, which contains the binding protein as described above.

In a fourth aspect, an embodiment of the present invention provides a method for detecting CA242, including: mixing a binding protein according to any one of the preceding embodiments with a sample to be tested.

In an alternative embodiment, the above method is for the purpose of non-disease diagnosis.

It should be noted that, those skilled in the art can perform qualitative or quantitative detection of the CA242 protein in the sample to be tested based on the characteristics of immune complex formed by antibody/antigen combination. The method for detecting an antigen or an antibody based on the formation of an immune complex upon binding of the antibody to the antigen comprises:

(1) The detection purpose is realized by a precipitation reaction, which comprises the following steps: a one-way immunodiffusion test, a two-way immunodiffusion test, an immunoturbidimetry, a countercurrent immunoelectrophoresis, an immunoblotting, and the like;

(2) The detection purpose is realized by marking an indicator for displaying the signal intensity, and the method comprises the following steps: immunofluorescence, radioimmunoassay, enzyme-linked immunoassay (e.g., double antibody sandwich, indirect or competitive assay), and chemiluminescence immunoassay.

The indicator may be selected appropriately according to different detection methods, including but not limited to the indicators described below:

(1) In immunofluorescence, the indicator may be a fluorescent dye, for example, a fluorescein dye (including Fluorescein Isothiocyanate (FITC), hydroxyphoton (FAM), tetrachlorofluorescein (TET), etc. or analogs thereof), a rhodamine dye (including rhodamine Red (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or analogs thereof), a Cy series dye (including Cy2, cy3B, cy3.5, cy5, cy5.5, cy3, etc. or analogs thereof), an Alexa series dye (including Alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or analogs thereof), a protein dye (including Phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polycyanoxanthin-chlorophyll protein (preCP), etc.);

(2) In radioimmunoassays, the indicator may be a radioisotope, for example: 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, 18F, and the like.

(3) In enzyme-linked immunoassays, the indicator may be an enzyme that catalyzes the development of a substrate (e.g., horseradish peroxidase, alkaline phosphatase, or glucose oxidase, etc.).

(4) In a chemiluminescent immunoassay, the indicator may be a chemiluminescent reagent such as acridinium ester, horseradish peroxidase and luminol, alkaline phosphatase and AMPPD, electrochemiluminescent agents such as ruthenium and tripropylamine terpyridyl, and the like.

Based on the disclosure of the above binding protein, those skilled in the art can easily think of using any one or a combination of several methods or other methods to achieve quantitative or qualitative detection of CA242 in a sample to be detected, and it is within the scope of the present invention to use the binding protein disclosed in the present invention to detect CA242 regardless of the method.

In alternative embodiments, the binding protein is labeled with an indicator that indicates the intensity of the signal, such that the complex of the binding protein bound to the CA242 protein is detected.

In a fifth aspect, embodiments of the invention provide an isolated nucleic acid encoding a binding protein according to any one of the preceding embodiments;

in alternative embodiments, the nucleic acid is DNA or RNA.

Based on the disclosure of the amino acid sequence of the binding protein, one skilled in the art can easily obtain the nucleic acid sequence encoding the binding protein according to the codon corresponding to the amino acid, and obtain various nucleic acid sequences encoding the binding protein according to the degeneracy of the codon, which are within the protection scope of the present invention as long as they encode the binding protein.

In a sixth aspect, embodiments of the invention provide a vector comprising a nucleic acid according to the previous embodiments.

Wherein the nucleic acid sequence is operably linked to at least one regulatory sequence. "operably linked" means that the nucleic acid sequence is linked to the regulatory sequence in a manner that allows expression. Regulatory sequences, including promoters, enhancers and other expression control elements, are selected to direct the expression of the protein of interest in a suitable host cell.

Herein, a vector may refer to a molecule or agent comprising a nucleic acid of the invention or a fragment thereof, capable of carrying genetic information and capable of delivering the genetic information into a cell. Typical vectors include plasmids, viruses, bacteriophages, cosmids and minichromosomes. The vector may be a cloning vector (i.e. a vector for transferring genetic information into a cell, which may be propagated and in which the presence or absence of said genetic information may be selected) or an expression vector (i.e. a vector comprising the necessary genetic elements to allow expression of the genetic information of said vector in a cell). Thus, a cloning vector may contain a selectable marker, as well as an origin of replication compatible with the cell type specified by the cloning vector, while an expression vector contains the regulatory elements necessary to effect expression in a specified target cell.

The nucleic acid of the invention or fragments thereof may be inserted into a suitable vector to form a cloning or expression vector carrying the nucleic acid fragment of the invention. Such novel vectors are also part of the present invention. The vector may comprise a plasmid, phage, cosmid, minichromosome, or virus, as well as naked DNA that is transiently expressed only in a particular cell. The cloning and expression vectors of the invention are capable of autonomous replication and thus provide high copy numbers for high level expression or high level replication purposes for subsequent cloning. The expression vector may comprise a promoter for driving expression of the nucleic acid fragment of the invention, optionally a signal peptide sequence encoding for secretion or integration of the protein expression product into a membrane, and optionally a nucleic acid sequence encoding for a terminator. When the expression vector is manipulated in a production strain or cell line, the vector, when introduced into a host cell, may or may not be integrated into the genome of the host cell. Vectors typically carry a replication site, as well as a marker sequence capable of providing phenotypic selection in transformed cells.

In a seventh aspect, embodiments of the present invention provide a host cell comprising a vector according to the previous embodiments.

The expression vectors of the invention are useful for transforming host cells. Such transformed host cells are also part of the invention and may be cultured cells or cell lines for propagation of the nucleic acid fragments and vectors of the invention, or for recombinant production of the binding proteins of the invention. Host cells of the present invention include microorganisms such as bacteria (e.g., escherichia coli, bacillus, etc.). Host cells also include cells from multicellular organisms such as fungi, insect cells, plant cells or mammalian cells, preferably from mammals, e.g., CHO cells.

In an eighth aspect, the embodiments provide a method of producing a binding protein according to any one of the preceding embodiments, comprising:

the host cell of the previous embodiment is cultured, and the binding protein is isolated and purified from the culture medium or from the cultured host cell.

The production method may be, for example, transfecting a host cell with a nucleic acid vector encoding at least a portion of the binding protein, and culturing the host cell under suitable conditions such that the binding protein is expressed. The host cell may also be transfected with one or more expression vectors, which may comprise, alone or in combination, DNA encoding at least a portion of the binding protein. The bound protein may be isolated from the culture medium or cell lysate using conventional techniques for purifying proteins and peptides, including ammonium sulfate precipitation, chromatography (e.g., ion exchange, gel filtration, affinity chromatography, etc.), and/or electrophoresis.

Construction of suitable vectors containing the coding and regulatory sequences of interest can be carried out using standard ligation and restriction techniques well known in the art. The isolated plasmid, DNA sequence or synthetic oligonucleotide is cleaved, tailed and religated as desired. Any method may be used to introduce mutations into the coding sequence to produce variants of the invention, and these mutations may comprise deletions or insertions or substitutions or the like.

Drawings

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

FIG. 1 is a reduced SDS-PAGE of the monoclonal antibody against CA242 of example 1.

FIG. 2 shows the results of OD450 of CA242 quality control samples with different concentrations in example 2, which were measured using anti-CA 242 antibody.

FIG. 3 is the result of the luminescence detection of CA242 quality control samples with different concentrations using anti-CA 242 antibody in example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit dosages herein, some are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (oligo Synthesis) (eds. M.j. Goal, 1984); animal Cell Culture (Animal Cell Culture), ed.r.i. freshney, 1987; methods in Enzymology (Methods in Enzymology), academic Press, inc. (Academic Press, inc.), "Handbook of Experimental Immunology" ("D.M.Weir and C.C.Black well"), gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos.), "Current Protocols in Molecular Biology" (F.M.Ausubel et al., 1987), "PCR, polymerase Chain Reaction (PCR: the Polymerase Chain Reaction) (Mullis et al., 1994), and" Current Protocols in Immunology "(blood), each of which is incorporated herein by reference, cold, 1991.

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One of ordinary skill in the relevant art will readily recognize, however, that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of activities or events, as some activities may occur in different orders and/or concurrently with other activities or events. Moreover, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Restriction enzyme, prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART ^TM RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were performed by Invitrogen corporation.

This example provides a method for preparing a recombinant antibody against CA242

1 construction of recombinant plasmid

(1) Primer and method for producing the same

Amplifying Heavy Chain and Light Chain 5' RACE primers:

(2) Antibody variable region gene cloning and sequencing

RNA is extracted from a hybridoma cell line secreting a monoclonal antibody against CA242, first strand cDNA synthesis is carried out by using SMARTERTM RACE cDNA Amplification Kit and SMARTER II A Oligonucleotide and 5' -CDS primers in the Kit, and an obtained first strand cDNA product is used as a PCR Amplification template. The Light Chain gene was amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP) and mIgG CKR primers, and the Heavy Chain gene was amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP) and mIgG CHR primers. The primer pair of Light Chain can amplify a target band about 0.8KB, and the primer pair of Heavy Chain can amplify a target band about 1.4 KB. The product was purified and recovered by agarose gel electrophoresis, and the product was inserted into pMD-18T vector after A-addition reaction with rTaq DNA polymerase, transformed into DH 5. Alpha. Competent cells, and after colonies were grown, the Heavy Chain and Light Chain genes were cloned, respectively, and sent to Invitrogen corporation for sequencing.

(3) Sequence analysis of variable region Gene of anti-CA 242 monoclonal antibody

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 339bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 357bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

(4) Construction of recombinant antibody expression plasmid

pcDNA ^TM 3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, bamHI, ecoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody gene in the pMD-18T, the light chain and heavy chain gene specific primers of the anti-CA 242 antibody are designed, wherein two ends of the primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and the primers are as follows:

a0.75 KB Light Chain gene fragment and a 1.42KB Heavy Chain gene fragment were amplified by PCR amplification. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the gene of the Heavy Chain and the gene of the Light Chain are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

2 Stable cell line selection

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells and determination of expression plasmid activity

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, the mixture is transferred into an electric rotating cup and is electrically rotated, the sampling counting is carried out on 3 rd, 5 th and 7 th days, and the sampling detection is carried out on 7 th day.

Diluting goat anti-mouse IgG1 mu g/ml with the coating solution to coat the microplate, wherein each well is 100 mu l, and the temperature is 4 ℃ overnight; the next day, washing with the washing solution for 2 times, and patting dry; blocking solution (20% BSA +80% PBS) was added, 120. Mu.l per well, 37 ℃,1h, patted dry; adding diluted cell supernatant at a concentration of 100 μ l/well at 37 deg.C for 60min; throwing off liquid in the plate, patting dry, adding 20% mouse negative blood, sealing, and sealing at 37 ℃ for 1h, wherein each hole is 120 mu l; throwing off the liquid in the plate, patting to dry, adding diluted CA242 human ascites, 100 mul per hole, 37 ℃,40min; washing with washing solution for 5 times, and drying; adding HRP-labeled CA242 monoclonal antibody into each well at a temperature of 37 ℃ for 30min in an amount of 100 mu l; adding a developing solution A (50 μ l/hole), adding a developing solution B (50 μ l/hole), and standing for 10min; adding stop solution into the mixture, wherein the concentration of the stop solution is 50 mu l/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results showed that the OD of the reaction after the cell supernatant was diluted 1000 times was still greater than 1.0, and the OD of the reaction without the cell supernatant was less than 0.1, indicating that the antibodies generated after the transient transformation of the plasmid were all active against the CA242 protein.

(2) Linearization of recombinant antibody expression plasmids

The following reagents were prepared: 50 mul Buffer, 100 mul DNA/tube, 10 mul Puv I enzyme and sterile water to 500 mul, enzyme digestion in water bath at 37 ℃ overnight; extraction was performed sequentially with equal volumes of phenol/chloroform/isoamyl alcohol (lower layer) 25, followed by chloroform (aqueous phase); precipitating with 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing with 70% ethanol, removing organic solvent, re-melting with appropriate amount of sterilized water after ethanol is completely volatilized, and finally measuring concentration.

(3) Stable transfection of recombinant antibody expression plasmid, pressurized screening of stable cell lines

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml are put into a centrifuge tube, 100 mul of plasmid is mixed with 700 mul of cells, and the mixture is transferred into an electric rotating cup and is electrically rotated, and the next day is counted; 25 u mol/L MSX 96 hole pressure culture about 25 days.

Observing the marked clone holes with cells under a microscope, and recording the confluence degree; taking culture supernatant, and sending the culture supernatant to a sample for detection; selecting antibody concentration, transferring cell strains with high relative concentration into 24 holes, and transferring into 6 holes after 3 days; after 3 days, the seeds were kept and cultured in batches, and the cell density was adjusted to 0.5X 10 ⁶ cells/ml,2.2ml, cell density 0.3X 10 ⁶ cell/ml, 2ml for seed preservation; and (4) 7 days, carrying out batch culture supernatant sample detection in 6 holes, and selecting cell strains with small antibody concentration and cell diameter, transferring the cell strains to TPP (thermoplastic vulcanizate) for seed preservation and passage.

3 recombinant antibody production

(1) Cell expanding culture

After the cells were recovered, they were cultured in 125ml size shake flasks, inoculated with 30ml Dynamis medium at a culture medium volume of 100%, and placed in a shaker at a rotation speed of 120r/min and a temperature of 37 ℃ with 8% carbon dioxide. Culturing for 72h, inoculating and expanding at inoculation density of 50 ten thousand cells/ml, and calculating the expanding volume according to production requirements, wherein the culture medium accounts for 100 percent. Then the culture is expanded every 72 h. When the cell amount meets the production requirement, the production is carried out by strictly controlling the inoculation density to be about 50 ten thousand cells/ml.

(2) Shake flask production and purification

Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8 percent. Feeding in a flowing mode: daily feeding was started when the culture was carried out for 72h in a shake flask, 3% of the initial culture volume was fed daily to HyCloneTM Cell BoostTM Feed 7a, and one thousandth of the initial culture volume was fed daily to Feed 7b, up to day 12 (day 12 feeding). Glucose was supplemented with 3g/L on the sixth day. Samples were collected on day 13. Affinity purification was performed using a proteinA affinity column. Mu.g of the purified antibody (i.e., the CA242 monoclonal antibody) was subjected to reducing SDS-PAGE, and the electrophoretogram thereof was as shown in FIG. 1. Two bands were shown after reducing SDS-PAGE, 1 with 50kD of Mr (i.e., heavy chain, SEQ ID NO: 14) and 28kD of Mr (i.e., light chain, SEQ ID NO: 12).

Example 2

Detection of antibody Performance

(1) Example 1 Activity assay of antibodies and mutants thereof

Further analysis revealed that the heavy chain variable region of the CA242 monoclonal antibody (WT) of example 1 is represented by SEQ ID NO 13, wherein the amino acid sequences of the complementarity determining regions of the heavy chain are as follows:

CDR-VH1：D-F(X1)-T-F-T(X2)-Y-Y-G-M-H(X3)；

CDR-VH2：W-I(X1)-D-T-N(X2)-T-G-E-P-S(X3)-Y-A-D(X4)-D-F-K-G；

CDR-VH3：A-R-R-A(X1)-P-Y-N(X2)-W-Y-F-D-I(X3)；

the light chain variable region is shown as SEQ ID NO. 11, wherein the amino acid sequences of the complementarity determining regions of the light chain are as follows:

CDR-VL1：R-S-S-R(X1)-S-IL(X2)-H-S-D(X3)-G-N-T-Y-L(X4)-Y；

CDR-VL2：K(X1)-M-S-N-I(X2)-I(X3)-S；

CDR-VL3：L(X1)-Q-H-I(X2)-E-F(X3)-P-F-T。

on the basis of the CA242 monoclonal antibody of example 1, a site involved in the activity of the antibody was mutated in the complementarity determining region, wherein X1, X2, X3, and X4 are all mutated sites. See table 1 below.

TABLE 1 mutant sites associated with antibody Activity

And (3) detecting the binding activity:

coating solution (PBS) dilutes goat anti-mouse IgG1 mug/ml to coat the microplate, each well is 100 mul, and the temperature is 4 ℃ overnight; the next day, washing with the washing solution for 2 times, and patting dry; blocking solution (20% BSA +80% PBS) was added, 120. Mu.l per well, 37 ℃,1h, patted dry; adding diluted CA242 monoclonal antibody (WT) or its mutant in Table 1at 100 μ l/well, 37 deg.C for 60min; throwing off liquid in the plate, patting dry, adding 20% mouse negative blood, sealing, and sealing at 37 ℃ for 1h, wherein each hole is 120 mu l; throwing off the liquid in the plate, patting to dry, adding diluted CA242 human ascites, 100 mul per hole, 37 ℃,40min; washing with washing solution (PBS) for 5 times, and drying; adding HRP-labeled CA242 monoclonal antibody (commercially available) at 37 deg.C for 30min in a volume of 100. Mu.l per well; adding developing solution A (50 μ L/well, 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L peroxide)Urea), adding developing solution B (50 μ L/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; stop solution (50. Mu.l/well, containing 0.75 g/EDTA-2 Na and 10.2ml/L concentrated H) was added ₂ SO ₄ ) (ii) a OD readings were taken at 450nm (reference 630 nm) on the microplate reader. The results are shown in Table 2.

TABLE 2 Activity data of antibodies and mutants thereof

Antibody concentration (ng/ml)	6.25	3.125	1.563	0.781	0.391	0
							WT	1.909	1.327	0.763	0.430	0.274	0.062
Mutation 1	2.305	2.166	1.584	0.989	0.591	0.053
							Mutation 2	2.108	2.007	1.681	1.082	0.607	0.064
Mutation 3	2.184	2.059	1.517	0.972	0.542	0.091
							Mutation 4	2.167	2.075	1.634	1.076	0.556	0.073
Mutation 5	1.987	1.925	1.534	0.975	0.453	0.083
							Mutation 6	0.552	0.012	-	-	-	-
Mutation 7	0.432	0.022	-	-	-	-

The data in table 2 show that WT, and mutations 1 through 5, have better binding activity, while mutations 6 and 7 have poorer binding activity, indicating that the mutation pattern at the mutation sites listed in table 1 is unpredictable.

(2) Example 1 affinity assays for antibodies and mutants thereof

(a) Based on mutation 1, other sites were mutated, and the sequence of each mutation is shown in table 3 below.

TABLE 3 mutation sites related to antibody affinity

And (3) affinity detection:

using AMC sensors, purified antibodies (each of the mutant antibodies in Table 3) were diluted to 10. Mu.g/ml with PBST, and CA242 human ascites was diluted with PBST in a gradient;

the operation flow is as follows: equilibration for 60s in buffer 1 (PBST), immobilized antibody for 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3, and data output. ( KD denotes the equilibrium dissociation constant, i.e. affinity; kon denotes the binding rate; kdis denotes the off-rate. )

Table 4 affinity assay data

The data in Table 4 show that K for mutation 1, and mutations 1-1 through 1-64 _D Lower, all had better affinity, indicating that the mutation pattern of the mutation sites listed in table 3 has no negative effect on affinity, and better affinity can be obtained after mutation according to the mutation pattern.

(b) Based on WT, mutation is carried out on other sites, and the affinity of each mutant is detected, the sequence of each mutation is shown in Table 5, and the corresponding affinity data is shown in Table 6.

TABLE 5 mutations with WT as backbone

TABLE 6 results of affinity assay of mutations with WT as backbone

The data in Table 6 show that WT, and WT 1-1 to WT 1-12, have better affinities, and also that the mutation pattern at the mutation sites listed in Table 5 has a less significant effect on affinity.

(3) Antibody auto-pairing assay

(a) The mutant 1 antibody is divided into two parts, one part is used as a coating antibody and coated on an ELISA plate, the other part is used as an enzyme-labeled antibody and labeled by HRP, CA242 quality control products with different concentrations are detected by adopting a double-antibody sandwich ELISA method, the OD value of 450nm is detected, the result is shown in table 7 and figure 2, and the result shows that the mutant 1 antibody can be self-paired to detect the CA242 antigen.

TABLE 7 detection of OD of CA242 quality control at different concentrations by auto-pairing of mutant 1 antibodies ₄₅₀ As a result, the

CA242 quality control concentration (U/mL)	OD ₄₅₀
		150.6	1.922
75.3	1.244
		37.7	0.634
18.8	0.320
		9.4	0.171
4.7	0.091
		2.4	0.047
0.0	0.012

As can be seen from Table 7, the mutant 1 antibody can be used in self-pairing mode, and has a wide linear range of 2.4-150U/mL when being detected in self-pairing mode ² Was 0.981.

(b) Taking the mutant 1 antibody, dividing into two parts, marking one part with biotin as a reagent 1 (with the concentration of 1 ug/ml), and coating magnetic beads with streptavidin; one part is marked by acridinium ester and used as a reagent 2 (with the concentration of 2 ug/ml), and CA242 quality control substances with different concentrations are detected on a chemiluminescence platform, and the detection results of the luminescence values are shown in Table 8 and figure 3.

TABLE 8 detection of OD of CA242 quality control at different concentrations by auto-pairing of mutant 1 antibodies ₄₅₀ Results

As can be seen from Table 8, in the luminescence platform, the mutant 1 antibody can also be used in self-pairing mode, the linear range can reach 0-1500U/ml, R ² Is 0.9988.

(4) Evaluation of stability against naked antibody

Placing the antibodies in the tables at 4 ℃ (refrigerator), -80 ℃ (refrigerator) and 37 ℃ (thermostat) for 21 days, taking samples at 7 days, 14 days and 21 days for state observation, and performing activity detection on the samples at 21 days, wherein the results show that under three assessment conditions, no obvious protein state change is seen in 21 days of antibody placement, and the activity is more likely to decrease with the increase of assessment temperature, which indicates that the self-produced antibody is stable. The following table shows the results of the 21-day enzyme immunity activity test OD of the mutant 1 antibody.

TABLE 9

Antibody concentration (ng/ml)	6.25	1.563	0
				Samples at 4 ℃ for 21 days	2.315	1.576	0.094
21 days samples at-80 deg.C	2.407	1.639	0.083
				21 day samples at 37 deg.C	2.353	1.554	0.067

As can be seen from table 9, after being stored at different temperatures for 21 days, the binding activity does not change significantly, and the antibody provided by the embodiment of the present invention can still detect the antigen, which indicates that the antibody provided by the embodiment of the present invention has better stability.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Dongguan City of Pengzhi Biotech Co., ltd

<120> CA242 binding protein and application, detection method and kit thereof

<160> 14

<170> PatentIn version 3.5

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

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Leu Gln Ile Arg Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

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Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr

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Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu

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Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

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

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Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

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Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

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Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

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Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

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Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

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Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

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Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

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Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

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

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Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

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Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

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Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

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Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

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Claims

1. A binding protein that specifically binds CA242, wherein the binding protein comprises an antigen binding domain; the antigen binding domain includes 6 complementarity determining regions:

a complementarity determining region CDR-VH1 having an amino acid sequence of D-X1-T-F-X2-Y-G-M-X3, wherein: x1 is Y;

a complementarity determining region CDR-VH2 having an amino acid sequence of W-X1-D-T-X2-T-G-E-P-X3-Y-A-X4-D-F-K-G, wherein: x3 is T;

a complementarity determining region CDR-VH3 having an amino acid sequence a-R-X1-P-Y-X2-W-Y-F-D-X3, wherein: x1 is G;

a complementarity determining region CDR-VL1 having the amino acid sequence R-S-S-X1-S-X2-H-S-X3-G-N-T-Y-X4-Y, wherein: x1 is K;

a complementarity determining region CDR-VL2 having the amino acid sequence X1-M-S-N-X2-X3-S, wherein: x1 is R;

a complementarity determining region CDR-VL3 having the amino acid sequence X1-Q-H-X2-E-X3-P-F-T, wherein: x3 is Y;

the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following mutation combinations 1-65:

site of the body CDR-VH1 X2/X3 CDR-VH2 X1/X2/X4 CDR-VH3 X2/X3 CDR-VL1 X2/X3/X4 CDR-VL2 X2/X3 CDR-VL3 X1/X2 Mutant combination 1 T/H I/N/D N/I IL/D/L I/I L/I Combination of mutations 2 S/H I/N/E GG/V IL/N/L L/I I/I Combination of mutations 3 T/N VN/D D/L II/D/L V/I L/V Combination of mutations 4 S/N V/N/E GG/I II/N/L I/L I/V Combination of mutations 5 T/S L/N/D N/I LI/D/L L/L L/L Combination of mutations 6 S/S L/N/E D/I LI/N/L V/L I/L Mutant combination 7 S/N I/T/D GG/V LL/D/L I/V I/L Combination of mutations 8 T/N I/T/E N/V LL/N/L L/V L/L Combination of mutations 9 S/H VT/D D/V IL/D/I V/V I/I Combination of mutations 10 T/H V/T/E GG/L IL/N/I V/I L/I Combination of mutations 11 S/S L/T/D N/L II/D/I V/V I/V Mutant combination 12 T/S L/T/E D/L II/N/I V/L L/V Mutant combinations 13 T/N I/Y/D GG/I LI/D/I I/I I/I Combination of mutations 14 S/N I/Y/E N/V LI/N/I L/V L/V Combination of mutations 15 T/H VY/D D/L LL/D/I I/L I/L Mutant combinations 16 S/H V/Y/E D/I LL/N/I I/I L/L Mutant combinations 17 T/S L/Y/D GG/V IL/D/L I/V I/V Mutant combinations 18 S/S L/Y/E N/L IL/N/I L/L L/I Combination of mutations 19 S/H I/N/D GG/V IL/N/L L/V I/L Combination of mutations 20 T/H I/T/D D/V II/D/I V/V L/L MutationsCombination 21 S/S I/Y/D N/V II/D/L I/V I/I Mutant combination 22 T/S VN/D D/L II/N/I V/L L/I Mutant combination 23 S/N VT/D GG/L II/N/L I/L I/V Mutant combinations 24 T/N VY/D N/L LI/D/I L/L L/V Mutant combinations 25 S/H I/N/E GG/I LI/D/L I/I I/I Mutant combinations 26 T/H I/T/E N/I LI/N/I L/L L/V Mutant combinations 27 S/S I/Y/E D/I LI/N/L V/I I/L Mutant combinations 28 T/S V/N/E GG/I LL/D/I V/I L/L Mutant combinations 29 S/N V/T/E N/I LL/D/L V/V I/V Combination of mutations 30 T/N V/Y/E D/I LL/N/I V/L L/I Combination of mutations 31 T/H L/N/D GG/V LL/N/L I/I L/I Mutant combinations 32 S/H L/T/D N/V IL/D/I L/V I/I Mutant combinations 33 T/N L/Y/D D/V IL/D/L I/L L/V Mutant combinations 34 S/N L/N/E GG/L IL/N/I I/I I/V Combination of mutations 35 T/S L/T/E N/L IL/N/L I/V L/L Combination of mutations 36 S/S L/Y/E D/L II/D/I L/L I/L Mutant combinations 37 S/N I/N/D N/I II/D/L L/V I/L Combination of mutations 38 T/N I/N/E GG/V II/N/I V/V L/L Mutant combinations 39 S/H VN/D D/L II/N/L I/V I/I Combination of mutations 40 T/H V/N/E GG/I LI/D/I V/L L/I Mutant combination 41 S/S L/N/D D/V LI/D/L I/L I/V Combination of mutations 42 T/S L/N/E N/L LI/N/I L/L L/V Combination of mutations 43 T/N I/T/D GG/I LI/N/L I/I I/I Mutant combinations 44 S/N I/T/E N/V LL/D/I L/L L/V Combination of mutations 45 T/H VT/D D/L LL/D/L V/I I/L Mutant combinations 46 S/H V/T/E D/I LL/N/I I/I L/L Mutant combinations 47 T/S L/T/D GG/V LL/N/L L/I I/V Mutant combinations 48 S/S L/T/E N/L IL/D/I V/I L/I Mutant combinations 49 S/H I/Y/D GG/I IL/D/I I/V I/L Mutant combinations 50 T/H I/Y/E N/V IL/N/I L/V L/L Combination of mutations 51 S/S VY/D D/L II/D/I V/V I/I Mutant combinations 52 T/S V/Y/E GG/V II/N/I I/L L/I Mutant combination 53 S/N L/Y/D D/V LI/D/I L/L I/V Mutant combinations 54 T/N L/Y/E N/V LI/N/I V/L L/V Mutant combinations 55 S/H I/N/D D/L LL/D/I V/I L/I Mutant combinations 56 T/H I/T/D GG/L LL/N/I V/V I/I Mutant combinations 57 S/S I/Y/D N/L IL/D/I V/L L/V Mutant combinations 58 T/S VN/D GG/I IL/D/L I/I I/V Mutant combinations 59 S/N VT/D N/I IL/N/L L/V L/L Mutant combinations 60 T/N VY/D D/I II/D/L I/L I/L Mutant combinations 61 T/H I/N/E N/I II/N/L I/I I/L Mutant combinations 62 S/H I/T/E GG/V LI/D/L I/V L/L Mutant combinations 63 T/N I/Y/E D/L LI/N/L L/L I/I Mutant combinations 64 S/N V/N/E GG/I LL/D/L V/V L/I Combination of mutations 65 T/S V/T/E D/V LL/N/L I/V I/V

。

2. A binding protein that specifically binds CA242, wherein the binding protein comprises an antigen binding domain; the antigen binding domain includes 6 complementarity determining regions:

a complementarity determining region CDR-VH1, the amino acid sequence of which is D-X1-T-F-X2-Y-Y-G-M-X3;

a complementarity determining region CDR-VH2, the amino acid sequence of which is W-X1-D-T-X2-T-G-E-P-X3-Y-A-X4-D-F-K-G;

a complementarity determining region CDR-VH3, the amino acid sequence of which is A-R-R-X1-P-Y-X2-W-Y-F-D-X3;

a complementary determining region CDR-VL1, the amino acid sequence of which is R-S-S-X1-S-X2-H-S-X3-G-N-T-Y-X4-Y;

a complementarity determining region CDR-VL2, the amino acid sequence of which is X1-M-S-N-X2-X3-S;

a complementarity determining region CDR-VL3, the amino acid sequence of which is X1-Q-H-X2-E-X3-P-F-T;

in the CDR-VH1, X1 is F;

in the CDR-VH2, X3 is S;

in the CDR-VH3, X1 is A;

in the complementarity determining region CDR-VL1, X1 is R;

in the complementarity determining region CDR-VL2, X1 is K;

in the complementarity determining region CDR-VL3, X3 is F;

the mutation site of each complementarity determining region of the antigen binding domain is selected from any one of the following combinations of mutations 66-78:

combination of mutations CDR-VH1 X2/X3 CDR-VH2 X1/X2/X4 CDR-VH3 X2/X3 CDR-VL1 X2/X3/X4 CDR-VL2 X2/X3 CDR-VL3 X1/X2 Mutant combinations 66 T/H I/N/D N/I IL/D/L I/I L/I Mutant combinations 67 S/H V/N/D GG/I LL/N/I V/L I/V MutationsCombination 68 T/N V/N/D GG/I IL/D/L L/I I/I Mutant combinations 69 S/H V/T/E D/L LL/D/I V/L I/V Mutant combinations 70 T/N V/T/D N/V II/D/L V/V L/L Mutant combinations 71 T/N L/N/E N/I II/N/I V/V I/I Mutant combinations 72 S/N I/T/E GG/I IL/N/I V/L L/L Mutant combinations 73 T/S V/T/E GG/L IL/D/I I/L L/L Mutant combinations 74 T/N I/Y/D D/V II/D/I L/I L/L Mutant combinations 75 S/H I/T/E D/V II/N/L V/I I/I Mutant combinations 76 S/N I/N/E D/L II/D/L L/I L/L Mutant combinations 77 S/H V/T/D D/I LI/D/I I/V I/I Mutant combinations 78 S/H L/Y/D D/I IL/N/L I/L L/V

。

3. The binding protein of any one of claims 1-2, wherein said binding protein is an antibody or a functional fragment thereof.

4. The binding protein of claim 3, wherein said binding protein is selected from any one of F (ab ') 2, fab', fab, fv, scFv, and diabody.

5. The binding protein according to any one of claims 1 to 2, wherein said binding protein comprises the light chain framework regions FR-L1, FR-L2, FR-L3 and FR-L4 as shown in SEQ ID NOs 1 to 4 in sequence, and/or the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 as shown in SEQ ID NOs 5 to 8 in sequence.

6. The binding protein according to any one of claims 1-2, wherein said binding protein further comprises an antibody constant region.

7. The binding protein of claim 6, wherein the antibody constant region is selected from the constant regions of any one of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD.

8. The binding protein of claim 6, wherein said antibody constant region is of a species of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin.

9. The binding protein according to claim 8, wherein said Niu Xuanzi dairy cow; alternatively, the chicken is selected from turkey or turkey.

10. The binding protein according to claim 8, wherein said antibody constant region is derived from a mouse;

the light chain constant region sequence of the antibody constant region is shown as SEQ ID NO. 9, and the heavy chain constant region sequence of the antibody constant region is shown as SEQ ID NO. 10.

11. A reagent or kit comprising a binding protein according to any one of claims 1 to 10.

12. Use of a binding protein according to any one of claims 1 to 10 in the preparation of a kit for detecting CA242 in a test sample.

13. The use according to claim 12, wherein the kit is for:

mixing the binding protein of any one of claims 1-10 with a sample to be tested;

the detection of CA242 is achieved by precipitation reaction or by labeling an indicator showing signal intensity.

14. The use according to claim 13, wherein the method for detecting CA242 by precipitation reaction is selected from any one or more of the following methods: one-way immunodiffusion assay, two-way immunodiffusion assay, immunoturbidimetry, immunoelectrophoresis, and immunoblotting.

15. Use according to claim 14, wherein said immunoelectrophoresis comprises convection immunoelectrophoresis.

16. The use according to claim 13, wherein the method for detecting CA242 by marking an indicator showing signal intensity is selected from any one or more of the following methods: immunofluorescence, radioimmunoassay, enzyme linked immunoassay, and chemiluminescent immunoassay.

17. The use according to claim 13, wherein the indicator is selected from any one of a fluorescent dye, a radioisotope, an enzyme catalyzing color development of a substrate, and a chemiluminescent reagent.

18. A vector comprising a nucleic acid encoding the binding protein according to any one of claims 1 to 10.

19. A host cell comprising the vector of claim 18.

20. A method of producing the binding protein of any one of claims 1 to 10, comprising:

culturing the host cell of claim 19, and isolating and purifying the binding protein from the culture medium or from the cultured host cell.