CN113493515B - anti-CLDN 18A2 antibody and drug for treating tumor - Google Patents

Abstract

The invention discloses an antibody of CLDN18A2 and a medicament for treating tumors. Relates to the technical field of biological medicine. The anti-CLDN 18A2 disclosed by the present invention has a heavy chain variable region and a light chain variable region, which can specifically recognize and bind to CLDN18A2, has good specificity, and can be used for treating CLDN18A2 positive tumors.

Description

anti-CLDN 18A2 antibody and drug for treating tumor

Technical Field

The invention relates to the technical field of biomedicine, in particular to an antibody of CLDN18A2 and a medicament for treating tumors.

Background

About 90% of malignant tumors are derived from epithelial cells, tight junctions between epithelial cells are closely related to the occurrence and development of malignant tumors, and the occurrence of tumors is often accompanied by reduction of tight junction function and change of Claudin protein expression.

The Claudin protein is a skeleton protein with a tightly connected structure and function, is positioned on the apical side of the gap between adjacent cells, has a synergistic effect with other structural proteins and signal proteins, and has the main biological functions of intercellular adhesion, cell acute maintenance, cell bypass permeability regulation, and participation in cell proliferation and differentiation regulation. Claudins proteins regulate permeability at cell junctions mainly by phosphorylation by kinase a (PKA) or Protein Kinase C (PKC) and amino acids mainly including serine and threonine. Abnormal expression of Claudin will disrupt the epithelial permeability barrier, leading to loss of cell polarity and decreased intercellular adhesion, leading to the development and progression of various tumors.

Claudin 18A2 belongs to the family of tight junction proteins (Claudin) and is an important component molecule found in 1998 in chicken liver to maintain cell polarity, cell-cell adhesion, and regulate cell bypass permeability. The Claudin 18A2 protein has 261 amino acids and a molecular weight of about 27.9kDa (27.7 kDa for Claudin 18A 1). Claudin18 A2 is a transmembrane protein complex comprising 4 transmembrane regions, two extracellular loops and one intracellular loop, with the N-and C-termini being intracytoplasmic. The Claudin18 gene has two alternative splice variants of Claudin 18A1 and Claudin 18A2, which are respectively and specifically expressed in lung tissues and gastric epithelial tissues. Claudin 18A1 and Claudin 18A2 differ only by 8 amino acids between the first extracellular domain, with greater similarity. The Claudin 18A2 target is not present in any healthy tissue except the stomach wall (gastric epithelial cells), making it a potential anti-tumor target.

Claudin18.2 is overexpressed in a variety of tumorigenesis processes, including 80% of gastrointestinal adenocarcinomas, 60% of pancreatic tumors, and a portion of colorectal, ovarian, lung, and the like. Claudin 18A2 expression profile in tumors (data source Ganymed IMAB-362 phase II clinical trial): 730 patients, 685 (94%) were immunohistochemically with 49% (n = 333/685) expressing CLDN18.2 (≧ 40% tumor cells CLDN18.2 ≧ 2 +).

The product IMAB362 of Ganymed is the first antibody targeting claudin18.2, the IMAB362 directly acts on Claudin18.2 to stimulate immune reactions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) and the like, and can play a role in recruiting T cells and changing tumor microenvironment together with chemotherapeutic drugs, thereby achieving the effect of killing Claudin18.2 positive tumors. In contrast, IMAB362 does not kill normal tissues that express little or no claudin18.2.

Disclosure of Invention

The present invention aims to provide an antibody having CLDN18A2 activity and a therapeutic agent for tumor. The present invention provides a novel antibody against CLDN18A2, which is capable of specifically recognizing and binding to CLDN18A2 with good specificity, and further, which can induce cell death by binding to CLDN18 A2-mediated CDC reaction, and can be used as a medicament for treating CLDN18 A2-positive tumors.

The invention is realized by the following steps:

in a first aspect, the present invention provides an antibody or a functional fragment thereof directed to CLDN18A2, the antibody or the functional fragment thereof having a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of complementarity determining regions HCDR1 (heavy chain complementarity determining region 1), HCDR2 and HCDR3 of the heavy chain variable region and the amino acid sequences of complementarity determining regions LCDR1 (light chain complementarity determining region 1), LCDR2 and LCDR3 of the light chain variable region are any one of the following (1) to (7):

(1): HCDR1 is shown as SEQ ID NO.28, HCDR2 is shown as SEQ ID NO.35, and HCDR3 is shown as SEQ ID NO. 42;

LCDR1 is shown as SEQ ID NO.49, LCDR2 is shown as SEQ ID NO.54, and LCDR3 is shown as SEQ ID NO. 59;

(2): HCDR1 is shown as SEQ ID NO.29, HCDR2 is shown as SEQ ID NO.36, and HCDR3 is shown as SEQ ID NO. 43; LCDR1 is shown as SEQ ID NO.50, LCDR2 is shown as SEQ ID NO.54, and LCDR3 is shown as SEQ ID NO. 60;

(3): HCDR1 is shown as SEQ ID NO.30, HCDR2 is shown as SEQ ID NO.37, and HCDR3 is shown as SEQ ID NO. 44;

LCDR1 is shown as SEQ ID NO.51, LCDR2 is shown as SEQ ID NO.55, and LCDR3 is shown as SEQ ID NO. 61;

(4): HCDR1 is shown as SEQ ID NO.31, HCDR2 is shown as SEQ ID NO.38, and HCDR3 is shown as SEQ ID NO. 45;

LCDR1 is shown as SEQ ID NO.52, LCDR2 is shown as SEQ ID NO.56, and LCDR3 is shown as SEQ ID NO. 62;

(5): HCDR1 is shown as SEQ ID NO.32, HCDR2 is shown as SEQ ID NO.39, and HCDR3 is shown as SEQ ID NO. 46;

LCDR1 is shown as SEQ ID NO.50, LCDR2 is shown as SEQ ID NO.54, and LCDR3 is shown as SEQ ID NO. 63;

(6): HCDR1 is shown as SEQ ID NO.33, HCDR2 is shown as SEQ ID NO.40, and HCDR3 is shown as SEQ ID NO. 47;

LCDR1 is shown as SEQ ID NO.53, LCDR2 is shown as SEQ ID NO.57, and LCDR3 is shown as SEQ ID NO. 64;

(7) HCDR1 is shown as SEQ ID NO.34, HCDR2 is shown as SEQ ID NO.41, and HCDR3 is shown as SEQ ID NO. 48;

LCDR1 is shown as SEQ ID NO.49, LCDR2 is shown as SEQ ID NO.58, and LCDR3 is shown as SEQ ID NO. 65.

CLDN18A1 and CLDN18A2 are highly homologous, the difference region is small, the difficulty of screening antibodies specific to CLDN18A2 is high, and in addition, CLDN18 is a multi-transmembrane protein, cannot directly express a cell-soluble ectodomain, and cannot use a protein antigen during immunization. However, the present inventors have conducted creative efforts to obtain an antibody having the CDR region structures shown in (1) to (7) above, which is capable of specifically recognizing and binding to CLDN18A2, does not bind to CLDN18A1, and has good specificity; in addition, the antibody provided by the invention can stimulate immune reactions such as Complement Dependent Cytotoxicity (CDC) and the like by binding to CLDN18A2, and has a killing effect on cells expressing CLDN18 A2; the antibody can be used for treating tumors with positive expression of CLDN18A2, and provides a medicine selection and a treatment idea for treating the tumors.

It should be noted that, on the basis of the CDR sequences of the above-mentioned antibody of the present invention, those skilled in the art can easily conceive of substitution, deletion, or addition of one or more amino acids to the CDR sequences (CDR 1, CDR2, and CDR 3) in the heavy chain or light chain variable region to obtain a variant sequence having the same or equivalent biological activity as the sequence shown in any one of (1) to (7) above, which also falls within the scope of the present invention.

Alternatively, in some embodiments of the present invention, the amino acid sequences of the heavy chain variable region and the light chain variable region are any one of the following (a) to (g):

(a) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.14, and the light chain variable region is shown as SEQ ID NO. 21;

(b) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.15, and the light chain variable region is shown as SEQ ID NO. 22;

(c) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.16, and the light chain variable region is shown as SEQ ID NO. 23;

(d) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.17, and the light chain variable region is shown as SEQ ID NO. 24;

(e) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.18, and the light chain variable region is shown as SEQ ID NO. 25;

(f) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.19, and the light chain variable region is shown as SEQ ID NO. 26;

(g) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.20, and the light chain variable region is shown as SEQ ID NO. 27.

It is to be noted that, in addition to the heavy chain or light chain variable region sequence of the above-mentioned antibody of the present invention, one skilled in the art can easily conceive of substitution, deletion, addition, or the like of one or more amino acids for the framework region (FR 1, FR2, FR3, and FR 4) sequence in the heavy chain or light chain variable region to obtain a variant sequence having the same or equivalent biological activity as the sequence shown in any one of (a) to (g) above, and the variant sequence is also within the scope of the present invention.

Alternatively, in some embodiments of the invention, the light chain constant region of the antibody is a kappa-type light chain constant region or a lambda-type light chain constant region.

Alternatively, in some embodiments of the invention, the heavy chain constant region of the antibody is a heavy chain constant region of an IgA, igD, igE, igG or IgM antibody.

Alternatively, in some embodiments of the invention, the heavy chain constant region of the antibody is a heavy chain constant region of an IgG antibody.

Alternatively, in some embodiments of the invention, the heavy chain constant region of the antibody is a heavy chain constant region of an IgG1, igG2, igG3, or IgG4 antibody.

Alternatively, in some embodiments of the invention, the light chain constant region of the antibody is set forth in SEQ ID No. 66;

alternatively, in some embodiments of the invention, the heavy chain constant region of the antibody is as set forth in SEQ ID No.67 or SEQ ID No.68.

It is to be noted that, based on the above-described antibody constant region sequences disclosed in the present invention, those skilled in the art can easily conceive of modification of the antibody constant region sequences, such as humanization modification, and that the resulting antibodies, regardless of the modification of the constant region sequences, are within the scope of the present invention.

Alternatively, in some embodiments of the invention, the functional fragment is a Fab, fab ', F (ab') 2, fv or ScFv fragment.

In a second aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof according to any one of the above conjugated to a therapeutic agent.

Optionally, in some embodiments of the invention, the therapeutic agent is a toxin, a radioisotope, or a cytotoxic agent.

The antibodies of the invention may be conjugated to other therapeutic agents, such as toxins, radioisotopes, or cytotoxic agents, and the like, to effect enhanced cell killing or to effect inhibition of cell proliferation. The type of therapeutic agent can be selected by those skilled in the art according to the actual needs without any inventive effort for those skilled in the art, and it is within the scope of the present invention to select any therapeutic agent to be conjugated to the antibody of the present invention.

In a third aspect, the present invention provides a fusion protein comprising an antibody or functional fragment thereof as defined in any one of the above.

Based on the antibody disclosed in the present invention, it is easy for those skilled in the art to think of fusing the antibody with other functional proteins or polypeptides to enhance the function of the antibody or to facilitate the purification of the antibody, etc., without the need of creative efforts for those skilled in the art, and therefore, the fusion protein obtained by fusing the antibody of the present invention with any type of protein or polypeptide is within the protection scope of the present invention.

In a fourth aspect, the present invention provides a medicament for treating tumors, which comprises the antibody or functional fragment thereof as described in any one of the above, the antibody conjugate as described in any one of the above, or the fusion protein as described above.

Alternatively, in some embodiments of the invention, the tumor is a tumor positive for CLDN18A2 expression.

Optionally, in some embodiments of the invention, the tumor positive for CLDN18A2 expression is selected from any one of gastric adenocarcinoma, pancreatic cancer, rectal cancer, breast cancer, ovarian cancer and lung cancer.

It is noted that the medicament provided by the present invention can treat any tumor positive for expression of CLDN18A2, including but not limited to gastric adenocarcinoma, pancreatic cancer, rectal cancer, breast cancer, ovarian cancer and lung cancer, and for other types of tumors positive for expression of CLDN18A2, the medicament of the present invention can also be used for treatment.

Optionally, in some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable excipient.

In a fifth aspect, the present invention provides a reagent for detecting CLDN18A2, comprising the antibody or functional fragment thereof according to any one of the above.

The antibody provided by the present invention specifically binds to CLDN18A2 protein, and thus, the antibody provided by the present invention can be used for detecting CLDN18A2 protein as a detection reagent for CLDN18A2 protein.

In a sixth aspect, the invention provides an isolated nucleic acid molecule encoding an antibody or functional fragment thereof as described in any one of the above.

Based on the degeneracy of the codons, the nucleic acid sequence encoding the antibody is easily obtained by those skilled in the art, and the nucleic acid sequence is within the protection scope of the present invention as long as the nucleic acid sequence encodes the antibody of the present invention.

In a seventh aspect, the present invention provides a recombinant vector comprising a nucleic acid molecule as described above.

In an eighth aspect, the present invention provides a recombinant cell containing the recombinant vector as described above.

In a ninth aspect, the present invention provides a method of preparing an antibody or functional fragment thereof as defined in any one of the above, comprising: culturing the recombinant cell as described above, and isolating and purifying the antibody or functional fragment thereof from the cultured species.

Based on the disclosure of the antibody sequence, the skilled in the art can easily think of using genetic engineering techniques to prepare the antibody of the present invention, which is not necessary for the skilled in the art to make creative efforts, and the method of preparing the antibody of the present invention is within the protection scope of the present invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required 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 those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

Fig. 1 shows the detection of transient expression of CLDN18A2 by HEK293 cells.

FIG. 2 shows the results of screening CHO-CLDN18A2 stable expression cell line, wherein A is the result of immunoblotting and B is the result of flow cytometry.

Fig. 3 is a preliminary screening result for a monoclonal antibody that binds to CLDN18.

Fig. 4 is a sequence alignment of human CLDN18A1 and CLDN18A2.

FIG. 5 shows the results of screening for murine monoclonal antibodies that specifically bind to CHO-CLDN18A2 cells but not to CHO-CLDN18A1 cells.

FIG. 6 is the results of screening for murine monoclonal antibodies that specifically bind to HEK293-CLDN18A2 cells but not to CHO-CLDN18A1 cells.

Fig. 7 shows the results of detecting the RNA expression levels of CLDN18A1 and CLDN18A2 before and after EOF treatment of tumor cell lines.

Fig. 8 is the result of detecting the protein expression level of CLDN18A1 and CLDN18A2 of tumor cell lines.

FIG. 9 shows the results of screening monoclonal antibodies that specifically bind to NUGC4-CLDN18A2 and NCI-SNU-16-CLDN18A 2.

FIG. 10 is a graph of binding curves of murine monoclonal antibodies (# 1, #26, #38, #49, #52, #60, # 63) and the antibody IMAB362-CH1 to CHO-CLDN18A2 cells.

FIG. 11 shows the results of the competitive detection of murine monoclonal antibodies (# 1, #26, #38, #49, #52, # 60) with IMAB361 antibody epitopes under the condition of first adding IMAB362 antibody.

FIG. 12 shows the results of the competitive detection of mouse monoclonal antibodies (# 1, #26, #38, #49, #52, # 60) with IMAB361 antibody epitopes under the condition that mouse monoclonal antibodies (# 1, #26, #38, #49, #52, # 60) are added first.

FIG. 13 shows the results of binding of murine monoclonal antibodies (# 1, #26, #38, #49, #52, #60, # 63) to peripheral blood lymphocytes.

FIG. 14 shows the results of binding of murine monoclonal antibodies (# 26, #38, #49, #52, #60, # 63) to non-small cell carcinomas.

FIG. 15 shows the results of binding of murine monoclonal antibodies (# 26, #38, #49, #52, #60, # 63) to breast cancer tissues.

FIG. 16 shows the results of binding of murine monoclonal antibodies (# 26, #38, #49, #52, #60, # 63) to gastric cancer tissues.

FIG. 17 shows flow assay results of murine monoclonal antibodies (# 26, #38, #49, #52, #60, # 63) mediated complement dependent cytotoxicity.

Fig. 18 shows the statistical results of the data in fig. 17.

FIG. 19 is a plasmid map of eukaryotic expression vector pcDNA3.4A.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in 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.

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

Example 1

Preparation of mouse antibodies to CLDN18

1. Immunization

Eukaryotic expression vector pcDNA3.4A (see FIG. 19) encoding human CLDN18A2 fragment (human CLDN18 isoform A2 precursor polypeptide, amino acid sequence shown in SEQ ID NO.1, coding sequence shown in SEQ ID NO. 2) was constructed, HEK293 cells were transiently transfected with the vector, and expression of CLDN18A2 in 293 cells was examined using CLDN18.2 antibody IMAB362 (self-produced, light chain amino acid sequence shown in SEQ ID NO.5, heavy chain amino acid sequence shown in SEQ ID NO. 6) from Ganzymed, as a result of which see FIG. 1.CLDN18A2 positive cells were 25.8%,41.96% and 44.60% at 24 hours, 48 hours and 72 hours, respectively, and the expression peak for CLDN18A2 appeared approximately at 48-72 hours.

At 2X 10 ⁷ At a cell count of one cell/time, 5-week-old Balb/c mice were immunized with HEK293 cells overexpressing human CLDN18A2. Immunization was performed in two ways (table 1).

Table 1: immunization protocols for the production of monoclonal antibodies

The first scheme is as follows: cells were injected into the abdominal cavity of mice on days 1, 16, 30, 43, 50, 57, 63 in the absence of adjuvant for the production of group 1 monoclonal antibodies.

Scheme two is as follows: cells were injected subcutaneously into the back of mice on day 1, cells were intraperitoneally boosted on day 16, and dorsal subcutaneous injections were performed again on day 30, followed by intraperitoneally boosting with cells on days 43, 50, 57, and 63, using freund's adjuvant, for the production of group 2 monoclonal antibodies. With Freund's complete adjuvant used on day 1, freund's incomplete adjuvant used on days 16 and 30, and the remaining 4 needles were not adjuvanted.

Mouse tail blood titers were measured by flow cytometry (FACS) between days 16 and 63, depending on the specific immunization protocol used. Mice were boosted 3 days prior to splenic removal with the aforementioned HEK293 cells overexpressing CLDN18A2 to generate group 1 and group 2 monoclonal antibodies.

2. Preparation of hybridomas producing human monoclonal antibodies against CLDN18

Spleen cells of immunized BAL b/C mice are fused with mouse myeloma cells, and the resulting hybridomas are then screened for antigen-specific antibodies. Mouse splenocytes were isolated based on standard protocols and cell fusion of a single cell suspension of splenocytes from immunized mice with PEG 1500 (Roche Cat # 10783641001) with one fifth number of mouse myeloma cells SP2/0 (ATCC, cat # CRL 1581) that did not secrete immunoglobulin. Fusing the cells at about 1x10 ⁵ One/well was plated on a 96-well cell culture plate, which was placed in an incubator (Panasonic MCO-18 AIC) at 37 ℃ with 5% CO ₂ . Subsequently, the cells were cultured in HAT selective medium 1640 medium containing 1 × streptomycin double antibody (Gibco, cat # 15140122), 1 × HAT (Sigma, cat # CRLP-7185) and 20% fetal bovine serum (Royacel, cat # RY-F11-01) for about one week. After 1 week, HAT was replaced with HT medium (1640 medium containing 1 × penicillin bisantib (Gibco, cat # 15140122), 1 × HT (Gibco, cat # 11067030) and 20% fetal bovine serum (Royacel, cat # RY-F11-01)) and then cell culture supernatants in the fusion plates were tested for binding to CHO-CLDN18A1 by FACS, and hybridomas secreting human CLD18A2 bound were screened. The antibody-secreting hybridomas were again transferred to 48 well plates and screened again, and the still positive anti-CLDN 18 monoclonal antibodies were subcloned by limiting dilution. At least one clone that retains the reactivity of the parental parent cells (as measured by FACS) is then selected from the hybridomas cultured in vitro, and 9-20 tubes are generated for each clone and stored in liquid nitrogen.

3. Selecting monoclonal antibodies that bind to CLDN18

Isotype ELISA assays were performed to determine the isotype of the antibody. The Ig subclasses of the identified CLDN18 reactive monoclonal antibodies were determined using mouse monoclonal antibody Ig class/subclass identification with ELISA kit (IgG 1\ IgG2a \ IgG2b \ IgG3\ IgM \ IgA) (Boolon, cat # BF 06001). The resulting antibody isotypes were identified as follows:

TABLE 2 murine monoclonal antibody subtypes

At 2X 10 ⁶ Individual cells/ml hybridoma cells were seeded into dialysis-based bioreactors and antibody-containing supernatants were harvested once a week. Mouse monoclonal antibodies were purified by FPLC using Protein A (GE-MabSelectSuRe LX, cat # 17-5438-03). Antibody concentration was determined by BCA kit or a280 absorbance, antibody purity was determined by SEC (size exclusion chromatography), and purity was checked by SDS (sodium dodecyl sulfate) gel electrophoresis and coomassie brilliant blue staining.

Example 2

Binding characteristics of monoclonal antibodies directed against CLDN18A2

1. Quality control of transfected cells

To generate cells stably expressing CLDN18A2, CHO cells were transfected with a vector encoding CLDN18A2 (the same eukaryotic expression vector as in example 1), and then stable cell lines were selected by flow cytometry using CHO cells as a negative control, via an antibody targeting the C-terminus of the CLDN18 protein (Invitrogen, cat # 700178) and an antibody IMAB362 targeting CLDN18A2.

CHO cells and CHO-CLDN18A2 cells stably expressing CLDN18A2 were separately collected, lysed with a loading buffer containing bromophenol blue, and subjected to sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The gel was immunoblotted and incubated with an antibody targeting the C-terminus of CLDN18 protein (Invitrogen, cat # 700178) followed by incubation with peroxidase (HRP) -labeled anti-rabbit antibody and the blot was developed with TMB reagent. As can be seen from a in fig. 2, a band of the predicted molecular weight of CLDN18 was seen only in cells transfected and stably expressing CLDN18A2, whereas no band was seen in the CHO cell negative control.

Flow cytometry (FACS) detection results (B in fig. 2) are consistent with immunoblot results, with a clear right shift in flow peak pattern after binding of the antibody IMAB362 targeting CLDN18A2 to CHO-CLDN18.2 compared to CHO empty cells.

2. Preliminary screening for monoclonal antibodies binding to CLDN18

CHO-CLDN18A2 cells expressing human CLDN18A2 at 2X 10 ⁵ The/well was plated in a U-bottom 96-well plate. Cells were incubated with hybridoma supernatant for 30 minutes at 4 ℃ and then washed once with 1x PBS containing 1% Bovine Serum Albumin (BSA). Either APC-labeled anti-murine Fc secondary antibody or PE-labeled anti-murine Fc secondary antibody was added and incubated at 4 ℃ for another 30 minutes, and the previous washing steps were repeated once. 1 XPBS was added to resuspend the cells and binding was assessed using a cytoflex flow cytometer (Beckman counter). Of the 64 mouse antibodies (Table 2), except for the fact that #10, #32, #45 did not bind to CHO-CLDN18A2 cells, specific binding to the surface of CHO-CLDN18A2 cells, such as hybridoma supernatants containing monoclonal antibodies #26, #38, #47 (FIG. 3, cells in V1R gate), was detected by the remaining mouse monoclonal antibodies. However, in these 63 antibodies, #8, #9, #21, #22, #27, #29, #30, #33, #36, #44, #46, #56 cross-reacted with CHO null cells.

Sequence alignment of human CLDN18A1 and human CLDN18 A2: human CLDN18A1 and human CLDN18A2 have some differences in amino acid sequences from 1 to 69 at the N-terminus in sequence comparison, and the sequences are completely identical after position 70. The sequence difference sites are shown in FIG. 4.

Comparison of binding of antibodies to human CLDN18A1 and CLDN18A2 by flow cytometry

Because CLDN18A1 is highly similar in sequence to CLDN18A2, it is desirable to exclude murine monoclonal antibodies that have a crossover with human CLDN18 A1. An expression vector for expressing a CLDN18A1 fragment (the amino acid sequence is shown as SEQ ID NO.3, and the coding sequence is shown as SEQ ID NO. 4) is constructed, and CHO cells are transfected to establish a CHO-CLDN18A1 cell line for stably expressing CLDN18A 1. CHO cells, CHO cells stably expressing human CLDN18A1 (CHO-CLDN 18 A1), and CHO cells stably expressing human CLDN18A2 (CHO-CLDN 18 A2) were incubated with the monoclonal antibody at 4 ℃ for 30 minutes, followed by washing once with 1 × PBS containing 1% Bovine Serum Albumin (BSA). After which incubation with APC-or PE-tagged secondary antibodies against mouse IgG for another 30 min, the previous washing steps were repeated once. Addition of 1 × PBS resuspended cells binding was assessed using a Cytoflex flow cytometer (Beckman Coulter). FIG. 5 shows several examples of sets of monoclonal antibodies identified in 48 murine monoclonal antibodies (64 strains removed of CHO-CLDN18A2 cells that cross and do not bind): monoclonal antibodies #1, #26, #38, #63 all bound well to CHO-CLDN18A2, but not to CHO-CLDN18A1, and monoclonal antibody #47 bound to both isoforms of CLDN18.

In addition, the specificity of murine monoclonal antibodies was also tested in the CLDN18 overexpression system. Expression vectors for CLDN18A1 and CLDN18A2 were transfected in HEK293 cells, respectively, and after 48 hours of transfection, HEK293-CLDN18A1 and HEK293-CLDN18A2 cells were plated, added with monoclonal antibodies, incubated at 4 ℃ for 30 minutes, and then washed once with 1 × PBS containing 1% Bovine Serum Albumin (BSA). After which incubation with APC-or PE-tagged secondary anti-mouse IgG antibody was performed for an additional 30 minutes and the previous washing steps were repeated once. Add 1x PBS heavy suspension cells with Beckman Countlerli flow cytometry assessment of binding. As shown in fig. 6, monoclonal antibody #38 can bind to HEK293-CLDN18A2 but not to HEK293-CLDN18A1, while monoclonal antibody #47 can bind to both HEK293CLDN18A1 and HEK293CLD 18A2.

From the results of these 48 murine mAbs, the murine mAbs that bind specifically to CLDN18A2 were designated #1, #4, #6, #8, #13, #14, #16, #18, #20, #24, #26, #28, #31, #35, #38, #49, #52, #60, #62 and #63.

3. Determination of expression of CLDN18 in gastric cancer cells and human pancreatic cancer cell lines

Expression of CLDN18A1 and CLDN18A2 in tumor cell lines was detected in a Real-Time PCR assay using a CLDN18A1 and CLDN18A2 gene specific primer pair (SEQ ID NO.7-13, SEQ ID NO.7-10 for CLDN18A1 gene, SEQ ID NO.11-13 for CLDN18A2 gene), and human gastric cancer cell lines NCI-SNU-16 (Beinai Chuangyi, cat # BNCC 332906), NUGC-4 (Nanjing family Bai biol, cat # CBP 60493), and KATO-III (Shanghai cell Bank of the Chinese academy, cat # SCSP-573) and human pancreatic cancer cell lines DAN-G (Beinai Chuangyi, cat # BNCC 340507) were found to have expression of CLDN18A2. Among them, NUGC-4 cells showed the highest level of RNA for CLDN18A2 and the lowest level of expression of CLDN18A2 by NCI-SNU-16 relative to Jurkat cells (from Beijing cooperative units). While NCI-SNU-16, NUGC-4, KATO-III, DAN-G did not detect expression of CLDN18A1 at the RNA level (A in FIG. 7 and B in FIG. 7).

After 96 hours of treatment with the combination of the chemotherapeutics EOF (10 ng/ml epirubicin +10ng/ml 5-fluorouracil +500ng/ml oxaliplatin), a significant increase in the transcription of CLDN18A2 by NUGC4, KATOIII and DAN-G was seen, with no significant change in expression of CLDN18A2 before and after NCI-SNU-16 treatment. For CLDN18A1, expression of CLDN18A1 of NCI-SNU-16, NUGC-4 and KATO-III also showed some increase after EOF treatment, but the transcription level was much lower than for CLDN18A2. Whereas DAN-G showed no significant change in CLDN18A1 before and after treatment (C in FIG. 7 and D in FIG. 7).

Samples of human gastric cancer cell lines SNU-16, NUGC-4 and KATO-III, human pancreatic cancer cell lines DAN-G, and lymphoma cells Jurkat cells and K562 cells (Shanghai cell Bank of China academy, cat # TCHU 191) were taken, the cells were lysed with a loading buffer containing sodium dodecyl sulfate, SDS-PAGE was performed, the gel was immunoblotted and incubated with an antibody targeting the C-terminus of CLDN18 protein (Invitrogen, cat # 700178), followed by incubation with horseradish peroxidase (HRP) -labeled anti-rabbit secondary antibody and then the blot was developed with TMB reagent. As shown in FIG. 8, cells KATOIII, K562, NUGC-4, raji and NCI-SNU-16 all expressed CLDN18 protein, and NUGC-4 was expressed in the highest amount. While Jurkat and DAN-G did not detect expression of CLDN18 at the protein level.

4. Detection of monoclonal antibodies screening for specific binding to CLDN18A2 in cells endogenously expressing CLDN18

To further screen for antibodies confirming specific binding to CLDN18A2, gastric cancer cells nucc-4 and NCI-SNU-16 were transfected with expression vectors encoding CLDN18A1 and CLDN18A2, respectively. After 48 hours, transfected cells were plated on U-bottom 96-well plates, incubated with murine monoclonal antibody for 30 minutes at 4 ℃, and then washed once with 1 × PBS containing 1% Bovine Serum Albumin (BSA). The incubation with APC-or PE-tagged secondary anti-mouse IgG antibody was repeated once for 30 min and the previous washing steps were repeated. 1 × PBS was added to resuspend the cells and binding was assessed using a Cytoflex flow cytometer (Beckman Coulter). As shown in fig. 9, the monoclonal antibodies finally confirmed without CLDN18A1 binding cross included #1, #26, #38, #49, #52, #60, #63 (sequence information see tables 3-5).

TABLE 3 sequence information of mouse monoclonal antibodies

TABLE 4 mouse monoclonal antibody CDR sequence information

TABLE 5 sequence identifiers corresponding to CDR sequences of mouse monoclonal antibodies

Serial number	Name (R)	HCDR1	HCDR2	HCDR3
						1	2CLD18.2-6	28	35	42
26	2CLDN18.2-5	29	36	43
					38	CLD18.2-1	30	37	44
49	4CLD18.2-3	31	38	45
					52	6CLD18.2-27	32	39	46
60	8CLD18.2-1	33	40	47
					63	4CLD18.2-1	34	41	48
Serial number	Name (R)	LCDR1	LCDR2	LCDR3
					1	2CLD18.2-6	49	54	59
26	2CLDN18.2-5	50	54	60
					38	CLD18.2-1	51	55	61
49	4CLD18.2-3	52	56	62
					52	6CLD18.2-27	50	54	63
60	8CLD18.2-1	53	57	64
					63	4CLD18.2-1	49	58	65

5. Binding Curve assay for monoclonal antibodies to CHO-CLDN18A2

To compare the affinity of murine monoclonal antibody to IMAB362, the Fc region of IMAB362 was replaced with mouse mIgG1 to construct an expression IMAB362-CH1.

The binding of 7 murine monoclonal antibodies specifically binding to CLDN18A2 of table 3 above to CHO-CLDN18A2 cells was concentration titrated and detected by flow cytometry to generate a binding curve (fig. 10). As can be seen, 7 murine mAbs bound well to CHO-CLDN18A2, of which at least two murine mAbs (# 52 and # 63) had a higher affinity for CHO-CLDN18A2 cells than the positive antibody IMAB362-CH1.

Example 3

Binding epitope competition detection of monoclonal antibodies against CLDN18A2

Murine monoclonal antibodies targeting CLDN18A2 were tested for epitope crossing with IMAB 362. The specific experimental steps are as follows:

(1) And (3) volume dilution: diluting murine mAb, IMAB362 and isotype control to a concentration of 20 μ g/ml with 1% BSA in1 XPBS;

(2) Cells were counted and plated: the CHO-CLDN18A2 cells to be detected were adjusted to a cell density of 4X 10 by FACS buffer ⁶ Evenly dividing each sample per mL into 96-hole V-shaped plates according to 100 mu L/tube, centrifuging and discarding supernatant;

(3) Adding the diluted primary antibody into CHO-CLDN18A2 cells, incubating at 2-8 ℃ for 30 minutes at 100. Mu.L/well, adding the secondary antibody, incubating at 2-8 ℃ for 30 minutes at 100. Mu.L/well;

(4) Taking out the 96-well plate, centrifuging for 5min at 250g, carefully removing the supernatant, adding FACS buffer 200 ul/well, centrifuging for 5min at 250g again, and carefully removing the supernatant;

(5) PE fluorescent secondary antibody (1 × 500x dilution) was prepared with 1 × PBS containing 1% bsa, added to the corresponding 96-well plate at 100 μ l/well, and incubated at 2-8 degrees for 30 minutes;

(6) Taking out the 96-well plate, centrifuging for 5min at 250g, carefully removing the supernatant, adding FACS buffer at 200 ul/well, centrifuging for 5min at 250g again, and carefully removing the supernatant;

(7) Resuspend with 1 XPBS 100. Mu.L/well and check on a flow cytometer.

The results are shown in FIGS. 11 and 12. When the first antibody was IMAB362, the binding of the second antibodies 1#, #26, #49, #52, #60 and #63 was not substantially affected, but the #38 murine monoclonal antibody binding was significantly blocked. Whereas, in the case of murine monoclonal antibodies #1, #26, #49, #60, #63, the first antibodies completely blocked the binding of the IMAB362 antibody, and the first antibodies #38, #49, #52 attenuated the binding of the IMAB362 antibody. The above results indicate that antibodies #38 and #52 may overlap with the IMAB362 binding epitope, whereas antibodies #1, #26, #49, #60, #63 recognize a different epitope than IMAB 362.

Example 4

Binding of monoclonal antibodies targeting CLDN18A2 to peripheral blood lymphocytes (PBMCs)

The separation of peripheral blood lymphocytes is carried out as follows:

(1) Fresh blood is extracted from healthy volunteers into a blood collection tube, centrifuged at 2000rpm at room temperature (acceleration 9, deceleration 5), serum is removed for 5min, and PBS is added for dilution;

(2) Adding 15ml of Ficoll lymph separating medium (Cat # AS1114546 in Dake) into a 50ml tube, adding diluted blood cells to the Ficoll liquid surface, centrifuging at 2000rpm at room temperature (5% acceleration and 0% deceleration), and 20min;

(3) The buffy coat layer was taken out into a 50ml tube, diluted three times more with PBS, and centrifuged at 1500rpm for 5min at room temperature to obtain peripheral blood lymphocytes (PBMC).

(4) PBMC cells were incubated with monoclonal antibodies for 30 minutes at 4 ℃ and then washed once with 1 XPBS containing 1% Bovine Serum Albumin (BSA). After which incubation with APC-or PE-tagged secondary anti-mouse IgG antibody was performed for an additional 30 minutes, and the previous washing steps were repeated once. 1 × PBS was added to resuspend the cells and binding was assessed using a Cytoflex flow cytometer (Beckman Countler). PBMC cells were stained and gated with CD14 (Biolegged, cat # 301808) antibody, CD3 (Biolegged, cat # 300312) antibody, and CD19 (Biolegged, cat # 392504) antibody in parallel.

As shown in FIG. 13, none of the murine monoclonal antibodies (# 1, #26, #38, #49, #52, #60, # 63) bound PBMC cells in the P1, P2, and P3 gates. Among them, the P1 gate is mainly CD14 positive cells, the P2 gate is CD3 and CD14 positive cells, and the P3 gate is CD19 and CD3 positive cells.

Example 5

Specificity of binding of monoclonal antibodies targeting CLDN18A2 to tumor tissue

In order to confirm the specificity of the monoclonal antibody binding to tumor tissues, the monoclonal antibody binding to gastric adenocarcinoma, non-small cell lung cancer and breast cancer tissues was examined.

Tumor tissue and tissues adjacent to the cancer, which were soaked in Hanks solution (Biyuntian, cat # C0218), were cut into small pieces with surgical scissors and placed in 10cm petri dishes. Adding 0.25% trypsin prepared with 0.1% collagenase (Sigma-Aldrich, cat # C5138) in1 XPBS buffer at 37 ℃ with 5% CO ₂ Digestion is carried out under conditions of 30 minutes to 1 hour. And filtering the digested tumor tissue by a cell filter membrane, centrifuging to collect digested single cells, respectively adding 2ml of erythrocyte lysate (Gibco, cat # A1049201) to resuspend, reacting for 5 minutes at room temperature, supplementing PBS to 20ml, and centrifuging to collect cells. Counting the cells to be detected, and adjusting the cell density to 2 × 10 ⁵ Perwell was placed in a 96-well U plate and centrifuged at 300g for 5 minutes at 4 ℃. The murine mAb and control antibody were diluted to 20. Mu.g/ml with 1 XPBS containing 3% Bovine Serum Albumin (BSA). Mu.l of diluted antibody was added to each well and incubated at 4 ℃ for 30 minutes. Thereafter, the cells were washed once with 1 XPBS containing 3% Bovine Serum Albumin (BSA). Thereafter, the second antibody against mouse was incubated with APC-labeled secondary antibody at 4 ℃ for another 30 minutes, and the previous washing step was repeated once. Then 100. Mu.l of 1 XPBS containing 3% bovine serum albumin was added to each well, followed by FITC-labeled anti-human CD45 antibody (Biolegend, cat # 304054), the control was set and incubated at 4 ℃ for 30 minutes. The previous washing steps were repeated once and then resuspended with 100. Mu.l of 1 XPBS per well. Antibody binding was assessed using a Cytoflex flow cytometer (Beckman Countler).

FIG. 14 shows the binding of murine mAbs to lung cancer tissues, murine mAbs #26 and #60 bound to lung cancer tissues, and murine mAbs #38, #49, #52, #60, #63 bound to lung cancer tissues similar to IMAB362-CH1.

FIG. 15 shows the binding of murine mAbs to breast cancer tissues, murine mAbs #26, #38, #49, IMAB362-CH1 bound to breast cancer tissues, and murine mAbs #52, #60, #63 bound weakly or not to breast cancer tissues.

FIG. 16 shows the binding of murine mAbs to gastric cancer tissues, and murine mAbs #26, #38, #49, #52, #60, #63 and IMAB362-CH1 bound to gastric cancer tissues.

Example 6

Complement Dependent Cytotoxicity (CDC)

Serum for complement lysis was prepared by drawing blood from healthy volunteers into blood collection tubes followed by centrifugation at 600g for 20 minutes and harvested serum was stored at 4 ℃. Cells (CHO, CHO-CLDN18A1, CHO-CLDN18A 2), monoclonal antibodies (30. Mu.g/ml), healthy volunteer serum (diluted 1.3 with optiMEM medium) were mixed in equal volumes (final concentration of monoclonal antibody 10. Mu.g/ml), incubated at 37 ℃ for 3 hours under 5% CO2 conditions, 15 minutes before harvest, incubated with propidium iodide (PI, final concentration 2.5. Mu.g/ml), washed once with 1% Bovine Serum Albumin (BSA) in1 XPBS, and resuspended with 1 XPBS. The killing effect was checked on a cytoflex flow cytometer (Beckman counter).

Cell debris was distinguished by adjusting the Forward scatter and side scatter thresholds. The percentage of PI cell positive cells is shown in figure 17. The results of complement dependent cytotoxicity of monoclonal antibodies #26, #38, #49, #52, #60, #63 and IMAB362, IMAB362-CH1 are summarized in FIG. 18.

In the serum-free case, monoclonal antibodies #52, #63 and IMAB362, IMAB362-CH1 did not mediate CDC of CHO cells and CHO-CLDN18A1 compared to isotype control groups. However, mouse monoclonal antibodies #26, #38, #49 and #60 caused some cell death in serum-free conditions, indicating that mouse monoclonal antibodies #26, #38, #49 and #60 may have other non-CDC cell death induction mechanisms.

In the presence of serum, the monoclonal antibody still produced no CDC effect on CHO cells compared to the isotype control. For CHO-CLDN18A2 cells, monoclonal antibodies #26, #38, #49, #60 and IMAB362 mediated high CDC response, monoclonal antibodies #52, #63 mediated moderate CDC response and IMAB362-CH1 mediated low CDC response. Low-grade CDC response was mediated by CHO-CLDN18A1, IMAB362 and murine monoclonal antibody #38, and moderate CDC response was mediated by murine monoclonal antibodies #26, #49, # 60. After serum addition, cell death by the #38 monoclonal antibody was reduced compared to serum-free conditions.

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> Shenzhen Fei Peng biopharmaceutical shares GmbH

<120> antibody having CLDN18A2 resistance and drug for treating tumor

<160> 68

<170> SIPOSequenceListing 1.0

<210> 1

<211> 261

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15

Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

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

115 120 125

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

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

<210> 2

<211> 786

<212> DNA

<213> Artificial sequence

<400> 2

atggccgtga ctgcctgtca gggcttgggg ttcgtggttt cactgattgg gattgcgggc 60

atcattgctg ccacctgcat ggaccagtgg agcacccaag acttgtacaa caaccccgta 120

acagctgttt tcaactacca ggggctgtgg cgctcctgtg tccgagagag ctctggcttc 180

accgagtgcc ggggctactt caccctgctg gggctgccag ccatgctgca ggcagtgcga 240

gccctgatga tcgtaggcat cgtcctgggt gccattggcc tcctggtatc catctttgcc 300

ctgaaatgca tccgcattgg cagcatggag gactctgcca aagccaacat gacactgacc 360

tccgggatca tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc tgtgtttgcc 420

aacatgctgg tgactaactt ctggatgtcc acagctaaca tgtacaccgg catgggtggg 480

atggtgcaga ctgttcagac caggtacaca tttggtgcgg ctctgttcgt gggctgggtc 540

gctggaggcc tcacactaat tgggggtgtg atgatgtgca tcgcctgccg gggcctggca 600

ccagaagaaa ccaactacaa agccgtttct tatcatgcct caggccacag tgttgcctac 660

aagcctggag gcttcaaggc cagcactggc tttgggtcca acaccaaaaa caagaagata 720

tacgatggag gtgcccgcac agaggacgag gtacaatctt atccttccaa gcacgactat 780

gtgtaa 786

<210> 3

<211> 261

<212> PRT

<213> Artificial sequence

<400> 3

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

1 5 10 15

Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

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

115 120 125

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

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

<210> 4

<211> 786

<212> DNA

<213> Artificial sequence

<400> 4

atgtccacca ccacatgcca agtggtggcg ttcctcctgt ccatcctggg gctggccggc 60

tgcatcgcgg ccaccgggat ggacatgtgg agcacccagg acctgtacga caaccccgtc 120

acctccgtgt tccagtacga agggctctgg aggagctgcg tgaggcagag ttcaggcttc 180

accgaatgca ggccctattt caccatcctg ggacttccag ccatgctgca ggcagtgcga 240

gccctgatga tcgtaggcat cgtcctgggt gccattggcc tcctggtatc catctttgcc 300

ctgaaatgca tccgcattgg cagcatggag gactctgcca aagccaacat gacactgacc 360

tccgggatca tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc tgtgtttgcc 420

aacatgctgg tgactaactt ctggatgtcc acagctaaca tgtacaccgg catgggtggg 480

atggtgcaga ctgttcagac caggtacaca tttggtgcgg ctctgttcgt gggctgggtc 540

gctggaggcc tcacactaat tgggggtgtg atgatgtgca tcgcctgccg gggcctggca 600

ccagaagaaa ccaactacaa agccgtttct tatcatgcct caggccacag tgttgcctac 660

aagcctggag gcttcaaggc cagcactggc tttgggtcca acaccaaaaa caagaagata 720

tacgatggag gtgcccgcac agaggacgag gtacaatctt atccttccaa gcacgactat 780

gtgtaa 786

<210> 5

<211> 220

<212> PRT

<213> Artificial sequence

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala 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 Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

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

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

<210> 6

<211> 448

<212> PRT

<213> Artificial sequence

<400> 6

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 Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

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

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

100 105 110

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

115 120 125

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

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

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

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

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

435 440 445

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<400> 7

tccaccacca catgccaagt g 21

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence

<400> 8

tccaccacca catgccaagt g 21

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

gtggcgttcc tcctgtccat 20

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

ccaatgcgga tgcatttcag g 21

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence

<400> 11

agagagctct ggcttcaccg agtg 24

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<400> 12

ttggggttcg tggtttcact 20

<210> 13

<211> 26

<212> DNA

<213> Artificial sequence

<400> 13

ccagaagtta gtcaccagca tgttgg 26

<210> 14

<211> 119

<212> PRT

<213> Artificial sequence

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Leu Arg Tyr Gly Gly Asp Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Ala Val Ser Ser

115

<210> 15

<211> 120

<212> PRT

<213> Artificial sequence

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Glu Gly Asp Tyr Tyr Tyr Gly Thr Leu Asp Asp Trp Gly Gln

100 105 110

Gly Thr Thr Leu Ser Val Ser Ser

115 120

<210> 16

<211> 120

<212> PRT

<213> Artificial sequence

<400> 16

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

1 5 10 15

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

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Arg Gly Ile Thr Thr Ala Pro Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 17

<211> 112

<212> PRT

<213> Artificial sequence

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Ser Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

100 105 110

<210> 18

<211> 119

<212> PRT

<213> Artificial sequence

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Tyr Ile Ser Tyr Tyr Asn Gly Ala Thr Thr Tyr Asn Gln Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 19

<211> 118

<212> PRT

<213> Artificial sequence

<400> 19

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Ser Ala Trp Thr Trp Ser Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Ala Tyr Ile Ser Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

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

85 90 95

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

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 20

<211> 119

<212> PRT

<213> Artificial sequence

<400> 20

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 Ser Tyr

20 25 30

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

35 40 45

Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Leu Ile 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 Thr Ala Met Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 21

<211> 114

<212> PRT

<213> Artificial sequence

<400> 21

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

1 5 10 15

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

20 25 30

Gly 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 Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys Arg

<210> 22

<211> 114

<212> PRT

<213> Artificial sequence

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

Pro Pro Lys Leu Phe Ile Tyr Trp Ala 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 Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

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

100 105 110

Lys Arg

<210> 23

<211> 112

<212> PRT

<213> Artificial sequence

<400> 23

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 Lys Ser Val Ser Thr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 24

<211> 106

<212> PRT

<213> Artificial sequence

<400> 24

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 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 Trp Ser Ser Asn Pro Thr Phe

85 90 95

Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 25

<211> 114

<212> PRT

<213> Artificial sequence

<400> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala 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 Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

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

100 105 110

Lys Arg

<210> 26

<211> 114

<212> PRT

<213> Artificial sequence

<400> 26

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

1 5 10 15

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

20 25 30

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

35 40 45

Pro Pro Lys Met Ser Ile Tyr Trp Ala Ser Thr Arg Asp Ser Gly Val

50 55 60

Pro Pro Arg Phe Thr 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 Val Tyr Phe Cys Gln Asn

85 90 95

Ala Tyr Ser Tyr Pro Phe Met Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg

<210> 27

<211> 114

<212> PRT

<213> Artificial sequence

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

85 90 95

Asp His Asn Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence

<400> 28

Gly Tyr Thr Phe Thr Ser Phe Tyr Ile Tyr

1 5 10

<210> 29

<211> 10

<212> PRT

<213> Artificial sequence

<400> 29

Gly Tyr Thr Phe Thr Asp Tyr Glu Met His

1 5 10

<210> 30

<211> 10

<212> PRT

<213> Artificial sequence

<400> 30

Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn

1 5 10

<210> 31

<211> 10

<212> PRT

<213> Artificial sequence

<400> 31

Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn

1 5 10

<210> 32

<211> 10

<212> PRT

<213> Artificial sequence

<400> 32

Gly Tyr Ser Phe Thr Gly Tyr Asn Met His

1 5 10

<210> 33

<211> 11

<212> PRT

<213> Artificial sequence

<400> 33

Gly Tyr Ser Ile Thr Ser Asp Ser Ala Trp Thr

1 5 10

<210> 34

<211> 10

<212> PRT

<213> Artificial sequence

<400> 34

Gly Phe Ser Leu Thr Ser Tyr Gly Val His

1 5 10

<210> 35

<211> 17

<212> PRT

<213> Artificial sequence

<400> 35

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

1 5 10 15

Gly

<210> 36

<211> 17

<212> PRT

<213> Artificial sequence

<400> 36

Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Ser Gln Lys Phe Lys

1 5 10 15

Gly

<210> 37

<211> 17

<212> PRT

<213> Artificial sequence

<400> 37

Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys

1 5 10 15

Gly

<210> 38

<211> 17

<212> PRT

<213> Artificial sequence

<400> 38

Leu Ile Asn Pro Tyr Asn Ser Gly Thr Thr Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 39

<211> 17

<212> PRT

<213> Artificial sequence

<400> 39

Tyr Ile Ser Tyr Tyr Asn Gly Ala Thr Thr Tyr Asn Gln Arg Phe Lys

1 5 10 15

Gly

<210> 40

<211> 16

<212> PRT

<213> Artificial sequence

<400> 40

Tyr Ile Ser Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 41

<211> 16

<212> PRT

<213> Artificial sequence

<400> 41

Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser

1 5 10 15

<210> 42

<211> 10

<212> PRT

<213> Artificial sequence

<400> 42

Leu Arg Tyr Gly Gly Asp Ala Met Asp Tyr

1 5 10

<210> 43

<211> 11

<212> PRT

<213> Artificial sequence

<400> 43

Glu Gly Asp Tyr Tyr Tyr Gly Thr Leu Asp Asp

1 5 10

<210> 44

<211> 11

<212> PRT

<213> Artificial sequence

<400> 44

Gly Arg Gly Ile Thr Thr Ala Pro Phe Ala Tyr

1 5 10

<210> 45

<211> 3

<212> PRT

<213> Artificial sequence

<400> 45

Gly Asp Tyr

1

<210> 46

<211> 10

<212> PRT

<213> Artificial sequence

<400> 46

Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Tyr

1 5 10

<210> 47

<211> 9

<212> PRT

<213> Artificial sequence

<400> 47

Phe Arg Thr Gly Asn Ala Met Asp Tyr

1 5

<210> 48

<211> 11

<212> PRT

<213> Artificial sequence

<400> 48

Asp Gln Phe Ser Ile Trp Gly Gly Phe Ala Tyr

1 5 10

<210> 49

<211> 17

<212> PRT

<213> Artificial sequence

<400> 49

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

1 5 10 15

Ala

<210> 50

<211> 17

<212> PRT

<213> Artificial sequence

<400> 50

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

1 5 10 15

Thr

<210> 51

<211> 15

<212> PRT

<213> Artificial sequence

<400> 51

Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His

1 5 10 15

<210> 52

<211> 10

<212> PRT

<213> Artificial sequence

<400> 52

Ser Ala Ser Ser Ser Val Asn Tyr Met His

1 5 10

<210> 53

<211> 17

<212> PRT

<213> Artificial sequence

<400> 53

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

1 5 10 15

Thr

<210> 54

<211> 7

<212> PRT

<213> Artificial sequence

<400> 54

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 55

<211> 7

<212> PRT

<213> Artificial sequence

<400> 55

Leu Val Ser Asn Leu Glu Ser

1 5

<210> 56

<211> 7

<212> PRT

<213> Artificial sequence

<400> 56

Asp Thr Ser Lys Leu Ala Ser

1 5

<210> 57

<211> 7

<212> PRT

<213> Artificial sequence

<400> 57

Trp Ala Ser Thr Arg Asp Ser

1 5

<210> 58

<211> 7

<212> PRT

<213> Artificial sequence

<400> 58

Gly Ala Ser Thr Arg Glu Ser

1 5

<210> 59

<211> 9

<212> PRT

<213> Artificial sequence

<400> 59

Gln Gln Cys Tyr Ser Tyr Pro Leu Thr

1 5

<210> 60

<211> 9

<212> PRT

<213> Artificial sequence

<400> 60

Gln Asn Asp Tyr Tyr Tyr Pro Phe Thr

1 5

<210> 61

<211> 9

<212> PRT

<213> Artificial sequence

<400> 61

Gln His Ser Arg Glu Leu Pro Pro Thr

1 5

<210> 62

<211> 8

<212> PRT

<213> Artificial sequence

<400> 62

Gln Gln Trp Ser Ser Asn Pro Thr

1 5

<210> 63

<211> 9

<212> PRT

<213> Artificial sequence

<400> 63

Gln Asn Asp Tyr Ser Tyr Pro Phe Thr

1 5

<210> 64

<211> 9

<212> PRT

<213> Artificial sequence

<400> 64

Gln Asn Ala Tyr Ser Tyr Pro Phe Met

1 5

<210> 65

<211> 9

<212> PRT

<213> Artificial sequence

<400> 65

Gln Asp Asp His Asn Tyr Pro Phe Thr

1 5

<210> 66

<211> 107

<212> PRT

<213> Artificial sequence

<400> 66

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> 67

<211> 330

<212> PRT

<213> Artificial sequence

<400> 67

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 Asp Glu

225 230 235 240

Leu 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> 68

<211> 326

<212> PRT

<213> Artificial sequence

<400> 68

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

1 5 10 15

Ser Thr Ser Glu Ser 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 Asn Phe Gly Thr Gln Thr

65 70 75 80

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

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

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

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

Claims (18)

1. An antibody having CLDN18A2 or a functional fragment thereof, wherein the antibody or the functional fragment thereof has a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of complementarity determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and the amino acid sequences of complementarity determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region are any one of the following (3), (5) to (7):

(3): HCDR1 is shown as SEQ ID NO.30, HCDR2 is shown as SEQ ID NO.37, and HCDR3 is shown as SEQ ID NO. 44;

LCDR1 is shown as SEQ ID NO.51, LCDR2 is shown as SEQ ID NO.55, and LCDR3 is shown as SEQ ID NO. 61;

(5): HCDR1 is shown as SEQ ID NO.32, HCDR2 is shown as SEQ ID NO.39, and HCDR3 is shown as SEQ ID NO. 46;

LCDR1 is shown as SEQ ID NO.50, LCDR2 is shown as SEQ ID NO.54, and LCDR3 is shown as SEQ ID NO. 63;

(6): HCDR1 is shown as SEQ ID NO.33, HCDR2 is shown as SEQ ID NO.40, and HCDR3 is shown as SEQ ID NO. 47;

LCDR1 is shown as SEQ ID NO.53, LCDR2 is shown as SEQ ID NO.57, and LCDR3 is shown as SEQ ID NO. 64;

(7) HCDR1 is shown as SEQ ID NO.34, HCDR2 is shown as SEQ ID NO.41, and HCDR3 is shown as SEQ ID NO. 48;

LCDR1 is shown as SEQ ID NO.49, LCDR2 is shown as SEQ ID NO.58, and LCDR3 is shown as SEQ ID NO. 65.

2. The antibody or the functional fragment thereof according to claim 1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are any one of the following (c), (e) to (g):

(c) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.16, and the light chain variable region is shown as SEQ ID NO. 23;

(e) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.18, and the light chain variable region is shown as SEQ ID NO. 25;

(f) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.19, and the light chain variable region is shown as SEQ ID NO. 26;

(g) The method comprises the following steps The heavy chain variable region is shown as SEQ ID NO.20, and the light chain variable region is shown as SEQ ID NO. 27.

3. The antibody or functional fragment thereof according to claim 2, wherein the light chain constant region of the antibody is a kappa-type light chain constant region or a lambda-type light chain constant region.

4. The antibody or functional fragment thereof according to claim 3, wherein the light chain constant region of said antibody is represented by SEQ ID No. 66.

5. The antibody or functional fragment thereof of claim 2, wherein the heavy chain constant region of the antibody is a heavy chain constant region of an IgA, igD, igE, igG, or IgM antibody.

6. The antibody or functional fragment thereof of claim 5, wherein the heavy chain constant region of the antibody is a heavy chain constant region of an IgG antibody.

7. The antibody or functional fragment thereof of claim 5, wherein the heavy chain constant region of the antibody is a heavy chain constant region of an IgG1, igG2, igG3, or IgG4 antibody.

8. The antibody or functional fragment thereof according to claim 5, characterized in that the heavy chain constant region of said antibody is as set forth in SEQ ID No.67 or SEQ ID No.68.

9. The antibody or functional fragment thereof according to any one of claims 1 to 8, wherein the functional fragment is a Fab, fab ', F (ab') 2, fv or ScFv fragment.

10. An antibody conjugate comprising an antibody or functional fragment thereof according to any one of claims 1 to 9 conjugated to a therapeutic agent.

11. The antibody conjugate of claim 10, wherein the therapeutic agent is a toxin, a radioisotope, or a cytotoxic agent.

12. A fusion protein comprising the antibody or functional fragment thereof according to any one of claims 1 to 9.

13. A medicament for the treatment of tumors comprising an antibody or functional fragment thereof according to any one of claims 1 to 9, an antibody conjugate according to claim 10 or 11, or a fusion protein according to claim 12; the tumor is a tumor with positive expression of CLDN18A2.

14. The medicament of claim 13, wherein the tumor positive for expression of CLDN18A2 is selected from any one of gastric adenocarcinoma, pancreatic cancer, rectal cancer, breast cancer, ovarian cancer and lung cancer.

15. The medicament of claim 13 or 14, further comprising a pharmaceutically acceptable excipient.

16. An isolated nucleic acid molecule encoding the antibody or functional fragment thereof of any one of claims 1-9.

17. A recombinant vector comprising the nucleic acid molecule of claim 16.

18. A recombinant cell comprising the recombinant vector of claim 17.

Images