CN112830969A - Monoclonal antibody specifically binding to human Claudin18.2, and medicine and kit containing monoclonal antibody - Google Patents

Abstract

The invention discloses a monoclonal antibody specifically binding to human Claudin18.2, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises: heavy chain variable region CDR1 shown in SEQ ID NO.1, heavy chain variable region CDR2 shown in SEQ ID NO.1, and heavy chain variable region CDR3 shown in SEQ ID NO. 3; the light chain variable region comprises: light chain variable region CDR1 shown in SEQ ID NO.4, light chain variable region CDR2 shown in SEQ ID NO.5, and light chain variable region CDR3 shown in SEQ ID NO. 6. The invention also discloses a bispecific antibody based on the monoclonal antibody, and a medicament or a kit for treating or relieving cancer based on the monoclonal antibody or the specific antibody.

Description

Monoclonal antibody specifically binding to human Claudin18.2, and medicine and kit containing monoclonal antibody

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a monoclonal antibody specifically binding to human Claudin18.2, a medicine containing the monoclonal antibody and a kit containing the monoclonal antibody.

Background

The tetraspanin protein coded by the Claudin family gene plays a very key structural and functional role in the tight junction of cells, and the tight junction is also important for the permeability and polarity of epithelial cells and endothelial cells. In mammals, the Claudin family has found 24 different proteins, each of which is specifically expressed on a tissue. The extracellular loop structure of the Claudin protein is connected with the structure of the adjacent cells, and plays a role in bridging the cell layer and regulating the parallel transportation between the cavity and the basal layer.

The Claudin18 protein has typical structural properties of this family of proteins with two splice variants, designated Claudin18.1(CLDN18.1) and Claudin18.2(CLDN18.2), respectively, the two spliceosomes differ in the first Transmembrane (TM) region and the N-terminal part of loop 1, while the C-terminal protein primary sequence is identical. In normal tissues, CLDN18.1 is selectively expressed in lung cells and CLDN18.2 is expressed only on stomach cells. While more and more tests indicate that CLDN18.2 is also expressed in a variety of tumor cells. Due to the selective expression characteristic of Claudin18.2, the Claudin is a brand new target point for tumor treatment.

Currently, no antibody medicine aiming at Claudin18.2 is on the market, IMAB362 (Zolbetximab) antibody of the Ansteela company is rapidly developed, clinical three-stage tests aiming at gastric cancer and gastroesophageal junction adenocarcinoma and clinical two-stage tests aiming at pancreatic cancer are carried out at present, and patients with gastric cancer and gastroesophageal junction adenocarcinoma are recruited in the clinical center of China, and the capacity of the antibody medicine is more than 200. The antibody is a chimeric antibody with a subtype IgG1, specifically binds to Claudin18.2 and does not bind to Claudin18.1, and in vitro activity studies show that the antibody mediates ADCC activity and CDC activity to play a role in inhibiting tumors. The publicly reported clinical data is a clinical secondary (NCT01630083) result: IMAB362 combined with standard chemotherapy scheme EOX for treating gastric adenocarcinoma, esophageal carcinoma and gastroesophageal junction adenocarcinoma, totalling 161 cases, remarkably prolongs the median PFS (7.5VS5.3 months; P <0.0005) and the median OS (13VS8.4 months; P ═ 0.0008), improves the ORR (39VS 25%; P ═ 0.022) curative effect, and has remarkable curative effect compared with the EOX standard chemotherapy scheme.

Stomach cancer and GEJ cancer are among the most undesirable malignancies that are currently in demand for medical treatment, and stomach cancer is also the third leading cause of cancer death worldwide. In addition, 5-year overall survival for metastatic gastric cancer and GEJ cancer is less than 20%. Chemotherapy and anti-HER 2 antibodies are widely used in the treatment of metastatic or recurrent gastric and GEJ cancers, however, there is a lack of effective targeted therapy especially in HER2 negative patients and therapeutic regimens are urgently needed. Therefore, the development of anti-Claudin18.2 antibody and other drugs such as bifunctional antibody thereof has great significance for the treatment of partial solid tumors, especially stomach related tumors.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a monoclonal antibody specifically binding to human Claudin18.2, a medicament and a kit containing the monoclonal antibody.

The species of the present invention specifically binds to the monoclonal antibody of human Claudin18.2, providing a Claudin18.2 antibody that exhibits the following properties but is not limited to: (1) has high affinity and specificity with human Claudin18.2; (2) can bind to the Claudin18.2 recombinant protein specifically; (3) can specifically bind to Claudin18.2 overexpression cells; (4) and does not bind to claudin18.1, a family member; (5) the target cell can be killed by combining the antigen site of the target cell and the FC receptor site of the effector cell to mediate ADCC activity.

The specific technical scheme is as follows:

one of the objects of the present invention is to provide a monoclonal antibody which specifically binds to human Claudin18.2.

A monoclonal antibody that specifically binds to human claudin18.2 comprising a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises: a heavy chain variable region CDR1 having an amino acid sequence shown in SEQ ID NO.1, a heavy chain variable region CDR2 having an amino acid sequence shown in SEQ ID NO.1, and a heavy chain variable region CDR3 having an amino acid sequence shown in SEQ ID NO. 3;

the light chain variable region comprises: light chain variable region CDR1 with an amino acid sequence shown in SEQ ID NO.4, light chain variable region CDR2 with an amino acid sequence shown in SEQ ID NO.5, and light chain variable region CDR3 with an amino acid sequence shown in SEQ ID NO. 6.

The beneficial effects of the above technical scheme are: the CDR region provided by the invention enables the Claudin18.2 antibody to have high affinity and specificity with human Claudin18.2, does not bind with other members of the family Claudin18.1, and can remarkably kill target cells.

On the basis of the technical scheme, the invention also comprises the following improved technical scheme:

further, the heavy chain variable region amino acid sequence of the monoclonal antibody comprises any one of the following sequences: 9, 10, 11, or 12; the light chain variable region amino acid sequence of the monoclonal antibody comprises any one of the following sequences: 13, 14, SEQ ID NO: 15, or SEQ ID NO: 16.

still further, the heavy chain variable region of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 9, and the light chain variable region of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 13.

Still further preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of said monoclonal antibody or antigen binding portion thereof are identical to the amino acid sequences of SEQ ID No. 9 and SEQ ID NO:13 have an overall sequence identity of at least 90%.

The advantage of using the above further scheme is to provide alternative antibodies with high affinity and specificity to human claudin18.2 and which do not bind to other members of the family claudin18.1, which can achieve the purpose of killing target cells by binding to the antigenic site of the target cells and the FC receptor site of the effector cells, mediating ADCC activity.

Still further preferably, the heavy chain amino acid sequence of the monoclonal antibody or the antigen binding portion thereof is shown as SEQ ID NO.17, and the light chain amino acid sequence is shown as SEQ ID NO. 18.

The beneficial effect of adopting the further scheme is that the heavy chain and light chain amino acid sequences provided by the invention enable the Claudin18.2 antibody to have high affinity and specificity with human Claudin18.2, and not to be combined with other members of the family Claudin18.1, and the antibody can mediate ADCC activity by combining an antigen site of a target cell and an FC receptor site of an effector cell, thereby achieving the purpose of killing the target cell.

Further, the monoclonal antibody is a full-length antibody of IgG1, IgG2 or IgG4 isotype.

The further scheme has the beneficial effects that the immunoglobulin is easier to diffuse into the extravascular space, so that the immunoglobulin plays an important role in combining complement, enhancing the capability of immune cells in phagocytosing pathogenic microorganisms and tumor cells and neutralizing bacterial toxins, and can effectively resist infection and tumors. On the other hand, immunoglobulins have a longer half-life and better stability in vivo.

Further, the monoclonal antibody is an antibody fragment or a single-chain antibody.

The advantage of using the above further protocol is to provide more alternative forms of antibodies.

It is a further object of the present invention to provide a bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain.

Wherein: a) the first antigen binding domain is from a monoclonal antibody that specifically binds human claudin18.2, described above;

and b) the second antigen-binding domain is from an anti-PDL 1 antibody that specifically binds human PDL 1.

On the basis of the technical scheme, the invention also comprises the following improved technical scheme:

further, the anti-PDL 1 antibody comprises the following CDR regions:

the amino acid sequence is shown as heavy chain variable region CDR1 of SEQ ID NO. 21, the amino acid sequence is shown as heavy chain variable region CDR2 of SEQ ID NO. 22, the amino acid sequence is shown as heavy chain variable region CDR3 of SEQ ID NO. 23, the amino acid sequence is shown as light chain variable region CDR1 of SEQ ID NO. 24, the amino acid sequence is shown as light chain variable region CDR2 of SEQ ID NO. 25, and the amino acid sequence is shown as light chain variable region CDR3 of SEQ ID NO. 26.

Still further, the heavy chain variable region of the anti-PDL 1 antibody comprises the amino acid sequence shown in SEQ ID No. 27, and the light chain variable region of the anti-PDL 1 antibody comprises the amino acid sequence shown in SEQ ID NO: 28, or a pharmaceutically acceptable salt thereof.

Further, the first antigen-binding domain and the second antigen-binding domain are connected by FC or by a linker fragment. Preferably, the FC ligation employs the Knobs intoholes technique; preferably, the linker fragment is preferably (GGGGS) 3.

Still further, the first and second antigen-binding domains are independently an immunoglobulin or antigen-binding fragment thereof, such as a half-antibody, Fab, F (ab)2, or single-chain antibody.

Still further preferably, the first antigen-binding domain is an immunoglobulin and the second antigen-binding domain is a single chain antibody.

The bifunctional antibody targeting Claudin18.2 and PDL1 simultaneously has the advantages of dual functional activities of anti-Claudin18.2 and anti-PDL 1, tumor cell binding activity provided by the targeted Claudin18.2 domain, and immune activation capability provided by the targeted PDL1 domain. Meanwhile, the ADCC activity is improved, and the capability of killing target cells is further increased.

A further object of the present invention is a medicament or pharmaceutical composition for the treatment or alleviation of cancer, said medicament or pharmaceutical composition comprising the above-mentioned monoclonal or bispecific antibody specifically binding to human claudin 18.2.

According to the binding specificity of the invention, it is a fourth object of the invention to provide a kit comprising the above-described monoclonal or bispecific antibody that specifically binds to human Claudin18.2.

The beneficial effect of adopting the further scheme is that the antibody or the antigen binding part thereof can be applied to the kit, and the related diseases can be effectively detected and diagnosed.

Drawings

FIG. 1 shows the results of the binding activity of the monoclonal antibody to cells overexpressing Claudin18.2 in example 1; the results showed that different monoclonal antibodies had different binding activities to cells overexpressing Claudin18.2, with EC50 of 213-m0306-4 reaching 86.09 ng/mL;

FIG. 2 shows the results of the binding activity of the monoclonal antibody to the cells overexpressing Caludin18.1 in example 1; the results show that the binding of five different monoclonal antibodies to the cells overexpressing Caludin18.1 shows different binding activities;

FIG. 3 shows the results of the binding activity of the humanized antibody of example 4 to Claudin18.2 overexpressing cells; panel A and B show the results that the EC50 values for the binding activity of the different humanized antibodies to Claudin18.2 overexpressing cells were all around 10 ng/mL;

FIG. 4 shows the results of the binding activity of the humanized antibody HA1LA1 with Claudin18.2 overexpressing cells and Claudin18.1 overexpressing cells in example 4; the results show that HA1LA1 bound to the control antibody and both bound to Claudin18.2 overexpressing cells, while both bound to Claudin18.1 overexpressing cells, and that the EC50 values for binding to Claudin18.2 overexpressing cells were 27.96ng/ml and 118.6ng/ml, respectively, and that HA1LA1 binding activity was significantly stronger than that of the control antibody;

FIG. 5 shows the results of the binding activity of the humanized antibody HA1LA1 to Claudin18.2 recombinant protein in example 5; the result shows that the binding activity of HA1LA1 and recombinant protein is higher than that of a control antibody, the EC50 value of the control antibody calculated because the reaction signal value does not reach a platform is very huge, and the EC50 value of HA1LA1 is 490 ng/mL;

FIG. 6 shows the ADCC activity of the humanized antibody HA1LA1 in example 6; the results show that the antibody HA1LA1 HAs significantly superior ADCC activity compared with the control antibody, and the ratio of killing target cells is represented by 0.01263nM HA1LA1 and 0.05754nM of the control antibody respectively on EC 50;

FIG. 7 is the in vivo tumor suppressive activity of antibody HA1LA1 in the MiaPaca2-Claudin 18.2 nude mouse model in example 7; the results show that HA1LA1 significantly inhibited the growth of MiaPaca2-Claudin 18.2 transplanted tumors;

FIG. 8 is the in vivo anti-tumor activity of antibody HA1LA1 in example 7 in a human gastric cancer PDX nude mouse model; the results show that HA1LA1 inhibits the growth of PDX transplantable tumor of human gastric cancer;

FIG. 9 shows the binding activity of the symmetric bispecific antibody of example 9 with PDL1 cell and Claudin18.2 cell, respectively; the results showed that symmetric bispecific antibodies 213H204CC, 204H213CC, 213CC204H and 204CC213H bound to PDL1 cells with EC50 of 0.1685nM,0.2342nM, 0.2387nM and 0.0825nM, respectively, and to claudin18.2 cells with EC50 of 0.4003nM, 0.6503nM, 0.3301nM and 0.5441nM, respectively;

FIG. 10 shows the binding activity of the asymmetric bispecific antibody of example 9 to PDL1 cells and Claudin18.2 cells, respectively; the results showed that 0413CC and 1304CC bound EC50 to PDL1 cells at 11.7ng/ml and 12.8ng/ml, respectively, and Claudin18.2 cells bound EC50 at 169.5ng/ml and 231.7ng/ml, respectively;

figure 11 is the ADCC activity of the symmetric bispecific antibody of example 9; the results showed that symmetric bispecific antibodies 213H204CC, 204H213CC, 213CC204H and 204CC213H had ADCC activity EC50 of 0.01986nM,0.1305nM,0.01864nM and 0.03851nM, respectively;

figure 12 is ADCC activity of the asymmetric bispecific antibody of example 9; the results showed that ADCC activity EC50 of asymmetric bispecific antibodies 0413CC and 1304CC was 0.04309nM and 0.03575nM, respectively;

FIG. 13 shows that the symmetric bispecific antibodies of example 9 inhibit the binding activity of PDL1 to PD1, and the results show that the symmetric bispecific antibodies 213H204CC, 204H213CC, 213CC204H and 204CC213H inhibit the binding activity with IC50 of 1.795nM,1.586nM, 1.663nM and 1.702nM, respectively;

FIG. 14 shows the activity of bispecific binding to Claudin18.2 and PDL1 in example 9, and the results indicate that bispecific antibodies 213H204CC, 204H213CC, 213CC204H and 204CC213H can all bind to Claudin18.2 and PD1 simultaneously.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Example 1: preparation of hybridoma cells expressing anti-human Claudin18.2 monoclonal antibody

Murine monoclonal antibodies were prepared by hybridoma cell technology. For experimental protocols see literature (Ed Harlow, David Lane. antibody: A laboratory Manual. 1988).

Firstly, a Claudin18.2 full-length gene is synthesized by a gene synthesis technology. Cloning the full-length gene of Caludin18.2 into PCDNA3.1(Invitrogen), transfecting the plasmid into a CHO-K1(ATCC No. CCL-60) cell strain, and performing G418(GIBCO) pressure screening to obtain a Caludin18.2 stable over-expression cell strain for detection and screening; the plasmid was transiently transferred into 293 cell line (TaKaRa, Lenti-X, 632180) for immunization.

By 2X10⁷A transient transformation of 293 cells was performed by Claudin18.2, i.e., intraperitoneal injection into BALB/c mice, followed by the first immunization, at day 14 and day 35, respectively, with 2X10⁷Mice were boosted by intraperitoneal injection of individual cells, 1X 10 on day 56⁶One BALB/c mouse was injected intravenously with 2X10⁷The cells were injected intraperitoneally for final immunization, and spleens were harvested 4 days later for fusion.

Mouse splenocytes were combined with SP2/0 cells (ATCC No. CRL-1581) at a ratio of 4: 1 ratio fusion, culturing in HAT (GIBCO) medium in 96-well plate (Corning), and then performing hybridoma cell selection, and the resulting cell clones are subjected to binding selection.

The identification and screening process is divided into three steps:

(1) a certain number of caludin18.2 stable overexpression cells were plated in a 96-well plate (Corning), and after 1 hour of incubation with the addition of clonal expression supernatant, washed 1 time with PBS, and cell clonal supernatants having binding activity to claudin18.2 were identified using goat anti-mouse IgG-hrp (jackson immuno), thereby obtaining positive clones that could bind directly to claudin 18.2.

(2) Subsequently, the positive clones obtained in step (2) were transferred to a 24-well plate (Corning) for culture, subcloned by limiting dilution, and subjected to multiple detection to obtain monoclonal positive cells.

(3) The obtained monoclonal antibody cell strain was frozen and cultured in SFM medium (Gibco) to obtain a monoclonal antibody for quality identification.

CHO-K1 cells stably expressing human Claudin18.2 were plated on cell culture plates, washed the next day and blocked, then a gradient of diluted mouse monoclonal antibody was added, incubated at room temperature for 1 hour, PBST washed and goat anti-mouse IgG-HRP (Jackson Immuno) added, after incubation at room temperature for 1 hour, washed with PBST, developed with TMB (thermo) and read at 450nm on a microplate reader after termination. The results are shown in FIG. 1, which shows that monoclonal antibody 213-m0306-4 binds to CHO-K1 cells stably expressing Claudin18.2 with an EC50 of 86.09 ng/ml.

The binding activity of the monoclonal antibody to human Claudin18.1 was tested by transient transfection of the expression plasmid human Claudin18.1 into 293 cells (TaKaRa, Lenti-X, 632180) with PEI transfection reagent, and the results are shown in FIG. 2, which shows that different levels of binding activity of the monoclonal antibody to 293 cells overexpressing human Claudin18.1 exist.

Example 2: mouse monoclonal antibody gene calling

And (3) cracking the screened hybridoma cells with positive clones, extracting mRNA, and performing reverse transcription to obtain cDNA. Using the cDNA as a template, respectively amplifying the light chain and heavy chain variable region nucleic acid sequences of a mouse IgG antibody by adopting a PCR method, analyzing the heavy chain variable region and the light chain variable region, wherein the encoded heavy chain variable region comprises a parent sequence (TYGVH) shown as SEQ ID NO.1, a parent sequence (VIWNGGNTDYNAAFIS) shown as SEQ ID NO.2 and a parent sequence (NRRGGGFGMDY) shown as SEQ ID NO. 3; the light chain variable region comprises the parent sequence (KSSQSLLNGGIQKNYLT) shown in SEQ ID NO.4, the parent sequence (WASTRES) shown in SEQ ID NO.5, and the parent sequence (QNAYTYPFT) shown in SEQ ID NO. 6.

Then, the heavy chain variable region or the light chain variable region and the corresponding constant region sequence of the human antibody are spliced by a sequence end complementation method, and the heavy chain and light chain fragments of the full-length chimeric antibody, both containing signal peptides, are cloned into pCDNA3.1(Invitrogen) plasmids respectively. Co-transfecting light and heavy chain plasmids to 293F cells, culturing for 5 days, collecting supernatant, purifying the supernatant by adopting protein A (GE) affinity chromatography, and finally obtaining the recombinant expression anti-human Claudin18.2 chimeric antibody.

The heavy chain variable region sequence of the antihuman Claudin18.2 chimeric antibody is shown in SEQ ID NO.7, and specifically comprises the following steps:

QVQLKQSGPDLVHPSQSLSISCTVSGFSLTTYGVHWIRQSPGKGLEWLGVIWNGGNTDYNAAFISRLSITKDNSKSQVFFKMNSLLPSDTAIYYCARNRRGGGFGMDYWGQGTSVTVSS；

the light chain variable region sequence of the antihuman Claudin18.2 chimeric antibody is shown in SEQ ID NO.8, and specifically comprises the following steps:

DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNGGIQKNYLTWYQQRPGQPPKLLFFWASTRESGVPVRFTGSGSGTDFTLTISSVQAEDLAVYYCQNAYTYPFTFGSGTKLEIK。

example 3: design and expression of humanization

Example 3 the monoclonal antibody of murine origin obtained in example 2, specifically binding to human claudin18.2, was humanized, in particular according to the following steps:

analyzing the sequence of the murine antibody, aligning with a human germline (germ line) gene of the IMGT, finally determining that IGKV4-1 × 01 or IGKV3-7 × 02 is a humanized framework sequence of a light chain, determining IGHV3-33 × 01 or IGHV4-4 × 08 is a humanized framework sequence of a heavy chain, then respectively selecting appropriate FR4 humanized templates, and juxtaposing CDRs of the heavy chain and the light chain to the framework sequences through CDR-grafting; meanwhile, the key sites on the framework regions are subjected to back mutation design to obtain a plurality of humanized antibody variable regions, and specific variable region sequences are shown in table 1.

TABLE 1 variable region sequences of Claudin18.2 humanized antibodies

Name of antibody	Heavy chain variable region	Light chain variable region
			HA1LA1	SEQ ID NO.9	SEQ ID NO.13
HA1LA2	SEQ ID NO.9	SEQ ID NO.14
			HA1LB1	SEQ ID NO.9	SEQ ID NO.15
HA1LB2	SEQ ID NO.9	SEQ ID NO.16
			HA2LA1	SEQ ID NO.10	SEQ ID NO.13
HA2LB1	SEQ ID NO.10	SEQ ID NO.15
			HA3LA1	SEQ ID NO.11	SEQ ID NO.13
HA3LA2	SEQ ID NO.11	SEQ ID NO.14
			HA3LB1	SEQ ID NO.11	SEQ ID NO.15
HA3LB2	SEQ ID NO.11	SEQ ID NO.16
			HB2LA2	SEQ ID NO.12	SEQ ID NO.14
HB2LB2	SEQ ID NO.12	SEQ ID NO.16

Respectively connecting the heavy and light chain variable regions of the antibody with corresponding antibody constant regions by an overlap-PCR method to obtain full-length heavy and light chain sequences of the antibody, and then respectively constructing the full-length heavy and light chain sequences of the antibody into pCDNA3.1 expression plasmids. The antibody heavy and light expression plasmid was transiently transferred to 293F cells, cultured for 5 days, and the supernatant was collected and purified by proteinA affinity chromatography.

Example 4: antibody binding Activity assay with overexpressing cells

Using PEI transfection, 293 cells (TaKaRa, Lenti-X, 632180) were transfected with human Claudin18.1 or human Claudin18.2 expression plasmids and used every other day. Placing the two over-expression cells in a reaction plate, blocking with 1% BSA, and incubating at room temperature for 1 hour; adding a gradient diluted antibody, adding 100 uL/hole, incubating at room temperature for 1 hour, washing with PBS once, then adding sheep anti-human IgG-HRP (Jackson immuno), adding 100uL of each hole, reacting at room temperature for 1 hour, washing with PBS twice, finally adding TMB for color development, stopping with 1M sulfuric acid, and reading at 450nm by using an enzyme-labeling instrument.

As shown in FIG. 3, cell binding experiments showed that the binding of the different anti-Claudin18.2 humanized antibodies to EC50 was around 10 ng/mL. In addition, compared to the binding activity of the control antibody (from patent CN20148000912, whose variable region sequences are SEQ ID No.19 and SEQ ID No.20, and whose full-length antibody was prepared as described in example 2), as shown in fig. 4, EC50(27.96ng/mL) of HA1LA1 was superior to EC50(118.6ng/mL) of the control antibody, while not binding to claudin18.1 expressing cells.

Example 5: determination of the binding Activity of antibodies to human Claudin18.2 recombinant proteins

Human Claudin18.2-His recombinant protein (Kactus, Cat. CLD-HE1821) was coated on an enzyme plate overnight at 4 ℃. After blocking, adding the antibody diluted in gradient, reacting at 37 ℃ for 1 hour, washing with PBST twice, adding Goat anti-human-IgG-HRP, reacting at 37 ℃ for 1 hour with 100uL per well, finally washing with PBST three times, adding TMB for color development, stopping, and reading at 450nm by an enzyme-labeling instrument.

The results show that HA1LA1(EC50 was 490ng/mL) HAs significantly better binding activity to recombinant protein than the control antibody, as shown in FIG. 5.

Example 6: determination of ADCC Activity of antibodies

Nugc4 gastric cancer cells overexpressing human claudin18.2 were plated at 5x10 in cell culture plates³The following day, the antibody was added in a gradient dilution, incubated at 37 ℃ for 30 minutes, and NK92 cells (effector cells) (ATCC), 2X10, were added⁴After incubation for 4 hours at 37 ℃ in the well, the percentage of lysis of the target cells was calculated by chromogenic reaction using the LDH kit (Dojido).

The results are shown in FIG. 6, where HA1LA1(EC50 is 0.01263nM) HAs a stronger ADCC activity than the control antibody (EC50 is 0.05754nM), whereas the nonspecific human IgG (hIgG) HAs no ADCC activity.

Example 7: in vivo pharmaceutical activity of anti-Claudin18.2 antibody

The in vivo effect of anti-Claudin18.2 antibody in inhibiting tumor growth was examined using a MiaPaca2-Claudin 18.2 cell subcutaneous nude mouse model and a human gastric carcinoma PDX nude mouse model. Inoculating human pancreatic cancer cell MiaPaca2-Claudin 18.2 cell or human gastric cancer tissue cell into female nude mouse subcutaneously, and selecting tumor with volume of 100mm when tumor volume grows to appropriate size³Left and right tumor mice, randomly grouped, were IgGisotype group (40mg/kg), HA1LA 140 mg/kg group or HA1LA 110 mg/kg group, respectively, and the body weight and tumor volume of the mice were measured on the day of administration, followed by 2 times weekly administration and measurement for 4 weeks by intravenous injection.

As a result, as shown in fig. 7 and 8, HA1LA1 significantly inhibited the growth of human pancreatic cancer cells and human gastric cancer tissue cell-transplanted tumors in the mouse tumor model.

Example 8: design and expression of bispecific antibodies

The bispecific antibody of the present invention employs IgG linked to the symmetric structure of scfv and the asymmetric structure of the nanobs into hole. Among them, the IgG of the invention is linked to the symmetric structure of scfv, i.e. a scfv fragment linked to another antigen binding domain via (GGGGS)3 at the C-or N-terminus of the heavy chain of an IgG antibody, as specifically designed in table 2.

TABLE 2 symmetrical Structure

The amino acid sequence of the anti-PDL 1 antibody scfv fragment shown in Table 2 is shown in SEQ ID NO.29 (the heavy chain variable region is shown in SEQ ID NO. 27, and the light chain variable region is shown in SEQ ID NO. 28), the amino acid sequence of the anti-PDL 1 antibody heavy chain is shown in SEQ ID NO.30, the amino acid sequence of the anti-PDL 1 antibody light chain is shown in SEQ ID NO.31, the amino acid sequence of the anti-Claudin18.2 antibody scfv fragment is shown in SEQ ID NO.32, the amino acid sequence of the anti-Claudin18.2 antibody heavy chain is shown in SEQ ID NO.17, and the amino acid sequence of the anti-Claudin18.2 antibody light chain is shown in SEQ ID NO. 18.

In addition, the asymmetric structure of the present invention, one antigen binding domain is designed as Fab structure, the other antigen binding domain is designed as scfv-FC structure, and the two are combined by disulfide bond between FC, which is specifically designed as shown in Table 3.

TABLE 3 design of asymmetric structures

Respectively constructing heavy and light chains of the bifunctional antibody and expression plasmids of scfv-FC structure by a molecular cloning method. Then, the heavy-light chain expression plasmid corresponding to the bifunctional antibody or the expression plasmid and the scfv-FC structure expression plasmid are transferred to 293F cells in a co-transient manner, cultured for 5 days, and the supernatant is collected and purified by protein A affinity chromatography.

Example 9: detection of Activity of bispecific antibodies

(1) Specific antibodies and binding activity of PDL1 cells to Claudin18.2 cells. The process is referred to example 4.

The results of the binding of the symmetric bispecific antibody and PDL1 cells to claudin18.2 cells are shown in fig. 9, and it can be seen from the figure that bispecific antibodies 213H204CC, 204H213CC, 213CC204H and 204CC213H all bind to the claudin18.2 cells in PDL1 cells with binding activity comparable to that of the mab control. Wherein the PDL1 monoclonal antibody contrast is 402F1, the amino acid sequence of the antibody heavy chain is shown as SEQ ID NO.30, and the amino acid sequence of the antibody light chain is shown as SEQ ID NO. 31. The control of the Claudin18.2 monoclonal antibody is HA1LA1, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.17, and the amino acid sequence of the light chain of the Claudin18.2 antibody is shown in SEQ ID NO. 18.

The results of the asymmetric bispecific antibody and the binding of PDL1 cells to Claudin18.2 cells are shown in FIG. 10, and it can be seen from the figure that the bispecific antibodies 1304CC and 0413CC both bind to Claudin18.2 cells from PDL1 cells and have binding activity to PDL1 cells comparable to that of mAb control 402F 1.

(2) ADCC Activity assay for bispecific antibodies

Target cells, 293 cells overexpressing Claudin18.2, were plated at 15000/50. mu.L/well, 37 ℃ with 5% CO₂The incubator was incubated overnight. The next day, the target cell culture supernatant was removed. The antibody was diluted with a gradient of medium, added to a target cell culture plate, and incubated at 37 ℃ for 1 hour. Effector cells Jurkat-ADCC (BPS Bioscience, 60541) were counted, 75000 cells/50. mu.L/well were added to 50. mu.L/well of the antibody, and the mixture was placed at 37 ℃ with 5% CO₂Incubate for 5 hours. 50 μ l of Bright-Glo (Promega, E2620) luciferase assay reagent was added, and the reaction was followed at room temperature by reading using a Pheragar microplate reader and related software program.

The ADCC activity results of the symmetric bispecific antibody are shown in fig. 11, and it can be seen from the figure that the bispecific antibodies 213H204CC, 204H213CC, 213CC204H and 204CC213H all can mediate stronger ADCC activity against target cells, wherein the activities of 213H204CC and 213CC204H are better than those of the mab control.

The ADCC activity results of the asymmetric bispecific antibody are shown in fig. 12, and it can be seen from the figure that both bispecific antibodies 1304CC and 0413CC can mediate stronger ADCC activity against target cells, the activity is equivalent to that of a monoclonal antibody control antibody, wherein HUT104 is a negative antibody control.

(3) Bispecific antibody Activity to inhibit binding of PDL1 to PD1

Coating PDL1-Fc recombinant protein on an enzyme label plate, and standing overnight at 4 ℃; after washing and blocking, adding the bispecific antibody and the control antibody which are diluted in a gradient way, and reacting for 15 minutes at 37 ℃; adding PD1-Fc-Biotin with a certain concentration into the antibody reaction plate, and reacting for 1 hour at 37 ℃; washing, adding Streptavidin-HRP (Jackson immuno), reacting for 1 hour at 37 ℃ with 100uL of each well; after washing, TMB was added and the development was stopped and the microplate reader read at 450 nm.

The results of the activity of the bispecific antibody in inhibiting the binding of PDL1 to PD1 are shown in fig. 13, and it can be seen from the figure that bispecific antibodies 213H204CC, 204H213CC, 213CC204H and 204CC213H inhibit the binding activity of PDL1 to PD1, which is comparable to PDL1 mab control 402F 1.

(4) Activity detection of bispecific antibody simultaneously binding PDL1 and Claudin18.2

Placing 293 cells overexpressing Claudin18.2 in a reaction plate; adding a gradient diluted antibody and incubating the cells for 10 minutes at room temperature; adding PDL1-Fc-Biotin with a certain concentration, and continuing the reaction at room temperature for 1 hour; after washing with PBS, adding Streptavidin-HRP (Jackson Immuno) and reacting at room temperature for 1 hour; finally, PBS wash, addition of TMB color after termination, plate reader at 450nm reading. The results are shown in fig. 14, bispecific antibodies 213H204CC, 204H213CC, 213CC204H and 204CC213H can bind both PDL1 and claudin18.2 targets simultaneously.

The antibody of the present invention can be produced by genetic engineering techniques, since the DNA sequence encoding the humanized antibody of the present invention can be obtained by conventional means well known to those skilled in the art, such as artificial synthesis of the amino acid sequence according to the disclosure of the present invention or amplification by PCR, and thus can be used by recombinant DNA methods, and the sequence can be ligated into an appropriate expression vector by various methods well known in the art.

Once the antibody molecule of the invention is prepared, it can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity chromatography of protein a and other chromatography columns), centrifugation, use of solubility differences, or by any other standard technique for purifying proteins. In many embodiments, the antibody is secreted from the cell into the culture medium, and the antibody is obtained by collecting the culture medium and purifying it.

In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and routine steps that are widely used in the relevant arts. Meanwhile, in order to better understand the present invention, the definitions and explanations of the related terms are provided below.

In the present invention, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH 3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The VH and VL regions can be further divided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant region of the antigen may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The extent of the framework regions and complementarity determining regions has been precisely defined (see "sequences of Proteins of Immunological Interest," E.Kabat et al, U.S. department of Health and human Services, 1991). All antibody amino acid sequences discussed herein are ordered with reference to the Kabat system. The term "antibody" is not limited by any particular method of producing an antibody, and includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies, among others.

In the present invention, the term "antigen binding domain" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" include: (1) fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL and CH1 domains; (2) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (3) an Fd fragment consisting of the VH and CH1 domains; (4) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (5) dAb fragments consisting of VH domains (Ward et al (1989) Nature 341: 544-546); and (6) an isolated Complementarity Determining Region (CDR).

In the present invention, the term "isotype" refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences. Other modifications may also be made within the human framework sequences. The term "chimeric antibody" is an antibody whose heavy and light chain genes have been constructed, particularly using genetically engineered antibody variable and constant region genes belonging to different species. For example, a variable region fragment of a murine monoclonal antibody gene can be ligated to a human antibody constant region fragment such as γ 1 and γ 3. Of course, other mammalian species may be used as the genetic source for the chimeric antibody.

In the present invention, the term "Knobs-into-holes technology" refers to a genetic engineering technique to make a knob (knob) mutation on one heavy chain CH3 and a hole (hole) mutation on the other heavy chain CH3 of an antibody, so as to facilitate the occlusion of the two heavy chains and form heterodimers.

The invention of at least 90% overall sequence identity means sequence identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, the monoclonal antibody or antigen binding portion thereof heavy chain variable region and light chain variable region of the amino acid sequences and the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:13, which is a monoclonal antibody or antigen-binding portion thereof, has at least 90% overall sequence identity to the amino acid sequences of SEQ ID No. 9 and SEQ ID NO:13 has at least 90% sequence identity to the sum of the sequences.

The heavy chain and the light chain of the Claudin18.2 antibody and the Claudin18.2 antibody can also be used for scientific researches related to the Claudin18.2, such as scientific researches in multiple fields of developmental biology, cell biology, metabolism, structural biology, functional genomics and the like, or medical and pharmaceutical application researches of tumors, autoimmune diseases and the like.

The Claudin18.2 antibody of the invention can be a single chain antibody, diabody, chimeric antibody, humanized antibody, and derivatives, functional equivalents and homologs of the foregoing, and also includes antibody fragments and any polypeptide comprising an antigen binding domain.

The pharmaceutical composition of the invention also comprises a pharmaceutically acceptable carrier and/or diluent.

The invention can also be a reagent or a chip comprising the Claudin18.2 antibody described above.

The invention also discloses a method for binding the antigen site of a target cell and the FC receptor site of an effector cell by using the Claudin18.2 antibody to mediate ADCC activity, and a method for treating related diseases by using the antibody or using a kit containing the antibody to carry out related diagnosis and detection.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Jiangsu Nuomibo biomedical science and technology Co., Ltd

<120> monoclonal antibody specifically binding to human Claudin18.2, and medicine and kit comprising same

<160> 36

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Thr Tyr Gly Val His

1 5

<210> 2

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile Ser

1 5 10 15

<210> 3

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr

1 5 10

<210> 4

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Thr

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gln Asn Ala Tyr Thr Tyr Pro Phe Thr

1 5

<210> 7

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

Lys Met Asn Ser Leu Leu Pro Ser Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 8

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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 Gly

20 25 30

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

35 40 45

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

50 55 60

Pro Val 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

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

100 105 110

Lys

<210> 9

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 10

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 11

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Thr Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

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

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 12

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 13

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

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

20 25 30

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

65 70 75 80

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

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 14

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 15

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 16

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 17

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 18

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

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

1 5 10 15

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

20 25 30

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

65 70 75 80

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

85 90 95

Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Gln 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> 19

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

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 Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu 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 Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 20

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

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 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 21

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Ser Gly Tyr Trp Asn

1 5

<210> 22

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

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

1 5 10 15

<210> 23

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr

1 5 10

<210> 24

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Lys Ala Ser Gln Asn Val Met Asp Asn Val Ala

1 5 10

<210> 25

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Ser Ala Ser Tyr Arg Phe Ser

1 5

<210> 26

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Gln Gln Tyr Asn Gly Tyr Pro Leu Thr

1 5

<210> 27

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Cys Leu Glu Tyr Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 28

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 29

<211> 241

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Cys Leu Glu Tyr Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

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

145 150 155 160

Gln Asn Val Met Asp Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

165 170 175

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

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Tyr Asn Gly Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 30

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 31

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Gly Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 32

<211> 247

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

145 150 155 160

Gln Ser Leu Leu Asn Gly Gly Ile Gln Lys Asn Tyr Leu Thr Trp Tyr

165 170 175

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

180 185 190

Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala

210 215 220

Val Tyr Tyr Cys Gln Asn Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Cys

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 33

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 34

<211> 487

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Cys Leu Glu Tyr Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

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

145 150 155 160

Gln Asn Val Met Asp Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

165 170 175

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

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Tyr Asn Gly Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

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

260 265 270

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

355 360 365

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

370 375 380

Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

385 390 395 400

Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ser Leu Ser Leu Ser Pro Gly

485

<210> 35

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Phe Ser Ser Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Tyr Arg Asp Trp Leu His Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 36

<211> 493

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Asn Gly Gly Asn Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asn Arg Arg Gly Gly Gly Phe Gly Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

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

130 135 140

Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser

145 150 155 160

Gln Ser Leu Leu Asn Gly Gly Ile Gln Lys Asn Tyr Leu Thr Trp Tyr

165 170 175

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

180 185 190

Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala

210 215 220

Val Tyr Tyr Cys Gln Asn Ala Tyr Thr Tyr Pro Phe Thr Phe Gly Cys

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

450 455 460

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

465 470 475 480

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

485 490

Claims (17)

1. A monoclonal antibody that specifically binds to human claudin18.2, comprising a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises: a heavy chain variable region CDR1 having an amino acid sequence shown in SEQ ID NO.1, a heavy chain variable region CDR2 having an amino acid sequence shown in SEQ ID NO.1, and a heavy chain variable region CDR3 having an amino acid sequence shown in SEQ ID NO. 3;

the light chain variable region comprises: light chain variable region CDR1 with an amino acid sequence shown in SEQ ID NO.4, light chain variable region CDR2 with an amino acid sequence shown in SEQ ID NO.5, and light chain variable region CDR3 with an amino acid sequence shown in SEQ ID NO. 6.

2. The monoclonal antibody according to claim 1,

the heavy chain variable region amino acid sequence of the monoclonal antibody comprises any one of the following sequences: 9, 10, 11, or 12;

the light chain variable region amino acid sequence of the monoclonal antibody comprises any one of the following sequences: 13, 14, SEQ ID NO: 15, or SEQ ID NO: 16.

3. the monoclonal antibody of claim 2, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 9 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 13.

4. The monoclonal antibody of claim 3, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of said monoclonal antibody are identical to the amino acid sequences of SEQ ID NO 9 and SEQ ID NO:13 have an overall sequence identity of at least 90%.

5. The monoclonal antibody of claim 3, wherein the heavy chain amino acid sequence of the monoclonal antibody is represented by SEQ ID NO.17 and the light chain amino acid sequence is represented by SEQ ID NO. 18.

6. The monoclonal antibody of any one of claims 1-5, wherein the monoclonal antibody is a full length antibody of the IgG1, IgG2, or IgG4 isotype.

7. The monoclonal antibody of any one of claims 1-5, wherein the monoclonal antibody is an antibody fragment or a single chain antibody.

8. A bispecific antibody comprising a first antigen-binding domain derived from the monoclonal antibody of any one of claims 1-7 and a second antigen-binding domain derived from an anti-PDL 1 antibody that specifically binds human PDL 1.

9. The bispecific antibody of claim 8, wherein said anti-PDL 1 antibody comprises the following CDR regions:

the amino acid sequence is shown as heavy chain variable region CDR1 of SEQ ID NO. 21, the amino acid sequence is shown as heavy chain variable region CDR2 of SEQ ID NO. 22, the amino acid sequence is shown as heavy chain variable region CDR3 of SEQ ID NO. 23, the amino acid sequence is shown as light chain variable region CDR1 of SEQ ID NO. 24, the amino acid sequence is shown as light chain variable region CDR2 of SEQ ID NO. 25, and the amino acid sequence is shown as light chain variable region CDR3 of SEQ ID NO. 26.

10. The bispecific antibody of claim 9, wherein the heavy chain variable region of the anti-PDL 1 antibody comprises the amino acid sequence set forth in SEQ ID No. 27, and the light chain variable region of the anti-PDL 1 antibody comprises the amino acid sequence set forth in SEQ ID NO: 28, or a pharmaceutically acceptable salt thereof.

11. The bispecific antibody of any one of claims 8-10, wherein the first antigen-binding domain and the second antigen-binding domain are connected by FC or by a connecting fragment.

12. The bispecific antibody according to claim 11,

the FC connection adopts a Knobs intoholes technology;

the connecting segment is (GGGGS) 3.

13. The bispecific antibody of claim 11, wherein the first and second antigen-binding domains are independently an immunoglobulin or antigen-binding fragment thereof; the antigen binding fragment is a half-antibody, Fab, F (ab)2 or single-chain antibody.

14. The bispecific antibody of claim 13, wherein said first antigen-binding domain is an immunoglobulin and said second antigen-binding domain is a single chain antibody.

15. The bispecific antibody of claim 14, wherein said second antigen-binding domain comprises a scfv single chain antibody having the amino acid sequence set forth in SEQ ID No. 29.

16. A medicament comprising a monoclonal antibody according to any one of claims 1-7 or a bispecific antibody according to any one of claims 8-15 for use in the treatment or alleviation of cancer.

17. A kit comprising a monoclonal antibody according to any one of claims 1-7 or a bispecific antibody according to any one of claims 8-15.

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