CN110317276A - A kind of recombinant antibodies sample T cell antigen receptor, T cell antigen coupled receptors drug and application - Google Patents

Abstract

The invention discloses a recombinant antibody-like T cell antigen receptor, T cell antigen receptor coupled drug, bispecific molecule and application thereof. In the present invention, by designing the fusion expression forms of various IgG1 constant regions and 1G4113, a fusion protein form that can be solublely expressed in mammalian cells is obtained through screening, and only one step of purification is required to obtain recombinant antibody-like T cell antigen receptors with high purity. body. The recombinant antibody-like T cell antigen receptor is coupled with the MMAE small molecule to prepare a T cell antigen receptor coupled drug, which can be used to prepare antitumor drugs.

Description

A recombinant antibody-like T cell antigen receptor, T cell antigen receptor conjugated drug and application

technical field

The invention relates to the technical field of biomedicine, in particular to a recombinant antibody-like T cell antigen receptor, T cell antigen receptor coupled drug and application thereof.

Background technique

Cancer has become an important disease that endangers human health. In recent years, tumor immunotherapy represented by tumor immune checkpoint inhibitors, adoptive immune cell therapy and individualized tumor vaccines has shown unprecedented anti-tumor therapeutic effects, and has become a hot spot in cancer research and clinical treatment. The selection of tumor antigen targets is a key factor for the success of tumor immunotherapy. Tumor neoantigens have attracted extensive attention from researchers due to their tumor specificity, and have become potential ideal targets for tumor immunotherapy.

Somatic mutation is an important driving factor for the development of tumors. There are a large number of point mutations, small fragment insertions and deletions, and gene fusions in tumor cells to form mutant proteins, which can be displayed on the cell surface to form tumor-specific antigens (Tumor- Specific Antigen, TSA). Unlike Tumor-Associated Antigen (TAA), which is highly expressed by tumor cells, TSA exists only in tumor cells and can effectively distinguish tumor cells from normal cells, making it an ideal drug target. Most of the proteins in the cell are located in the cell, and the mutated intracellular protein can be processed by the intracellular pathway to become an antigenic peptide, which is presented to the cell surface by the Major Histocompatibility Complex (MHC) class I molecules to form MHC - Polypeptide complex (pMHC), which becomes tumor-specific neoantigen (Tumor-Specific Neoantigen), is recognized by TCR (T cell receptor, T cell receptor) to mediate T cell immune response. At present, studies have found tumor-specific neoantigens that can be recognized by specific T cells, such as the classic drug-resistant mutations EGFR ^T790M and HLA-A*0201 in non-small cell lung cancer, and the oncogenic mutations G12D and HLA-C in KRAS. *0802, pMHC formed by the Philadelphia chromosome fusion gene BCR-ABL nascent polypeptide and mutant polypeptides such as HLA-A*0301 and MHC class I molecules in chronic myelogenous leukemia.

Antibody drugs play a pivotal role in the field of anti-tumor. In recent years, the screening of antibodies against pMHC (TCR-mimic or TCR-like Antibody, TCRm) has been favored by researchers. Although the antibody affinity (generally 10-100nM) obtained by screening is already at a relatively high level, it is still difficult to reach the level of antibody drugs (generally <10nM), which requires further modification and maturation. Among the more than 50 published TCRm, there are only 2 cases of antibodies targeting tumor neoantigens, respectively targeting the G12V mutation of KRAS and HLA-A*0201, the L858R mutation of EGFR and the pMHC formed by HLA-A*0301 , and the specificity of the latter is not ideal. There are only a small number of amino acid residue changes in the pMHC formed between the mutant protein and the normal protein, and the amino acid residues of the antigenic peptide are easily buried by MHC, which makes the screening of TCRm antibodies targeting tumor neoantigens extremely difficult and workload-intensive. Disadvantages such as poor specificity. The recognition of TCR to pMHC is highly specific, and the specific TCR that can recognize tumor neoantigens is expected to become a tumor-specific TCR targeting tumor cells, but the affinity of TCR is not satisfactory (generally 1-100 μM), which is also a limitation of TCR. As a bottleneck for the widespread application of drugs.

On the other hand, TCR is a membrane protein, which is difficult to express solublely, which also limits the application of TCR as a protein drug and increases the difficulty of evaluating TCR activity in vitro. Jonathan (BOULTER J M, GLICK M, TODOROV P T, et al. Stable, soluble T-cell receptor molecules for crystallization and therapeutics [J]. Protein Eng, 2003, 16 (9): 707-711.) etc. T48C and TRBC of TRAC S57C was mutated and expressed in Escherichia coli, and a variety of soluble TCRs were successfully obtained through renaturation. Although this method is suitable for the preparation of a variety of soluble TCRs, there are the following problems: 1. The renaturation efficiency is low (≈40 %); 2. Escherichia coli has endotoxin and other toxic substances that are difficult to remove and are harmful to the human body; 3. The method includes expression and purification of E. coli inclusion bodies, renaturation and purification by ion exchange columns and molecular sieves after renaturation , the process is very cumbersome, and the technical requirements are high; 4. The purity of the obtained product is about 95%, which still needs to be further improved to meet the follow-up medicine. Therefore, the application of TCR as a drug molecule is currently limited. Exploring safe and effective soluble expression methods suitable for different TCRs is the prerequisite for TCRs to become protein drugs.

Contents of the invention

The present invention provides a soluble recombinant antibody-like T cell antigen receptor expressed by mammalian cells.

A recombinant antibody-like T cell antigen receptor, which is a heterotetramer, including 2 long chains and 2 short chains, the long chains include sequentially linked ECDβ-IgG1 HC, and the short chains include sequentially linked ECDα-LC, wherein ECDα and ECDβ are the extracellular domains of the α chain and β chain of T cell antigen receptor 1G4113, respectively, and IgG1 HC and LC are the heavy chain constant region and light chain constant region of IgG1, respectively. This heterotetramer forms a "Y"-shaped structure similar to antibodies.

Preferably, the C-terminus of the long chain and/or the short chain is also connected with a tether LPETG.

sortase A is an enzyme isolated from Staphylococcus aureus that catalyzes the transpeptidation reaction. The cysteine at position 184 nucleophilically attacks threonine (T) and glycine in the LPXTG (X represents any one of the natural amino acids) tags The peptide bond between (G) forms a covalent thio intermediate with simultaneous release of glycine and its carboxy-terminal peptide. This intermediate captures the glycine substrate in the solvent to form a new peptide bond between threonine and the captured glycine substrate, releasing the sortase A enzyme for the next step of catalysis. Due to the high reaction specificity, simple operation and mild reaction conditions, sortase A has been widely used in protein engineering and protein site-directed modification.

More preferably, the amino acid sequence of the long chain is shown in SEQ ID No.11; the amino acid sequence of the short chain is shown in SEQ ID No.10.

Most preferably, the coding gene sequence of the first subunit is shown in SEQ ID No.7; the coding gene sequence of the second subunit is shown in SEQ ID No.6.

The preparation method of the recombinant antibody-like T cell antigen receptor of the present invention comprises the following steps:

(1) respectively cloning the coding gene of the long chain and the coding gene of the short chain into the expression vector to obtain two expression plasmids;

(2) co-transfecting the two expression plasmids into mammalian cells for expression;

(3) Purify by column chromatography using Protein A filler to obtain the recombinant antibody-like T cell antigen receptor.

Wherein, the expression vector used is pMH3 plasmid, and the mammalian cells used for expression are 293F cells.

The present invention further provides the application of the recombinant antibody-like T cell antigen receptor in the preparation of antitumor drugs.

The present invention further provides a T cell antigen receptor conjugate drug, in which vcMMAE is coupled to the C-terminus of the long chain and/or short chain of the recombinant antibody-like T cell antigen receptor through sortase A enzyme-catalyzed transpeptide reaction , where vc is a valine-citrulline dipeptide linker, and MMAE is dolastatin. The valine-citrulline dipeptide linker can be cleaved by the lysosomal enzyme cathepsin B.

The present invention also provides the application of the T cell antigen receptor coupled drug in the preparation of antitumor drugs.

In the present invention, by designing the fusion expression forms of various IgG1 constant regions and 1G4113, a fusion protein form that can be solublely expressed in mammalian cells is obtained through screening, and only one step of purification is required to obtain recombinant antibody-like T cell antigen receptors with high purity. body. The recombinant antibody-like T cell antigen receptor is coupled with the MMAE small molecule to prepare a T cell antigen receptor coupled drug, which can be used to prepare antitumor drugs.

Description of drawings

Figure 1 is a schematic diagram of different forms of 1G4113-IgG fusion proteins, in which Figures a to e are 5 different forms of fusion proteins, named 1G4113-1, 1G4113-2, 1G4113-3, 1G4113-4, 1G4113-5 respectively.

Figure 2 is a graph of affinity and specificity analysis results, in which graphs a and b are the results of T2 cell analysis, the arrow marks the peak: NY-ESO-1 _157-165 , and the two overlapping peaks are blank control and irrelevant Peptide control (RMFPNAPYL).

Figure 3 is the results of HPLC analysis, in which Figures a and b are TOSOH Butyl-NPR HPLC analysis of unreduced 1G4113 and 1G4113-vcMMAE respectively; Figures c and d are RP-HPLC analysis of unreduced 1G4113 and 1G4113-vcMMAE, DAR =0: 1G4113, DAR=1: 1 1G4113 molecule coupled to 1 MMAE, DAR=2: 1 1G4113 molecule coupled to 2 MMAE.

Figure 4 is the results of affinity and endocytosis analysis of 1G4113 and 1G4113-vcMMAE, wherein (a) affinity analysis of 1G4113 and 1G4113-vcMMAE to A375-NY-ESO-1 _157-165 cells; (b) fluorescence Confocal microscope analysis of endocytosis of 1G4113 and 1G4113-vcMMAE; (c) flow cytometry analysis of endocytosis efficiency of 1G4113 and 1G4113-vcMMAE, endocytosis efficiency (%)=(MFI of 4°C-MFI of 37°C)/ MFI of 4°C x 100%.

Figure 5 is a graph showing the in vitro inhibitory activity test results of 1G4113-vcMMAE on tumor cells with different expression levels of HLA-A2/NY-ESO-1 _157-165 . -Inhibitory activity of NY-ESO-1 _157-165 cells; Figures b and d are the inhibitory activities of two kinds of 1G4113-vcMMAE on three kinds of K562-derived tumor cells, respectively.

Detailed ways

The animal experiments related to this application have been approved by the Experimental Animal Welfare Ethics Review Committee of Zhejiang University, and the application number is 12435. The relevant experiments involving humans in this application have been approved by the Medical Ethics Committee of the School of Pharmacy, Zhejiang University, and the approval document number is: (2018) Lunyan Pi No. 003.

Example 1

1G4113 mammalian cell expression plasmid construction.

TCR selection 1G4113 (affinity-matured clone of TCR (named 1G4) targeting the complex of SLLMWITQC polypeptide and HLA-A*0201), from patent: US20110014169A1. A total of 5 different truncated and combined 1G4113 and IgG1 antibody constant regions were designed (the gene sequence of the heavy chain constant region and knob-into-hole mutation is shown in SEQ ID No.14, and the gene sequence of the light chain constant region is shown in SEQ ID No.15) fusion expression form (Figure 1), and the GGGGSLPETGG polypeptide sequence (G ₄ S-LPETGG) was introduced at the carboxy-terminus of the IgG1 antibody constant region for subsequent catalytic coupling of sortase A to construct human kidney epithelial cells (293F ) 9 expression plasmids.

1G4113-1: Vα-(G ₄ S) ₃ -ECDβ (containing C-terminal cysteine)-IgG1HC-G ₄ S-LPETGG (the gene sequence is shown in SEQ ID No.1), the fusion protein forms homology dimer.

1G4113-2: Vα-GS-LC (gene sequence shown in SEQ ID No.2), Vβ-GS-IgG1HC-G ₄ S-LPETGG (gene sequence shown in SEQ ID No.3), fusion protein formation is similar Tetramer of antibody "Y" form.

1G4113-3: ECDα (containing C-terminal cysteine)-GS-LC (gene sequence shown in SEQ ID No.4), ECDβ (containing C-terminal cysteine)-GS-IgG1HC-G ₄ S-LPETGG (gene sequence shown in SEQ ID No.5), the fusion protein forms a tetramer similar to the "Y" type of antibody.

1G4113-4: ECDα (without C-terminal cysteine)-GS-LC (gene sequence shown in SEQ ID No.6), ECDβ (without C-terminal cysteine)-GS-IgG1HC- G ₄ S-LPETGG (gene sequence shown in SEQ ID No.7), the fusion protein forms a tetramer similar to the "Y" type of antibody.

1G4113-5: ECDα (containing C-terminal cysteine)-GS-hinge region-CH2-CH3(hole)-G ₄ S-LPETGG (gene sequence shown in SEQ ID No.8), ECDβ (containing C Cysteine at the end)-GS-hinge region-CH2-CH3(knob)-G ₄ S-LPETGG (gene sequence shown in SEQ ID No.9), the fusion protein forms a heterodimer.

Among them, ECDα represents the extracellular region of 1G4113-α, Vα represents the variable region of 1G4113-α, Cα represents the extracellular constant region of 1G4113-α, ECDβ represents the extracellular region of 1G4113-β, Vβ represents the variable region of 1G4113-β, Cβ denotes the extracellular constant region of 1G4113-β, IgG1HC denotes the H constant region of IgG1, and LC denotes the L constant region.

By overlapping PCR, each section of gene is combined as shown in Figure 1, and EcoR I restriction site (GAATTC), kozaka sequence (ACCACC) and signal peptide are introduced upstream of the recombinant gene, and a stop codon ( TGA) and Not I enzyme cutting site (GCGGCCGC), the carboxy terminus of Fc is introduced into G ₄ S-LPETGG polypeptide sequence. After the PCR product was digested by EcoR I and Not I, it was connected to the pMH3 plasmid by T4 ligase (the plasmid was also digested by EcoR I and Not I). The primer information is shown in Table 1 below.

Table 1 Nucleotide sequence of each primer.

The ligation product was directly used to transform DH5α competent cells, and spread on Amp ⁺ LB plates. After colonies were clearly visible, single clones were picked and cultured in Amp ⁺ LB liquid medium for 8-10 hours, a part was added to 15% sterile glycerol, frozen at -20°C, and the rest were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The DH5α Escherichia coli containing the correctly sequenced recombinant plasmid was extracted with the endotoxin-free plasmid large-scale extraction kit and frozen at -20°C for later use.

Example 2

Expression and purification of 1G4113.

The above five kinds of plasmids containing different forms of 1G4113 (wherein 1G4113-1 is a plasmid, and 1G4113-2~5 include two plasmids for expressing two subunits respectively) were transfected into 293F cells for 4 days, 4000g, Centrifuge for 15 minutes, take the supernatant, filter it through a 0.45 μm filter membrane, and use Purification instrument and HiTrap Protein A HP prepacked column (purchased from GE, catalog number: 17-0403-01) for purification. Purification steps: HiTrap Protein A HP prepacked column was equilibrated with 100% Protein A loading buffer (50mM Tris-NaCl, 150mM NaCl, pH=7.4, filtered through a 0.45μm filter) and started to inject samples. After the sample injection, wash the prepacked column with 100% Protein A loading buffer until the unbound impurities are removed; finally use 100% Protein A elution buffer (50mM citric acid, pH=3.2, 0.45μm filter membrane filter) to collect the target protein. Different forms of 1G4113 were ultrafiltered with ultrafiltration membranes with a pore size of 30kDa (purchased from Millipore, product number: UF503096), and after the solvent was replaced with Tris-NaCl buffer, the protein concentration was measured with Nanodrop ND-1000, and then frozen. Store at -80°C for later use.

Expressed in 293F cells and purified to 1G4113-4 (the expression level is about 6 mg/L, the amino acid sequences of the two subunits are respectively SEQ ID No.10 (short chain) and SEQ ID No.11 (long chain)), 1G4113 -5 (the expression level is about 20mg/L, and the amino acid sequences of the two subunits are SEQ ID No.12 and SEQ ID No.13 respectively), while the other three 1G4113 failed to express in 293F. The Fc carboxy terminus of 1G4113-4 and 1G4113-5 introduced the G ₄ S-LPETGG sequence for the subsequent catalytic coupling of sortase A (srtA); the mutation was introduced into the Fc of 1G4113-5, and the knob-into-hole technique was used to achieve Facilitates the formation of heterodimers.

Example 3

T2 cells validate the specificity and affinity of 1G4113.

T2 cells (purchased from ATCC and cultured in IMDM medium containing 20% serum) are TAP-deficient cell lines, and only empty HLA-A*0201 molecules exist on the cell surface. Form a specific pMHC complex for subsequent detection.

Collect the T2 cells in the logarithmic growth phase, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Wash twice with serum-free RPMI-1640 medium. Resuspend in serum-free IMDM medium, spread 5×10 ⁵ cells/well on 12-well plate, 1 mL/well. Add peptide (synthesized by Hefei Guopeptide Biotechnology Co., Ltd., purity>95%) to a final concentration of 25 μg/mL, and β2m (purchased from Sigma, catalog number: M4890) to a final concentration of 5 μg/mL. After mixing evenly, place in a constant temperature incubator at 37° C., 5% CO ₂ , and saturated humidity to incubate for 6 hours.

After the incubation, the cells were collected by centrifugation at 500 g for 5 min, and washed once with stain buffer (PBS solution containing 0.5% BSA, 2 mmol/L EDTA). Add 200 μL stain buffer and block at 4°C for 30 min, then centrifuge to discard the stain buffer. Add 200 μL of 1G4113 antibody (5 μg/mL dissolved in stain buffer), and incubate at 4°C for 30 min. Wash three times with stain buffer, add 200 μL FITC-labeled goat anti-human IgG antibody (purchased from Beyontine Biotechnology Co., Ltd., catalog number: A0556, diluted 1:200 in stain buffer), and incubate at 4°C for 30 min. Wash three times with stain buffer, add 400 μL stain buffer to resuspend the cells, and measure the intensity of FITC with ACEA NovoCyte ^TM flow cytometer.

After co-incubating T2 cells with β2m and polypeptides (NY-ESO-1 _157-165 polypeptides as the experimental group, RMFPNAPYL, an irrelevant polypeptide derived from WT1 protein as the negative control, and unincubated polypeptides as the blank control), by flow cytometry The verification found that T2 cells were incubated with NY-ESO-1 _157-165 polypeptide and detected with 1G4113-4 or 1G4113-5, and the average fluorescence intensity of FITC was significantly shifted compared with the blank control; T2 cells were incubated with WT1 polypeptide and detected with 1G4113-4 or 1G4113- 5 detection, the average fluorescence intensity of FITC did not shift relative to the blank control (Figure 2), indicating that 1G4113-4 or 1G4113-5 maintained a certain affinity for the target HLA-A2/NY-ESO-1 _157-165 , It has no affinity for HLA-A2/WT1 complex, and two kinds of 1G4113 can specifically recognize target HLA-A2/NY-ESO-1 _157-165 .

Example 4

Identification and construction of HLA-A2/NY-ESO-1 _157-165 positive cell lines.

1) Identification of NY-ESO-1 and HLA-A*02 expression in K562 and A375 cells

The mRNA of K562 and A375 cells was extracted and reverse transcribed into cDNA. Design primers to verify whether the two cells express NY-ESO-1. Primers (5' to 3'): NY-ESO-1-up: GCTTGAGTTTCTACCTCGCCA, NY-ESO-1-down: AGCCAAAAACACGGGCAGAA. After the PCR product was recovered by gel, it was sent to Sangon Biotechnology Co., Ltd. for sequencing to further verify whether it was a NY-ESO-1 fragment.

Collect A375 cells in the logarithmic growth phase, and use an antibody targeting HLA-A*02 (BB7.2, purchased from ThermoFisher, catalog number: MA1-10344) to detect whether A375 expresses HLA-A*02 on a flow cytometer . The incubation concentration of BB7.2 was referred to the instruction manual, and FITC-labeled goat anti-mouse IgG antibody (purchased from Beyond Biotechnology Co., Ltd., item number: A0568) was selected as the detection antibody.

A375 and K562-A2 cells in the logarithmic growth phase were collected, and 1G4113-5 was used to detect whether the two cells expressed HLA-A2/NY-ESO-1 _157-165 on a flow cytometer. A PE-labeled goat anti-human IgG antibody (purchased from China, Cat. No.: 12-4998-82) was selected as the detection antibody.

It was verified at the RNA level that K562 cells do not express NY-ESO-1, and A375 expresses NY-ESO-1; using the BB7.2 antibody specifically targeting HLA-A*02, A375 cells were determined to be HLA-A*02 positive cell line. HLA-A*0201 was stably transfected in K562 cells, and the HLA-A*02-positive K562-A2 cell line was constructed, so A375 cells with NY-ESO-1 and HLA-A*02 double-positive, NY- ESO-1 and HLA-A*02 double negative K562 cells, NY-ESO-1 negative and HLA-A*02 positive K562-A2 cells (Table 2). 1G4113-5 was used to verify the expression of the HLA-A2/NY-ESO-1 _157-165 complex on the surface of the above three types of cells, and it was found that K562 and K562-A2 were HLA-A2/NY-ESO-1 _157-165 negative, and The theory agrees; however the HLA-A2/NY-ESO-1 _157-165 complex was also not detected by A375 (Table 2).

2) Construction of HLA-A2/NY-ESO-1 _157-165 positive cell lines

The fusion expression of ubiquitin (Ub) and polypeptide was used to construct cell lines with high expression of antigen presenting. EcoR I-EGFP-Ub-NY-ESO-1 _157-165 -Not I gene was synthesized by Sangon Biotechnology Co., Ltd. After the gene and pMHC vectors were digested by EcoR I and Not I, they were ligated by T4 ligase and transfected into DH5α competent cells. Single clone colonies were picked for sequencing verification, and endotoxin-free plasmids were extracted from clones with correct sequencing. The plasmid was transfected into A375 cells and K562-A2 cells. The day after transfection, discard the old medium and add fresh medium containing 800 μg/mL G418 sulfate. After the G418 drug-resistant cells fill the culture vessel, take about 100 cells and evenly spread them on a 96-well cell culture plate, select monoclonal cells, and use the green fluorescent signal of EGFP and 1G4113-5 to screen for stable expression of EGFP by flow cytometry - The cell lines of Ub-peptide are reserved (the cell lines are named A375-NY-ESO-1 _157-165 and K562-A2-NY-ESO-1 _157-165 ).

EcoR V-signal peptide-NY-ESO-1 _157-165 -(G ₄ S) ₃ -β2m-(G ₄ S) ₄ -(HLA-A*0201)-Xho I Gene Sangong Biotechnology Co., Ltd. synthesized and Inserted into pCDNA3.1-hygro(+) plasmid. The endotoxin-free plasmid was extracted and transfected into K562 cells. The day after transfection, discard the old medium and add fresh medium containing 250 μg/mL hygromycin B. After the hygromycin B-resistant cells covered the culture vessel, about 100 cells were taken and spread evenly on a 96-well cell culture plate, and monoclonal cells were selected, and 1G4113-5 was used to screen for stably expressing NY-ESO- 1 _157-165 -(G ₄ S) ₃ -β2m-(G ₄ S) ₄ -(HLA-A*0201) cell line (the cell line is named K562-NY-ESO-1 _157-165 pMHC). The expression of HLA-A2/NY-ESO-1 _157-165 complex of K562-NY-ESO-1 _157-165 pMHC was about 12.8 times higher than that of K562-A2-NY-ESO-1 _157-165 (Table 2) .

Table 2 NY-ESO-1 _157-165 , HLA-A*02 expression and 1G4113-5 affinity analysis of different cell lines.

cell line NY-ESO-1_157-165 HLA-A*02 1G4113-5 Affinity Strength K562 feminine feminine feminine K562-A2 feminine positive feminine K562-A2-NY-ESO-1_157-165 positive positive 4.67 K562-NY-ESO-1_157-165pMHC positive positive 59.70 A375 positive positive not detected A375-NY-ESO-1_157-165 positive positive 10.17

Example 5

Preparation of 1G4113-vcMMAE.

GGG-vcMMAE (synthesized by Lianning (Suzhou) Biopharmaceutical Co., Ltd.) was coupled to the carboxyl terminus of 1G4113 via srt A catalysis. The coupling method is the same as the preparation of 1G4113-biotin. After coupling for 12 hours at 37°C, 100 μL was taken, filtered through a 0.22 μm filter membrane, and analyzed by RP-HPLC. The concentration of purified 1G4113-vcMMAE was adjusted to 1 mg/mL with PBS. Take 100 μL, filter through a 0.22 μm filter membrane, and perform RP-HPLC and HIC-HPLC analysis.

RP-HPLC analysis method is as follows: chromatographic column: PLRP-S ( 8μm, 2.1×150mm). Flow rate: 0.6mL/min. Column temperature: 50°C. Injection volume: 30 μL.

The HIC-HPLC analysis method is as follows: Chromatographic column: TSKgel Butyl-NPR. Flow rate: 0.8mL/min. Column temperature: 25°C. Injection volume: 10 μL. Solution A: 25mM sodium phosphate, 2M ammonium sulfate, pH=7.0, solution B: 25mM sodium phosphate, pH=7.0.

The uncoupled free small molecules, srt A, etc. were purified by HiTrap Protein A HP prepacked column, and the 1G4113-vcMMAE coupling product was isolated, and the DAR was further analyzed by TOSOH Butyl-NPR HPLC and RP-HPLC. 1G4113-4-vcMMAE contains two components, namely DAR=1 and DAR=2, with an average DAR≈1.6; 1G4113-5-vcMMAE has a small amount of DAR in addition to the two components of DAR=1 and DAR=2 = 0, the average DAR≈1.4 (Figure 3). From the analysis results of TOSOH Butyl-NPR HPLC, it was found that the DAR=1 component of 1G4113-5-vcMMAE contained two peaks. In addition, 1G4113-4 and 1G4113-5 had only one peak in the analysis of TOSOH Butyl-NPR HPLC and RP-HPLC, which also indicated that the purified soluble 1G4113 had a higher purity.

Example 6

Affinity verification of 1G4113-vcMMAE.

The affinity and endocytosis of ADC to tumor cell surface antigens are the key steps to exert its activity, so it is necessary to verify the affinity and endocytosis efficiency of 1G4113-vcMMAE.

First, the affinity of 1G4113 and 1G4113-vcMMAE was verified on A375-NY-ESO-1 _157-165 cells by flow cytometry, and PE-labeled goat anti-human IgG antibody was used as the detection antibody. It was found that within the detected concentration range, the affinity of 1G4113 and 1G4113-vcMMAE to A375-NY-ESO-1 _157-165 cells was concentration-dependent, and the higher the concentration, the more molecules bound to the cell surface (Figure 4). When 1G4113 was coupled with MMAE small molecules, the affinity decreased significantly, about 1/2 of that of unconjugated (Figure 4).

The endocytosis of 1G4113 and 1G4113-vcMMAE was detected qualitatively by fluorescent confocal method. K562-NY-ESO-1 _157-165 pMHC was spread at 1×10 ⁴ per well in a 24-well cell culture plate containing a clean 6×6mm round cover glass. On the next day, two kinds of 1G4113 and its conjugates were added respectively to a final concentration of 5 μg/mL, and cultured at 37° C., 5% CO ₂ for 4 hours. Discard the supernatant, gently add PBS to wash twice, fix the cells with 4% paraformaldehyde and punch with 0.1% Triton X-100. After blocking with 2% BSA, rabbit anti-LAMP1 antibody (purchased from abcam, product number: EPR4204) was diluted 1:1000 in PBS buffer containing 1% BSA, and incubated at room temperature for 30 min in 200 μL. PBS washed 3 times, Cy3-labeled goat anti-rabbit IgG (purchased from Beyond Biotechnology Co., Ltd., product number: A0516) and FITC-labeled goat anti-human IgG antibody 1:200 were co-diluted in PBS buffer containing 1% BSA, 200 μL was incubated at room temperature in the dark for 30 min. After washing with PBS for 3 times, DAPI staining and mounting were performed. The cells after fluorescent staining were observed and imaged and recorded under the DU-897D-CS0 spinning disk confocal microscope. The results showed that both 1G4113 and 1G4113-vcMMAE could observe obvious FITC fluorescence in the cells, indicating that both of them could be endocytosed by K562-NY-ESO-1 _157-165 pMHC cells. Part of the FITC fluorescence overlapped with Cy3 fluorescence, indicating that 1G4113 and 1G4113-vcMMAE endocytosed into cells could be transported to lysosomes for degradation and release of small MMAE molecules (Figure 4).

In order to further determine the endocytosis efficiency of 1G4113 and 1G4113-vcMMAE, the endocytosis of 1G4113-vcMMAE was quantitatively detected by flow cytometry. Take 5×10 ⁵ K562-NY-ESO-1 _157-165 pMHC cells, collect the cells by centrifugation at 500 g for 5 min, and wash twice with PBS. Two kinds of 1G4113 and its conjugates at 5 μg/mL were incubated at 4°C for 30 min (2 copies for each group, 3 replicates for each copy), washed with PBS for 3 times, one of them was bathed in water at 37°C for 2 hours, and the other was placed in an ice bath ( 4°C) 2h. FITC-labeled goat anti-human IgG was diluted 1:200 in PBS, 200 μL, and incubated at 4°C for 30 min. After washing with PBS for 3 times, the cells were resuspended in 400 μL of PBS, and the MFI value was detected by ACEANovoCyte ^TM flow cytometer. Endocytosis efficiency (%)=(average MFI at 4°C-average MFI at 37°C)/average MFI at 4°C×100%. It was found that the endocytic efficiency of 1G4113 was about 40%. After coupling MMAE, although the affinity of 1G4113 decreased, the endocytic efficiency increased instead, indicating that coupling MMAE at the carboxy-terminus of 1G4113 by srtA catalysis can effectively increase the endocytic efficiency (Figure 4).

Example 7

In vitro antitumor activity of 1G4113-vcMMAE.

Tumor cells were plated in 96-well cell culture plates according to 3000-5000 cells/well, and drugs of different concentrations were incubated (3 replicates for each concentration, 0 ng/mL was the control group). After culturing for 96 hours at 37°C in 5% CO ₂ , add CCK-8 to a final concentration of 10%, incubate at 37°C in 5% CO ₂ for 0.5-2 h, and use BioRad Model 680 Microplate Reader to detect the OD ₄₅₀ value. According to the formula: OD ₄₅₀ value of different concentrations of wells/average value of OD ₄₅₀ of the control group × 100%, the survival rate of cells in each well was calculated, and according to the survival rate, the IC ₅₀ was calculated by using GraphPad Prim 6.01 software (IC ₅₀ means that the cell survival rate is 50 % drug concentration).

The results showed that although the killing activity of the two 1G4113-vcMMAEs on A375-NY-ESO-1 _157-165 cells was better than that on A375 cells, both of them had poor activities (IC ₅₀ >20 μg/mL, Figure 5 ). The expression of HLA-A2/NY-ESO-1 _157-165 complex was not detected on the surface of A375 cells, but because of its NY-ESO-1 and HLA-A*02 double positive, it is speculated that the surface of A375 slightly expresses HLA-A2/ NY-ESO-1 _157-165 complex, expression levels below the detection limit of flow cytometry. The weak toxicity of 1G4113-vcMMAE to A375 cells in vitro may be mediated by its micro-expressed targets on the cell surface. Since the expression of HLA-A2/NY-ESO-1 _157-165 complex in A375-NY-ESO-1 _157-165 cells is much higher than that in A375 cells, it is speculated that 1G4113-vcMMAE has an effect on A375-NY-ESO-1 _157-165 The difference in activity between cells and A375 cells was caused by the difference in expression of HLA-A2/NY-ESO-1 _157-165 complex.

In order to further verify the effect of antigen expression on the anti-tumor activity of 1G4113-vcMMAE in vitro, K562-A2 cells, K562-A2-NY-ESO-1 _157-165 cells and K562-NY-ESO-1 _157-165 pMHC cells were homogeneously were spread in 96-well cell culture plates, and a series of concentrations of 1G4113-vcMMAE were added. After 4 days of drug treatment, CCK-8 was used to detect the activity of cells in each well, and the relative survival rate of cells in the administration group was calculated. Since the three kinds of cells used in this part of the experiment are all stable cell lines derived from K562 cells, except for the difference in the expression of the HLA-A2/NY-ESO-1 _157-165 complex, the cell background is the same, which is more conducive to 1G4113-vcMMAE activity comparison in vitro. It was found that 1G4113-vcMMAE was negative for HLA-A2/NY-ESO-1 _157-165 complex K562-A2 cells and HLA-A2/NY-ESO-1 _157-165 complex low expression K562-A2-NY -ESO-1 _157-165 cells have no significant activity (IC ₅₀ >10μg/mL), express higher K562-NY-ESO-1 _157-165 pMHC to HLA-A2/NY-ESO-1 _157-165 complex Cells were significantly viable (Fig. 5). The differences in the activity of 1G4113-vcMMAE on the three stable cell lines derived from K562 indicated that it had specific killing activity on antigen-positive cells and was positively correlated with the expression level of the active antigen.

sequence listing

<110> Zhejiang University

<120> A recombinant antibody-like T cell antigen receptor, T cell antigen receptor conjugated drug and its application

<160> 46

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2199

<212>DNA

<213> Artificial Sequence

<400> 1

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

caggaggtga cccagatccc cgccgccctg agcgtgcccg agggcgagaa cctggtgctg 120

aactgcagct tcaccgacag cgccatctac aacctgcagt ggttccgcca ggaccccggc 180

aagggactga cctccctgct gctgatcacc ccctggcaga gggagcagac cagcggcaga 240

ctgaacgcca gcctggacaa gagcagcggc agaagcaccc tgtacatcgc cgccagccag 300

cccggcgaca gcgccaccta cctgtgcgcc gtgcggcccc tgctcgacgg cacctacatc 360

cccaccttcg ggcgcggcac cagcttgatc gtgcacccct acggtggtgg tggtagcggc 420

ggcggcggct ctggtggtgg tggatccatg ggcgtgacac agacacctaa atttcaggtg 480

ctgaagacag gccagagtat gacactgcag tgtgcccagg atatgaatca tgagtacatg 540

tcttggtata gacaggaccc cggcatgggc ctgagactga tccactacag cgtggccatc 600

cagaccaccg acaggcga ggtgcccaac ggctacaacg tgagcagatc caccatcgag 660

gacttccccc tgcggctgct gagcgccgcc cccagccaga ccagcgtgta cttctgcgcc 720

agcagctacc tgggcaacac cggcgagctg ttcttcggcg agggcagcag actgaccgtg 780

ctggaggacc tgaagaacgt gttccccccc gaggtggccg tgttcgagcc cagcgaggcc 840

gagatcagcc acacccagaa ggccaccctg gtgtgcctgg ccaccggctt ctaccccgac 900

cacgtggagc tgagctggtgggtgaacggc aaggaggtgc acagcggcgt gagcaccgac 960

ccccagcccc tgaaggagca gcccgccctg aacgacagca ggtacgccct gagcagccgc 1020

ctgagagtgt ccgccacctt ctggcaggac cccaggaacc acttcagatg ccaggtgcag 1080

ttctacggcc tgagcgagaa cgacgagtgg accccaggaca gggccaagcc cgtgacccag 1140

atcgtgagcg ccgaggcctg gggcagagcc gacgctagca ccaagggccc atcggtcttc 1200

cccctggcac cctcctccaa gagcacctct gggggcacag cggccctggg ctgcctggtc 1260

aaggactact tccccgaacc ggtgacggtg tcgtggaact caggcgccct gaccagcggc 1320

gtgcacacct tcccggctgt cctacagtcc tcaggactct actccctcag cagcgtggtg 1380

accgtgccct ccagcagctt gggcacccag acctacatct gcaacgtgaa tcacaagccc 1440

agcaacacca aggtggacaa gaaagttgag cccaaatctt gtgacaaaac tcacacatgc 1500

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa 1560

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 1620

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 1680

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 1740

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 1800

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1860

caggtgtaca ccctgccccc atcccggggag gagatgacca agaaccaggt cagcctgacc 1920

tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1980

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 2040

tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 2100

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 2160

aaaggcggtg gaggctctct gccggagact ggtggttaa 2199

<210> 2

<211> 732

<212>DNA

<213> Artificial Sequence

<400> 2

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

caggaggtga cccagatccc cgccgccctg agcgtgcccg agggcgagaa cctggtgctg 120

aactgcagct tcaccgacag cgccatctac aacctgcagt ggttccgcca ggaccccggc 180

aagggactga cctccctgct gctgatcacc ccctggcaga gggagcagac cagcggcaga 240

ctgaacgcca gcctggacaa gagcagcggc agaagcaccc tgtacatcgc cgccagccag 300

cccggcgaca gcgccaccta cctgtgcgcc gtgcggcccc tgctcgacgg cacctacatc 360

cccaccttcg ggcgcggcac cagcttgatc gtgcacccct acggttctcg tacggtggct 420

gcaccatctg tcttcatctt cccgccatct gatgagcagt tgaaatctgg aactgcctct 480

gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg gaaggtggat 540

aacgccctcc aatcgggtaa ctcccaggag agtgtcacag agcaggacag caaggacagc 600

acctacagcc tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc 660

tacgcctgcg aagtcaccca tcagggcctg agctcgcccg tcacaaagag cttcaacagg 720

ggagagtgtt aa 732

<210> 3

<211> 1425

<212>DNA

<213> Artificial Sequence

<400> 3

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

ggcgtgacac agacacctaa atttcaggtg ctgaagacag gccagagtat gacactgcag 120

tgtgcccagg atatgaatca tgagtacatg tcttggtata gacaggaccc cggcatgggc 180

ctgagactga tccactacag cgtggccatc cagaccaccg acagaggcga ggtgcccaac 240

ggctacaacg tgagcagatc caccatcgag gacttccccc tgcggctgct gagcgccgcc 300

cccagccaga ccagcgtgta cttctgcgcc agcagctacc tgggcaacac cggcgagctg 360

ttcttcggcg agggcagcag actgaccgtg ctgggttctg ctagcaccaa gggcccatcg 420

gtcttccccc tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc 480

ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 540

agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc 600

gtggtgaccg tgccctccag cagcttgggc accccagacct acatctgcaa cgtgaatcac 660

aagcccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtga caaaactcac 720

acatgcccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 780

ccaaaaccca aggacacccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 840

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 900

cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 960

gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1020

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1080

gaaccacagg tgtacacccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 1140

ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1200

gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1260

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1320

tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1380

ccgggtaaag gcggtggagg ctctctgccg gagactggtg gttaa 1425

<210> 4

<211> 1014

<212>DNA

<213> Artificial Sequence

<400> 4

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

caggaggtga cccagatccc cgccgccctg agcgtgcccg agggcgagaa cctggtgctg 120

aactgcagct tcaccgacag cgccatctac aacctgcagt ggttccgcca ggaccccggc 180

aagggactga cctccctgct gctgatcacc ccctggcaga gggagcagac cagcggcaga 240

ctgaacgcca gcctggacaa gagcagcggc agaagcaccc tgtacatcgc cgccagccag 300

cccggcgaca gcgccaccta cctgtgcgcc gtgcggcccc tgctcgacgg cacctacatc 360

cccaccttcg ggcgcggcac cagcttgatc gtgcacccct acatccagaa ccccgacccc 420

gccgtgtacc agctgcggga tagcaagagc agcgataagt ccgtgtgcct gttcaccgac 480

ttcgacagcc agaccaacgt gagccagagc aaggacagcg acgtgtacat caccgacaag 540

accgtgctgg acatgagatc catggacttc aagtccaaca gcgccgtggc ctggagcaac 600

aagagcgact tcgcctgcgc caatgccttt aacaatagca ttatccctga ggatacattt 660

ttccctagcc ctgaatctag ctgtggttct cgtacggtgg ctgcaccatc tgtcttcatc 720

ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 780

aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 840

aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 900

accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 960

catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttaa 1014

<210> 5

<211> 1818

<212>DNA

<213> Artificial Sequence

<400> 5

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

ggcgtgacac agacacctaa atttcaggtg ctgaagacag gccagagtat gacactgcag 120

tgtgcccagg atatgaatca tgagtacatg tcttggtata gacaggaccc cggcatgggc 180

ctgagactga tccactacag cgtggccatc cagaccaccg acagaggcga ggtgcccaac 240

ggctacaacg tgagcagatc caccatcgag gacttccccc tgcggctgct gagcgccgcc 300

cccagccaga ccagcgtgta cttctgcgcc agcagctacc tgggcaacac cggcgagctg 360

ttcttcggcg agggcagcag actgaccgtg ctggaggacc tgaagaacgt gttccccccc 420

gaggtggccg tgttcgagcc cagcgaggcc gagatcagcc acacccagaa ggccaccctg 480

gtgtgcctgg ccaccggctt ctaccccgac cacgtggagc tgagctggtg ggtgaacggc 540

aaggaggtgc acagcggcgt gagcaccgac ccccagcccc tgaaggagca gcccgccctg 600

aacgacagca ggtacgccct gagcagccgc ctgagagtgt ccgccacctt ctggcaggac 660

cccaggaacc acttcagatg ccaggtgcag ttctacggcc tgagcgagaa cgacgagtgg 720

accccaggaca gggccaagcc cgtgacccag atcgtgagcg ccgaggcctg gggcagagcc 780

gactgtggtt ctgctagcac caagggccca tcggtcttcc ccctggcacc ctcctccaag 840

agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 900

gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 960

ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 1020

ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacccaa ggtggacaag 1080

aaagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 1140

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 1200

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1260

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcggggag 1320

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1380

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1440

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1500

tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1560

cccagcgaca tcgccgtgga gtgggagc aatgggcagc cggagaacaa ctacaagacc 1620

acgcctcccg tgctggactc cgacggctcc ttcttcctct acaagcaagct caccgtggac 1680

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1740

aacccataca cgcagaagag cctctccctg tctccgggta aaggcggtgg aggctctctg 1800

ccggagactg gtggttaa 1818

<210> 6

<211> 1011

<212>DNA

<213> Artificial Sequence

<400> 6

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

caggaggtga cccagatccc cgccgccctg agcgtgcccg agggcgagaa cctggtgctg 120

aactgcagct tcaccgacag cgccatctac aacctgcagt ggttccgcca ggaccccggc 180

aagggactga cctccctgct gctgatcacc ccctggcaga gggagcagac cagcggcaga 240

ctgaacgcca gcctggacaa gagcagcggc agaagcaccc tgtacatcgc cgccagccag 300

cccggcgaca gcgccaccta cctgtgcgcc gtgcggcccc tgctcgacgg cacctacatc 360

cccaccttcg ggcgcggcac cagcttgatc gtgcacccct acatccagaa ccccgacccc 420

gccgtgtacc agctgcggga tagcaagagc agcgataagt ccgtgtgcct gttcaccgac 480

ttcgacagcc agaccaacgt gagccagagc aaggacagcg acgtgtacat caccgacaag 540

accgtgctgg acatgagatc catggacttc aagtccaaca gcgccgtggc ctggagcaac 600

aagagcgact tcgcctgcgc caatgccttt aacaatagca ttatccctga ggatacattt 660

ttccctagcc ctgaatctag cggttctcgt acggtggctg caccatctgt cttcatcttc 720

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 780

ttctatccca gagaggccaa aagtacagtgg aaggtggata acgccctcca atcgggtaac 840

tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 900

ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 960

cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta a 1011

<210> 7

<211> 1815

<212>DNA

<213> Artificial Sequence

<400> 7

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

ggcgtgacac agacacctaa atttcaggtg ctgaagacag gccagagtat gacactgcag 120

tgtgcccagg atatgaatca tgagtacatg tcttggtata gacaggaccc cggcatgggc 180

ctgagactga tccactacag cgtggccatc cagaccaccg acagaggcga ggtgcccaac 240

ggctacaacg tgagcagatc caccatcgag gacttccccc tgcggctgct gagcgccgcc 300

cccagccaga ccagcgtgta cttctgcgcc agcagctacc tgggcaacac cggcgagctg 360

ttcttcggcg agggcagcag actgaccgtg ctggaggacc tgaagaacgt gttccccccc 420

gaggtggccg tgttcgagcc cagcgaggcc gagatcagcc acacccagaa ggccaccctg 480

gtgtgcctgg ccaccggctt ctaccccgac cacgtggagc tgagctggtg ggtgaacggc 540

aaggaggtgc acagcggcgt gagcaccgac ccccagcccc tgaaggagca gcccgccctg 600

aacgacagca ggtacgccct gagcagccgc ctgagagtgt ccgccacctt ctggcaggac 660

cccaggaacc acttcagatg ccaggtgcag ttctacggcc tgagcgagaa cgacgagtgg 720

accccaggaca gggccaagcc cgtgacccag atcgtgagcg ccgaggcctg gggcagagcc 780

gacggttctg ctagcaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 840

acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 900

acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 960

cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 1020

accccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 1080

gttgagccca aatcttgtga caaaactcac acatgccccac cgtgcccagc acctgaactc 1140

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacacccct catgatctcc 1200

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 1260

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 1320

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 1380

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1440

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1500

cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1560

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1620

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1680

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1740

cactacacgc agaagagcct ctccctgtct ccgggtaaag gcggtggagg ctctctgccg 1800

gagactggtg gttaa 1815

<210> 8

<211> 1419

<212>DNA

<213> Artificial Sequence

<400> 8

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

caggaggtga cccagatccc cgccgccctg agcgtgcccg agggcgagaa cctggtgctg 120

aactgcagct tcaccgacag cgccatctac aacctgcagt ggttccgcca ggaccccggc 180

aagggactga cctccctgct gctgatcacc ccctggcaga gggagcagac cagcggcaga 240

ctgaacgcca gcctggacaa gagcagcggc agaagcaccc tgtacatcgc cgccagccag 300

cccggcgaca gcgccaccta cctgtgcgcc gtgcggcccc tgctcgacgg cacctacatc 360

cccaccttcg ggcgcggcac cagcttgatc gtgcacccct acatccagaa ccccgacccc 420

gccgtgtacc agctgcggga tagcaagagc agcgataagt ccgtgtgcct gttcaccgac 480

ttcgacagcc agaccaacgt gagccagagc aaggacagcg acgtgtacat caccgacaag 540

accgtgctgg acatgagatc catggacttc aagtccaaca gcgccgtggc ctggagcaac 600

aagagcgact tcgcctgcgc caatgccttt aacaatagca ttatccctga ggatacattt 660

ttccctagcc ctgaatctag ctgtggttct cccaaatctt gtgacaaaac tcacacatgc 720

ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa 780

cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 840

agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 900

gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 960

accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 1020

gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1080

caggtgtaca ccctgccccc atcccgggag gagatgacca agaaccaggt cagcctgtcc 1140

tgcgcggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1200

ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1260

gtcagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1320

gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccgggt 1380

aaaggcggtg gaggctctct gccggagact ggtggttaa 1419

<210> 9

<211> 1521

<212>DNA

<213> Artificial Sequence

<400> 9

atggactgga cctggcggat cctgttcctg gtggccgccg ccaccggcgc ccactccatg 60

ggcgtgacac agacacctaa atttcaggtg ctgaagacag gccagagtat gacactgcag 120

tgtgcccagg atatgaatca tgagtacatg tcttggtata gacaggaccc cggcatgggc 180

ctgagactga tccactacag cgtggccatc cagaccaccg acagaggcga ggtgcccaac 240

ggctacaacg tgagcagatc caccatcgag gacttccccc tgcggctgct gagcgccgcc 300

cccagccaga ccagcgtgta cttctgcgcc agcagctacc tgggcaacac cggcgagctg 360

ttcttcggcg agggcagcag actgaccgtg ctggaggacc tgaagaacgt gttccccccc 420

gaggtggccg tgttcgagcc cagcgaggcc gagatcagcc acacccagaa ggccaccctg 480

gtgtgcctgg ccaccggctt ctaccccgac cacgtggagc tgagctggtg ggtgaacggc 540

aaggaggtgc acagcggcgt gagcaccgac ccccagcccc tgaaggagca gcccgccctg 600

aacgacagca ggtacgccct gagcagccgc ctgagagtgt ccgccacctt ctggcaggac 660

cccaggaacc acttcagatg ccaggtgcag ttctacggcc tgagcgagaa cgacgagtgg 720

accccaggaca gggccaagcc cgtgacccag atcgtgagcg ccgaggcctg gggcagagcc 780

gactgtggtt ctcccaaatc ttgtgacaaa actcacacat gcccaccgtg cccagcacct 840

gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 900

atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 960

gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 1020

gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1080

tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1140

gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc 1200

ccatcccggg aggagatgac caagaaccag gtcagcctgt ggtgcctggt caaaggcttc 1260

tatcccagcg acatcgccgt ggagtggggag agcaatgggc agccggagaa caactacaag 1320

accacgcctc ccgtgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1380

gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1440

cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaaggcgg tggaggctct 1500

ctgccggaga ctggtggtta a 1521

<210> 10

<211> 317

<212> PRT

<213> Artificial Sequence

<400> 10

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

1 5 10 15

Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn

20 25 30

Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu

35 40 45

Leu Ile Thr Pro Trp Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala

50 55 60

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

65 70 75 80

Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Pro Leu Leu

85 90 95

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

100 105 110

His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp

115 120 125

Ser Lys Ser Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser

130 135 140

Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp

145 150 155 160

Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala

165 170 175

Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn

180 185 190

Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315

<210> 11

<211> 585

<212> PRT

<213> Artificial Sequence

<400> 11

Met Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln

1 5 10 15

Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser

20 25 30

Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser

35 40 45

Val Ala Ile Gln Thr Thr Asp Arg Gly Glu Val Pro Asn Gly Tyr Asn

50 55 60

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

65 70 75 80

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

85 90 95

Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu

100 105 110

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

115 120 125

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

130 135 140

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

145 150 155 160

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

165 170 175

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu

180 185 190

Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Gly Gly Ser Leu Pro Glu Thr Gly Gly

580 585

<210> 12

<211> 453

<212> PRT

<213> Artificial Sequence

<400> 12

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

1 5 10 15

Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn

20 25 30

Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu

35 40 45

Leu Ile Thr Pro Trp Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala

50 55 60

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

65 70 75 80

Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Pro Leu Leu

85 90 95

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

100 105 110

His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp

115 120 125

Ser Lys Ser Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser

130 135 140

Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp

145 150 155 160

Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala

165 170 175

Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn

180 185 190

Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Leu

435 440 445

Pro Glu Thr Gly Gly

450

<210> 13

<211> 487

<212> PRT

<213> Artificial Sequence

<400> 13

Met Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln

1 5 10 15

Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser

20 25 30

Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser

35 40 45

Val Ala Ile Gln Thr Thr Asp Arg Gly Glu Val Pro Asn Gly Tyr Asn

50 55 60

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

65 70 75 80

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

85 90 95

Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu

100 105 110

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

115 120 125

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

130 135 140

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

145 150 155 160

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

165 170 175

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu

180 185 190

Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

435 440 445

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

450 455 460

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

465 470 475 480

Ser Leu Pro Glu Thr Gly Gly

485

<210> 14

<211> 990

<212>DNA

<213> Artificial Sequence

<400> 14

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720

atgaccaaga accagtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa 990

<210> 15

<211> 321

<212>DNA

<213> Artificial Sequence

<400> 15

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtgt 321

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 16

Ser Leu Leu Met Trp Ile Thr Gln Cys

1 5

<210> 17

<211> 11

<212> PRT

<213> Artificial Sequence

<400> 17

Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly

1 5 10

<210> 18

<211> 50

<212>DNA

<213> Artificial Sequence

<400> 18

gaattcacca ccatggactg gacctggcgg atcctgttcc tggtggccgc 50

<210> 19

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 19

atcctgttcc tggtggccgc cgccaccggc gcccactcca tgcaggaggt gacccagat 59

<210> 20

<211> 53

<212>DNA

<213> Artificial Sequence

<400> 20

accagagccg ccgccgccgc taccaccacc accgtagggg tgcacgatca agc 53

<210> 21

<211> 53

<212>DNA

<213> Artificial Sequence

<400> 21

agcggcggcg gcggctctgg tggtggtgga tccatgggcg tgacacagac acc 53

<210> 22

<211> 40

<212>DNA

<213> Artificial Sequence

<400> 22

gatgggccct tggtgctagc gtcggctctg ccccaggcct 40

<210> 23

<211> 40

<212>DNA

<213> Artificial Sequence

<400> 23

aggcctgggg cagagccgac gctagcacca agggcccatc 40

<210> 24

<211> 58

<212>DNA

<213> Artificial Sequence

<400> 24

gcggccgctt aaccaccagt ctccggcaga gagcctccac cgcctttacc cggagaca 58

<210> 25

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 25

gatggtgcag ccaccgtacg agaaccgtag gggtgcacga tcaagc 46

<210> 26

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 26

gcttgatcgt gcacccctac ggttctcgta cggtggctgc accatc 46

<210> 27

<211> 31

<212>DNA

<213> Artificial Sequence

<400> 27

gcggccgctt aacactctcc cctgttgaag c 31

<210> 28

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 28

atcctgttcc tggtggccgc cgccaccggc gcccactcca tgggcgtgac acagacacc 59

<210> 29

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 29

gatgggccct tggtgctagc agaacccagc acggtcagtc tgctgc 46

<210> 30

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 30

gcagcagact gaccgtgctg ggttctgcta gcaccaaggg cccatc 46

<210> 31

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 31

gatggtgcag ccaccgtacg agaaccacag ctagattcag ggctaggga 49

<210> 32

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 32

tccctagccc tgaatctagc tgtggttctc gtacggtggc tgcaccatc 49

<210> 33

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 33

gatgggccct tggtgctagc agaaccacag tcggctctgc cccaggcct 49

<210> 34

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 34

aggcctgggg cagagccgac tgtggttctg ctagcaccaa gggcccatc 49

<210> 35

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 35

gatggtgcag ccaccgtacg agaaccgcta gattcagggc taggga 46

<210> 36

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 36

tccctagccc tgaatctagc ggttctcgta cggtggctgc accatc 46

<210> 37

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 37

gatgggccct tggtgctagc agaaccgtcg gctctgcccc aggcct 46

<210> 38

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 38

aggcctgggg cagagccgac ggttctgcta gcaccaaggg cccatc 46

<210> 39

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 39

gttttgtcac aagatttggg agaaccacag ctagattcag ggctaggga 49

<210> 40

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 40

tccctagccc tgaatctagc tgtggttctc ccaaatcttg tgacaaaac 49

<210> 41

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 41

gttttgtcac aagatttggg agaaccacag tcggctctgc cccaggcct 49

<210> 42

<211> 49

<212>DNA

<213> Artificial Sequence

<400> 42

aggcctgggg cagagccgac tgtggttctc ccaaatcttg tgacaaaac 49

<210> 43

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 43

Arg Met Phe Pro Asn Ala Pro Tyr Leu

1 5

<210> 44

<211> 4

<212> PRT

<213> Artificial Sequence

<400> 44

Gly Gly Gly Lys

1

<210> 45

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 45

gcttgagttc tacctcgcca 20

<210> 46

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 46

agccaaaaac acgggcagaa 20

Claims (6)

1. a kind of recombinant antibodies sample T cell antigen receptor, which is characterized in that be the heterologous tetramer, including 2 long-chains and 2 short Chain, the long-chain include sequentially connected ECD β-IgG1 HC, and the short chain includes sequentially connected ECD α-LC, wherein ECD α It is respectively the α chain of T cell antigen receptor 1G4113 and the extracellular domain of β chain with ECD β, IgG1 HC and LC are respectively IgG1's Heavy chain constant region and constant region of light chain.

2. recombinant antibodies sample T cell antigen receptor as described in claim 1, which is characterized in that the amino acid sequence of the long-chain Column are as shown in SEQ ID No.11；The amino acid sequence of the short chain is as shown in SEQ ID No.10.

3. recombinant antibodies sample T cell antigen receptor as claimed in claim 2, which is characterized in that the coding of first subunit Gene order is as shown in SEQ ID No.7；The coding gene sequence of second subunit is as shown in SEQ ID No.6.

4. the recombinant antibodies sample T cell antigen receptor application in preparation of anti-tumor drugs as described in claims 1 to 3 is any.

5. a kind of T cell antigen coupled receptors drug, which is characterized in that in the recombinant antibodies sample as described in claims 1 to 3 is any The C-terminal of the long-chain of T cell antigen receptor and/or short chain is coupled upper vcMMAE by sortase A enzymatic transpeptidation reaction, Middle vc is two peptide linker of valine-citrulline, and MMAE is aplysiatoxin.

6. T cell antigen coupled receptors drug application in preparation of anti-tumor drugs as claimed in claim 5.