TCR, polypeptide, expression vector, host cell, pharmaceutical composition and method for obtaining TCR

Abstract

Disclosed in the present application is a T cell receptor (TCR) capable of specifically recognizes MAGE-A4 antigenic peptide, including a TCR Polypeptide having at least 90% identity with any one selected from a group consisting of sequences of SEQ ID No: 1 to SEQ ID No: 30 and a TCR polypeptide having at least 90% identity with any one selected from a group consisting of sequences of SEQ ID No: 31 to SEQ ID No: 60, in which the TCR polypeptides are in one-to-one correspondence with the TCR polypeptides in order.

Claims

1. A T cell receptor (TCR), comprising a TCR polypeptide comprising CDR1 having an amino acid sequence comprising SEQ ID No: 64, CDR2 having an amino acid sequence comprising SEQ ID No:94, and CDR3 having an amino acid sequence comprising SEQ ID No: 124; and a TCR polypeptide comprising CDR1 having an amino acid sequence comprising SEQ ID No: 154, CDR2 having an amino acid sequence comprising SEQ ID No: 184, and CDR3 having an amino acid sequence comprising SEQ ID No: 214, wherein the T cell receptor (TCR) further comprises a humanized C domain and/or a murine C domain and/or a modified humanized C domain, wherein the humanized C domain comprises SEQ ID No: 243 or 244, the murine C domain comprises SEQ ID No: 245 or 246, and the modified humanized C domain comprises SEQ ID No: 247 or 248.

2. The T cell receptor (TCR) according to claim 1, wherein the TCR polypeptide has an amino acid sequence comprising SEQ ID No: 4, and the TCR polypeptide has an amino acid sequence comprising SEQ ID No: 34.

3. A cell, comprising a recombinant expression vector containing a polynucleotide encoding a T cell receptor (TCR) operatively cloned therein, wherein the TCR comprises: a TCR polypeptide comprising CDR1 having an amino acid sequence comprising SEQ ID No: 64, CDR2 having an amino acid sequence comprising SEQ ID No:94, and CDR3 having an amino acid sequence comprising SEQ ID No: 124; and a TCR polypeptide comprising CDR1 having an amino acid sequence comprising SEQ ID No: 154, CDR2 having an amino acid sequence comprising SEQ ID No: 184, and CDR3 having an amino acid sequence comprising SEQ ID No: 214.

4. The cell according to claim 3, wherein the TCR polypeptide has an amino acid sequence comprising SEQ ID No: 4, and the TCR polypeptide has an amino acid sequence comprising SEQ ID No: 34.

5. The T cell receptor (TCR) according to claim 1, wherein the TCR is capable of specifically recognizing MAGE-A4 antigenic peptide and the MAGE-A4 antigenic peptide is HLA-A2 restricted.

6. The T cell receptor (TCR) according to claim 5, wherein the HLA-A2 is HLA-A*0201 typing.

7. The T cell receptor (TCR) according to claim 1, wherein the T cell receptor (TCR) further comprises a modified linker domain comprising SEQ ID No: 241 or 242.

8. The T cell receptor (TCR) according to claim 1, wherein the humanized C domain or the murine C domain or the modified humanized C domain linked to the TCR polypeptide and the humanized C domain or the murine C domain or the modified humanized C domain linked to the TCR polypeptide are of the same source.

9. The cell according to claim 3, wherein the recombinant expression vector comprises a viral vector.

10. The cell according to claim 9, wherein the viral vector comprises a retroviral vector and a lentiviral vector.

11. The cell according to claim 3, wherein the cell comprises an immune cell.

12. The cell according to claim 3, wherein the cell comprises T cell, NK cell, NKT cell, constant NK cell or other lymphocyte in peripheral blood.

13. The cell according to claim 3, wherein the cell is autologous or allogeneic cell isolated from umbilical cord or blood.

14. A method for treating a tumor in a subject in need thereof, comprising administrating the cell according to claim 3 into the subject, wherein the tumor expresses MAGE-A4, and wherein the cell is an immune cell.

15. The method according to claim 14, wherein the tumor comprises a cancer.

16. The method according to claim 14, wherein the tumor comprises an esophageal cancer, a gastric cancer, a bladder cancer, a head and neck cancer and a sarcoma.

17. The method according to claim 14, wherein the subject is human or a mouse.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1A to 1D show the scanning and screening results of MAGE-A4 positive tumor sections. Among them, FIG. 1A shows the scanning results of PA001 esophagus moderately differentiated squamous cell carcinoma in clinical phase IA; FIG. 1B shows the scanning results of PA002 esophagus basaloid squamous cell carcinoma with medium to low differentiation in clinical phase IIIB; FIG. 1C shows the scanning results of PA003 glottic moderately differentiated squamous cell carcinoma in clinical phase III; and FIG. 1D shows the scanning results of PA004 bladder invasive urothelial carcinoma in clinical phase II.

(2) FIG. 2 is a diagram showing a killing effect of a cell line.

(3) FIGS. 3A to 3D are diagrams showing tumor clearance effect in an experiment, and FIG. 3E is a diagram showing the change in weight of experimental mice.

(4) FIGS. 4A to 4C are diagrams show results of IFN ELISA experiments.

(5) FIG. 5 shows 6PA2021-TCR01 MAGE-A4 IHC pathological staining results (40) in Example 6.

(6) FIGS. 6A-6B are pictures showing a therapeutic effect of TCR-T cells screened according to an embodiment on tumor patients, in which FIG. 6A shows a MR result of intravenously infusing TCR01-05 cell preparation (5e.sup.10 TCR-T cells) on day 0, and FIG. 6B shows a MR result on day 45, that is, 45 days after intravenous infusion.

(7) In the Sequence Listing:

(8) SEQ ID NO:1-SEQ ID NO:30: sequences of TCR polypeptide chain

(9) SEQ ID NO:31-SEQ ID NO:60: sequences of TCR polypeptide chain

(10) SEQ ID NO:61-SEQ ID NO90:

(11) SEQ ID NO:91-SEQ ID NO120: sequences of CDR2 of TCR polypeptide chain

(12) SEQ ID NO:121-SEQ ID NO:150: sequences of CDR3 of TCR polypeptide chain

(13) SEQ ID NO:151-SEQ ID NO:180: sequences of CDR1 of TCR polypeptide chain

(14) SEQ ID NO:181-SEQ ID NO:210: sequences of CDR2 of TCR polypeptide chain

(15) SEQ ID NO:211-SEQ ID NO:240: sequences of CDR3 of TCR polypeptide chain

(16) SEQ ID NO:241-SEQ ID NO:242: Joint domain

(17) SEQ ID NO:243: humanized C domain linking chain

(18) SEQ ID NO:244: humanized C domain linking chain

(19) SEQ ID NO:245: murine C domain linking chain

(20) SEQ ID NO:246: murine C domain linking chain

(21) SEQ ID NO:247: modified humanized C domain linking chain

(22) SEQ ID NO:248: modified humanized C domain linking chain

DETAILED DESCRIPTION

(23) In view of the problems present in existing technologies, the present inventor conducted a lot of research, and found a new technical path, which includes selecting a tumor tissue of a patient with esophageal cancer, bladder cancer or laryngeal cancer showing MAGE A4+ and HLA-A02+, obtaining T cells therefrom, and screening a natural TCR sequence that can recognize MAGE A4+. The TCR obtained by this method is a TCR subjected to the patient's own thymus negative selection, which excludes the TCRs that can react with a normal human tissue, and greatly reduces an off-target toxicity from a source.

(24) In addition, in some related technologies, the TCR-T technology for MAGE A4 primarily selects sarcoma as an indication for clinical development and research in practice, but a proportion of this indication in tumor patients in China is very low. In order to expand the application scope of this technology, the present inventor measured the expression of MAGE A4 in 20 kinds of cancer by immunohistochemical method, and found that the target was highly expressed and positive in esophageal cancer, gastric cancer, bladder cancer and laryngeal cancer of tumor patients in China. Considering that an incidence rate of the above cancers in China is higher than that of sarcomas, a TCR sequence targeting esophageal cancer, gastric cancer, bladder cancer, laryngeal cancer and sarcomas, an expression vector, an excipient, cells and preparations containing the TCR sequence, and a clinical application of a product containing the same in cancer patients are developed in the present application.

(25) The present application is made based on the above discovery.

(26) Unless clearly indicated otherwise in the context, nouns without quantifier modification used in the present application represent one or more, for example, the reference to cell includes a plurality of such cells, and the reference to peptide includes one or more peptides and their equivalents (for example, polypeptides) known to those skilled in the art.

Example 1

(27) Screening T cells potentially capable of recognizing MAGE-A4 positive solid tumors (HLA-A*02:01 positive patients) by using MAGE-A4 antigen peptide tetramer included the following steps.

(28) (1) Human MAGE-A4 Positive (HLA-A*02:01) Tumor Surgical Specimens or Puncture Tumor Tissues were Obtained by Immunohistochemistry and HLA Sequencing Typing Screening.

(29) In this example, 2 esophageal cancer tissues showing MAGE-A4+, 1 bladder cancer showing MAGE-A4+ and 1 head and neck tumor showing MAGE-A4+ were obtained, and a strong positive expression of MAGE-A4 was confirmed by immunohistochemical method.

(30) The specific methods were as follows:

(31) 4 m-thick sections were cut from a FFPE specimen, attached on glass slides, baked and dewaxed. Antigen repair was performed on these sections.

(32) After blocking endogenous peroxidase and nonspecific binding protein (goat serum blocking solution), MAGE-A4 primary antibody was diluted by a 1:200, and incubated at 4 C. overnight. An enzyme-labeled secondary antibody was incubated for the next day, subjected to DAB color development, counterstained with Harris hematoxylin staining solution, and finally sealed with a neutral gum. Scanning was performed on Vectra3 multispectral scanner under 40equivalent objective lens. The scanning results are shown in FIGS. 1A-1D. After scanning, the images were analyzed with inForm software to confirm results of screening.

(33) Then HLA-A*02:01 typing was confirmed. In particular, the HLA-A*02:01 typing was confirmed as follows.

(34) Original expression data obtained by single cell sequencing was aligned to a reference genome GRCh38 via a count module of CellRanger (v6.0.2) to generate a BAM file sorted_genome_bam. BAM and a cell-gene expression matrix.

(35) Based on the BAM file and a cell barcode file generated by alignment, a software scHLAcount was used to perform HLA typing of single cells, which generated a file of all HLA typing types detected in the sample (labels. tsv), a depth statistics file of individual HLA typing of the specimens (summary.tsv), and a depth matrix file of cell-HLA typing (count_matrix. MTX), for confirming the typing of the specimens.

(36) The HLA typing of 4 patients measured in this Example is shown in Table 1-Table 4.

(37) TABLE-US-00001 TABLE 1 PA001 HLA Type Read_counts HLA_cell_number Total_cell_num Pos_Cell_ratio A*02:01:176 123094 11594 12342 0.9394 B*38:01:01:06 116695 11248 12342 0.9114 C* 12:227 107872 11254 12342 0.9118 DPA1*01:03:01:34 56519 7251 12342 0.5875 DPAl1*02:02:08 30281 5544 12342 0.4492 DPB1*02:01:19:01 23787 5365 12342 0.4347 DPB1*05:01:01:14 27268 5804 12342 0.4703 DQA1*03:02:01:02 28750 5348 12342 0.4333 DQB1*03:19:01:01 31422 5475 12342 0.4436 DRB1*09:01:02:07 168840 9347 12342 0.7573

(38) TABLE-US-00002 TABLE 2 PA002 HLA Type Read_counts HLA_cell_number Total_cell_num Pos_Cell_ratio A*02:01:175 175081 9859 10452 0.9433 B*54:01:09 69756 9170 10452 0.8773 C*01:03:01 81025 9388 10452 0.8982 DPA1*04:01:01:02 53980 5697 10452 0.5451 DPB1*19:01:01:03 38738 5669 10452 0.5424 DQA1*01:03:01:09 33252 4424 10452 0.4233 DQB1*04:01:01:02 18702 4677 10452 0.4475 DRB1*08:03:02:01 82962 6894 10452 0.6596

(39) TABLE-US-00003 TABLE 3 PA003 HLA Type Read_counts HLA_cell_number Total_cell_num Pos_Cell_ratio A*02:01:176 84405 5362 5886 0.911 B*08:18 84061 5065 5886 0.8605 C*01:17 57872 5181 5886 0.8802 DPA1*01:03:01:34 40401 3787 5886 0.6434 DPB1*05:01:01:14 12676 2536 5886 0.4309 DPB1*124:01:02:01 14969 2441 5886 0.4147 DQA1*03:02:01:02 12375 2092 5886 0.3554 DQB1*03:03:02:04 6243 1529 5886 0.2598 DRB1*09:01:02:07 52190 4147 5886 0.7046

(40) TABLE-US-00004 TABLE 4 PA004 HLA Type Read_counts HLA_cell_number Total_cell_num Pos_Cell_ratio A*02:01:175 28510 2703 2983 0.9061 B*08:18 31832 2671 2983 0.8954 C*03:47 2300 1287 2983 0.4314 C*07:429 10576 2289 2983 0.7673 DPA1*01:03:17 955 531 2983 0.178 DPA1*02:06 20972 1499 2983 0.5025 DPB1*22:01:01:01 6010 918 2983 0.3077 DPB1*105:01:01:10 6470 980 2983 0.3285 DQA1*03:02:01:02 18009 1350 2983 0.4526 DQB1*03:01:01:35 10154 1177 2983 0.3946 DRB1*09:01:02:07 50977 1964 2983 0.6584
(2) Preparation of Tetramer Containing MAGE-A4 Peptide HLA-A*02:01
Step S1. 10 mM MAGE-A4 Peptide Stock Solution was Selected, which has the Following Amino Acid Sequence:

(41) TABLE-US-00005 1) (SEQIDNo:249) KVLEHVVRV; 2) (SEQIDNo:250) GVYDGREHTV; 3) (SEQIDNo:251) ALLEEEEGV; 4) (SEQIDNo:252) KVDELAHFL; 5) (SEQIDNo:253) ALAETSYVKV; and 6) (SEQIDNo:254) ALSNKVDEL.

(42) HLA-A*02:01 with an amino acid sequence of GVAGDVSAV (SEQ ID No:255, non naturally present) was selected as a negative control.

(43) S2. Peptide Exchange

(44) S2-1. 5 l peptide stock solution was mixed with 120 l PBS buffer to dilute the 10 mM peptide stock solution to 400 M to obtain a diluted peptide which was stored on ice;

(45) S2-2. 20 l of diluted peptide prepared in step s2-1 and 20 l of peptide Flex-t HLA-A *02:01 monomer UVX (200 G/ml) were injected into a 96-well V-shaped plate and mixed by using a pipette;

(46) S2-3. the plate was sealed, and centrifuged at 2500g at 4 C. for 2 min to obtain a supernatant;

(47) S2-4. the plate was unsealed, placed on the ice and irradiated with an ultraviolet lamp for 30 min, with a distance between the ultraviolet lamp and the plate of 4 cm; and

(48) S2-5. the plate was sealed again, and cultured in the dark at 37 C. for 30 min to collect a liquid, which is s peptide exchange monomer.

(49) In order to evaluate the efficiency of peptide exchange, a protocol of HLA class I ELISA was followed.

(50) S3. Preparation of Tetramer

(51) S3-1. 30 l of the peptide exchange monomer obtained in step S2 was transferred to 1.5 ml micro centrifuge tube or new plate, then added with 3.3 l of PE streptavidin (purchased from Biolegend under article No. 405204), mixed with a pipette, and incubated in the dark on ice for 30 min;

(52) S3-2. during incubation in step S3-1, 1.6 l of 50 mM D-biotin and 6 l of 10% (w/v) NaN.sub.3 were added to 192.4 l PBS buffer to prepare a blocking solution, 2.4 l of which was added to the solution incubated in step S3-1 under vortex mixing, and mixed with a pipette to stop the reaction; and

(53) S3-3. the micro centrifuge tube or plate was incubated at 5 C. overnight to obtain tetramer.

(54) S4. Preparation of Target Group:

(55) S4-1. before dyeing, the tetramer obtained in step S3 was centrifuged in a micro centrifuge tube or plate under 2500g at 4 C. for 5 min, and then stored on ice in the dark;

(56) S4-2. 2*10.sup.6 cells were added to 12*75 mm tube or 96-well U-shaped base plate. The volume was adjusted to 200 l with cell staining buffer. Each Flex-t sample was added with 2 l tetramer obtained in s4-1, mixed and cultured in the dark on ice for 30 min;

(57) S4-3. the cells were washed with the staining buffer twice and recultured in the staining buffer; and

(58) S4-4. positive cell samples were collected by using a flow cytometry within 2 hours.

(59) (3) Digesting of a Patient's Tumor Tissue into a Single Cell Suspension

(60) After removing connective tissues from a surface of a tumor issue, the tumor issue was cut into 1 cm*1 cm tissue pieces, fully shredded with a scissor, placed into a 15 ml centrifuge tube. Each tube was added with 6 ml digestive solution of Miltenyi gentleMACS discociator KIT or a mixed solution of self-developed tissue digestive enzyme to digest the tissue.

(61) Cell quality detection: Bio-Rad TC20 automatic cell counter was used in combination with a microscope to accurately detect the quality of the cell suspension, so as to ensure an experimental cell activity of greater than 70% and a cell diameter of less than 40% m.

(62) (4) Obtaining of TCR V(d)J Full-Length Sequence Capable of Recognizing MAGE-A4 Peptide

(63) The TCR V(d)J full-length sequence capable of recognizing the MAGE-A4 peptide segment was obtained from the above four patients' tumors (2 MAGE-A4 positive esophageal cancer tissues, 1 MAGE-A4 positive bladder cancer tissue, and 1 MAGE-A4 positive head and neck tumor tissue).

(64) A total of 30 TCR V(d)J full-length sequences were obtained, numbered TCR01 to TCR30. Each of the TCR V(d)J full-length sequences contains one TCR polypeptide and one TCR polypeptide, the sequence of which is shown in Table 5.

(65) TABLE-US-00006 TABLE 5 TCR TCR polypeptide chain TCR polypeptide chain TCR01 SEQ ID NO:1 SEQ ID NO:31 TCR02 SEQ ID NO:2 SEQ ID NO:32 TCR03 SEQ ID NO:3 SEQ ID NO:33 TCR04 SEQ ID NO:4 SEQ ID NO:34 TCR05 SEQ ID NO:5 SEQ ID NO:35 TCR06 SEQ ID NO:6 SEQ ID NO:36 TCR07 SEQ ID NO:7 SEQ ID NO:37 TCR08 SEQ ID NO:8 SEQ ID NO:38 TCR09 SEQ ID NO:9 SEQ ID NO:39 TCR10 SEQ ID NO:10 SEQ ID NO:40 TCR11 SEQ ID NO:11 SEQ ID NO:41 TCR12 SEQ ID NO:12 SEQ ID NO:42 TCR13 SEQ ID NO:13 SEQ ID NO:43 TCR14 SEQ ID NO:14 SEQ ID NO:44 TCR15 SEQ ID NO:15 SEQ ID NO:45 TCR16 SEQ ID NO:16 SEQ ID NO:46 TCR17 SEQ ID NO:17 SEQ ID NO:47 TCR18 SEQ ID NO:18 SEQ ID NO:48 TCR19 SEQ ID NO:19 SEQ ID NO:49 TCR20 SEQ ID NO:20 SEQ ID NO:50 TCR21 SEQ ID NO:21 SEQ ID NO:51 TCR22 SEQ ID NO:22 SEQ ID NO:52 TCR23 SEQ ID NO:23 SEQ ID NO:53 TCR24 SEQ ID NO:24 SEQ ID NO:54 TCR25 SEQ ID NO:25 SEQ ID NO:55 TCR26 SEQ ID NO:26 SEQ ID NO:56 TCR27 SEQ ID NO:27 SEQ ID NO:57 TCR28 SEQ ID NO:28 SEQ ID NO:58 TCR29 SEQ ID NO:29 SEQ ID NO:59 TCR30 SEQ ID NO:30 SEQ ID NO:60

Example 2

(66) The difference of this example from Example 1 lies that, a different method for confirming HLA-A*02:01 typing in step (2) is adopted. In particular, the method for confirming HLA-A*02:01 typing in this example was as follows:

(67) The sample was freeze stored as PBMC in cell cryopreservation solution. After thawing, the sample was washed twice with PBS to remove the cell cryopreservation solution, and centrifuged to remove the PBS buffer. RNA was extracted from obtained cell precipitate using Qiagen total RNA extraction kit. The RNA was subjected to denaturation, and reversely transcribed using Vazyme HiScript to obtain cDNA.

(68) The cDNA was target enriched twice using Abclonal Gloria HS PCR Kit and corresponding primers of HAL1 and HLA2. 50 ng of the enriched product was subjected to fragmentation, terminal repair, linker connection and Index PCR amplification to construct a sequencing library using Abclonal FS DNA Lib Prep Kit.

(69) The library was subjected to quality inspection and then sequencing on Illumina Novaseq6000 sequencing platform. A target data volume of each sample was 9G.

Example 3 Construction of Plasmid, Virus and Cells from TCR-T

(70) (1) Construction of Plasmid

(71) A complete TCR sequence (TRA V(d)J+C domain linking chain+linker domain+TRB V(d)J+C domain linking chain) was cloned into a target plasmid of lentivirus system (for example, in PGK), and transfected into E. coli. Positive clones with kanamycin resistance were selected for amplification and culture, and plasmid was extracted by using Qiagen Plasma Maxi Kit.

(72) The linker domain was modified P2A (SEQ ID No: 241) or P2A linker sequence (SEQ ID No: 242).

(73) In this example, C domain sequences selected for TCR01-TCR05 were SEQ ID No: 243 and SEQ ID No: 244, and the linker domain was modified P2A (SEQ ID No: 241);

(74) C domain sequences selected for TCR06-TCR10 were SEQ ID No: 245 and SEQ ID No: 246, and the linker domain was modified P2A (SEQ ID No: 241);

(75) C domain sequences selected for TCR11-TCR15 were SEQ ID No: 243 and SEQ ID No: 244, and the linker domain was P2A linker sequence (SEQ ID No: 242);

(76) C domain sequences selected for TCR15-TCR20 were SEQ ID No: 245 and SEQ ID No: 246, and the linker domain was P2A linker sequence (SEQ ID No: 242);

(77) C domain sequences selected for TCR16-TCR20 were SEQ ID No: 247 and SEQ ID No: 248, and the linker domain was modified P2A (SEQ ID No: 241);

(78) C domain sequences selected for TCR21-TCR25 were SEQ ID No: 243 and SEQ ID No: 244, and the linker domain was modified P2A (SEQ ID No: 241); and

(79) C domain sequences selected for TCR26-TCR30 were SEQ ID No: 243 and SEQ ID No: 244, and the linker domain was C linker sequence (SEQ ID No: 242).

(80) (2) Virus Packaging

(81) 1) 24 h before packaging, 8*10.sup.6 293 T cells were spread out in a 10 cm Petri dish, supplemented with 10 ml DMEM medium containing 10% FBS, and then cultured in a 5% CO.sub.2 incubator at 37 C.; 2) the cells were observed on the day of packaging, and lentivirus packaging was performed when it was confirmed that the cells reached a confluence of 80% (2%) and assumed a transparent state; 3) reagents used during packaging were removed and balanced to room temperature; 4) 10 ml of the medium in Petri dish and 9 ml serum-free DMEM medium added along the dish wall were transfer to CO.sub.2 incubator for use; 5) 450 l of Opti-MEM was added to a 1.5 ml centrifuge tube, and then added with 7.5 g of psPAX2, 5 g of pMD2. G and 10 g of target plasmid, and mixed with a pipette; 6) another 1.5 ml centrifuge tube was add with 450 l of Opti-MEM and 22.5 ul of PEIpro reagent, and mixed with a pipette; 7) a mixture obtained in step 5) with a mixture obtained in step 6) were mixed with a pipette, and incubated at room temperature for 10-15 min; 8) a mixture obtained in step 7) was gently dripped into a 10 cm Petri dish, and then transferred to a CO.sub.2 incubator for further culturing; 9) after 6 h, the medium in the 10 cm culture dish obtained in step 8) was removed, 10 ml of DMEM medium containing 10% FBS was added to the dish along the dish wall, and the dish was transferred to CO.sub.2 incubator for further culturing; 10) After culturing the medium obtained in step 9) for 48 h, supernatant was collected into a 50 ml centrifuge tube, 10 ml of DMEM medium containing 10% FBS was added along the dish wall, the dish was transferred to CO.sub.2 incubator for further culturing, and the supernatant containing virus was stored in a refrigerator at 4 C.; 11) after 72 h, the supernatant stored in step 10) was collected into a 50 ml centrifuge tube, and centrifuged at 500g and 4 C. for 10 min to remove cell debris; 12) the supernatant obtained upon centrifuging in step 11) was filtered through 0.45 m filter into a new 50 ml centrifugal tube; 13) the supernatant obtained upon filtering in step 12) was added with a quarter volume of PEG8000 concentration reagent and incubated in a shaking table at 4 C. for more than 3 h; 14) the virus solution incubated in step 12) was centrifuged at 2000g and 4 C. for 40 min to obtain a precipitate, which was added with PBS precooled at 4 C., and gently mixed with a pipette; and 15) The virus obtained in step 14) is divided an packaged into cryopreservation tubes and stored at 80 C. for use.

(82) The virus obtained in the above steps was subjected to virus titration test as follows: 1) on the day of test, the cell density of Jurkat was adjusted to 3*10.sup.5 cells/ml, added to a 24-well plate by 1 ml cell suspension/each well, and then cultured in 5% CO.sub.2 incubator at 37 C. for use; 2) the virus was gradient diluted according to the ratios listed in Table 6 below, in which an additional group of secondary wells was provided for each group;

(83) TABLE-US-00007 TABLE 6 Diluting Volume of Volume of 1640 ) 10 mg/mL ratio virus (L) medium (L polybrene(L) 1:50 30 468.5 1.5 1:100 15 483.5 1.5 1:200 7.5 483.5 1.5 1:400 3.75 494.75 1.5 3) the diluted virus solution was added to cells, centrifuged at 800 g and 21 C. for 2 h, and then cultured in 5% CO.sub.2 incubator at 37 C.; 4) after 60 h, the cells were subjected to FACS test.

(84) Titer was calculated by:
Tu/ml=((number of infected cells)positive rate(times of dilution))/infected volume

(85) For example, when the positive rate of one well with a dilution ratio of 1:100 is 25%, the titer is (3*10.sup.5)0.25100)/(1.5 ml)=5*106 TU/ml

(86) When calculating the titer, only the wells with a positive rate of less than 40% are considered, for the reason that, when the positive rate is higher than 40%, the possibility of multiple virus particles repeatedly infecting one cell will result in inaccurate titer calculation.

(87) (3) Thawing of PBMC:

(88) 1) 30 ml of L500 medium was added into 50 ml centrifuge tube and restored to room temperature for use; 2) frozen PBMC was removed from 80 C. refrigerator, quickly placed into a 37 C. water bath, and quickly shaked until there was no visible ice in the frozen tube; 3) thawed cryopreservation tube was disinfected with 75% alcohol, transferred to a biosafety cabinet, slowly dripped into the thawed L500 medium with a 1 ml pipette, and centrifuged at 400 g and 25 C. for 5 min; 4) supernatant was discarded after centrifugation, the cells were resuspended in 30 ml of pre-heated L500 medium, from which 10 ul of the sample was taken for living cell counting, followed by centrifuging at 25 C. and 400 g for 5 min.
(4) Stimulation and Activation of PBMC 1) based on counting results, cells were resuspended in L500 medium and the density of living cells was adjusted to 1-2e.sup.6/ml; 2) a suspension obtained in step 1) was added with 300 IU/ml IL-2 and 50 ng/ml CD3 antibody, mixed, and transferred to CO.sub.2 incubator where they were stimulated and activated for 60 h.
(5) Lentivirus Transduction 1) activation of PBMC 48 hours after activation was observed, and lentivirus transduction was carried out; 2) virus was added by MOI=2 and cells were transferred into carbon dioxide incubator and cultured for 48 h; 3) the cells were transferred into a 50 ml centrifuge tube from which 10 l was taken for living cell counting, centrifuged at 25 C. and 400 g for 5 min, and washed; 4) based on the results of counting, the transfected cells were resuspended in L500 medium, the cell concentration was adjusted to 1e.sup.6/ml, IL-2 was added by 100 IU/ml, and the cells were transferred to CO.sub.2 incubator for culturing.
(6) Expanding and Culturing of Cells 1) after lentivirus transduction and solution replacing, cells were observed and counted every day, and controlled to have a number of 1e.sup.6/ml and IL-2 concentration of 100 IU/ml by supplementing medium; and 2) the cells were harvested on the ninth day of culturing.

Example 4 Cell Line Killing Experiment

(89) Positive: A375, h1755, h1395-(MAGE-A4+, HLA-A02+)

(90) Negative: H1299-(MAGE-A4, HLA-A02)

(91) T cells screened in this application that can specifically recognize MAGE-A4 positive tumors and target cells, by a ratio of 5:1, were subjected to relevant experiments. The results are shown in FIG. 2, where the abscissa 1-30 represents TCR01-TCR30, respectively, and the ordinate represents a percentage of killing.

(92) The T cells prepared using TCR01-TCR30 were mixed with the target cells with positive A375, H1755 and H1395 respectively by a ratio of T cells to target cells of 5:1. The results are shown in FIG. 2, where the abscissa 1-30 represents TCR01-TCR30, respectively, and the ordinate represents the percentage of killing.

(93) The results show that, TCR-T cells constructed using TCRs screened in this application have the most significant killing effect on target cells with positive H1755 and H1395, with a cell killing percentage of 70%-90%. TCR-T cells constructed using TCRs screened in this application have a relatively significant killing effect on cells with positive A375, with a cell killing percentage of 50%-70%. The killing effect of T cells screened in this application on cells with H1299 showing HLA-A*02:01 negative and having MAGE-A4 knockout is very low, that is, less than 10%.

Example 5 Tumor Clearance Experiment in Mice

(94) NCG mice were inoculated with 1e.sup.7 A375, H1395, H1755 and H1395 (MAGE-A4), in which the first three cell lines were natural HLA-A*02:01 and MAGE-A4 positive, and H1395 (MAGE-A4) has MAGE-A4 knocking-out. When the tumor grew to a volume of 100 mm.sup.3, the mice were infused with 200 l suspension containing 3*10.sup.6 TCR-T cells at caudal vein for one time, and then subjected to tumor size measurement and weight detection. FIG. 3A-FIG. 3D show the results of tumor clearance, in which the abscissa represents days from medication and the ordinate represents a change in tumor size with the increase of days from medication.

(95) FIG. 3A shows that, in the NCG H1395 tumor mice having an initial tumor size of about 100 mm.sup.3, there is a small increase in 1-5 days after TCR-T injection, that is, the tumor size being increased to 200 mm.sup.3; and during 5-18 days, the tumor size is decreased significantly. The tumor size in the control group MOCK is increased to 800 mm.sup.3 on the 18th day, decreased to 100 mm.sup.3 or below after injection treatment, and after TCR05 injection, remains at about 100 mm.sup.3. The tumor size of other cells after TCR-T injection was decreased significantly, approaching 0.

(96) FIG. 3B shows that, in the NCG A375tumor mice having an initial tumor size of about 100 mm.sup.3, there is a small increase in 1-8 days after TCR-T injection, that is, the tumor size being increased to 250 mm.sup.3; and during 8-18 days, the tumor size is decreased significantly. The tumor size in the control group MOCK is increased to 1000 mm.sup.3 or above on the 18th day, and decreased to 100 mm.sup.3 or below after injection treatment. The tumor size of other cells after TCR07 and TCR12 injection approached 0, showing a good result.

(97) FIG. 3C shows that, in the NCG H1755 tumor mice having an initial tumor size of about 100 mm.sup.3, the tumor size in the control group MOCK is increased to 900 mm.sup.3 during 1-18 days. In the TCR-T treatment group, the tumor size fluctuates greatly, except for TCR17 group. In other groups, the tumor size fluctuates gently after TCR-T cell injection, and an overall volume of cells is not increased within 18 days and remains at or below 100 mm.sup.3.

(98) FIG. 3D shows that, in the tumor mice of H1395 (MAGE-A4 and HLA-A02+) with MAGE-A4 knockout or wild-type H1395 (MAGE-A4+ and HLA-A02+) tumor mice having an initial tumor size of about 100 mm.sup.3, treatment of TCR01-05-T cells mixture are significantly impaired when the tumor cells assume MAGE-A4 negative, and the tumor size reaches 800-900 mm.sup.3 18 days after injection. When the tumor cells assume MAGE-A4 positive, injection of TCR-T cells according to the present application can significantly inhibit the increase of cell volume, which is controlled to be 100 mm.sup.3 or below on the 18.sup.th day from injection.

(99) FIG. 3E shows a weight change of mice after injection. It can be seen that, there is no significant change in the weight of mice during the whole injection period, indicating that TCR-T cells according to the present application will not cause obvious toxic reaction after injection.

(100) The results from FIG. 3A to FIG. 3E show that, the constructed TCR-T cells have good tumor inhibition effect and low toxicity in vivo.

Example 6 IFN ELISA Test Confirming No Reaction with Normal Cell Lines

(101) TCR01-TCR15 were selected to react with 11 kinds of normal tissue cells and positive h1755 (MAGE-A4+ and HLA-A02+) cells. INF content in individual cells were detected after 3 days. The selected normal tissue cells are shown in Table 7 below:

(102) TABLE-US-00008 TABLE 7 Chinese name of primary cells Article No. Pulmonary type II alveolar CTCC-A005-PC epithelial cells Cardiomyocyte CTCC-C002-PC Esophageal epithelial cells CTCC-D001-PC Gastric mucosal epithelial cells CTCC-D004-PC Intestinal mucosal epithelial cells CTCC-D007-PC Colonic mucosal epithelial cells CTCC-D011-PC Gallbladder epithelial cells CTCC-D014-PC Hepatic parenchymal cells CTCC-D017-PC Renal tubular epithelial cells CTCC-U002-PC Bladder epithelial cells CTCC-U008-PC Islet cells CTCC-G004-PC

(103) The test results are shown in FIG. 4A-FIG. 4C, in which the abscissa is TCR01-TCR10 and the ordinate is INF content. In FIG. 4A, pulmonary type II alveolar epithelial cells, cardiomyocytes, esophageal epithelial cells and gastric mucosal epithelial cells are illustrated; and in FIG. 4B, small intestinal mucosal epithelial cells, colonic mucosal epithelial cells, gallbladder epithelial cells and hepatic parenchymal cells are illustrated; and in FIG. 4C, renal tubular epithelial cells and bladder epithelial cells are illustrated.

(104) When TCR-T cells according to the present application are injected, INF content in 11 normal tissue cells is maintained at 0-5 ng/ml, approaching 0 ng/ml, indicating that TCR-T cells are basically non-toxic to normal cells, while in positive H1755 (MAGE-A4+, HLA-A02+) tumor cells, the INF content can reach up to 15-255 ng/ml.

Example 7 Effect of TCR-T Screened in this Application in Treating Tumor Patients

(105) Patient PA2021-TCR01, male, 75 years old, was diagnosed with high-grade papillary urothelial carcinoma of the bladder in September 2020 due to gross hematuria. After performing relevant examinations, bladder space occupation was considered. Transurethral resection of bladder tumor was performed in October 2020. Postoperative pathology showed that high-grade papillary urothelial carcinoma of the bladder infiltrated the muscular layer. PET CT showed positive FDG uptake in multiple pelvic lymph nodes, and metastasis was considered. After diagnosis, the patients were treated with gemcitabine+cisplatin+teplizumab for 4 times. The tumor still progressed after reexamination in March 2021. After being treated with MAGE-A4 TCR-T cells, IHC staining result of tumor pathological white film showed MAGE-A4 positive (FIG. 5), and the HLA typing result of peripheral blood was HLA-A*02:01, as shown in Table 8 below.

(106) TABLE-US-00009 TABLE 8 PA2021- PA2021- PA2021- PA2021- PA2021- TCRO1_HLA TCRO l_Read_counts TCROl_HLA_cell_num TCR01_Total_cell_num TCROl_Cell_ratio A*02:01:175 28510 2703 2983 0.9061 B*08:18 31832 2671 2983 0.8954 C*03:47 2300 1287 2983 0.4314 C*07:429 10576 2289 2983 0.7673 DPAl*01:03:17 955 531 2983 0.178 DPA1*02:06 20972 1499 2983 0.5025 DPB1*22:01:01:01 6010 918 2983 0.3077 DPB1*105:01:01:10 6470 980 2983 0.3285 DQA1*03:02:01:02 18009 1350 2983 0.4526 DQB1*03:01:01:35 10154 1177 2983 0.3946 DRB1*09:01:02:07 50977 1964 2983 0.6584

(107) TCR01-05 cell preparation containing 5e.sup.9 TCR-T cells were infused intravenously. MR reexamination results on the day of injection (FIG. 6A) and 45 days after injection (FIG. 6B) showed that the focus was reduced and the diffusion was weakened, and the maximum diameter of the tumor was reduced from 48.52 mm to 34.48 mm.

(108) The above examples are only provided for the purpose of explaining the technical concept and characteristics of the application, so as to enable those skilled in the art familiar with the technology and understand the content of the application and implement it. They are not intended to limit the scope of protection of the application. Without departing from the concept, spirit and scope of the application, the method described in the application and the steps or step sequence of the method can be changed. All equivalent changes or modifications made according to the spirit and essence of this application shall be covered by the scope of protection of this application.