WO2020133050A1

WO2020133050A1 - Ebv epitope high affinity t cell receptor

Info

Publication number: WO2020133050A1
Application number: PCT/CN2018/124294
Authority: WO
Inventors: 王飞; 赵正琦; 王石雷; 尹悦露; 李波; 侯勇
Original assignee: 深圳华大生命科学研究院
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2020-07-02
Also published as: CN113423724A; CN113423724B

Abstract

Disclosed is an EBV epitope high affinity T cell receptor. The EBV epitope high affinity T cell receptor provided by the present invention comprises a chain α and a chain β. The chain α comprises three complementarity determining regions, and comprises the sequences of positions 43-49, 67-71, and 106-116 of SEQ ID No: 3, respectively. The chain β comprises three complementarity determining regions, and comprises amino acid sequences of 46-50, 68-73, and 111-118 of SEQ ID No: 4, respectively. The TCR-expressing T cell provided by the present invention can effectively recognize an EBV antigen polypeptide loaded on a T2 cell and secrete IFN-γ, thereby demonstrating the functionality thereof. The EBV epitope high affinity T cell receptor provided by the present invention has important applications.

Description

EBV Epitope High Affinity T Cell Receptor Technical Field

The present invention belongs to the field of biotechnology and specifically relates to an EBV epitope high affinity T cell receptor.

Background technique

Nasopharyngeal carcinoma (NPC) is a tumor derived from epithelial cells, which is highly correlated with EBV virus (Epstein-Barr virus, EBV) infection. All EBV-positive NPC malignant tumors are accompanied by latent EBV infection. Chemotherapy and radiotherapy are the traditional treatments for NPC, which can effectively control the development of the disease, but can not completely remove the small lesions and metastatic tumor cells in the circulation. As immunotherapy has achieved more and more attention in the treatment of cancer, the combination of traditional NPC therapy and immunotherapy will have better results in terms of improving efficacy, reducing side effects, and removing small lesions.

In terms of tumor immunity, cytotoxic T lymphocytes (CTL) are known to play a key role in fighting infection and tumor-specific immune responses. It is not the entire antigen molecule that initiates the immune response program, but the short peptide of amino acids that binds to the MHC molecule, the CTL epitope. With the development of bioinformatics, more and more MHC class I molecule-restricted CTL epitopes related to viruses and tumor antigens have been successfully predicted and identified. Nasopharyngeal cancer cells can present internally expressed viral antigens through human leukocyte antigen (HLA) class I molecules to generate specific CTL, which provides an opportunity for immunotherapy of nasopharyngeal cancer.

The viral proteins expressed during EBV latent infection include 6 nuclear antigens (EBNAsl, 2, 3A, 3B,

3C and LP), three latent membrane proteins (LMPsl, 2A and 2B) and BawM-A open reading frame transcripts (BARTs) to the right. These latent gene products of EBV present different expression patterns in different tumor tissues, including latent type 0, latent type I, latent type II, and latent type III. They exist in latent type II in NPC, and only express EBNA1, LMP1, LMP2A , LMP2B and BARF1. Among them, the LMP2A sequence is relatively conservative, can be continuously expressed in tumor-related tissues, and contains a variety of CTL epitopes restricted by HLA. It is currently an ideal target antigen for EBV-related NPC immunotherapy. With the deepening of research, many epitope peptides of LMP2A have been identified. Among them, many epitope peptides have achieved good results in in vivo and in vitro immunotherapy experiments against NPC.

The current immunotherapy for NPC mainly focuses on active immunotherapy. Design the corresponding epitope peptide vaccine according to the target antigen, select the appropriate antigen to present to the patient, and induce a specific immune response to clear the tumor cells. Lin et al. reported on the treatment of 16 cases of refractory nasopharyngeal carcinoma with LMP2 peptide-loaded autologous DCs. DCs were loaded with autologous derived HLA-A1 101, HLA-A2402 and HLA-B4001 1 restricted epitope peptides and then returned to the patients. LMP2-specific CD8 ⁺ T lymphocytes were used as the evaluation criteria. 9 patients (56 %) Showed a strong immune response against LMP2 peptides. In addition, the tumors of two patients regressed, and the tumor-free periods were 10 months and 12 months respectively. This study shows that the related epitope polypeptides are feasible for stimulating the immune response of NPC patients, and have great reference value for other immunotherapy in the later stage.

Invention Disclosure

The object of the present invention is to provide a T cell receptor that recognizes EBV antigens. Among them, the EBV antigen The amino acid sequence is shown in SEQ ID No. 5 (LMP2 epitope).

The T cell receptor that recognizes the EBV antigen provided by the present invention includes the a chain and the P chain. Wherein, the a chain contains three complementarity determining regions, and the amino acid sequences are respectively positions 43-49, 67-71, and 106-1 16 of SEQ ID No. 3; or up to three of these sequences , 2 or 1 amino acid variants. The P chain includes three complementarity determining regions, and the amino acid sequences are 46th to 50th, 68th to 73th, and 11th to 1st to 18th positions of SEQ ID No. 4; or up to 3 of these sequences , 2 or 1 amino acid variants.

Further, the amino acid sequence of the variable region of the a chain is positions 18 to 127 of SEQ ID No. 3; or variants of these sequences having at most 3, 2 or 1 amino acid changes. The amino acid sequence of the variable region of the P chain is positions 20-128 of SEQ ID No. 4; or variants of these sequences with up to 3, 2 or 1 amino acid changes.

The amino acid sequence of the constant region of the a chain is 128-268 of SEQ ID No. 3. The amino acid sequence of the constant region of the P chain is 129-307 of SEQ ID No. 4.

Further, the amino acid sequence of the a chain is specifically SEQ ID No. 3; the amino acid sequence of the P chain is specifically SEQ ID No. 4 _o

The nucleic acid molecule encoding the T cell receptor also belongs to the protection scope of the present invention.

The nucleic acid molecule encoding the T cell receptor includes a nucleic acid molecule encoding the a chain of the T cell receptor and a nucleic acid molecule encoding the P chain of the T cell receptor.

Wherein, the sequences of the nucleic acid molecules encoding the three complementarity determining regions in the a chain of the T cell receptor are 127-147, 199-213, and 316-348 of SEQ ID No. 1, respectively; or Sequences having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with these sequences and encoding the same amino acid residues. The sequences of the nucleic acid molecules encoding the three complementarity determining regions in the P chain of the T cell receptor are 136-150, 202-219, and 331-354 of SEQ ID No. 2, respectively; or The sequence has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity and encodes the same amino acid residue.

Further, the sequence of the nucleic acid molecule encoding the variable region of the a chain is positions 52-381 of SEQ ID No. 1; or 99% or more, 95% or more, 90% or more, 85% or more of these sequences Or sequences with more than 80% identity and encoding the same amino acid residues. The sequence of the nucleic acid molecule encoding the variable region of the P chain is positions 58-384 of SEQ ID No. 2; or 99% or more, 95% or more, 90% or more, 85% or 80% or more of these sequences Sequences of the above identity and encoding the same amino acid residues.

The nucleotide sequence of the constant region of the a chain is 382-804 of SEQ ID No. 1. The nucleotide sequence of the constant region of the P chain is 385-921 of SEQ ID No. 2.

Further, the sequence of the nucleic acid molecule encoding the a chain is specifically SEQ ID No. 1. The sequence of the nucleic acid molecule encoding the P chain is specifically SEQ ID No. 2 _o

The expression cassette, vector or cell containing the nucleic acid molecule also belongs to the protection scope of the present invention.

Further, the vector may be a lentiviral vector.

The vector may be a multiple cloning site of the vector pRRLSIN.cPPT.PGK-GFP.WPRE (limited A nucleic acid molecule encoding the a chain of the T cell receptor and a nucleic acid molecule encoding the P chain of the T cell receptor are inserted between the systemic endonucleases BamHI and Sail) to obtain a recombinant plasmid.

The vector may specifically be a recombinant plasmid obtained by inserting a DNA sequence between multiple cloning sites of the vector pRRLSIN.cPPT.PGK-GFP.WPRE (such as restriction enzymes BamHI and Sail); the DNA sequence is The nucleic acid molecule encoding the a chain and the nucleic acid molecule encoding the P chain are formed by connecting a coding sequence of a linking peptide (such as a P2A peptide).

In an embodiment of the present invention, the carrier may be a DNA molecule shown in SEQ ID No. 6 inserted between the restriction enzyme BamHI and Sail of the vector pRRLSIN.cPPT.PGK-GFP.WPRE.

Further, the cell may be a T cell.

T cells having any of the T cell receptors mentioned above also fall within the protection scope of the present invention.

A pharmaceutical composition containing the carrier or the cell, or a T cell containing any of the T cell receptors described above also falls within the protection scope of the present invention.

Among them, the pharmaceutical composition can be used to prevent and/or treat diseases caused by EBV infection.

The T cell receptor, or, the nucleic acid molecule, or, the carrier or cell, or the T cell containing any of the T cell receptors described above are prepared to prevent and/or treat diseases caused by EBV infection The application in the medicine also belongs to the protection scope of the present invention.

The T cell receptor, or, the nucleic acid molecule, or, the vector or cell, or, the T cell containing any of the T cell receptors described above is used in the prevention and/or treatment of diseases caused by EBV infection Application also belongs to the protection scope of the present invention.

The present invention also claims a method for preventing and/or treating diseases caused by EBV infection. The method may include the steps of: preventing the T cell receptor described above, or, the nucleic acid molecule, or, the vector or cell, or, T cells containing any of the T cell receptors described above to prevent and /Or treat diseases caused by EBV infection.

Among them, the diseases caused by EBV infection mentioned above may specifically be nasopharyngeal carcinoma and/or oropharyngeal squamous cell tumors and/or T cell malignancies.

Experiments show that the present invention stimulates specific T cells in vitro by EBV antigen polypeptides to obtain specific T cell populations, and uses single cell pairing TCR sequencing technology to obtain TCR sequence sets of effective T lymphocytes corresponding to EBV antigen polypeptides, and then these sequences The collections are sorted by abundance to perform in vitro functional verification, and finally the TCR claimed by the present invention is obtained. Experiments show that the TCR-expressing T cells provided by the present invention can effectively recognize the EBV antigen polypeptide (target cell) loaded on T2 cells and secrete positive N-y, proving that this group of T cells is functional. The EBV epitope high affinity T cell receptor provided by the present invention has important application value.

BRIEF DESCRIPTION

Figure 1 shows the results of flow cytometry after the first round of EBV antigen peptide stimulation. The left picture shows the control group of T cells without peptide stimulation; the right picture shows the T cells stimulated by EBV peptide for one round.

Figure 2 shows the results of Elispot detection of EB V antigen peptide-specific T cells. The left picture shows the specific T cells stimulated by EBV peptide reacting with antigen (T2+EBV peptide) can effectively secrete INF-Y, the right picture shows the unrelated peptide The control group cannot secrete IFN-Y.

Figure 3 is a single-cell TCR amplification electrophoresis diagram, the frame marked amplified TCRa/p target band.

Figure 4 is a PCR electrophoresis diagram of colonies of TA clones. Different lanes represent different positive single cell clones amplified.

The best way to implement the invention

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the test materials used in the following examples are all purchased from conventional biochemical reagent stores. In the quantitative tests in the following examples, three repeated experiments are set, and the results are averaged.

The experimental reagent items used in the following examples are as follows:

Experimental items: blood collection tube (containing ACD anticoagulant), syringe, centrifuge tube, 0.2pm filter membrane, MS sorting column, magnetic stand, low-adsorption six-well plate; 0.2pm filter membrane, Dayu cryopreservation kit, PCR tube.

Experimental reagents: Sterile saline solution (DPBS), RPMI1640 medium, Ficoll, AIM-V medium, sterile ultrapure water (O. lum filter filtration), MACS running buffer; AIM-V medium, GM-CSF , IL-4, IFN-y, LPS, IL-7. Antigen presentation beads, AIM-V medium, IL-21, IL-2, IL-15. Sterile saline solution (PBS), Human IFN-y ELISpot kit (MABTECH), tetramer (EBV tetramer). The vector pRRLSIN.cPPT.PGK-GFP.WPRE is a product of Addgene.

Example 1. Obtaining high-affinity T cell receptor full-length sequence of EBV epitope

1. EBV-specific T cell stimulation

1. Peripheral blood PBMC separation in healthy people

1) Collect blood sample in 50ml, centrifuge at room temperature 100g, 15min (ACC 2, DEC2);

2) Collect the upper layer plasma and lower layer blood cells separately. The layer plasma is 1100g at room temperature, centrifuge at 20min, and discard the pellet;

3) After inactivation at 56°C (30min), put it in the frozen layer at -20°C and let it stand for 15min;

4) Centrifuge at 3800 rpm at room temperature for 20 minutes. After centrifugation, the supernatant is human serum, which can be used as a spare;

5) Take DPBS to make up the lower blood cells to 50ml, mix upside down and mix up;

6) Pipette Ficoll 20mL into 50mL centrifuge tube;

7) Carefully add 25mL of mixed blood sample above Ficoll and centrifuge at room temperature;

8) After the centrifugation is completed, the liquid is divided into four layers, from top to bottom are the plasma layer, the white membrane layer, the Ficoll layer and the blood cell layer. The white membrane layer is carefully aspirated with a Pasteur pipette and transferred to a Bacteria in the centrifuge tube;

9) Add 3 times the volume of 1640 medium to wash the aspirated white membrane layer, and gently pipette several times, at room temperature 500g, centrifuge at lOmin, carefully aspirate the supernatant, the precipitate is PBMC;

10) Add DNAase to digest the clumped cells, visually judge as a single cell suspension and add 5 -6ml 4° CMACS running buffer to terminate.

11) Add the terminated single-cell suspension to the cell screen of 70^m, wash the tube and screen three times with l-2ml of MACS running buffer, centrifuge at room temperature 300g, lOmin, and count after resuspending.

2. CD8 ⁺ T cell sorting 1) After counting, PBMC is resuspended by adding 80 _[j, l buffer/ 10 ⁷ cells to MACS running buffer, and adding 20 _[j l CD8 magnetic beads/ 10 ⁷ cells, mixing, and incubating at 4°C for 15 min;

2) After incubation, add 1-2mL buffer/ 10 ⁷ cells to wash;

3) Aspirate the supernatant completely, the PBMC is scattered, and add 500 (J buffer (0-10 ⁸ total cells) to resuspend;

4) The sorting column is placed on a magnetic stand, and MACS running buffer is added to balance the sorting column. (MS: 500^1, LS: 3mL) Add the cell suspension, wash the tube and sorting column three times with MACS running buffer, each volume is the same as above;

5) Remove the column, add lml MACS running buffer and push the piston of the column, the liquid pushed out is CD8 ⁺ T cells. After counting, freeze at 10 ⁷ /ml;

6) CD8-T cells (negative cells) adhere to DC cells (1.5-2h or overnight).

3. DC load EBV antigen peptide

1) Negative cells are resuspended in 5% human serum AIM-V and plated;

2) Shake the petri dish to resuspend the non-adherent cells in the supernatant, aspirate the supernatant, and then add AIM-V medium to drip wash;

3) Adherent cells are added to DC medium, and after 48h, a half amount of 5% human serum AIM-V medium is added, and after 24h, the cells are blown down with cold DPBS (the original medium is separated from the cell liquid after subsequent addition of DPBS to wash In different tubes), follow the 12-well plate 5x l0 ⁵ cdls, lml medium per well, the medium is 5% human serum AIM-V, and add cytokines to induce DC cell maturation to obtain mature DC cells.

4) Add the polypeptide (EBV antigen polypeptide, LMP2 epitope, aa426-aa434, SEQ ID No. 5) to the mature DC cells to obtain the system. In this system, the concentration of EBV antigen polypeptide is 10ug/ml.

5) Take the system obtained in step 4) and incubate at 37°C for 4 hours to obtain DC cells loaded with EBV antigen polypeptide.

4. Co-culture of DC cells loaded with EBV antigen polypeptide and CD8 ⁺ T cells

1) After 16h, DC cells loaded with EBV antigen polypeptide were pipetted with cold DPBS and co-cultured with the recovered CD8 ⁺ T;

2) Purge CD8 ⁺ down, and purge the well plate with AIM-V medium at least 3 times, room temperature 400g, centrifuge for 5 min;

3) Resuspend 1ml AIM-V, add DNAase to digest to single-cell suspension 0-5min, then add 5ml AIM-V to terminate, centrifuge at room temperature 400g, 5min;

4) T cells were resuspended with 5% human serum AIM-V and plated at 6.25 X 10 ⁵ cells/cm ² .

5) Add DC cells loaded with EBV antigen peptide according to the proportion, and add IL-21;

6) After 72h, add fluid every 2-3 days or change the fluid in half, and add the total volume of cytokines IL2, IL-7 and IL-15;

7) Every 2-3 days, supplement cytokines or change fluid;

8) At the 5th day of culture, prepare the antigen presentation beads for the second round of stimulation: add equal volume of boric acid solution to wash twice, resuspend in equal volume, add CD28 and HLA-A2:Ig, shake at 4°C overnight. After culture to day 10, the cells were resuspended, and the number of cells used for detection and flow sorting was taken out, and the remaining cells were used for the second round of co-culture. 2. Single cell TCR sequencing of EBV antigen peptide specific T cells

1. Synthesis of HLA-A*02 tetramer loaded with EBV epitope polypeptide

The tetramer is formed by connecting four monomers. The complex formed by a single HLA molecular protein and a peptide is called a monomer. The four monomers are connected by biotin-affinity streptomycin to form a tetramer. Monomer replacement refers to the process of peptide exchange on monomers. Due to different experimental needs, it is necessary to construct tetramers for different antigens. The replacement of different antigens (polypeptide sequences) is called monomer replacement.

mM EBV epitope polypeptide (LMP2 epitope, aa426-aa434, SEQ ID No. 5),

Place on ice;

2) Add 20^1 diluted target polypeptide and HLA-A*02 monomer to the 96-well U-bottom plate, and mix them;

3) Seal the board with tin box paper and the reaction solution to the bottom of the board;

4) UV lamp cross-linking at 365nm for 30min, and incubating at 37°C in the dark for 30min;

5) Take 30M EBV epitope peptide to replace HLA-A*02 monomer in a new plate, add 3.3M fluorescent coupled streptavidin, and place on ice for 30min;

6) During the incubation on ice, prepare the stop solution. Overnight at 4°C, protected from light for 30 minutes on ice, to obtain HLA-A*02 tetramer (hereinafter referred to as tetramer) loaded with EBV epitope polypeptide.

2. EBV antigen peptide specific T cell flow cytometry

Resuspend the stimulated cells in step 1 and count, take out the number of cells for flow sorting 2x l0 ⁵ /tube, add 1ml PBS to resuspend, centrifuge at 500g at 4°C, 5min, carefully discard the supernatant , Add 200M PBS to resuspend; add the tetramer (10^d/ml) prepared in step 1 to the tube, mix and react at 4°C for 30min; after the reaction time is over, add 1ml PBS to resuspend, 500g at 4°C After centrifugation for 5 min, carefully discard the supernatant, add 200 (^1 PBS to resuspend, and place on ice for flow cytometry; after flow cytometry, select a positive population for sorting single cells.

The flow cytometry results after the first round of EBV antigen peptide stimulation are shown in Figure 1, which can be seen: Compared with the control group (the control group is cultured under the same conditions, unstimulated T cells), after stimulation with EBV antigen polypeptide antigen Of T cells can be detected by tetramer 8.9% of positive tumor-specific T cells.

3. EBV antigen peptide specific T cell Elispot detection

Prepare target cells: Count T2 cells, take out the required number of cells, centrifuge at 400g at room temperature, and resuspend in serum-free IMDM medium.

Target cells loaded with EBV antigen polypeptide: Determine the appropriate well plate and loading volume according to the number of cells removed. The EBV antigen polypeptide (LMP2 epitope, aa426-aa434, SEQ ID No. 5) is formulated into 10/(^1, added at 1000X In the loading volume, resuspend and mix, and incubate at 37°C in a 5% CO ₂ incubator for 4 hours. A control group loaded with unrelated peptides is also set.

Prepare effector cells (EBV antigen-peptide-specific T cells selected by flow cytometry after the second round of stimulation): Count the effector cells, take out the desired cells, centrifuge at room temperature 300g, lOmin, and use 5% human serum AIM-V medium Resuspend and place on ice.

Plate washing: When the antigen load remains 45 minutes, remove the reaction well plate in the Human IFN-y ELISpot kit from the ultra-clean table, add PBS, let stand for 30s and take off the liquid in the well. After repeating this action five times, add Incubate with 10% FBS RPMI1640 medium 100M/well, 37°C, 5% CO ₂ incubator for 30min. Sample addition: After the antigen loading time is over, the T2 cells in the well plate are flushed down with 5% human serum AIM-V medium, room temperature 400g, centrifuged for 5 minutes, and resuspended with 5% human serum AIM-V medium. After adding 50M/well of effector cells to the reaction well plate, add 50M/well of the target cell suspension, put it in 37°C, 5% C0 ₂ incubator and incubate for 16-48h; after the incubation time, add PBS 150 well After standing for 30 seconds, the liquid in the well was taken off. After this action was repeated five times, an anti-human IFN-y detection antibody solution (7-b6-l-ALP) was prepared with 0.5% FBS in PBS (0.2 filter filtration). After adding 7-b6-l-ALP antibody to 200X and thoroughly mixing 0.5% FBS in PBS, 100 wells were added to the reaction well plate. The reaction plate was placed in 37°C, 5% C0 ₂ incubator and reacted for 2h; reaction time After the end, add PBS 1500/well, let stand for 30s and take off the liquid in the well. After repeating this action five times, add NBT/BCIP (0.2pm filter membrane filtration) 10 (^1/well into the reaction well, avoid light Avoid light for 30s-5min (take the positive control spot as the end point of the reaction), rinse with plenty of tap water, and observe the results after drying.

The results are shown in Figure 2. Compared with the control group (the control group is the reaction of EBV-specific T cells with irrelevant T2 loaded peptides), EBV antigen peptide-specific T cells can effectively recognize T2-loaded peptides (target cells) and secrete IFN-y. This group of cells is functional.

4. Single cell TCR sequencing

The reagents used are shown in Table 1.

Table 1. Reagents required for single-cell TCR full-length sequencing

The primer sequences used are shown in Table 2.

Table 2. Primers required for single cell TCR full-length sequencing of EBV antigen peptide-specific T cells

(1) Cell lysis

Prepare cell lysis mixture according to Table 3.

When preparing, prepare according to the number of samples 1 10% (if there are 10 cell samples, then prepare 11 tubes). Mix the prepared lysate into a clean PCR tube by pipetting and mixing, centrifuge at 4°C 14000rpm, 30s (centrifuge the droplet to the bottom of the tube and remove air bubbles), place it in an ice box, and divide it into cells afterwards; select positive population (That is, the EBV antigen peptide-specific T cells obtained in step 1) and divide the single cells into the PCR tube containing the lysate; after sorting, cover the tube, centrifuge for a short time, and debug the PCR instrument to prepare the single Cell lysis.

Place the 0.2ml PCR tube in the PCR instrument, incubate at 72°C for 3min (the cell cells are increased to 5min for bulk samples), the temperature of the hot lid is 75°C, and immediately place on ice for 1min after lysis; 10000 rpm 4° C Centrifuge for 30 s, and immediately transfer to ice; after this step, all mRNAs are released from single cells, and Oligo-dT primers have also bound to mRNAs.

(2) Prepare a reverse transcription system according to Table 4.

When preparing, prepare according to the number of samples +0.5 (if there are 9 cell samples, prepare 9.5 tubes). After the prepared Mix is mixed well, add it to the centrifuge tube of the previous step in sequence;

(3) After pipetting and mixing, and instantaneous centrifugation, perform reverse transcription reaction under the conditions shown in Table 5 (hot cover at 75°C).

Table 5. Reverse transcription reaction conditions

After this step, the first strand cDNA synthesis of all mRNAs is completed;

(4) Prepare the first round of PCR Mix according to Table 6.

Table 6. The first round of PCR Mix

When preparing, prepare according to the number of samples +0.5 (if there are 9 cell samples, prepare 9.5 tubes). After the prepared Mix is thoroughly mixed, take 15 _{| i} l in sequence and add it to the centrifuge tube in the previous step. After mixing by pipetting and instantaneous centrifugation, pre-amplify according to the conditions shown in Table 7.

Table 7. Conditions for the first PCR preamplification

(5) Prepare the second round of PCR Mix according to Table 8.

Table 8. Second round PCR Mix

When preparing, prepare according to the number of samples +0.5 (if there are 9 cell samples, prepare 9.5 tubes). After the prepared Mix is thoroughly mixed, take 24 _{| i} l in sequence and add it to the centrifuge tube in the previous step. Pre-amplify according to the conditions shown in Table 9.

Table 9. Pre-amplification conditions for the second round of PCR

(6) Prepare the third round of PCR Mix according to Table 10.

Table 10. Third round PCR Mix

When preparing, prepare according to the number of samples +0.5 (if there are 9 cell samples, prepare 9.5 tubes). After the prepared Mix is thoroughly mixed, take 24 _{| i} l in sequence and add it to the centrifuge tube of the previous step. After mixing by pipetting and instantaneous centrifugation, pre-amplify according to the conditions shown in Table 11.

(7) Electrophoresis detection: After completion of PCR, electrophoresis detection, using 2% agarose gel, take 15 ^ L product, add 3ul Loading buffer mixed hook, 130V electrophoresis for 45min, the target band is cut and recovered. After connecting with T carrier, colony PCR identification was carried out.

Figure 3 is a single-cell TCR amplification electrophoresis diagram; Figure 4 is a TA clone colony PCR electrophoresis diagram. The results in the figure show that the amplified TCR fragment is successfully connected to the T vector, and the successfully constructed insert vector can be effectively detected by colony PCR.

(8) TA clone positive target clone, sent to sanger sequencing to obtain TCRa/p sequence, the sequence is in IMGT The database is compared.

3. Screening and functional verification of EBV antigen peptide specific TCR sequences

1. Sort the collection of single-cell TCR sequences of EBV antigen-peptide-specific T cells obtained in step two according to the order of abundance, select the sequence with the highest abundance (top3) for preliminary functional verification to determine the final Full-length sequence of TCR for treatment. Among them, a functional paired TCR a/p sequence with an abundance of 3%, after finding the start codon, is spliced according to the actual sequence of the constant region (TRAC/TRBC) to form a new sequence. The complete coding gene of a chain The sequence is SEQ ID No. 1 (encoding the a chain shown in SEQ ID No. 3), and the sequence of the complete coding gene of the P chain is SEQ ID No. 2 (encoding the P chain shown in SEQ ID No. 4). Among them, positions 52-381 of SEQ ID No. l are the coding genes of the a chain variable region (positions 127-147, positions 199-213 and positions 316-348 are the coding genes of the three CDRs), Positions 58-384 of SEQ ID No. 2 are the coding genes of the P-chain variable region (positions 136-150, 202-219, and 331-354 are the coding genes of the three CDRs, respectively). The amino acid sequence of the variable region of a chain is the 18th to 127th positions of SEQ ID No. 3 (the 43th to 49th positions, the 67th to 71th positions, and the 106th to 16th positions are three complementary determining regions). The amino acid sequence of the variable region of the P chain is SEQ ID No. 4 positions 20-128 (positions 46-50, 68-73, and positions 11 1-1 18 are three complementary determining regions).

2. Connect the gene encoding the a chain shown in SEQ ID No. 1 and the gene encoding the P chain shown in SEQ ID No. 2 through the gene sequence of the P2A peptide to construct the vector pRRLSIN.cPPT.PGK-GFP.WPRE In this way, a recombinant viral vector is obtained. The structure of the recombinant viral vector is described as follows: The DNA molecule shown in SEQ ID No. 6 is inserted between the restriction enzyme BamHI and Sail of the vector pRRLSIN.cPPT.PGK-GFP.WPRE.

3. Infect the recombinant virus vector constructed in step 2 with T cells, and then perform Elispot detection according to the method in step 2 in step 3.

4. Infect the T cell with the vector pRRLSIN.cPPT.PGK-GFP.WPRE, and then perform Elispot detection (as a control group) according to the method in step 2 of step 2.

The results show that compared with the control group, T cells infected with recombinant viral vectors can effectively recognize T2-loaded EBV antigen polypeptides and secrete IFN-Y, that is, they can effectively react with target cells.

Industrial applications

In the present invention, specific T cells are stimulated in vitro by EBV antigen polypeptides to obtain specific T cell populations, and single cell pair TCR sequencing technology is used to obtain TCR sequence collections of effective T lymphocytes corresponding to EBV antigen polypeptides, and then these sequence collections are passed through Sorted by degree, and the functional verification in vitro was carried out, and finally the TCR claimed by the present invention was obtained. Experiments show that the TCR-expressing T cells provided by the present invention can effectively recognize the EBV antigen polypeptide (target cell) loaded by T2 cells and secrete IFN-Y, proving that this group of T cells is functional. The effective population can be quickly verified by the TCR sequence collection, which can provide more comprehensive and effective information than previous methods. This information can be pushed to the clinic after in vitro functional verification and functional verification of humanized mice, and has a very high clinical Value.

Claims

Rights request

1. A T cell receptor that recognizes EBV antigens, including a chain and P chain;

The a chain contains three complementarity determining regions, and the amino acid sequences are respectively positions 43-49, 67-71, and positions 106-1 16 of SEQ ID No. 3; or these sequences have at most 3, 2 Variants with one or one amino acid change;

The P chain contains three complementarity determining regions, the amino acid sequences are positions 46-50, 68-73, and positions 1 1 1-1 18 of SEQ ID No. 4, respectively; or up to 3 of these sequences , 2 or 1 amino acid variants.

2. The T cell receptor according to claim 1, characterized in that: the amino acid sequence of the variable region of the a chain is positions 18-127 of SEQ ID No. 3; or these sequences have at most 3 , 2 or 1 amino acid variants;

The amino acid sequence of the variable region of the P chain is the 20th to 128th positions of SEQ ID No. 4; or a variant of these sequences having at most 3, 2 or 1 amino acid changes.

3. The T cell receptor according to claim 1 or 2, characterized in that: the amino acid sequence of the constant region of the a chain is positions 128-268 of SEQ ID No. 3; the constant region of the P chain The amino acid sequence of is 129-307 of SEQ ID No. 4.

4. The T cell receptor according to any one of claims 1-3, wherein: the amino acid sequence of the a chain is SEQ ID No. 3; the amino acid sequence of the P chain is SEQ ID No. 4 .

5. A nucleic acid molecule encoding the T cell receptor of any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, wherein: the nucleic acid molecule encoding the T cell receptor comprises a nucleic acid molecule encoding the a chain of the T cell receptor and P encoding the T cell receptor Stranded nucleic acid molecule;

The sequences of the nucleic acid molecules encoding the three complementarity determining regions in the a chain of the T cell receptor are 127-147, 199-213, and 316-348 of SEQ ID No. 1, respectively; or The sequence has a sequence of 99% or more, 95% or more, 90% or more, 85% or more or 80% identity and encodes the same amino acid residue;

The sequences of the nucleic acid molecules encoding the three complementarity determining regions in the P chain of the T cell receptor are 136-150, 202-219, and 331-354 of SEQ ID No. 2, respectively; or The sequence has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity and encodes the same amino acid residue.

7. The nucleic acid molecule according to claim 5 or 6, characterized in that: the sequence of the nucleic acid molecule encoding the variable region of the a chain is positions 52 to 381 of SEQ ID No. 1; or Sequences with 99% or more, 95% or more, 90% or more, 85% or more or 80% identity and encoding the same amino acid residues;

The sequence of the nucleic acid molecule encoding the variable region of the P chain is positions 58-384 of SEQ ID No. 2; or 99% or more, 95% or more, 90% or more, 85% or 80% or more of these sequences Above identity And encode sequences of the same amino acid residues.

8. The nucleic acid molecule according to any one of claims 5-7, characterized in that: the sequence of the nucleic acid molecule encoding the a chain is SEQ ID No. l; the sequence of the nucleic acid molecule encoding the P chain is SEQ ID No. 2.

9. An expression cassette, vector or cell containing the nucleic acid molecule of any one of claims 5-8.

10. The vector according to claim 9, wherein the vector is a nucleic acid encoding the a chain of the T cell receptor inserted between the multiple cloning sites of the vector pRRLSIN.cPPT.PGK-GFP.WPRE The molecule and the nucleic acid molecule encoding the P chain of the T cell receptor, the resulting recombinant plasmid.

11. The cell according to claim 9, wherein the cell is a T cell.

12. A T cell having the T cell receptor of any one of claims 1-4.

13. A pharmaceutical composition containing the carrier or cell according to any one of claims 9 to 11, or containing the T cell according to claim 12.

14. The T cell receptor according to any one of claims 1 to 4, or the nucleic acid molecule according to any one of claims 5 to 8, or the vector according to any one of claims 9 to 11. The use of cells, or, the T cells of claim 12 in the preparation of a medicament for preventing and/or treating diseases caused by EBV infection.

15. The T cell receptor according to any one of claims 1 to 4, or the nucleic acid molecule according to any one of claims 5 to 8, or the vector according to any one of claims 9 to 11. The use of cells, or, the T cells of claim 12 in the prevention and/or treatment of diseases caused by EBV infection.

16. A method for preventing and/or treating diseases caused by EBV infection, comprising the steps of: using the T cell receptor according to any one of claims 1-4, or, any one of claims 5-8 The nucleic acid molecule, or, the vector or cell according to any one of claims 9 to 11, or the T cell according to claim 12, to prevent and/or treat a disease caused by EBV infection.

17. The use according to claim 14 or 15 or the method according to claim 16, characterized in that the disease caused by EBV infection is nasopharyngeal carcinoma and/or oropharyngeal squamous cell tumor and/or T Cell malignancy.