JP6508873B2 - Method for obtaining antigen-specific T cell receptor - Google Patents

Description

  The present invention relates to methods for detecting antigen-specific T cells and obtaining T cell receptors (TCRs). The present invention is useful in the fields of analysis of T cells, analysis of the efficacy of pharmaceuticals such as peptide vaccines, and diagnosis / treatment of diseases.

  In T cell receptor (TCR) gene therapy, which is mainly considered for application to specific cancers, cancer antigen-specific TCR genes are introduced into lymphocytes of cancer patients. The transduced lymphocytes are cultured in large quantities and then returned to the cancer patient, but because the TCR that recognizes the tumor antigen peptide is expressed on the lymphocytes, the cancer cells that present the tumor antigen It can be expected that they will specifically attack and recognize cancer cells eventually.

  In order to obtain an antigen-specific TCR gene for use in gene therapy, a T cell capable of recognizing a cancer antigen is identified from T cells in peripheral blood lymphocytes (PBL) recovered from a patient, and the TCR gene is identified. Need to be cloned.

Non-Patent Document 1 describes a method of stimulating human T cells with an antigen and a cytokine to obtain T cell lines and T cell clones. However, the method requires about 3 months to obtain T cell clones. In addition, Non-Patent Document 2 discloses that an α-chain of an antigen-specific TCR is obtained by capturing a genomic DNA fragment encoding a TCR gene of T cell, decoding a base sequence, and analyzing a large amount of data obtained. It is described that a pair of β chain is predicted, expressed in T cell line, and confirmation of antigen specificity is performed.

On the other hand, we establish a TCR cloning system that not only analyzes the non-biased TCR repertoire but also recovers antigen-specific TCR α / β cDNA pairs and also allows their function to be evaluated. I tried. In other words, we separated antigen-specific T cells with a cell sorter, put them one by one into tubes, and tried to amplify TCR cDNA from single T cells by RT-PCR, but with this method amplification efficiency is very low. , The cloning turned out to be difficult. Then, antigen-specific T cells were placed one by one in a tube as in the previous method, and T cells were stimulated with a stimulator and then RT-PCR was applied, but the low efficiency was not improved. Therefore, when T cells are stimulated in the whole population with a stimulator, and then antigen-specific T cells are sorted one by one into a tube by a cell sorter, RT-PCR is applied, surprisingly, 70-80%. It was found that TCR cDNA can be recovered from cells, and a patent application was filed earlier (Patent Document 1).

PCT / 2013/070028

Current Protocols in Immunology (2002) 7.19.1-7.19.12 Edited by JE Coligan et al Generation and Maintenance of Cloned Human T Cell Lines. High-throughput identification of antigen-specific TCRs by TCR gene capture-Nature Medicine 19: 1534-1541, 2013

Patent Document 1 discloses a method for identifying T cells specific to known antigenic peptides from peripheral blood lymphocytes, performing single sorting with a flow cytometer, and obtaining antigen-specific TCRs from the T cells. It is disclosed. However, after that, in the method, the antigen is specific to cancer cells among the T cells with unknown antigens, such as T cells with unknown antigens, for example, T cells invading cancer tissues of cancer patients. It has proved difficult to identify healthy T cells.

Stimulate T cells directly with antigen-presenting cells such as cancer cells without using peptides etc. Recognize antigen-presenting cells such as cancer cells using the expression of cytokines such as interferon γ or activation markers as indicators The above-mentioned problems can be solved by identifying T cells that react.
Hereinafter, the present invention will be described in detail.

The present invention provides the following:
[1] 1) Stimulating a T cell group containing T cells specific for antigen a with antigen presenting cells;
2) identifying T cells specific for antigen a from T cells including T cells specific for antigen a and sorting them into containers one by one; and 3) activating one in the container A method for producing a TCR gene specific to antigen a, comprising the step of subjecting antigen a-specific T cells to PCR to amplify a TCR gene specific to antigen a.
[2] Furthermore,
4) The production method according to 1, which comprises the steps of expressing the obtained TCR gene in a T cell line and analyzing its antigen specificity.
[3] The production method according to [1] or [2], wherein all the steps are performed within 10 days.
[4] The antigen-presenting cell of step 1) is a cancer cell; a virus-infected cell; a cell artificially expressing a cancer antigen protein, a virus protein or a fragment thereof [1] to [3] The manufacturing method described in 1).
[5] The method according to claim 4, wherein the stimulation on the antigen-presenting cells is co-culture of a T cell group and an antigen-presenting cells.
[6] The production method according to any one of [1] to [5], wherein step 2) is performed by flow cytometry or immunospot-array assay on a chip (ISAAC) method.
[7] The production method according to [6], wherein step 2) is a step of sorting by flow cytometry using CD137.
[8] [1] to [7], and a TCR gene specific to the obtained antigen a is introduced into another T cell to recombine specific for the antigen a. A method of producing a recombinant T cell, further comprising the step of obtaining T cells.

  According to the present invention, a T cell specific to a cancer cell is identified from among T cells in which an antigen infiltrates an unknown cancer tissue in a short period of time, for example, within about 10 days, and its TCR is It became easy to get it.

FIG. 1 is a schematic view of the hTEC 10 system, one aspect to which the present invention is applied. FIG. 8 shows the TCR of donor B (Donor B) cloned by MHC tetramer using hTEC10. FIG. 5 shows detection of antigen specific T cells by artificial antigen presenting cells. The BRLF1 expression vector is introduced into artificial antigen-presenting cells, and BRLF1 protein is expressed in cells. Stimulate donor B T cells with these cells and evaluate the activation marker CD137. It is a figure which shows the structure of an expression vector. The expression vector to be introduced into artificial antigen-presenting cells was added to the BRLF1 expression vector, and a BRLF1-DNA-free Mock vector as a negative control and a BRLF1 antigen peptide expression vector as a positive control FIG. 5 is a diagram of flow cytometry for detecting antigen-specific T cells activated by stimulation of artificial antigen-presenting cells. In the upper panel, PBLs are identified, and in the upper panel, the CD8 positive CD137 positive cells activated by the lower panel are identified (P4 gate) except for the antigen-presenting cells which slightly enter. It is the figure which amplified the TCR gene by single cell RT-PCR from the collect | recovered single cell. Four out of 30 are amplified in pairs by the TCRα / β gene (3, 9, 16, 25).

In the present invention and the specification, when “「 ”represents a numerical range, the range includes the numerical values at both ends, unless otherwise specified.
Further, in the present invention, the terms "alone" and "one" in the context of cell status mean that the environment is free from cells other than the cells, unless otherwise specified.
Moreover, when saying "group" and "group", it means that it exists in the environment where several cells exist.

The present invention provides methods for detecting antigen-specific T cells and obtaining T cell receptors (TCRs). More specifically, the present invention provides a method for producing a TCR gene specific to antigen a, which comprises the following steps:
1) Stimulation of T cells including T cells specific for antigen a with antigen presenting cells;
2) identifying T cells specific for antigen a from T cells including T cells specific for antigen a and sorting them one by one into a container; and 3) activating one in the container Subjecting the selected antigen a-specific T cells to PCR to amplify a TCR gene specific for antigen a

  Step 1) is a step of stimulating a T cell group with antigen-presenting cells. Specifically, as antigen-presenting cells, for example, cancer cells; virus-infected cells; cells selected from cells artificially expressing cancer antigen proteins, virus proteins or fragments thereof and T cell groups It is to co-culture.

Co-culture may be performed according to a known method, but in one embodiment of the present invention, the T cell group and the antigen-presenting cells have the same number of cell densities, specifically 1 × 10 ⁵ to 1 × 10 ⁷ , for example. Suspend both cells at cells / mL, preferably 5 × 10 ^{5 to} 5 × 10 ⁶ cells / mL, and culture it under conditions such as normal temperature for culturing T cells using an appropriate incubator. Good. The culture time may be 3 hours to 24 hours, preferably 6 hours to 12 hours.

  Step 2) is a step of identifying T cells of interest and sorting them one by one into a container for PCR. This step can be performed by various existing means such as flow cytometry or immunospot assay (ISAAC) method using a chip.

  In one embodiment of the present invention, step 2) is performed by sorting by flow cytometry using CD137 of the activation marker as an index.

  Step 2) can also be performed by the ISAAC method. This method is carried out using a microwell array having a plurality of wells on one major surface of a substrate, the wells being sized to receive only one T cell. With regard to the method using a microwell array, reference may be made to Patent 4148367 and the like.

  Step 3) is a step of subjecting one activated (which is the same as stimulated) antigen a-specific T cell in a container to PCR to amplify an antigen A-specific TCR gene. . Although the identified cells may be proliferated and then subjected to PCR, various PCR methods capable of amplifying cDNA for one or several cells are known, and in the present invention, Known methods can be applied. Typically, cell lysis is performed first, followed by cDNA synthesis from mRNA by reverse transcription using dT adapter primer (RT dT Primer 2). The obtained cDNA may be amplified or used as it is as a template and real time PCR (quantitative PCR, qPCR) is performed using appropriately designed primers.

  In one aspect of the invention, steps 1), 2) and 3) are carried out in this order.

  The method of the present invention may further comprise, as step 4), introducing the obtained TCR into a T cell line not expressing the TCR, expressing it, and verifying its antigen specificity.

According to the method of the present invention, T cells specific to cancer cells and the like can be identified from T cell groups whose antigens are unknown. Also, a method for producing a recombinant T cell, further comprising the step of introducing a TCR gene specific for the antigen a obtained by the method of the present invention into another T cell to obtain a recombinant T cell specific for the antigen a. The implementation time can be shortened.
Hereinafter, the present invention will be described by way of examples.

〔Method〕
Experiments with healthy donors and classified humans of HLA were carried out with the approval of the Ethics Committee of Toyama University. Informed consent was obtained from all subjects. Peripheral blood lymphocytes (PBL) were separated from heparinized blood samples by density gradient centrifugation using Ficoll-Hypaque (Immuno-BioloGical Laboratories). Screening for HLA-A24 haplotype positivity was carried out by flow cytometry analysis after staining PBL with anti-HLA-A24 antibody (One Lambda) followed by FITC-labeled anti-mouse IgG antibody (ICN / Cappel) .

The cell line RPMI 1640 and DMEM medium (Wako Pure Chemical) were supplemented with 10% heat-inactivated fetal bovine serum (Biowest), 100 μg / ml streptomycin, and 100 U / ml penicillin to culture the cells. K562 cells (kindly provided by Dr. Mizukoshi of Kanazawa University) were maintained in RPMI 1640 medium. Phoenix-A cells (kindly provided by Dr. G. Nolan of Stanford University) were maintained in DMEM medium.

Preparation of artificial antigen-presenting cells HLA-A * 2402 gene (kindly provided by Dr. Kuzushima of Aichi Cancer Center Research Institute), human CD80 gene (purchased from Origene), human CD137L gene (Origene) The cDNA encoding the gene is inserted into the pMX vector (provided kindly to Dr. Kitamura, The University of Tokyo) and then into the retroviral packaging cell Phoenix-A, FuGENE 6 ( Each was transfected using Roche). Seventy-two hours after transfection, culture supernatants containing recombinant retrovirus were collected from transfected Phoenix-A. Collected retroviral supernatants were added together to plates that had been coated overnight with 50 μg / ml retronectin (TaKaRa). The retrovirus in the supernatant was spin-loaded onto the plate by centrifugation at 1900 xg for 2 hours at 32 ° C. 0.2 × 10 ⁶ / mL K562 cells were added to the wells in the retrovirus loaded plate. The plate was centrifuged at 1000 × g for 10 minutes at 32 ° C., and then cultured at 37 ° C. in 5% CO ₂ . On the 5th day of culture, cells expressing HLA-A * 2402, CD80 and CD137L were sorted by flow cytometry and used as artificial antigen presenting cells (aAPC).

Preparation of artificial antigen-presenting cells presenting antigen expressed in cells DNA encoding BRLF1 which is Epstein-Barr (EB) virus related antigen and DNA encoding BRLF1 peptide (TYPVLEEME) as positive control pMX vector (The University of Tokyo (PMX vector without any introduction as a negative control) and then into the retroviral packaging cell Phoenix-A with FuGENE 6 (Roche) Each was used for transfection. Seventy-two hours after transfection, culture supernatants containing recombinant retrovirus were collected from transfected Phoenix-A. Collected retroviral supernatants were added together to plates that had been coated overnight with 50 μg / mL retronectin (TaKaRa). The retrovirus in the supernatant was spin-loaded onto the plate by centrifugation at 1900 xg for 2 hours at 32 ° C. 0.2 × 10 ⁶ / mL of aAPC was added to the wells in the retrovirus loaded plate. The plate was centrifuged at 1000 × g for 10 minutes at 32 ° C., and then cultured at 37 ° C. in 5% CO ₂ . On the fifth day of culture, cells into which GFP had been introduced were each sorted by flow cytometry, and this was used as aAPC to present an antigen expressed intracellularly (FIG. 3 and FIG. 4).

Detection of T cells that respond to artificial antigen-presenting cells presenting antigens expressed in cells Peripheral blood lymphocytes of Donor B (Donor B) are revealed to have one type of BRLF1-specific T cells (FIG. 3: Patent Document 1). Therefore, in order to evaluate this system, a TCR gene that responds to aAPC (aAPC-BRLF1-DNA) expressing BRLF1 in cells from peripheral blood lymphocytes of donor B is cloned, and this is a known donor B-derived TCR. It verified that it matched. That is, 1 × 10 ⁵ cells of donor B-derived peripheral blood lymphocytes were co-cultured with 1 × 10 ⁵ cells aAPC-BRLF1-DNA in a 96-well plate at 37 ° C. for 8 hours. Thereafter, CD137 positive cells were detected using flow cytometry.

Single cell RT-PCR
CD137-positive cells activated by co-culture with aAPC-BRLF1-DNA were analyzed using FACSAria II (Becton Dickinson) in 29.2 μg of Dynabeads Oligo (dT) (Invitrogen), 2.9 μL of lysis / binding buffer The cells were single cell-sorted into MicroAmp® reaction tubes (Applied Biosystems) containing cell lysis solution consisting of a solution (Invitrogen) and primers specific for 0.29 pmol of each gene.

The sequences of the primers were as follows:
alpha-RT (5'-AGCAGTGTTTGTGCAGCTCTT-3 ', SEQ ID NO: 1),
beta1-RT (5'-CTGGCAAAAGAAGAATGTGT-3 ', SEQ ID NO: 2), and beta2-RT (5'-ACACAGATTGGGAGCAGGTA-3', SEQ ID NO: 3).

The cells were lysed in a tube. Then, poly-A RNA was bound to Oligo (dT) on Dynabead. The Dynabeads were then compared to 4.0 U SuperScript III (Invitrogen), 0.3 U mouse RNase inhibitor (New England Biolabs), 0.5 mM each dNTP, 5 mM DTT, 0.2% Triton X-100, and 1 × First Transfer into a solution containing Strand Buffer (Invitrogen). Reverse transcription (RT) reaction was performed at 50 ° C. for 40 minutes. After RT reaction, Dynabeads in 8 U terminal deoxynucleotidyl transferase (Roche), 0.5 mM dGTP, 0.4 U mouse RNase inhibitor, 4 mM MGCl ₂ , 0.2% Triton-X 100, 5% PK buffer It was transferred into another solution containing [1 M K ₂ HPO ₄ and 1 M KH ₂ PO ₄ , pH 7.0] and incubated at 37 ° C. for 40 minutes to add poly dG to the 3 'end of the cDNA. The Dynabeads were then transferred into a new PCR tube containing the first PCR reaction mixture. The first PCR was performed using PrimeSTAR HS DNA polymerase (TaKaRa) and the AP-1, alpha-1st, beta1-1st and beta2-1st primers according to the manufacturer's instructions.

  AP-1 (5'-ACAGCAGGTCAGTCAAGCAGTAGCAGCAGTTCGATAACTTCGAATTCTGCAGTCGACGGTACCGCGGGCCGGGATCCCCCCCCCCCCCDCN-3 ', SEQ ID NO: 4), alpha-1st (5'-AGAGGGAGAGAGAGGGGGCAAT-3', SEQ ID NO: 5), beta1-1st (5'-CCATGACGG GGT, SEQ ID NO: 4) And the PCR cycle with beta2-1st (5'-GGATGAAGAATGACCTGGGAT-3 ', SEQ ID NO: 7) was as follows: 5 minutes at 95 ° C, followed by 30 cycles of 15 seconds at 95 ° C, 60 ° C For 5 seconds, and 1 minute 30 seconds at 72 ° C.

  The resulting PCR product was diluted 100-fold with water, and 2 μL of the diluted PCR product was added as a template DNA to a 23 μL nested PCR reaction solution. Nested PCR is performed using adapter primer AP-2 (5'-AGCAGTAGCAGCAGTTCGATAA-3 ', SEQ ID NO: 8) and a primer specific for the constant region of TCRα (alpha-Nest; 5'-GGTGAATAGGCAGACAGACTT-3', SEQ ID NO: 9) or The reaction was carried out using the same reaction solution as in the first PCR except that a primer specific to the constant region of TCRβ (beta-Nest; 5'-GTGGCCAGGCACACCACCGTGT-3 ', SEQ ID NO: 10) was used. The PCR cycles were as follows: 98 ° C for 1 minute, followed by 35 cycles of 98 ° C for 15 seconds, 60 ° C for 5 seconds, and 72 ° C for 45 seconds.

The PCR product was then directly sequenced using alpha-nest or beta-nest primers, or alternatively, the DNA sequence was determined after subcloning of the PCR product into an expression vector. The TCR repertoire was analyzed using the IMGT / V-Quest tool (http://www.imgt.org/) (Reference 1).
Reference 1: Giudicelli,
V., Chaume, D. & Lefranc, MP IMGT / V-QUEST, an integrated software program
for immunoglobulin and T cell receptor VJ and VDJ rearrangement analysis. Nucleic acid
Acids Res 32, W435-440 (2004).

<Result>
1 × 10 ⁵ cells of donor B-derived peripheral blood lymphocytes were co-cultured with 1 × 10 ⁵ cells aAPC-BRLF1-DNA in a 96-well plate at 37 ° C. for 8 hours. Thereafter, CD137 positive cells were detected using flow cytometry. As a result of detection, the frequency of CD137 positive cells in CD8 positive T cells was 0.5%. The negative control was 0.1% and the positive control was 0.8%.

Then, 15 CD137 positive cells were single-cell separated using FACS. Furthermore, 4 pairs of TCR α and β cDNAs were amplified from isolated single cells using the 5 ′ RACE method (FIG. 6).
The amplification efficiency of RT-PCR was α: 17/90 (18.9%), β: 26/90 (28.9%), and the pair ratio: 17/90 (18.9%).

Next, it was verified whether each TCR gene matched with a known TCR gene. As a result, two out of the four pairs obtained matched the known TCR gene (Table 1).

Claims (2)

1) The T cells by co-culturing a T cell group containing T cells specific for cancer cells and an antigen-presenting cell in which a gene encoding a human CD80 gene and a gene encoding a human CD137L gene has been introduced into the cancer cells Stimulating the group;
2) sorting the CD137 positive cells from the T cell group containing the stimulated T cells by flow cytometry using the activation marker CD137 as an index;
3) subjecting the single cells of the sorted CD137 positive cells to PCR to amplify a TCR gene specific to the cancer cell, and a method for producing a TCR gene specific to a cancer cell.   A method for producing a recombinant T cell, wherein a TCR gene specific to a cancer cell obtained by the method according to claim 1 is introduced into another T cell to obtain a recombinant T cell.