Method for producing human t-cell receptor specific for epitope 100-108 vldgldvll prame

Abstract

FIELD: biotechnology.SUBSTANCE: method for producing a human T-cell receptor specific to the epitope 100–108 VLDGLDVLL, obtained from the PRAME protein. The claimed method involves sequencing the RNA of single cells, which allows for obtaining complete information about the structure of a specific T-cell receptor on each cell and using a significantly smaller number of target cells for this purpose.EFFECT: obtaining, in a short period of time, more accurate information on the structure and affinity of the human T-cell receptor specific to the epitope 100–108 VLDGLDVLL PRAME, which has a high affinity for the target peptide and minimal affinity for non-target peptides.1 cl, 3 dwg

Description

[0001] The invention relates to the field of biotechnology, specifically to immunology, and is aimed at obtaining a construct encoding a sequence of a T-cell receptor TCR that recognizes an antigen epitope of the PRAME protein and is capable of inducing antigen-specific destruction of a target cell expressing PRAME.

[0002] In the structure of mortality of the population of Russia, malignant neoplasms rank second (15.4%) after diseases of the circulatory system. The leading localizations of malignant tumors in both sexes are skin (12.3%, with melanoma - 14.0%), mammary gland (11.4%), trachea, bronchi, lung (10.5%), stomach (7.0%). At the same time, the leading oncological pathology in the female population is breast cancer (20.9%), the mortality rate from which ranks first (17.0%) (Ed. by A.D. Kaprin, V.V. Starinsky, G.V. Petrova Malignant neoplasms in Russia in 2013 (incidence and mortality) M .: FGBU "P.A. Herzen Moscow Research Institute of Oncology" of the Ministry of Health of the Russian Federation. - 2015. ill. 250 p.).

[0003] The currently accepted methods of cancer treatment are surgery, radiation therapy, and chemotherapy. These methods effectively reduce tumor burden in a short period of time, but they do not eliminate all tumor cells disseminated within the patient's body. A minimal tumor burden, remaining after removal of the main portion of the tumor, is a cause of tumor recurrence and the development of metastases, which in turn leads to increased mortality and disability in cancer patients.

[0004] Thus, traditional approaches to cancer treatment reveal shortcomings in eliminating minimal tumor load, and the need for new technologies aimed at improving the processes of recognition and destruction of individual tumor cells remaining in the patient’s body after radiation therapy, chemotherapy, or surgical removal of the bulk of the tumor becomes obvious.

[0005] Basic and clinical studies of recent years confirm that using the potential of the immune system can be very promising for eliminating tumor cells (Palucka K., Ueno H., Banchereau J. Recent developments in cancer vaccines. / / JImmunol. - 2011. - Vol. 186. - №3. - P. 1325-31; Qian X., Wang X., Jin H. Cell transfer therapy for cancer: past, present, and future. / / JImmunolRes. - 2014. doi: 10.1155 / 2014 / 525913). Immunotherapeutic approaches make it possible to activate the cellular component of immunity, targeting a specific immune response against tumor cells carrying tumor antigens on their surface, without damaging healthy cells. In view of the above, it is advisable to develop approaches that allow for the effective generation of a specific antitumor immune response in the patient’s body.

[0006] To date, several different immunotherapeutic approaches have been developed aimed at various forms of melanoma and other neoplasms expressing PRAME (Goodison S, Urquidi V. The cancer testis antigen PRAME as a biomarker for solid tumor cancer management. Biomark Med. 2012 Oct;6(5):629-32. doi: 10.2217 / bmm.12.65.). Immunotherapy against the PRAME antigen is aimed at activating effector cells.

[0007] Cytotoxic T lymphocytes play a key role in the development of a specific antitumor immune response. The presence of antitumor cytotoxic T lymphocytes (CTLs), both in terms of their quantity and normal functioning, is a prerequisite for the destruction of tumor cells by the immune system (Aerts J., Hegmans J. Tumor-specific cytotoxic T cells are crucial for the efficacy of immunomodulatory antibodies in patients with lung cancer. / / Cancer Res. - 2013. - Vol. 73. - P. 2381-2388). Immunotherapeutic approaches aimed at combating specific tumor-associated antigens also use activated antigen-specific cytotoxic T lymphocytes as the main antitumor agent.

[0008] The closest method to the claimed one is for producing a human T-cell receptor specific to the epitope 100-108 VLDGLDVLL, obtained from the PRAME protein, including the isolation of peripheral blood mononuclear cells (MNCs) of HLA-A0201+ donors, followed by stimulation with a synthetic short peptide VLDGLDVLL (SEQ ID No.; Peking Saibance Gene technology, Inc.). The short peptide VLDGLDVLL and HLA-A0201 with a biotin label were annealed to obtain the pHLA haploid. These haploids were combined with PE-labeled streptavidin (BD Co.) to form PE-labeled tetramers, which were sorted into double-positive anti-CD8-APC cells. The sorted cells were expanded and re-sorted, followed by single cloning by limiting dilution. Monoclonal cells were stained with tetramer, and double-positive clones were selected. Further analysis was performed using Quick-RNA. TMMiniPrep (ZYMO Research) was used to extract total RNA from a T-cell clone specific for the short antigenic peptide VLDGLDVLL and restricted to HLA-A0201. cDNA was synthesized from the obtained RNA using the SMART RACE cDNA amplification kit from clontech, using primers designed to preserve the C-terminal region of the human TCR gene. The sequences were cloned into the T vector (TAKARA) and sequenced. It should be noted that the sequence is complementary, excluding introns. Sequence structures of the TCR alpha chain and beta chain expressed by the double-positive clone. The resulting CD8+ T cells, transduced with lentiviruses carrying the TCR gene specific for the short antigenic peptide VLDGLDVLL, were then stimulated with anti-CD3 / CD28-coated beads (T-cell amplicons, life technologies). Also, the resulting TCR T cells were cultured in the presence of growth factors: 10% FBS with 50 IU / ml IL-2 and 10 ng / ml IL-7 for 9-12 days after transduction.The function of the obtained effector cells transduced with TCR specific to the antigen short peptide VLDGLDVLL and restricted to HLA-A0201 was assessed by the specific reaction (IFN-γ production by the ELISPOT method) of activation of TCR-transduced T cells to target cells. PRAME-positive cell lines (293T-PRAME, SW620-PRAME, K562-A2, MEL526) were used as target cells, while PRAME-negative ones (SW620 and K526-A24) served as controls (patent "TCR (T cell receptor) for recognizing PRAME (platelet-derived antigen) antigen short peptide", CN108264550B, C07K 14 / 435, 2018).

[0009] A disadvantage of the prototype is the lack of use of antigen-presenting cells and the use of only the synthetic short peptide VLDGLDVLL as a stimulator. Evaluation of IFN-γ production by the resulting TCR T cells in response to co-culture with tumor cell lines can only indirectly reflect the cytotoxic cellular response. These shortcomings preclude obtaining complete information on the antitumor cytotoxic activity of cells transduced with the T cell receptor to the epitope 100-108 of VLDGLDVLL PRAME.

[0010] The objective of the invention is to obtain, in a short period of time, accurate information about the human T-cell receptor specific to the epitope 100-108 VLDGLDVLL PRAME.

[0011] The problem is solved in that in the method for producing a human T-cell receptor specific to the epitope 100-108 VLDGLDVLL, obtained from the PRAME protein, including the isolation of mononuclear cells (MNC) of peripheral blood HLA-A0201+ donors, then MNC are cultured in the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 4 days; on the 5th day, the epitope 100-108 VLDGLDVLL is added to the dendritic cell culture as a priming factor, followed by their incubation at a temperature of 37 ° C; on the 6th day, a maturation factor is added to complete the maturation of dendritic cells; then the obtained dendritic cells are collected, washed with a nutrient medium, the number of dendritic cells is counted, their viability is assessed and they are co-cultured with autologous CD8+ T lymphocytes in a certain ratio, and after 7 days of co-cultivation, antigen-specific CD8+ T cells are isolated,then, the TCR gene sequence is identified and the variable region of the TCRα and TCRβ chains, including CDR3, isolated from T cell clones is amplified by sequencing, wherein the TCRα and TCRβ chains are connected by a 2A peptide linker, in the form of TCRb-P2A-TCRa, and the complete TCR construct is cloned into the backbone of a viral plasmid to obtain TCR complexes, wherein the donors are conditionally healthy individuals, from whose blood monocytes were isolated on a Ficoll gradient, on the 5th day, the epitope 100-108 VLDGLDVLL is added as a priming factor at a dose of 100 μg / ml, followed by incubation for 24 hours, on the 6th day, TNF-α is added as a maturation factor at a concentration of 25 ng / ml for 24 hours, and co-cultivation dendritic cells are carried out with CD8+ T lymphocytes in the presence of 0.5 μg / ml antibody to CD3, 1 μg / ml antibody to CD28 and IL-2, IL-7, IL-15 at 10 ng / ml each, in a ratio of 1:10,After 7 days of co-cultivation, antigen-specific CD8+ T cells are isolated using Flex-T reagents, followed by identification of the TCR gene sequence using single-cell RNA sequencing (scRNA-seq) and selection of TCR clones specific to the 100-108 VLDGLDVLL PRAME epitope using ERGO-II neural networks and the ClonoSort program. In addition to connecting the TCRα and TCRβ chains with a 2A peptide linker, in the form of TCRb-P2A-TCRa, 5' and 3' untranslated regions of beta-globulin at the ends of the insert are used. The HIV-1-based lentivirus pLenti_hPGK is used as a viral plasmid, obtaining one dominant TCR with one alpha and one beta chain originating from the same cell.

[0012] We believe this invention is novel. A key feature of this method is its rapid, more accurate information on the structure and affinity of the human T-cell receptor specific to the 100-108 VLDGLDVLL PRAME epitope, which exhibits high affinity for the target peptide and minimal affinity for non-target peptides.

[0013] This is achieved, in particular, by using a more accurate method of cellular RNA analysis (single-cell RNA sequencing), which allows for obtaining complete information on the structure of a specific T-cell receptor (α-chain and β-chain) on each cell and requires a significantly smaller number of target cells, allowing for the necessary information to be obtained using donor biological material. This method eliminates the need for additional procedures (vaccination of donors with cells loaded with the target antigen and prolonged cultivation of the test cells to obtain the required number) and reduces the time required to obtain the T-cell receptor. Using ERGO-II neural networks and the ClonoSort program (https: / / github.com / Perik-Zavodskaia / ClonoSort) allows for the selection of a TCR clone for the PRAME antigen with high affinity for the target peptide and minimal affinity for non-target peptides.Using Flex-T, which is a more specific method for detecting antigen-specific cells.

[0014] The method is as follows.

[0015] 1. Obtaining antigen-specific T cells.

[0016] Mononuclear cells (MNCs) were isolated from the peripheral blood of healthy donors with the HLA-A0201 genotype. MNCs were separated into adherent and non-adherent fractions by adhesion to plastic for 30 minutes at +37°C in a CO2 incubator. Mature dendritic cells (DCs) were isolated from the adherent fraction using 100 ng / ml GM-CSF and 50 ng / ml IL-4. The VLDGLDVLL 100-108 peptide, derived from the PRAME protein, was used for antigen loading at a dose of 100 μg / ml on the 5th day of culture, followed by DC maturation using 25 ng / ml TNF-alpha on the 6th day of culture. Non-adherent MNC cells were cultured for 6 days. On day 7, DC and CD8+ cell cultures were combined (1:10 ratio) with the addition of 0.5 μg / ml anti-CD3 antibody, 1 μg / ml anti-CD28 antibody, and IL-2, IL-7, and IL-15 at 10 ng / ml each to maintain the viability and proliferation of CD8+ cytotoxic T lymphocytes.

[0017] After 7-8 days of co-culture, antigen-specific T cells were isolated from the co-culture of DCs and CD8+ cells using Flex-T reagents and corresponding peptides, with simultaneous staining of positive specific cells with two fluorochromes and anti-CD8+ antibodies for identification by flow sorting.

[0018] After a full cycle of the antigen-specific T cell production protocol, two samples specific for the 100-108 VLDGLDVLL epitope, the PRAME antigen, were obtained. Cell viability was assessed using 7AAD and calcein stains. The 7AAD-negative / calcein-positive cell content in the sample was at least 80%. The obtained cells were labeled with SampleTag antibodies for multiplexing biological samples for use on the BD Rhapsody single-cell multiome platform.

[0019] Twenty-seven thousand T cells labeled with multiplexing antibodies were loaded onto a BD Rhapsody Express, where metal beads with molecular barcodes were added to capture poly-A-carrier molecules (mRNA, including the alpha and beta chains of the TCR and SampleTag), cells were lysed, and poly-A-carrier molecules from the lysed cells were hybridized to the metal beads. The poly-A-carrier molecules were then reverse transcribed to generate cDNA. Template Switch Oligo (TSO) was added to the cDNA, and another round of reverse transcription was performed, allowing reads to be generated from the 5' end of the cDNA. The cDNA was amplified in two rounds of semi-nested PCR. Then, random primer annealing and extension were performed for the TCR library.To obtain the final immune transcriptome libraries (379 genes associated with the immune response), TCR, and SampleTag, the products of the second semi-nested PCR of the SampleTag and immune transcriptome libraries, and the products of random annealing and elongation of the TCR library were amplified in nested PCR with primers containing adapters for Illumina sequencers. The resulting libraries were pooled at 40,000 loaded cells and 10,000 reads per cell for the immune transcriptome library, 15,000 reads per cell for the TCR library, and 1,200 reads per cell for the SampleTag library. They were sequenced on a NovaSeq 6000 instrument on the S2 cell with 150 paired-end reads.

[0020] The resulting FASTQ files were processed using the BD pipeline version 1.11.1L, which performs quality control of reads, their alignment to the reference transcriptome, including T-cell receptors, builds libraries of cell indices, eliminates PCR effects using UMI RSEC (unique molecular identifier recursive substitution error correction) and UMI DBEC (unique molecular identifier distribution-based error correction), builds cell libraries, eliminates technical noise using second derivative analysis and produces final gene expression matrices and Adaptive Immune Receptor Repertoire (AIRR) - matrices of sequences of the alpha and beta chains of the T-cell receptor.

[0021] We obtained 7,700 single cells. Bioinformatics analysis of the sequencing data was performed using the ClonoSort software tool (https: / / github.com / Perik-Zavodskaia / ClonoSort). ClonoSort is a tool for determining dominant (by the number of single cells) T-cell receptor clonotypes by CDR3 loop or by complete amino acid sequences. In the first stage, CPM (Counts Per Million) normalization of the gene expression matrix is ​​performed, then the matrix is ​​transformed by log2. In the second stage, only CD8 + Using quadrant gating for the CD4 and CD8a genes, TCR contigs are selected, cross-linked via the alpha and beta chain spacer (CDR3 / full-length TCR), and the number of cells with identical pairs of cross-linked sequences is counted. Approximately 30 clonotypes were detected for the PRAME epitope.

[0022] It is known that many CAR-T and TCR-T cells have an off-target effect due to their potential to interact with related proteins. To mitigate this effect, a computer analysis of the interactions of the resulting TCRs with peptides of related proteins was performed in silico. Using the ERGO-II neural network, the probability of TCR binding to a peptide / MHC complex (peptides from PRAME proteins) was predicted in silico. Using ERGO-II, we can predict the affinity of the resulting TCRs for a peptide, facilitating the selection of candidates from the TCR pool depending on the desired application.

[0023] After downloading the ERGO-II neural network repository, the tool was launched from the terminal, selecting the input file and the database (McPAS) on which the model was trained. McPAS-TCR is a manually curated database based on published literature, containing information on approximately 40,000 T-cell receptor sequences, the antigens they bind to, the T-cell type (CD4+ / CD8 + ) and MHC type (MHC-I / MHC-II class). McPAS-TCR includes information on T lymphocytes expanding in various human or mouse pathological conditions (including viral infections, cancer, and autoimmune reactions). The input CSV file for running ERGO-II contains information on the TCR CDR3α and CDR3β sequence, peptide sequence, MHC type (MHC-I / MHC-II class), V and J genes, and T cell type (CD4 + / CD8 + ). The output file contains a predicted probability of T-cell receptor binding to the peptide / MHC complex (normalized affinity score), which ranges from 0 to 1, where 0 represents minimum affinity and 1 represents maximum affinity. Clones with a normalized affinity score greater than 0.5 were selected.

[0024] Based on the analysis of clonotype dominance using ClonoSort, their affinity to the target peptide and minimum affinity to non-target peptides using ERGO-II neural networks, the clone with the highest cell number was selected.

[0025] Next, a lentiviral vector based on HIV-1 (pLenti_hPGK_gfp) with an insert instead of the EGFP gene was constructed. The pLenti_hPGK plasmid scheme is shown in Fig. 1 and used to generate TCR-T cells specific for the PRAME epitope. The lentiviral vector transfer plasmid included the TCRa and TCRb sequences in a single reading frame, separated by a signal for the polypeptide ejection with the P2A polysem. The scheme for constructing the insert for the plasmid is shown in Fig. 2.

[0026] The following protocol was used to synthesize and clone the specific gene encoding the PRAME epitope-specific TCR:

[0027] 1. Gene synthesis by PCR (polymerase chain reaction).

[0028] The gene encoding TCR was synthesized by PCR amplification. Primers were first synthesized, the sequences of which are shown.

[0029] PCR was performed using the ThermoFisherScientific kit (#F549), which includes Phusion Hot Start II DNA polymerase, 5x HF and GC buffers, 50 mM MgCl2, and DMSO. PCR was performed on a Bio Rad Thermal cycler C-1000. Flanking primers For and Rev were used at a concentration of 10 μM. The remaining primers were used at a concentration of 2 μM. The reaction mixture for the first PCR was prepared as follows (final volume 50 ml): 10 μl 5X GC buffer, 10 μl equimolar primer solution, 0.5 μl dNTP mixture (25 mM each), 1 μl Phusion HS II polymerase, 28.5 μl mQ. Mode: initial denaturation (1 cycle): 98 °C - 2 min; amplification (15 cycles): 98 °C - 10 sec, 60 °C - 15 sec, 72 °C - 2.5 min; presynthesis (1 cycle): 72 °C - 5 min; storage at + 4 °C.

[0030] After completion of the first PCR, the second PCR was performed to obtain amplicons of the target length (2050 bp). The reaction mixture for the second PCR was prepared as follows (final volume 50 ml): 10 μl 5X GC buffer, 2 μl working solution of flanking primers (10 μM each), 6 μl reaction mixture obtained after the first stage PCR, 0.5 μl dNTP mixture (25 mM each), 1 μl Phusion HS II polymerase, 30.5 μl mQ. Mode: initial denaturation (1 cycle): 98 °C- 2 min; amplification (25 cycles): 98 °C - 15 sec, 50 °C - 15 sec, 72 °C - 2.5 sec; presynthesis (1 cycle): 72 °C - 5 min; storage at + 4 °C.

[0031] After the second PCR, horizontal gel electrophoresis was performed in a 1% agarose gel at 6 V / cm for at least 30 minutes. PCR fragments of 2050 bp in length were visible in the gel. The fragment was excised from the gel and purified using the commercial LumiPure agarose gel DNA extraction kit.

[0032] 2. Cloning into the intermediate vector pUC57:

[0033] The pUC57 vector, linearized at the EcoRV site, was used as the cloning vector. Ligation reactions were performed using Thermo Scientific™ T4 DNA ligase (5 units / µl) (#EL0011). The vector:purified PCR product ratio was 1:3. Incubation was carried out for 16-18 hours at +4°C.

[0034] The ligase mixture was transformed into E. coli XL1-blue cells for chemical transformation Eurogene CC001 according to the manufacturer's protocol. Then, these transformed cells were plated on LB agar Petri dishes containing X-gal, IPTG and ampicillin at a dose of 100 μg / ml. The dishes were incubated for 16-18 h at +37C. Verification of white clones was performed by PCR screening using specific primers for the insert (M13 for (5-GTAAAACGACGGCCAGT-3), M13_rev (5-AGCGGATAACAATTTCACACAGGA-3).). For screening, the reaction mixture for PCR Basic, 2x Lumiprobe #5024 were used. The expected size of the PCR product was 2196 bp. Clones that yielded positive PCR results were cultured in liquid LB medium (5 ml, ampicillin 100 μg / ml). Plasmid isolation was performed the following day (16 hours later) using the LumiPure Plasmid DNA Isolation Kit (Spin Miniprep), Lumiprobe #1583. Suitable clones were then selected by sequencing.

[0035] 3. Recloning the desired gene into the final lentiviral vector pLenti_hPGK_gfp:

[0036] The insert was prepared for recloning by cutting out the pUC57_M plasmid with a sequential restriction reaction, first at the BstAUI site (SibEnzyme) (1 h at 37C in ThermoScientific Fast Digest buffer, No. B72), then at the BamHI site (SibEnzyme) (also in ThermoScientific Fast Digest buffer, No. B72). Then, horizontal gel electrophoresis of the reaction mixture was performed in a 1% agarose gel at a voltage of 5-6 V / cm for at least 45 min, and sections with the desired fragment size (2044 bp) were excised from the gel. The fragment was purified using the LumiPuree DNA agarose gel extraction kit, Lumiprobe, No. 5793.

[0037] pLenti_hPGK_gfp vector was prepared for recloning by removing the gfp-encoding gene from the vector with simultaneous restriction digestion at the BstAUI (SibEnzyme) and BamHI (SibEnzyme) sites using ThermoScientific Fast Digest, #B72, for 1 h at 37°C. The reaction mixture was then subjected to horizontal gel electrophoresis in a 1% agarose gel at 5-6 V / cm for at least 45 min, and sections containing the desired 7594 bp fragment, corresponding to the vector without the insert, were excised from the gel. The fragment was purified using the LumiPuree Agarose Gel DNA Extraction Kit, Lumiprobe, #5793.

[0038] 4. Cloning of the gene into the pLenti_hPGK_gfp vector using BstAUI, BamHI sites:

[0039] The ligation reaction was performed using Thermo Scientific™ T4 DNA Ligase (5 U / µL) (No. EL0011). The vector:fragment ratio was 1:5. Incubation was carried out for 16-18 hours at +4°C.

[0040] The ligase mixture was transformed into E. coli XL1-blue cells for chemical transformation Eurogene CC001 according to the manufacturer's protocol. Then, these transformed cells were plated on Petri dishes with LB agar containing ampicillin at a dose of 100 μg / ml. The dishes were incubated overnight at + 37 ° C. Clones were verified by PCR screening using specific primers hPGK-F (5- GTGTTCCGCATTCTGCAAG-3), WPRE-R (5- CATAGCGTAAAAGGAGCAACA-3), PCR reaction mixture Basic, 2x Lumiprobe # 5024 were used for screening. The expected size of the PCR product was 2168 bp. Clones that yielded positive PCR results were cultured in liquid LB medium (5 ml, ampicillin 100 μg / ml, overnight culture). Plasmid isolation was performed the following day (16 hours later) using the LumiPure Plasmid DNA Isolation Kit (Spin Miniprep), Lumiprobe #1583. Suitable clones were then selected by sequencing.

[0041] The transfer plasmid, VSV-G-encoding plasmid, and third-generation lentivirus packaging plasmids were produced in E. coli (NEB Stable strain), and the resulting plasmids were verified by restriction enzymes and gel electrophoresis. The above plasmids were delivered to HEK-293T packaging cells using Lipofectamine 2000 / 3000. The produced lentiviruses were concentrated using a commercial TransLv™ Lentivirus Precipitation Solution kit (5×) and titrated by quantitative (with diluted standards) PCR for proviral DNA (TransLv™ Lentivirus qPCR Titration Kit) in target T cells. The required multiplicity of infection (MOI) for the target cells was selected to transduce the largest number of cells with the highest vitality and the fewest exhaustion markers. Target T cells were then transduced with the resulting lentiviral particles according to the optimal MOI, after which CD8 cells were sorted.To assess the cytotoxic activity of transduced TCR-T cells, they were cocultured with tumor cell lines (ratio 1:10) differing in PRAME expression (SK-MeL-5 − PRAME / MHC-I-positive, SK-MeL-37 − PRAME / MHC-1-positive, and HCT-116 − PRAME-negative / MHC-1-positive). Untransduced T cells served as a control.

[0042] Figure 3 shows the percentage of tumor cell death of PRAME-positive (SK-MeL-5 and SK-Mel-37) and PRAME-negative (HCT-116) lines when co-cultured with CD8+ T cells transduced with a lentiviral construct encoding the TCR-T cell receptor to the epitope 100-108 VLDGLDVLL PRAME, and with non-transduced CD8+ T cells (control), n=6. Arrows indicate statistically significant differences between the experimental and control groups (p≤0.05).

[0043] Thus, the proposed method for obtaining a human T-cell receptor specific for the epitope 100-108 VLDGLDVLL of the PRAME protein makes it possible to create in a short time, without additional lengthy procedures (obtaining dendritic cell vaccines, vaccinating donors), and having an accurate description of the structure of the T-cell receptor specific for the epitope 100-108 VLDGLDVLL PRAME. In vitro studies of CD8+ cells transduced with a lentiviral construct encoding the developed cellular receptor to the epitope 100-108 VLDGLDVLL PRAME, using a direct cytotoxic test against tumor cells expressing the epitope 100-108 VLDGLDVLL PRAME, demonstrated a high rate of cytotoxicity against tumor cells expressing the epitope 100-108 VLDGLDVLL PRAME, in contrast to the corresponding (antigen-negative) control.

Claims

A method for producing a human T-cell receptor specific to the 100-108 VLDGLDVLL epitope derived from the PRAME protein, comprising isolating peripheral blood mononuclear cells (MNCs) from HLA-A0201+ donors, then separating the MNCs into adherent and non-adherent fractions, and obtaining mature dendritic cells from the adherent fraction using 100 ng / ml GM-CSF and 50 ng / ml IL-4 for 4 days; on the 5th day, adding the 100-108 VLDGLDVLL epitope as a priming factor to the dendritic cell culture, followed by incubation at 37°C; on the 6th day, adding a maturation factor to complete the maturation of dendritic cells; then the obtained dendritic cells are collected, washed with a nutrient medium, the number of dendritic cells is counted, their viability is assessed and they are co-cultured with autologous CD8+ T lymphocytes obtained from the non-adherent fraction of MNCs in a certain ratio, and after 7 days of co-cultivation, antigen-specific CD8+ T cells are isolated,then, the TCR gene sequence is identified by sequencing the variable region of the TCRα and TCRβ chains, including CDR3, isolated from T cell clones, wherein the TCRα and TCRβ chains are connected by a 2A peptide linker, in the form of TCRb-P2A-TCRa, and the complete TCR construct is cloned into the backbone of a viral plasmid to obtain TCR complexes, characterized in that the donors are conditionally healthy individuals, from whose blood monocytes are isolated on a Ficoll gradient, on the 5th day, the epitope 100-108 VLDGLDVLL is added as a priming factor at a dose of 100 μg / ml and subsequent incubation for 24 hours, on the 6th day, TNF-α is added as a maturation factor at a concentration of 25 ng / ml for 24 hours, and co-cultivation dendritic cells are carried out with CD8+ T lymphocytes in the presence of 0.5 μg / ml antibody to CD3, 1 μg / ml antibody to CD28 and IL-2, IL-7, IL-15 at 10 ng / ml each, in a ratio of 1:10,After 7 days of co-cultivation, antigen-specific CD8+ T cells are isolated using Flex-T reagents, followed by identification of the TCR gene sequence using single-cell RNA sequencing (scRNA-seq) and selection of a TCR clone specific to the 100-108 VLDGLDVLL epitope of the PRAME protein using the ERGO-II and NetMHCpan-4.1 neural networks and the ClonoSort program, with the 5' and 3' untranslated regions of beta-globulin at the ends of the insert being used in addition to connecting the TCRα and TCRβ chains with the 2A peptide linker in the form of TCRb-P2A-TCRa, and the HIV-1-based lentivirus pLentih_PGK being used as the viral plasmid, one dominant TCR with one alpha and one beta chain originating from the same cell is obtained.

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