RU2706554C1 - Method for creating anti-infectious immunological protection against salmonella typhimurium and listeria monocytogenes using transgenesis of t-lymphocytes - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry, in particular to a method of adoptin cell therapy for preventing or treating infectious diseases caused by S.thyphimurium, L.monocytogenes.

EFFECT: invention provides effective treatment of diseases, caused by Salmonella thyphimurium, Listeria monocytogenes.

1 cl, 8 dwg, 1 ex

Description

The invention relates to the field of molecular biotechnology and relates to T-lymphocytes having a new receptor.

In the process of an immune response to antigens of bacteria, viruses and tumors in the body, a population of memory cells is formed - lymphocytes capable of an accelerated and enhanced response to the same antigen, re-introduced. The meeting of an organism with an antigen leads to the differentiation of naive T-lymphocytes into effector cells, and then into memory cells. This is a pool of cells that makes up about 5% of the total number of lymphocytes capable of long-term persisting and an effective immune response when the body meets the antigen again (Rocha B., 1997; Jacob J. et al., 1997, Rocha B., 1999, Murali KK et al., 1998). All known vaccination methods are based on the emergence of memory cells.

Specific antigen recognition by T-lymphocytes occurs due to the presence of a T-cell receptor (TCR). TCR consists of alpha and beta chains, which are unique to each clone of T-lymphocytes. The pool of body T-lymphocytes is a mixture of cells, on the surface of each of which there is a unique combination of alpha and beta TCR chains, which is transmitted to its descendants resulting from division (clones).

Obtaining T-lymphocytes in laboratory conditions with TCR of the desired specificity occurs in several stages:

1. Carrying out T-cell cloning. In the pool of T-lymphocytes, the multiplication of individual cells with the desired TCR specificity is induced by adding a specific antigen to them. To obtain a sufficient number of such cells, several rounds of restimulation of their proliferation by a specific antigen are carried out. The result is the required number of T lymphocytes that carry the same combination of alpha and beta TCR chains as the original cell (clone). This procedure requires significant time and technical costs associated with the duration of growth of individual clones and the need to select and provide fairly complex (sometimes individual) conditions for their maintenance and further testing.

2. Gene cloning, identification and determination of the nucleotide sequences of the alpha and beta chains of each clone of T-lymphocytes. The obtained genetic sequences are introduced into the activated T-lymphocytes by means of expression vectors, as a result of which their descendants acquire the ability to express TCR with a new specified specificity. (Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CC, Levy CL, Li YF, El-Gamil M, Schwarz SL, Laurencot C, Rosenberg SA. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-lJ Clin Oncol. 2011 Mar 1; 29 (7): 917-24 .; Blankenstein T, Leisegang M, Uckert W, Schreiber H. Targeting cancer-specific mutations by T cell receptor gene therapy. Curr Opin Immunol. 2015; 33: 112-9).

Previously, we found that among the pool of T-lymphocytes there are T-lymphocytes, the specificity of which to the desired antigen is determined only by the TCR alpha chain [Kazan DB Transgenic technologies for creating an immunological defense of the body. In the collection of reports of the seminar of the Foundation for Advanced Research “Problematic Issues of Immunology”, October 3, 2014, Moscow, B-Print LLC, 2015, pp. 17-25.]. Such TCRs recognize an antigen, regardless of which combination with which beta chain they are present on the T-lymphocyte membrane. Thus, transgenesis of the TCR alpha chain avoids the procedure of cell cloning and rejects the transgenic transfer of beta chains upon receipt of TCR with the desired specificity.

Brduscha-Riem К, Hayday AC, Owen MJ, Hengartner H, Pircher H. T cell development and repertoire of mice expressing a single T cell receptor alpha chain. Eur J Immunol. 1995 Sep;25(9):2650-5).In this method, the technology for identifying the required TCR is reduced to obtaining libraries of complementary DNA (cDNA) from polyclonal memory cells of immune animals, identifying and sequencing the genes of biologically active alpha chains by new generation sequencing (NGS sequencing). This approach can significantly reduce the duration of the search and identification of the desired receptors and facilitates the technical implementation of the transfer of genetic constructs into the cellular genome due to the reduction of their size. As one of the approaches to protect against infectious diseases, transgenic mice were obtained, all T-lymphocytes of which expressed only one type of TCR α-chain specific for lymphocytic choriomeningitis virus (LCMV) (

Brduscha-Riem K, Hayday AC, Owen MJ, Hengartner H, Pircher H. T cell development and repertoire of mice expressing a single T cell receptor alpha chain. Eur J Immunol. 1995 Sep; 25 (9): 2650-5).

The disadvantage of this approach: the animals obtained had a sharply limited TCR repertoire compared with wild-type mice, which did not allow them to successfully fight the virus.

There is a known approach aimed at the clinical use of T-lymphocytes transduced with a T-cell receptor (Jin J, Gkitsas N, Fellowes VS, Ren J, Feldman SA, Hinrichs CS, Stroncek DF, Highfill SL. Enhanced clinical-scale manufacturing of TCR transduced T -cells using closed culture system modules. J Transl Med. 2018 24; 16 (1): 13).

The disadvantage of this approach: the specificity of the transduced T-cell receptor is aimed at antitumor antigens.

Another method of treatment with transgenic TCR is retroviral transduction of T-lymphocytes in an in vitro culture, followed by their activation and production of lymphokinactivated killers (Schendel DJ. Expression of transgenic t cell receptors in lak-T cells. Patent US 20110020308 Al. Priority date: 2006- 12-12).

The difference between this approach and the proposed use of the full TCR configuration, consisting of alpha and beta chains, and the clinical focus of the approach to the treatment of tumors.

As one of the approaches for the treatment of certain cancers (melanoma, leukemia), lymphocytes with a genetically modified chimeric receptor (CAR) are used (C. Slaney, B. Scheidt, A. Davenport, P. Beavis, J. Westwood, S. Mardiana, D.Tscharke, S. Ellis, HM Prince, J.Trapani, R. Johnstone, M. Smyth, M.Teng, A. Ali, Z. Yu, S. Rosenberg, N. Restifo, P. Neeson, P. Darcy , M. Kershaw. Dual-specific Chimeric Antigen Receptor T Cells and an Indirect Vaccine Eradicate a Variety of Large Solid Tumors in an Immunocompetent, Self-antigen Setting. Clin Cancer Res; 23 (10) May 15, 2017; Kershaw MH, Westwood JA, Darcy PK. Gene-engineered T cells for cancertherapy. Nat Rev Cancer 2013; 13: 525-41.).

The disadvantage of this approach: CAR is a completely artificial receptor that combines the elements of T and B cell receptors. Fragments of several different molecules have to be used to make a chimeric receptor, and the resulting protein often becomes immunogenic [Maus MV, Haas AR, Beatty GL, Albelda SM, Levine BL, Liu X, Zhao Y, Kalos M, June CH. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res. 2013 Jul; l (l): 26-31. doi: 10.115 8 / 2326-6066.CIR-13-0006.].

The objective of the invention is: the development of a new method of creating anti-infection immunological protection using transgenesis of T-lymphocytes.

The problem is solved by obtaining T-lymphocytes with expression of the TCR α-chain. Mouse T-lymphocytes were transduced with retroviral particles containing a TCR α-chain gene specific for Salmonella typhimurium (S. typhimurium strain IV147) or Listeria monocytogenes (L. monocytogenes strain EGD), followed by adaptive transfer of the modified cells to infected mouse recipients for the purpose assessing the functional activity of transduced cells in in vitro and in vivo systems.

The technical result of the invention is to obtain individual transgenic alpha TCR chains specific for the pathogen S.thyphimurium, L.monocytogenes (represented by the sequence SEQ ID NO 35-39 and SEQ ID NO 40-41), which form a full functional TCR for the protection and treatment of infectious diseases caused by these pathogens.

A brief description of the drawings.

In FIG. 1 shows an alpha chain amplification scheme. Among the DNA isolated from the spleen of immunized mice, there is a set of a large number of different α-chains. Using the nucleotide sequence of the hypervariable CDR3 and the 5'-end for each alpha chain, a set of primers was obtained that individually amplified three sections of the gene and then combined them.

In FIG. 2 shows a vector scheme for the expression of alpha chains.

In FIG. Figure 3 presents the results of checking the correctness of assembled structures by analytical restriction of TCR alpha chains in the MigRl-PGK vector (using the α-chains SM # 1 and SM # 2 as an example).

The AgeI site is located at the 5'-end of the gene reading frame, SalI is at the 3'-end. In addition, the SM # 2 reading frame contains an additional SalI site at the 367th position. Two BamHI sites are located at the 5 'and 3' ends of the PGK promoter. The reading frames of both chains SM # 1 and SM # 2 contain two Ncol sites in the 184th position and the 530th (536th) position.

Thus, restriction by AgeI and SalI was supposed to give one fragment ~ 830 bp in length. for SM # 1 and two fragments for SM # 2: ~ 370 and -480 bp In addition, in both cases, a band corresponding to the remaining vector, 5358 bp in length, should be observed.

Restriction by BamHI and NcoI should produce three relatively short fragments: ~ 550 bp corresponding to the PGK promoter, ~ 350 bp corresponding to the middle part of the reading frame, and ~ 180 bp In addition, in both cases, a band corresponding to the remaining vector with a length of ~ 4800 bp should be observed.

In FIG. Figure 4 presents the results of an assessment of the functional activity of in vitro T-lymphocytes transduced with alpha chains of SM-1, SM14, SM16, SM20, SM21 TCR specific for S. typhimurium.

Background proliferation of T lymphocytes transduced by a genetic construct carrying GFP or TCR alpha chain (open bars). Proliferation of T lymphocytes transduced with a genetic construct carrying GFP or TK alpha chain when cultured with antigen-presenting cells in the presence of a specific antigen (filled bars). The ordinate shows the number of pulses per minute (CPM).

In FIG. 5 presents an assessment of the functional activity of in vitro T-lymphocytes transduced with alpha chains of TKR LM2 and LM3 specific for L. monocytogenes.

Proliferation of T-lymphocytes transduced by the genetic construction of TKP alpha chains LM2 or LM3, when they are cultured with splenocytes of intact mice in the absence (empty columns) or in the presence (filled columns) of a specific antigen. The ordinate shows the number of pulses per minute (CPM).

In FIG. Figure 6 presents an assessment of the functional activity in vivo of T-lymphocytes transduced with alpha chains of SM-1, SM14, SM20 TCR specific for S. typhimurium (a preventive effect in which T-lymphocytes were introduced into the mouse before infection with bacteria).

The abscissa shows: 1 - mice with adoptively transferred transduced T-lymphocytes and infected with S. typhimurium.

2 - mice with adoptively transferred non-transduced (activated) T-lymphocytes and infected with S.typhimurium, 3 - mice without adoptively transferred T-lymphocytes infected with S.typhimurium.

The ordinate shows the number of bacterial colonies seeded from mouse spleen.

In FIG. Figure 7 presents the assessment of the functional activity in vivo of T-lymphocytes transduced with alpha chains of SM-16, SM21 TKP specific for S. typhimurium (therapeutic effect in which T-lymphocytes were introduced into the mouse after infection with bacteria). The abscissa shows: 1 - mice infected with S. typhimurium and treated with transduced T-lymphocytes. 2 - mice infected with S. typhimurium and treated with non-transduced (activated) T-lymphocytes.

3 - mice infected with S. typhimurium and not treated with T-lymphocytes. The ordinate shows the number of bacterial colonies seeded from mouse spleen.

In FIG. Figure 8 shows the number of L. monocytogenes in the spleens of R101 mice with adoptively transferred T cells transduced with a mixture of TCR alpha chains (LM2 and LM3, prophylactic effect).

The abscissa axis shows: 1- mice infected with L. monocytogenes and treated with transduced T-lymphocytes. 2 - mice infected with L.monocytogenes and treated with non-transduced (activated) T-lymphocytes. 3 - mice infected with L.monocytogenes and not treated with T-lymphocytes.

The ordinate shows the number of bacterial colonies seeded from the liver of infected mice.

The method is as follows. Memory cells specific for the pathogen S.typhimurium or L.monocytogenes were enriched by in vitro restimulation in culture with an appropriate killed by warming bacterium. All bacterial strains used were obtained from the collection of the Honorary Academician N.F. Gamalei National Research Center for Epidemiology and Microbiology. At the end of restimulation, the cells were placed in a reagent, for example, TRIzol (ThermoFisher Scientific, USA) to isolate total matrix RNA (mRNA). Next, the method of polymerase chain reaction with reverse transcription (RT PCR) was performed synthesis of complementary DNA (cDNA). The resulting cDNA was sequenced and libraries were formed that contained sequences of V and J segments of TCR alpha chains specific for pathogens (Fig. 1). Then, a full-length cDNA of a pathogen specific TCR α-chain was amplified by PCR, which was then cloned into a MigR1 retroviral vector containing the PGK promoter (Fig. 2). With this genetic construct, together with the auxiliary plasmid pCLeco, cells of the packing line HEK293T were transfected and retroviral particles containing the TCR α chain gene were obtained. The obtained retroviral particles were used for transduction of activated mouse lymphocytes by spinoculation at 2000 × g for 90 min. The functional activity of T lymphocytes transduced with TCR α-chain was tested in vitro to increase the proliferative response in the presence of a specific pathogen and in vivo to determine the elimination of the corresponding pathogen from the organism of infected mice.

1. Obtaining and enrichment of memory cells of mice of B10.D2 (R101) mice to S.typhimurium. B10D2 (R101) mice were immunized with S. typhimurium cells (avirulent strain 274 at a dose of 1 × 10 ⁶ CFU / mouse. After 21 days, the animals developed immunological memory for the antigens of this bacterium. To enrich the pool of specific memory T cells, stimulation in vitro of the total population of splenocytes of immune mice by inactivated (heated at 60 ° C for 60 min) bacteria of the virulent S. typhimurium strain (strain IV 147).

To this end, inactivated S. typhimurium culture cells were added to the spleen cells in an amount of 2.0 × 10 ⁶ CFU and incubated at 37 ° C in an atmosphere with 5% CO ₂ and absolute humidity for 72 hours. Then, the culture was added further 2,0 × ¹⁰ May CFU S.typhimurium inactivated cell culture and incubated for an additional 72 hours. The total duration of in vitro restimulation was 144 hours. As a control, spleen cells of immunized mice that were cultured in the absence of bacteria were used. At the end of the cultivation time, 0.5-1 × 10 ⁶ cells were selected and placed in 1 ml of reagent, for example, TRIzol (ThermoFisher Scientific, USA) for RNA isolation.

2. Obtaining and enrichment of memory cells in mice B10.D2 (R101) to L.monocytogenes. Female B10D2 (R101) mice weighing 16-18 grams were intraperitoneally immunized with L. monocytogenes cells (EGD strain). The formation of memory cells occurred 45 days after the introduction of the pathogen. To enrich the pool of specific memory T cells, in vitro stimulation of splenocytes of immune mice was carried out with inactivated (heated at 74 ° C for 90 min) L.monocytogenes cells. For this, inactivated cells of L.monocytogenes in the amount of 3.0 × 10 ⁷ CFU were added to the spleen cells and incubated in RPMI 1640 nutrient medium, for example, PanEco, Russia, with the addition of 10% serum of cattle embryos, for example, PanEco, Russia , 5 × 10 ^-5 M 2-mercaptoethanol, for example, Merck, USA, 2 mM L-glutamine, for example, Sigma, USA, 20 mM HEPES, for example, PanEco, Russia, and 10 μg / ml ciprofloxacin, for example firms (KRK, Slovenia) (PS) at 37 ° C for 72 hours in an atmosphere with 5% CO ₂ and absolute humidity. As a control, spleen cells of immunized mice that were cultured in the absence of bacteria were used. For in vitro stimulation completion selected 0,5-1 × 10 ⁶ cells for RNA extraction.

3. Creation of cDNA libraries of TCR alpha chains specific for pathogens.

1) Obtaining total RNA of memory cells of mice that are immune to pathogens.

RNA isolation was performed using a reagent, for example, TRIzol (ThermoFisher Scientific, USA) according to the standard manufacturer's protocol. The resulting RNA was dissolved in sterile water and the concentration was determined on a spectrophotometer, for example, NanoDrop 2000 (Thermo Scientific, USA). Samples of isolated REPS were stored at -70 ° C.

2) Obtaining cDNA of memory cells of mice that are immune to pathogens. The cDNA synthesis was performed using primers for the synthesis of cDNA α and β-chains of TCR and SMARTNNNa adapter, which contained a unique sequence of 12 degenerate nucleotides (SEQ ID NO: 4-6; E. Egorov, E. Merzlyak, A. Shelenkov, O. Britanova, G. Sharonov, D. Staroverov, D. Bolotin, A. Davydov, E. Barsova, Y. Lebedev, M. Shugay, D. Chudakov Quantitative Profiling of Immune Repertoires for Minor Lymphocyte Counts Using Unique Molecular Identifiers. - The Journal of Immunology, 2015).

3) Amplification of cDNA alpha and beta TCR chains specific for pathogens.

PCR was performed in two sequential steps (E. Egorov, E. Merzlyak, A. Shelenkov, O. Britanova, G. Sharonov, D. Staroverov, D. Bolotin, A. Davydov, E. Barsova, Y. Lebedev, M. Shugay , D. Chudakov, Quantitative Profiling of Immune Repertoires for Minor Lymphocyte Counts Using Unique Molecular Identifiers. - The Journal of Immunology, 2015).

For the first PCR step, the reverse transcription reaction product was used as a template and a mixture of direct adapter primers (SEQ ID NO: 7-8) for rapid amplification of terminal fragments from target transcripts and reverse primers for amplification of TCR α and β chains (SEQ ID NO : 9-10). PCR was carried out for 18 cycles at temperature conditions: 95 ° C - 10 seconds; 60 ° C - 20 seconds; 72 ° C - 40 seconds. The products of the first PCR step were purified and used as templates for the second PCR step.

The second stage of PCR was performed using primers with a unique nucleotide sequence at the 5'-end ("barcode"). As a result, each product of the second round of amplification contained a known nucleotide sequence at the 5'- and 3'-ends. PCR was carried out for 10-12 cycles at temperature conditions: 95 ° C - 10 seconds; 60 ° C - 20 seconds; 72 ° C - 40 seconds.

4) Purification of PCR products.

The products of the first and second stages of PCR were purified using a kit, for example, QIAquick PCR purification kit (Qiagen, Germany) according to the standard manufacturer's protocol.

5) Sequencing of the obtained cDNA samples was carried out by NGS sequencing on the basis of the Central European Institute of Technology (Masaryk University, Brno, Czech Republic). Before sequencing, TrueSeq adapters (Illumina, USA) were ligated to the second amplification products using a reagent kit, for example, DNA sample prep kit (Illumina, USA) according to the manufacturer's standard protocol. Sequencing was performed on a sequencer, for example, MiSeq (Illumina, USA). To carry out the sequencing cycle, a one-time set of reagents was used, for example, MiSeq Reagent Kit v2 (Illumina, USA), which allows reading of 150 bp cDNA fragments from the 5 'and 3' ends of one DNA molecule.

6) Bioinformatics processing of data obtained as a result of sequencing was performed using the software MiGEC (http://milaboratory.com), MiXCR (http://milaboratory.com), MiTCR (D.Bolotin, M. Shugay, I. Mamedov, E. Putintseva, M. Turchaninova, I. Zvyagin, O. Britanova, D. Chudakov, MiTCR: software for T-cell receptor sequencing data analysis. - Nature Methods 10, 813-814 (2013)).

At the first stage, using the MiGEC software (http://milaboratory.com), data related to individual libraries was extracted. For this, in the sequence of each DNA molecule, pairs of barcodes were analyzed, which were part of the primers and were unique for each individual cDNA sample. Then, using molecular barcoding of individual cDNA molecules, the primary correction of errors that could appear during PCR and / or sequencing was performed. For this, a sequence consisting of 12 degenerate nucleotides embedded in a SmartNNNa adapter was used as a barcode. Thus, the 5'-end portion of each obtained cDNA molecule was represented by a unique sequence. The use of such a molecular barcode makes it possible to normalize the data for the obtained samples, that is, to arbitrarily select and conduct a quantitative analysis of molecular events.

For subsequent data processing procedures, the MiXCR program (http://milaboratory.com) was used. After dividing the library data, a CDR3 hypervariable region was determined in each sequence, defined as the region from the last conserved cysteine in the V segment to the first conserved phenylalanine in the FGXG motif of the J segment. In addition, the boundaries of the V, J, and D segments for β chains and the V and J segments for α chains were determined by aligning with genomic sequences using the MiTCR software (D. Bolotin, M. Shugay, I. Mamedov, E. Putintseva, M. Turchaninova, I. Zvyagin, O. Britanova, D. Chudakov, MiTCR: software for T-cell receptor sequencing data analysis. - Nature Methods 10, 813-814 (2013)).

Further, the same nucleotide sequences with high quality sequencing, i.e. with an error rate of not more than 1: 10,000 read nucleotides, combined into one clone, while maintaining information about the number of combined sequences. Sequences with low reading quality were attached to sequences with high reading, with several nucleotide mismatches being allowed. Poor-quality readings were deleted if no similar high-quality sequences were found.

After clone formation, error correction was performed, since reverse transcription and amplification of cDNA during PCR could lead to nucleotide substitutions and the appearance of very similar variants of the same sequence. In order to reduce the artificial diversity of TCR, clones were combined that differed from each other by one nucleotide substitution in the region of CDR3, while it was assumed that the number of clone molecules with errors in the library was at least three times less than the number of clone molecules from which they originated (D. Bolotin, S. Poslavsky, I. Mitrophanov, M. Shugay, I. Mamedov, E. Putintseva, D. Chudakov. MiXCR: software for comprehensive adaptive immunity profiling. - Nature Methods, 12, 380-381 (2015) )

As a result, databases were formed with full-length cDNA sequences of α and β chains of TCR of memory cell clones specific for the pathogens of S. typhimurium and L. monocytogenes.

The analysis of α-chains of TCR of memory cell clones specific for S. typhimurium or L. monocytogenes was carried out in the Exel program (Microsoft Office, USA). For this, a pool of memory cells formed during immunization of animals with a pathogen was compared with clones formed during stimulation of memory cells in vitro. As a result of sequence analysis of the most common clones, it was found that in vitro reactivation of memory cells with a specific pathogen led to their enrichment. Two types of clones of enriched memory cells were identified: 1) clones whose frequency increased compared to an unactivated pool of memory cells; 2) clones unique to the enriched pool of memory cells and absent in the inactive pool of memory cells.

The sequences of the hypervariable regions (CDR3 region) of α-chains of both types of clones were established. The sequences of α-chains of TCR memory cells with established CDR3 regions were inserted into expression vectors, which were then used for subsequent modification (transduction) of mouse T-lymphocytes.

7. Creation of genetic constructs of TCR alpha chains specific to pathogens.

1) Genetic cloning of TCR alpha chains. To clone the α-chains of T-cell receptors of memory cells specific for S. typhimurium or L.monocytogenes antigens, primers for the constant region of the α-chain were synthesized, common to all variants of the cloned α-chains (SEQ ID NO: 1-3 ) In addition, primers were synthesized that are unique to each variant of the cloned α chain (SEQ ID NO: 11-31).

When setting up PCR, the conditions for the amplification of individual V and J fragments were chosen so that the PCR product appeared if mRNA from T lymphocytes from the immunized mouse pathogen was used as starting material, but did not appear if mRNA from T was used as starting material lymphocytes of an unimmunized mouse. For the synthesis of V fragments, a direct primer with the AgeI restriction site (SEQ ID NO: 11, 14.17, 20, 23, 26, 29) and a unique reverse primer (SEQ ID NO: 13, 16, 19, 22, 25, 28, 31). For the synthesis of the J fragment, a unique forward primer (SEQ ID NO: 12,15,18,21,24,27, 30) and a reverse primer to the constant region of the a chain (SEQ ID NO: 2) were used. For all chains, a constant C fragment was synthesized using primers C-forw (SEQ ID NO: 1) and C-Sall (SEQ ID NO: 3). As a matrix for the synthesis of the C fragment, the already existing cDNA of the alpha chain of TKR of memory cells (1D1) specific for the histocompatibility molecule of mouse H-2b (SEQ ID NO: 34) was used.

All fragments were amplified, analyzed on an agarose gel and isolated from the gel using a QIAEX II Gel Extraction Kit (Qiagen) in accordance with the manufacturer's instructions. Then, V, J, and C segments of the same chain were combined in one GSHR mixture and a direct primer reaction was performed with the AgeI restriction site (SEQ ID NO: 11, 14, 17, 20, 23, 26, 29) for this chain and the reverse primer C-Sall (SEQ ID NO: 3), (Fig. 1). The reaction results were analyzed by gel electrophoresis, a fragment of the desired molecular weight was excised and isolated using a QIAEX II Gel Extraction Kit (Qiagen) in accordance with the manufacturer's instructions. Then, Taq polymerase was used to add A nucleotides at the 3-stroke ends of both DNA chains to the selected fragment, the fragment was purified using QIA quick PCR Purification Kit (Qiagen) according to the manufacturer's instructions and ligated with pTZ57 vector (InsTAclone PCR Cloning Kit, ThermoScientific). The ligase mixture was transformed into E. coli, the transformed bacteria were seeded on a Petri dish and grown at 37 ° C for 16-18 hours. Colonies were checked for insert using PCR. The insert-bearing colony was grown in 5 ml of LB medium for 16-18 hours, after which plasmid DNA was isolated using the QI Aprep Spin Miniprep Kit (Qiagen) in accordance with the manufacturer's instructions. The obtained plasmid DNA was sequenced (LLC Evrogen) using primers (SEQ ID NO: 32 and 33). In addition, to verify the correct assembly of the genetic construct, analytical restriction of plasmid DNA was performed at the restriction sites BamHI and NcoI, as well as SalI and AgeI, and its products were analyzed by gel electrophoresis. Two bands were obtained: one corresponding to the alpha chain sequence and the other corresponding to the PGK promoter sequence (Fig. 2). For each alpha chain variant, one completely correct clone was transferred to the next stage of work. The sequences of the chains used are represented by SEQ ID N0: 35-41.

8. Transfer of alpha chains into an expression vector.

The MigRl-PGK retroviral vector based on MigRl plasmids (AddgenePlasmid # 27490) and pLenti PGK GFP Blast (AddgenePlasmid # 19069), in which transgene expression was controlled by the phosphoglycerate kinase (PGK) promoter, was created by us. To clone the transgene reading frame, it contains AgeI sites at the 5'-end of the putative transgene and SalI at the 3'-end of the putative transgene. At the SalI and AgeI sites, during the preparative restriction, an alpha chain reading frame was cut from the correct clone and connected to the MIGR1-PGK vector prepared by restriction at the same sites (Fig. 3).

9. The production of plasmids for transfection and transduction. To produce plasmids and genetic constructs, competent E. coli bacteria cells (XL 1 blue) were used. Transformable plasmid DNA in an amount of 10 ng was mixed with E. coli cells, incubated on ice for 20 minutes, then the mixture was heat shocked at 42 ° C for 30 seconds and again incubated for 5 minutes in ice. Then, 0.5 ml of LB medium (AppliChem GmbH, Germany) was added to the mixture and incubated for 30 min at 37 ° C.

Next, the bacterial cells were applied to a plate with LB agar (AppliChem GmbH, Germany, Sigma agar agar, USA) and incubated in an incubator at a temperature of 37 ° C. The grown single colonies were transferred to LB medium and cultured for 16-18 h on a thermal shaker (150-200 rpm) at a temperature of 37 ° C. Isolation of plasmid DNA using commercial reagent kits was performed according to the manufacturer's protocols — HiSpeed Plasmid MaxiKit (Qiagen, USA).

10. Obtaining transduced T-lymphocytes.

1) Transfection of HEK293T cells and obtaining a medium containing viral particles.

Cells of the HEK293T packaging line were grown in culture until confluence of 60-80% was reached and transfected with MigRl-PGK-a plasmids containing the gene for the studied TCR α-chain and pCl-Eco (1: 1 ratio) by calcium phosphate method for 24 hours.

Transfection efficiency was monitored using GFP protein. For this, transfection of HEK293T cells with plasmids MigRl-PGK-GFP containing the GFP gene and pCL-Eco was performed in parallel under the above conditions. The presence of GFP protein expression in HEK293T cells was evaluated using a fluorescence microscope (Nikon, Japan). The obtained level of transfection efficiency with the GFP gene was assumed to be similar for HEK293T cells transfected with the studied α-chain gene. Transfection was considered successful if fluorescence of GFP protein was observed in at least 60% of the cells of the packaging line.

The growth medium of HEK293T cells was collected 48 and 72 hours after transfection of HEK293T cells with plasmids MigR1-PGK-a and pC1-Eco. The freshly obtained virus-containing medium was used for transduction of mouse T cells.

2) Obtaining lymphocytes from the spleen and lymph nodes of the mouse. The work was carried out using male and female mice of the B10.D2 (R101) line of mice weighing 18-20 grams obtained from a dilution of the Federal State Budget Scientific Research Center for Oncology named after N.N. Blokhin »Ministry of Health of Russia. Animals were sacrificed by cervical dislocation and the spleen and lymph nodes (mesenteric, 2 axillary and 2 inguinal) were removed. The spleen was homogenized in a Potter homogenizer and hypotonic erythrocyte lysis was performed. Then the cells were precipitated by centrifugation at 1500 rpm for 5 min at 4 ° C, the supernatant was removed and the pellet was resuspended in 3 ml of RPMI-1640 medium, for example, PanEco, Russia, containing 10 μg / ml ciprofloxacin (KRK, Slovenia) .

Lymph nodes were homogenized as described above and introduced into a splenocyte suspension. The resulting cell mixture was precipitated by centrifugation at 1500 rpm for 5 min at 4 ° C, the supernatant was removed and resuspended in 3 ml of PS. The number of lymphocytes in the suspension was calculated in the Goryaev chamber in the presence of a 1: 1 ratio of 1% trypan blue and 1% eosin dyes and adjusted to a concentration of 10 ⁷ cells / ml.

3) Activation of mouse T-lymphocytes.

A mixture of spleen cells and mouse lymph nodes in an amount of 5 × 10 ⁷ cells was placed in 5 ml of PS in culture bottles with a growth area of 25 cm ² , for example, Costar, USA. In PS, a concanavalin A (ConA) mitogen of T-lymphocytes was added, for example, from Sigma, USA, to a final concentration of 1.5-3 μg / ml and interleukin-2 (IL-2) to a final concentration of 10 IU / ml (Neto E.N., Coelho AL, Sampaio AL et al. Activation of human T lymphocytes via integrin signaling induced by RGD-disintegrins. Biochim. Biophys. Acta. 2007, 1773 (2): 176-84). Lymphocytes were cultured in a CO ₂ incubator at 37 ° C, 5% CO ₂ for 24 hours (Neveu PJ, Perdoux D. Polyclonal activation of guinea pig spleen lymphocytes. Int. Arch. Allergy. Appl. Immunol.l985; 78 ( 4): 401-405).

4) Transduction of mouse T-lymphocytes by viral particles.

Transduction of activated mouse T-lymphocytes by viral particles containing the gene of the studied TCR α-chain was carried out in two stages.

A) After activation, the concentration of lymphocytes was calculated using a Goryaev camera in a mixture of trypan blue and eosin dyes and transferred to a 6-well plate (Costar, USA) at a rate of 12 × 10 ⁶ cells per well in a volume of 9 ml of which 6 ml was virus-containing cell growth medium HEK293T and 3 ml PS. ConA (1.5 μg / ml), IL-2 (10 IU / ml) and polybrene to a final concentration of 8 μg / ml were also added to the plate wells. Cells were transduced by spinoculation, which was carried out by centrifugation at 2000 g for 90 min at room temperature. Then the lymphocytes were placed in a CO ₂ incubator.

B) After 16-20 hours of cultivation, the lymphocyte growth medium was changed to a similar one and transduction was performed again with the same spinoculation parameters. At the end of the second spinoculation, lymphocytes were cultured in a CO ₂ incubator at 37 ° C, 5% CO ₂ for 2 hours, after which the virus-containing medium was replaced with fresh PS containing IL-2 at the above concentration, and cultivation continued within 48 hours under the same conditions.

B) Assessment of the level of transduction of T-lymphocytes was carried out by flow cytometry, for example, using FacsCantoII, Beckton Dickenson, USA, using fluorescently labeled antibodies to the CD3 molecule, which is a specific marker of T-lymphocytes. The level of transduction was determined by expression of the GFP protein.

Analysis of cells on a flow cytometer. When analyzed on a flow cytometer, adherent and dead cells stained with propidium iodide were excluded. Among single living lymphocytes, T-lymphocytes were determined by the presence of a CD3 marker, for which the level of expression of the GFP protein by fluorescence in the spectral region of 520 nm was evaluated. The results of cell analysis on a flow cytometer were processed using FlowJo software (Beckton Dickenson, USA). T-lymphocytes activated in a similar way (ConA and IL-2) but not transduced (activated T-lymphocytes) were used as control.

12. A functional assessment of transduced T-lymphocytes was carried out in vitro and in vivo systems. In vitro evaluation was performed to enhance the proliferative response of transduced T-lymphocytes in response to a specific antigen in a mixed lymphocyte culture (SCR) reaction. The level of transduction of T-lymphocytes was 50-80%.

1) Assessment of the functional activity in vitro of T-lymphocytes transduced with alpha chains of TCR specific for S. typhimurium.

As antigen-presenting cells, macrophages previously incubated with S. typhimurium and syngenic splenocytes of intact mice treated with a cytostatic agent were used.

Preparation of macrophage layer. Mice were injected intraperitoneally with 3 ml of 3% peptone. After 3 days, the animals were euthanized by cervical dislocation and cells were removed from the abdominal cavity. For this, 5 ml of RPMI-1640 ice-cold medium was injected into the peritoneal cavity of the mouse and 2 ml of washout were taken. The resulting lavage was transferred to a test tube previously placed on ice. In the resulting suspension, the number of cells was counted as described above. SS cells were diluted to a concentration of 1,0 × 10 ⁵ cells / ml and transferred into wells of a 96-well plate, e.g., Costar company, USA, the rate of 1,0 × 10 ⁴ cells per 1 well. Then, inactivated S.typhimurium bacteria cells (strain IV 147) were added to macrophages at the rate of 1.0 × 10 ⁵ bacterial cells per well.

The final volume of growth medium was 200 μl per 1 well. Macrophages with bacterial cells were cultured for 72 hours in a CO ₂ incubator at 37 ° C, 5% CO ₂ .

Treatment of splenocytes of intact mice with a cytostatic agent. The spleen cells of mice were obtained and counted as described above. 30.0 × 10 ⁶ spleen cells were selected and transferred into 4.5 ml of PS. To the resulting suspension, mitomycin C was added, for example, from Kyowa, Japan, at a concentration of 25 μg / ml, thoroughly mixed and incubated in a water bath at 37 ° C for 30 min. At the end of the incubation, the cells were washed three times in 4 ml of RPMI-1640 growth medium by centrifugal deposition, for example, from Hettich, Germany, at 1500 rpm for 5 min at 4 ° C. The supernatant was removed, the cell pellet was resuspended in 3 ml of PS, the number of cells were counted as described above and the concentration was adjusted to 5,0 × 10 ⁶ cells / ml.

Setting SCR. After 72 h of culturing macrophages with S. typhimurium cells from a 96-well plate, for example, Costar, USA, the growth medium was removed and cytostatic-treated splenocytes were added to a portion of the wells containing the macrophage layer at a rate of 5.0 × 10 ⁵ cells per 1 well. Then, T-lymphocytes transduced with constructs of TCR α-chains, GFP protein or control (activated) T-lymphocytes at the rate of 3.0 × 10 ⁵ cells per well were introduced into the culture. Cells were incubated in 200 μl PS at 37 ° C, 5% CO ₂ for 72 hours. 18 hours before the end of the cultivation period, a solution of ³ N-thymidine in the amount of 1 μCi per well was added. The level of inclusion of the label was evaluated on a scintillation counter, for example, the company LKB, Sweden, by the number of recorded pulses per minute (CPM). In the process of testing, a specific increase in the proliferation of lymphocytes transduced with α-chains of TKR SMI, SM14, SM16, SM20, SM21 was demonstrated (Fig. 4).

2) Assessment of the functional activity in vitro of T-lymphocytes transduced with alpha TCR chains specific for L. monocytogenes. A functional in vitro evaluation of T lymphocytes transduced with alpha-TKR chains specific for L. monocytogenes (LM2 and LM3) was performed using the SCR reaction.

Setting SCR. The culture was added transduced genes LM2, LM3, GFP or netransdutsirovannye control (activated) T lymphocytes based 7,0 × 10 ⁴ cells / well. Syngenic splenocytes of intact mice treated with a cytostatic agent (12 × 10 ⁴ / well), to which inactivated bacteria L.monocytogenes (10 ³ -10 ⁴ / well) were used, were used as antigen-presenting cells. No bacteria were added to the control wells. Cells were cultured in a 96-well plate (Costar, USA) in PS with the addition of IL-2 (10 IU / ml) at 37 ° C, 5% CO ₂ for 72 hours. 8 hours before the end of the cultivation period, the wells were added a solution of ³ N-thymidine in an amount of 1 µci per well. The level of inclusion of the label was evaluated on a scintillation counter (LKB, Sweden) by the number of detected pulses per minute (CPM). During in vitro testing, specific proliferation of lymphocytes transduced by LM2 and LM3 chains was demonstrated (Fig. 5).

3) Functional assessment in vivo of T-lymphocytes transduced with TCR α-chains specific for S. typhimurium.

A) Assessment of the protective (prophylactic) action of transduced T-lymphocytes.

A mixture of T-lymphocytes transduced with alpha chains of SM1, SM14, SM20, in equal proportions of 100 × 10 ^{3 of} each type of transduced cells at a transduction level of 68.5%, was adoptively transferred to the B10D2 (R101) mice by intravenous administration. 3 days after the transfer of lymphocytes, enterogastric animals were infected with S. typhimurium at a dose of 3.0-5.0 × 10 ⁶ CFU / mouse using a gavage needle. Each group consisted of 3 animals. On the 7th day after infection (10th day after lymphocyte transfer), the mice were sacrificed by cervical dislocation, an autopsy was performed under sterile conditions, and the spleen was removed. The organs were homogenized in phosphate-buffered saline, a series of 10-fold dilutions were prepared in phosphate-buffered saline, and plated on Petri dishes coated with 6.0% SS agar (BD, USA). Visual colony counting was performed 18-24 hours after incubation of Petri dishes at a temperature of 37 ° C.

It was shown that the number of salmonella was reduced in the spleens of infected mice that were previously injected with transduced T-lymphocytes, compared with control intact mice and mice that had previously been administered with non-transduced activated T-lymphocytes (Fig. 6).

B) Evaluation of the therapeutic effect of T-lymphocytes transduced with alpha chains of SM16 and SM21 TCR specific for S. typhimurium.

Mice were enterogastrally using a saucer needle infected with cells of a virulent strain of S. typhimurium at a dose of 6.0 × 10 ⁶ CFU / mouse. Each group consisted of 5 animals. On the 4th day after infection, the mixture of T-lymphocytes transduced with alpha chains of SM16, SM21, in equal proportions of 150 × 10 ³ transduced cells of each type at a transduction level of 63.5% and the total number of introduced cells 470 × 10 ³ cells / mouse. 3 days after lymphocyte transfer and 7 days after infection, the mice were sacrificed by cervical dislocation, the spleen was removed, and bacterial cultures on Petri dishes were prepared as described above. A clear therapeutic potential of T-lymphocytes transduced with the α-chains of SMI6 and SM21 was shown, expressed in the complete eradication of the pathogen (Fig. 7).

4) Functional evaluation in vivo of T-lymphocytes transduced with alpha-TKR chain LM2 and LM3 specific for L.monocytogenes.

Evaluation of the prophylactic effect of transduced T-lymphocytes.

A mixture of T-lymphocytes transduced with alpha-chains of TKR LM2 and LM3 (transduction level 60%) in equal ratios of 360 × 10 ³ cells with a total number of introduced cells 1.2 × 10 ⁶ / mouse was adoptively transferred to mice by intravenous administration. Three hours after the transfer of lymphocytes, enterogastric animals were infected with L. monocytogenes at a dose of 4.5 × 10 ⁶ CFU / mouse using a gavage needle. Each group consisted of 5 animals. On the 5th day after infection, mice were sacrificed by cervical dislocation, autopsy was performed under sterile conditions, and the liver and spleen were removed. The organs were homogenized in phosphate-buffered saline using an IKA 10T homogenizer, a series of 10-fold dilutions were prepared in phosphate-buffered saline, and plated on Petri dishes containing PALCAM agar (HiMedia). Visual colony counting was performed 48 hours after incubation of Petri dishes at a temperature of 37 ° C.

It was shown that the number of L.monocytogenes was reduced in the liver and spleen of mice that were previously injected with transgenic T-lymphocytes compared to control intact mice and mice that had previously been injected with non-transduced activated T-lymphocytes (Fig. 8).

Claims (1)

A method of adaptive cell therapy for the prophylaxis or treatment of infectious diseases caused by S. thyphimurium, L. monocytogenes, by introducing T lymphocytes transduced with the TCR alpha chain specific to the S. thyphimurium pathogen, L. monocytogenes, characterized in that T lymphocytes were performed with retroviral particles containing the TCR α chain gene represented by the sequence of SEQ ID NOS 35-39 and SEQ ID NOS: 40-41, respectively.

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