RU2708558C1 - Method for producing anti-tumor immunological protection to el-4 lymphoma cells - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry, in particular to a method for producing anti-tumor immune defense of an organism to EL-4 lymphoma cells.

EFFECT: invention enables effective resistance to EL-4 lymphoma cells.

1 cl, 1 dwg, 3 tbl, 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 α- and β-chains, which are unique for 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 α and β TCR chains, which is transmitted to its descendants resulting from division (clones).

Obtaining T-lymphocytes 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. As a result, the required number of T lymphocytes is obtained that carry the same combination of α and β 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 α and β chains of each T lymphocyte clone. The obtained genetic sequences are introduced into the activated T-lymphocytes using expression vectors, as a result of which their descendants acquire the ability to express TCR with a new specified specificity.

This approach has been used for adoptive tumor immunotherapy in oncology [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-1. J 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].

The disadvantages of this approach: the need for a cell cloning procedure. The complete TCR configuration consisting of α and β chains was used.

Earlier, we found that among T-lymphocytes there are T-lymphocytes with TCR, the specificity of which for a given antigen is determined only by the α-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 β-chain it is present on the T-lymphocyte membrane. This makes it possible to avoid the cell cloning procedure and refuse transgenic transfer of α-chains upon receipt of TCR with the desired specificity. In this method, the technology for identifying the desired receptor is reduced to obtaining cDNA libraries from polyclonal memory cells and determining and sequencing the genes of biologically active α-chains by new generation sequencing (NGS sequencing). This approach significantly reduces 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. This method involves the use of individual transgenic α-chains, which, when interacting with endogenous β-chains present in the cell, form TCR to protect against cancer.

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; 1 (1): 26-31. doi: 10.1158 / 2326-6066.CIR-13-0006].

A known approach aimed at enhancing the immune response by obtaining high-affinity TKR by selection and mutagenesis of TKR chains [Schendel D, Wilde S, Frankenberger B, Uckert W. High affinity T cell receptor and use thereof. Patent US 8697854 B2. Priority date 2008-11-24; Jakobsen BK, Harwood N, Liddy NR T cell receptors. Patent WO 2011001152 A1; Priority date 2009-07-03].

The difference between this approach and the claimed one consists in using the full configuration of TCR, consisting of α and β chains.

Another method of treating tumors with transgenic TCR is retroviral transduction of T-lymphocytes in an in vitro culture with their subsequent 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 one proposed in using the full TCR configuration, consisting of α and β chains.

A known method of enhancing the immune response to cancerous tumors and pathogens, which describes modified T-lymphocytes simultaneously expressing TCR or a chimeric antigenic receptor and antibody (Patent WO 2017147383A1 (2017-08-31)).

The disadvantage of this method is the need for the expression of the transgenic TCR β-chain and the T-cell cloning procedure to identify the α and β chains of such TCR.

A method is proposed for enhancing the immune response by modifying various TCRs, leading to an increase in their expression on the surface of a T-lymphocyte (Patent WO 2016170320 A1 (2016-10-27)).

The disadvantage of this method is the need for the presence of a β-chain in the TKR genetic construct and the need for T-cell cloning to identify α and β-chains of such TKR.

The objective of the invention is: the development of a new method of creating antitumor immunological protection using transgenesis of T-lymphocytes in vitro and in vivo.

The problem is solved by obtaining T-lymphocytes with expression of the TCR α-chain. Mouse T-lymphocytes were transduced with retroviral particles containing the TKR 1D1 α-chain gene (SEQ ID NO 1, GenBank: DQ983579.1), followed by adaptive transfer of the modified cells to the recipient mice in order to evaluate the functional activity of the transduced cells in vitro and in vivo.

The technical result of the invention: a method for creating anti-tumor immunological protection using transgenesis of T-lymphocytes.

The invention is illustrated in FIG. 1, tab. 1-3 and a list of sequences used.

In FIG. Figure 1 presents an assessment of the level of cell transduction by Migr1-PGK-1-GFP virus by flow cytometry. A- non-transduced cells (control); B-T cells transduced by the virus.

On the table. 1 presents an assessment of the functional activity of transduced T-lymphocytes in the in vitro system. The killer-target ratio is 10: 1.

On the table. 2 presents an assessment of the functional activity of transduced T-lymphocytes in the in vivo system. The absolute number of EL-4 lymphoma cells on day 6 after adoptive transfer of T-lymphocytes. The killer-target ratio is 10: 1.

On the table. 3 presents an assessment of the protective activity of transduced T-lymphocytes in an in vivo system. Analysis of EL-4 cells in the peritoneal cavity of B10.D2 mice (R101) with adoptively transferred transduced T-lymphocytes 7, 14 and 28 days before the introduction of tumor cells. The killer-target ratio is 10: 1.

The method is as follows.

1. Creation of genetic constructs.

Two DNA vectors with genetic constructs were used in the work to assemble particles of a retrovirus. To obtain the first DNA vector, the commercial MigR1 genetic construct (Addgene, USA) containing the green fluorescent protein (GFP) gene was modified by removing the GFP gene sequence from it and introducing the PGK promoter and TCR 1D1 α-chain gene (SEQ ID NO 1). The created vector is called MigR1-PGK-1D1a. Since there are no commercial antibodies to the 1D1a chain, a second genetic construct was created to evaluate the level of transduction, which was also obtained on the basis of MigR1 (Addgene, USA) by inserting the PGK promoter into the region of the site for multiple cloning of this plasmid. Thus obtained vector MigR1-PGK-GFP containing the GFP gene was used in further work to control the effectiveness of the procedure for introducing genetic constructs into target cells.

The pCL-Eco commercial genetic construct (Addgene, USA) was used as the second DNA vector. This plasmid contains all the protein genes necessary for the assembly of a viral particle with an ecotropic membrane (membrane protein (env), polymerase (pol), revertase (rev), integrase).

2. Obtaining particles of retroviruses.

2.1. Transfection of HEK293T cells and production of a medium containing viral particles.

2.1.1 Preparation of HEK293T cells packaging viral particles.

HEK293T packaging line cells were grown in a 75 cm ² culture vial (Costar, USA) in 12 ml of DMEM standard growth medium (Paneko, Russia) containing 4.5 g / l glucose and 10% fetal calf serum (Paneko, Russia) until confluence of 60-80% is reached.

2.1.2. Transfection of HEK293T, obtaining a virus-containing medium.

MigR1-PGK-1D1a plasmids containing the TCR 1D1 α-chain gene (SEQ ID NO 1) and pCl-Eco in a 1: 1 ratio of 20 μg of each plasmid were mixed in 1.5 ml of sterile water. To the mixture was added 75 μl of 2M CaCl ₂ and stirred by pipetting. Then, 750 μl of double HBS buffer (50 mM HEPES, pH 7.05; 10 mM KCl, 12 mM dextrose, 280 mM NaCl, 1.5 mM Na ₂ HPO ₄ ) was added dropwise with stirring. The mixture was incubated for 5 min at room temperature and was added dropwise to the growth medium contained in culture bottles with cells of the packaging line. HEK293T cells were transfected for 24 hours, after which the modified growth medium was removed and replaced with standard DMEM growth medium.

Transfection efficiency was monitored using GFP protein. For this, under the same conditions, HEK293T cells were transfected with the plasmids MigR1-PGK-GFP containing the GFP gene and pCL-Eco. The presence of GFP protein expression in HEK293T cells was evaluated using a fluorescence microscope (Nikon, Japan). The transfection level was extrapolated to HEK293T cells with the 1D1 gene (SEQ ID NO 1). Transfection is considered successful if GFP fluorescence is 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 infection of HEK293T cells with plasmids MigR1-PGK-1D1a and pCl-Eco. The freshly obtained virus-containing medium was used for transduction of mouse T cells. It is possible to freeze the virus-containing medium at a temperature of -70 ° C. Single defrosting is allowed.

3. Obtaining lymphocytes from the spleen and lymph nodes of the mouse.

B10.D2 (R101) mice were sacrificed by cervical dislocation and the spleen and lymph nodes (mesenteric, 2 axillary and 2 inguinal) were removed. The spleen was placed in a Potter homogenizer with 3 ml of RPMI-1640 growth medium (Paneko, Russia) containing 10 μg / ml of ciprofloxacin (KRK, Slovenia). After homogenization of the spleen, the cell suspension was transferred to a centrifuge tube and precipitated by centrifugation at 1500 rpm for 5 min at 4 ° C. The supernatant was removed, the cell pellet was resuspended, and hypotonic erythrocyte lysis was performed. For this, 360 μl of sterile distilled water was added to the splenocyte pellet, the tube was shaken for 10-20 seconds, and 40 μl of 10-fold phosphate-saline buffer and 6-7 ml of 1-fold phosphate-saline buffer were added. 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 growth medium (Paneko, Russia) containing 10 μg / ml ciprofloxacin (KRK, Slovenia).

Lymph nodes were homogenized as described above, precipitated by centrifugation at 1500 rpm for 5 min at 4 ° C. The supernatant was removed, the cell pellet was resuspended in 2 ml of RPMI-1640 growth medium (Paneko, Russia) and introduced into the 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 complete medium (PS) for culturing lymphocytes consisting of RPMI 1640 growth medium (Paneko, Russia) with the addition of 10 % serum of cattle embryos (Paneko, Russia), 5 × 10 ^-5 M 2-mercaptoethanol (Merck, USA), 2 mM L-glutamine (Sigma, USA), 20 mM HEPES (Paneko, Russia) and 10 μg / ml of ciprofloxacin (KRK, Slovenia). The number of splenocytes in the suspension was calculated in a 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.

3.1. Mouse lymphocyte activation.

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 (Costar, USA). On Wednesday, concanavalin A mitogen T-lymphocytes (ConA, Sigma, USA) were additionally introduced to a final concentration of 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. This approach provided activation and proliferation of only spleen T-lymphocytes and mouse lymph nodes (Neveu PJ, Perdoux D. Polyclonal activation of guinea pig spleen lymphocytes - Int. Arch. Allergy. Appl. Immunol. 1985; 78 (4): 401-405 )

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

The transduction of activated mouse T-lymphocytes with viral particles containing the TCR 1D1 α-chain gene (SEQ ID NO 1) was carried out in two stages.

1) 24 hours after activation, the number of splenocytes was counted using a Goryaev camera and transferred to a 6-well plate (Costar, USA) at a rate of 12 × 10 ⁶ cells per well. Then the medium was selected and the virus-containing HEK293T cell growth medium was added at the rate of 6 ml of medium per well and 3 ml of PS. The final volume of medium in each well was 9 ml. ConA (3 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 centrifuged (spinoculated) at 2000 g for 90 min at room temperature. Then the lymphocytes were placed in a CO ₂ incubator.

2) After 16-20 hours of cultivation, another change of the splenocyte growth medium to a virus containing medium was carried out and transduction of splenocytes was repeated with the same spinoculation parameters. At the end of the second spinoculation, splenocytes 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 and ConA in the above concentrations, and continued cultivation for 48 hours under the same conditions.

5. The level of transduction of T-lymphocytes was assessed by flow cytometry analysis (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 cells were excluded, as well as dead cells stained with propidium iodide. Among single living lymphocytes, T-lymphocytes were determined by the presence of a CD3 marker, for which the level of expression of GFP protein by fluorescence in the spectral region of 520 nm was evaluated.

5.1. Processing the results of cytofluorimetric analysis.

The results of cell analysis on a flow cytometer were processed using FlowJo software (Beckton Dickenson, USA). The functional efficacy of transduced T-lymphocytes was determined with a transduction level of at least 50% (Fig.).

6. Assessment of the functional activity of transduced T-lymphocytes in the in vitro system.

T-lymphocytes transduced with the 1D1 α-chain gene (SEQ ID NO 1), in an amount of 500 × 10 ³ cells, were placed in a culture with EL-4 lymphoma cells in the ratio of transduced T-lymphocytes: EL-4 = 10: 1. Cultivation was carried out in 96 well plastic plates (Costar, USA) in PS in a humid atmosphere in a CO ₂ incubator at 37 ° C, 5% CO ₂ for 48 hours. As control, activated non-transduced cells were used, as well as T cells transduced with GFP, in a similar ratio.

After the incubation time, flow cytofluorimetry analyzed the relative number of tumor cells, which were determined using fluorescently labeled antibodies to marker K ^b (Table 1).

It has been shown that T lymphocytes transduced with the 1D1a α-chain gene (SEQ ID NO 1) significantly eliminate tumor cells of EL-4 lymphoma in vitro compared to non-transduced cells (control). Moreover, this effect was specific, because in a culture with T lymphocytes transduced by GFP, the detected amount of EL-4 was comparable with the control (14.1% and 17.2%, respectively, Table 1).

7. Assessment of the functional activity of transduced T-lymphocytes in the in vivo system.

7.1. Adaptive transfer of transduced T-lymphocytes. After assessing the level of transduction, T-lymphocytes transduced with retroviral particles containing the TCR 1D1 α-chain gene (SEQ ID NO 1) were injected intraperitoneally to B10.D2 (R101) mice at a rate of 5.0 × 10 ⁶ transduced cells in 1 ml phosphate salt buffer. Non-transduced T-lymphocytes were used as a control, which were activated and cultured similarly to transduced cells. Control T lymphocytes were administered in an amount equal to the number of adoptively transferred transduced T lymphocytes.

7.2. Preparation of lavage of the abdominal cavity.

6 days after the introduction of tumor cells and adoptive transfer of T-lymphocytes, mice were euthanized by cervical dislocation and cells were removed from the abdominal cavity. For this, 2 ml of chilled RPMI-1640 medium was introduced into the peritoneal cavity of mice and a liquid was taken. The resulting cell suspension was washed by centrifugation (200 × g, 5 min) and the number of cells was counted using a Goryaev chamber in the presence of a mixture of trypan blue and eosin dyes. Part of the cells (1 × 10 ⁶ ) was used for analysis on a flow cytometer, staining with fluorescently labeled antibodies to the K ^b molecule to determine EL-4 tumor cells. Statistical data processing was performed according to the statistical criterion for small Mann-Whitney samples.

Adoptive transfer of transduced lymphocytes in the killer: target ratio of 10: 1 showed 25 times less tumor cells in the lavage of recipient mice on day 6 after immunization with EL-4 compared with animals that were injected with non-transduced lymphocytes (Table 2).

8. Determination of the duration of maintaining the state of immunological memory in animals with transgenic T-lymphocytes transferred.

To assess the protective potential of transduced T-lymphocytes, adaptive transfer of transduced or non-transduced (control) T-lymphocytes was performed in an amount of 5.0 × 10 ⁶ cells / mouse. After 7, 14, or 28 days, mice were immunized with EL-4 lymphoma cells at a dose of 5.0 × 10 ⁵ cells / mouse. Thus, the ratio of introduced T cells to EL-4 cells was 10: 1. Groups were made up of 5 animals.

The protective effect of transduced T-lymphocytes was manifested as follows: the introduction of lymphocytes 7 and 14 days before immunization with EL-4 contributed to complete or accelerated rejection of tumor cells by day 6 and did not appear in the case of adoptive transfer 28 days before immunization (Table 3). A similar protective effect was observed at a ratio of killer: target = 20: 1.

Thus, with the intraperitoneal adoptive transfer of transduced T-lymphocytes in a ratio of 10: 1 or 20: 1 to EL-4 lymphoma cells, immunological protection of wild-type B10.D2 (R101) mice is formed for a period of 14 days.

Claims (1)

A method of creating an antitumor immunological defense of an organism to EL-4 lymphoma cells by transduction of T-lymphocytes with retroviral particles containing the TCR gene represented by the sequence SEQ ID NO: 1, characterized in that transgenesis of mouse T-lymphocytes in vitro is carried out by introducing only the α gene -chain TKR-specific to tumor cells, and then adoptive transfer of T-lymphocytes modified by retroviral particles containing the TKR gene represented by the sequence SEQ ID NO: 1, followed by guide functional assessment of T-lymphocytes in vivo.

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