RU2689558C1 - Method of constructing allogenic and drug resistant t-cells for immunotherapy - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: present invention relates to biotechnology, specifically to cell technologies, and can be used in medicine for cancer immunotherapy. Ex-vivo method for producing clofarabine and / or fludarabine-resistant T-cells involves the steps of: (a) transfecting T cells with a nucleotide sequence encoding a low-boiling endonuclease, specifically directed to a gene expressing an enzyme having deoxycytidine kinase activity (dcK – EC 2.7.1.74); (b) expressing said endonuclease in said T-cells to achieve targeted inactivation of said dcK gene; (c) propagation of said engineered T-cells obtained in step (b), optionally in the presence of clofarabine and / or fludarabine. Said T-cells represent allogenic T-cells from the donor, in which the gene coding the T-cell receptor component (RTL), which is selected from RTL alpha and RTL beta, is inactivated, or are autologous T-cells from the patient diagnosed with cancer; and wherein said cells express a chimeric receptor antigen (CAR) specific to the cancer antigen.EFFECT: invention provides higher effectiveness of antineoplastic immunotherapy.18 cl, 27 dwg, 4 ex

Description

Technical field

The present invention relates to the use of "ready-to-use" allogeneic therapeutic cells for immunotherapy in combination with chemotherapy for the treatment of patients with cancer. In particular, the authors of the present invention have developed a method for constructing allogenic T cells resistant to chemotherapeutic agents. The therapeutic utility achieved through such a strategy should be enhanced by a synergistic action between chemotherapy and immunotherapy. In particular, the present invention relates to a method for modifying T cells by inactivating at least one gene encoding a component of the T cell receptor and by modifying said T cells to make them resistant to drugs. The present invention opens up possibilities for standard and affordable adoptive immunotherapy strategies for the treatment of cancer.

The level of technology

Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells obtained ex vivo, is a promising strategy for treating cancer. T cells used for adoptive immunotherapy can be created either by propagating antigen-specific T cells or by redirecting T cells by means of genetic engineering (Park, Rosenberg et al. 2011). The transfer of viral antigen-specific T cells is a generally accepted method used for the treatment of viral infections associated with transplants and rare malignant tumors associated with viruses. Similarly, it was shown that the isolation and transfer of tumor-specific T cells can be successful in treating melanoma. New types of specificity in T-cells were successfully generated by genetic transfer of transgenic T-cell receptors or chimeric antigen receptors (CAR). CARs are synthetic receptors consisting of a directional component that binds to one or more signal domains in a single hybrid molecule. CAR allowed T-lymphocytes to be successfully redirected to antigens expressed on the surface of tumor cells from various types of malignant tumors, including lymphomas and solid tumors (Jena, Dotti et at. 2010).

The current protocol for treating patients with adoptive immunotherapy is based on the transfer of autologous cells. According to this approach, T-lymphocytes are taken from the patient, genetically modified or subjected to ex vivo selection, cultured in vitro to increase the number of cells, if necessary, and, at the end, they are administered by infusion to the patient. When treating autologous cells, technical and logistical difficulties arise with specific applications, their creation requires expensive specialized facilities and experienced staff, they must be created in a short time after the patient is diagnosed, and in many cases, prior treatment of the patient leads to impaired immune function. for example, patient's lymphocytes may function poorly and be present in very small quantities. Because of these difficulties, each drug in a patient’s autologous cells is essentially a new product, which leads to significant variations in efficacy and safety.

In the ideal case, it would be desirable to apply a standardized treatment tool in which allogeneic therapeutic cells could be obtained in advance, characterized in detail, and available for immediate administration to patients. However, allogeneic T cells are obtained from individuals belonging to the same species, but genetically distinct. Thus, specific endogenous T-cell receptors (RTLs) of allogeneic cells recognize host tissue as foreign tissue, which leads to graft versus host disease (TSH), which can lead to severe tissue damage and death. T-cell receptors (RTLs) are cell surface receptors that are involved in T-cell activation in response to antigen presentation. As with immunoglobulin molecules, the alpha and beta chain variable region is generated by V (D) J recombination, which leads to the creation of a wide variety of specificities for antigens within a specified T cell population. However, unlike immunoglobulins that recognize an intact antigen, T-lymphocytes are activated by processed peptide fragments in association with the major histocompatibility complex (MHC) molecule, introducing an additional parameter to T-cell recognition of the antigen, known as MHC restriction. Recognizing the difference between the SIT between the donor and the recipient using the T-cell receptor leads to the proliferation of T-cells and the potential development of TPC. In order to efficiently use allogeneic cells, the inventors inactivated the gene for RTL alpha or RTL beta, which leads to the elimination of RTL from the surface of T cells and, thus, prevents the recognition of alloantigen and, as a result, the development of TPC.

Although noticeable progress has been made in the areas of cancer detection and biology of tumor cells, the treatment of advanced cancer and metastatic cancer remains a big unsolved problem. Cytotoxic chemotherapeutic agents remain one of the most widely used and successfully implemented anticancer agents. Several cytotoxic agents, such as antimetabolites, alkylating agents, anthracyclines, DNA methyltransferase inhibitors, platinum compounds and spindle poisons, have been developed to destroy cancer cells. However, they do not demonstrate equally high efficacy, and the introduction of these agents with new therapeutic agents, such as immunotherapy, is problematic. For example, chemotherapeutic agents can be detrimental to the formation of resistant anticancer immunocompetent cells due to the non-specific toxic profile of these agents. Small molecule therapies aimed at cell proliferation can also impede the formation of antitumor immunity. However, if chemotherapeutic regimens that have temporary efficacy could be combined with new immunocompetent cell-based agents, a significant improvement in antitumor therapy could be achieved (for review (Dasgupta, McCarty et al. 2011)).

Thus, to use “ready-to-use” allogeneic therapeutic cells in combination with chemotherapy, the authors of the present invention are developing a method for constructing allogeneic T-cells resistant to chemotherapeutic agents. The therapeutic utility achieved through such a strategy should be enhanced by a synergistic action between chemotherapy and immunotherapy. In addition, drug resistance can also be beneficial due to the ability of the designated T-cells to selectively multiply, thus avoiding problems with inefficient gene transfer to these cells.

Summary of Invention

In accordance with one aspect of the present invention, methods are provided for constructing immune cells with imparting resistance to chemotherapeutic drugs from the class of purine nucleotide analogs (APNs), such as clofarabin and fludarabine, so that they can be used in cancer immunotherapy in patients who have previously undergone traditional chemotherapy. Immune cells can be obtained from a patient, as in the case of TIL (tumor-penetrating cells), for autologous treatment, or from donors for allogeneic cells that can be used for allogeneic treatment.

In the latter case, when the immune cells are T-cells, according to the present invention, methods for constructing T-cells are also proposed, which are obtained both resistant to chemotherapeutic drugs and allogeneic. Such methods include the step of inactivating at least one gene encoding a component of the T-cell receptor (RTL), in particular the RTL alpha genes, RTL beta, along with the inactivation of a drug susceptibility gene, such as the dcK genes and HPRT.

According to another aspect, drug resistance can be imparted to T cells by expressing the drug resistance gene. It was found that drug resistance to cells according to the present invention is conferred by variant alleles of several genes, such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT).

The present invention includes isolated cells or cell lines obtained using the method of the present invention, more specifically, isolated immune cells containing any proteins, polypeptides, allelic variants, modified or deleted genes or vectors described in this application.

The immune cells of the present invention or cell lines may additionally contain exogenous recombinant polynucleotides, in particular CAR or “suicide” genes, or they may contain altered or deleted genes encoding nodal proteins or their ligands that contribute to their efficacy as a therapeutic product, ideally, as a “finished product”. According to another aspect, the present invention relates to a method of treating or preventing cancer in a patient by introducing a constructed immune cell obtained by the methods described above.

- Figure 1 shows a diagram of the metabolic pathways and cellular toxicity of purine nucleotide analogs (APS); inactivation of the enzyme deoxycytidine kinase (dCK) confers resistance to clofarabine and fludarabine;

- Figure 2 shows that inactivation of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGFT) confers resistance to 6-mecraptopurin (6MR) and 6-thioguanine (6TG) preparations;

- Figure 3A depicts the general architecture of the dCK gene for exons and introns, and

- Figure 3B shows the sequences of target TALE nuclease sites located for pairs of TALE nucleases in exon 2 dCK;

- Figure 4 shows the sequence of actions performed when generating and describing the properties of T-lymphocytes with a HGFT gene knockout; D0 denotes day 0, Dn denotes day n; T7 is Endo T7;

- Figure 5 shows the results obtained in the analysis with endo T7 to verify the processing of the dCK gene; the upper band corresponds to the unprocessed wild-type dCK gene, and the 2 lower bands correspond to the processed dCK gene;

- Figure 6 shows the expansion of T-cells with a knockout of the dCK gene, treated with 5 μg and 10 μg of mRNA, encoding dCK2 TALE-nuclease, and control wild-type T-cells 1 and 2 for a period of 14 days after electroporation.

- Figure 7 shows the endo T7 assay performed on Day 8 (D8) to check for inactivation of dCK in T-cells (using 5 µg of the tALE-nuclease dCK 2 pair) in the presence of 1 µM clofarabine (+) or in the absence of clofarabine (-);

- Figure 8 shows the percentage of viability of wild-type cells with a knockout of the dCK gene (treated with 5 μg and 10 μg of mRNA coding for a pair of dCK2-TALE-nuclease) cultured for two days in the presence of an increasing amount of clofarabine (10 nM - 10 μM) . This graph allows the IC50 (half maximal inhibition concentration) of clofarabine to be determined for both cell populations;

- Figure 9 shows 2 sequences of actions used in generating and describing the properties of clofarabine-resistant allogeneic T-cells; the top panel shows the case when the selection was carried out on the sensitivity to the drug, in contrast to the bottom panel, where the selection on the sensitivity to the drug was not carried out; Day 0 (D0) is the day when double electroporation was performed with TALE nuclease TRAC and dCK;

- Figure 10 shows the analysis with endo T7 for genetic verification of the efficiency of double knockout of dCK / TRAC genes in T cells at different times after electroporation (D1, D3 and D6). The primers used for each locus are given in the example, for T-cells with simple dCK (+ -) knockout and T-cells with simple TRAC (- +) knockout, T-cells with double dCK / TRAC (++) and Wild type T cells (-); the lower bands indicate the correct processing of the dCK and TRAC genes;

- Figure 11 shows an analysis with endo T7 and deep sequencing data to test the effectiveness of dCK inactivation in the presence of (+) or absence (-) clofarabine, with (+) or without (-) inactivation of TRAC, the legend is the same as in Figure ten; to assess the percentage of insertions / deletions in the dCK locus, an estimate of the frequency of insertions was performed;

- Figure 12A shows an experiment to control labeling conducted with T-cells in the presence of (labeled T-cells) or no anti-RTL mAb-PE (unlabeled T-cells); in Figure 12B, TCAR-negative cells are monitored after incubation with or without clofarabine, before and after purification of TRAC knockout T-cells. These cells were also inactivated by the dCK gene;

- Figure 13 shows the growth rate for T-cells with a simple knockout of dCK and TRAC and T-cells with double knockout of dCK / TRAC compared to wild-type T-cells in the absence of clofarabine for 12 days after electroporation;

- Figure 14 shows growth rate curves for T cells with a double knockout of CAR dCK / TRAC in a medium containing different doses of clofarabine (from 0.1 to 10 μM) compared to CAR T cells (with or without clofarabine) for 11 days;

- Figure 15 shows the percent viability for T cells with a simple knockout of dCK or TRAC, T cells with double knockout of dCK / TRAC compared to wild type T cells in a medium containing different doses of clofarabine (from 0.1 to 100 μM ); This chart allows you to determine the IC50 for clofarabine for each population of T cells;

- Figure 16 shows the percentage of specific cytotoxicity for T cells with a double knockout of CAR dCK / TRAC compared to T cells of the CAR FMC63 (and those and others express a CD19 antigen) relative to T cells with a double knockout of dCK / TRAC (without CAR, i.e., do not express CD19 antigen) and wild-type T cells (without knockout and without CAR);

- Figure 17 shows the percent viability for T cells with a double knockout CAR dCK / TCAR compared with control T cells with CAR, when these cells were incubated with increasing doses of clofarabine (10 ng - 10 μg, upper graph), and fludarabine (10 μM - 100 μM, lower graph). These graphics allow you to determine the IC50 for both clofarabine and fludarabine;

- Figure 18 shows the analysis with endo T7 on day 2 (D2) for the purpose of genetic verification of the effectiveness of dCK inactivation in Dowdy (+) cells (5 μg of mRNA encoding TALE-nucase dCK was used) in comparison with wild-type (-) cells. The upper band corresponds to the unprocessed dCK gene, while the 2 lower bands correspond to the inactivation products of dCK;

- Figure 19 shows the growth rate (expressed as × 10 ⁶ cells) for 7 days in Daudi cells with simple dCK knockout and for wild type Daudi cells in the absence or presence of increasing amounts of clofarabine (0.1-1 μM);

- Figure 20 shows the architecture of the whole HGFT gene (for exons and introns) and the localization of different target sites of TALE nucleases (all of them in exon 2);

- Figure 21 shows the sequence of actions used to generate and describe the properties of T-cells with a HGFT gene knockout;

- Figure 22 shows the analysis with endo T7 in order to check the inactivation of the HGFT gene in T cells by TALE-nuclease-HGFT pairs No. 1 and T-pair No. 2 (2 doses were studied: 5 μg and 10 μg), Day 4 (D4) ;

- Figure 23 shows the growth rate (expressed as × 10 ⁶ cells) for 13 days in T-cells with a simple GGFT knockout, using 5 or 10 μg of a TALE-nuclease-GGFT1 (GGFT1) pair or a GHFT2 pair of TALE nucleases in comparison with wild-type T-cells - control 1 and control 2;

- Figure 24 shows the analysis with endo T7 in order to check the inactivation of the HGFT gene in T-cells using 5 or 10 μg of the TALE-nuclease-GGFT No. 1 pair (TALE-nuclease HGTPT 1) in comparison with wild-type T cells [labeled as (-)] in D8 / and D18 when these cells were incubated in 1 μM of TG preparation 6;

- Figure 25 shows the analysis with endo T7 in order to check the inactivation of the HGFT gene in T cells in the presence or absence of 4G7 CAR, this analysis was performed without selection on 6TG;

- Figure 26 shows the percentage of specific cytotoxicity for CAR T cells with knockout of the HGFT gene compared with CAR 4G7 T cells (both types express the C019 antigen) and wild type T cells (without and without CAR);

- Figure 27 shows the percent viability for CAR T cells with knockout of the HGFT gene compared to wild type T cells with increasing doses of the 6TG preparation (10 ng-50 μM).

Detailed Description of the Invention

Unless otherwise indicated in this application, all technical and scientific terms used have the same meaning as commonly understood by experts in the field of gene therapy, biochemistry, genetics and molecular biology.

All methods and materials similar or equivalent to the methods and materials described in this application can be applied in implementing or testing the present invention, together with the appropriate methods and materials described in this application. All publications, patent applications, patents and other references referred to in this application are incorporated by reference to their full content. In the event of a conflict, the present description, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting unless otherwise indicated.

The implementation of the present invention should be achieved, unless otherwise indicated, by traditional methods of cell biology, cell cultures, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the competence of specialists in this field of technology. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001; Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Harries & S.J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), in particular, Vols.154 and 155 (Wu et al. eds.) and Vol .185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell & Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

- drug resistant T cells

In this application, the terms "therapeutic agent", "chemotherapeutic agent" or "drug" refer to a compound or its derivative that can interact with a cancer cell, and as a result, reduce the cell proliferative status and / or destroy the cell. Examples of chemotherapeutic agents include, without limitation, alkylating agents (for example, cyclophosphamide, ifosamide), metabolic antagonists (for example, anti-metabolites of purine nucleosides, such as clofarabine, fludarabine or 2'-deoxyadenosine, methotrexate (MTX), 5-fluorouracil or its derivatives, or its derivatives. antibiotics (eg, mitomycin, adriamycin), plant derived antitumor agents (eg, vincristine, vindesine, taxol), cisplatin, carboplatin, etopoxide, and the like. Such agents may also include, without limitation, antineoplastic agents TRIMETOTRIXAT ™ (TMTH), Temozolomide ™, RALTRITREXED ™, S- (4-nitrobenzyl) -6-thioinosine (NBMPR), 6-benzyl-guanidine (6-BG), bis-chloro-nitrosine (6-BG), bis-chloro-nitrosine (6-BG), bis-chloro-nitro-benzine (6-BG), 6-benzyl-guranidine (6-BG), bis-chloro-nitrosine (6-BG), 6-benzylozine (NBMPR), 6-benzyl-guanidine (6-BG), bis-chlorobrosine. (BCNU) and CAMPTOTOCINE ™, or a therapeutic derivative of any of them.

In this application, a cell that is “resistant or tolerant” to an agent is understood to mean a cell that has been genetically modified so that said cell proliferates in the presence of a certain amount of agent that inhibits or prevents cell proliferation without modification.

Gene expression of drug resistance

According to a specific implementation variant specified drug resistance can be given to a T-cell by expressing at least one drug resistance gene. By said drug resistance gene is meant a nucleotide sequence that encodes "resistance" to an agent, such as a chemotherapeutic agent (for example, methotrexate). In other words, expression of the gene of drug resistance in the cell allows cells to proliferate in the presence of the specified agent to a greater extent than the corresponding cell proliferates without the gene of drug resistance. The drug resistance gene of the present invention can encode resistance to an antimetabolite, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, their analogs or derivatives, and the like.

Several drug resistance genes have been discovered that can potentially be used to impart drug resistance to target cells (Takebe, Zhao et al. 2001; Sugimoto, Tsukahara et al. 2003; Zielske, Reese et al. 2003; Nivens, Felder et al. 2004; Bardenheuer, Lehmberg et al. 2005; Kushman, Kabler et al. 2007).

One example of a drug resistance gene can also be a mutant or a modified form of dihydrofolate reductase (DHFR). DHFR is an enzyme that is involved in regulating the amount of tetrahydrofolate in a cell and is important for DNA synthesis. In the clinic, folate analogues such as methotrexate (MTX), which inhibit DHFR, are used as antitumor agents. Various mutant forms of DHFR have been described, which are highly resistant to inhibition by antifolates used in therapy. According to a specific embodiment, the drug resistance gene according to the present invention may be a nucleotide sequence encoding a wild type human mutant form of human DHFR (SEQ ID no: 14, GenBank: AAN71996.1), which contains at least one mutation that confers resistance to antifolate preparations, such as methotrexate. According to a specific embodiment, the mutant form of DHFR contains at least one mutated amino acid at position G15, L22, F31 or F34, preferably at position L22 or F31 ((Schweitzer, Dicker et al. 1990); international patent application WO 94/24277 ; US patent US 6,642,043). According to a specific embodiment, said mutant form of DHFR contains two mutated amino acids at positions L22 and F31. The correspondence of the positions of the amino acids described in this application is often expressed for the positions of the amino acids in the form of a wild type DHPF polypeptide, the sequence of which is indicated in SEQ ID NO: 14. According to a specific embodiment, the serine residue in position 15 is preferably replaced by a tryptophan residue. In another specific embodiment, the leucine residue at position 22 is preferably replaced with an amino acid that will break the binding of the mutant DHFR to antifolates, preferably uncharged amino acid residues, such as phenylalanine or tyrosine. According to another specific implementation variant, the phenylalanine residue in positions 31 or 34 is preferably replaced with a small hydrophilic amino acid, such as alanine, serine or glycine.

In this application, the term "antifolate agent" or "folate analogs" refers to a molecule that specifically disrupts the folate metabolic pathway at a certain level. Examples of antifolate agents include, for example, methotrexate (MTX); aminopterin; trimetrexate (Neutrexin ™); edatrexate; N10-propargyl-5,8-dideazofolic acid (CB3717); ZD1694 (Tumodex), 5,8-dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid (DDATHF); 5-deazafolic acid; PT523 (N alpha- (4-amino-4-deoxypretroyl) -N delta-hemiphthaloyl-L-ornithine); 10-ethyl-10-deazaminopterin (DDATHF, lomatrexol); pyriterexim; 10-EDAM; ZD1694; GW1843; and pemetrexate and PDX (10-propargyl-10-deazaaminopterin).

Another example of a drug resistance gene is also a mutant or modified form of ionisin-5'-monophosphate dehydrogenase II (IMPDH2), which limits the rate of the enzyme in de novo synthesis of guanosine-nucleotides. The mutant or modified form is the IMPDH gene resistant to inhibitors. IMPDH inhibitors can be mycophenolic acid (IFC) or its prodrug-mycophenolate motefil (MMF). Mutant IMPDH2 may contain at least one, preferably two mutations in the wild-type human MAP IMPDH2 binding site (SEQ ID no: 15; NP_000875.2), which lead to a significant increase in resistance to the IMPDH inhibitor. Mutations are preferably present in the provisions of T333 and / or S351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al. 2007; Jonnalagadda, Brown et al. 2013). According to a specific embodiment, the threonine residue at position 333 is replaced with an isoleucine residue and the serine residue at position 351 is replaced with a tyrosine residue. The correspondence of the positions of the amino acids described in this application is often expressed relative to the positions of the amino acids in the form of a wild-type human IMPDH2 polypeptide, the sequence of which is given in SEQ ID no: 15.

Another genome of drug resistance is the mutant form of calcinerin. Calcineurin (PP2B) is an abundantly expressed serine / threonine phosphatase protein that is involved in many biological processes and which plays a central role in the activation of T cells. Calcineurin is a heterodimer consisting of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). After contacting the T cell receptor, calcineurin dephosphorylates the transcription factor NFAT, allowing it to translocate into the nucleus and activate a key target gene, such as IL2. FK506 in combination with FKBP12 or cyclosporin A (CsA) in combination with CyPA block NFAT access to the active center of calcineurin, preventing its dephosphorylation and thereby inhibiting T-cell activation (Brewin, Mancao et al. 2009). The genome of drug resistance according to the present invention may be a nucleotide sequence encoding a calcineurin mutant form resistant to a calcineurin inhibitor, such as FK506 and / or CsA. According to a specific embodiment, the mutant form may contain at least one mutated amino acid in the wild type calcineurin heterodimer in the V314, Y341, M347, T351, W352, L354, K360 positions, preferably double mutations in the T351 and L354 or V314 and Y341 positions. According to a specific embodiment, the valine residue in position 341 can be replaced by a lysine residue or arginine, the tyrosine residue in position 341 can be replaced by a phenylalanine residue; the methionine at position 347 may be replaced by a glutamic acid, arginine or tryptophan residue; threonine at position 352 may be replaced by a cysteine, glutamic acid or alanine residue; the serine residue at position 353 may be replaced by a histidine residue or asparagine; the leucine at position 354 may be replaced by an alanine residue; lysine at position 360 can be replaced by an alanine or phenylalanine residue in the sequence SEQ ID no: 16. The consistency of the amino acid positions described in this application is often expressed relative to the amino acid positions in the form of a wild-type human calcineurin heterotrimer polypeptide, the sequence of which is given in SEQ ID no : 16 (GenBank: АСХ34092.1).

According to a specific embodiment, the mutant form may contain at least one mutated amino acid in wild type calcineurin b heterodimer in the following positions: V120, N123, L124 or K125, preferably double mutations in the L124 and K125 positions. According to a specific embodiment, the valine at position 120 can be replaced with a serine residue, aspartic acid, phenylalanine or leucine; asparagine at position 123 can be replaced by a tryptophan, lysine, phenylalanine, arginine, histidine or serine residue; leucine at position 124 can be replaced with a threonine residue; lysine at position 125 can be replaced by an alanine residue, glutamic acid, tryptophan, or after lysine at position 125 in the amino acid sequence of SEQ ID NO: 17, two residues can be added, such as leucine-arginine or isoleucine-glutamic acid. The consistency of the positions of the amino acids described in this application is often expressed relative to the positions of the amino acids in the form of a wild-type human calcineurinum b heterodimer polypeptide, the sequence of which is given in SEQ ID NO: 17 (GenBank: АХХ34095.1).

Another drug resistance gene is the O- (6) -methylguanine methyltransferase (MGMT) gene, which encodes the human alkylguanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic action of alkylating agents such as nitrosurea and temozolomide (TMZ). 6-benzylguanine (6- BFG) is an AGT inhibitor that potentiates the toxicity of nitrosourea, and is co-administered with TMZ to potentiate the cytotoxic action of this agent. Several mutant forms of MGMT that encode AGT variants are highly resistant to inactivation under the action of 6-BG, but retain their ability to repair DNA (Maze, Kurpad et al. 1999). According to a specific embodiment, the mutant form of AGT may contain the mutated amino acid in wild-type AGT at position P140, in the amino acid sequence of SEQ ID NO: 18 (UniProtKB: P16455). According to preferred embodiments, said proline at position 140 is replaced with a lysine residue.

Another gene for drug resistance may be the protein 1 gene for multidrug resistance (MDR1). This gene encodes a membrane glycoprotein known as P-glycoprotein (P-GP), which is involved in the transport of metabolic by-products through the cell membrane. The P-GP protein exhibits broad specificity for several structurally unrelated chemotherapeutic agents. Thus, cells can be made resistant to drugs by expressing the nucleotide sequence that encodes MDR-1 (NP_000918).

The genome of drug resistance can be genes of cytotoxic antibiotics, such as the ble gene or the mcrA gene. Ectopic expression of the ble or mcrA gene in an immune cell gives a selective advantage when a chemotherapeutic agent acts on the cell, respectively, bleomycin or mitomycin C.

The most practical approach to gene therapy is to add a gene to a constructed T-cell by efficient delivery using vectors, preferably a viral vector. Thus, according to a specific embodiment, said drug resistance gene can be expressed in the cell by introducing a transgene, preferably encoded by at least one vector, into the cell.

Accidental insertion of genes into the genome can lead to inadequate expression of the inserted gene or a gene located close to the insertion site. Specific gene therapy using homologous recombination of exogenous nucleic acid containing endogenous sequences with target genes at specific sites in the genome allows the construction of resistant T cells to be achieved. As described above, the step of genetic modification in this method may include the step of introducing an exogenous nucleic acid into cells containing at least one sequence encoding a drug resistance gene and a part of the endogenous gene, such as homologous recombination, which occurs between the endogenous gene and exogenous nucleic acid acid. According to a specific embodiment, said endogenous gene may be a wild-type “drug resistance” gene, for example, as after homologous recombination, the wild type gene is replaced with a mutant form of the gene that confers resistance to the drug.

It is known that cleavage of bonds between nucleotides increases the frequency of homologous recombination. Thus, according to a specific embodiment, the method according to the present invention also includes the step of expressing in the cell a rarely-releasing endonuclease that is capable of cleaving the target sequence in the endogenous gene. The specified endogenous gene can encode, for example, DHFR, IMPDH2, calcineurin or AGT. Specified rare endonuclease can be TALE-endonuclease, zinc finger nuclease, CRISPR / Cas9 endonuclease, MBBBD nuclease or meganuclease.

Inactivation of drug sensitivity genes

According to another specific implementation variant specified resistance to drugs can be given to the T-cell by inactivating the genes of drug sensitivity. For the first time, the author of the present invention attempted to inactivate a potential drug sensitivity gene in order to construct T cells for immunotherapy.

It is assumed that when inactivating the gene in question, it will not be expressed to form a functional protein. According to a specific implementation variant, the genetic modification underlying the method is based on the expression, in the cells proposed for construction, of one rarely ending endonuclease, such as the indicated rarely ending endonuclease that specifically catalyzes cleavage in one target gene and, as a result, inactivates said target gene. According to a specific embodiment, the step of inactivating at least one drug susceptibility gene comprises introducing a rarely-releasing endonuclease into a cell that is able to cleave at least one drug susceptibility gene. According to a more specific implementation variant, said cells are transformed with a nucleic acid encoding a rarely-releasing endonuclease that is able to cleave the drug-susceptible gene, and the indicated rare-ending endonuclease is expressed in said cells. Specified rare endonuclease can be a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 nuclease, a MBBBD nuclease or a TALE nuclease.

According to a preferred embodiment, the genome of drug sensitivity, which can be inactivated to confer resistance to the drug T-cell, is the human deoxycytidine kinase (dCK) gene. This enzyme is needed for the phosphorylation of deoxycytidine (dC), deoxyguanosine (dG) and deoxyadenosine (dA) deoxyribonucleotides. Analogs of purine nucleotides (PNA) are metabolized with the participation of dCK to mono-, di- and triphosphate-PNA. Their triphosphate forms and in particular clofarabine triphosphate compete with ATP for DNA synthesis, act as a proapoptotic agent and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in the production of trinucleotides (cf-putative mechanism of action in Figure 1).

Preferably, inactivation of dCK in T cells is mediated by TALE nuclease. To accomplish this task several pairs of TALE nucleases dCK were developed, which are assembled at the level of polynucleotide and verified by sequencing. Examples of pairs of TALE nucleases that can be used according to the present study are displayed in the sequences SEQ ID No. 63 and SEQ ID No. 64. When a pair of TALE nucleases is used, the target dCK sequence corresponds to SEQ ID No. 62.

As shown in the examples, such inactivation of dCK in T cells confers resistance to purine nucleoside analogs (PNA), such as clofarabin and fludarabine.

According to another preferred embodiment, the inactivation of dCK in T-cells is combined with the inactivation of TRAC genes, and T-cells with a knockout of two of these genes resistant to a drug such as clofarabine and allogeneic are obtained. Such dual characteristics are particularly useful for therapeutic purposes and allow the use of “ready-made” allogeneic cells for immunotherapy in combination with chemotherapy for treating patients with cancer. Such double inactivation by knockout of dCK / TRAC genes can be carried out simultaneously or sequentially. One example of a TALE nuclease dCK / TRAC pair that leads to a successful outcome according to the present invention is the use of SEQ ID No. 63 and SEQ ID No. 64, as well as SEQ ID No. 66 and No. 67, respectively. The target sequences in 2 loci (dCK and TRAC) are displayed in SEQ ID No. 62 and SEQ ID No. 65, respectively.

Another example of an enzyme that can be inactivated is the human hypoxanthine-guanine phosphoribosyltransferase (HGFT) gene (Genbank: M26434.1). In particular, HGFT can be inactivated when constructing T cells to make them resistant to the cytotoxic metabolite 6-thioguanine (6TG), which is converted with the participation of HGTP to cytotoxic thioguanine nucleotide, and which is currently used to treat patients with cancer, in particular leukemias Hacke, Treger et al., 2013). Guanine analogs are metabolized by the HGFT enzyme, which catalyzes the addition of a phosphorybozyl group and allows the formation of thioGMP (Figure 2). Guanine analogs, including 6-mercaptopurine (6MP) and 6-thioguanine (6TG) are usually used as anti-lymphomas for the treatment of ALL (acute lymphoblastic leukemia). They are metabolized by GPT (hypoxanthine phosphoribosyltransferase), which catalyzes the addition of the phosphoribosyl group and allows the formation of thioGMP. Their subsequent phosphorylation leads to the formation of their phosphorylated forms, which are eventually incorporated into the DNA. Being included in the DNA of thio-GTP, violates the accuracy of reproduction of DNA replication due to its thiol groups and generates a random point mutation, which is largely harmful to the integrity of the cell.

In yet another embodiment, inactivation of normal T-cells expressed on the surface of the cell may confer resistance to anti-CD3 antibodies, such as replicumab.

Daudi-resistant CD19 + / luc + drug-resistant cells for assessing the cytotoxicity of drug-resistant allogeneic T-cells with CAR

The present invention also includes a method for producing target cells that express both a T cell-specific surface receptor with CAR, and a resistance gene. These target cells are particularly useful in evaluating the cytotoxicity of T cells with CAR. These cells are highly resistant to a clinically significant dose of clofarabine and possess luciferase activity. This combination of properties allows you to trace them in vivo in model mice. More specifically, they can be used to evaluate the cytotoxic properties of drug resistant T cells in mice in the presence of clofarabine or other PNAs. Dowdy-resistant cells with a clofarabine model simulate the physiological state of patients with acute lymphoblastic leukemia (ALL), who have relapses during induction of therapy, and have drug-resistant B-cell tumors. Thus, these cells are of great interest for assessing the reliability and cytotoxicity of drug resistant T cells with CAR. Preferably, said target cells are Daudi cells CD19 + luciferase +.

Isolated (isolated) cells

The present invention also relates to isolated cells that can be obtained using the method described above. In particular, the present invention relates to a drug resistant T-cell that contains at least one disrupted gene encoding a component of the T-cell receptor. According to a specific embodiment, said T cell expresses at least one drug resistance gene, preferably the ble gene or the mcrA gene, or a gene encoding mutant DHFR, mutant IMPDH2, mutant AGT or mutant calcineurin. According to another specific implementation variant specified T-cell contains at least one disrupted gene drug sensitivity, such as the dCK gene or GGFT. According to a more specific embodiment, said isolated T-cell contains an impaired HGFT gene and expresses mutant DHFR; said isolated T-cell contains an abnormal HGFT gene and expresses mutant IMPDH2; said isolated T-cell contains a disturbed HGFT gene and expresses mutant calcineurin; indicated

an isolated T cell contains an aberrant HGFT gene and expresses mutant AGT. According to a preferred embodiment, said isolated cell expresses a chimeric antigen receptor (CAR), which may be CD19 or CD123.

Allogeneic T-cell resistant to the drug

In particular, the present invention relates to a drug-resistant allogeneic T-cell, particularly suitable for immunotherapy. Drug resistance can be imparted by inactivating drug sensitivity genes or by expressing drug resistance genes that have been described above. Some examples of drugs that are suitable for implementing the present invention include purine nucleodide analogs (PNA), such as clofarabine or fludarabine, or other drugs, such as 6-mercaptopurine (6MP) and 6-thioguanine (6TG).

Under the cell according to the present invention understand the cell of hematopoietic origin, functionally involved in the initiation and / or actuation of a congenital and / or acquired immune response. The cell of the present invention is preferably a T-lymphocyte obtained from a donor. The specified T-lymphocyte according to the present invention can be obtained from a stem cell. These stem cells can be adult stem cells, embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, totipotent stem cells or poetic stem cells. Representative human stem cells include CD34 + cells. The isolated cell may also be a dendritic cell, killer dendritic cell, mast cell, NK cell (natural killer), B-lymphocyte or T-cells selected from the group consisting of inflammatory T-cells, cytotoxic T-cells, regulatory T cells or helper T cells. According to another implementation variant of the specified cell can be obtained from the group consisting of CD4 + T cells and CD8 + T cells. Before reproduction and genetic modification of cells according to the present invention, the cell source can be obtained from the subject using various non-limiting methods. Cells can be obtained from a variety of sources, including, without limitation, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the source of infection, ascites, pleural effusion, spleen tissue and tumors. According to certain embodiments of the present invention, any number of T-cell lines available and known to those skilled in the art can be used. According to another implementation variant of the specified cell preferably receive from a healthy donor. According to another implementation variant of this cell is part of a mixed population of cells that exhibit different phenotypic characteristics.

Multiple drug resistance

According to another specific implementation variant, the authors of the present invention attempted to develop a “standard” immunotherapy strategy using allogeneic T cells resistant to several drugs to facilitate the selection of engineered T cells when a patient receives different drugs. Therapeutic efficacy can be significantly improved by the genetic design of multidrug-resistant allogeneic T cells. Such a strategy can be particularly effective in treating tumors that respond to combinations of drugs that have a synergistic effect. In addition, designed multidrug-resistant T cells can multiply and undergo selection with a minimum dose of drugs.

Thus, the method according to the present invention may include the implementation of the modification of T-cells to impart multi-drug resistance of the specified T-cell. This multidrug resistance can be imparted either by expressing more than one drug resistance gene, or by inactivating more than one drug susceptibility gene. According to another specific implementation variant, multidrug resistance can be imparted to said T-cell by expressing at least one drug-resistance gene and by inactivating at least one drug-sensitive gene. In particular, multidrug resistance can be imparted to said T-cell by expressing at least one drug resistance gene, such as the mutant form DHFR, the mutant form IMPDH2, the mutant form calcineurin, the mutant form MGMT, the ble gene and the mcrA gene, and by inactivating at least one drug susceptibility gene, such as the GMP gene. In a preferred embodiment, multidrug resistance can be imparted by inactivating the HGFT gene and by expressing the mutant form of DHFR; or by inactivating the HGFT gene and by expressing the mutant form of IMPDH2; or by inactivating the HGFT gene and by expressing the mutant form of calcineurin; due to inactivation of the gene GGFT and due to the expression of the mutant form of MGMT; due to the inactivation of the gene GGFT and due to the expression of the gene ble; due to the inactivation of the gene HGFT and due to the expression of the gene mcrA.

A method for constructing drug-resistant allogeneic T-cells:

To improve anticancer therapy and selective engraftment of allogeneic T cells, these cells are drug resistant to protect them from the toxic side effects of the chemotherapeutic agent. Drug-resistant T cells also allow them to enrich their pool in vivo or ex vivo, since T cells that express the drug resistance gene will survive and reproduce compared to cells sensitive to the drug. In particular, the present invention relates to the design of allogeneic and drug-resistant T-cells for immunotherapy, including:

(a) Providing T cells;

(b) Selection with at least one drug;

(c) Realizing the modification of said T-cells by inactivating at least one gene encoding a component of the T-cell receptor (RTL);

(d) Implementation of the modification of T-cells to give them resistance to the drug;

(e) Ensuring the reproduction of these engineered T-cells in the presence of the indicated drug.

- Allogenic T-cells

The present invention relates to allogeneic immunotherapy. Engraftment of allogenic T cells is possible by inactivating at least one gene encoding a component of the RTL. RTLs are made non-functional in cells by inactivating the RTL-alpha gene and / or the RTL-beta gene. Inactivation of RTL allows to exclude TPH. It is assumed that when the gene is inactivated, the gene in question is not expressed to form a functional form of the protein. According to specific embodiments, the genetic modification according to this method is based on the expression of one rare endonuclease obtained in the construction of cells, so that the rare rare endonuclease specifically catalyzes cleavage in one target gene, thereby inactivating the indicated target gene. The gaps in the nucleotide chain, caused by the rare-cleaving endonuclease, are usually recovered by different mechanisms of homologous recombination or non-homologous ends (NHEJ). However, NHEJ is an imperfect repair process that often leads to changes in the DNA sequence at the cleavage site. Mechanisms imply reconnecting what remains at the two ends of the DNA through direct re-ligation or through the so-called connection of the ends mediated by microhomology (Betts, Brenchley et al. 2003; Ma, Kim et al. 2003). Reparation by connecting non-homologous ends (NHEJ) often leads to small insertions or deletions and can be used to obtain directional knockout of genes. This modification can be a substitution, deletion or addition of at least one nucleotide. The cells in which the mutagenesis event occurred, i.e. a mutagenesis event with a subsequent NHEJ event can be identified and selected using the method well known in the art. According to a specific embodiment, the step of inactivating at least one gene encoding a component of the T-cell receptor (RTL) in a cell of a sample of each individual, comprises introducing into the said cell a rarely ending endonuclease capable of disrupting at least one gene encoding a component of the T-cell receptor ( RTL). According to a more specific implementation variant, said cells of a sample of each individual are transformed with a nucleic acid encoding a component of the T-cell receptor (RTL), and said rare-cleaving endonuclease is expressed in said cells.

Said rare-cutting endonuclease can be a meganuclease, a zinc finger nuclease, a CRISPR / Cas9 nuclease, a TALE nuclease, or an MBBBD nuclease. According to a preferred embodiment, said rare-cleaving endonuclease is a TALE nuclease. TALE nuclease is understood to mean a fusion protein consisting of a DNA-binding domain derived from an activator-like effector transcription (TALE) and a single nuclease catalytic domain for cleaving the target nucleotide sequence (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Cermak, Doyle et al. 2011; Geissler, Scholze et al. 2011; Huang, Xiao et al. 2011; Li, Huang et al. 2011; Mahfouz, Li et al. 2011; Miller, Tan et al. 2011; Morbitzer, Romer et al. 2011; Mussolino, Morbitzer et al. 2011; Sander, Cade et al. 2011; Tesson, Usal et al. 2011; Weber, Gruetzner et al. 2011; Zhang, Cong et al. 2011; Deng, Yan et al. 2012; Li, Piatek et al. 2012; Mahfouz, Li et al. 2012; Mak, Bradley et al. 2012). According to the present invention, new TALE-nucleases have been developed with precise targeting of the corresponding genes for adoptive immunotherapy strategies.

Preferred TALE nucleases according to the present invention are nucleases that recognize and cleave a target sequence selected from the group consisting of: SEQ ID no: 1-5 (RTL-alpha), SEQ ID no: 6 and 7 (RTL-beta). These TALE nucleases preferably contain a polypeptide sequence selected from SEQ. ID no: 8 - SEQ ID no: 13. According to another embodiment, an additional catalytic domain can be introduced into the cell along with this rarely-releasing endonuclease to enhance mutagenesis to increase its ability to inactivate target genes. In particular, said additional catalytic domain is an enzyme processing the ends of the DNA. Examples of enzymes for processing DNA ends include 5-3'-exonucleases, 3-5 'exonucleases, 5-3' alkaline exonucleases, 5'-flap endonucleases, helicases, cospases, hydrolases, and matrix-independent DNA polymerases. Non-limiting examples of such catalytic domains include a protein domain or a catalytically active derivative of a protein domain selected from the group consisting of hExol (EXO1_HUMAN), Exol yeast (EXO1_YEAST), Exol E. coli, TREX2 human, TREX1 mouse, TREX1 human, TREX1 bov, TREX1 human rats, TdT (terminal deoxyncleotidyl transferase) human DNA2, yeast DNA2 (DNA2_YEAST). According to a preferred embodiment, said additional catalytic domain has 3'-5'-endonuclease activity, and according to a more preferred embodiment, said additional catalytic domain is TREX, more preferably the catalytic domain TREX2 (WO 2012/058458). According to another preferred embodiment, the catalytic domain is encoded by a single-stranded TREX2 polypeptide. Specified additional catalytic domain can be integrated into the hybrid protein or chimeric protein nuclease according to the present invention, optionally using a protein linker.

It is known that gaps between nucleotides increase the percentage of homologous recombination. Thus, according to another embodiment, the step of genetic modification of the method also includes the step of introducing exogenous nucleic acid into the cells containing a sequence homologous to at least part of the target nucleotide sequence so that homologous recombination occurs between the target nucleotide sequence and exogenous nucleic acid. According to specific embodiments, said exogenous nucleic acid comprises first and second parts that are homologous to the 5 'and 3' regions of the target nucleotide sequence, respectively. This exogenous nucleic acid according to these embodiments also contains a third part located between the first and second parts, which does not have homology with the 5 ′ and 3 ′ regions of the target nucleotide sequence. After cleavage of the target nucleotide sequence, an event of homologous recombination between the target nucleotide sequence and exogenous nucleic acid is stimulated. Preferably, homologous sequences of at least 50 in length, preferably more than 100 in and more preferably more than 200 in, are used within the specified donor matrix. According to a particular embodiment, the homologue starting sequence may be from 200 in each to 6000 in, more preferably in the range from 1000 in to 2000 in. In fact, nucleic acid-shared homology is located in the regions flanking the cleavage site above and below in the sequence, and the nucleotide sequence that needs to be entered must be located between the two arms.

According to a specific implementation variant specified exogenous nucleic acid may contain a transgene encoding a gene for drug resistance according to the present invention.

Construction of additional possible properties of T-cells

The immune cells according to the present invention can also be designed so that they acquire additional properties that would facilitate their more specific or more effective use in therapy.

- Chimeric antigen receptors (CAR)

и обладает способностью, когда экспрессируется в Т-клетках, перенаправлять распознавание, основанное на специфичности моноклонального антитела. Одним примером CAR, применяемого согласно настоящему изобретению, является CAR, направленный на антиген CD19, который может содержать в качестве неограничивающих примеров аминокислотную последовательность: SEQ ID №: 19 или 20.Chimeric antigen receptors (CAR) are able to redirect the specificity and reactivity of immune cells and target them to a chosen target, based on the properties of ligand-binding domains. Thus, according to another embodiment, this method also includes the step of introducing the chimeric antigen receptor into the lymphocytes. Said chimeric antigen receptor combines a binding domain for a component present on a target cell, for example, antibody-based specificity for the desired antigen (eg, tumor antigen) with the intracellular domain that activates the T-cell receptor, to generate a chimeric protein that exhibits specific immune activity against the target cell. As a rule, CAR consists of an extracellular single cell anti-antibody (scFv), which is connected to the extracellular signaling domain of the zeta chain of the T-cell receptor complex for antigen recognition

and has the ability, when expressed in T cells, to redirect recognition based on the specificity of the monoclonal antibody. One example of a CAR used according to the present invention is CAR directed to a CD19 antigen, which may contain, as non-limiting examples, the amino acid sequence: SEQ ID NO: 19 or 20.

- Inactivation of immune checkpoint genes

T-cell mediated immunity includes several successive stages, including selection of clones of antigen-specific cells, their activation and proliferation in the secondary lymphoid tissue, their directed migration to the foci of antigen and inflammation, direct effector function and assistance (through cytokines and membrane ligands) ) a large number of immune effector cells. Each of these stages is regulated by balancing stimulating and suppressing signals, which produce a fine adjustment of the response. Experts in the art should understand that the term "immune checkpoints" refers to a group of molecules expressed by T-cells. These molecules effectively perform the function of "stoppers" for negative modulation or suppression of the immune response. Immune checkpoint molecules include, without limitation, programmed death molecule 1 (PD-1, also called PDCD1 or CD279, access number: NM_005018), cytotoxic T-cell antigen 4 (CTLA-4, also called CD152, GenBank access number AF414120.1) , LAG3 (also called CD223, access number: NM_002286.5), Tim3 (also called HAVCR2, GenBank access number: JX049979.1), BTLA (also called CD272, access number: NM_181780.3), BY55 (also called CD160 , GenBank access number: CR541888.1), TIG IT (also called VSTM3, access number: NM_173799), LAIR1 (also called CD305, GenBank access number: CR542051.1, (Meyaard, Adem a et al. 1997)), SIGLEC10 (GeneBank access number: AY358337.1), 2B4 (also called CD244, access number: NM_001166664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 (Nicoll, Ni et al. 1999), SIGLEC9 (Zhang, Nicoll et al. 2000; Ikehara, Ikehara et al. 2004), TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, CASP7, FADD, FAS, TGFBRII, TGFRBRI, CASP7, FADD, FAS, TGFBRII, TGFRBRI, CASP7, FADD, FAS, TGFBRII, TGFRBRI, CASP7, FADD; SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF (Quigley, Pereyra et al. 2010), GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, which directly suppress the immune response. For example, CTLA-4 is a cell surface antigen expressed on certain CD4 and C08 T cells; when it binds to its ligand (B7-1 or B7-2) on antigen-presenting cells, the activation of T cells and their effector function is inhibited. Thus, the present invention relates to a method for constructing an allogeneic T-cell resistant to a drug, also comprising modifying T-cells by inactivating at least one protein involved in an immune checkpoint, in particular PD1 and / or CTLA-4. According to a preferred embodiment, the step of inactivating at least one protein involved in an immune checkpoint is realized by expressing a rare-cutting endonuclease that can specifically cleave the target sequence in the immune checkpoint gene. According to a preferred embodiment, the rare-cleaving endonuclease is TALE-endonuclease. For example, said TALE nuclease can specifically cleave the target sequence selected from the group consisting of: SEQ ID NO: 21-23 (CTLA-4) and SEQ ID NO: 24 and SEQ ID NO: 25 (PDCD1), and according to more the preferred implementation variant specified TALE-nuclease contains an amino acid sequence selected from the group consisting of SEQ ID no: 26 - SEQ ID no: 35.

- Immunosuppression resistant T cells

Allogeneic cells are quickly rejected by the host’s immune system. It has been demonstrated that allogeneic leukocytes in unirradiated blood products can last no more than 5-6 days (Boni, Muranski et al. 2008). Thus, to prevent rejection of allogeneic cells, the host’s immune system should generally be suppressed to a certain extent. However, in the case of adoptive immunotherapy, the use of immunosuppressive drugs also has a detrimental effect on the administered therapeutic T cells. Therefore, in order to effectively apply the approach of immunotherapy under these conditions, the introduced cells must also be resistant to immunosuppressive treatment. Thus, according to a specific embodiment, the method of the present invention further comprises the step of modifying T cells to render them resistant to an immunosuppressive agent, preferably by inactivating at least one gene encoding a target for an immunosuppressive agent. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. In other words, the immunosuppressive agent plays the role of a compound that exhibits the ability to reduce the degree of immune response. The method according to the present invention makes it possible to impart immunosuppressive resistance to T-cells for immunotherapy by inactivating a target for the indicated immunosuppressive agent in T-cells. As non-limiting examples, targets for an immunosuppressive agent may be a receptor for an immunosuppressive agent, such as: CD52, glucocorticoid receptor (GR), a member of the FKBP gene family, and a member of the cyclophillin gene family. According to a specific implementation variant, the genetic modification according to the present method is based on the expression, in the cells proposed for construction, of one rarely ending endonuclease, such as this rarely ending endonuclease that specifically catalyzes cleavage in one target gene, thus inactivating the indicated target gene. Specified rare endonuclease can be a meganuclease, a zinc finger nuclease or a TALE endonuclease. Preferred TALE nucleases according to the present invention are endonucleases that recognize and cleave a target sequence selected from the group consisting of: SEQ ID no: 36-41 (GR), and SEQ ID no: 54-59 (CD52). These TALE-nucleases preferably include a polypeptide sequence selected from the group consisting of SEQ ID no: 42 - SEQ ID no: 53 and SEQ ID no: 60-SEQ ID no: 61.

- Suicide genes

According to another aspect, since the engineered T-cells multiply and persist for several years after administration, it is desirable to include a security mechanism that would allow the selective removal of the introduced T-cells. Thus, in some embodiments, the method of the present invention involves the transformation of said T cells with a recombinant suicide gene. The indicated recombinant suicide gene is used to reduce the risk of direct toxicity and / or uncontrolled proliferation of these T cells when they have already been introduced to the subject (Quintarelli C, Vera F, blood 2007; Teu SK, Dotti G., Rooney CM, boil blood marrow transplant 2007 ). Suicide genes can selectively remove transformed cells in vivo. In particular, the suicide gene has the ability to transform from a non-toxic prodrug into a cytotoxic drug or to be expressed to form a toxic product. In other words, the “suicide gene” is a nucleic acid encoding a product, and this product causes cell death by itself or in the presence of another compound. A typical example of such a suicide gene is the gene encoding the herpes simplex virus kinase. Additional examples include varicella zoster virus thymidine kinase and the bacterial cytosine deaminase gene, which can convert 5-fluorocytosine to a highly toxic compound 5-fluorouracil. Suicide genes also include, as non-limiting examples, caspase-9 or caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical dimerization inducer (CID). Also, suicide genes can be polypeptides that are expressed on the cell surface and can give the cell sensitivity to therapeutic monoclonal antibodies. In this application, the term "prodrug" refers to any compound useful for implementing the methods of the present invention, which can be converted into a toxic product. The prodrug is converted into a toxic product by the action of a suicide gene product when implementing the method of the present invention. A typical example of such a prodrug is ganciclovir, which is converted in vivo into a toxic compound by HSV-thymidine kinase. Therefore, ganciclovir derivative is toxic to tumor cells. Other typical examples of prodrugs include acyclovir, FIAU [1- (2-deoxy-2-fluoro-β-D-arabinofuranosyl) -5-ioduracil, 6-methoxypurine-arabinoside for VZV-TK, and 5-fluorocytosine for cytosine deaminase.

- Delivery methods

The different methods described above imply the expression of the protein under consideration, such as the gene for drug resistance, a rarely ending endonuclease, a chimeric antigen receptor (CAR), a suicide gene, in a cell. As a non-limiting example, the protein in question can be expressed in a cell by introducing it as a transgene, preferably encoded by at least one plasmid vector. The polypeptides can be expressed in the cell as a consequence of the introduction of polynucleotides encoding these polypeptides into the cell. Alternatively, these polypeptides can be obtained outside the cell, and then enter them into it. Methods for introducing a polynucleotide construct into cells are known in the art and include as non-limiting examples of stable transformation methods in which the polynucleotide construct integrates into the genome of a specified cell, temporary transformation methods in which the polynucleotide construct does not integrate into the genome of the specified cell, and methods mediated by viruses. These polynucleotides can be introduced into a cell, for example, using recombinant viral vectors (for example, retroviruses, adenoviruses), liposomes, and the like. For example, temporal transformation methods include, for example, microinjection, electroporation, or particle bombardment. These polynucleotides can be incorporated into vectors, more specifically into plasmids or a virus, so that they are expressed in cells. The specified plasmid vector may contain a selection marker that allows the identification and / or selection of cells that received the specified vector. Different transgenes can be included in one vector. The specified vector may contain a nucleotide sequence encoding a sequence of jumps of ribosomes, such as a sequence encoding a peptide 2A. Peptides 2A, which were found in the subgroup of picornaviruses - avtoviruses, cause a "jump" of the ribosome from one codon to the next without the formation of a peptide bond between two amino acids encoded by these codons (see Donnelly et al., J. of General Virology 82: 1013- 1025 (2001); Donnelly et al., J. of Gen. Virology 78: 13-21 (1997); Doronina et al., Mol. And. Cell. Biology 28 (13): 4227-4239 (2008); Atkins et al., RNA 13: 803-810 (2007)). By “codon” is meant three nucleotides in mRNA (or in the sense strand of the DNA molecule), which are translated by the ribosome into an amino acid residue. Thus, when polypeptides are separated by an oligopeptide sequence 2A, which are in a frame, two polypeptides can be synthesized from one adjacent open reading frame within the mRNA. Such mechanisms of ribosome jumps are well known in the art, and it is known that they are used with the help of several vectors for the expression of several proteins encoded by a single messenger RNA.

According to a more specific embodiment, the polynucleotides encoding the polypeptides of the present invention can be mRNA that is introduced directly into the cells, for example, by electroporation. The authors of the present invention have determined the optimal conditions for electroporation of mRNA into a T-cell. The author of the present invention applied the cytoPulse technology, which allows, by applying pulsed electric fields, to temporarily permeabilize living cells to deliver material to the cells. This technology, based on the use of PulseAgile electroporation pulse shapes (BTX Havard Apparatus, 84 October Hill Road, Holliston, MA 01746, USA), allows precise control of the duration, pulse intensity, and pulse spacing (US Pat. WO 2004083379). All of these parameters can be modified to achieve the best conditions for high transfection efficiency with minimal mortality. Essentially, the first high impulses of the electric field allow the formation of a pore, and the subsequent lower impulses of the electric field allow the polynucleotide to be moved into the cell.

- Activation and reproduction of T-cells

Either before or after genetic modification, T cells can be activated and multiplied, as a rule, using methods described, for example, in US patents 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; 6867041; and the publication of applications for US patent No. 20060121005. It is possible to provide an increase in the number (reproduction) of T-cells in vitro or in vivo. Typically, the T cells of the present invention are propagated by contacting an agent that stimulates the CD3-PT / 1 complex and a costimulatory molecule on the surface of the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenetic lectins such as phytohemagglutinin (PHA) can be used to create an activating signal for a T-cell. As a non-limiting example, T cell populations can be stimulated in vitro, for example, by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or with an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase activator C ( for example, briostatin) in combination with calcium ionophore. For joint stimulation of the auxiliary molecule on the surface of T-cells, a ligand is used that binds the auxiliary molecule. For example, a population of T cells may be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions suitable for stimulating the proliferation of T cells. To stimulate the proliferation of either CD4 + T cells or CD8 + T cells, an anti-CD3 antibody and an anti-CD28 antibody are used. For example, agents providing each signal may be in solution or bound to a surface. As can be easily understood by those skilled in the art, the ratio of the number of particles to the number of cells may depend on the particle size relative to the target cell.

Conditions suitable for the cultivation of T cells include an appropriate medium (for example, minimal supporting medium or RPMI 1640 or X-vivo 5, (Lonza) medium), which may contain factors necessary for proliferation and viability, including serum (for example, fetal bovine serum or human serum), interleukin-2 (IL-2), IFN-g, 1L-4, 1L-7, GM-CSF, -10, - 2, 1L-15, TGFp, IL-21 and TNF or any other cell growth supplements known to those skilled in the art. Other supplements for cell growth include, without limitation, a surfactant, plasmatane and reducing agents, such as N-acetylcysteine and 2-mercaptoethanol. Medium may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate and vitamins, or without serum, or with an appropriate amount of serum (or plasma), or a given set of hormones, and / or the amount of cytokine (s), sufficient for the growth and reproduction of T-cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures, but not in cell cultures, which will be administered by infusion to a subject. Target cells are stored under conditions necessary to maintain growth, for example, at an appropriate temperature (eg, 37 ° C) and atmosphere (eg, air plus 5% CO2). T cells that have been stimulated for different times may exhibit different properties.

Therapeutic applications

According to another implementation variant of these isolated T cells, obtained as described above, can be used for allogeneic adoptive cellular immunotherapy. In particular, said T cells of the present invention can be used to treat cancer, infections, or an autoimmune disease in patients in need of such treatment. According to another aspect, the present invention is based on methods for treating patients in need of such treatment, said method comprising at least one of the following steps:

(a) Providing an isolated T cell that can be obtained by any of the methods described previously;

(b) Injecting said cells into a patient.

According to one implementation variant of these T cells according to the present invention can undergo reliable reproduction in vivo, and can persist for a long period of time.

The specified treatment may be facilitating, therapeutic or prophylactic. The present invention is particularly suitable for allogeneic immunotherapy, in so far as it allows the transformation of T-cells, usually obtained from donors, to produce non-alloreactive cells. This can be done in accordance with standard protocols and reproduced as many times as necessary. The resulting T-cells are administered to one or more patients, and they become available as a “ready-made” therapeutic drug.

Cells that can be used to implement the disclosed methods are described in the previous section. This tool can be used in the treatment of patients diagnosed with cancer, viral infection, autoimmune disorders. Cancer that can be treated includes tumors that are not vascularized, or that are not yet substantially vascularized, as well as vascularized tumors. Types of cancer may include unsightly tumors (such as hematologic tumors, for example, leukemias and lymphomas) or may include solid tumors. Cancer types that can be treated with drug-resistant allogeneic T-cells according to the present invention include, without limitation, carcinoma, blastoma and sarcoma, and certain leukemias and lymphoid malignancies, benign and malignant tumors, as well as cancers, such as sarcomas , carcinomas and melanomas. Also included are tumors / types of adult cancer and tumors / types of children. According to one embodiment of the present invention, typically, allogeneic drug resistant T cells according to the present invention treat children's acute lymphoblastic leukemia (ALL) and amyotrophic myeloid leukemia (AML). This can be achieved with the use of drug-resistant CD19 ⁺ T cells with CAR with knockout of TRAC gene and drug-resistant CD123 ⁺ T cells with CAR with knockout of TRAC gene.

It can be treated in combination with one or more anti-cancer therapeutic agents selected from the group of antibodies, chemotherapy, cytokines, dendritic cell therapy, gene therapy, hormone therapy, laser therapy and radiation therapy.

According to a preferred embodiment of the present invention, said agent is administered to patients undergoing immunosuppressive therapy. The present invention preferably involves the use of cells or populations of cells that have been rendered resistant to at least one drug in accordance with the present invention either by expressing the drug resistance gene or by inactivating the drug susceptibility gene. According to this aspect, drug therapy should facilitate the selection and expression of T-cells according to the present invention in the patient's body.

The introduction of cells or cell populations according to the present invention can be carried out by any conventional method, including aerosol inhalation, injection, oral administration, transfusion, implantation or transplantation. The compositions described in this application can be administered to a patient subcutaneously, intracutaneously, into a tumor, into lymph nodes, intramedullary, intramuscularly, intracranially, by intravenous or intralymphotic injection, or intraperitoneally. According to one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

The introduction of cells or cell populations can consist in the introduction of 10 ³ -10 ¹⁰ cells per kg of body weight, preferably 10 ⁵ -10 ⁶ cells / kg of body weight, including all integer values of the numbers of cells within this range. Cells or cell populations can be administered in one or more doses. According to another implementation variant of the specified effective amount of cells injected in a single dose. According to another implementation variant of the specified effective number of cells injected in more than one dose over a period of time. The time of administration is at the discretion of the attending physician and depends on the clinical condition of the patient. Cells or cell populations can be obtained from any source, such as a blood bank or donor. Although individual needs vary, the determination of the optimal ranges of an effective number of cells of a particular type for a particular disease or pathological condition is within the competence of those skilled in the art. By an effective amount is meant an amount that provides therapeutic or prophylactic benefit. The dose administered will depend on the age, state of health and mass of the recipient, type of concomitant disease, if any, frequency of treatment and the nature of the desired action.

According to another implementation variant specified effective amount of cells or pharmaceutical compositions containing these cells, is administered parenterally. Said administration may be by intravenous administration. The specified introduction can be carried out directly by injection into the tumor. According to certain embodiments of the present invention, cells are administered to a patient in combination (for example, before, simultaneously or after) with any number of relevant therapeutic modalities, including without limitation treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalisimab for patients with multiple sclerosis or efalizimab for patients with psoriasis or other drugs for patients with promyelocytic leukemia. According to an additional embodiment, the T cells according to the present invention can be used in combination with chemotherapy, radiation, immunosuppresra, such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunodestructive agents, such as CAMPATH, anti-CD3 antibodies or other agents based on antibodies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and irradiation. These drugs either inhibit calcium-dependent phosphatase-calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase, which is important for the signaling growth factor induced (rapamycin) (Liu et al., Cell 66: 807-815, 1 1; Henderson et al., Immun. 73: 316-321, 1991; Bierer et al., Citr. Opin. mm n. 5: 763-773, 93). According to an additional embodiment, the composition of cells according to the present invention is administered to a patient in combination (for example, before, simultaneously or after) with bone marrow transplantation, ablative therapy for T-cells using chemotherapeutic agents such as fludarabine, external radiation exposure (OVL), by administering cyclophosphamide or other antibodies, such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after ablative therapy for B cells, such as agents that react with CD20, for example, Rituxan. For example, according to one embodiment, subjects can undergo standard treatment with a high dose of a chemotherapeutic agent, and then transplantation of peripheral blood stem cells. According to certain embodiments, after transplantation, subjects are infused with propagated immune cells of the present invention. According to one additional embodiment, the multiplied cells are administered before or after the operation.

Pharmaceutical composition

The isolated T cells of the present invention can be administered alone or in the form of a pharmaceutical composition in combination with solvents and / or with other components, such as IL-2 or other cytokines, or with cell populations. Briefly, the pharmaceutical compositions of the present invention may contain T-cells, which are described in this application, in combination with one or more pharmaceutically or physiologically acceptable carriers, solvents or excipients. Such compositions may contain buffers, such as a neutral buffered saline solution, phosphate buffered saline and the like; carbohydrates such as glucose, sucrose or dextrans, mannitol; squirrels; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as ethylenediaminetetraacetic acid (EDTA) or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives. Compositions according to the present invention are preferably converted to intravenous administration forms. The pharmaceutical compositions according to the present invention can be administered in a manner appropriate to the disease that is to be treated (or prevented). The volume and frequency of administration should be determined by factors such as the patient’s condition and the type and severity of the patient’s illness, although appropriate dosages can be determined by clinical studies.

A method for assessing the cytotoxicity of isolated CAR T cells and a kit for its use

Another embodiment of the present invention includes a method for evaluating the cytotoxicity of isolated T cells with a chimeric antigen receptor (CAR), such as the drug resistant cells described above; and these isolated T cells with CAR expressing the chimeric antigen receptor (CAR), and target cells expressing at least one specific surface antigen (and optionally a marker gene, such as luciferase), including:

(a) Providing populations of both indicated T cells and target cells;

(b) Incubating the population of said T cells with at least one population of said target cells;

(c) Determination of the percent viability of the indicated specific target cells.

The resistance gene can be selected from the genes listed in the previous section.

Preferably said resistance gene is dCK. The surface antigen, which should be selected according to the present invention, includes one of those antigens that can be expressed in T-cells through chimeric antigen receptors (CAR), and depends on the cell to which the action should be directed, and is usually specific in against cancer cells. Preferably, the surface antigen that needs to be applied in T cells with CAR is CD19, since this antigen appears to be expressed specifically for certain lymphomas and leukemias, such as acute lymphocytic leukemia (ALL).

Finally, the present invention relates to a kit for implementing a method for evaluating the cytotoxicity of CAR T cells in relation to target cells, including:

(d) The population of the indicated T cells endowed with CARs specific for a particular antigen;

(e) These target cells expressing said antigen;

(f) Optional nutrient medium;

both T-cells and target cells were given resistance to the chemotherapeutic drugs of the present invention.

In this application, patent protection is requested not only for the general method of constructing T-cells resistant to purine nucleotide analogs (PNA) and 6GT. In a broader sense, it relates to methods for producing T-cells that are resistant to chemotherapy and allogenic, including at least the following objects:

1) A method for constructing allogeneic and drug resistant T cells for immunotherapy, comprising:

(a) Providing T cells;

(b) Selection with at least one drug to which said T-cell is sensitive;

(c) Implementing a modification of said T-cell by inactivating at least one gene encoding a component of the T-cell receptor (PTL);

(d) The implementation of the modification of the specified T-cells to give it resistance to the drug;

(e) The implementation of the reproduction of the specified modified T-cells, optionally in the presence of the specified drug.

2) The method according to claim 1, wherein at least one gene encoding a component of the RTL is inactivated by expressing a rarely ending endonuclease that can cleave the target sequence within at least one gene encoding a component of the RTL.

3) The method according to claim 1 or 2, characterized in that said resistance to the drug is imparted to said T-cell by inactivating at least one drug-sensitive gene.

4) The method according to claim 3, characterized in that said drug sensitivity gene is inactivated by expressing a rarely ending endonuclease that is capable of cleaving the target sequence within the specified drug susceptibility gene.

5) The method according to p. 4, characterized in that said rarely-cleaving endonuclease is a TALE-nuclease.

6) The method according to p. 3-5, characterized in that said drug susceptibility gene is dCK.

7) The method according to p. 6, characterized in that the dCK gene is inactivated with TALE nucleases.

8) The method according to claim 7, characterized in that the inactivation of the dCK gene by the TALE nucleases is performed using TALE nucleases with the sequence SEQ ID No. 63 and SEQ ID No. 64, and the target sequence dCK is SEQ ID No. 62.

9) The method according to p. 3-5, characterized in that the specified gene of drug sensitivity is GHFR.

10) The method according to claim 1, characterized in that said resistance to the drug is attached to a T-cell by expressing at least one drug resistance gene.

11) The method of claim 10, wherein said drug resistance gene is mutated with dihydrofolate reductase (DHFR), which confers resistance to treatment with antifolates, preferably methotrexate (MTX).

12) The method according to claim 11, characterized in that said mutated DHFR contains at least one amino acid mutation selected from the group consisting of: G15, L22, F31, or F34 in the SEQ ID no: 14.

13) The method of claim 12, wherein said mutated DHFR contains two amino acid mutations at position L22 and F31 in the sequence SEQ ID NO: 14.

14) The method according to claim 10, wherein said drug resistance gene is mutated inosine-5'-monophosphate dehydrogenase II (IMPDH2), which confers resistance to an IMPDH inhibitor, preferably mycophenolate motefil (MMF).

15) The method of claim 14, wherein said mutated IMPDH2 contains at least one amino acid mutation at position T333 and / or S351 in SEQ ID NO: 15.

16) The method of claim 10, wherein said drug resistance gene is a mutated calcineurin heterodimer (KN) a and / or b, which confers resistance to a calicenavrin inhibitor, preferably FK506 and / or CsA.

17) The method according to claim 16, wherein said mutated calcineurin heterodimer contains at least one amino acid mutation at a position selected from the group consisting of: V314, Y341, M347, T351, W352, L354 and K360 in SEQ ID no. : sixteen.

18) The method according to claim 17, characterized in that said mutated calcineurin heterodimer contains amino acid mutations in the positions: T351 and L354 in SEQ ID NO: 16.

19) The method of claim 17, wherein said mutated calcineurin heterodimer contains amino acid mutations in positions: V314 and Y341 in SEQ ID NO: 17.

20) The method of claim 16, wherein said mutated calcineurin b heterodimer contains at least one amino acid mutation at a position selected from the group consisting of: V120, N123, L124 and K125 in SEQ ID NO: 17.

21) The method of claim 20, wherein said heterodimer of mutated calcineurin b contains amino acid mutations in positions: L124 and K125 in SEQ ID NO: 17.

22) The method according to any one of claims. 10-21, characterized in that the specified gene drug resistance is expressed in the T-cell as a result of the introduction into the specified T-cell transgene encoding the specified gene drug resistance.

23) The method according to any one of claims. 10-21, characterized in that the specified gene drug resistance is expressed in the T-cell as a result of the introduction into the specified T-cell donor matrix, which contains at least one sequence homologous to the endogenous gene, and the sequence encoding the drug resistance gene, so that homologous recombination occurs between the endogenous gene and the indicated donor matrix.

24) The method of claim 23, further comprising introducing a rarely-releasing endonuclease into said T-cell, which is capable of selectively cleaving the target sequence within said endogenous gene, so that the rate of homologous recombination is stimulated.

25) The method according to p. 24, characterized in that the rarely-cleaving endonuclease is TALE-nuclease.

26) The method according to any one of claims. 1-25, further comprising the implementation in T-cells of the expression of the chimeric receptor antigen.

27) The method according to any one of claims. 1-26, in which the specified chimeric receptor antigen is CD19 + or CD123 +.

28) The method according to any one of claims. 1-27, further comprising the implementation of the inactivation of the gene of the immune checkpoint.

29) The method according to any one of claims. 1-28, characterized in that the specified designed T-cells multiply in the blood of the patient.

30) The method according to any one of claims. 1-28, characterized in that said engineered T-cells multiply in-vitro.

31) The method according to any one of claims. 1-30, characterized in that the specified designed T-cells multiply in the presence of the specified drug.

32) Isolated T-cell or cell line, which can be obtained using the method according to any one of paragraphs. 1-31.

33) An isolated drug resistant T-cell that contains at least one disrupted gene encoding a component of the T-cell receptor.

34) The isolated T cell of claim 33, expressing at least one drug resistance gene.

35) The isolated T-cell according to claim 33, wherein said drug resistance gene is selected from the group consisting of: the ble gene, the mcrA gene, and the genes encoding mutant DHFR, mutant IMPDH2, mutant calcineurin, and mutant AGT.

36) The isolated T-cell of claim 33, comprising at least one disturbed drug susceptibility gene, preferably the HGFT gene.

37) An isolated T-cell according to any one of paragraphs. 32-36, characterized in that said isolated T-cell is endowed with a chimeric antigen receptor (CAR) specific for a specific antigen.

38) The isolated T cell according to claim 37, wherein said CAR is targeted to CD19 + cells or CD123 + cells;

39) An isolated T-cell according to any one of paragraphs. 32-38 for use as a drug.

40) An isolated T-cell according to any one of paragraphs. 32-39 for the treatment of cancer, an autoimmune disease or infection caused by a pathogen.

41) The isolated T-cell of claim 40 for use in the treatment of acute lymphoblastic leukemia (ALL) or amyotrophic myeloid leukemia (AML).

42) A pharmaceutical composition comprising at least one isolated T-cell according to any one of claims. 32-41.

43) A method for treating a patient in need of such treatment, comprising:

(a) Obtaining a population of T cells according to the method according to any one of paragraphs. 1-27;

(b) the Introduction of the specified transformed T-cells to the specified patient.

44) The method according to p. 36, characterized in that said patient is treated with said preparation applied according to the method of claims. 1-31.

45) A method for evaluating the cytotoxicity of isolated T cells with a chimeric antigen receptor (CAR) according to any one of claims. 32-41 for drug resistant target cells; moreover, isolated CAR T cells express a chimeric antigen receptor (CAR), and target cells express at least one specific surface antigen (and not necessarily a marker gene, such as luciferase), including:

(a) Obtaining the specified populations of T-cells and target cells;

(b) Incubating the population of said T cells with at least the indicated specific target cells;

(c) Determination of the percent viability of the specific target cells indicated.

46) The method of claim 45, wherein said drug resistance gene is dCK.

47) The method according to claim 44 or 45, characterized in that said surface antigen is CD19.

48) The method of claim 47, wherein said target is Daudi cells CD19 + luciferase +.

49) A kit for implementing a method for evaluating the cytotoxicity of T cells with CAR for a target cell, comprising:

(a) A population of T cells endowed with CAR specific for a specific antigen;

(b) Target cells expressing the indicated antigen;

moreover, these T cells and the target cells were given resistance to the chemotherapeutic drug.

DEFINITIONS

In the above description, some terms are widely used. To facilitate understanding of the embodiments of the present invention, definitions of some terms are given below.

- Amino acid residues in the polypeptide sequence are designated in this application according to a single letter code, in which, for example, Q denotes Gln or a glutamine residue, R denotes Arg or an arginine residue, and D denotes Asp or an aspartic acid residue.

- Nucleotides are designated as follows: a single-letter code is used to designate the base of a nucleotide; and means adenine, t means thymine, s means cytosine, and g - guanine. For degenerate nucleotides, g means g or a (purine nucleotides), k means g or t, s matches g or c, w matches a or t, m matches a or c, y corresponds to t or c (pyrimidine nucleotides), d corresponds to g , a or t, v corresponds to g, a or c, b corresponds to g, t or c, h corresponds to a, t or c, and n corresponds to g, a, t or c.

- In this application, the terms "nucleic acid" or "nucleic acid nucleotide" refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by ligation, cleavage, action of endonucleases or action of exonucleases. Molecules of nucleic acids may consist of monomers that are natural nucleotides (such as DNA and RNA) or analogues of natural nucleotides (for example, the enantiomeric forms of natural nucleotides), or a combination of both. Nucleic acids can be single-stranded or double-stranded.

- By “gene” is understood the basic unit of inheritance, consisting of a DNA segment aligned linearly along a chromosome, which encodes a specific protein or protein segment, small RNA, or the like. Typically, a gene includes a promoter, a 5 ′ untranslated region, one or more coding sequences (exons), optionally introns, a 3 ′ untranslated region. A gene may also contain a terminator, enhancers, and / or silencers.

- The term "transgene" means a nucleotide sequence (encoding, for example, one or more polypeptides) that is partially or completely heterologous, i.e. a foreign host cell into which it is introduced, or homologous to the endogenous host cell is introduced, but which can be designed to embed it, or it can be inserted into the genome of the cell in such a way that the genome of the cell into which it is inserted changes (for example, it is embedded in a position that differs from that in the natural gene, or in traction leads to inactivation (knockout) of a gene. A transgene may contain one or more sequences for regulating transcription and any other nucleic acid, such as introns, which may be necessary for optimal expression of the selected nucleic acid that encodes a polypeptide. The polypeptide encoded by the transgene may not expressed, or expressed, but not be biologically active, in cells into which the specified transgene is introduced.

- The term “genome” means all the genetic material contained in a cell, such as the nuclear genome, the chloroplast genome, and the mitochondrial genome.

- The term “mutation” means a substitution, deletion, insertion of one or more nucleotides / amino acids in a polynucleotide (cDNA, gene) or polypeptide sequence. This mutation may affect the coding sequence of a gene or its regulatory sequence. It can also affect the structure or genomic sequence or structure / stability of the encoded mRNA.

- The term "rarely-ending endonuclease" refers to a wild-type enzyme or an enzyme variant capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids in a DNA or RNA molecule, preferably in a DNA molecule. In particular, this nuclease can be an endonuclease, more preferably a rarely-releasing endonuclease that has high specificity, recognizes target nucleic acid sites from 10 to 45 base pairs in length (by), usually from 10 to 35 base pairs. The endonuclease according to the present invention recognizes and cleaves nucleic acid in specific polynucleotide sequences, also called "target sequence". A rare-bone endonuclease can recognize and generate single- or double-stranded breaks in specific polynucleotide sequences.

According to a particular embodiment, the rare-cleaving endonuclease according to the present invention can be endonuclease Cas9. In fact, a new genome design tool based on Cas9 RNA-driven nuclease Cas9 from prokaryotic type II CRISPR (short polindromic repeats regularly arranged in groups) in the acquired immunity system (for review, see (Sorek, Lawrence et al. 2013)) was recently developed. The CRISPR-associated system (CAS) was first discovered in a bacterium, and it functions as protection against foreign DNA, viral or plasmid. For CRISPR-mediated genome design, first select the target sequence, often flanked by a short motif, called a motif adjacent to the protospeacer (PAM). After selecting the target sequence, a specific crRNA is constructed that is complementary to this target sequence. The transactivating crRNA (tracrPHK), which is required in CRISPR type II systems, is paired with crRNA and is associated with the desired Cas9 protein. Cas9 acts as a molecular anchor that facilitates the coupling of tracPHK bases to cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the double structure of the tracrPHK: crPHK acts as an RNA conductor that guides the Cas9 endonuclease during recognition of the target sequence. Target recognition by the Cas9-tracrPHK: crPHK complex begins with scanning the target sequence for homology between the target sequence and the crRNA. In addition to complementarity of the target sequence crRNA, DNA targeting requires the presence of a short motif adjacent to the protospeacer (the motif adjacent to the protospeacer is PAM). After pairing between the double-RNA and the target sequence, Cas9 consistently produces a double-stranded gap with sticky ends 3 bases higher than the PAM motif (Garneau, Dupuis et al. 2010). According to the present invention, an RNA conductor can be constructed, for example, for a specific targeting of a gene encoding a component of the RTL. After mating between the conductor RNA and the target sequence, Cas9 causes cleavage in the RTL gene.

The homing endonuclease, also known as meganuclease, can also be a rare-tissue endonuclease. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases have high specificity, recognizing target sites of DNA from 12 to 45 base pairs () in length, usually from 14 to 40 in length. The homing endonuclease according to the present invention may, for example, correspond to the endonuclease LAGLIDADG, the endonuclease HNH or the endonuclease GIY-YIG. A preferred endonuclease according to the present invention may be the I-Crel variant. "Variant" endonuclease, i.e. an endonuclease that does not exist in nature and which is obtained by genetic engineering or random mutagenesis can be linked to DNA sequences other than those recognized by wild-type endonuclease (see international patent application WO 2006/097854).

The rare-cleaving endonuclease can be a block nuclease that binds DNA. The term DNA-binding block endonuclease means any chimeric proteins containing at least one catalytic domain of the endonuclease and at least one DNA-binding domain formed by an independently twisting polypeptide or protein domain that contains at least one motif that recognizes two or single-stranded polynucleotides. The technique has described many such polypeptides with the ability to bind to specific nucleotide sequences. Such binding domains often contain, for non-limiting examples, leucine zipper domains, winged helix domains, helix-loop-helix domains, HMG box domains, immunoglobulin domains, B3 domain or a designed zinc finger domain.

According to a preferred embodiment of the present invention, the NK binding domain is derived from an activator-like effector transcription (TALE), with sequence specificity determined by a series of 33-35 amino acid repeats derived from Xanthomonas or Ralstonia bacteria proteins. These repeats differ significantly in two amino acid positions that determine the specificity of the interaction with the base pair (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). Each base pair in the target DNA sequence is contacted with a single repeat, and specificity arises from two variants of amino acids in repeats (the so-called dipeptide with variable repeats, RDV). TALE-binding domains may additionally contain an N-terminal translocation domain responsible for the requirement of the first thymine base (T ₀ ) in the target sequence and the C-terminal domain that contains cellular intranuclear localization signals (NLS).

The nucleic acid binding domain TALE corresponds to the constructed TALE central framework containing a plurality of repeated TALE sequences, each repeat containing an RDV specific to each nucleotide base of the TALE recognition site. According to the present invention, each repeated TALE sequence of said central framework is made of 30-42 amino acids, more preferably of 33 or 34, with two critical amino acids (the so-called variable repeat dipeptide, RDV) located at positions 12 and 13, mediate recognition of one nucleotide the specified binding site TALE; equivalent two critical amino acids may be located at positions other than 12 and 13, especially in a repeated TALE sequence that is longer than 33 or 34 amino acids. Preferably, RVDs associated with the recognition of different nucleotides are HD for recognition C, NG for recognition T, NI for recognition A, NN for recognition G or A. According to another implementation variant, critical amino acids 12 and 13 can be mutated and replaced by other amino acid residues in order to modulate their specificity against nucleotides A, T, C and G, and in particular to increase such specificity. The nucleic acid binding domain of TALE typically contains from 8 to 30 repeated TALE sequences. More preferably, the specified Central frame according to the present invention contains from 8 to 20 repeated sequences TALE; even more preferably 15 repeated TALE sequences. It may also contain an additional single processed TALE repeated sequence formed by 20 amino acids located at the C-terminus of the specified set of repeated TALE sequences, i.e. additional C-terminal semi-TALE repeated sequence.

Other engineered DNA binding domains are block-specific nucleic acid binding domains (MBBBD) specific to each base (PCT / US2013 / 051783). Said MBBBD can be constructed, for example, newly discovered proteins, namely proteins EAV36_BURRH, E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH of recently sequenced genome endosymbiotic fungus Burkholderia Rhizoxinica (Lackner, Moebius et al. 2011). MBBBD proteins contain blocks of about 31-33 amino acids that are specific to bases. These blocks exhibit less than 40% sequence identity with frequent repeats of TALE Xanthomonas, but they exhibit more pronounced variability in the polypeptide sequence. When they come together, such block polypeptides can, however, be targeted to specific nucleotide sequences quite similar to TALE nucleases of Xanthomonas. According to a preferred embodiment of the present invention, said DNA binding domain is a constructed MBBBD binding domain containing from 16 to 20 modules.

Different domains of the above proteins (blocks, N- and C-termini) from Burkholderia and Xanthomonas are useful in constructing new proteins or scaffolds that have binding properties with specific nucleotide sequences. In particular, additional N-terminal and C-terminal domains constructed by MBBBD can be obtained from natural TALE, such as AvrBs3, PthXo1, AvrHah1, PthA, Tal1c as non-limiting examples.

- The terms "TALE-nuclease" or "MBBBD-nuclease" refer to proteins obtained by combining a DNA-binding domain, usually derived from proteins - similar to the effectors transcription activator (TALE) or MBBBD binding domain, to the catalytic endonuclease domain. Such a catalytic domain is preferably a nuclease domain and more preferably an domain with endonuclease activity, such as, for example, I-Tevl, ColE7, NucA and Fok-I. According to a specific embodiment, said nuclease is a monomeric TALE nuclease or MBBBD nuclease. Monomeric nuclease is a nuclease that does not need to dimerize for specific recognition and cleavage, for example, as chimeras from a constructed DNA-binding domain with the I-Tevl catalytic domain described in WO 2012138927. According to another specific implementation variant, this rarely ending endonuclease is a dimeric TALE -nuclease or MBBBD-nuclease, preferably containing a DNA-binding domain joined to Fokl. TALE nuclease has already been described and applied to stimulate gene targeting and gene modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010). Such constructed TALE nucleases are commercially available under the trade name TALEN ™ (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

- The term "cleavage" refers to the cleavage of the covalent core of a polynucleotide. Cleavage can be initiated using a variety of methods, including, without limitation, enzymatic or chemical hydrolysis of the phosphodiester bond. Splitting of both single-stranded and double-stranded polynucleotides is possible, and splitting of a double strand may occur due to two different splitting events of single strands. Cleavage of double-stranded DNA, RNA, or a DNA / RNA fusion may result in sticky or stepped ends.

- By "chimeric antigen receptor" (CAR) is meant a chimeric receptor that contains an extracellular ligand-binding domain, a transmembrane domain, and a signal transduction domain.

- In this application, the term "extracellular ligand-binding domain" means an oligo- or polypeptide that is capable of binding to a ligand. Preferably, the specified domain may be able to interact with a molecule on the surface of the cell. For example, the extracellular ligand-binding domain can be chosen so that it recognizes a ligand that acts as a cell surface marker for target cells associated with a particular pathological condition.

According to a preferred embodiment, said extracellular domain contains a single-chain antibody fragment (scFv) containing a variable fragment of light (V _L ) and heavy chain (V _H ) of a specific monoclonal antibody on a target antigen attached by a flexible linker. According to a preferred embodiment, said scFv is obtained from an antibody on CD19 or CD123. Preferably, the specified scFV according to the present invention includes scFV derived from a monoclonal antibody on CD19-4G7 (Peipp, Saul et al. 2004).

- The domain of signal transduction or the intracellular domain of signal transduction CAR according to the present invention is responsible for the intracellular signal transmission after binding the extracellular ligand-binding domain to the target, leading to the activation of the immune cell and the immune response. Preferred examples of a signal transduction domain for use in CAR may be the cytoplasmic T-cell receptor and co-receptor sequences that act in concert, initiating signal transmission after the antigen receptor has been activated. The signal transduction domain contains two different classes of cytoplasmic signal sequences — sequences that initiate antigen-dependent primary activation, and sequences that act in an antigen-independent way, providing a secondary or costimulatory signal. The primary cytoplasmic signal sequence may contain signaling motifs, which are known as immunoreceptor tyrosine activating motifs or ITAM. According to a specific embodiment, the signal transduction domain CAR according to the present invention contains a costimulatory signaling molecule. A costimulatory molecule is a molecule on the cell surface, different from the antigenic receptor or their ligands, which is needed for an effective immune response. The costimulatory molecules include, without limitation, the MHC class I molecule, BTLA and the ligand receptor Toll. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1 BB (CD137), OH40, CD30, CD40, PD-1, ICOS, lymphocytic functional antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7 -H3 and ligand, which binds specifically to CD38, and the like.

The CAR of the present invention is expressed on the cell surface membrane. Thus, CAR may contain a transmembrane domain. Distinctive features of the respective transmembrane domains include the ability to be expressed on the cell surface, preferably according to the present invention on the surface of an immune cell, in particular cells or natural killer cells (NK), and interact together to direct the response of the immune cell to a given target cell.

The transmembrane domain may further comprise a “stem” region between the said extracellular ligand-binding domain and the specified transmembrane domain. In this application, the term “stem region” generally refers to an oligo- or polypeptide that functions by binding a transmembrane domain to an extracellular ligand-binding domain. In particular, the stalk region is used to achieve greater flexibility in the availability of the extracellular ligand-binding domain. The stem region can contain up to 300 amino acids, preferably from 10 to 100 amino acids, and most preferably from 25 to 50 amino acids. The region of the "stem" can be obtained from a whole or part of a naturally occurring molecule, for example, from a whole or part of the extracellular region of CD8, CD4 or CD28, or from the whole or part of the constant region of an antibody. Alternatively, the “stem” region may be a synthetic sequence that matches the natural sequence of the “stem”, or it may be a completely synthetic sequence of the stem.

In cancer cells, there is usually a negative regulation or mutation in the target antigens, which leads to the formation of “escape” variants with the lost antigen. Thus, to compensate for the tumor's escape and make immune cells more specific for a target, CD19-specific CAR may contain other extracellular ligand-binding domains to simultaneously bind different elements in the target, thereby enhancing the activation and function of immune cells. Examples of CD19-specific CAR include ScFv FMC63 (Kochenderfer JN, Wilson WH, Janik JE, et al. Eradication of B-lineage cells and autologous T cells genetically engineered to recognize CD19. Blood 2010; 116 ( 20): 4099-410) or ScFv 4G7 CAR (described in the application filed under the number PCT / EP2014 / 059662). In one embodiment, the extracellular ligand binding domains can be located in tandem in a single transmembrane polypeptide, and need not be separated by a linker. In yet another embodiment, said other extracellular ligand binding domains may be located on different transmembrane polypeptides containing CAR. According to another embodiment, the present invention relates to a CAR population, each of which contains different extracellular ligand binding domains. In particular, the present invention relates to a method for constructing immune cells, including obtaining an immune cell and expressing a CAR population on the surface of said cell, each of which contains different extracellular ligand-binding domains. In yet another embodiment, the present invention relates to a method for constructing immune cells, including obtaining an immune cell and introducing into said immune cell polynucleotides encoding polypeptides containing CAR populations, each of which contains different extracellular ligand-binding domains. By a CAR population, we mean at least two, three, four, five, six, or more CARs, each of which contains different extracellular ligand-binding domains. Different extracellular ligand binding domains according to the present invention may preferably simultaneously bind to different elements of the target, which enhances the activation and function of immune cells. The present invention also relates to an isolated immune cell that contains a population of CARs, each of which contains different extracellular ligand binding domains.

- The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid with which it is associated. A vector of the invention includes, without limitation, a viral vector, a plasmid, an RNA vector or a linear or circular DNA-based vector, or an RNA molecule, which may contain chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. Preferred vectors are vectors that are capable of autonomous replication (episomal vector) and / or expression of the nucleic acids to which they are connected (expression vectors). Specialists in this field of technology know a large number of suitable vectors, and they are on sale.

- “Delivery vector” means any delivery vector that can be used according to the present invention to establish contact with a cell (i.e., “contacting”) or delivering cells inside or subcellular compartments (i.e., “introduction”) of agents / chemicals and molecules (proteins or nucleic acids) required by the present invention. These include, without limitation, liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions or other suitable means of transfer.

- Viral vectors include retroviruses, adenoviruses, parvoviruses (for example, adeno-associated viruses), coronoviruses, negative chains of RNA viruses, such as orthomyxoviruses (for example, influenza virus) rdovirus (for example, rabies virus or vesicular stomatitis virus), paramyxovirus (for example, measles virus and Sendai virus), RNA positive strand viruses, such as picornavirus and alphavirus, and double-stranded DNA viruses, including adenovirus, herpes virus (for example, type 1 and 2 herpes simplex virus, Epstein-Barr virus, cytomeg lovirus) and poxvirus (for example, vaccinia virus, fulpox and canarypox). Other viruses include, for example, norovirus, togavirus, lavivirus, reovirus, papovavirus, hepadnavirus and hepatitis virus. Examples of retroviruses include avian leukemia-sarcoma virus, mammalian viruses of type C, type B, type D, group HTLV-BLV, lentivirus, spumavirus (Coffin, J.M., Retroviridae), Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

The "lentiviral vector" is understood to be HIV-based lentiviral vectors that are very promising in terms of gene delivery due to their relatively high packaging ability, reduced immunogenicity and their ability to stable transduction with high efficiency of a wide range of cell types. Usually lentiviral vectors are generated as a result of transient transfection of three (packaging, sheath and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter a target cell through the interaction of glycoproteins on the surface of the virus with receptors on the cell surface. After virus entry, viral RNA is subjected to reverse transcription, which is mediated by the reverse transcriptase viral complex. The product of reverse transcription is a double-stranded linear viral DNA, which is suitable for integrating the virus into the DNA of an infected cell. By "integrative lentiviral vectors (or LV)" is meant such vectors which, by way of non-limiting example, are capable of integrating into the genome of the target cell. On the contrary, by “non-integrative lentiviral vectors (or NLV)” is meant the vectors of efficient gene delivery that are not integrated into the genome of the target cell under the action of viral integrase.

- Under the cell or cells understand any living cells of eukaryotes, primary cells and cell lines obtained from these organisms in cultures in vitro.

- “Primary cell” or “primary cells” means cells taken directly from living tissue (i.e. biopsy material) and intended for growth in vitro, which have undergone a small number of population doubling and, therefore, are more typical for basic functional components and characteristics of the tissues from which they are derived, compared with continuous tumor-forming or artificially immobilized cell lines. As non-limiting examples, cell lines can be selected from the group consisting of: CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-93 cells; MRC5 cells; IMR90 cells; Yurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; H cells u-h7; Huvec cells; Molt 4 cells.

- Since some variability may occur on the basis of genomic data, on the basis of which these polypeptides are obtained, and also to take into account the possibility of replacing some amino acids present in these polypeptides without significant loss of activity (functional variants), the invention includes variants of the polypeptides listed above that have at least 70%, preferably at least 80%, more preferably at least 90% and even more preferably at least 95% identity with the sequence proposed in this patent application.

Thus, the present invention relates to polypeptides containing an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, 95% 97% or 99% identity with the amino acid sequence selected from group consisting of SEQ ID no: 8 - SEQ ID no: 20 and SEQ ID no: 26 - SEQ ID no: 35.

- The term “identity” refers to sequence identity between two amino acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When the position in the compared sequence is occupied by the same base, then the molecules are identical in this position. The degree of similarity between nucleotide or amino acid sequences is a function of the number of identical or corresponding nucleotides in the positions common to the indicated nucleotide sequences. Different alignment algorithms can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of a package for analyzing GCG sequences (University of Wisconsin, Madison, WI) and can be used, for example, with default parameters. For example, the invention includes polypeptides having identity with the specific polypeptides described in this application, at least 70%, 85%, 90%, 95%, 98% or 99%, as well as a polynucleotide encoding such a polypeptide;

- “gene knockout” means that the gene is mutated to such an extent that it cannot be expressed;

- The term "TRAC" refers to the "constant T-cell alpha receptor" and corresponds to the TCRa subunit of the constant gene.

In addition to the properties described above, the present invention includes additional properties that will become clear from the following examples illustrating a method for constructing allogeneic and resistant T cells for immunotherapy, as well as from the accompanying drawings.

Example 1: Creating and describing the properties of T-cells resistant to clofarabine

TALE-nuclease mediated inactivation of dCK

For the inactivation of dCK, two pairs of TALE-nucleases of dCK were developed, assembled and tested by sequencing; the subsequent work was carried out only with a pair called TALE-nuclease dCK2, with the sequence SEQ ID NO: 63 and SEQ ID NO: 64. Detailed information on the general architecture of the dCK gene (exons and introns) and the sequences of target TALE nuclease sites located in exon 2 are shown in Figure 3.

The target dCK sequence for a pair of TALE nucleases dCK2 corresponds to SEQ ID No. 62.

After verification, the mRNA encoding the two TALE nucleases was obtained, polyadenylated, and used for T-cell electroporation using fast pulse modulation technology (5 or 10 μg of the left and right TALE nuclease mRNA were used) as described in WO 2013/176915. Cold temperature shock was produced by incubating T cells at 30 ° C immediately after electroporation and for 24 hours. Reactivation (12.5 μl of granules / 10 ⁶ cells) was performed on day 8 (8 days after electroporation).

The resulting T-cells were allowed to grow and, as a result, their genotypic properties were described (by analysis with endo T7 and deep sequencing in the dCK and TRAC loci), as well as phenotypic properties. A description of their phenotypic properties included (i) - testing the ability to grow in the presence or absence of a drug, (ii) - determining the IC50 of PNA, clofarabine and fludarabine for T-cells and (III) determining the degree of inactivation of TRAC using FACS analysis conducted a double gene knockout.

Description of the genotypic properties of T-cells with the knockout of the dCK gene

To evaluate the efficiency of inactivation of the dCK gene, cells transfected with 5 or 10 μg of TALE-nuclease mRNA were grown for 4 days (D4, 4 days after electroporation) and selected for analysis with T7 at the dCK locus (Figure 5).

The primer sequences used in this assay with T7 correspond to SEQ ID No. 68 and SEQ ID No. 69. An assay protocol with T7 is described in Reyon, D., Tsai, S.Q., Khayter, C, Foden, J.A., Sander, J. D., and Joung, J.K. (2012) FLASH assembly of TALE-nucleases for high-throughput genome editing. Nat Biotechnologies.

The results of this analysis with endo T7 show that when 5 and 10 μg of left and right dCK2 TALE nuclease were transfected, significant gene processing showed that dCK was effectively inactivated.

Determination of the growth rate of T-cells with knockout dCK

As shown in Figure 6, dCK knockout cells exhibit a growth rate similar to that of wild-type cells. In addition, they can be re-activated on day 8 with the same efficiency as wild-type T cells.

Selection of T-cells with knockout dCK in the presence of clofarabine

T cells with knockout dCK or wild type were allowed to grow from day 8 to day 13, and then incubated with 1 μM clofarabine or without it until day 18. Cells were selected on day 8 (before adding the drug) and on day 18 (after incubation with the drug) and used for analysis with endo T7.

The results shown in Figure 7 show that the presence of 1 μM clofarabine in the medium by day 18 selectively enriched the culture of T-cells with dCK knockout compared to wild-type T-cells (2 lower molecular weight bands for T-cells with knockout dCK compared to a single higher molecular weight band for wild-type T cells). This indicates that TALE-nuclease-mediated inactivation of dCK allows selection of drug-resistant T-cells relative to wild-type T-cells. Thus, dCK knockout T cells are able to withstand the presence of 1 µM clofarabine, which corresponds to a clinically significant dose for the treatment of acute lymphoblastic leukemia (ALL) according to C _max reported by the European Medicines Agency (EMA).

IC50 determination for clofarabine against dCK knockout T cells compared to wild type T cells

To examine in more detail the ability of T cells to withstand the presence of clofarabine, the IC50 for this drug was determined by the action on T cells with dCK knockout and wild type T cells. Cells were harvested 3 days after transfection and incubated for 2 days in the presence of increasing clofarabine concentration (0-10 μM). At the end of the incubation with clofarabin, the viability of the T cells was determined by FACS2 analysis.

The results presented in Figure 8 clearly show that the processing of the dCK gene, mediated by TALE nucleases, effectively inactivates the activity of dCK in T-cells. Such inactivation correlates with resistance to clofarabin, in contrast to the sensitivity of wild-type T cells to it. The IC50 values (the amount of drug that needs to be added to the medium to reduce cell viability up to 50%) correspond to approximately 100 nM and 10 μM for wild-type T cells and dCK knockout T cells.

Taken together, this first data set allows us to conclude that TALE-nuclease mediated inactivation of the dCK gene is sufficient. Inactivation of dCK does not disrupt the growth of constructed T cells, but allows them to withstand a clinically significant dose of clofarabine.

Example 2. The creation and description of the properties of allogenic T-cells resistant to clofarabine

To develop and obtain clofarabine-resistant allogeneic T cells with CAR, the dCK and TRAC genes were simultaneously inactivated. After the demonstration in Example 1 that the inactivation of dCK was successful, T-cells with a double knockout of TRAC / dCK were generated and their properties were described. In parallel, two sequences of actions were performed, shown in Figure 9. One of them corresponds to the period of cell incubation in the presence of clofarabine - 5 days.

Genotype description

In order to first assess the efficacy as well as the kinetics of inactivation of the TRAC and / or dCK genes, the transfected cells were grown for 6 days and selected on day 1, day 3 and day 6 for analysis with T7 at the dCK and TRAC loci. To achieve this, 2 pairs of primers with the sequences SEQ ID nos 68 and 69; and SEQ ID No. 70 and No. 71, respectively, were used in the analysis with T7 for the dCK and TRAC loci.

The protocol used was described in Reyon, D., Tsai, S.Q., Khayter, C, Foden, J.A., Sander, J. D., and Joung, J.K. (2012) FLASH assembly of TALE-nucleases for high-throughput genome editing. Nat Biotechnol

The results presented in Figure 10 show that the TALE-nuclease-mediated single knockout of TRAC and dCK is highly effective on day 1. Even though cells with inactivated genes cannot be characterized as a homogeneous population, it appears that a double knockout of TRAC / dCK also highly effective.

Cells were then grown in the presence or absence of 1 μM clofarabine. On day 6 (six days after transfection) and after 3 days of cultivation in the presence or absence of clofarabine, the cells were selected, and the efficiency of the knockout of the dCK gene was determined by analysis with T7 and highly efficient DNA sequencing.

The protocol used for deep sequencing is described in Shendure, J., & Ji, N. (2008). Next-generation DNA sequencing. Nature biotechnology, 26 (10), 1135-1145.

The results presented in Figure 11 show that the frequency of insertion-deletion mutations generated in the dCK locus in all experiments is about 80-90%. This again indicates that TALE-nuclease-mediated inactivation of dCK is highly effective, even when combined with simultaneous inactivation of TRAC. The presence of 1 μM clofarabine in the nutrient medium for 5 days does not lead to an increase in the T7 band, which is specific for dCK knockout, as seen in the first series of experiments. This suggests that in this particular experiment, the inactivation of dCK was sufficiently successful that the designed T cells could grow in the presence of clofarabine. Interestingly, this indicates that if dCK knockout is effective enough, there is no need to select T cells in the presence of clofarabine to obtain drug resistant T cells. Therefore, this property is a clear advantage in obtaining drug-resistant allogeneic T-cells.

Phenotypic evaluation of the effectiveness of TCAR knockout

TRAC knockout T cells selected for the double knockout experiment were analyzed and purified by FACS (fluorescently activated cell sorting) (CliniMACS). The results presented in Figure 12B show an experiment with labeling T-cells with anti-RTL mAb-PE (monoclonal antibody-polyethylene) or without it. Also Figure 12B refers to the labeling of mAb-PE T-cells in an environment with or without clofarabin, before and after purification of T-cells with a TRAC knockout.

The results show that the efficiency of RTL knockout in T cells treated with TRAC mRNA and dCK (double dCK / TRAC knockout) is high (approximately 85%). The purification method allows for efficient selection / purification of RTL-negative cells with obtaining purity up to 99.3%.

Description of T-cell phenotype with TRAC / dCK knockout

Figure 13 shows the growth rate of T cells in the absence of clofarabine. Even if dCK knockout T cells show a small growth defect, they can be reactivated on day 10 with the same efficiency as wild type T cells.

Figure 14 shows the growth rate of T cells in the presence of clofarabine. This experiment was performed on T-cells with dCK / TCAR double knockout CAR (FMC63, which are described in patent application PCT / EP2014 / 059662) by culturing cell data for 11 days in medium containing different concentrations of clofarabine ( from 0.1 μM to 10 μM). The results presented in Figure 14 clearly show that an increase in the number of cells for T cells with CAR with double knockout of dCK / TCAR occurs to a clonoarabine concentration of 1 μM (which corresponds to Сmax), even if the growth is less pronounced than the growth of these cells in the absence of drug.

IC50 determination of clofarabine for engineered T-cells versus wild-type T-cells

To investigate the ability of T-cells with double knockout to withstand the presence of clofarabine, the IC50 for this drug was determined. T cells were grown with or without clofarabine from day 3 to day 8 (see step 2 in Figure 9), then they were incubated for 2 days (day 15 - day 17) in medium with different concentrations of clofarabine. T cell viability was determined by FACS analysis using a brightness quantification kit.

The results presented in Figure 15 show that T-cells with dCK knockout and dCK / TRAC show significant ability to transfer clofarabine compared with negative control T-cells and T-cells with only TRAC knockout. It is noteworthy that cell selection using 1 μM clofarabine for 5 days from day 3 to day 8 (see step 2 in Figure 9) did not improve their ability to tolerate clofarabine. This suggests that inactivation of dCK is sufficiently effective, and that 5-day incubation for selection with the drug to obtain allogenic T-cells resistant to Klofarabin with CAR is not required.

The cytotoxicity of obtaining drug-resistant allogeneic T-cells with CAR

The cytotoxicity analysis was performed as follows: 10 T cells with CAR (FMC63, for reference, see above) were incubated with Daudi cells (specific targets) and K562 cells (non-specific targets) for 5 hours. The cells were then selected and the viability of the Daudi and K562 cells was determined by counting the frequency of targeted lysis of the cells.

The results presented in Figure 16 show that T cells with double knockout dCK / TRAC CAR exhibit similar directional cytotoxicity, as do wild type T cells (35% directional cytotoxicity). This indicates that inactivation of the dCK and TRAC genes does not affect the cytotoxicity of T cells with CAR FMC63.

Then these cells were used to determine their sensitivity to clofarabine and fludarabine, as was previously the case. The results presented in Figure 17 show that T cells with CAR with knockout dCK / TRAC have significant ability to tolerate clofarabin compared with T cells with CAR negative control (IC ₅₀ = 500 nM and 0.1 nM, respectively). Similar results were obtained with fludarabine (IC ₅₀ = 400 μM and 10 μM for T cells with double knockout CAR and T cells with CAR, respectively).

findings

Taken together, these experiments show that the simultaneous inactivation of the dCK and TRAC genes is highly effective and allows you to generate more than 70% of T-cells with double knockout in one electroporation cycle. Interestingly, due to such high efficiency there is no need for a long selection stage. Designed T cells exhibit a pronounced ability to tolerate clofarabine and remain at their maximum viability under the pressure of a clinically significant dose of clofarabine.

Example 3- Creation of Daudi cells resistant to clofarabine

The goal is to obtain drug-resistant Daudi target cells CD19 ⁺ / Luc ⁺ to assess the cytotoxicity of clofarabin-resistant allogeneic T cells with CAR.

Description of the genotype of cells Daudi with knockout dCK

TALE-nuclease dCK mRNA was prepared and electrodation of Daudi cells with TALE-nuclease dCK mRNA was carried out in accordance with the protocols described in WO 2013/176915.

To assess the efficiency of the dCK knockout analysis, an analysis was performed with T7 as in Example 1. The analysis was performed 2 days after transfection. The primer sequences corresponded to SEQ ID Nos. 68 and 69.

The results presented in Figure 18 show a high degree of inactivation of the dCK gene.

Daudi cell phenotype description with dCK knockout

Daudi cells were cultured in medium containing different concentrations of clofarabine (0; 0.1; 0.25; 0.5 and 1 μM) for several days, and cells were counted in each passage.

The results presented in Figure 19 show that Daudi's dCK knockout cells are able to grow in the presence of clofarabine at a concentration of up to 1 μM. Their growth rate was similar to the growth rate of wild-type T cells in the absence of clofarabine, which indicates that inactivation of dCK does not impair growth of Daudi cells. As expected, the growth of wild-type Daudi cells was clearly impaired. These results demonstrate that CD19 ⁺ -Luc ⁺ -GFP ⁺ cells with dCK knockout were successfully generated.

Example 4. Creating a description of the properties of T-cells resistant to 6TG

To obtain T-cells resistant to 6MR and 6TG (T-cells with GGFT knockout), the GGFT gene was inactivated using TALE nuclease as follows. The overall architecture of the GGFT gene (exons and introns) and the position of the different target sites of TALE nuclease are shown in Figure 20.

Mediated by TALE-nuclease inactivation of the gene GGFT

The Figure 21 shows the sequence of actions used in this experiment to generate and describe the properties of T-cells with GGFT knockout. For the inactivation of the GGFT gene, 2 pairs of TALE nucleases were developed for GGFT, they were collected and verified by sequencing (for GGFT 1: SEQ ID No. 74 and SEQ ID No. 75; for GGFT2: SEQ ID No. 77 and SEQ ID No. 78). Detailed information on the general architecture of the HGFT gene (exons and introns) and the location of the target sites of the TALE nuclease is shown in Figure 20. The target sequences of the pairs of TALE nucleases GGFT1 and GGFT2 correspond to SEQ ID No. 76 and SEQ ID No. 79, respectively.

Description of T-cell genotype with GGFT knockout

The genotype of T-cells with HGFT knockout was characterized on day 47 by analysis with T7, and inactivation of the HGFT gene in T-cells was shown. A pair of primers in this analysis had the sequences SEQ ID No. 72 and SEQ ID No. 73. The results presented in Figure 22 show that a pair of T-Nucleases HGFT was able to process the HGFT gene with high efficiency.

Growth rate of T-cells with GGFT knockout

According to the results presented in Figure 23, GGFT knockout cells exhibit a growth rate similar to the growth rate of wild-type T cells, despite the fact that the growth is slightly reduced for the GHFT2 TALE nuclease pair (experiment was performed with 10 μg TALE nuclease). However, T-cells inactivated with 10 μg of TG-nuclease HGFT2 were re-activated on day 10 with the same efficiency as wild-type T-cells, which indicates that inactivation of HGFT does not significantly disrupt T-growth lymphocyte. In the following experiments, the pair of TALE-nuclease GGFT1 was selected.

Selection of T-cells with GGFT knockout in the presence of 6TG

T-cells with GGFT or wild type knockout were allowed to grow from day 8 to day 13, and then incubated in the presence or absence of 1 μM 6TG until day 18 (the sequence of actions is shown in Figure 22). Cells were collected on day 8 (before the addition of the drug) and day 18 (after incubation with the drug) and used for analysis with endo T7. A pair of primers in this analysis had the sequences SEQ ID No. 72 and SEQ ID No. 73. The results presented in Figure 24 show that the presence of 1 μM 6TG in the medium allows the culture to be selectively enriched for T-cells with GGFT knockout (as seen by the lower density of the wild-type band in the presence of 6TG on day 18).

Generation of T cells with CAR with GGFT knockout

To study the effect of HGFT inactivation on the cytotoxic activity of T-cells with CAR, T-cells were transduced with a lentiviral vector with CAR 4G7 (such as described in the application filed under registration number PCT / EP2014 / 059662) and then their electroporation of mRNA encoding TALE - GGFT1 nucleus. All experiments described below were performed with constructed T-cells generated without any selection on 6TG. The efficiency of GGFT processing was evaluated by analysis with endo T7. The primer pair used in this assay had the sequences SEQ ID No. 72 and SEQ ID No. 73. The results presented in Figure 25 show that the HGFT gene was successfully inactivated in the presence or absence of CAR 4G7. In T-cells, a more pronounced inactivation of HGFT was achieved than in T-cells with CAR.

Cytotoxic properties in T cells with CAR with GGFT knockout for DAUDI cells

The cytotoxicity assay was performed as shown schematically in Figure 27. A set of 10 T cells with CAR were incubated for 5 hours with Daudi cells (specific targets) and K562 cells (non-specific targets). The cells were then taken, and the cell viability was determined by counting the frequency of directed lysis of the cells. The results presented in Figure 26 show that T-cells with CAR with GGFT knockout show similar directional cytotoxicity, as do T-cells with wild-type CAR. This suggests that inactivation of the HGFT gene does not affect the cytotoxicity of T cells with CAR 4G7

ICT determination for 6TG in relation to designed T-cells compared to wild-type T-cells

The results presented in Figure 27 show that the processing of the HGFT gene (as was seen earlier in the analysis with T7) effectively suppresses the activity of HGFT in T-cells. This inactivation confers resistance to 6TG, in contrast to the sensitivity of wild-type T cells to this drug. The IC50 can be roughly defined as 10 nM and> 100 μM for wild-type T-cells with GGFT knockout, respectively.

findings

Taken together, these results show that inactivation of the HGFT gene is effective. Such inactivation allows T cells to tolerate a high dose of 6TG without the need for purification, which is a time consuming process. It is also shown that inactivation of HGFT can be carried out in T cells with CAR to a slightly lesser extent. Such inactivation does not impair the cytotoxic properties of T cells with CAR against Daudi cells.

links

Bardenheuer, W., K. Lehmberg, et al. (2005). "Resistance to cytarabine and gemcitabine in vitro selection of transduced cells after retroviral expression in human hematopoietic progenitor cells." Leukemia 19 (12): 2281-8.

Betts, M.R., J.M. Brenchley, et al. (2003). "Cytometric assay for degranulation." J Immunol Methods 281 (1-2): 65-78.

Boch, J., H. Scholze, et al. (2009). "Breaking the code for DNA binding specificity of TAL-type III effectors." Science 326 (5959): 1509-12.

Brewin, J., C. Mancao, et al. (2009). "EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease." Blood 114 (23): 4792-803.

Cermak, T., E.L. Doyle, et al. (2011). "TALEN Efficient design and assembly of TALEN and other TAL effector-based constructs for DNA targeting." Nucleic Acids Res 39 (12): e82.

Christian, M., T. Cermak, et al. (2010). "Targeting DNA double-strand breaks with TAL effector nucleases." Genetics 186 (2): 757-61.

Cong, L., F.A. Ran, et al. (2013). "Multiplex genome engineering using CRISPR / Cas systems." Science 339 (6121): 819-23.

Critchlow, S.E. and S.P. Jackson (1998). "DNA end-joining: from yeast to man." Trends Biochem Sci 23 (10): 394-8.

Dasgupta, A., D. McCarty, et al. (2011). "Engineered drug-resistant immunocompetent cells enhance tumor cell killing during a chemotherapy challenge." Biochem Biophys Res Commun 391 (1): 170-5.

Deltcheva, E., K. Chylinski, et al. (2011). "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Nature 471 (7340): 602-7.

Deng, D., C. Yan, et al. (2012). "Structural basis for sequence-specific recognition by DNA effectors." Science 335 (6069): 720-3.

Garneau, J.E., M.E. Dupuis, et al. (2010). "The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA." Nature 468 (7320): 67-71.

Gasiunas, G., R. Barrangou, et al. (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria." Proc Natl Acad Sci USA 109 (39): E2579-86.

Geissler, R., H. Scholze, et al. (2011). "Transcriptional activators of human genes with programmable DNA-specificity." PLoS One 6 (5): e19509.

Hacke, K., J.A. Treger, et al. (2013). "Genetic modification of the mouse bone marrow by lentiviral vector-mediated delivery of short hairpin RNA confers chemoprotection against 6-thioguanine cytotoxicity." Transplant Proc 45 (5): 2040-4.

Huang, P., A. Xiao, et al. (2011). "Heritable targeting in zebrafish using customized TALENs." Nat Biotechnol 29 (8): 699-700.

Ikehara, Y., S.K. Ikehara, et al. (2004). "Negative regulation of the cell receptor signaling by Siglec-7 (p70 / AIRM) and Siglec-9." J Biol Chem 279 (41): 43117-25.

Jena, B., G. Dotti, et al. (2010). "Redirecting the T-cell specificity by introducing a tumor-specific chimeric antigen receptor." Blood 116 (7): 1035-44.

Jinek, M., K. Chylinski, et al. (2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337 (6096): 816-21.

Jonnalagadda, M., C.E. Brown, et al. (2013). "Engineering T cells for resistance to mytrexate and mycophenolate as an in vivo cell selection strategy." PLoS One 8 (6): e65519.

Kushman, M.E., S.L. Kabler, et al. (2007). "Expression of human glutathione S-transferase P1 confers resistance to benzo [a] pyrene or benzo [a] pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in staged transfected V79MZ cells- expressing hCYP1A1. " Carcinogenesis 28 (1): 207-14.

Lackner, G., N. Moebius, et al. (2011). "Complete genome sequence of Burkholderia rhizoxinica, an Endosymbiont of Rhizopus microsporus." J Bacteriol 193 (3): 783-4.

Li, L., M.J. Piatek, et al. (2012). "TALE-based transcriptional regulators for genome modification." Plant Mol Biol 78 (4-5): 407-16.

Li, T., S. Huang, et al. (2011). "Modularly assembled TAL designer for eukarvotes." Nucleic Acids Res 39 (14): 6315-25.

Ma, J.L, E.M. Kim, et al. (2003). "Breaking up the lack of overlapping end sequences." Mol Cell Biol 23 (23): 8820-8.

Mahfouz, M.M., L. Li, et al. (2012). "Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein." Plant Mol Biol 78 (3): 311-21.

Mahfouz, M.M., L. Li, et al. (2011). "De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks." Proc Natl Acad Sci USA 108 (6): 2623-8.

Mak, A.N., P. Bradley, et al. (2012). "The crystal structure of TAL effector PthXo1 bound to its DNA target." Science 335 (6069): 716-9.

Mali, P., L. Yang, et al. (2013). "RNA-guided human genome engineering via Cas9." Science 339 (6121): 823-6.

Maze, R., C. Kurpad, et al. (1999). "Retroviral-mediated expression of the P140A, but not P140A / G156A, mutant form of O6-methylguanine DNA methyltransferase protects hematopoietic cells against O6-benzylguanine sensitization to chloroethylnitrosourea treatment." J Pharmacol Exp Ther 290 (3): 1467-74.

Meyaard, L., G.J. Adema, et al. (1997). "LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes." Immunity 7 (2): 283-90.

Miller, J.C., S. Tan, et al. (2011). "A TALE nuclease architecture for efficient genome editing." Nat Biotechnol 29 (2): 143-8.

Morbitzer, R., P. Romer, et al. (2011). "Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE) -type transcription factors." Proc Natl Acad Sci S A 107 (50): 21617-22.

Moscou, M.J. and A.J. Bogdanove (2009). "A simple cipher governs DNA recognition by TAL effectors." Science 326 (5959): 1501.

Mussolino, C, R. Morbitzer, et al. (2011). "A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity." Nucleic Acids Res 39 (21): 9283-93.

Nicoll, G., J. Ni, et al. (1999). "Siglec-7, siglec-7, expressed by human natural killer cells and monocytes. J Biol Chem 274 (48): 34089-95.

Nivens, M.C., T. Felder, et al. (2004). "Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase." Cancer Chemother Pharmacol 53 (2): 107-15.

Park, T.S., S.A. Rosenberg, et al. (2011). "Treating cancer with genetically engineered T cells." Trends Biotechnol 29 (11): 550-7.

Quigley, M., F. Pereyra, et al. (2010). "Transcriptional analysis of HIV-specific CD8 + T cells shows that PD-1 inhibits cell function by upregulating BATF." Nat Med 16 (10): 1147-51.

Sander, J.D., L. Cade, et al. (2011). "Targeted gene disruption in somatic zebrafish cells using engineered TALENs." Nat Biotechnol 29 (8): 697-8.

Sangiolo, D., M. Lesnikova, et al. (2007). "Lentiviral vector conferring resistance to mycophenolate and in vivo T-cell selection." Gene Ther 14 (21): 1549-54.

Schweitzer, V.I., A.P. Dicker, et al. (1990). "Dihydrofolate reductase as a therapeutic target." Faseb J 4 (8): 2441-52.

Sorek, R., C.M. Lawrence, et al. (2013). "CRISPR-mediated Adaptive Immune Systems in Bacteria and Archaea." Annu Rev Biochem.

Stoddard, B.L. (2005). "Homing endonuclease structure and function." Q Rev Biophys 38 (1): 49-95.

Sugimoto, Y., S. Tsukahara, et al. (2003). "Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDRl-bicistronic retrovirus vector Ha-MDR-IRES-gp91." J Gene Med 5 (5): 366-76.

Takebe, N., S.C. Zhao, et al. (2001). "Generation of dual resistance to 4-hydroperoxycyclophosphamide and aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reductase gene." Mol Ther 3 (1): 88-96.

Tesson, L., C. Usal, et al. (2011). "Knockout rats generated by embryo microinjection of TALENs." Nat Biotechnol 29 (8): 695-6.

Weber, E., R. Gruetzner, et al. (2011). "Assembly of designer TAL effectors by Golden Gate cloning." PLoS One 6 (5): e19722.

Yam, P., M. Jensen, et al. (2006). "Inosine monophosphate dehydrogenase 2 after inosine monophosphate 2 after HIV vector transduction of effects on lymphocytes, monocytes, and CD34 + stem cells." Mol Ther 14 (2): 236-44.

Zhang, F., L. Cong, et al. (2011). "Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription." Nat Biotechnol 29 (2): 149-53.

Zhang, J.Q., G. Nicoll, et al. (2000). "Siglec-9, a superfamily expressed opinion of the immunoglobulin of a binding member of the broadly on human blood leukocytes." J Biol Chem 275 (29): 22121-6.

Zielske, S.P., J.S. Reese, et al. (2003). "In vivo selection of MGMT (P140K) lentivirus-transduced human NOD / SCID repopulating cells without pretransplant irradiation conditioning." J Clin Invest 112 (10): 1561-70.

Claims (22)

1. An ex-vivo method of producing T cells for use in cancer therapy, wherein said cells are resistant to clofarabine and / or fludarabine, comprising the steps of: (a) transfecting T-cells with a nucleotide sequence that encodes a rarely ending endonuclease specifically directed to a gene expressing an enzyme that has deoxycytidine kinase activity (dcK - EC 2.7.1.74); (b) expressing said endonuclease in said T-cells to achieve targeted inactivation of said dcK gene; (c) reproducing said engineered T cells obtained in step (b), optionally in the presence of clofarabine and / or fludarabine, however, these T-cells are allogeneic T-cells from the donor, in which the gene encoding the component of the T-cell receptor (RTL), selected from RTL alpha and RTL beta, is inactivated, or autologous T-cells from a patient in whom cancer diagnosed; while these cells express the chimeric antigen receptor (CAR) specific for the cancer antigen. 2. The method according to p. 1, characterized in that said T-cells are primary cells. 3. The method according to p. 1, characterized in that said T cells are CD8 + cells. 4. The method according to p. 1, characterized in that said T-cells are TIL (tumor infiltrating lymphocytes). 5. The method according to p. 1, characterized in that said T-cells originate from the patient's body, who was diagnosed with cancer. 6. The method according to p. 1, characterized in that said T-cells originate from the body of the donor. 7. The method according to p. 1, characterized in that the rare-cleaving endonuclease is a TALE-nuclease. 8. The method according to p. 7, characterized in that the inactivation of the dSC gene of the TALE-nucleases is carried out using TALE-nucleases with sequence SEQ ID No. 63 or SEQ ID No. 64 and the target sequence dCK is SEQ ID No. 62. 9. The method according to p. 1, characterized in that the chimeric antigen receptor is a CD19 + or CD123 +. 10. Dedicated autologous T-cell for use in cancer immunotherapy, obtained by the method according to any one of paragraphs. 1 - 9, while this cell is resistant to clofarabine and / or fludarabine and in this cell, the drug sensitivity gene dCK is inactivated using a rare-digestive endonuclease. 11. The selected allogeneic T-cell for use in cancer immunotherapy, obtained by the method according to any one of paragraphs. 1 - 9, while the specified cell is resistant to clofarabine and / or fludarabine, and in this cell, the drug sensitivity gene dCK is inactivated using a rare-cleaving endonuclease. 12. Pharmaceutical composition for use in the treatment of cancer, containing an effective amount of T-cells according to any one of paragraphs. 10 or 11. 13. A method of treating a patient diagnosed with cancer, comprising administering an effective amount of T-cells according to any one of claims. 10, 11 to the indicated patient. 14. The method according to p. 13, characterized in that said cancer is an acute lymphoblastic leukemia (ALL) or amyotrophic myeloid leukemia (AML). 15. A method of treating a patient diagnosed with cancer, comprising administering an effective amount of T-cells according to any one of claims. 10 or 11 to the specified patient, while these T-cells are administered in combination with the introduction of the drug, which is a purine analogue. 16. The method according to p. 15, characterized in that said cancer is an acute lymphoblastic leukemia (ALL) or amyotrophic myeloid leukemia (AML). 17. A population of autologous T cells for use in cancer immunotherapy, consisting of T cells according to claim 10. 18. A population of allogenic T cells for use in cancer immunotherapy, consisting of T cells according to claim 11.

Images