Methods for using engineered T-cells resistant to chemotherapy drugs for immunotherapy

Abstract

This invention relates to the use of "off-the-shelf" allogeneic therapeutic cells in combination with chemotherapy for the treatment of cancer patients. In particular, the inventors have developed a method for engineering allogeneic T-cells resistant to chemotherapy agents. The therapeutic benefits provided by this strategy should be enhanced through the synergistic effect between chemotherapy and immunotherapy. Specifically, this invention relates to a method for modifying T-cells by inactivating at least one gene encoding a T-cell receptor component and by modifying said T-cells to confer drug resistance. This invention opens the door to standard and affordable adoptive immunotherapy strategies for treating cancer.

Description

Technical Field

[0001] This invention relates to the use of "off-the-shelf" allogeneic therapeutic cells for immunotherapy combined with chemotherapy to treat cancer patients. Specifically, the inventors have developed a method for engineering allogeneic T-cell resistance to chemotherapy agents. The therapeutic benefits obtained through this strategy should be enhanced by a synergistic effect between chemotherapy and immunotherapy. In particular, this invention relates to a method for modifying T-cells by inactivating at least one gene encoding a T-cell receptor component and by modifying the T-cells to confer resistance. This invention opens the door to standard and affordable adoptive immunotherapy strategies for treating cancer. Background Technology

[0002] Adoptive immunotherapy, involving the transfer of ex vivo-generated autoantigen-specific T cells, is a promising cancer treatment strategy. T cells used in adoptive immunotherapy can be generated through the expansion of antigen-specific T cells or through the retargeting of T cells via genetic engineering (Park, Rosenberg et al. 2011). The transfer of viral antigen-specific T cells is a well-established treatment for transplant-associated viral infections and rare virus-associated malignancies. Similarly, the isolation and transfer of tumor-specific T cells has proven successful in the treatment of melanoma. Novel intracellular specificity has been successfully generated through gene transfer of transgenic T-cell receptors or chimeric antigen receptors (CARs). CARs consist of a targeting portion that binds to one or more signaling domains in a single signaling fusion molecule. CARs have successfully allowed T cells to be retargeted for antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0003] Current adoption immunotherapy for patients is based on autologous cell transfer. In this approach, T lymphocytes are harvested from the patient, genetically modified or selected in vitro, cultured in vitro to expand cell numbers if necessary, and finally infused into the patient. Autologous therapy faces significant technical and logistical hurdles for practical application. Its production requires expensive specialized facilities and expert personnel; it must be generated very quickly after cancer diagnosis; and in many cases, pre-treatment can lead to immune degradation, potentially resulting in impaired lymphocyte function and very low cell counts. Because of these obstacles, the preparation of autologous cells for each patient is essentially a new product, leading to significant variations in efficacy and safety.

[0004] Ideally, standardized therapies are desired, where allogeneic therapeutic cells are pre-generated, meticulously characterized, and readily administered to the patient. However, allogeneic T cells, derived from individuals of the same species, are genetically distinct. Consequently, the endogenous TCR specificity of allogeneic cells can recognize host tissue as foreign, leading to graft-versus-host disease (GvHD), which can cause severe tissue damage and death. T-cell receptors (TCRs) are cell surface receptors involved in T-cell activation in response to the presence of antigens. For immunoglobulin molecules, variable regions of the α and β chains are generated through V(D)J recombination, resulting in a diverse range of antigen specificities within the T-cell population. However, in contrast to immunoglobulins that recognize intact antigens, T-cells are activated via processed peptide fragments linked to MHC molecules, introducing another dimension of antigen recognition through T-cells, known as MHC restriction. The recognition of MHC differences between donor and recipient T-cell receptors can lead to T-cell proliferation and the potential development of GvHD. In order to effectively use allogeneic cells, the inventors inactivated the TCRα or TCRβ gene, which resulted in the elimination of TCR from the surface of T-cells, thereby preventing the recognition of allogeneic antigens and thus GVHD.

[0005] Despite significant advancements in cancer detection and tumor cell biology, the treatment of advanced and metastatic cancers remains a major challenge. Cytotoxic chemotherapy drugs remain the most commonly used and have been successfully employed in anticancer therapy. A variety of cytotoxic drugs, such as antimetabolites, alkylating agents, anthracycline antibiotics, DNA methyltransferase inhibitors, platinum compounds, and spindle toxins, have been developed to kill cancer cells. However, they are not consistently effective, and the introduction of these drugs into novel therapies, such as immunotherapy, presents challenges. For example, chemotherapy drugs may impair the establishment of robust antitumor immune cells due to their nonspecific toxicity profiles. Small molecule therapies targeting cell proliferation pathways may also hinder the establishment of antitumor immunity. However, significant advances in antitumor therapy could be achieved if transiently effective chemotherapy regimens could be combined with novel immunotherapies (review (Dasgupta, McCarty et al. 2011)).

[0006] Therefore, in order to utilize "off-the-shelf" allogeneic therapeutic cells in conjunction with chemotherapy, the inventors have developed a method for engineering allogeneic T-cells resistant to chemotherapy drugs. The therapeutic benefits obtained through this strategy should be enhanced through the synergistic effect between chemotherapy and immunotherapy. Moreover, drug resistance can also benefit from the ability of selectively expanded engineered T-cells, thereby avoiding problems caused by ineffective gene transfer to these cells. Summary of the Invention

[0007] In one aspect, the present invention provides a method for engineering immune cells to tolerate purine nucleotide analog (PNA) chemotherapy agents, such as clofarabine and fludarabine, enabling these immune cells to be used for immunotherapy of cancer in patients pretreated with conventional chemotherapy. Considering the autologous treatment being performed, the immune cells can be derived from the patient, such as in the case of TILs (tumor-infiltrating lymphocytes), or considering the generation of allogeneic cells, the immune cells can be derived from a donor and can be used for allogeneic therapy.

[0008] In the latter case, when the immune cells are T cells, the present invention also provides a method for engineering T cells to be resistant to both chemotherapeutic drugs and allogeneic T cells. This method, in addition to inactivating drug-sensitizing genes such as dcK and HPRT genes, also includes inactivating at least one gene encoding a component of the T-cell receptor (TCR), particularly the TCRα and TCRβ genes.

[0009] According to another approach, drug resistance can be conferred on T cells by expressing drug resistance genes. Variant alleles of various genes according to the present invention, such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT), have been identified as conferring drug resistance to cells.

[0010] This invention covers isolated cells or cell lines, more particularly isolated immune cells, that can be obtained by the methods of this invention, containing any of the proteins, peptides, allele variants, altered or deleted genes or vectors described herein.

[0011] The immune cells or cell lines of the present invention may further comprise exogenous recombinant polynucleotides, particularly CAR or suicide genes, or they may include altered or deleted genes encoding checkpoint proteins or their ligands, which contributes to their efficiency as a therapeutic product, ideally as an "off-the-shelf" product. In another aspect, the present invention relates to a method for treating or preventing cancer in a patient by administering engineered immune cells obtainable via the methods described above. Attached Figure Description

[0012] - Figure 1 A schematic diagram corresponding to the pathway and cytotoxicity of purine nucleoside analogs (PNA); inactivation of the enzyme deoxycytidine kinase (dCK) conferred tolerance to the drugs clofarabine and fludarabine.

[0013] - Figure 2 The inactivation of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) conferred tolerance to the drugs 6-mercaptopurine (6MP) and 6-thio-guanine (6TG).

[0014] - Figure 3 A describes the overall dCK gene structure in terms of exons and introns, and

[0015] Figure 3 B shows the sequence of the TALE-nuclease target site located for the 2TALE-nuclease pair in exon 2 of dCK;

[0016] - Figure 4 The workflow for generating and characterizing HPRT KO T-cells is shown; D0 represents day 0, Dn represents day n; T7 corresponds to the endonuclease T7 assay;

[0017] - Figure 5The results of the auto-inverse T7 analysis are shown to examine the treatment of the dCK gene; the upper band corresponds to the untreated WT dCK gene and the two lower bands correspond to the treated dCK gene;

[0018] - Figure 6 This indicates cell expansion of dCK KO T-cells and WT T-cells (controls 1 and 2) treated with 5 μg or 10 μg of mRNA encoding dCK2TALE-nuclease during a 14-day period following electroporation.

[0019] - Figure 7 This indicates that on day 8 (D8), an endonuclease T7 assay was performed in the presence or absence of 1 μM chlorofarabine (+) or chlorofarabine (-) (using 5 μg of TALE-nuclease dCK 2 pairs) to examine dCK inactivation in T-cells;

[0020] - Figure 8 This figure represents the percentage of cell viability for WT and dCK KO T-cells (treated with 5 μg or 10 μg TALE-nuclease dCK 2 pairs) cultured for 2 days in the presence of incremental clofarabine (10 nM to 10 μM). This figure allows for the determination of clofarabine IC50 for both cell populations.

[0021] - Figure 9 Two workflows for generating and characterizing allogeneic T-cells resistant to clofarabine are shown; the one above corresponds to the case when drug selection is performed, compared to the one below when no drug selection is performed; day 0 (D0) is the day when double electroporation via TRAC and dCK TALE-nuclease is achieved.

[0022] - Figure 10 The efficacy of double KO dCK / TRAC in T-cells was examined at different time points (D1, D3, and D6) following electroporation, corresponding to the T7 restriction enzyme assay. Primers for each locus (+-) are shown in the examples for simple KO dCK T-cells (+-) and simple KO TRAC T-cells (-+), double KO dCK / TRAC T-cells (++), and WT T-cells (--); the bands below indicate correct dCK and TRAC gene treatment.

[0023] - Figure 11 Endonuclease T7 analysis and deep sequencing data corresponding to the presence or absence of clofarabine (+) or the presence or absence of TRAC inactivation (+ or -) are used to examine the efficacy of dCK inactivation. (Figure and legend are provided.) Figure 10 The same applies; the insertion / deletion frequency is used to evaluate the insertion or deletion rate at the dCK locus;

[0024] - Figure 12 A indicates a labeling control experiment performed using T cells in the presence of anti-TCRmAb-PE (labeled T cells) or in the absence of anti-TCRmAb-PE (unlabeled T cells); Figure 12 B monitors TCAR-negative cells collected before and after TRAC KO T-cell purification following culture with or without clofarabine. These cells also showed inactivation of the dCK gene;

[0025] - Figure 13 The growth rates of simple KO dCK and TRACT- cells and dual KO dCK / TRAC T- cells relative to WTT- cells were shown 12 days after electroporation in the absence of chlorofarapine.

[0026] - Figure 14 The growth rate curves of dCK / TCAR dual-KO CART-cells relative to CAR T-cells (with or without clofarabine) over 11 days are shown.

[0027] - Figure 15 The percentage of cell viability of simple KO dCK or TRAC T-cells, and dual KO dCK / TCART-cells relative to WTT-cells in media with different clofarabine doses (1 nM to 100 μM) is shown; the curve allows for the determination of the IC50 of clofarabine for each T-cell population.

[0028] - Figure 16 The percentage of specific cytotoxicity of double KO TRAC / dCK CAR T cells compared to CAR FMC63 T cells (both expressing CD19 antigen) versus double KO TRAC / dCK T cells (without CAR, therefore not expressing CD19 antigen) and WT T cells (without KO and without CAR);

[0029] - Figure 17 The percentage of cell survival of dual KO dCK / TCAR CAR T-cells relative to CAR T-cell controls in these T-cell cultures cultured with increasing doses of clofarabine (10 ng to 100 μg, top panel) and fludarabine (10 μM to 100 μM, bottom panel) is shown. These graphs allow for the determination of the IC50 of both drugs, clofarabine and fludarabine.

[0030] - Figure 18The T7 endonuclease assay corresponds to the dCK inactivation efficacy of Daudi cells (+) (using 5 μg of mRNA encoding dCK TALE-nuclease) relative to WT- cells (-) on day 2 (D2). The upper band corresponds to the untreated dCK gene, while the two lower bands correspond to the dCK inactivation products;

[0031] - Figure 19 This represents the 7-day growth rate of KO dCK Daudi cells relative to WT Daudi cells in the absence or presence of incremental chlorofarabine (0.1 to 1 μM) (in × 10⁻¹⁰). 6 (cell representation);

[0032] - Figure 20 The total HPRT gene structure of exons and introns and the location of different TALE-nuclease target sites (all of them in exon 2) are shown.

[0033] - Figure 21 The workflow for generating and characterizing HPRT KO T-cells is described;

[0034] - Figure 22 The endonuclease T7 analysis of HPRT gene inactivation in T-cells by TALE-nuclease HPRT to n°1 and T pair n°2 (tested at two doses: 5 μg and 10 μg) is shown on day 4 (D4).

[0035] - Figure 23 This represents the 13-day growth rate (in × 10⁻¹⁰) of KO HPRT T-cells relative to WT T-cell controls 1 and 2, achieved by using 5 or 10 μg of TALE-HPRT 1 pair (HPRT1) or TALE-HPRT 2 pair (HPRT2). 6 (cell representation);

[0036] - Figure 24 This indicates the endonuclease T7 assay used to examine HPRT gene inactivation in these T-cells (TALE-HPRT 1) cultured in 1 μM of drug 6TG relative to WT T-cells [symbolized par (-)] at D8 and D18.

[0037] - Figure 25 This indicates the T7 endonuclease assay used to examine HPRT gene inactivation in T-cells in the presence or absence of 4G7CAR; this assay was performed without 6TG selection.

[0038] - Figure 26The percentage of specific cytotoxicity of KO HPRT CAR T-cells relative to CAR 4G7 T-cells (both expressing CD19 antigen) and WTT-cells (without KO and without CAR) is shown.

[0039] - Figure 27 The percentage of cell survival of KO HPRT CART-cells relative to WT T-cells in increasing doses of 6TG drug (10 ng to 50 μM) is shown. Detailed Implementation

[0040] Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0041] All methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, wherein suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including definitions, shall prevail. Furthermore, unless otherwise specified, materials, methods, and examples are illustrative only and not intended to be limiting.

[0042] Unless otherwise stated, the practice of this invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which fall within the scope of this art. These techniques are fully explained in the literature. 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 (MJ Gaited., 1984); Mullis et al.USPat.No.4,683,195; Nucleic Acid Hybridization (BD Harries&S.J.Higgins eds.1984); Transcription And Translation (BDHames&S.J.Higgins eds.1984); Culture Of Animal Cells (RIFreshney, 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), especially, Vols.154 and 155 (Wuet al.eds.) and Vol.185, "Gene Expression Technology" (D. Goeddel, ed.); GeneTransfer Vectors For Mammalian Cells (JHMiller and MPCalos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DMWeir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0043] -Drug-resistant T cells

[0044] As used herein, the terms “therapeutic agent,” “chemotherapeutic agent,” or “drug” refer to compounds or derivatives thereof that can interact with cancer cells to reduce their proliferative state and / or kill them. Examples of chemotherapeutic agents include, but are not limited to: alkylating agents (e.g., cyclophosphamide, ifosfamide), metabolic antagonists (e.g., purine nucleoside antimetabolites such as clofarabine, fludarabine, or 2'-deoxyadenosine, methotrexate (MTX), 5-fluorouracil, or derivatives thereof), antitumor antibiotics (e.g., mitomycin, doxorubicin), plant-derived antitumor agents (e.g., vincristine, vinblastine, paclitaxel), cisplatin, carboplatin, etoposide, etc. This class of drugs may further include, but is not limited to, the anticancer agent TRIMETHOTRIXATE. TM (TMTX), TEMOZOLOMIDE TM RALTRITREXED TM S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis(chloronitrosourea) (BCNU), and CAMPTOTHECIN TM , or any of their therapeutic derivatives.

[0045] As used in this article, "resistant or tolerant" cells to a reagent are those that have been genetically modified to proliferate in the presence of a reagent that inhibits or prevents the proliferation of unmodified cells.

[0046] Expression of drug resistance genes

[0047] In a particular embodiment, the drug resistance can be conferred on T-cells by expressing at least one drug resistance gene. The drug resistance gene is a nucleic acid sequence encoding resistance to an agent, such as a chemotherapeutic agent (e.g., methotrexate). In other words, expression of the drug resistance gene in cells allows for greater cell proliferation in the presence of the drug than corresponding cells without the drug resistance gene. The drug resistance gene of the present invention can encode resistance to: antimetabolites, methotrexate, vincristine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, immunoaffinity, their analogues or derivatives, etc.

[0048] Several drug resistance genes have been identified as potentially useful for conceiving drug resistance in 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).

[0049] An example of a drug resistance gene can also be a mutant or modified form of dihydrofolate reductase (DHFR). DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in cells and is essential for DNA synthesis. Folate analogs, such as methotrexate (MTX), inhibit DHFR and are therefore used clinically as antitumor agents. Different mutant forms of DHFR have been described that enhance tolerance to the inhibitory effects of antifolates used in therapy. In a particular embodiment, the drug resistance gene according to the invention can be a nucleic acid sequence encoding a mutant form of human wild-type DHFR (SEQ ID NO: 14, gene bank: AAH71996.1) containing at least one mutation conferring resistance to antifolate therapy, such as methotrexate. In a particular embodiment, the mutant form of DHFR contains at least one mutated amino acid at positions G15, L22, F31, or F34, preferably at position L22 or F31 (Schweitzer, Dicker et al. 1990; International Application WO94 / 24277; US Patent 6,642,043). In a particular embodiment, the mutant form of DHFR contains two mutated amino acids at positions L22 and F31. The amino acid position correspondences herein are often expressed based on the amino acid positions of the wild-type DHFR polypeptide form described in SEQ ID NO: 14. In a particular embodiment, the serine residue at position 15 is preferably substituted with a tryptophan residue. In another particular embodiment, the leucine residue at position 22 is preferably substituted with an amino acid that will interrupt the binding of the mutant DHFR to the antifolate agent, preferably using an uncharged amino acid residue such as phenylalanine or tyrosine. In another particular embodiment, the phenylalanine residue at positions 31 or 34 is preferably substituted with a small hydrophilic amino acid such as alanine, serine, or glycine.

[0050] As used herein, "antifolate" or "folate analogue" refers to molecules that interfere with the folic acid metabolic pathway to some extent. Examples of antifolate agents include, for example, methotrexate (MTX); aminopterin; and trimethyltroxane (Neutrexin). TM); Idatrix; N10-propargyl-5,8-dideazafoliacid (dideazafoliacid) (CB3717); ZD1694 [Tumodex], 5,8-dideazafoliacid (IAHQ); 5,10-dideazatetrahydrofolate (DDATHF); 5-deazafoliacid; PT523 (N-α-(4-amino-4-deoxypteroyl) teroyl))-Nδ-hemiphthaloyl (hemiphthaloyl)-L-ornithine); 10-ethyl-10-deazaminopterin (DDATHF, lomatrexol); pyrithione; 10-EDAM; ZD1694; GW1843; pemetrexate and PDX (10-propargyl-10-deazaminopterin).

[0051] Another example of a resistance gene can be a mutant or modified form of inosine-5'-monophosphate dehydrogenase II (IMPDH2) (the rate-limiting enzyme in the de novo synthesis of guanosine nucleotides). Mutants or modified forms of IMPDH2 are IMPDH inhibitor resistance genes. IMPDH inhibitors can be mycophenolic acid (MPA) or its prodrug, mycophenolate mofetil (MMF). Mutant IMPDH2 may contain at least one, preferably two, mutations at the MAP-binding site of wild-type human IMPDH2 (SEQ ID NO: 15; NP_000875.2) that result in a significant increase in resistance to IMPDH inhibitors. These mutations are preferably located at positions T333 and / or S351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al. 2007; Jonnalagadda, Brown et al. 2013). In a particular 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 amino acid positional correspondences described herein are often based on the amino acid positional expression of the wild-type human IMPDH2 polypeptide as described in SEQ ID NO:15.

[0052] Another resistance gene is a mutant form of calcineurin. Calcineurin (PP2B) is a widely expressed serine / threonine protein phosphatase involved in many biological processes and central to T-cell activation. Calcineurin is a heterodimer composed of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). Upon T-cell receptor binding, calcineurin dephosphorylates the transcription factor NFAT, allowing it to translocate to the nucleus and activate key target genes such as IL2. FK506, complexed with FKBP12, or cyclosporine A (CsA), complexed with CyPA, blocks NFAT from entering the active site of calcineurin, preventing its dephosphorylation and thus inhibiting T-cell activation. (Brewin, Mancao) (etal. 2009). The drug resistance gene of the present invention may be a nucleic acid sequence encoding a mutant form of calcineurin that is resistant to calcineurin inhibitors such as FK506 and / or CsA. In a particular embodiment, the mutant form may contain at least one mutant amino acid of at least the wild-type calcineurin heterodimer at positions V314, Y341, M347, T351, W352, L354, and K360, preferably a double mutation at positions T351 and L354 or V314 and Y341. In a particular embodiment The valine residue at position 341 can be replaced with a lysine or arginine residue; the tyrosine residue at position 341 can be replaced with a phenylalanine residue; the methionine at position 347 can be replaced with a glutamic acid, arginine, or tryptophan residue; the threonine at position 351 can be replaced with a glutamic acid residue; the tryptophan residue at position 352 can be replaced with a cysteine, glutamic acid, or alanine residue; the serine at position 353 can be replaced with a histidine or asparagine residue; the leucine at position 354 can be replaced with an alanine residue; and the lysine at position 360 can be replaced with an alanine or phenylalanine residue from SEQ ID NO:16. The corresponding amino acid positions described herein are often expressed according to the amino acid positions of the wild-type human calcineurin heterodimer (the polypeptide described in SEQ ID NO:16 (gene bank: ACX34092.1)).

[0053] In another specific embodiment, the mutant form contains at least one mutant amino acid of wild-type calcineurin heterodimer b at positions V120, N123, L124, or K125, preferably a double mutation at positions L124 and K125. In a specific embodiment, valine at position 120 can be replaced with a serine, aspartic acid, phenylalanine, or leucine residue; asparagine at position 123 can be replaced with tryptophan, lysine, phenylalanine, arginine, histidine, or serine; leucine at position 124 can be replaced with a threonine residue; and lysine at position 125 can be replaced with alanine, glutamic acid, tryptophan, or by adding two residues after lysine at position 125 in the amino acid sequence SEQ ID NO:17, such as leucine-arginine or isoleucine-glutamic acid. The amino acid positions described herein are often expressed according to the amino acid positions of the wild-type human calcineurin heterodimer b polypeptide as described in SEQ ID NO:17 (gene bank: ACX34095.1).

[0054] Another resistance gene is O(6)-methylguanine methyltransferase (MGMT), which encodes human alkylguanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents such as nitrosoureas and temozolomide (TMZ). 6-Benzylguanine (6-BG) is an AGT inhibitor that enhances nitrosourea toxicity and is co-administered with TMZ, which enhances the cytotoxic effects of this drug. Various mutant forms of MGMT encoding AGT variants exhibit high resistance to inactivation induced by 6-BG while retaining their ability to repair DNA damage (Maze, Kurpad et al. 1999). In a particular embodiment, the mutant form of AGT may contain a mutant amino acid at position P140 of the wild-type AGT in the amino acid sequence SEQ ID NO:18 (UniProtKB: P16455). In a preferred embodiment, the proline at position 140 is replaced with a lysine residue.

[0055] Another drug resistance gene is the multidrug resistance protein 1 (MDR1) gene. This gene encodes a membrane glycoprotein called P-glycoprotein (P-GP), which is involved in the transmembrane transport of metabolic byproducts. P-GP proteins exhibit broad-spectrum specificity to several structurally unrelated chemotherapeutic agents. Therefore, drug resistance can be conferred on cells by expressing a nucleic acid sequence encoding MDR-1 (NP_000918).

[0056] Drug resistance genes can also be cytotoxic antibiotic genes, such as the ble or mcr genes. Ectopic expression of the ble or mcrA genes in immune cells provides a selective advantage when exposed to chemotherapeutic agents, bleomycin, or mitomycin C, respectively.

[0057] The most practical method of gene therapy is to add genes to engineered T cells using efficient gene delivery via vectors, preferably viral vectors. Thus, in a particular embodiment, drug resistance genes can be expressed in cells by introducing a transgene, preferably encoded by at least one vector.

[0058] Random gene insertion into the genome can lead to inappropriate expression of the inserted gene or genes near the insertion site. Specific gene therapy using homologous recombination of exogenous nucleic acids can allow for the engineering of safe T-cells containing endogenous sequences to target these genes to specific sites in the genome. As described above, the genetic modification step of this method can include the introduction of an exogenous nucleic acid containing at least a sequence encoding a drug resistance gene and a portion of an endogenous gene into the cell, such that homologous recombination occurs between the endogenous gene and the exogenous nucleic acid. In a particular embodiment, the endogenous gene may be a wild-type "drug resistance" gene, such that after homologous recombination, the wild-type gene is replaced with a mutated form of the gene conferring drug resistance.

[0059] It is known that the breaking of endonucleases stimulates the rate of homologous recombination. Therefore, in a particular embodiment, the method of the present invention further includes expressing a rare-cut endonuclease in the cell that can break a target sequence in an endogenous gene. The endogenous gene may encode, for example, DHFR, IMPDH2, calcineurin, or AGT. The rare-cut endonuclease may be a TALE-nuclease, a zinc finger nuclease, a CRISPR / Cas9 endonuclease, an MBBBD-nuclease, or a meganuclease.

[0060] Inactivation of drug-sensitizing genes

[0061] In another specific embodiment, the drug resistance can be conferred on T cells by inactivating drug-sensitizing genes. The inventors are the first to seek to inactivate potential drug-sensitizing genes to engineer T cells for immunotherapy.

[0062] By inactivating genes, it is desirable that the gene of interest is no longer expressed as a functional protein. In a particular embodiment, the gene modification of this method, in the provided engineered cells, depends on the expression of a rare-cut endonuclease, such that the rare-cut endonuclease specifically catalyzes the cleavage of a target gene, thereby inactivating the target gene. In a particular embodiment, the step of inactivating at least one drug-sensitizing gene includes introducing a rare-cut endonuclease capable of cleaving at least one drug-sensitizing gene into the cells. In a more particular embodiment, the cells are transformed with nucleic acid encoding a rare-cut endonuclease capable of cleaving a drug-sensitizing gene, and the rare-cut endonuclease is expressed in the cells. The rare-cut endonuclease may be a giant nuclease, a zinc finger nuclease, a CRISPR / Cas9 nuclease, an MBBBD-nuclease, or a TALE-nuclease. In a preferred embodiment, the rare-cut endonuclease is a TALE-nuclease.

[0063] In a preferred embodiment, the drug-sensitizing gene that can be inactivated and confer T-cell resistance is the human deoxycytidine kinase (dCK) gene. This enzyme is essential for the phosphorylation of deoxyribonucleosyl deoxycytidine (dC), deoxyguanosine (dG), and deoxyadenosine (dA). Purine nucleotide analogs (PNAs) are metabolized by dCK into mono-, di-, and tri-phosphate PNAs. Their triphosphate forms, especially chlorofarabine triphosphate, compete with ATP for DNA synthesis, acting as apoptosis-promoting agents and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production (also as...). Figure 1 (The hypothetical mechanism of action in the middle).

[0064] Preferably, dCK inactivation in T cells is mediated by TALE nucleases. To achieve this, multiple pairs of dCK TALE-nucleases have been designed, assembled at the polynucleotide level, and validated by sequencing. Examples of TALE-nuclease pairs that can be used according to the present invention are described by SEQ ID N°63 and SEQ ID N°64. When using this pair of TALE-nucleases, the dCK target sequence corresponds to SEQ ID N°62.

[0065] As demonstrated in these examples, inactivation of this dCK in T-cells confers resistance to purine nucleoside analogs (PNAs), such as clofarabine and fludarabine.

[0066] In another preferred embodiment, inactivation of dCK in T-cells, combined with inactivation of the TRAC gene, confers both resistance to drugs such as clofarabine and tolerance to allogeneic drugs to these double knockout (KO) T-cells. This dual function is particularly useful for therapeutic targets, enabling “off-the-shelf” allogeneic cells for immunotherapy combined with chemotherapy to treat cancer patients. This dual KO inactivation of dCK / TRAC can be performed simultaneously or sequentially. An example of a successful TALE-nuclease dCK / TRAC pair in this invention is described in SEQ ID N°63 and SEQ ID N°64 and SEQ ID N°66 and N°67, with target sequences (dCK and TRAC) at two gene loci (LOCs) depicted in SEQ ID N°62 and SEQ ID N°65, respectively.

[0067] Another example of an inactivated enzyme is the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (gene bank: M26434.1). Specifically, HPRT can be inactivated in engineered T cells to confer resistance to the cell growth inhibitory metabolite 6-thioguanine (6TG), which is converted to cytotoxic thioguanine nucleotides via HPRT and is currently used to treat cancer patients, particularly those with leukemia (Hacke, Treger et al. 2013). Guanine analogues are metabolized by HPRT transferases, which catalyze the addition of the phosphoribosyl moiety and enable the formation of TGMP (…). Figure 2 Guanine analogues, including 6-mercaptopurine (6MP) and 6-thioguanine (6TG), are commonly used as lymphodepleting agents in the treatment of ALL. They are metabolized by HPRT (hypoxanthine phosphoribosyltransferase, which catalyzes the addition of the phosphoribosyl moiety and enables TGMP to form). Their subsequent phosphorylation leads to the formation of their triphosphate form, which eventually integrates into DNA. Once introduced into DNA, thioGTP impairs the fidelity of DNA replication via its thiolate group and generates random point mutations that are highly detrimental to cellular integrity.

[0068] In another implementation, inactivation of CD3, which is normally expressed on the surface of T cells, can confer resistance to anti-CD3 antibodies, such as teplizumab.

[0069] CD19+ / luc+ resistant Daudi cells were tested for allogeneic CAR T-cell cytotoxicity.

[0070] This invention also covers methods for preparing target cells that express surface receptors and resistance genes specific to CAR T cells. These target cells are particularly suitable for testing the cytotoxicity of CAR T cells. These cells readily develop resistance to clinically relevant doses of clofarabine and possess luciferase activity. This combination of characteristics allows for in vivo tracking in mouse models. 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. Clofarabine-resistant Daudi cells mimic the physiological state of relapsing form induction therapy in patients with drug-resistant B-cell malignancies in acute lymphoblastic leukemia (ALL). Therefore, these cells are of great interest for evaluating the reliability and cytotoxicity of drug-resistant CAR T cells. Preferably, these target cells are CD19+ luciferase+ Daudi cells. isolated cells

[0071] This invention also relates to isolated cells obtainable by the above methods. In particular, the invention relates to isolated drug-resistant T-cells containing at least one disrupted gene encoding a T-cell receptor component. In a particular embodiment, the T-cell expresses at least one drug resistance gene, preferably the ble gene or mcr gene, or a gene encoding mutant DHFR, mutant IMPDH2, mutant AGT, or mutant calcineurin. In another particular embodiment, the T-cell contains at least one disrupted drug sensitization gene such as the dCK or HPRT gene. In a more particular embodiment, the isolated T-cell contains a disrupted HPRT gene and expresses a DHFR mutant; the isolated T-cell contains a disrupted HPRT gene and expresses an IMPDH2 mutant; the isolated T-cell contains a disrupted HPRT gene and expresses a calcineurin mutant; the isolated T-cell contains a disrupted HPRT gene and expresses an AGT mutant. In another preferred embodiment, the isolated cells express a chimeric antigen receptor (CAR), which may be CD19 or CD123.

[0072] Drug-resistant allogeneic T cells

[0073] In particular, this invention relates to drug-resistant allogeneic T-cells that are particularly suitable for immunotherapy. Drug resistance can be conferred by inactivation of drug-sensitizing genes or by expression of resistance genes as previously known. Some examples of drugs suitable for this invention are purine nucleoside analogs (PNAs) such as clofarabine or fludarabine, or other drugs such as 6-mercaptopurine (6MP) and 6-thioguanine (6TG).

[0074] The cells according to the invention refer to hematopoietic cells that functionally participate in the initiation and / or execution of innate and / or adaptive immune responses. The cells according to the invention are preferably T cells obtained from a donor. The T cells according to the invention can be derived from stem cells. Stem cells can be adult stem cells, embryonic stem cells, more particularly non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, pluripotent stem cells, or hematopoietic stem cells. Representative human stem cells are CD34+ cells. The isolated cells can also be dendritic cells, cytotoxic dendritic cells, mast cells, NK cells, B cells, or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another embodiment, the cells can be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. Prior to cell expansion and gene modification according to the invention, the cell source can be obtained from the subject by various non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissues from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, any number of T-cell lines available and known to those skilled in the art can be used. In another embodiment, the cells are preferably derived from a healthy donor. In yet another embodiment, the cells are part of a mixed population of cells exhibiting different phenotypic characteristics.

[0075] Multidrug resistance

[0076] In another specific embodiment, when a patient is treated with different drugs, the present invention aims to develop an "off-the-shelf" immunotherapy strategy by using multidrug-resistant allogeneic T cells to mediate the selection of engineered T cells. Treatment efficiency can be significantly enhanced by genetically engineered multidrug-resistant allogeneic T cells. This strategy is particularly effective in treating tumors that respond to combinations of drugs exhibiting synergistic effects. Moreover, multidrug-resistant engineered T cells can be expanded and selected using minimal doses of drug reagents.

[0077] Therefore, the method according to the invention may include modifying T-cells to confer multidrug resistance. This multidrug resistance can be conferred by expressing more than one resistance gene or by inactivating more than one drug-sensitizing gene. In another specific embodiment, multidrug resistance can be conferred on T-cells by expressing at least one resistance gene and inactivating at least one drug-sensitizing gene. Specifically, multidrug resistance can be conferred by expressing at least one resistance gene, such as a mutant of DHFR, a mutant of IMPDH2, a mutant of calcineurin, a mutant of MGMT, the BLE gene, and the mgr gene, and inactivating at least one drug-sensitizing gene, such as the HPRT gene. In a preferred embodiment, multidrug resistance can be conferred by inactivating the HPRT gene and expressing a mutant of DHFR; or by inactivating the HPRT gene and expressing a mutant of IMPDH2; or by inactivating the HPRT gene and expressing a mutant form of calcineurin; by inactivating the HPRT gene and expressing a mutant of MGMT; by inactivating the HPRT gene and expressing the BLE gene; or by inactivating the HPRT gene and expressing the mgr gene.

[0078] Methods for engineering drug-resistant allogeneic T cells:

[0079] To improve cancer treatment and selective implantation of allogeneic T-cells, cells are conferred drug resistance, thus protecting them from the toxic side effects of chemotherapy agents. Drug resistance in T-cells also allows for their accumulation in vitro and in vivo, as T-cells expressing drug resistance genes can survive and proliferate relative to drug-sensitive cells. In particular, this invention relates to a method for engineering allogeneic, drug-resistant T-cells resistant to immunotherapy, comprising:

[0080] (a) Provide T-cells;

[0081] (b) Choose at least one drug;

[0082] (c) Modifying T cells by inactivating at least one gene encoding a component of the T-cell receptor (TCR);

[0083] (d) Modifying T-cells to confer drug resistance;

[0084] (e) Expand the engineered T cells in the presence of the drug.

[0085] -Allogeneic T-cells

[0086] This invention relates to allogeneic immunotherapy. Implantation of allogeneic T-cells may be performed by inactivating at least one gene encoding a component of the TCR. The TCR is rendered not functional in the cell by inactivating the TCRα gene and / or the TCRβ gene. TCR inactivation in allogeneic T-cells avoids GvHD. By inactivating the gene, it is desirable that the gene of interest is not expressed as a functional protein. In a particular embodiment, the gene modification of this method depends on the expression of a rare-cut endonuclease in the provided engineered cells, such that the rare-cut endonuclease specifically catalyzes a break in a target gene, thereby inactivating the target gene. Nucleic acid strand breaks induced by rare-cut endonucleases are typically repaired through different mechanisms, such as homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that typically results in changes to the DNA sequence at the cleavage site. These mechanisms involve reconnecting the remainder of two DNA ends via direct re-ligation (Critchlow and Jackson 1998) or via so-called microhomology-mediated end joining (Betts, Brenchley et al. 2003; Ma, Kim et al. 2003). Repair via non-homologous end joining (NHEJ) typically results in small insertions or deletions and can be used to create specific gene knockouts. The modification can be a substitution, deletion, or addition of at least one nucleotide. Cells that have experienced break-induced mutations, i.e., consecutive mutations that constitute NHEJ events, can be identified and / or selected using methods well known in the art. In a particular embodiment, the step of inactivating at least one gene encoding a component of the T-cell receptor (TCR) into cells of each individual sample includes introducing a rare endonuclease into the cells that can interrupt at least one gene encoding a component of the T-cell receptor (TCR). In a more specific embodiment, the cells of each individual sample are transformed with nucleic acid encoding a rare-cut endonuclease capable of interrupting at least one gene encoding a component of the T-cell receptor (TCR), and the rare-cut endonuclease is expressed into the cells.

[0087] The rare-cut endonuclease can be a giant nuclease, zinc finger nuclease, CRISPR / Cas9 nuclease, TALE-nuclease, or MBBBD-nuclease. In a preferred embodiment, the rare-cut endonuclease is a TALE-nuclease. For TALE-nucleases, the expectation is that they are fusion proteins composed of a DNA-binding domain derived from transcription activator-like effectors (TALEs) and a nuclease catalytic domain that cleaves nucleic acid target sequences (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. (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). In this invention, the novel TALE-nuclease has been designed to precisely target relevant genes for adoptive immunotherapy strategies.

[0088] The preferred TALE-nuclease according to the invention is that which recognizes and cleaves target sequences selected from the group consisting of SEQ ID NO:1 to 5 (TCRα), SEQ ID NO:6, and 7 (TCRβ). The TALE-nuclease preferably comprises a polypeptide sequence selected from SEQ ID NO:8 to SEQ ID NO:13. In another embodiment, additional catalytic domains may be further introduced into cells containing sparse-cleavage endonucleases to increase mutagenesis and enhance their ability to inactivate target genes. In particular, the additional catalytic domain is a DNA end-processing enzyme. Non-limiting examples of DNA end-processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' basic exonucleases, 5' valve endonucleases, helicases, phosphatases, hydrolases, and template-independent DNA polymerases. Non-limiting examples of such catalytic domains include protein domains or catalytically active derivatives of protein domains selected from the group consisting of: hExoI (EXO1_HUMAN), yeast ExoI (EXO1_YEAST), Escherichia coli ExoI, human TREX2, mouse TREX1, human TREX1, bovine TREX1, rat TREX1, TdT (terminal deoxynucleotidyl transferase) human DNA2, and yeast DNA2 (DNA2_YEAST). In a preferred embodiment, the additional catalytic domain has 3'-5' exonuclease activity, and in a more preferred embodiment, the additional catalytic domain is TREX, more preferably the TREX2 catalytic domain (WO2012 / 058458). In another preferred embodiment, the catalytic domain is encoded by a single-chain TREX2 polypeptide. The additional catalytic domain according to the invention may optionally be fused to a nuclease fusion protein or a chimeric protein via a linker peptide.

[0089] Endonucleotide breaks are known to stimulate the rate of homologous recombination. Therefore, in another embodiment, the gene modification step of this method further includes the introduction of a foreign nucleic acid into the cell, the foreign nucleic acid comprising at least one sequence homologous to a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target nucleic acid sequence and the foreign nucleic acid. In a particular embodiment, the foreign nucleic acid comprises first and second portions, respectively, homologous to the 5' and 3' regions of the target nucleic acid sequence. In these embodiments, the foreign nucleic acid also comprises a third portion located between the first and second portions, which is not homologous to the 5' and 3' regions of the target nucleic acid sequence. Following the breakage of the target nucleic acid sequence, homologous recombination is stimulated between the target nucleic acid sequence and the foreign nucleic acid. Preferably, a homologous sequence of at least 50 bp, more preferably more than 100 bp, and more preferably more than 200 bp is used in the donor matrix. In a particular embodiment, the homologous sequence can be from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. In fact, the shared nucleic acid homologous region is located in the regions upstream and downstream of the lateral break site, while the nucleic acid sequence to be introduced should be located between the two arms.

[0090] In a particular embodiment, according to the present invention, the exogenous nucleic acid may contain a transgene encoding a drug resistance gene.

[0091] Other possible engineering modifications of T-cell properties

[0092] The immune cells according to the invention can be further engineered to acquire additional properties that enable them to participate in more specific or effective therapeutic uses.

[0093] -Chimeric antigen receptor

[0094] Chimeric antigen receptors (CARs) utilize ligand-binding domain properties to redirect immune cell specificity and reactivity toward selected targets. Therefore, in another specific embodiment, the method further includes the step of introducing a chimeric antigen receptor into the lymphocytes. The chimeric antigen receptor binds its binding domain, for example, to an antibody-based specificity for a desired antigen (e.g., a tumor antigen), to an intracellular domain of a T-cell receptor-activated component present on the target cell, resulting in a chimeric protein exhibiting specific anti-target cell immune activity. Generally, a CAR consists of an extracellular single-chain antibody (scFv) fused to an intracellular signaling domain of the Zeta chain (scFv:ζ) of the T-cell antigen receptor complex, and when expressed on T-cells, has the ability to redirect antigen recognition based on monoclonal antibody specificity. One example of a CAR used in this invention is a CD19 antigen-targeted CAR and may contain the amino acid sequence: SEQ ID NO:19 or 20 as a non-limiting example.

[0095] - Inactivation of immune checkpoint genes

[0096] T-cell-mediated immunity involves multiple sequential steps involving the clonal selection of antigen-specific cells, their activation and proliferation in secondary lymphoid tissues, their transport to antigens and inflammatory sites, the execution of direct effector functions, and the provision of aid to numerous effector immune cells (via cytokines and membrane ligands). Each of these steps is modulated by counterbalancing of stimulatory and inhibitory signals that fine-tune the response. Those skilled in the art will understand that the term "immune checkpoint" refers to a set of molecules expressed by T-cells. These molecules effectively act as a "brake" on the immune response, either downregulating or inhibiting it.Molecules used as immune checkpoints include, but are not limited to, programmed death 1 (PD-1, also known as PDCD1 or CD279, accession number: NM_005018), cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152, accession number: AF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, accession number: JX049979.1), BTLA (also known as CD272, accession number: NM_181780.3), BY55 (also known as CD160, accession number: CR541888.1), TIGIT (also known as VSTM3, accession number: NM_173799), LAIR1 (also known as CD305, accession number: CR542051.1), (Meyaard, Adema SIGLEC10 (GenBank accession number: AY358337.1), 2B4 (also known as CD244, accession 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. 1997), SIGLEC10 (GenBank accession number: AY358337.1), 2B4 (also known as CD244, accession number: NM_001166664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 (Nicoll, Ni et al. 1999), SIGLEC9 (Zhang, Nicoll et al. 2000; Ikehara et al. 2000). al.2004), TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD3, SMAD4, SM AD10, 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 inhibit immune cells. For example, CTLA-4 is a cell surface protein expressed on certain CD4 and CD8 T cells; when bound by its ligands (B7-1 and B7-2) on antigen-presenting cells, T cell activation and effector function are inhibited. Therefore, the present invention relates to a method for engineering drug-resistant allogeneic T cells, further comprising modifying T cells by inactivating at least one protein involved in immune checkpoints, particularly PD1 and / or CTLA-4.In a preferred embodiment, the step of inactivating at least one protein involved in the immune checkpoint is achieved by expressing a rare-cut endonuclease capable of specifically cleaving target sequences in the immune checkpoint gene. In a preferred embodiment, the rare-cut endonuclease is a TALE-nuclease. For example, the TALE-nuclease can specifically cleave target sequences selected from the group consisting of SEQ ID NO:21 to 23 (CTLA-4) and SEQ ID NO:24 and SEQ ID NO:25 (PDCD1), and in a more preferred embodiment, the TALE-nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NO:26 to SEQ ID NO:35.

[0097] -Immune suppression and tolerance T-cells

[0098] Allogeneic cells are rapidly rejected by the host immune system. It has been shown that allogeneic leukocytes present in non-irradiated blood products will persist for no more than 5 to 6 days (Boni, Muranski et al. 2008). Therefore, to prevent rejection of allogeneic cells, the host immune system usually has to be suppressed to some extent. However, in the case of adoptive immunotherapy, immunosuppressive drugs also have an adverse effect on the introduced therapeutic T-cells. Therefore, for adoptive immunotherapy to be used effectively under these conditions, the introduced cells also need to be resistant to immunosuppressive therapy. Therefore, in certain embodiments, the method according to the invention further includes the step of modifying T-cells (preferably by inactivating at least one gene encoding a target for an immunosuppressant) to make them resistant to immunosuppressants. An immunosuppressant is a drug that inhibits immune function through one of a variety of mechanisms of action. In other words, an immunosuppressant acts as a compound that exhibits the ability to reduce the degree of immune response. The method according to the present invention allows for the conferral of T-cell immunosuppressive tolerance for immunotherapy by inactivating the target of an immunosuppressant in T-cells. As a non-limiting example, the target of the immunosuppressant can be a receptor for the immunosuppressant, such as CD52, glucocorticoid receptor (GR), FKBP family gene members, and cyclin family gene members. In a particular embodiment, the gene modification of this method depends on the expression of a rare-cut endonuclease in the provided engineered cells, such that the rare-cut endonuclease specifically catalyzes a break in a target gene, thereby inactivating the target gene. The rare-cut endonuclease can be a giant nuclease, a zinc finger nuclease, or a TALE-nuclease. Preferably, the TALE-nuclease according to the invention recognizes and breaks target sequences selected from the group consisting of SEQ ID NO:36 to 41 (GR) and SEQ ID NO:54 to 59 (CD52). The TALE-nuclease preferably comprises a polypeptide sequence selected from SEQ ID NO:42 to SEQ ID NO:53 and SEQ ID NO:60 to SEQ ID NO:61.

[0099] -Suicide gene

[0100] On the other hand, since engineered T-cells can proliferate and persist for years after administration, it is ideal to include a safety mechanism to allow selective deletion of the administered T-cells. Therefore, in some embodiments, the method of the present invention may include transforming the T-cells with a recombinant suicide gene. Once administered to a subject, the recombinant suicide gene is used to reduce the risk of direct toxicity and / or uncontrolled proliferation of T-cells (Quintarelli C, Vera F, blood 2007; Tey SK, Dotti G., Rooney CM, boil blood marrowtransplant 2007). The suicide gene is capable of selectively deleting transformed cells in the body. In particular, the suicide gene has the ability to convert a non-toxic prodrug into a cytotoxic drug or to express a toxic gene expression. In other words, a “suicide gene” is a nucleic acid encoding a product that causes cell death by itself or in the presence of other compounds. A representative example of such a suicide gene is the gene encoding thymidine kinase of herpes simplex virus. Other examples are thymidine kinase of varicella-zoster virus and bacterial gene cytosine deaminase, which can convert 5-fluorocytosine into the 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 inducer (CID) for dimerization. Suicide genes can also be polypeptides expressed on the cell surface that can sensitize cells to therapeutic monoclonal antibodies. As used herein, "prodrug" refers to any compound suitable for conversion into a toxic product in the methods of this invention. A representative example of such a prodrug is ganciclovir, which is converted in vivo into a toxic compound via HSV thymidine kinase. Ganciclovir derivatives are subsequently toxic to tumor cells. Other representative examples of prodrugs include acyclovir, FIAU [1-(2-deoxy-2-fluoro-β-D-furanarabinosyl)-5-iodouracil], 6-methoxypurine arabinoside of VZV-TK, and 5-fluorocytosine of cytosine deaminase.

[0101] -Delivery method

[0102] The aforementioned methods involve expressing proteins of interest, such as drug resistance genes, rare endonucleases, chimeric antigen receptors (CARs), and suicide genes, into cells. As a non-limiting example, the protein of interest can be expressed in cells by introducing it as a transgene (preferably encoded by at least one plasmid vector). Peptides can be expressed in cells, thereby introducing polynucleotides encoding the polypeptide into the cells. Alternatively, the polypeptide can be generated extracellularly and subsequently introduced. Methods for introducing polynucleotide constructs into cells are known in the art and, as non-limiting examples, include stable transformation methods that integrate the polynucleotide construct into the cell's genome, transient transformation methods that do not integrate the polynucleotide construct into the cell's genome, and virus-mediated methods. The polynucleotide can be introduced into cells, for example, by recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. For example, transient transformation methods include, for example, microinjection, electroporation, or particle bombardment. The polynucleotide, given its expression in cells, can be contained in a vector, more particularly in a plasmid or virus. The plasmid vector may contain selection markers for recognizing and / or selecting cells that receive the vector. Different transgenes may be contained in the vector. The vector may contain a nucleic acid sequence encoding a ribosomal skip sequence, such as a sequence encoding a 2A peptide. The 2A peptide is identified in the foot-and-mouth disease virus subunit of a piconerivirus, which causes a ribosomal "skip" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codon (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)). A "codon" refers to three nucleotides on mRNA (or on the sense strand of a DNA molecule) that are translated into one amino acid residue by a ribosome. Therefore, when a polypeptide is separated by a 2A oligopeptide sequence within a frame, two polypeptides can be synthesized from a single, adjacent open reading frame in the mRNA. This ribosome skipping mechanism is well known in the art and is known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.

[0103] In a more preferred embodiment of the invention, the polynucleotide encoding the polypeptide according to the invention can be, for example, mRNA introduced directly into cells via electroporation. The inventors have determined the optimal conditions for mRNA electroporation in T-cells. The cytoPulse technology used by the inventors allows for the delivery of material into cells by transiently penetrating living cells using pulsed electric fields. This technology, based on the use of PulseAgile (BTX Havard Apparatus, 84 October Hill Road, Holliston, MA 01746, USA) electroporation waveform pulses, provides precise control over the pulse duration, intensity, and interval between pulses (US Patent 6,010,613 and International PCT Application WO2004083379). All these parameters can be modified to achieve optimal conditions for high transfection efficiency and low mortality. Essentially, a first high-field pulse allows pore formation, while a subsequent low-field pulse allows the polynucleotide to enter the cell.

[0104] Activation and expansion of T cells

[0105] Whether before or after T-cell gene modification, T-cells can generally be activated and expanded using methods described in the following patent documents: for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T-cells can be expanded in vitro or in vivo. Generally, the T-cells of the present invention are expanded by contacting reagents that stimulate the CD3TCR complex and co-stimulatory molecules on the T-cell surface to generate an activation signal for the T-cells. For example, chemicals such as calcium ionophores (ion channels) A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins, such as phytohemagglutinin (PHA), can be used to generate an activation signal for the T-cells. As a non-limiting example, T-cell populations can be stimulated in vitro by binding calcium ionophores, such as by contacting anti-CD3 antibodies, their antigen-binding fragments, or surface-immobilized anti-CD2 antibodies, or by contacting protein kinase C activators (e.g., bryophytein). For co-stimulation of helper molecules on the T-cell surface, ligands binding helper molecules are used. For example, T-cell populations can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T-cell proliferation. To stimulate the proliferation of CD4+ T- cells or CD8+ T- cells, contact with anti-CD3 and anti-CD28 antibodies is performed. For example, the reagents providing each signal can be in solution or coupled to a surface. As will readily be understood by those skilled in the art, the particle-to-cell ratio can depend on the particle size relative to the target cells.

[0106] Suitable conditions for T-cell culture include appropriate culture media (e.g., minimum essential medium or RPMI 1640 or X-vivo 5, (Lonza)) that may contain factors essential for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, interferon, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFP, IL-21, and TNF-α, or any other additives known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma proteins, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-vivo 1 and X-vivo 20, Optimizer, and added amino acids, sodium pyruvate and vitamins, serum-free or supplemented with adequate serum (or plasma) or defined hormones, and / or a sufficient amount of cytokines for the growth and expansion of T cells. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and not in the cell culture to be injected into the subject. Target cells are maintained under the necessary conditions to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to different stimulation times may exhibit different characteristics.

[0107] Therapeutic applications

[0108] In another embodiment, the isolated T-cells obtained as described above can be used in allogeneic adoptive cell immunotherapy. Specifically, the T-cells according to the invention can be used to treat cancer, infection, or autoimmune diseases in patients requiring this treatment. In another aspect, the invention relies on a method for treating patients requiring this treatment, the method comprising at least one of the following steps:

[0109] (a) Provide isolated T cells that are obtainable by any of the methods described above;

[0110] (b) Administer the cells to the patient.

[0111] In one embodiment, the T-cells of the present invention can undergo robust in vivo expansion and can persist for a long period of time.

[0112] The treatment can be remission-relieving, curative, or preventative. This invention is particularly suitable for allogeneic immunotherapy because it can convert T-cells, normally obtained from a donor, into non-allogeneic reactive cells. This can be performed under standard protocols and repeated as needed. The resulting modified T-cells are administered to one or more patients, making them available as an "off-the-shelf" therapeutic product.

[0113] Cells that can be used with the disclosed methods are described in the previous section. The treatment can be used to treat patients diagnosed with cancer, viral infections, or autoimmune diseases. Treatable cancers include non-vascurized tumors, or tumors that are not yet significantly vasculized, as well as vasculized tumors. Cancers can include non-solid tumors (such as hematologic malignancies, e.g., leukemia and lymphoma) or can include solid tumors. Types of cancer treated with the drug-resistant allogeneic T-cells of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or malignant lymphomas, benign and malignant tumors, and malignant tumors, such as sarcomas, epithelial malignancies, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment of the invention, childhood acute lymphoblastic leukemia (ALL) and amyotrophic myeloid leukemia (AML) are typically treated with the allogeneic drug-resistant T-cells according to the present invention. This can be achieved by using drug-resistant KOTRAC CD19. + CAR T-cells and drug-resistant KO TRAC CD123 + T-cells are involved.

[0114] It can be a combination of one or more anticancer therapies selected from the group consisting of: antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiation therapy.

[0115] According to a preferred embodiment of the invention, the treatment is administered to a patient undergoing immunosuppressive therapy. The invention preferably relies on cells or cell populations that have developed resistance to at least one pharmaceutical agent according to the invention due to the expression of drug resistance genes or the inactivation of drug sensitization genes. In this respect, the pharmaceutical treatment according to the invention should facilitate the selection and expansion of T cells in the patient.

[0116] Administration of the cells or cell populations according to the invention can be performed in any convenient manner, including by aerosol inhalation, injection, uptake, perfusion, implantation, or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodularly, intramedullaryly, intramuscularly, intracranially, intravenously or intralymphaticly, or intraperitoneally. In one embodiment, the cell compositions of the invention are preferably administered intravenously.

[0117] The cells or cell populations to be administered may include 10 3 -10 10 Cells / kg body weight, preferably 10 5- 10 6 Cells / kg body weight, including all integer values ​​within those ranges. Cells or cell populations may be administered in one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered at more than one dose over a period of time. The timing of administration is within the judgment of the administering physician and depends on the patient's clinical condition. Cells or cell populations may be obtained from any source, such as a blood bank or donor. While individual needs may vary, determining the optimal range of effective amounts of cell types to be administered for a particular disease and condition is within the scope of the art. An effective amount refers to the amount that provides therapeutic or preventative benefit. The dose administered will depend on the recipient's age, health status and weight, any concurrent treatments, if present, the frequency of treatments, and the nature of the desired effect.

[0118] In another embodiment, the effective amount of the cell or pharmaceutical composition containing those cells is administered via the gastrointestinal tract. The administration may be intravenous. The administration may be performed directly by injection into the tumor.

[0119] In some embodiments of the invention, cell-bound (e.g., before, simultaneously with, or after) administration of any number of relevant therapeutic modalities to the patient, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nataliziimab for MS patients or efaliztimab for psoriasis patients or other treatments for PML patients. In a further embodiment, the T-cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit calcium-dependent phosphatases such as calcineurin (cyclosporine and FK506) or p70S6 kinase (rapamycin), which is important for growth factor induction signaling (Liu et al., Cell 66:807-815, 11; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Cirr. Opin. mm n. 5:763-773, 93). In a further embodiment, the cell composition of the present invention is administered to a patient in conjunction with (e.g., before, during, or after) bone marrow transplantation, T-cell ablation therapy with chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after B-cell ablation therapy, such as with an agent that reacts with CD20, for example, rituximab. For example, in one embodiment, the subject may receive standard treatment with high-dose chemotherapy, followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives perfusion of the expanded immune cells of the present invention. In other embodiments, the expanded cells are administered before or after the procedure.

[0120] Pharmaceutical Composition

[0121] The isolated T-cells of the present invention can be administered alone or in combination with diluents and / or other components, such as IL-2 or other cytokines or cell populations, as a pharmaceutical composition. In short, the pharmaceutical compositions of the present invention may comprise T-cells as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration. The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's physical condition and the type and severity of the patient's disease, but an appropriate dose can be determined through clinical trials.

[0122] Methods for testing the cytotoxicity of isolated CAR T cells and kits for their intended use.

[0123] Another embodiment of the invention includes a method for testing the cytotoxicity of isolated chimeric antigen receptor (CAR) T-cells as previously described against: drug-resistant target cells; both the isolated CAR T-cells expressing a chimeric antigen receptor (CAR) and target cells expressing at least one specific surface antigen (and optionally a marker gene, such as luciferase), comprising:

[0124] (a) Simultaneously preparing the cell population of T-cells and target cells;

[0125] (b) The T-cell population is incubated with at least the specific target cells;

[0126] (c) Determine the survival rate of the specific target cells.

[0127] Resistance genes can be selected from those described in the preceding sections. Preferred resistance genes are...

[0128] dCK.

[0129] The surface antigen selected in this invention is one that can be expressed in T-cells via a chimeric antigen receptor (CAR) and is dependent on the target cell and is typically specific to cancer cells. Preferably, the surface antigen used in CAR T-cells is CD19, because this antigen is specifically expressed in certain lymphomas or leukemias, such as acute lymphoblastic leukemia (ALL).

[0130] Finally, this invention relates to a kit for testing the cytotoxicity of CAR T-cells against target cells, comprising:

[0131] (d) Confer antigen-specific CARs to the T-cell population;

[0132] (e) The target cells expressing the antigen;

[0133] (f) Optionally, culture medium;

[0134] According to the present invention, both T-cells and target cells have developed resistance to chemotherapeutic agents.

[0135] This invention application does not merely seek protection for general methods of engineering T-cells to be resistant to purine nucleotide analog (PNA) drugs and 6TG. It should be more broadly extended to methods for obtaining allogeneic T-cells that are both resistant to chemotherapeutic agents, encompassing at least one of the following objectives:

[0136] 1) A method for engineering allogeneic drug-resistant T cells for immunotherapy, comprising:

[0137] (a) Provide T-cells;

[0138] (b) Select at least one chemotherapeutic agent to which T-cells are sensitive;

[0139] (c) Modifying the T-cell by inactivating at least one gene encoding a component of the T-cell receptor (TCR);

[0140] (d) Modifying the T cells to confer resistance to the chemotherapeutic agents;

[0141] (e) Optionally, the engineered T cells may be expanded in the presence of the drug.

[0142] 2) The method according to claim 1, wherein at least one gene encoding a TCR component is inactivated by expressing a rare endonuclease capable of cleaving a target sequence within at least one gene encoding a TCR component.

[0143] 3) The method according to claim 1 or 2, wherein the drug resistance is conferred on T cells by inactivating at least one drug sensitization gene.

[0144] 4) The method according to claim 3, wherein the drug sensitization gene is inactivated by expressing a rare endonuclease that can cleave the target sequence within the drug sensitization gene.

[0145] 5) The method according to claim 4, wherein the sparse endonuclease is a TALE-nuclease.

[0146] 6) The method according to claims 3 to 5, wherein the drug sensitization gene is dCK.

[0147] 7) The method according to claim 6, wherein the dCK gene is inactivated by TALE-nuclease.

[0148] 8) The method of claim 7, wherein the inactivation of the TALE-nuclease dCK gene is carried out by using the TALE-nucleases of SEQ ID N°63 and SEQ ID N°64, and the dCK target sequence is SEQ ID N°62.

[0149] 9) The method according to claims 3 to 5, wherein the drug sensitizing gene is HPRT.

[0150] 10) The method of claim 1, wherein the drug resistance is conferred on T-cells by expressing at least one drug resistance gene.

[0151] 11) The method of claim 10, wherein the drug resistance gene is a mutant dihydrofolate reductase (DHFR) protein conferred with resistance to folic acid treatment, preferably methotrexate (MTX).

[0152] 12) The method of claim 11, wherein the mutated DHFR, at a position selected from the group consisting of G15, L22, F31 or F34 in SEQ ID NO:14, comprises at least one amino acid mutation.

[0153] 13) The method of claim 12, wherein the mutated DHFR at positions L22 and F31 in SEQ ID NO:14 comprises two amino acid mutations.

[0154] 14) The method according to claim 10, wherein the resistance gene is a mutant inosine-5'-monophosphate dehydrogenase II (IMPDH2) that confers resistance to an IMPDH inhibitor, preferably mycophenolate mofetil (MMF).

[0155] 15) The method of claim 14, wherein the mutant IMPDH2 at position T333 and / or S351 in SEQ ID NO:15 comprises at least one amino acid mutation.

[0156] 16) The method according to claim 10, wherein the resistance gene is a mutant calcineurin (CN) heterodimer a and / or b that confers resistance to calcineurin inhibitors, preferably FK506 and / or CsA.

[0157] 17) The method according to claim 16, wherein the mutated calcineurin heterodimer a 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:16.

[0158] 18) The method of claim 17, wherein the mutated calcineurin heterodimer a contains an amino acid mutation at positions T351 and L354 in SEQ ID NO:16.

[0159] 19) The method of claim 17, wherein the mutated calcineurin heterodimer a contains an amino acid mutation at positions V314 and Y341 in SEQ ID NO:17.

[0160] 20) The method of claim 16, wherein the mutated calcineurin heterodimer b 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.

[0161] 21) The method of claim 20, wherein the mutant calcium phosphatase heterodimer b contains an amino acid mutation at positions L124 and K125 in SEQ ID NO:17.

[0162] 22) The method according to any one of claims 10 to 21, wherein the drug resistance gene is expressed in T-cells by introducing a transgene encoding the drug resistance gene into T-cells.

[0163] 23) The method of any one of claims 10 to 21, wherein the drug resistance gene is expressed in T-cells by introducing a donor matrix into the T-cells to cause homologous recombination between the endogenous gene and the donor matrix, the donor matrix comprising at least one homologous sequence of the endogenous gene and a sequence encoding the drug resistance gene.

[0164] 24) The method of claim 23 further comprises introducing a rare endonuclease into T-cells that can selectively cleave target sequences in the endogenous gene, thereby stimulating homologous recombination.

[0165] 25) The method of claim 24, wherein the sparse endonuclease is a TALE-nuclease.

[0166] 26) The method of any one of claims 1 to 25, further comprising expressing a chimeric antigen receptor in T cells.

[0167] 27) The method according to any one of claims 1 to 26, wherein the chimeric antigen receptor is CD19+ or CD123+.

[0168] 28) The method according to any one of claims 1 to 27, further comprising inactivating immune checkpoint genes.

[0169] 29) The method according to any one of claims 1 to 28, wherein the engineered T-cells are expanded in the patient's blood.

[0170] 30) The method according to any one of claims 1 to 28, wherein the engineered T-cells are expanded in vitro.

[0171] 31) The method according to any one of claims 1 to 30, wherein the engineered T-cells are expanded in the presence of the drug.

[0172] 32) An isolated T-cell or cell line obtainable by the method according to any one of claims 1 to 31.

[0173] 33) An isolated drug-resistant T-cell containing at least one broken gene encoding a component of the T-cell receptor.

[0174] 34) The isolated T-cells according to claim 33 express at least one drug resistance gene.

[0175] 35) The isolated T-cells according to claim 33, wherein the drug resistance gene is selected from the group consisting of the BLE gene, the MCR gene, and genes encoding mutant DHFR, mutant IMPDH2, mutant calcineurin, and mutant AGT.

[0176] 36) The isolated T-cells according to claim 33 contain at least one broken drug sensitization gene, preferably the HPRT gene.

[0177] 37) The isolated T-cell according to any one of claims 32 to 36, wherein the isolated T-cell is conferred an antigen-specific chimeric antigen receptor (CAR).

[0178] 38) The isolated T-cells according to claim 37, wherein the CAR targets CD19+ cells or CD123+ cells;

[0179] 39) An isolated T-cell according to any one of claims 32 to 38, which is suitable for use as a drug.

[0180] 40) An isolated T-cell according to any one of claims 32 to 39, suitable for treating cancer, autoimmune diseases or infections caused by pathogens.

[0181] 41) The isolated T-cells according to claim 40 are suitable for the treatment of acute lymphoblastic leukemia (ALL) or myeloid leukemia (AML).

[0182] 42) A pharmaceutical composition comprising at least one isolated T-cell according to any one of claims 32 to 41.

[0183] 43) Methods for treating patients who require this include:

[0184] (a) Preparing a population of T cells according to any one of claims 1 to 27;

[0185] (b) The transformed T-cells are administered to the patient.

[0186] 44) The method of claim 36, wherein the patient is treated with the drug used in the methods of claims 1 to 31.

[0187] 45) A method for testing the cytotoxicity of isolated chimeric antigen receptor (CAR) T-cells according to any one of claims 32 to 41 against: drug-resistant target cells; isolated CAR T-cells expressing chimeric antigen receptor (CAR) and target cells expressing at least a specific surface antigen (and optionally a marker gene, such as luciferase), comprising:

[0188] (a) Preparation of the population of T-cells and target cells;

[0189] (b) The T-cell population is incubated with at least the specific target cells;

[0190] (c) Determine the survival rate of the specific target cells.

[0191] 46) The method according to claim 45, wherein the resistance gene is dCK.

[0192] 47) The method according to claim 44 or claim 45, wherein the surface antigen is CD19.

[0193] 48) The method of claim 47, wherein the target is a CD19+ luciferase+ Daudi cell.

[0194] 49) A kit for performing a method to test the cytotoxicity of CAR T-cells against target cells, comprising:

[0195] (a) A population of T cells that confer CAR antigen specificity;

[0196] (b) Target cells expressing the antigen;

[0197] Both the T cells and target cells in question were already resistant to chemotherapy drugs.

[0198] definition

[0199] In the above description, many terms are used extensively. The following definitions are provided to aid in understanding this embodiment.

[0200] - Amino acid residues in the polypeptide sequence are designated in this paper according to single-letter codes, where, for example, Q represents Gln or glutamine residues, R represents Arg or arginine residues, and D represents Asp or aspartic acid residues.

[0201] Nucleotides are specified as follows: Single-letter codes are used to specify the bases of nucleosides: a for adenine, t for thymine, c for cytosine, and g for guanine. For denatured nucleotides, r represents g or a (purine nucleotide), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotide), d represents g, a, or t, v represents g, a, or c, b represents g, t, or c, h represents a, t, or c, and n represents g, a, t, or c.

[0202] As used herein, “nucleic acid” or “nucleic acid molecule” refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules may contain monomers of naturally occurring nucleotides (such as DNA and RNA), analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations thereof. Nucleic acids can be single-stranded or double-stranded.

[0203] - A "gene" is the basic unit of heredity, consisting of a segment of DNA arranged linearly along a chromosome that encodes a specific protein or protein segment, small RNA, etc. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), and optionally introns and a 3' untranslated region. Genes may further include terminators, enhancers, and / or silencers.

[0204] The term "transgenic" refers to a nucleic acid sequence (encoding, for example, one or more polypeptides) that is partially or completely heterologous (i.e., foreign) to the endogenous gene of the host cell into which it is introduced, or homologous to the endogenous gene of the host cell into which it is introduced, but which can be designed to be inserted, or can be inserted into the genome of a cell in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location different from the natural gene or its insertion results in gene knockout). Transgenics may include one or more transcriptional regulatory sequences and any other nucleic acids, such as introns, which may be necessary for the optimal expression of the selected nucleic acid encoding the polypeptide. The polypeptide encoded by the transgenic may not be expressed in the cell into which the transgenic is inserted, or may be expressed but inactive.

[0205] - The term "genome" refers to the complete genetic material contained within a cell, such as the nuclear genome, chloroplast genome, and mitochondrial genome.

[0206] A "mutation" refers to the substitution, deletion, or insertion of one or more nucleotides / amino acids in a polynucleotide (cDNA, gene) or polypeptide sequence. Mutations can affect the coding sequence of a gene or its regulatory sequence. They can also affect the structure of the genome sequence / the structure / stability of the encoded mRNA.

[0207] The term "sparse-cut endonuclease" refers to a wild-type or variant enzyme that can catalyze the hydrolysis (breakage) of bonds between nucleic acids within DNA or RNA molecules, preferably DNA molecules. Specifically, the nuclease can be an endonuclease, more preferably a sparse-cut endonuclease with high specificity, recognizing nucleic acid target sites ranging from 10 to 45 base pairs (bp) in length, typically ranging from 10 to 35 base pairs. The endonuclease according to the invention recognizes and breaks nucleic acids on specific polynucleotide sequences, further referred to as "target sequences." Sparse-cut endonucleases can recognize and generate single-strand or double-strand breaks on specific polynucleotide sequences.

[0208] In a particular embodiment, the rare-cut endonuclease according to the present invention may be a Cas9 endonuclease. In fact, recent novel genome engineering tools have been developed from the type II prokaryotic CRISPR (regularly clustered short palindromic repeats) adaptive immune system based on RNA-guided Cas9 nucleases (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) (see review (Sorek, Lawrence et al. 2013)). The CRISPR-associated (Cas) system was first discovered in bacteria and functionally serves as a defense against exogenous DNA (whether viral or plasmid). CRISPR-mediated genome engineering begins by selecting a target sequence typically flanked by a short sequence motif called a preintermediate sequence adjacent motif (PAM). After selecting the target sequence, a specific crRNA complementary to the target sequence is engineered. In the CRISPR-II system, the required trans-activated crRNA (tracrRNA) pairs with crRNA and binds to the provided Cas9 protein. Cas9 acts as a molecular anchor that facilitates base pairing between tracrRNA and cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the dual tracrRNA:crRNA structure acts as a guide RNA to direct the endonuclease Cas9 to its homologous target sequence. Target recognition via the Cas9-tracrRNA:crRNA complex begins by scanning the target sequence for homologous regions between the target sequence and crRNA. In addition to target-crRNA complementarity, DNA targeting requires the presence of a short motif adjacent to the interstitial region (interstitial region adjacent motif - PAM). Following pairing between the dual RNA and the target sequence, Cas9 then introduces blunt double-strand break 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010). In this invention, the guide RNA can be designed, for example, to specifically target genes encoding TCR components. After pairing between the guide RNA and the target sequence, Cas9 induces a break within the TCR gene.

[0209] The homing endonuclease can also be a homing endonuclease, also known as a giant nuclease. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, typically from 14 to 40 bp. The homing endonuclease according to the present invention can, for example, correspond to the LAGLIDADG endonuclease, the HNH endonuclease, or the GIY-YIG endonuclease. A preferred homing endonuclease according to the present invention can be an I-CreI variant. A "variant" endonuclease, i.e., an endonuclease not naturally occurring in nature but obtained through engineering or random mutagenesis, can bind to DNA sequences different from those recognized by wild-type endonucleases (see International Application WO2006 / 097854).

[0210] The sparse-cut endonuclease can be a modular DNA-binding nuclease. A modular DNA-binding nuclease is any fusion protein comprising a catalytic domain of at least one endonuclease and at least one DNA-binding domain or a protein specifying a nucleic acid target sequence. The DNA-binding domain is typically an RNA or DNA-binding domain formed by an independently folded polypeptide or protein domain containing at least one motif that recognizes a double-stranded or single-stranded polynucleotide. Many such polypeptides have been described in the art and possess the ability to specifically bind nucleic acid sequences. Such binding domains typically include, as non-limiting examples: helical-turn-helical domains, leucine zipper domains, winged helical domains, helical-loop-helical domains, HMG-box domains, immunoglobulin domains, B3 domains, or engineered zinc finger domains.

[0211] In a preferred embodiment of the invention, the DNA-binding domain is derived from a transcription activator-like effector (TALE), wherein sequence specificity is driven by a series of 33-35 amino acid repeat sequences derived from proteins of *Xanthomonas* or *Ralstonia solanacearum*. These repeat sequences are substantially distinguished by two amino acid positions specifying the interaction with the base pair (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). Each base pair on the DNA target is contacted via a single repeat sequence, wherein specificity is generated by two variant amino acids of the repeat sequence (so-called repeat sequence variable dipeptide, RVD). The TALE-binding domain may further include an N-terminal transposition domain responsible for the first thymine base (TO) of the target sequence and a C-terminal domain containing a nuclear localization signal (NLS). The TALE nucleic acid-binding domain generally corresponds to an engineered core TALE scaffold comprising multiple TALE repeat sequences, each repeat sequence containing an RVD specific to each nucleotide base of the TALE recognition site. In this invention, each TALE repeat sequence of the core scaffold consists of 30 to 42 amino acids, more preferably 33 or 34 amino acids, wherein two key amino acids at positions 12 and 13 (i.e., so-called repeat variable dipeptides, RVDs) mediate the recognition of a nucleotide in the TALE binding site sequence; the corresponding two key amino acids may be specifically located at positions other than positions 12 and 13 in a TALE repeat sequence longer than 33 or 34 amino acids. Preferably, the RVDs associated with the recognition of different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, and NN for recognizing G or A. In another embodiment, key amino acids 12 and 13 may be mutated toward other amino acid residues to modulate their specificity for nucleotides A, T, C, and G, and in particular, to enhance this specificity. The TALE nucleic acid binding domain typically contains 8 to 30 TALE repeat sequences. More preferably, the core scaffold of the present invention contains 8 to 20 TALE repeat sequences; more preferably, 15 TALE repeat sequences. It may also contain an additional truncated TALE repeat sequence consisting of 20 amino acids located at the C-terminus of the TALE repeat sequence group, i.e., an additional C-terminal hemi-TALE repeat sequence.

[0212] Other engineered DNA-binding domains are modular base-per-base-specific nucleic acid-binding domains (MBBBDs) (PCT / US2013 / 051783). These MBBBDs can be engineered, for example, from recently identified proteins such as EAV36_BURRH, E5AW43_BURRH, E5AW45_BURRH, and E5AW46_BURRH from the recently sequenced genome of the endosymbiotic fungus *Burkholderia rhizoxinica* (Lackner, Moebius et al. 2011). MBBBD proteins contain base-specific modules of approximately 31 to 33 amino acids. These modules exhibit less than 40% sequence identity with *Xanthomonas rhizoxinica* TALE co-repetitive sequences, while displaying greater polypeptide sequence variability. When assembled together, these modular polypeptides can target specific nucleic acid sequences in a manner very similar to *Xanthomonas rhizoxinica* TALE-nucleases. According to a preferred embodiment of the invention, the DNA-binding domain is an engineered MBBBD-binding domain comprising 10 to 30 modules, preferably 16 to 20 modules. Different domains (modules, N- and C-terminals) of the aforementioned proteins from Burkholderia and Xanthomonas are suitable for designing novel proteins or scaffolds with binding properties specific to nucleic acid sequences. In particular, the additional N-terminal and C-terminal domains of the engineered MBBBD can be derived from natural TALE-like AvrBs3, PthXo1, AvrHah1, PthA, and Tal1c as non-limiting examples.

[0213] "TALE-nuclease" or "MBBBD-nuclease" refers to an engineered protein typically derived from the fusion of a DNA-binding domain and a nuclease catalytic domain, where the DNA-binding domain is usually derived from a transcription activator-like effector protein (TALE) or an MBBBD-binding domain. This catalytic domain is preferably a nuclease domain, and more preferably a domain with nuclease activity, such as I-TevI, ColE7, NucA, and Fork-I. In a particular embodiment, the nuclease is a monomeric TALE-nuclease or MBBBD-nuclease. A monomeric nuclease is a nuclease that does not require dimerization for specific recognition and cleavage, such as the engineered DNA-binding domain fused to the catalytic domain of I-TevI ​​described in WO2012138927. In another particular embodiment, the sparse-cleavage endonuclease is a dimerized TALE-nuclease or MBBBD-nuclease, preferably comprising a DNA-binding domain fused to Fork. TALE-nucleases have been described and used to stimulate gene targeting and gene modification (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermake et al. 2010). This engineered TALE-nuclease can be marketed under the trade name TALEN. TM Acquired through commercial purchase (Cellectis, 8ruede la Croix Jarry, 75013 Paris, France).

[0214] The term "break" refers to the detachment of the covalent backbone of a polynucleotide. Breaks can be initiated by various methods, including but not limited to enzymatic or chemical hydrolysis via phosphodiester bonds. Both single-strand and double-strand breaks are possible, and a double-strand break can occur due to two distinct single-strand break events. Breaks in double-stranded DNA, RNA, or DNA / RNA hybrids can result in the formation of blunt ends or cross-ended ends.

[0215] - The term "chimeric antigen receptor" (CAR) refers to a chimeric receptor that includes an extracellular ligand-binding domain, a transmembrane domain, and a signal transduction domain.

[0216] - As used herein, the term "extracellular ligand-binding domain" is defined as an oligopeptide or polypeptide capable of binding ligands. Preferably, the domain can interact with cell surface molecules. For example, an extracellular ligand-binding domain can be selectively configured to recognize ligands that act as cell surface markers on target cells associated with specific disease states.

[0217] In a preferred embodiment, the extracellular ligand-binding domain comprises a single-chain antibody fragment (scFv) containing a light chain (V) of a target antigen-specific monoclonal antibody linked by a flexible linker.L ) and heavy chain (V H ) Variable fragment. In a preferred embodiment, the scFV is derived from a CD19 or CD123 antibody. Preferably, the scFV of the present invention comprises an scFV derived from the CD19 monoclonal antibody 4G7 (Peipp, Saul et al. 2004).

[0218] - The signal transduction domain or intracellular signal domain of the CAR according to the present invention is responsible for intracellular signal transduction following binding of the extracellular ligand-binding domain to the target, thereby leading to activation of immune cells and an immune response. A preferred example of a signal transduction domain suitable for a CAR species may be a cytoplasmic sequence of a co-receptor that synergistically initiates signal transduction after binding to a T-cell receptor and an antigen receptor. The signal transduction domain includes two distinct types of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals. The primary cytoplasmic signal sequence may contain a signal motif of an activation motif of an immune receptor tyrosine class known as ITAM. In a particular embodiment, the signal transduction domain of the CAR of the present invention includes a co-stimulatory signaling molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or ligands required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of co-stimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0219] The CAR according to the invention is expressed on the surface membrane of a cell. Therefore, the CAR may contain a transmembrane domain. A key characteristic of a suitable transmembrane domain is its ability to be expressed on the surface of a cell, preferably an immune cell, particularly a lymphocyte or natural killer (NK) cell, and to interact with it to guide a cellular response of the immune cell to a predefined target cell. The transmembrane domain may further contain a stalk region between the extracellular ligand-binding domain and the transmembrane domain. The term "stalk region" as used herein generally refers to any oligo- or polypeptide that functionally links the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk region provides greater flexibility and accessibility to the extracellular ligand-binding domain. The stalk region may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The stalk region may be wholly or partially derived from natural molecules, such as all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of the antibody constant region. Alternatively, the stem region may correspond to a synthetic sequence of a naturally occurring stem sequence, or it may be a completely synthetic stem sequence.

[0220] Downregulation or mutation of target antigens is commonly observed in cancer cells, resulting in antigen-loss escape mutants. Therefore, to counteract tumor escape and confer greater specificity to the target on immune cells, CD19-specific CARs can include another extracellular ligand-binding domain and simultaneously bind different elements to the target, thereby increasing the activation and function of immune cells. Examples of CD19-specific CARs are ScFV FMC63 (Kochenderfer JN, Wilson WH, Janik JE, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 2010; 116(20):4099-410) or ScFv 4G7CAR (described in application filed under serial number PCT / EP2014 / 059662). In one embodiment, the extracellular ligand-binding domain can be confluently (tandemly) on the same transmembrane polypeptide. In another embodiment, the different extracellular ligand-binding domains may be positioned on different transmembrane polypeptides constituting the CAR. In another embodiment, the invention relates to a group of CARs comprising each of different extracellular ligand-binding domains. In particular, the invention relates to a method of engineering immune cells, comprising providing an immune cell and expressing each group of CARs comprising different extracellular ligand-binding domains on the cell surface. In another specific embodiment, the invention relates to a method of engineering immune cells, comprising providing an immune cell and introducing a polynucleotide into the cell encoding a polypeptide comprising each group of CARs comprising different extracellular ligand-binding domains. A CAR group refers to at least two, three, four, five, six or more CARs each comprising a different extracellular ligand-binding domain. The different extracellular ligand-binding domains according to the invention can preferably bind different elements simultaneously in the target, thereby increasing the activation and function of the immune cell. The invention also relates to isolated immune cells comprising each group of CARs containing a different extracellular ligand-binding domain.

[0221] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. In this invention, "vector" includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules, which may be composed of chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acids. Preferred vectors are those capable of automatically replicating (free vectors) and / or expressing the nucleic acids linked to them (expression vectors). A large number of suitable vectors are known to those skilled in the art and are commercially available.

[0222] - The term "delivery carrier" refers to any delivery carrier that can be used in this invention to induce cell contact (i.e., "contact type") or to deliver (i.e., "introduce") the drugs / chemicals and molecules (proteins or nucleic acids) required in this invention within cells or subcellular regions. It includes, but is not limited to, liposome delivery carriers, viral delivery carriers, drug delivery carriers, chemical substance carriers, polymer carriers, lipid complexes, polymers, dendritic polymers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions, or other suitable transport carriers.

[0223] Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as positive myxoviruses (e.g., influenza virus), rod-shaped viruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., cowpox, fowlpox, and canarypox). Other viruses include, for example, norovirus, enveloped viruses, flaviviruses, reoviruses, polymorphonuclear papillomaviruses, hepatotropic DNA viruses, and hepatitis viruses. Examples of retroviruses include: avian leukosis sarcoma, mammalian C, B, and D viruses, HTLV-BLV groups, lentiviruses, and foam viruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0224] - The term "lentiviral vector" refers to HIV-like lentiviral vectors that are highly promising for gene delivery due to their relatively large encapsulation capacity, reduced immunogenicity, and efficient and stable transduction of a wide range of different cell types. Lentiviral vectors are typically produced after transient transfection of three or more plasmids (packaging, capsid, and transporter) into production cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, viral RNA undergoes reverse transcription, mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of the infected cell. The term "integrating lentiviral vector (or LV)" refers to such vectors, as a non-restrictive example, that can integrate into the genome of the target cell. Conversely, "non-integrating lentiviral vector (or NILV)" refers to highly efficient gene delivery vectors that do not integrate into the genome of the target cell through the action of viral integrase.

[0225] - For cells or multiple cells, the expectation is that it refers to any eukaryotic living cells, primary cells, and cell lines of these organisms derived from in vitro cultures.

[0226] - The term "native cells" or "primary cells" is intended to refer to cells directly collected from living tissue (i.e., biopsy material) and intended for in vitro growth, having undergone several population multiplications and thus being more representative of the major functional components and the characteristics of tissues derived from them compared to continuous tumorigenic or artificially immortalized cell lines. As a non-limiting example, cell lines may 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-937 cells; MRC5 cells; IMR90 cells; JurkaT- cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.

[0227] Because some variability can be derived from genomic data derived from these peptides, and in order to take into account the possibility of replacing some amino acids present in these peptides without significantly reducing activity (functional variants), the present invention includes polynucleotide variants of the aforementioned peptides that share at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identity with the sequence provided in this patent application.

[0228] The present invention therefore relates to polypeptides comprising a polypeptide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO:20 and SEQ ID NO:26 to SEQ ID NO:35.

[0229] "Identity" refers to the sequence similarity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence that can be aligned for comparison. When the positions in the aligned sequences are occupied by the same bases, the molecules are considered identical (identical) at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at shared positions in the nucleic acid sequences. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA, or BLAST, which is available as part of the GCG sequence analysis software package (University of Wisconsin, Madison, Wis.) and can be utilized, for example, by default. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity with the specific polypeptides described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are considered.

[0230] -<<Knockout>> refers to a gene mutation to the point that it cannot be expressed;

[0231] - "TRAC" refers to "T-cell receptor α constant region (conserved region)" and corresponds to the constant gene of the TCRα subunit.

[0232] In addition to the foregoing features, the present invention includes further features disclosed in the following embodiments illustrating a method for using engineered allogeneic and drug-resistant T-cells to treat resistance to immunotherapy, as well as in the accompanying drawings.

[0233] Example 1: Production and characterization of clofarabine-resistant T cells

[0234] dCK's TALE-nuclease-mediated inactivation

[0235] To inactivate dCK, two pairs of dCK TALE-nucleases were designed, assembled, and verified by sequencing; subsequent work was carried out using only the two pairs named TALE-nuclease dCK2 and those with SEQ ID NO: 63 and SEQ ID NO: 64. Details regarding the overall dCK gene architecture (exons and introns) and the sequence indication of the TALE-nuclease target site located in exon 2 are provided. Figure 3 .

[0236] The dCK target sequence of the TALE-nuclease dCK2 pair corresponds to SEQ ID N°62.

[0237] Once validated, mRNA encoding two TALE-nucleases was produced, polyadenylated, and used to electroporate T-cells using the agile pulse technique as described in WO2013 / 176915 (left and right, using 5 or 10 μg of TALE-nuclease mRNA). Following electroporation, T-cells were subjected to an immediate cold shock by culturing at 30°C for 24 h. Reactivation was then performed (12.5 μL beads / 10 cells). 6 The procedure was performed on day 8 (8 days after electroporation).

[0238] The obtained cells were allowed to grow and were eventually genotyped (analyzed by T7 restriction enzyme and deep sequencing at dCK and TRAC sites) and phenotypically characterized. Phenotypic characterization consisted of (i) examining their ability to grow in the presence or absence of drugs, and (ii) determining the IC50 of PNA, clofarabine, and fludarabine on T-cells. 50 (iii) When a double KO is performed, the extent of TRAC inactivation is determined by FACS analysis.

[0239] Genotypic characterization of dCK KO T-cells

[0240] To evaluate the efficiency of dCK gene inactivation, cells transfected with 5 or 10 μg TALE-nuclease mRNA were grown for 4 days (day 4 after electroporation, D4) and collected for T7 analysis at the dCK site. Figure 5 ).

[0241] The primer sequences used in these T7 analyses correspond to SEQ ID N°68 and SEQ ID N°69. The T7 assay protocol is described in Reyon, D., Tsai, SQ, Khayter, C., Foden, JA, Sander, JD, and Joung, JK (2012) FLASH assembly of TALE-nucleases for high-throughput genomeediting. Nat Biotechnologies.

[0242] The results of this endonuclease T7 analysis showed that significant gene processing indicated effective inactivation of dCK when transfected with approximately 5 and 10 μg (left and right) of dCK2TALE-nuclease.

[0243] Determination of the growth rate of dCK KO T-cells

[0244] As Figure 6 As shown, dCK KO cells exhibit a similar growth rate to WT-cells. Furthermore, they can be reactivated at D8 with the same efficiency as WT T-cells.

[0245] Selection of dCK KO T-cells in the presence of clofarabine

[0246] dCK KO or WT T-cells were allowed to grow from D8 to D13 and then cultured with or without 1 μM clofarabine until D18. Cells were collected at D8 (before drug addition) and at D18 (after drug culture) for the performance of T7 restriction enzyme assays.

[0247] Figure 7 The results shown indicate that the presence of 1 μM clofarabine in the culture medium at D18 selectively enriched dCK KO T- cells compared to wild-type (WT) T- cells (dCK KO T- cells had a two-band low molecular weight compared to the single-band high molecular weight of WT T- cells). This suggests that TALE-nuclease-mediated inactivation of dCK cells allows for the selection of drug-resistant T- cells superior to WT T- cells. Therefore, dCK KO T- cells can tolerate the presence of 1 μM clofarabine, which corresponds to C according to the European Medecines Agency (EMA) report. 最大 (C max Clinically relevant doses of ) for the treatment of acute lymphoblastic leukemia (ALL).

[0248] Determination of the IC50 of clofarabine against dCK KO T-cells relative to WT T-cells

[0249] To further investigate the tolerance of T cells to clofarabine, the IC50 of this drug was determined in dCK KO and WT T cells. Cells were collected three days after transfection and cultured for two days in the presence of gradually increasing concentrations of clofarabine (0–10 μM). T cell viability was determined by FACS analysis at the end of clofarabine culture.

[0250] Figure 8 The results shown clearly demonstrate that treatment of the dCK gene mediated by TALE-nuclease effectively inactivates dCK activity in T-cells. This inactivation is associated with clofarabine resistance, in contrast to the sensitivity of WT T-cells. The IC50 values ​​(the amount of drug added to the culture medium that reduces cell viability to 50%) correspond to approximately 100 nM and 10 μM for WT and dCK KO T-cells, respectively.

[0251] In summary, this first set of data allows us to conclude that TALE-nuclease-mediated inactivation of the dCK gene is effective. Inactivation of dCK does not impair the growth rate of engineered T cells while simultaneously enabling them to tolerate clinically relevant doses of clofarabine.

[0252] Example 2. Production and characterization of allogeneic T cells resistant to clofarabine

[0253] To develop and produce allogeneic CAR T-cells resistant to clofarabine, both dCK and TRAC genes were inactivated. Following successful dCK inactivation demonstrated in Example 1, TRAC / dCK dual-KO T-cells were generated and characterized. Figure 9 The two workflows shown are performed in parallel. One of them corresponds to a 5-day period in which cells are cultured in the presence of chlorofarabine.

[0254] Genotype characterization

[0255] To first evaluate the efficiency and kinetics of TRAC and / or dCK gene inactivation, transfected cells were grown for 6 days and collected on days 1, 3, and 6 for T7 analysis at the dCK and TRAC loci. To achieve this, two pairs of primers, SEQ IDs N°68 and N°69, and SEQ IDs N°70 and N°71, were used for the dCK and TRAC loci, respectively, for T7 analysis.

[0256] The scheme uses the one described in the literature Reyon, D., Tsai, SQ, Khayter, C., Foden, JA, Sander, JD, and Joung, JK (2012) FLASH assembly of TALE-nucleases for high-throughput genome editing. Nat Biotechnol.

[0257] Figure 10 The results shown indicate that TALE-nuclease-mediated single TRAC and dCK KO are highly efficient even on day 1. Although double KO cells can no longer be characterized as a homologous population, TRAC / dCK double KO is also highly efficient.

[0258] Cells were then grown with or without 1 μM chlorofarabine. Cells were harvested on day 6 (6 days post-transfection) and cultured for 3 days with or without chlorofarabine, and dCK KO efficiency was determined by T7 restriction enzyme assay and high-throughput DNA sequencing.

[0259] The protocol for deep sequencing is described in the literature Shendure, J., & Ji, H. (2008). Next-generation DNA sequencing. Nature biotechnology, 26(10), 1135-1145.

[0260] Figure 11 The results shown indicate that the frequency of insertions and deletions generated at the dCK site was approximately 80% to 90% in all experiments. This again demonstrates that TALE-nuclease-mediated inactivation of dCK is highly efficient, even when combined with TRAC inactivation. The presence of 1 μM clofarabine in the culture medium for 5 days did not enhance the dCK KO-specific T7 band, as observed in the first set of experiments. This indirectly suggests that, in this particular experiment, the success of dCK inactivation was sufficient to allow the growth of engineered T-cells in the presence of clofarabine. Interestingly, this suggests that if dCK KO is sufficiently effective, there is no need to select T-cells in the presence of clofarabine to obtain drug-resistant T-cells. Therefore, this characteristic represents a significant advantage in the production of drug-resistant allogeneic T-cells.

[0261] Phenotypic evaluation of TCAR KO efficiency

[0262] TRAC KO T-cells collected from the double KO assay were analyzed and purified by FACS (CliniMACS). Figure 12 The results shown in A illustrate a labeling experiment of T-cells with or without anti-TCR mAb-PE. Figure 12 B also involves the presence or absence of chlorofarabine in the culture medium, and the mAb-PE markers of T cells before and after TRAC KO T-cell purification.

[0263] The results showed that the efficiency of TCR knockout was high (approximately 85%) in T cells treated with both TRAC and dCK mRNA (dCK / TRAC double knockout). The purification method allowed for efficient selection / purification of TCR-negative cells up to 99.3% purity.

[0264] Phenotypic characterization of TRAC / dCK KO T-cells

[0265] The growth rate of T cells in the absence of clofarabine is as follows: Figure 13 As shown in the figure. Even though KO dCKT cells exhibit slight growth defects, they can be reactivated on day 10 with the same efficiency as WT T-cells.

[0266] The growth rate of T cells in the presence of clofarabine is as follows: Figure 14As shown in the figure. This experiment was performed on double KO dCK / TCART CART-cells (FMC63 described in patent application PCT / EP2014 / 059662) by culturing these cells for 11 days in media with different chlorofarabine (0.1 μM to 10 μM). Figure 14 The results shown clearly demonstrate that, although growth was not as significant as in the drug-free form of these cells, T-cell expansion of double KO dCK / TCAR CAR T-cells was achieved at up to 1 μM clofarabine (which corresponds to C...). 最大 The following is correct.

[0267] Determination of the IC50 of clofarabine on engineered T cells relative to WTT cells

[0268] To further investigate the ability of double-KO T-cells to respond to clofarabine, the IC50 of this drug was determined. T-cells were cultured for up to D3 to D8 with and without clofarabine (see [link to study]. Figure 9 In workflow 2), T-cells were cultured for 2 days (from D15 to D17) in media containing different concentrations of clofarabine. T-cell viability was then evaluated using a count bright kit via FACS analysis.

[0269] Figure 15 The results shown indicate that dCK and dCK / TRAC KO T-cells exhibited a significant ability to tolerate clofarabine compared to negative control T cells and TRAC simple KO T-cells. Notably, cell selection was performed using 1 μM over 5 days from D3 to D8 (see [link to study]). Figure 9 Workflow 2) in the process does not improve their tolerance to clofarabine. This indirectly suggests that dCK inactivation is sufficiently effective and that clofarabine-tolerant allogeneic CAR T-cells do not require 5 days of culture for drug selection to obtain clofarabine-tolerant allogeneic CAR T-cells.

[0270] Drug-resistant allogeneic T-CAR T-cell cytotoxicity

[0271] The cytotoxicity analysis was performed as follows: Ten CAR T-cells (FMC63, see reference above) were cultured for 5 hours in DAUDI cells (specific target) and K562 cells (non-specific target). Cells were then collected and the viability of DAUDI and K562 cells was determined by calculating the frequency of targeted cell lysis.

[0272] Figure 16The results shown indicate that dCK / TRAC dual-KO CAR T-cells exhibited similar targeted cytotoxicity (35% targeted cytotoxicity) to WT CAR T-cells. This suggests that inactivation of the dCK and TRAC genes does not affect the cytotoxicity of CAR FMC63T-cells.

[0273] Then, as previously performed, these cells were used to determine their sensitivity to chlorofarabine and fludarabine. Figure 17 The results shown indicate that dCK / TRAC KO CAR T-cells exhibit significantly greater anti-clofarabine activity compared to the CAR T-cell negative control (IC50, IC50, and IC50, respectively). 50 =500 nM and 0.1 nM). Similar results were obtained using fludarabine (IC50 for dual KOCAR T-cells and T CARs, respectively). 50 =400μM and 10μM).

[0274] in conclusion

[0275] In summary, these experiments demonstrate that simultaneous inactivation of the dCK and TRAC genes is highly efficient and allows for the generation of over 70% double-KO T-cells using single-round electroporation. Interestingly, due to this high efficiency, time-consuming selection steps are unnecessary. The engineered T-cells exhibited significant resistance to clofarabine and maintained their maximum survival rate under clinically relevant clofarabine doses.

[0276] Example 3. Production of Daudi cells resistant to clofarabine

[0277] The aim is to prepare drug-resistant CD19 + / Luc + Daudi target cells were used to evaluate the cytotoxicity of allogeneic CAR T-cells resistant to clofarabine.

[0278] dCK KO Daudi genotype characterization

[0279] dCK TALE-nuclease mRNA was prepared and Daudi cells were electroporated with dCK TALE-nuclease mRNA according to the protocol described in WO2013 / 176915.

[0280] As described in Example 1, endonuclease T7 assay was performed to evaluate dCK KO efficiency. Analysis was performed 2 days after transfection. Primers have SEQ IDs N°68 and N°69.

[0281] Figure 18 The results shown indicate high inactivation of the dCK gene.

[0282] Phenotypic characterization of dCK KO Daudi cells

[0283] Daudi cells were cultured for several days in media containing different concentrations of chlorofarabine (0; 0.1; 0.25; 0.5 and 1 μM) and counted at each passage.

[0284] Figure 19 The results shown indicate that dCK KO Daudi cells can grow in the presence of 1 μM clofarabine. Their growth rate is similar to that of WT T-cells in the absence of clofarabine, indirectly indicating that dCK inactivation does not impair Daudi cell growth. As expected, WT Daudi cell growth was significantly impaired. This result confirms the successful production of dCK KO-CD19. + -Luc + -GFP + cell.

[0285] Example 4. Generation and characterization of 6TG-resistant T cells

[0286] To develop T-cells resistant to 6MP and 6TG (HPRT KO T-cells), the HPRT gene is inactivated via TALE-nuclease-mediated inactivation as follows. The complete HPRT gene architecture (exons and introns) and the locations of different TALE-nuclease target sites are shown in [the diagram]. Figure 20 middle.

[0287] TALE-nuclease-mediated inactivation of the HPRT gene

[0288] The workflow for generating and characterizing HPRT single-KO T cells in this experiment is reported in Figure 21 To inactivate the HPRT gene, two pairs of HPRT TALE-nucleases were designed and assembled, and validated by sequencing (for HPRT1: SEQ ID N°74 and SEQ ID N°75; for HPRT2: SEQ ID N°77 and SEQ ID N°78). Details regarding the complete HPRT gene structure (exons and introns) and the location of the TALE-nuclease target sites are as follows: Figure 20 As shown in the figure. The target sequences for HPRT1 and HPRT2TALE-nucleases correspond to SEQ ID N°76 and SEQ ID N°79, respectively.

[0289] Genotypic characterization of HPRT KO T cells

[0290] HPRT KO T cells were genotyped on day 4 by T7 restriction enzyme assay, which showed that the HPRT gene was inactivated in the T cells. The primer pair used in this analysis has SEQ ID N°72 and SEQ ID N°73. Figure 22The results shown indicate that the HPRT TALE-nuclease pair can efficiently process the HPRT gene.

[0291] HPRT KO T-cell growth rate

[0292] according to Figure 23 The results showed that KO HPRT-cells exhibited a growth rate similar to WT T-cells, although slightly lower for the TALE-HPRT2 pair (implemented with 10 μg TALE-HPRT2). However, T cells inactivated by the 10 μg TALE-HPRT2 pair were reactivated on day 10 with the same efficiency as WT T-cells, indicating that HPRT inactivation did not significantly impair T-cell growth. The TALE-HPRT1 pair was selected for the following experiments.

[0293] HPRT KO T-cell selection in the presence of 6TG

[0294] HPRT KO or WT T-cells were allowed to grow from D8 to D13 and then cultured in the presence or absence of 1 μM 6TG until D18. Figure 22 (The workflow is shown in the diagram). Cells were collected on D8 (before drug addition) and D18 (after drug culture) and used for endonuclease T7 analysis. The primer pairs used have sequences SEQ ID N°72 and SEQ ID N°73. Figure 24 The results shown indicate that the presence of 1 μM 6TG in the culture medium allows for the selective enrichment of HPRT KO T-cells (as indicated by the less dense WT bands at D18 in the presence of 6TG).

[0295] HPRT KO CAR T-cell generation

[0296] To investigate the effect of HPRT inactivation on the cytotoxic activity of CAR T-cells, T-cells transduced with the CAR 4G7 lentiviral vector (as described in application filed under PCT / EP2014 / 059662) were electroporated using the TALE-nuclease HPRT1 encoding the mRNA. All experiments described below were performed using engineered T-cells generated without any 6TG selection. The efficiency of HPRT processing was evaluated by endonuclease T7 assay. The primer pairs used for this assay correspond to SEQ ID N°72 and SEQ ID N°73. Figure 25 The results shown indicate that the HPRT gene was successfully inactivated in the presence or absence of CAR 4G7. HPRT inactivation was more complete in T cells than in CAR T cells.

[0297] Cytotoxic properties of HPRT KO CAR-T cells against Daudi cells

[0298] like Figure 27 The cytotoxicity analysis is illustrated schematically. Groups of 10 CAR T cells were cultured for 5 hours using Daudi cells (specific target) and K562 cells (non-specific target). Cells were then collected, and the viability of Daudi and K562 cells was determined to calculate the frequency of targeted cell lysis. Figure 26 The results shown indicate that HPRT KO CAR T cells exhibit targeted cytotoxicity similar to WT CAR T cells. This suggests that HPRT gene inactivation does not affect the cytotoxicity of CAR 4G7T cells.

[0299] IC50 assay of 6TG on engineered T cells relative to WT T cells

[0300] Figure 27 The results shown demonstrate that treatment with the HPRT gene (as previously observed via T7 analysis) effectively inactivates HPRT activity in T-cells. This inactivation confers 6TG tolerance relative to the sensitivity of WT T-cells to this drug. For WT and HPRT KO T-cells, IC50 can be approximated to 10 nM and >100 μM, respectively.

[0301] in conclusion

[0302] In summary, these results demonstrate that HPRT gene inactivation is effective. This inactivation allows T-cells to tolerate high doses of 6TG without requiring a time-consuming purification process. It also indicates that HPRT inactivation can be implemented in CAR T-cells to a slightly lower degree. This inactivation does not impair the cytotoxic properties of CAR T-cells against Daudi cells.

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Claims

1. A method for preparing ex vivo immune T-cells or NK-cells suitable for cancer immunotherapy and resistant to purine analog drugs, wherein the purine analog drug is clofarabine or fludarabine, the method comprising the following steps: (a) Provide immune T-cells or NK-cells; (b) Transfect the immune T-cells or NK-cells with a nucleic acid sequence encoding a rare-cut endonuclease, wherein the rare-cut endonuclease specifically targets and expresses the gene of the enzyme dcK-EC 2.7.1.74 with deoxycytidine kinase activity; (c) Express the nuclease in the immune T-cells or NK-cells to achieve targeted inactivation of the dcK gene; (d) Optionally, in the presence of the purine analog drug, the engineered immune T-cells or NK-cells obtained in step (c) are amplified.

2. The method of claim 1, wherein, The immune T-cells or NK-cells mentioned are native cells.

3. The method according to claim 1, wherein the immune T-cells or NK-cells are CD8+ cells.

4. The method according to any one of claims 1 to 3, wherein the immune T-cells or NK-cells are TIL tumor-infiltrating cells.

5. The method according to any one of claims 1 to 3, wherein the immune T-cells or NK-cells are derived from a patient diagnosed with cancer.

6. The method according to any one of claims 1 to 3, wherein the immune T-cells or NK-cells are derived from a donor.

7. The method of claim 1, wherein the immune T-cell or NK-cell is a T-cell and is further inactivated in the gene encoding TCR α or TCR β such that the T-cell is allogeneic.

8. The method according to any one of claims 1 to 3, wherein the sparse-cut endonuclease is a TALE-nuclease.

9. The method according to claim 8, wherein the TALE-nuclease dCK gene inactivation is performed using the TALE-nuclease of SEQ ID N°63 or SEQ ID N°64, and the dCK target sequence is SEQ ID N°62.

10. The method according to any one of claims 1 to 3, wherein the engineered cells are expanded in vitro.

11. The method of claim 1, further comprising expressing a chimeric antigen receptor in the T-cells or NK-cells.

12. The method of claim 11, wherein the chimeric antigen receptor is CD19+ or CD123+.

13. The method according to any one of claims 1 to 3, further comprising inactivating the immune checkpoint gene.

14. An isolated immune T-cell or NK-cell that can be obtained by the method according to any one of claims 1 to 13, which is resistant to purine analog drugs and has a drug sensitization gene dCK that is inactivated by using a rare endonuclease.

15. An isolated T-cell resistant to a purine analogue, wherein the cell has a drug sensitization gene dCK that is inactivated by using a rare endonuclease, and the cell contains at least one broken gene encoding a T-cell receptor component, wherein the purine analogue is clofarabine or fludarabine.

16. The isolated T-cells according to claim 15, wherein TCR inactivation is performed by a rare endonuclease.

17. The T-cell according to claim 14 or 16, wherein the rare nuclease is a TALE nuclease.

18. An isolated T-cell resistant to a purine analogue, wherein the cell has a drug sensitization gene dCK inactivated by using a rare endonuclease, and the cell is conferred an antigen-specific chimeric antigen receptor CAR, wherein the purine analogue is clofarabine or fludarabine.

19. The isolated T-cells of claim 18, wherein the CAR targets CD19+ cells or CD123+ cells.

20. Use of T-cells according to any one of claims 14 to 19 in the preparation of a medicament for treating cancer, wherein the cancer is acute lymphoblastic leukemia or myeloid leukemia.

21. Use of T-cells according to any one of claims 14 to 19 in the preparation of pharmaceutical preparations for use in combination with purine analog drugs.

22. A pharmaceutical composition comprising at least one T-cell according to any one of claims 14 to 19.

23. Use of a population of T-cells according to any one of claims 14 to 19 in the preparation of a medicament for treating cancer in patients in need of it, wherein the cancer is acute lymphoblastic leukemia or myeloid leukemia.

24. The use according to claim 23, wherein the treatment comprises administering the agent to the patient in combination with administration of a purine analogue drug.

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