Methods for engineering allogeneic and highly active t cell for immunotherapy

Abstract

The present invention relates to methods for developing engineered T-cells for non-alloreactive immunotherapy. The present invention relates to methods for modifying T-cells by inactivating both immune checkpoint genes and genes encoding T-cell receptors to unlock the potential of an immune response. This method involves the use of polynucleotides encoding these polypeptides and specific rare cleavage endonucleases, particularly TALE-nucleases (TAL effector endonucleases), to precisely target the selection of key genes in T-cells available from cultures of primary cells or from donors. The present invention opens the way to standard and available adoptive immunotherapy strategies for treating viral infections and cancer.

Description

Technology Field

[0001] The present invention relates to methods for developing engineered non-alloreactive T-cells for immunotherapy and, more specifically, to methods for modifying T-cells by inactivating both genes encoding at least one immune checkpoint and a T-cell receptor to trigger the potential for an immune response. The method comprises the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonucleases) and polynucleotides encoding these polypeptides, to precisely target a selection of key genes in T-cells, which are available from cultures of primary cells or from donors. The present invention also relates to additional attributes that can be taken into these engineered T cells, such as preTCRα ("pTalpha") and its functional derivatives, Chimeric Antigen Receptor (CAR), multichain CAR, and their use to enhance the efficiency of immunotherapy. The present invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancers and viral infections. Background Technology

[0002] Adoptive immunotherapy involving the migration of autologous antigen-specific T cells generated ex vivo is a promising strategy for treating viral infections and cancers. The T cells used in adoptive immunotherapy can be generated through the redirection of T cells via genetic engineering or the expansion of antigen-specific T cells (Park, Rosenberg et al. 2011). The migration of viral antigen-specific T cells is a well-established procedure used in the treatment of rare virus-associated malignancies and transplants associated with viral infections. Similarly, the migration and isolation of tumor-specific T cells have been shown to be successful in treating melanoma.

[0003] Novel specificities of T cells have been successfully created through the genetic transfer of chimeric antigen receptors (CARs) or transgenic T cell receptors (Jena, Dotti et al. 2010). CARs are synthetic receptors composed of a targeting moiety associated with one or more signaling domains within a single fusion molecule. Generally, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv) containing light-variable fragments of a monoclonal antibody joined by a flexible linker. Binding moietyes based on receptor or ligand domains have also been successfully used. Signaling domains for first-generation CARs are derived from the cytoplasmic regions of Fc receptor gamma chains or CD3 zeta. While first-generation CARs were shown to successfully redirect T-cell cytotoxicity, they failed to provide anti-tumor activity and long-term extension in vivo. Signaling domains from co-stimulatory molecules, including CD28, OX-40 (CD134), and 4-1BB (CD137), were added alone (second generation) or in combination (third generation) to increase the proliferation and enhance the survival of CAR-modified T cells. CARs have successfully allowed T cells to be redirected toward antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0004] Current CAR architectures are based on a design in which all relevant domains are contained within a single polypeptide. This design necessitates the sequential appending of signaling domains, thereby requiring some domains from their natural juxtamembrane positions. Architectures in which ligands and signaling domains are separated in this way can allow them to be located on different chains at their normal juxtamembrane positions rather than appended with some domains located far from the plasma membrane, for improved function of the co-stimulating domains. Natural receptors, which are high-affinity receptors for IgE (FcεRI), would provide such an architecture. FcεRI, present on basophils and mast cells, binds to IgE with high affinity. FcεRI is a tetrameric receptor complex composed of a homodimer of a ligand-binding alpha subunit, a beta subunit, and two transducing gamma subunits (Metzger, Alcaraz et al. 1986). The FcεRI alpha domain consists of an extracellular domain containing two Ig-like domains that bind to IgE, a transmembrane domain, and a short cytoplasmic tail. The beta subunit contains four transmembrane segments separating the amino and carboxyl-terminal cytoplasmic tails.The gamma chain fundamentally contains a cytoplasmic tail and a transmembrane region containing a single immunoreceptor tyrosine-based activation motif (ITAM) (Cambier 1995). The zeta chain of the TCR complex is closely related to the gamma chain and can substitute for the gamma chain of FcεRI (Howard, Rodewald et al. 1990).

[0005] Current protocols for treating patients with adoptive immunotherapy are based on autologous cell transfer. In this approach, T lymphocytes are recovered from patients, selected ex vivo or genetically modified, cultured in vitro if necessary to amplify the number of cells, and finally infused into the patient. In addition to lymphocyte infusion, the host may be treated in other ways to support their participation in the immune response or T cell engraftment, such as the administration of lymphocyte growth factors (like IL-2) and pre-conditioning (with radiation or chemotherapy). Each patient receives individually engineered treatment using their own lymphocytes (i.e., autotherapy). Autologous therapies face significant technical and logistical hurdles before application; their generation requires expensive, dedicated facilities and specialized personnel, and they must occur quickly after a patient's diagnosis. Furthermore, in many cases, pretreatment can lead to reduced immune function, resulting in poorly functioning lymphocytes present in very low numbers. Due to these obstacles, the preparation of each patient's own cells is effectively a novel product, causing significant variations in efficacy and safety. Ideally, standardized therapies would be used in which allogeneic therapeutic cells are pre-manufactured, detailed, and available for immediate administration to patients. By allogeneic, it means that the cells are obtained from genetically different individuals belonging to the same species.However, the use of allogeneic cells currently presents many challenges. In immune-competent hosts, allogeneic cells are rapidly rejected, a process known as host-versus-graft rejection (HvG), which significantly limits the efficacy of the transferred cells. In immune-incompetent hosts, allogeneic cells can be engrafted, but their endogenous TCR specificities cause them to recognize the host tissue as foreign, leading to graft-versus-host disease (GvHD), which can result in severe tissue damage and death. Both of these problems must be overcome to effectively utilize allogeneic cells.

[0006] In immunocompetent hosts, allogeneic cells are rapidly rejected by the host immune system. It has been claimed that allogeneic leukocytes present in unirradiated blood products persist for five to six days or less (Boni, Muranski et al. 2008). To prevent this rejection of allogeneic cells, the host immune system must be effectively suppressed. Glucocorticoid steroids are widely used therapeutically for immunosuppression (Coutinho and Chapman 2011). Among steroid hormones, this type binds to glucocorticoid receptors (GRs) present in the cytosol of T cells, causing the binding of specific DNA motifs that regulate the expression of numerous genes involved in nuclear localization and immunological processes. Treating T cells with glucocorticoid steroids interferes with T cell activation and causes reduced levels of cytokine production, leading to T cell anergy. Alemtuzumab, also known as CAMPATH1-H, is a humanized monoclonal antibody that targets CD52, a 12-amino acid glycosylphosphatidyl-inositol-(GPI) linked glycoprotein (Waldmann and Hale 2005). While CD52 is absent on granulocytes and myeloid precursors, it is expressed at high levels on T and B lymphocytes and at lower levels on monocytes.Treatment with alemtuzumab, a humanized monoclonal antibody targeting CD52, has been shown to induce rapid depletion of circulating lymphocytes and monocytes. It is frequently used in certain cases as part of a conditioning regimen for transplantation and in the treatment of T-cell lymphomas. However, in cases of adoptive immunotherapy, the use of immunosuppressants will also have a detrimental effect on the introduced therapeutic T cells. Therefore, to effectively utilize the adoptive immunotherapy approach in these diseases, the introduced cells will need to be resistant to immunosuppressive therapy.

[0007] In contrast, T cell receptors (TCRs) are cell surface receptors that participate in the activation of T cells in response to antigen presentation. TCRs are typically constructed from two chains, alpha and beta, which assemble to form a heterodimer and associate with CD3-transducing subunits to form a T-cell receptor complex on the cell surface. Each of the TCR's alpha and beta chains consists of immunoglobulin-like N-terminal variable (V) and constant (C) regions, a hydrophobic transmembrane domain, and a short cytoplasmic region. For immunoglobulin molecules, the variable regions of the alpha and beta chains are formed by V(D)J recombination, which creates significant variability in antigen specificity within the T cell population. However, in contrast to immunoglobulins that recognize intact antigens, T cells are activated by processed peptide fragments associated with MHC molecules, known as MHC restriction, which introduce an extra dimension to antigen recognition by T cells. The recognition of MHC differences between donors and recipients via T cell receptors leads to the potential development of GVHD and T cell proliferation. It has been shown that normal surface expression of the TCR depends on the assembly and coordinated synthesis of all seven components of the complex (Ashwell and Klusner 1990). Inactivation of TCR alpha or TCR beta can lead to the removal of the TCR from the surface of T cells, which prevents the recognition of alloantigens, and thus cause GVHD.However, TCR disruption causes the removal of CD3 signaling components and alters the means of further T cell expansion.

[0008] T-cell-mediated immunity involves multiple sequential steps regulated by a balance between co-stimulatory and inhibitory signals that fine-tune the immune response. Inhibitory signals, referred to as immune checkpoints, are critical for limiting immune-mediated collateral tissue damage and for auto-tolerance. The expression of immune checkpoint proteins can be deregulated by tumors. The ability of tumors to co-opt these inhibitory pathways represents a critical mechanism of immune resistance and limits the success of immunotherapy. One promising approach to activating a therapeutic T-cell immune response is the blockade of these immune checkpoints (Pardoll 2012). Immune checkpoints represent important barriers to the activation of functional cellular immunity in cancer, and antagonistic antibodies specific to T cell-onset inhibitory ligands, including programmed death-1 (PD-1) and CTLA4, are examples of targeted agents being evaluated in clinical practice.

[0009] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4; also known as CD152) downregulates the amplitude of T-cell activation, and antagonist CTLA4 antibodies (ipilimumab) have shown survival benefits in patients with melanoma (Robert and Mateus 2011). Programmed cell death protein 1 (PDCD1, also known as PD1 or CD279) represents another very promising target for immunotherapy (Pardoll and Drake 2012; Pardoll 2012). In contrast to CTLA-4, PD1 limits T-cell effector function in peripheral tissues to suppress autoimmunity and during inflammatory responses to infection. The first clinical trial with PD1 antibodies showed several cases of tumor regression (Brahmer, Drake et al. 2010). Multiple additional immune checkpoint proteins represent promising targets for therapeutic blockade based on recent studies.

[0010] In normal T cells, T cell receptors are expressed by immature thymocytes and originate from pre-T cell receptors (pTCRs), which are critical for T cell development from double-negative (CD4-CD8-) to double-positive (CD4+CD8+) stages. Pre-T cells that have successfully undergone productive rearrangement of the TCR beta locus express functional TCR beta, which pairs with CD3 signaling elements and an invariant preTalpha chain to form the pre-TCR complex. The expression of preTCRs on the cell surface is necessary to induce the expansion of developing T cells, to enforce allelic exclusion at the TCR-beta site, and to trigger beta-selection, a process that causes the induction of rearrangements at the TCR-alpha site (von Boehmer 2005). After productive TCR-alpha rearrangements and the substitution of pT-alpha by TCR-alpha to form a mature TCR, thymocytes undergo a second stage of selection called TCR-alpha / beta selection or positive when self-peptide MHC complexes expressed on the thymic epithelial cells bind. Thus, mature T cells recognize and respond to antigen / MHC complexes through their TCRs. The most immediate consequence of TCR activation is the initiation of signaling pathways via associated CD3 subunits, which lead to a number of events including T cell clonal expansion, upregulation of cell surface activation markers, and the induction of cytotoxicity or cytokine secretion.

[0011] Through mating with preTalpha during thymic development Due to the selectivity of TCR beta chains, the heterologous introduction of a pTalpha transgene in T cells in which TCR alpha is inactivated can lead to the formation of a pre-TCR. This pTCR can serve as a means of stimulation or T cell activation in a non-MHC-dependent manner, thereby allowing, for example, the sustained expansion of alpha / beta T-cells following TCR alpha inactivation. Importantly, the pTCR complex exhibits a biochemical composition similar to that of a TCR in terms of associated CD3 subunits (Carrasco, Ramiro et al. 2001). Furthermore, in contrast to TCRs, pre-TCR signaling can occur to some extent through ligand-independent events. The crystal structure of the pTCR extracellular domain provided a structural basis for the possible ligand-independence of pTCR signaling. pTCR has been shown to form a head-to-tail dimer involving two pT-alpha-TCR-beta heterodimers (Pang, Berry et al. 2010).

[0012] In the present invention, the inventors have achieved the production of genetically modified T-cells that overcome the limitations of current immunotherapy strategies and allow them to be non-alloreactive and highly active. Although the blockade of immune checkpoints has been recognized using antibodies, another method of achieving suppression is by inactivating the expression of immune checkpoint genes within T cells, which ideally enables the production of engineered allogeneic T cells as "off-the-shelf" products. This was made possible by gene inactivation using specific TALE-nucleases directed toward TCR-beta or TCR-alpha, coupled with the inactivation of genes encoding immune checkpoint proteins such as PD1 and CTLA-4.

[0013] In particular, the inactivation of TCR alpha or TCR beta, coupled with the inactivation of immune checkpoint genes within T lymphocytes derived from allogeneic donors, enables the activation and proliferation of the introduced lymphocytes, while significantly reducing the risk of GVHD by eliminating the TCR responsible for the recognition of MHC differences. Therefore, these modified allogeneic T cells are expected to be highly active in the patient's blood, capable of targeting tumor cells or infected cells.

[0014] In addition to the above concept of genetically modified T cells, both of which are non-alloreactive and can be highly active, the inventors caused and inactivated these other genes within the T cells by designing and using specific TALE-nucleases, thereby obtaining double mutations. In fact, double gene targeting by DSBs has not been achieved in T cells until now due to the difficulty of maintaining and producing T cells in culture over time, their low transformation rates, and losses during selection procedures. These difficulties resulted in a low probability of success in obtaining these cells.

[0015] Therefore, one important part of the present invention is to have specific TALE-nucleases designed to enable higher rates of DSB events in T-cells that are well tolerated by cells (particularly after co-transfection), which can target a selection of genes according to the present invention. By using rare-cutting endonucleases such as the TALE-nucleases described herein, the probability of obtaining double inactivation of genes in transfected T-cells is significantly increased, so it appears that engineered T-cells available from donors can now be produced regularly using standard procedures.

[0016] Furthermore, the present invention proposes an example in which T-cells are manipulated to enable proliferation when TCR alpha is inactivated. A significant problem with T-cells that have undergone TCR subunit inactivation is that the cells can no longer expand through the CD3 complex. To overcome this problem, the inventors provide a means for expanding T-cells in which TCR alpha is inactivated through the CD3 complex by the expression of preTalpha within the cells, and the functional CD3 complex is restored in the absence of the functional alpha / beta TCR.

[0017] Finally, T cells are further transformed into CARs to redirect allogeneic cells specifically toward tumors associated with independent MHC antigens. In particular, the present invention relates to a multi-chain CAR in which co-stimulating domains are located at their juxtamembrane sites to enhance their functions, thereby increasing the proliferation and enhancing the survival of engineered T-cells. As a result, the present invention provides polynucleotides, polypeptides, and methods that enable the effective transformation of allogeneic T cells for adoptive immunotherapy, and an easy extension thereof. The problem to be solved

[0018] The present invention relates to methods for developing engineered T-cells for non-alloreactive immunotherapy. means of solving the problem

[0019] The present invention relates to methods for developing engineered T-cells for non-alloreactive immunotherapy. The present invention relates to methods for modifying T-cells by inactivating both immune checkpoint genes and genes encoding T-cell receptors to unlock the potential of an immune response. The method involves the use of polynucleotides encoding these polypeptides and specific rare cleavage endonucleases, in particular TALE-nucleases (TAL effector endonucleases), to precisely target the selection of key genes in T-cells available from cultures of primary cells or from donors. Effects of the invention

[0020] The present invention paves the way for standard and available adoptive immunotherapy strategies for treating viral infections and cancer. Brief explanation of the drawing

[0021] Brief description of drawings and tables In addition to the foregoing features, the present invention further comprises other features derived from the accompanying drawings and the following description. A more complete evaluation of the present invention and its accompanying many benefits will be readily obtained, as the same can be better understood by referring to the following drawings linked to the detailed description. Fig. 1: Schematic illustration of the normal relationship between T-cells and antigen-presenting cells. FIG. 2: Schematic representation of the patient's tumor cells and genetically modified therapeutic T-cells according to the present invention. Fig. 3: Schematic representation of a multi-chain CAR. FIG. 4: Schematics of different versions of multi-chain CARs. A. Schematics of an FcεRI receptor. Different versions of multi-chain CARs (csm1 to csm10) comprising a CD8 Stork domain and scFv fused to a transmembrane domain of a BC FcεRI alpha chain. At least one 41BB, CD28 and / or CD3 zeta domain may be fused to the FcεRI alpha, beta and / or gamma chain. Fig. 5: Schematic illustration of an example of a method for manipulating human allogenic cells for immunotherapy. Fig. 6: Concentration in cells per milliliter of living CD52-positive or CD52-negative cells after treatment with the anti-CD52 antibody (CAMPATH1-H) as a control or complement. Fig. 7: Comparison of cell size indications and forward-side scatter (FSC) distribution between inactivated cells as a control and between TCR-positive and TCR-negative cells, or between CD52-positive and CD52-negative cells. Fig. 8: Flow cytometry analysis of CD107a expression (marker of degranulation) on targeted CD52 and TCR-alpha inactivated T cells. CD107 expression was analyzed on CD52+TCRαβ+ cells (first column), CD52-TCRαβ- cells (second column), CD52-TCRαβ+ cells (third column), and CD52+TCRαβ- cells (fourth column) before (A) and after (B) culture with Daudi cells. C) shows the flow cytometry analysis of T cells further transfected with CAR and inactivated with Daudi cells; D) shows the flow cytometry analysis of T cells transfected with CAR but not cultured with Daudi cells, and E) shows the flow cytometry analysis of T cells transfected with CAR and treated with PMA / ionomycin (positive control). Fig. 9: Deep sequencing analysis of potential off-site targets of CD52 and TRAC TALE nucleases. Fig. 10: Analysis of PDCD1 and CTLA-4 genomic loci by T7-endonuclease analysis. Arrows indicate digested PCR products. Fig. 11: Schematic representation of some examples of preTalpha structures. Fig. 12: Flow cytometry analysis of the activity (% CD3 surface expression) and transduction efficiency (% BFP+ cells) of FL, △18, and △48 pTalpha constructs in TCR alpha-inactivated Jurkat cells. Fig. 13: Schematic representation of the pTalpha protein (a lentiviral construct encoding preTCRα). Fig. 14: A: Indication of the experimental protocol. B: Flow cytometry analysis of TCR alpha / beta, CD3 expression, and BFP expression on TCR alpha-inactivated T cells (KO) transduced with the control BFP lentiviral vector (KO / BFP) or BFP-2A-pTalpha△48 (KO / △48) before and after purification. C: Flow cytometry analysis of TCR alpha / beta and CD3 expression on purified TCR alpha-inactivated cells transduced with the BFP-2A-pTalpha△48 lentiviral vector (BFPpos) or not transduced (BFPneg). NEP indicates cells not electroporated with TRAC TALE nucleases. Fig. 15: AB. Flow cytometry analysis of early activation marker CD69 (A) and late activation marker CD25 (B) expression 24 and 48 hours after reactivation with anti-CD3 / CD28 beads, respectively, on non-electroporated cells (NEP) and TCR-alpha-inactivated cells (KO) transfected with the control BFP vector (BFP) or BFP-2A-pTα-△48 lentiviral vector (pTα-△48) and BFP-2A-pTα-△48.41BB lentiviral vector (pTα-△48.BB). The pTα-△48 bar graphs correspond to signals detected in TCR-inactivated cells expressing pTα-△48 (BFP+ cells), while the KO bar graphs correspond to TCR-alpha-inactivated cells that do not express pTα-△48 (BFP- cells), and the pTα-△48.BB bar graphs correspond to signals detected in TCR-inactivated cells expressing pTα-△48.41BB (BFP+ cells), while the KO bar graphs correspond to TCR-alpha-inactivated cells that do not express pTα-△48.41BB (BFP- cells). The NEP (non-electroporated) bar graphs correspond to signals detected in unmanipulated cells. C. Flow cytometry analysis of cell size 72 hours after reactivation with anti-CD3 / CD28 beads in TCR-alpha-inactivated (KO) and non-electroporated (NEP) cells transduced with the control BFP vector (BFP) or BFP-2A-pTα-△48 lentiviral vector (pTα-△48) and BFP-2A-pTα-△48.41BB lentiviral vector (pTα-△48.BB).The values ​​displayed at the top of each graph correspond to the geometric mean of the fluorescence of each population. Fig. 16: Cell growth analysis of TCR alpha-inactivated cells (KO) transduced with anti-CD3 / CD28 beads at different time points (x-axis) in IL2 or with a control BFP vector (BFP) or pTalpha-△48 (pTa△48) maintained in IL2. The number of BFP+ cells was estimated at different time points for each condition, and the fold induction of these cells was estimated for values ​​obtained 2 days after reactivation. Results were obtained from two independent donors. For the second donor, cell growth was also determined for cells transduced with full-length pTalpha- (pTa-FL) and pTalpha-△48.41BB (pTa-△48.BB). Fig. 17: Flow cytometry analysis of GFP-positive cells on PBMCs electroporated with five different Cytopulse programs. The top line is 6x10 per cuvette. 6 It corresponds to the transfection of cells, whereas the bottom line is 3x10 per cuvette 6 It corresponds to the transfection of cells. Fig. 18: GFP-positive cells from a population alive after electroporation with control pUC DNA and GFP mRNA, GFP DNA, and cells purified using the viability dye (eFluor-450). This is a flow cytometry analysis of T cell mortality. NEP corresponds to cells maintained in the electroporation buffer but not electroporated, and NT corresponds to cells maintained in the culture medium that are not electroporated. Fig. 19: Flow cytometry analysis of TCR alpha / beta and CD3 expression in human primary T cells after TRAC TALE-nuclease mRNA electroporation (top). Deep sequencing analysis of genomic DNA extracted from human primary T cells after TRAC TALE-nuclease mRNA electroporation (bottom). Fig. 20: A. Flow cytometry analysis of CAR expression (anti-F(ab')2) in T cells after electroporation with or without mRNA encoding a single-stranded CAR. B. Flow cytometry analysis of CD107a expression (marker of degranulation) in electroporated T cells co-cultured with daudi cells. Fig. 21: A. Labeling of mRNA encoding a multi-chain CAR. B. Flow cytometry analysis of CAR expression (anti-F(ab')2) on electroporated viable T cells with or without polycystron mRNA encoding a multi-chain CAR. C. Flow cytometry analysis of CD107a expression (marker of degranulation) on electroporated T cells co-cultured with daudi cells. Fig. 22: Expression of multi-chain CARs in human T cells after electroporation of polycistron mRNAs. Fig. 23: The expression of multi-subunit CARs is conditioned by the expression of three chains: α, β, and γ. Fig. 24: Human T cells transiently expressing multi-chain CARs are degranulated after co-culture with target cells. A: csm1 to csm5 CAR constructs. B: csm6 to csm10 CAR constructs. Fig. 25: Human T cells transiently expressing multi-chain CARs silverSecretes cytokines after co-culture with target cells (T cells vs. Daudi cells or K562). A: IL8 release. B: IFNγ release. C: IL5 release. Fig. 26: Human T cells transiently expressing multi-chain CARs (scm1 to csm10 structures) lyse target cells. Fig. 27: CTLA4 inactivation in primary T cells measured by intracellular staining using fluorescent antibodies and flow cytometry analysis. Fig. 28: Distribution of fluorescent T-cells expressing CTLA4 transfected with TALENs T1, T2, and T3. The proportion of cells expressing CTLA4 is dramatically reduced compared to control cells. Fig. 29: PD1 inactivation in primary T cells measured by intracellular staining using fluorescent antibodies and flow cytometry analysis. The proportion of cells expressing PD1 is dramatically reduced compared to control cells. Fig. 30: Illustration showing the frequencies of deletions observed in T-cells transfected with TALEN T01 and T03 targeting the PD1 gene. Fig. 31: Illustration showing that cytotoxic activity is enhanced in T-cells degraded for PD1 according to the experiment described in Example 3. Table 1: List of immune checkpoint genes identified by the inventors as suitable for producing allogeneic T-cells that are more active in immunotherapy. Table 2: Sequences of TALE-nuclease target sites within the human GR gene and description of GR TALE-nuclease. Table 3: Cleavage activity of GR TALE-nucleases in yeast. Values ​​range between 0 and 1. The maximum value is 1. Table 4: Percentage of targeted mutagenesis at endogenous TALE-nuclease target sites in 293 cells. Table 5: Percentage of targeted mutagenesis at endogenous TALE-nuclease target sites in primary T lymphocytes. Table 6: Description of CD52, TRAC, and TRBC TALE-nucleases and their sequences at TALE-nuclease target sites in corresponding human genes. Table 7: Additional target sequences for TRAC and CD52 TALE-nucleases. Table 8: Percentages of insertions and deletions for TALE nucleases targeting CD52_T02, TRAC_T01, TRBC_T01, and TRBC_T02 targets. Table 9: Percentages of CD52-negative, TCR-negative, and CD52 / TCR-double-negative T lymphocytes after transfection with corresponding TALE-nuclease-expressing polynucleotides. Table 10: Percentage of TCR-negative T lymphocytes after transfection with TRBC TALE-nuclease-expressing polynucleotides. Table 11: Description of CTLA4 and PDCD1 TALE-nucleases and sequences at TALE-nuclease target sites within corresponding human genes. Table 12: Description of a subset of pTalpha structures. Table 13: Activity of other pTalpha constructs in Jurkat TCR alpha-inactivated cells. Activity was measured by flow cytometry analysis of CD3 expression on Jurkat TCR alpha-inactivated cells transfected with other preTalpha constructs. Table 14: Different cytopulse programs used to determine the minimum voltage required for electroporation in PBMC-derived T-cells. Table 15: Cytopulse program used to electroporate purified T-cells. Specific details for implementing the invention

[0022] Overview of the present invention

[0023] In one aspect, the present invention discloses methods for manipulating T cells, particularly allogeneic T cells obtainable from donors, in order to make them suitable for immunotherapy purposes. More specifically, the methods of the present invention enable precise modification of the genome of cells relevant to immunotherapy by replacing or inactivating immune checkpoint proteins and / or genes related to MHC recognition. In certain examples, the modified cells relevant to immunotherapy further comprise exogenous recombinant polynucleotides encoding CARs for specific cell recognition. Current CARs are signal fusion molecules that require the sequential addition of signaling domains. Moving signaling domains from their natural juxtamembrane locations can interfere with their function. Therefore, to overcome this problem, the inventors design a multi-chain CAR derived from FcεRI to enable the normal juxtamembrane location of all relevant signaling domains. The high-affinity IgE binding domain of the FcεRI alpha chain is replaced by an extracellular ligand-binding domain such as scFv to redirect T-cell specificity to cellular targets, and the N and / or C-terminal tails of the FcεRI beta chain are used to localize co-stimulatory signals to normal juxtamembrane locations.

[0024] In another aspect, to promote the activation or stimulation of T cells in which TCR-alpha is inactivated, pT-alpha or its functional variant is introduced into engineered T-cells. The pT-alpha or its functional variant used may be full-length pT-alpha, a splice variant (Saint-Ruf, Lechner et al. 1998), or a C-terminal truncated version that has been shown to increase pre-TCR cell surface expression (Carrasco, Ramiro et al. 2001). Other additional truncations smaller or larger than those described may be used. Other preTalpha versions may additionally include signaling moiety from other molecules (CD28, CD137, CD8, TCRalpha, etc.) to promote proliferation, or may include mutations affecting the ability to dimerize, such as the W46R mutation described in humans (Pang, Berry et al. 2010) or the D22A, R24A, R102A, or R117A mutations previously described in mice (Yamasaki, Ishikawa et al. 2006), to reduce proliferation potential. A portion of the scFv portion of the CAR may also be fused to the extracellular domain of pTalpha or its functional variant, thereby directly linking specificity to target antigens to the proliferation activity of preTCR.

[0025] In another aspect, the present invention relates to polypeptides and polynucleotides encoding rare-cleavage endonucleases that enable genetic modification of T-cells for immunotherapy by precisely targeting the said genes of interest, particularly TCR alpha, TCR beta, and immune checkpoint genes. The present invention further provides TALE-nucleases designed to target each of these genes and specific target sequences within these genes.

[0026] The present invention also relates to isolated cells or cell lines comprising any of the proteins, polypeptides, or vectors described herein. In certain examples, the T cells of the present invention comprise TCR alpha, TCR beta, and immune checkpoint genes inactivated for their use in immunotherapy. The isolated cells or cell lines of the present invention may further comprise exogenous recombinant polynucleotides, in particular pT alpha or its functional variant CARs or multi-chain CARs.

[0027] In a desirable example, modified T cells are used as a therapeutic product, ideally as an "off-the-shelf" product.

[0028] In another aspect, the present invention relates to a method for preventing or treating cancer or infections in a patient by administering engineered T-cells obtainable by the above methods.

[0029] Detailed description of the present invention

[0030] Unless otherwise specifically 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.

[0031] All materials and methods similar to or equivalent to those described herein may be used to test or practice the invention in conjunction with the suitable materials and methods described herein. All documents, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the definitions provided herein shall apply. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting unless otherwise specified.

[0032] Practice of the present invention will utilize general techniques within the art, cell biology, cell culture, molecular biology, gene transfer biology, microbiology, recombinant DNA, and immunology, unless otherwise indicated. These techniques are fully described in the literature. 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 Gait ed., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harries & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol.185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JH Miller and M.P. Calos 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 (DM Weir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0033] In a general sense, the present invention relates to methods for novel adoptive immunotherapy strategies for treating cancers and infections.

[0034] Allergic and highly active T cells for immunotherapy

[0035] In a specific aspect, the present invention relates to a method of manipulating T-cells, in particular to immunotherapy.

[0036] In certain examples, the invention includes the following:

[0037] (a) Step of providing T cells,

[0038] (b) introducing a rare-cleavage endonuclease capable of selectively inactivating immune checkpoint genes by DNA cleavage into the T cells; and

[0039] (c) A step of expanding the above cells.

[0040] In particular, this method includes the following:

[0041] (a) A step of modifying T-cells by inactivating at least one:

[0042] - The first gene encoding the immune checkpoint protein, and

[0043] - The second gene encoding a component of the T-cell receptor (TCR)

[0044] (b) A step of expanding the above cells.

[0045] T cell-mediated immunity involves multiple sequential steps, including the clonal selection of antigen-specific cells, their activation and proliferation in secondary lymphoid tissues, their trafficking to sites of inflammation and antigens, the performance of direct effector functions, and the presentation of assistance (via cytokines and membrane ligands) to many effector immune cells. Each of these steps is regulated by counterbalancing stimulating and inhibitory signals that fine-tune the response. Those skilled in the art will understand that the term "immune checkpoints" refers to a group of molecules expressed by T cells. These molecules effectively act as "brakes" to suppress or down-modulate the immune response. Immune checkpoint molecules are Programmed Death 1 (also known as PD-1, PDCD1, or CD279, accession number: NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, GenBank accession number AF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM_181780.3), BY55 (also known as CD160, GenBank accession number: CR541888.1), TIGIT (also known as VSTM3, accession number: NM_173799), and B7H5 (also known as C10orf54, mouse vista). Homolog of the gene, registry number: NM_022153.1), LAIR1 (also known as CD305, GenBank registry number: CR542051).1), SIGLEC10 (GeneBank registration number: AY358337.1), 2B4 (also known as CD244, registration number: NM_001166664.1), and directly inhibit immune cells, including but not limited to these methods. For example, CTLA-4 is a cell-surface protein expressed on certain CD4 and CD8 T cells; when engaged with its ligands (B7-1 and B7-2) on antigen-presenting cells, T-cell activation and effector functions are inhibited. Therefore, the present invention relates to a method of manipulating T-cells, particularly immunotherapy, comprising the step of genetically modifying T-cells by inactivating at least one protein associated with immune checkpoints, in particular PD1 and / or CTLA-4.

[0046] In a specific example, the genetic modification step of the method relies on the inactivation of one gene, preferably two genes selected from the group consisting of PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCR alpha, and TCR beta. In another example, the genetic modification step of the method relies on the inactivation of two genes selected from the group consisting of PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, and 2B4 and TCR beta. In another example, the genetic modification step of the method relies on the inactivation of more than two genes. The genetic modification preferably operates ex-vivo.

[0047] Table 1 below shows immune checkpoint genes that can be inactivated according to the teachings of the present invention to improve the suitability and efficiency of engineered T-cells without exhaustion. The immune checkpoint genes are preferably selected from genes having identity with those listed in this table, which are associated with co-inhibitor receptor function, cell death, cytokine signaling, arginine tryptophan starvation, TCR signaling, induced T-reg repression, transcription factors controlling anergy or exhaustion, and hypoxia-mediated tolerance.

[0048]

[0049]

[0050]

[0051] Table 1: Immune checkpoint genes suitable for producing allogeneic T-cells more active in immunotherapy

[0052] By inactivating a gene, it is intended that the gene of interest is not expressed in the form of a functional protein. In a specific example, the genetic modification of the method relies on the expression of a rare-cleavage endonuclease in the provided cells to be manipulated, so that the rare-cleavage endonuclease specifically catalyzes a cleavage within a single target gene, thereby inactivating the target gene. Nucleic acid strand breaks caused by the rare-cleavage endonuclease are usually repaired through distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an incomplete repair process that often causes changes in the DNA sequence at the cleavage site. Mechanisms involve the rejoining of the remaining two DNA ends, either through direct ligation (Critchlow and Jackson 1998) or through so-called microhomology-mediated end joining (Ma, Kim et al. 2003). Repairs via non-homologous end joining (NHEJ) sometimes result in small insertions or deletions and can be used to create specific gene knockouts. Such modifications may be the substitution, deletion, or addition of at least one nucleotide. Cells that have undergone a cleavage-induced mutagenesis event, i.e., a mutagenesis event following an NHEJ event, can be selected and / or identified by methods well known in the art.

[0053] In a specific example, the above method for manipulating (engineer) cells includes at least one of the following steps:

[0054] (a) a step of providing T-cells, preferably from a cell culture or a blood sample;

[0055] (b) introducing into the T-cell a rare-cleavage endonuclease capable of selectively inactivating each of the following by DNA cleavage, preferably double-strand break;

[0056] - The above gene encoding an immune checkpoint protein, and

[0057] - At least one gene encoding a component of the T-cell receptor (TCR).

[0058] (c) A step of expanding the above cells.

[0059] In a more preferred example, the above method comprises the following:

[0060] (a) a step of providing T-cells, preferably from a cell culture or a blood sample;

[0061] (b) transforming the T cells with a nucleic acid encoding a rare-cleavage endonuclease capable of selectively inactivating each of the following by DNA cleavage, preferably by double-strand break:

[0062] - The above gene encoding the immune checkpoint protein and

[0063] - At least one gene encoding a component of the T-cell receptor (TCR)

[0064] (c) a step of expressing the rare-cleavage endonucleases into the T-cells;

[0065] (d) a step of sorting transformed T-cells that do not express TCR on their cell surface;

[0066] (e) A step of expanding the above cells.

[0067] In a specific example, the rare-cleavage endonuclease specifically targets one gene selected from the group consisting of: PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCR alpha, and TCR beta. In another example, the genetic modification of the present method depends on the expression of two rare-cleavage endonucleases in the provided cells to be manipulated, wherein each of the two rare-cleavage endonucleases specifically and pairs of genes selected from the group consisting of PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, and 2B4 and TCR beta Each catalyzes cleavage, thereby inactivating the targeted genes. In another example, more than two rare-cleavage endonucleases may be expressed in cells to engineer them to inactivate and / or target more than two genes.

[0068] In another example, the rare-cleavage endonuclease may be a meganuclease, a zinc finger nuclease, or a TALE-nuclease. In a preferred example, the rare-cleavage endonuclease is a TALE-nuclease. A fusion protein is intended to consist of a DNA-binding domain derived from a Transcription Activator-Like Effector (TALE) by TALE-nuclease and a single nuclease catalytic domain for cleaving a nucleic acid target sequence (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Cermak, Doyle et al. 2011; Geissler, Scholze et al. 2011; Huang, Xiao et al. 2011; Li, Huang et al. 2011; Mahfouz, Li et al. 2011; Miller, Tan et al. 2011; Morbitzer, Romer et al. 2011; Mussolino, Morbitzer et al. 2011; Sander, Cade et al. 2011; Tesson, Usal et al. 2011; Weber, Gruetzner et al. 2011; Zhang, Cong et al. 2011; Deng, Yan et al. 2012; Li, Piatek et al. 2012; Mahfouz, Li et al. 2012; Mak, Bradley et al. 2012).

[0069] In the present invention, novel TALE-nucleases are designed to accurately target relevant genes for adoptive immunotherapy strategies. Preferred TALE-nucleases according to the present invention are those that recognize and cleave target sequences selected from the group consisting of: SEQ ID NO: 77 and SEQ ID NO: 78 (PD1), SEQ ID NO: 74 to SEQ ID NO: 76 (CTLA-4), SEQ ID NO: 37, 57 to 60 (TCR alpha), SEQ ID NO: 38 or 39 (TCR beta). The present invention also relates to TALE-nuclease polypeptides comprising amino acid sequences selected from the group consisting of SEQ ID NO: 79 to SEQ ID NO: 88 and SEQ ID NO: 41 to 46.

[0070] The present invention also relates to polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 79 to 88 and an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity. The scope of the present invention also includes polynucleotides and vectors encoding the rare-cleavage endonucleases described above according to the present invention. This method may be associated with any one of the other methods described in this disclosure.

[0071] In another example, additional catalytic domains may be further introduced into the cell to enhance mutagenesis, thereby increasing their ability to inactivate target genes. In particular, the additional catalytic domains are DNA end-processing enzymes. Non-limited examples of DNA end-processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' alkaline exonucleases, 5' flap endonucleases, helicases, hosphatases, hydrolases, and template-independent DNA polymerases. Non-limited 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), E. 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 example, the additional catalytic domain has 3'-5'-exonuclease activity, and in a more preferred example, the additional catalytic domain is TREX, and more preferably, TREX2 catalytic domain (WO2012 / 058458). In another preferred example, the catalyst domain is encoded by a single-chain TREX polypeptide (WO2013 / 009525).The above additional catalyst domain can be selectively fused to a nuclease fusion protein or chimeric protein according to the present invention by a peptide linker.

[0072] Endonucleolytic breaks are known to stimulate the rate of homologous recombination. Therefore, in another example, the genetic modification step of the present method further comprises the step of introducing an exogenous nucleic acid into cells, wherein the exogenous nucleic acid comprises at least a homologous sequence to a portion of the target nucleic acid sequence such that homologous recombination occurs between the nucleic acid sequence and the exogenous nucleic acid. In certain examples, the exogenous nucleic acid comprises first and second portions homologous to the 5' and 3' regions of the target nucleic acid sequence, respectively. In these examples, the exogenous 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. After cleavage of the target nucleic acid sequence, a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid. Preferably, at least 50 bp, preferably more than 100 bp, and more preferably more than 200 bp, homologous sequences are used within the donor matrix. Therefore, the exogenous nucleic acid is preferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. Indeed, the shared nucleic acid homologies are located in regions on the downstream and upstream sides of the break site, and the nucleic acid sequence to be introduced must be located between the two arms.

[0073] In particular, the exogenous nucleic acid comprises, in succession, a first region homologous to the upstream sequences of the cleavage, a sequence for inactivating one targeted gene selected from the group consisting of immune checkpoint genes, TCR alpha, and TCR beta, and a second region homologous to the downstream sequences of the cleavage. The polynucleotide introduction step may occur simultaneously with, before, or after the expression or introduction of the rare-cleavage endonuclease. Depending on the location of the target nucleic acid sequence where the break event occurs, such exogenous nucleic acid may be used to knock out a gene, for example, when the exogenous nucleic acid is located within the open reading frame of the gene, or to introduce genes of interest or novel sequences. Sequence insertions using such exogenous nucleic acids may be used to modify an existing target gene by replacing or correcting the said gene (an allele swap, not limited to an example), or to upregulate or downregulate the expression of the target gene by correcting or replacing the said target gene (an promoter swap, not limited to an example).In a preferred example, the inactivation of genes from the group consisting of immune checkpoint genes, TCR alpha and TCR beta, may be performed at a precise genomic location targeted by a specific TALE-nuclease, wherein the specific TALE-nuclease catalyzes cleavage and the exogenous nucleic acid comprises a sequence for inactivating one targeted gene selected from the group consisting of immune checkpoint genes, TCR alpha and TCR beta, which is incorporated by at least one homology region and homologous recombination. In another example, several genes may be inactivated, either sequentially or simultaneously, by several TALE-nucleases, and several specific polynucleotides and one defined gene may be specifically targeted for specific gene inactivation.

[0074] By an additional genome modification step, the inactivation of another gene selected from the group consisting of immune checkpoint genes, TCR alpha, and TCR beta may also be intended. As previously mentioned, the additional genome modification step may be an inactivation step comprising the following:

[0075] (a) introducing at least one rare-cleavage endonuclease into the cells, wherein the rare-cleavage endonuclease specifically catalyzes a cleavage within one targeted sequence of the genome of the cells.

[0076] (b) selectively introducing an exogenous nucleic acid into the cells, wherein the exogenous nucleic acid comprises, in succession, a first region of homology to the upstream sequences of the cleavage, a sequence inserted into the genome of the cell, and a second region of homology to the downstream sequences of the cleavage.

[0077] In this case, the introduced exogenous nucleic acid inactivates the gene and integrates at least one exogenous polynucleotide sequence encoding at least one recombinant protein of interest. In another example, the exogenous polynucleotide sequence is integrated into a gene selected from the group consisting of immune checkpoint genes, TCR alpha and TCR beta.

[0078] In certain examples, the method of manipulating cells further comprises an additional genome modification step. By the additional genome modification step, one protein of interest may be intended to be introduced into the cells to be manipulated. The protein of interest may be, as non-limiting examples, pTalpha or its functional variant, Chimeric Antigen Receptor (CAR), multi-chain CAR, or bispecific antibody, as described in this disclosure. The method of manipulating cells may also further comprise the introduction of a rare-cleavage endonuclease capable of selectively inactivating the gene encoding the target for the immunosuppressant by DNA cleavage, as described in this disclosure.

[0079] The present invention also relates to TALE-nucleases. In general, the present invention relates to TALE-nucleases comprising the following:

[0080] (a) Transcription activator-like effector (TALE) DNA binding domain engineered to bind to target sequences within genes selected from the group consisting of immune checkpoint genes, TCR alpha, and TCR beta;

[0081] (b) cleavage domain or cleavage half-domain.

[0082] Preferred TALE-nucleases according to the present invention are those that recognize and cleave a target sequence selected from the group consisting of the following:

[0083] - Sequence No.: 77 and Sequence No.: 78 (PD1)

[0084] - Sequence No. 74 to Sequence No. 76 (CTLA-4),

[0085] - Sequence Nos. 37, 57 to 60 (TCR alpha), and

[0086] - Sequence number: 38 or 39 (TCR beta),

[0087] The above TALE-nucleases comprise polypeptide sequences selected from the group consisting of SEQ ID NOs: 79 to 88 to cleave each target SEQ ID NO: 74 to 78 and, preferably, SEQ ID NOs: 41 to 46 to cleave each target sequence SEQ ID NO: 37 to 39.

[0088] Because some variability may arise from the genomic data from which these polypeptides originate, and also to consider substituting some of the amino acids present in these polypeptides without significant loss of activity (functional variants), the present invention includes polypeptide variants that share at least 70%, preferably at least 80%, more preferably at least 90%, and much more preferably at least 95% identity with the sequences provided in this patent application.

[0089] 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 79 to 88 and SEQ ID NOs: 41 to 46.

[0090] Vectors and polynucleotides encoding the aforementioned rare-cleavage endonucleases according to the present invention are also included within the scope of the present invention.

[0091] The scope of the present invention also includes isolated cells or cell lines that are readily obtained by a method of manipulating the cells, in particular, T cells in which immune checkpoint genes, preferably selected from the group below, are inactivated: PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCR alpha, and TCR beta. Preferably, two genes selected from the group consisting of the following were inactivated: PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, 2B4 and TCR beta.

[0092] According to the present invention, the genes are preferably inactivated by at least one rare-cleavage endonuclease. It has been shown by the inventors that the use of TALE-nucleases is particularly advantageous for achieving double inactivation within T-cells. The present invention comprises isolated T-cells containing at least two polynucleotides, said polynucleotides encoding at least a first and a second TALE-nuclease, preferably the first TALE-nuclease directs directly to a gene encoding a TCR and the second directs directly to a gene encoding an immune checkpoint protein such as PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4. In another example, said isolated cell further comprises one additional genomic modification. In another example, said additional genomic modification is the integration of at least one exogenous polynucleotide sequence. In another example, the exogenous sequence is incorporated into a single gene selected from the group consisting of PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCR alpha, and TCR beta.

[0093] Allergic and immunosuppressive resistant T cells:

[0094] In a particular aspect, the present invention relates to a method for manipulating T-cells, in particular to immunotherapy. In particular, the method comprises the following:

[0095] (a) A step of modifying T-cells by inactivating at least one:

[0096] - The first gene expressing the target for an immunosuppressant, and

[0097] - The second gene encoding the element of the T-cell receptor (TCR)

[0098] (b) a step of optionally expanding the cells in the presence of the immunosuppressant.

[0099] Immunosuppressants are agents that suppress immune function by one of several mechanisms of action. In other words, immunosuppressants are compounds that play a role demonstrated by their ability to reduce the voracity and / or magnitude of the immune response. Examples, without limitation, include calcineurin inhibitors, rapamycin, interleukin-2α-chain blockers, inosine monophosphate dehydrogenase inhibitors, dihydrofolic acid reductase inhibitors, corticosteroids, or immunosuppressive antimetabolites. Classic cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act by inhibiting the activation of helper cells or through the activation of T-cells. The method according to the present invention makes it possible to confer immunosuppressive resistance to T cells for immunotherapy by inactivating targets of immunosuppressants within T cells. As an example, but not limited to, targets of immunosuppressants may be receptors for immunosuppressants such as: CD52, glucocorticoid receptor (GR), FKBP family gene members, and cyclophilin family gene members.

[0100] In a specific example, the genetic modification step of the method relies on the inactivation of a single gene selected from the group consisting of CD52, GR, TCR alpha, and TCR beta. In another example, the genetic modification step of the method relies on the inactivation of two genes selected from the group consisting of CD52 and GR, CD52 and TCR alpha, CD52 and TCR beta, GR and TCR alpha, GR and TCR beta, and TCR alpha and TCR beta. In yet another example, the genetic modification step of the method relies on the inactivation of more than two genes. The genetic modification is preferably performed ex-vivo.

[0101] By inactivating the gene, it is intended that the gene of interest is not expressed in the form of a functional protein. In a specific example, the genetic modification of the method relies on the expression of a rare-cleavage endonuclease in cells provided for manipulation, said rare-cleavage endonuclease specifically catalyzes cleavage in a targeted gene and thereby inactivates said targeted gene. In a specific example, the method of manipulating said cells comprises at least one of the following steps:

[0102] (a) preferably, a step of providing T-cells from a cell culture or a blood sample;

[0103] (b) a step of selecting a gene within the T-cell that expresses a target for an immunosuppressant;

[0104] (c) a step of introducing a rare-cleavage endonuclease, which can be selectively inactivated by DNA cleavage, preferably by double-strand break, into each of the T-cells:

[0105] - The gene encoding a target for the above immunosuppressant, and

[0106] - At least one gene encoding an element of the T-cell receptor (TCR).

[0107] (d) a step of optionally expanding the cells in the presence of the immunosuppressant.

[0108] In a more preferred example, the above method comprises the following:

[0109] (a) preferably, a step of providing T-cells from a cell culture or a blood sample;

[0110] (b) a step of selecting a gene in the T-cells that express a target for an immunosuppressant;

[0111] (c) transforming the T cells with a nucleic acid encoding a rare-cleavage endonuclease capable of selectively inactivating each of the following by DNA cleavage, preferably by double-strand break:

[0112] - The gene encoding a target for the above-mentioned immunosuppressant, and

[0113] - At least one gene encoding an element of the T-cell receptor (TCR);

[0114] (d) a step of expressing the rare-cleavage endonucleases into the T cells;

[0115] (e) a step of sorting transformed T-cells that do not express TCR on their cell surface;

[0116] (f) a step of selectively expanding the cells in the presence of the immunosuppressant.

[0117] In a specific example, the rare-cleavage endonuclease specifically targets one gene selected from the group consisting of CD52, GR, TCR alpha, and TCR beta. In another example, the genetic modification of the method depends on the expression of two rare-cleavage endonucleases in the cells provided for processing, wherein each of the two rare-cleavage endonucleases specifically catalyzes cleavage in each pair of genes selected from the group consisting of CD52 and GR, CD52 and TCR alpha, CD52 and TCR beta, GR and TCR alpha, GR and TCR beta, and TCR alpha and TCR beta, thereby inactivating the targeted genes. In another example, more than two rare-cleavage endonucleases may be expressed in the cells to be manipulated to inactivate and / or target more than two genes.

[0118] In another example, the gene in step (b) specific to the immunosuppressive treatment is CD52, and the immunosuppressive treatment in step (d) or (e) comprises a humanized antibody targeting the CD52 antigen.

[0119] In another example, the gene in step (b) specific to the immunosuppressive treatment is a glucocorticoid receptor (GR), and the immunosuppressive treatment in step d) or (e) includes a corticosteroid such as dexamethasone.

[0120] In another example, the target gene of step (b) specific to the immunosuppressive treatment is an FKBP family gene member or a variant thereof, and the immunosuppressive treatment of step (d) or (e) includes FK506, also known as tacrolimus or fujimycin. In another example, the FKBP family gene member is FKBP12 or a variant thereof.

[0121] In another example, the gene in step (b) specific to the immunosuppressive treatment is a member of the cyclophilin family of genes or a variant thereof, and the immunosuppressive treatment in step (d) or (e) comprises cyclosporine.

[0122] In another example, the rare-cleavage endonuclease may be a meganuclease, a zinc finger nuclease, or a TALE-nuclease. In a preferred example, the rare-cleavage endonuclease is a TALE-nuclease. Preferred TALE-nucleases according to the present invention are those that recognize and cleave a target sequence selected from the group consisting of:

[0123] - Sequence No.: 1 to 6 (GR),

[0124] - Sequence No.: 37, 57 to 60 (TCR alpha),

[0125] - Sequence number: 38 or 39 (TCR beta), and

[0126] - Sequence No.: 40, 61 to 65 (CD52)

[0127] The above TALE-nucleases preferably comprise polypeptide sequences selected from SEQ ID NOs: 7 to 18 and SEQ ID NOs: 41 to 48 to cleave each of the target sequences SEQ ID NOs: 1 to 6 and SEQ ID NOs: 37 to 40.

[0128] In another example, an additional catalytic domain may be further introduced into the cell with the rare-cleavage endonucleases to increase mutagenesis and enhance their ability to inactivate targeted genes. In particular, the additional catalytic domain is a DNA end-processing enzyme. Non-limited examples of DNA end-processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' alkaline exonucleases, 5' flap endonucleases, helicases, hosphatases, hydrolases, and template-independent DNA polymerases. Non-limited examples of such catalytic domains include catalytically active derivatives or protein domains selected from the group consisting of hExoI (EXO1_HUMAN), Yeast ExoI (EXO1_YEAST), E. 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 example, the additional catalytic domain has 3'-5'-exonuclease activity, and in a more preferred example, the additional catalytic domain is TREX, and more preferably is the TREX2 catalytic domain (WO2012 / 058458). In another preferred example, the catalytic domain is encoded by a single-chain TREX polypeptide.The above additional catalyst domain can be selectively fused to a chimeric protein or nuclease fusion protein according to the present invention by a peptide linker.

[0129] Endonucleolytic breaks are known to stimulate the rate of homologous recombination. Therefore, in another example, the genetic modification step of the present method further comprises the step of introducing an exogenous nucleic acid into cells that contains a sequence homologous to at least a portion of the target nucleic acid sequence. In certain examples, the exogenous nucleic acid comprises first and second portions that are homologous to regions 5' and 3' of the target nucleic acid sequence, respectively. In these examples, the exogenous nucleic acid also comprises a third portion located between the first and second portions that is not homologous to regions 5' and 3' of the target nucleic acid sequence. After cleavage of the target nucleic acid sequence, homologous recombination events are stimulated between the target nucleic acid sequence and the exogenous nucleic acid. Preferably, at least 50 bp, preferably more than 100 bp, and more preferably more than 200 bp, homologous sequences are used within the donor matrix. Therefore, the exogenous nucleic acid is preferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. Indeed, the shared nucleic acid homologies are located in the flank regions upstream and downstream of the break site, and the nucleic acid sequence to be introduced must be located between the two arms.

[0130] In particular, the exogenous nucleic acid comprises, in succession, a first region homology to the upstream sequences of the cleavage, a sequence for inactivating one targeted gene selected from the group consisting of CD52, GR, TCR alpha, and TCR beta, and a second region homology to the downstream sequence of the cleavage. The polynucleotide introduction step may be performed simultaneously with, before, or after the expression or introduction of the rare-cleavage endonuclease. In this case, depending on the location of the target nucleic acid sequence where the break event occurs, such exogenous nucleic acid may be used to knock out a gene or to introduce genes of interest or novel sequences, for example, when the exogenous nucleic acid is located within the open reading frame of the gene. By utilizing such exogenous nucleic acids, sequence insertions may be used to modify a target existing gene by correction or replacement of the said gene (an allelic swap, not limited to an example), or to upregulate or downregulate the expression of the target gene by correction or replacement of the said target gene (a promoter replacement, not limited to an example).In a preferred example, inactivation of genes from the group consisting of CD52, GR, TCR alpha, and TCR beta may be carried out at a specific genomic location targeted by a specific TALE-nuclease, wherein the specific TALE-nuclease catalyzes cleavage, and wherein the exogenous nucleic acid is a sequence for inactivating one targeted gene selected from the group consisting of CD52, GR, TCR alpha, and TCR beta that includes at least a homologous region in succession and is incorporated by homologous recombination. In another example, several genes may be inactivated sequentially or simultaneously using several specific polynucleotides for specific gene inactivation and several TALE-nucleases that specifically target one defined gene, respectively.

[0131] By an additional genome modification step, the inactivation of another gene selected from the group consisting of CD52, GR, TCR alpha, and TCR beta may also be intended. As previously stated, the additional genome modification step may be an inactivation step comprising the following:

[0132] (a) introducing at least one rare-cleavage endonuclease into the cells, wherein the rare-cleavage endonuclease specifically catalyzes cleavage of one targeted sequence of the genome of the cells.

[0133] (b) selectively introducing an exogenous nucleic acid into the cells, wherein the exogenous nucleic acid comprises, in succession, a first region homologous to the upstream sequences of the cleavage, a sequence to be inserted into the genome of the cell, and a second region homologous to the downstream sequences of the cleavage.

[0134] At this time, the introduced exogenous nucleic acid inactivates the gene and incorporates at least one exogenous polynucleotide sequence encoding at least one recombinant protein of interest. In another example, the exogenous polynucleotide sequence is incorporated into a gene selected from the group consisting of CD52, GR, TCR alpha, and TCR beta.

[0135] In certain examples, the method of manipulating cells further includes an additional genome modification step. The additional genome modification step is intended to introduce one protein of interest into the cells to manipulate it. The protein of interest may be, as non-limiting examples, pTalpha or its functional variant, a Chimeric Antigen Receptor (CAR), a multi-chain CAR, a bispecific antibody, a rare-cleavage endonuclease targeting PDCD1, or CTLA-4 as described in this disclosure.

[0136] The present invention also relates to TALE-nucleases. In general, the present invention relates to TALE-nucleases comprising the following:

[0137] (a) Transcription activator-like effector (TALE) DNA binding domain engineered to bind to target sequences within genes selected from the group consisting of CD52, GR, TCR alpha, and TCR beta;

[0138] (b) cleavage domain or cleavage half-domain.

[0139] Preferred TALE-nucleases according to the present invention are those that recognize and cleave a target sequence selected from the group consisting of:

[0140] - Sequence No.: 1 to 6 (GR),

[0141] - Sequence No.: 37, 57 to 60 (TCR alpha),

[0142] - Sequence number: 38 or 39 (TCR beta), and

[0143] - Sequence No.: 40, 61 to 65 (CD52)

[0144] The above TALE-nucleases preferably comprise polypeptide sequences selected from SEQ ID NOs: 7 to 18 and SEQ ID NOs: 41 to 48 in order to cleave each of the target sequences SEQ ID NOs: 1 to 6 and SEQ ID NOs: 37 to 40.

[0145] Since some variability arises from the genomic data from which these polypeptides are derived, and also considering the ability to substitute some of the amino acids present in these polypeptides without significant loss of activity (functional variants), the present invention includes polypeptide variants that share at least 70%, preferably at least 80%, more preferably at least 90%, and much more preferably at least 95% identity with the sequences provided in this patent application.

[0146] Therefore, the present invention 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 18 and SEQ ID NOs: 41 to 48.

[0147] Within the scope of the present invention, vectors and polynucleotides encoding the aforementioned rare-cleavage endonucleases according to the present invention are included.

[0148] Within the scope of the present invention, isolated cells or cell lines that can be obtained by the method for manipulating cells, particularly T cells, are also included, in which at least one gene selected from the group consisting of CD52, GR, TCR alpha, and TCR beta is inactivated. Preferably, two genes selected from the group consisting of CD52 and GR, CD52 and TCR alpha, CD52 and TCR beta, GR and TCR alpha, GR and TCR beta, and TCR alpha and TCR beta are inactivated.

[0149] According to the present invention, the genes are preferably inactivated by at least one rare-cleavage endonuclease. It has been shown by the inventors that the use of TALE-nucleases is particularly advantageous for achieving double inactivation in T-cells. The present invention comprises isolated T-cells comprising at least two polynucleotides, wherein the polynucleotides are said to be at least two polynucleotides, wherein the first TALE-nuclease is directed against a gene encoding a TCR and the second is directed against a gene encoding a receptor for an immunosuppressant, such as CD52 or GR.

[0150] In another example, the isolated cell further comprises one additional genomic modification. In another example, the additional genomic modification is the incorporation of at least one exogenous polynucleotide sequence. In another example, the exogenous sequence is incorporated into a gene selected from the group consisting of CD52, GR, TCR alpha, and TCR beta.

[0151] preTalpha

[0152] In another aspect, the present invention relates to a method for expanding TCR alpha-deficient T-cells or their functional variants, comprising the step of introducing said cells into said T-cell pTalpha (also referred to as preTCRα), and selectively expanding said cells through stimulation of the CD3 complex. In a preferred example, the present method comprises the following:

[0153] a) transforming the cells with a nucleic acid encoding at least a fragment of pTalpha to support CD3 surface expression

[0154] b) a step of expressing the pTalpha into the cells

[0155] c) A step of expanding the cells by selectively stimulating the CD3 complex.

[0156] The present invention also relates to a method for preparing T-cells for immunotherapy, comprising steps of an expansion method for T-cells.

[0157] In certain examples, the pTalpha polynucleotide sequence may be introduced randomly or otherwise through homologous recombination, and in particular, the insertion may be associated with the inactivation of the TCRalpha gene.

[0158] According to the present invention, other functional variants of pTalpha are used. A “functional variant” of a peptide refers to a molecule that is substantially similar to the whole peptide or a fragment thereof. A “fragment” of pTalpha or its functional variant of the present invention represents any subset of the molecule, i.e., a shorter peptide. Preferred pTalpha or functional variants may be full-length pTalpha or C-truncated pTalpha versions. C-truncated pTalpha lacks one or more residues at the C-terminus. As non-limiting examples, the C-truncated pTalpha version lacks 18, 48, 62, 78, 92, 110, or 114 residues from the C-terminus of the protein (SEQN: 107 to SEQN: 114). Furthermore, amino acid sequence variants of the peptide can be prepared by mutations within the DNA encoding the peptide. These functional variants include, for example, deletions from residues within the amino acid sequence, or insertions or substitutions of residues. Any combination of deletions, insertions, and substitutions can also be produced to achieve the desired activity, in particular the restoration of the functional CD3 complex, in the final construct. In a preferred example, at least one mutation is introduced into other pTalpha methods as described above to affect dimerization.As an example not limited to, the mutated residues may be at least W46R, D22A, K24A, R102A, or R117A of the human pTalpha protein or positions aligned with CLUSTALW on the pTalpha family or homologous members. Preferably, pTalpha or its variant as described above comprises the mutated residue W46R (SEQ No. 123) or the mutated residues D22A, K24A, R102A, and R117A (SEQ No. 124). In certain examples, the pTalpha or variants are also fused to a single-transducing domain such as CD28, OX40, ICOS, CD27, CD137 (4-1BB), and CD8, as non-limiting examples (SEQN: 115 to 120). As previously described, the extracellular domain of pTalpha or variants may be fused to a fragment of the TCR-alpha protein, in particular to the transmembrane and intracellular domains of TCR-alpha (SEQN: 122). pTalpha variants may also be fused to the intracellular domain of TCR-alpha (SEQN: 121).

[0159] In another example, the versions of pTalpha are fused to an extracellular ligand-binding domain, and more preferably, pTalpha or its functional variant is fused to a single chain antibody fragment (scFV) comprising light (VL) and heavy (VH) variable fragments of a target antigen-specific monoclonal antibody joined by a flexible linker. As an example, but not limited to, the amino acid sequence of pTalpha or its functional variant is selected from the group consisting of SEQ ID NOs 107 to 124.

[0160] Since some variability arises from the genomic data from which these polypeptides originate, and also considering the ability to substitute some of the amino acids present in these polypeptides without significant loss of activity (functional variants), the present invention includes polypeptide variants of said polypeptides that share at least 70%, preferably at least 80%, more preferably at least 90%, and much more preferably at least 95% identity with the sequences provided in this patent application.

[0161] Therefore, the present invention 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 107 to SEQ ID NO: 124.

[0162] Isolated T cells lacking expression of functional TCR alpha chains TA cell is intended. This can be achieved by other means, such as, as non-limiting examples, by manipulating a T cell so that it does not express any functional TCR alpha on its cell surface, or by manipulating a T cell so that it produces very little functional TCR alpha chain on its surface, or by manipulating a T cell to express a mutated or truncated form of the TCR alpha chain.

[0163] TCR alpha-deficient cells can no longer expand through the CD3 complex. In this way, to overcome this problem and to allow the proliferation of TCR alpha-deficient cells, pTalpha or a functional variant thereof is introduced into said cells to thus restore the functional CD3 complex. In a preferred example, the present invention comprises the step of introducing into said T cells rare-cleavage endonucleases that can be selectively inactivated by DNA cleavage of a gene encoding a component of the T-cell receptor (TCR). In certain examples, said rare-cleavage endonucleases are TALE-nucleases. As examples, but not limited to, TALE-nucleases are directed toward one of the gene target sequences of TCR alpha selected from the group consisting of SEQ ID NO: 37 and SEQ ID NOs: 57 to 60. Preferably, TALE-nucleases are selected from the group consisting of SEQ ID NO: 41 and SEQ ID NO: 42.

[0164] In certain examples, the method for expanding TCR alpha-deficient T-cells comprises an additional genomic modification step. By the additional genomic modification step, introduction into the cells may be intended for the manipulation of one protein of interest. The protein of interest may be, as non-limiting examples, a chimeric antigen receptor (CAR), particularly a CAR containing amino acid sequence SEQ NO: 73, a multi-chain CAR, particularly a multi-chain CAR containing amino acid sequence SEQ NO: 125, a bispecific antibody, rare-cleavage endonucleases targeting CTLA-4 or PDCD1, particularly targeting nucleic acid sequences SEQ NO: 74 to SEQ NO: 78, or a rare-cleavage endonucleases targeting a target for an immunosuppressant as described in this disclosure.

[0165] The present invention also includes polypeptides encoding pTalpha, particularly the aforementioned functional variants. In a preferred example, the present invention relates to pTalpha or its functional variants fused to transducing domains such as CD28, OX40, ICOS, CD137, and CD8. More particularly, the present invention relates to pTalpha functional variants comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 107 to 124. The present invention also includes vectors and polynucleotides encoding pTalpha or the aforementioned functional variants thereof.

[0166] The scope of the present invention also includes isolated cells or cell lines that can be obtained by the above method. In particular, said isolated cells or cell lines may be obtained by introducing pTalpha or a functional variant thereof into said cells to support CD3 surface expression. In a preferred example, said isolated cells or cell lines are further genetically modified by inactivating the TCRalpha gene. This gene is preferably inactivated by at least one rare-cleavage endonuclease. In a preferred example, said rare-cleavage endonuclease is a TALE-nuclease.

[0167] Multi-chain Chimeric Antigen Receptor (CAR)

[0168] In another example, the present invention relates to a multi-chain chimeric antigen receptor (CAR) adapted for the expansion and production of engineered T-cells, in particular.

[0169] Multi-chain CAR comprising at least two of the following elements:

[0170] a) A polypeptide comprising an FcεRI alpha chain and a transmembrane domain of an extracellular ligand-binding domain,

[0171] b) A polypeptide comprising a transmembrane domain of the FcεRI beta chain and a pair of N- and C-terminal cytoplasmic tails and / or

[0172] c) Two polypeptides comprising each portion of the transmembrane domain and the intracytoplasmic tail of the FcεRI gamma chain, thereby spontaneously multimerizing with each other to form a dimer, trimer, or tetrameric CAR.

[0173] An example of a tetrameric CAR is shown in Fig. 3. Other versions of multichain CARs are shown in Fig. 4. An example of a multichain CAR includes the amino acid sequence SEQ ID NO: 125. As used herein, the term “part of” refers to any subset of a molecule that is a shorter peptide. Alternatively, functional variants of the polypeptide's amino acid sequence can be prepared by mutations within the DNA encoding the polypeptide. These functional variants include, for example, deletions from residues within the amino acid sequence, or insertions or substitutions of residues. Any combination of deletions, insertions, and substitutions can also be made to arrive at the final construct, provided that the final construct possesses the desired activity, particularly specific anti-target cell immune activity.

[0174] In a preferred example, the extracellular ligand-binding domain is a scFv. Other binding domains other than scFv may also be used for predetermined targeting of lymphocytes, such as antibody binding domains, antibody hypervariable loops or CDRs, receptor ligands such as integrin-binding peptides, heregulin, IL-13 mutein or vascular endothelial growth factor polypeptide, or camelid single-domain antibody fragments, as examples but not limited to.

[0175] In a preferred example, the polypeptide of a) further comprises a stalk region between the extracellular ligand-binding domain and the transmembrane domain. As used herein, the term "stalk region" usually refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the stalk region is used to provide 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 derived from part or all of naturally occurring molecules, such as from part or all of the antibody constant region or from part or all of the extracellular region of CD8, CD4, or CD28. Alternatively, the Stokes region may be a synthetic sequence corresponding to a naturally occurring Stokes region, or a completely synthetic Stokes sequence. In a preferred example, the polypeptide of a), b), and / or c) further comprises at least one transducing domain. In the most preferred example, the transducing domain is selected from the group consisting of CD28, OX40, ICOS, CD137, and CD8.

[0176] In a preferred example, the C-terminal cytoplasmic tail of the FcεRI alpha, beta, and / or gamma chain fragment further comprises TNFR-associated Factor 2 (TRAF2) binding motifs. In the most preferred example, the C-terminal cytoplasmic tail of the FcεRI alpha, beta, and / or gamma chain is replaced by the cytoplasmic tail of a costimulatory TNFR member family. The cytoplasmic tail of the costimulatory TNFR family member comprises TRAF2 binding motifs, consisting of a major conserved motif (P / S / A)X(Q / E)E) or a minor motif (PXQXXD), wherein X is any amino acid. TRAF proteins are formed in the intracellular tails of many TNFRs in response to receptor trimerization.

[0177] In another preferred example, the cytoplasmic domain of the FcεRI alpha, beta, and / or gamma chain is replaced by the cytoplasmic domain of the TCR zeta chain (also named CD3 zeta). In another preferred example, the cytoplasmic domain of the FcεRI alpha, beta, and / or gamma chain comprises at least one additional immunoreceptor tyrosine-based activation motif (ITAM). ITAMs are well-defined signal motifs found in the cytoplasmic tails of various receptors that act as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention include those derived from TCR zeta, FCR gamma, FCR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0178] As a non-limiting example, other versions of multi-chain CAR are shown in Fig. 4.

[0179] In a preferred example, the multi-chain CAR comprises the amino acid sequence SEQ ID NO: 125. The present invention 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 125.

[0180] The scope of the present invention also includes vectors and polynucleotides encoding the aforementioned multi-chain CAR according to the present invention.

[0181] In particular examples included, the present invention relates to a method for preparing T-cells for immunotherapy, comprising the steps of expanding said cells and introducing other polypeptides composing said multi-chain CAR into said T-cells.

[0182] In another example, the method further comprises the step of genetically modifying the cells by inactivating at least one gene that expresses a target for an immunosuppressant and / or one element of the TCR. In a preferred example, the gene is selected from the group consisting of TCR alpha, TCR beta, CD52, and GR. In a preferred example, the method further comprises the step of introducing the genes into the T cells with a rare-cleavage endonuclease capable of selectively inactivating them by DNA cleavage. In a more preferred example, the rare-cleavage endonuclease is a TALE-nuclease. Preferred TALE-nucleases according to the present invention are those that recognize and cleave a target sequence selected from the group consisting of: SEQ ID NOs: 1 to 6 (GR), SEQ ID NOs: 37, 57 to 60 (TCR alpha (alpha)), SEQ ID NO: 38 or 39 (TCR beta (beta)), and SEQ ID NO: 40, SEQ ID NOs: 61 to 65 (CD52).

[0183] In certain examples, the above method further comprises an additional genome modification step. By the additional genome modification step, introduction into cells may be intended to manipulate one protein of interest. The protein of interest may be, as non-limiting examples, a bispecific antibody, a rare-cleavage endonuclease targeting CTLA-4 or PDCD1, pTalpha, or a functional variant thereof described in this disclosure.

[0184] The present invention also relates to isolated cells or cell lines that are easily obtained by the above method for manipulating cells. In particular, the isolated cells comprise exogenous polynucleotide sequences encoding polypeptides composing the multi-chain CAR.

[0185] bispecific antibodies

[0186] According to additional examples, engineered T cells obtained by other methods as described above may be further exposed to bispecific antibodies. The T cells may be exposed to bispecific antibodies in vivo after administration to the patient or ex vivo before administration to the patient. The bispecific antibodies comprise two variable regions having distinct antigenic characteristics that allow engineered cells to be brought close to the target antigen. As a non-limiting example, the bispecific antibodies are directed against lymphocyte antigens such as CD3 and tumor markers and have the potential to activate and redirect any circulating T cells against the tumor.

[0187] Delivery methods

[0188] The other methods described above include the step of selectively introducing pTalpha or its functional variants, rare cleavage endonucleases, TALE-nucleases, CARs, or multi-chain CARs into a cell using exogenous nucleic acids or DNA-terminal processing enzymes.

[0189] As a non-limiting example, the above pTalpha or its functional variants, rare cleavage endonucleases, TALE-nucleases, CAR or multi-chain CAR may optionally be introduced as DNA-terminal processing enzymes or exogenous nucleic acids as other plasmid vectors or as transgenes encoded by one. Other transgenes may be included in one vector containing a nucleic acid sequence encoding a ribosomal skip sequence, such as the sequence encoding the 2A peptide. 2A peptides identified in the aphthovirus subgroup of picornaviruses cause a ribosome "skip" from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (see Donnelly et al., J. of General Virology 82: 1013-1025 (2001); Donnelly et al., J. of Gen. Virology 78: 13-21 (1997); Doronina et al., Mol. And. Cell. Biology 28(13): 4227-4239 (2008); Atkins et al., RNA 13: 803-810 (2007)). A "codon" refers to three nucleotides on mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into a single amino acid residue. Therefore, two polypeptides can be synthesized from a single, contiguous open reading frame within the mRNA when the polypeptides are separated by a 2A oligopeptide sequence in which they are framed.These ribosome skip mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA. As an example, but not limited to, the 2A peptides in the present invention are used to express rare-cleavage endonucleases and DNA end-processing enzymes or other polypeptides of multi-chain CARs into cells.

[0190] The above plasmid vector may include a selection marker provided for the identification and / or selection of cells receiving the vector.

[0191] Polypeptides may be synthesized in situ within the cell as a result of the introduction of polynucleotides encoding said polypeptides into the cell. Alternatively, said polypeptides may be produced outside the cell and then introduced therein. Methods for introducing polynucleotide constructs into animal cells are known in the art and, as examples but not limited to, include stable transformation methods in which the polynucleotide construct is incorporated into the cell genome, transient transformation methods in which the polynucleotide construct is not incorporated into the cell genome, and virus-mediated methods. said polynucleotides may be introduced into the cell, 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 polynucleotides may be included in vectors, more particularly in plasmids or viruses, in terms of being expressed in cells.

[0192] - electroporation

[0193] In a more preferred embodiment of the present invention, polynucleotides encoding polypeptides according to the present invention may be mRNAs that are directly introduced into cells, for example, by electroporation. The present invention determined optimal conditions for mRNA electroporation in T-cells.

[0194] The inventors used a cytopulse technique that allows living cells to be temporarily permeable for material transfer into cells by using pulsed electric fields. Based on PulseAgile (Cellectis property) electroporation waveforms, the technique allows for precise control of pulse duration, intensity, and interval between pulses (U.S. Patent 6,010,613 and International PCT Application WO2004083379). These parameters can all be modified to achieve optimal conditions for high transfection efficiency with minimal mortality. While subsequent lower electric field pulses allow for the transport of polynucleotides into the cell, the first high electric field pulses essentially enable pore formation. In one aspect of the invention, the inventors describe steps leading to the achievement of >95% transfection efficiency of mRNA in T cells, and the use of an electroporation protocol for transiently expressing other types of proteins in T cells. In particular, the invention comprises the steps of contacting RNA with said T cells and... The present invention relates to a method for transforming T cells comprising the step of applying to a configured agile pulse;

[0195] (a) a single electrical pulse having a voltage range of 2250 to 3000 V per centimeter, a pulse width of 0.1 ms, and a pulse interval of 0.2 to 10 ms between the electrical pulses of step (a) and (b);

[0196] (b) a voltage interval from 2250 to 3000 V with a pulse width of 100 ms and one electrical pulse having a pulse interval of 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and

[0197] (c) With a pulse width of 0.2 ms 3 25 V voltage and 4 electrical pulses with a pulse interval of 2 ms between the 4 electrical pulses.

[0198] In particular, the method for transforming T cells includes the step of contacting the T cells with RNA and the step of applying the T cells to an agile (pulse) sequence composed of the following;

[0199] (a) a single electrical pulse having a voltage of 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V per centimeter, a pulse width of 0.1 ms, and a pulse interval of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ms between the electrical pulses of steps (a) and (b);

[0200] (b) a voltage range from 2250, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000V, having a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and

[0201] (c) Four electrical pulses having a voltage of 325 V with a pulse interval of 2 ms and a pulse width of 0.2 ms between each of the four electrical pulses.

[0202] Any numerical values ​​included within the aforementioned numerical ranges are disclosed in this application. The electroporation medium may be any suitable medium known in the art. Preferably, the electroporation medium has a conductivity spanning from 0.01 to 1.0 milliSiemens.

[0203] In certain examples, and as non-limited examples, the RNA codes for a rare-cleavage endonuclease, a monomer of a rare-cleavage endonuclease such as Half-TALE-nuclease, a chimeric antigen receptor, at least one element of a multi-chain chimeric antigen receptor, pTalpha or a functional variant thereof, an exogenous nucleic acid, and one additional catalytic domain.

[0204] Activation and expansion of T cells

[0205] Before or after genetic modification of T cells, T cells can generally be activated and expanded using methods as described in, for example, US 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 US Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

[0206] In general, the present invention T cells expand through contact with surfaces to which ligands stimulating co-stimulatory molecules on the surface of T cells and agents stimulating CD3 TCR complex-related signals attach.

[0207] In particular, T cell populations can be stimulated in vitro by contact with an anti-CD3 antibody, or its antigen-binding fragment, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For the co-stimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. To stimulate the proliferation of CD4+ T cells or CD8+ T cells, anti-CD3 antibody and anti-CD28 antibody. For example, the respective signaling agents can be coupled into solution or to the surface. As a person of routine skill in the art, it is readily apparent that the ratio of particles to cells may depend on particle size proportional to the target cells. In further examples of the present invention, cells such as T cells are combined with formulation-coated beads, the beads and cells are then separated, and then the cells are cultured. In an alternative example, prior to culture, the formulation-coated beads and cells are not separated but cultured together. Cell surface proteins can be ligated by enabling paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached to come into contact with T cells. In one example, cells (e.g., 4 to 10 T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, preferably PBS (free of divalent cations such as calcium and magnesium).Again, those skilled in the art will readily acknowledge that any cell concentration may be used. The mixture may be cultured for a few hours (about 3 hours) to about 14 days or for any integer value of every hour in between. In another example, the mixture may be cultured for 21 days. Conditions suitable for T cells include a suitable medium (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 5, (Lonza)) which may contain factors necessary for proliferation and viability, including serum (e.g., bovine fetal or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2, 1L-15, TGFp, and TNF- or other additives for the growth of cells known to those skilled in the art. Other additives for the growth of cells include, but are not limited to, surfactants, plasmanates, and reducing agents such as 2-mercaptoethanol and N-acetyl-cysteine. The media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, which are serum-free, having vitamins, sodium pyruvate, and added amino acids, or supplemented with a prescribed set of cytokine(s) sufficient for the growth and expansion of T cells, and / or an appropriate amount of serum (or plasma). Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in the cultures of cells infused into the subject. The target cells are maintained under conditions necessary to support growth, such as, for example, an appropriate temperature (e.g., 37°C) and air (e.g., air plus 5% CO2).T cells exposed to various stimulation times may exhibit different characteristics.

[0208] In another specific example, the cells may be expanded by co-culture with tissues or cells. The cells may also be expanded in vivo, for example, in the blood of a subject after administering the cells into the subject.

[0209] Transformed T-cells

[0210] The scope of the present invention includes isolated T cells obtained according to any one of the methods described above. The T cells according to the present invention may be derived from stem cells. The stem cells may be adult stem cells, embryonic stem cells, more particularly non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Representative human cells are CD34+ cells. The isolated cells may also be T cells selected from the group consisting of dendritic cells, NK cells, B cells, or inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another example, the cells may be derived from a group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. Before genetic modification and expansion of the cells of the present invention, the source of the cells may be obtained from the subject through various non-limiting methods. T cells may be obtained from non-limiting sources, including hemorrhagic blood remnants, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, infection, ascites, pleural effusion, spleen tissue, and tissue from the site of tumors. In specific examples of the present invention, any number of T cell lines known and available in the art may be used. In another example, the cells may be derived from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In another example, the cells are part of a mixed population of cells exhibiting different phenotypic characteristics.The scope of the present invention includes cell lines obtained from T-cells transformed according to the method described above. Transformed cells that are resistant to immunosuppressive treatment and allow to be obtained by the aforementioned method are included within the scope of the present invention.

[0211] In another example, the isolated cell according to the present invention comprises one inactivated gene selected from the group consisting of CD52, GR, PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCR alpha and TCR beta, and / or expresses a CAR, multi-chain CAR and / or pTalpha transgene. In another example, the isolated cells according to the present invention are two selected from the group consisting of CD52 and GR, CD52 and TCR alpha, CDR52 and TCR beta, GR and TCR alpha, GR and TCR beta, TCR alpha and TCR beta, PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, and 2B4 and TCR beta. It contains an inactivated gene and / or expresses a CAR, multi-chain CAR and / or pTalpha transgene.

[0212] In another example, the TCR is rendered non-functional in cells according to the present invention by inactivating the TCR alpha gene and / or TCR beta gene(s). These strategies are used more particularly to avoid GvHD. In a specific aspect of the present invention, a method of obtaining modified cells derived from an individual, wherein the cells can be proliferated independently of the major histocompatibility complex signaling pathway. The method comprises the following steps:

[0213] (a) A step of recovering cells from the above individual;

[0214] (b) a step of genetically modifying the cells in ex-vivo by inactivating TCR alpha or TCR beta genes;

[0215] (c) A step of amplifying said cells by culturing genetically modified T-cells in vitro under appropriate conditions.

[0216] Modified cells capable of independently proliferating of the major histocompatibility complex signaling pathway, which are obtainable by this method, are included in the scope of the present invention. Such modified cells may be used in a particular aspect of the present invention to treat patients who need it against host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD); and thus, within the scope of the present invention is a method for treating patients who need it against host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising the step of treating said patient by administering an effective amount of modified cells containing inactivated TCR alpha and / or TCR beta genes to said patient.

[0217] Therapeutic Applications

[0218] In another example, the separated parts obtained by other methods Cells or cell lines derived from the isolated cells as described above may be used as a medicine. In another example, the medicine may be used to treat infections or cancer in patients who need it. In another example, the isolated cells or cell lines derived from the isolated cells according to the present invention may be used to manufacture a medicine for treating viral infections or cancer in patients who need it.

[0219] In another aspect, the present invention relies on methods for treating patients in need of the method, comprising at least one of the following steps:

[0220] (a) a step of providing T-cells obtainable by any one of the aforementioned methods;

[0221] (b) a step of administering the transformed T-cells to the patient,

[0222] In one example, the T cells of the present invention may undergo robustness in in vivo T cell expansion and may persist for an extended period of time.

[0223] The above treatment may be corrective, therapeutic, or prophylactic. It may be part of autologous immunotherapy or part of allogenic immunotherapy. By autologous, it means that the population, cell line, or cells used in the treatment of patients may originate from the patient or from a Human Leukocyte Antigen (HLA) compatible donor. By allogenic, it means that the population or cells used in the treatment of patients do not originate from said patient but originate from a donor.

[0224] The present invention is particularly suitable for allogenic immunotherapy insofar as it enables the transformation of T-cells, typically obtained from donors, into non-alloreactive cells. This can be done under standard protocols and can be reproduced as many times as necessary. The resulting transformed T-cells are pooled and made available as an "off-the-shelf" therapeutic product, which can be administered to one or several patients.

[0225] The disclosed methods and cells that may be used are described in the previous section. The above treatment may be used to treat patients diagnosed with cancer, viral infections, autoimmune disorders, or Graft-versus-Host (GvHD) disease. Cancers that may be treated include non-vascularized tumors, or tumors that are not yet primarily vascularized and vascularized. Cancers may include non-solid tumors (such as blood tumors, e.g., leukemias and lymphomas) or solid tumors. The types of cancers treated with the CARs of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0226] It may be a treatment in combination with one or more therapies against cancer selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0227] According to a preferred embodiment of the present invention, the treatment may be administered to patients undergoing immunosuppressive therapy. Indeed, the present invention preferably relies on a population of cells or cells that are made resistant to at least one immunosuppressant due to the inactivation of a gene encoding a receptor for such immunosuppressant. In this regard, the immunosuppressive therapy should facilitate the selection and expansion of T-cells according to the present invention within the patient.

[0228] The population of cells or the administration of cells according to the present invention may be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, intralymphatic injection, or intraperitoneally. In one example, the cell compositions of the present invention are preferably administered by intravenous injection.

[0229] The population of cells or the administration of cells is 10 per kg of body weight. 4 -10 9 of cells, preferably 10 5 to 10 6It may consist of the administration of cells per kg body weight, comprising all integer values ​​of the number of cells within their ranges. Cells or populations of cells may be administered in one or more doses. In one example, the effective amount of cells is administered as a single dose. In another example, the effective amount of cells is administered in more than one dose over a period of time. The timing of administration is at the discretion of the attending physician and depends on the patient's clinical condition. Cells or populations of cells may be obtained from any source, such as a blood bank or a donor. While individual needs differ, determining the optimal ranges of effective amounts of a defined cell type for specific diseases or conditions is within the art of the art. An effective amount refers to the amount that provides therapeutic or prophylactic benefit. The administered dose will depend on the recipient's age, health, and weight, the type of concurrent treatment, the frequency of treatment if any, and the characteristics of the desired effect.

[0230] In another example, the composition containing these cells or the effective amount of the cells is administered parenterally. The administration may be intravenous. The administration may be made directly by injection into the tumor.

[0231] In specific examples of the present invention, cells are administered to a patient (e.g., before, simultaneously with, or after) any number of relevant modes of treatment, including but not limited to antiviral therapy, agents such as cidofovir and interleukin-2, Cytarabine (also known as ARA-C), or nataliziimab treatment for MS patients or efaliztimab treatment for psoriasis patients or other treatments for PML patients. In additional examples, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit calcium-dependent phosphatases calcineurin (cyclosporine and FK506) or inhibit p70S6 kinases important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1 1; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Citrr. Opin. mm n. 5:763-773, 93).In additional examples, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) T-cell ablative therapy using bone marrow transplantation, antibodies such as OKT3 or CAMPATH, or fludarabine, external-beam radiation therapy (XRT), or chemotherapy agents such as cyclophosphamide. In another example, the cell compositions of the present invention are administered after B-cell ablative therapy, such as agents that react with CD20, e.g., Rituxan. For example, in one example, a subject may receive standard treatment with high-dose chemotherapy and then receive a peripheral blood stem cell transplant. In specific examples, after transplantation, subjects receive an infusion of the expanded immune cells of the present invention. In additional examples, the expanded cells are administered before or after surgery. The modified cells obtained by any one of the methods described herein may be used in a specific aspect of the present invention to treat patients who need it against host versus graft (HvG) rejection and graft versus host disease (GvHD); therefore, within the scope of the present invention, a method for treating patients who use it as a treatment against host versus graft (HvG) rejection and graft versus host disease (GvHD) comprises the step of treating said patient by administering an effective amount of modified cells containing inactivated TCR alpha and / or TCR beta genes to said patient.

[0232] Method of manipulating human allogeneic cells for immunotherapy yes

[0233] For a better understanding of the present invention, an example of a method for manipulating human allogenic cells for immunotherapy is shown in FIG. 5. A method comprising one or a combination of some of the following steps:

[0234] 1. A step of providing T-cells from a blood sample from a single individual patient or from a blood bank or from a cell culture, and activating said T cells using anti-CD3 / C28 activator beads. The beads provide primary and co-stimulatory signals required for the activation and expansion of the T cells.

[0235] 2. a) Transducing the cells with pTalpha or its functional variant transgene to support CD3 surface expression and enabling cell expansion through stimulation of the CD3 complex. TCR disruption is expected to result in the removal of the TCR complex and alloreactivity (GvHD), but may alter allogenic cell expansion due to the loss of CD3 signaling elements. The transduced cells are expected to express the pTalpha chain or its functional variant. This pTalpha chain pairs with the TCR beta chain and CD3 signaling elements to form a preTCR complex, and thus restores the functional CD3 complex and supports the stimulation or activation of inactivated TCR-alpha cells. Transduction of T-cells with a pT-alpha lentiviral vector can be realized before or after TCR-alpha inactivation.

[0236] b) Transducing the cells with multi-chain CARs allows T cells to be redirected toward antigens expressed on the surface of target cells from various malignancies, including lymphomas and solid tumors. To improve the function of the co-stimulatory domain, the inventors designed a multi-chain CAR derived from FcεRI as described above. Transduction can be performed before or after the inactivation of other genes, such as TCR-alpha and CD52 genes.

[0237] 3. Step of manipulating non-alloreactive and immunosuppressant-resistant T cells:

[0238] a) It is possible to avoid GvHD by inactivating TCR alpha in the cells to remove TCR from the cell surface and preventing the host tissue from being recognized as foreign by allogenic TCRs.

[0239] b) It is also possible to inactivate a single gene encoding a target for an immunosuppressant in order to render said cells resistant to immunosuppressive treatment in order to prevent graft rejection without affecting the transplanted T cells. In this example, the target of the immunosuppressant is CD52 and the immunosuppressant is a humanized monoclonal anti-CD52 antibody.

[0240] The inventors have shown that the use of TALE-nucleases is particularly advantageous for achieving the aforementioned double inactivation of T-cells by allowing a higher rate of DSB events within the T-cells. Preferably, TCR-alpha and CD52 genes are inactivated by electroporating T-cells with mRNA encoding a TALE-nuclease that targets said genes. The inventors have found that using mRNA that causes a high transformation rate is less harmful to T-cells and is very important in the process of manipulating T-cells. The inactivated T-cells are then sorted using magnetic beads. For example, T-cells expressing CD52 are removed by fixation on a solid surface, and the inactivated cells are not exposed to stress as they pass through a column. This mild method increases the concentration of appropriately manipulated T-cells.

[0241] 4. Expansion of engineered T-cells in vitro after administration to a patient or before administration to a patient via stimulation of the CD3 complex. Prior to the administration step, patients may be treated with immunosuppressive agents such as CAMPATH1-H, humanized monoclonal antibody anti-CD52.

[0242] 5. Cells selectively exposed to bispecific antibodies in vivo after administration to the patient or ex vivo before administration to the patient in order to bring into the cells engineered to be close to the target antigen.

[0243] Other definitions

[0244] - Amino acid residues in the polypeptide sequence are designated here according to a single letter code, for example, Q means Gln or glutamine residue, R means Arg or arginine residue, and D means Asp or aspartic acid.

[0245] - Amino acid substitution refers to the replacement of one amino acid residue with another. For example, the replacement of an arginine residue with a glutamine residue in a peptide sequence is an amino acid substitution.

[0246] - Nucleotides are designated as follows: A single character code is used to designate the base of a nucleotide: a is adenine, t is thymine, c is cytosine, and g is guanine. For the designated nucleotides, r represents g or a (purine nucleotides), 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 nucleotides), 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.

[0247] - As used herein, “nucleic acid” or “polynucleotides” refers to polynucleotides and / or nucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules may comprise monomers that are naturally occurring nucleotides (such as DNA and RNA), analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified Nucleotides may have alterations in pyrimidine or purine base moiety and / or sugar moiety. Sugar alterations include, for example, the replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars may be functionalized as ethers or esters. Furthermore, the entire sugar moiety may be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.Examples of changes within the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers may be linked by phosphodiester bonds or analogues of these linkages. Nucleic acids may be single-stranded or double-stranded.

[0248] A matrix or DNA structure is intended to comprise first and second portions homologous to the 5' and 3' regions of a DNA target in situ, by means of a polynucleotide comprising sequentially a first region homologous to the upstream sequences of the double-strand break, a sequence inserted into the genome of the cell, and a second region homologous to the downstream sequences of the double-strand break. The DNA structure also comprises a third portion located between the first and second portions having partial homology to the corresponding DNA sequence in situ, or alternatively, not having homology to the 5' and 3' regions of the DNA target in situ. After cleavage of the DNA target, a homologous recombination event is stimulated between this matrix and the genome containing the targeted gene contained within the locus of interest, wherein the genome sequence containing the DNA target is, by the third portion of the matrix and various portions of the first and second portions of the matrix It is replaced.

[0249] - A polynucleotide sequence that can be targeted and processed by a rare-cleavage endonuclease according to the present invention is intended by "DNA target," "DNA target sequence," "target DNA sequence," "nucleic acid target sequence," "target sequence," or "processing site." These terms refer to a specific DNA location, preferably a genomic location in a cell, but also, as an example but not limited to, organelles such as mitochondria, or a portion of genetic material that may exist independently of the body of genetic material such as transposons, viruses, episomes, and plasmids. As non-limiting examples of TALE-nuclease targets, the targeted genomic sequences usually consist of two 17-bp length sequences (referred to as half targets) separated by a 15-bp spacer. Each half-target is recognized by repeats of the TALE-nucleases listed in Tables 2, 6, 7, and 11, as non-limiting examples, coded within plasmids under the control of the EF1-alpha promoter or T7 promoter. The nucleic acid target sequence is defined by the sequence from 5' to 3' of one strand of the target, as indicated in Tables 2, 6, 7, and 11.

[0250] - To create a chimeric protein that exhibits specific anti-target cell immune activity by means of a chimeric antigen receptor (CAR), a molecule is intended that combines a binding domain against elements present on the target cell, antibody-based specificity against a desired antigen (e.g., a tumor antigen), and an intracellular domain that activates the T cell receptor. Generally, a CAR consists of an extracellular monoclonal antibody (scFvFc) that fuses to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (scFvFc:ζ) and, when expressed in T cells, has the ability to redirect to antigen recognition based on the specificity of the monoclonal antibody. An example of a CAR used in the present invention is a CAR directed against the CD19 antigen and may include, as an example but not limited to, amino acid sequence : SEQ ID NO: 73.

[0251] Any delivery vector that can be used in the present invention to deliver preparations / chemicals and molecules (proteins or nucleic acids) into subcellular compartments (i.e., “introducing”) or cells, or to insert them into cellular structures (i.e., “contacting”) by means of a “delivery vector” or “delivery vectors” is intended. It includes, but is not limited to, liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions, or other suitable transfer vectors. These delivery vectors enable the delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as peptides and plasmids developed by Diatos. In these cases, the delivery vectors are molecular carriers. Delivery methods for performing transfection are intended by "delivery vectors" or "delivery vectors".

[0252] - The terms "vector" or "vectors" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In the present invention, "vector" includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vectors) and / or the expression of the nucleic acids to which they are linked (expression vectors). Many suitable vectors are known in the art and are commercially available.

[0253] Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adenoassociated viruses), negative-strand RNA viruses such as coronaviruses and orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as alphaviruses and picornaviruses, and double-stranded DNA viruses including herpesviruses (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), adenoviruses, and poxviruses (e.g., vaccine, fowlpox and Includes canarypox. Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include: avian leukosis-sarcoma, mammalian type C, type B, and type D viruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0254] - "Lentiviral vectors" are very promising for gene delivery due to their relatively large packaging capacity, reduced immunogenicity, and the ability to stably transduce a wide range of different cell types with high efficiency. H IV-based lentiviral vectors are intended. Lentiviral vectors are typically produced after the transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter target cells through interactions with viral surface glycoproteins on 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 serves as a template for viral integration into the DNA of infected cells. These vectors, which can be integrated into the genome of target cells, are intended by "integrative lentiviral vectors" (or LVs), but are not limited to such examples. In contrast to "non-integrative lentiviral vectors (or NILVs)," effective gene delivery vectors that are not integrated into the genome of target cells through the action of viral integrase are referred to.

[0255] - The delivery vectors and vectors may be associated with or combined with any cell permeabilization techniques, such as sonoporation or electroporation or derivatives of these techniques.

[0256] Any eukaryotic living cells, primary cells, and cell lines derived from these organisms are intended for in vitro cultures by cells or cells.

[0257] - Cells established for in vitro growth are intended to be taken directly from living tissue (i.e., biopsy material) that has undergone very few population doublings compared to serial tumorigenic or artificially immortalized cell lines by "primary cells" or "primary cells," and therefore is very representative of the characteristics and main functional elements of the tissues from which they originated.

[0258] As examples but not limited to, 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; and Molt 4 cells.

[0259] All of these cell lines may be modified by the method of the present invention to provide cell line models for producing, expressing, quantifying, detecting, and studying a gene or protein of interest: these models may also be used to screen biologically active molecules of interest in production and research in various fields such as chemicals, biofuels, therapeutics, and agronomy, with examples not limited to these.

[0260] - Substitution, deletion, or insertion of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, forty, fifty (fifty) or more nucleotides / amino acids is intended by a "mutation." A mutation can affect the coding sequence of a gene or its regulatory sequence. It can also affect the structure / stability of the encoded mRNA or the structure of the genome sequence.

[0261] - By "variant(s)," it is intended to be a polypeptide variant, TALE-nuclease variant, DNA binding variant, variant, or repeat variant obtained by mutation or replacement of at least one residue within the amino acid sequence of the parent molecule.

[0262] - By "functional variant," a catalytically active mutant of a protein or protein domain is intended; such mutant may have the same activity as its parent protein or protein domain or additional characteristics, or higher or lower activity.

[0263] - The term "gene" refers to the basic unit of heredity, consisting of a portion of DNA arranged linearly along a chromosome that codes for a specific protein or segment of a protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, and a 3' untranslated region. A gene may further include a terminator, enhancers, and / or silencers.

[0264] - As used herein, the term “locus” is a specific physical location of a DNA sequence (e.g., of a gene) on the genome. The term “locus” may refer to a specific physical location of a rare-cleavage endonuclease target sequence on the genome. Such a locus may include a target sequence recognized and / or cleaved by a rare-cleavage endonuclease according to the present invention. It is understood that the locus of interest of the present invention may qualify as a nucleic acid sequence existing within the main body of the genetic material of a cell (i.e., within the genome), as well as, as not limited to, a portion of the genetic material that may exist independently of said main body of the genetic material, such as within organelles like mitochondria or, as examples, plasmids, episomes, viruses, and transposons.

[0265] - The term "endonuclease" refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably within a DNA molecule. Endonucleases do not cleave DNA or RNA molecules despite their sequences, but recognize and cleave DNA or RNA molecules at specific polynucleotide sequences, which are further referred to as "target sequences" or "target sites." Endonucleases may be classified as rare-cleaving endonucleases when they have polynucleotide recognition sites that are typically longer than 12 base pairs (bp), more preferably 14 to 55 bp. Rare-cleavage endonucleases significantly increase HR by inducing DNA double-strand breaks (DSBs) at defined locus (Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007). Rare-cleavage endonucleases can be homing endonucleases (Paques and Duchateau 2007), chimeric zinc-finger nucleases (ZFNs) resulting from the fusion of engineered zinc-finger domains of the catalytic domains of restriction enzymes such as FokI (Porteus and Carroll 2005), or chemical endonucleases (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). In chemical endonucleases, a chemical or peptide cleaver is conjugated to another DNA or a polymer of nucleic acids that recognizes a specific target sequence.Chemical endonucleases also include synthetic nucleases such as triplex-forming oligonucleotides (TFOs), DNA cleaving molecules, and conjugates of orthophenanthroline, which are known to bind to specific DNA sequences (Kalish and Glazer 2005). These chemical endonucleases are included within the term "endonuclease" according to the present invention.

[0266] Rare-cleavage endonucleases may also be TALE-nucleases, a novel class of chimeric nucleases utilizing DNA binding domains derived from Transcription Activator-Like Effector (TALE) and FokI catalytic domains, families of proteins used in the infection process by plant pathogens of the genus Xanthomonas, for example (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al.). The functional layout of FokI-based TALE-nucleases (TALE-nucleases) is a zinc-finger DNA binding domain replaced by a TALE domain, which is essential to that of ZFNs. As such, DNA cleavage by TALE-nucleases requires two DNA recognition regions flanking a non-specific central region. The rare-cleavage endonucleases included in the present invention may also be derived from TALE-nucleases.

[0267] Rare-cleavage endonucleases may be homing endonucleases and may also be known by the name meganuclease. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and create single- or double-strand breaks. Homing endonucleases are highly specific and recognize DNA target sites ranging in length from 12 to 45 base pairs (bp), usually in the range of 14 to 40 bp. The homing endonucleases according to the present invention correspond, for example, to LAGLIDADG endonucleases, HNH endonucleases, or GIY-YIG endonucleases. The preferred homing endonuclease according to the present invention may be an I-CreI variant.

[0268] - A fusion protein is intended by "TALE-nuclease" (TALEN), comprising a nucleic acid-binding domain typically derived from a Transcription Activator-like Effector (TALE) and a nuclease-catalytic domain for cleaving a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, such as I-TevI, ColE7, NucA, and Fok-I. In certain examples, the TALE domain may be fused to a meganuclease or its functional variant, such as I-CreI and I-OnuI. In a more preferred example, the nuclease is a monomeric TALE-nuclease. Monomer TALE-nucleases are TALE-nucleases that do not require dimerization for specific recognition and cleavage, such as the catalytic domain of I-TevI ​​described in WO2012138927 and fusions of engineered TAL repeats. Transcription activator-like effector (TALE) is a protein from the bacterial species Xanthomonas that contains numerous repeated sequences, each repeat containing two (di) residues at positions 12 and 13 (RVD) specific to each nucleotide base of the nucleic acid target sequence. Binding domains having similar modular base-per-base-nucleic acid binding properties (MBBBD) may also be derived from novel modular proteins recently discovered by the applicant in other bacterial species.Novel modular proteins have the advantage of exhibiting more sequence variability than TAL repeats. Preferably, the RVDs associated with the recognition of other nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A, YG for recognizing T, TL for recognizing A, VT for recognizing A or G, and SW for recognizing A. In another example, important amino acids 12 and 13 can be mutated toward other amino acid residues to regulate their specificity toward nucleotides A, T, C, and G, and particularly to enhance this specificity. TALE-nucleases have already been described and used to stimulate gene targeting and genetic modification (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al.). Engineered TAL-nucleases are commercially available under the trademark TALENTM (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

[0269] The term "cleavage" refers to the breakage of the covalent backbone of a polynucleotide. Cleavage can be initiated by various methods, including but not limited to the enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-stranded and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. Cleavage of double-stranded DNA, RNA, or DNA / RNA hybrids can result in the production of blunt or staggered ends.

[0270] - Translation of the "fusion gene" is intended to result in a single polypeptide having functional characteristics derived from each of the original proteins, as a result of a process well known in the art consisting of the joining of two or more genes that originally code for proteins or parts thereof separated by the "fusion protein."

[0271] "Identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions within each sequence that can be aligned for comparison purposes. When positions within the sequences being compared are occupied by the same base, then the molecules are 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 positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs, including BLAST or FASTA (University of Wisconsin, Madison, Wis.), available as part of the GCG sequence analysis package, can be used to calculate the identity between two sequences, for example, with default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity with the specific polypeptides listed herein and preferably exhibiting significantly similar functions, and polynucleotides encoding these polypeptides are considered.

[0272] - "Similarity" describes the relationship between the amino acid sequences of two or more polypeptides. BLASTP can also be used to identify amino acid sequences having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, and 99% sequence similarity to a reference amino acid sequence using a similarity matrix such as BLOSUM45, BLOSUM62, or BLOSUM80. Unless otherwise indicated, the similarity score will be based on the use of BLOSUM62. When BLASTP is used, percent similarity is based on the BLASTP positives score, and percent sequence identity is based on the BLASTP identities score. BLASTP "identities" show the fraction and number of total residues within identical, high-score sequence pairs; and BLASTP "positives" show the fraction and number of residues that are similar to each other and have positive alignment scores. Any intermediate degree of identity or amino acid sequences having such degree of identity or similarity to the amino acid sequences disclosed herein are considered and included in this disclosure. Polynucleotide sequences of similar polypeptides may be estimated using a genetic code and may be obtained by conventional means. For example, the functional variant of pTalpha has 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, and 97% of the amino acid sequence of SEQ ID NO: 107.It can have 5%, 98%, and 99% sequence similarity. Polynucleotides encoding these functional variants will be produced by back-translating their amino acid sequences using the genetic code.

[0273] - "Signal transduction domain" or "co-stimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a co-stimulatory molecule on the T-cell and provides a signal that mediates a T-cell response, including but not limited to proliferation, activation, differentiation, etc., in addition to the primary signal provided by the binding of the TCR / CD3 complex to an MHC molecule loaded with a peptide, for example. Co-stimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligands (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptors, 3 / TR6, ILT3, ILT4, antibodies binding to agonist or Toll ligand receptors, and ligands specifically binding to B7-H3. Co-stimulatory ligands also include, among others, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, It includes antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83.

[0274] "Co-stimulatory molecules" refer to cognate binding partners on T cells that specifically bind to co-stimulatory ligands and mediate co-stimulatory responses by cells, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to, HC class I molecules, BTLAs, and Toll ligand receptors.

[0275] As described herein, "co-stimulatory signals" refer to signals that, in combination with primary signals such as TCR / CD3 ligation, lead to the upregulation or downregulation of key molecules and / or T cell proliferation.

[0276] - "Bispecific antibody" refers to an antibody having binding sites for two different antigens within a single antibody molecule. It will be recognized by those skilled in the art that, in addition to the canonical antibody structure, other molecules can be constructed to have two binding specificities. Furthermore, it will be recognized that antigen binding by bispecific antibodies can be simultaneous or sequential. Bispecific antibodies can all be produced by chemical techniques that are themselves well known (e.g., Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78, 5807), by “polydoma” techniques (see US Pat. No. 4,474,893), or by recombinant DNA techniques. As a non-limiting example, each binding domain comprises at least one variable region ("VH or H region") from the heavy chain of the antibody, wherein the VH region of the first binding domain specifically binds to a lymphocyte marker such as CD3, and the VH region of the second binding domain specifically binds to a tumor antigen.

[0277] - As used herein, the term "extracellular ligand-binding domain" is defined as an oligo- or polypeptide capable of binding to a ligand. Preferably, the domain will be able to interact with cell surface molecules. For example, the extracellular ligand-binding domain may be selected to recognize ligands that act as cell surface markers on target cells associated with specific disease states. Examples of cell surface markers that can act as ligands in this way include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0278] The terms "subject" or "patient" as used herein include all members of the animal kingdom, including non-human primates and humans.

[0279] The above-described description of the present invention provides a process and method of using and making it, so that a person skilled in the art can make and use the same as this enablement, which is particularly provided for the subject matter of the appended claims forming part of the original specification.

[0280] When numerical limits or ranges are mentioned herein, their endpoints are also included. Additionally, all values ​​and subranges within the numerical limits or ranges are specifically included as explicitly stated.

[0281] The foregoing description is provided for those skilled in the art to make and use the present invention and is provided within the scope of specific applications and their requirements. Various variations of the preferred examples will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to applications and other examples without departing from the scope and subject matter of the present invention. In this way, the present invention is not intended to be limited to the examples shown, but will be in accordance with the broadest scope consistent with the features and principles disclosed herein.

[0282] The invention is described generally, and further understanding will be obtained by referring to specific embodiments, which are provided herein for illustrative purposes only and are not intended to be limited unless otherwise specified.

[0283] Examples

[0284] Example 1: TALE-nucleases that cleave the human GR gene

[0285] Six heterodimeric TALE-nucleases targeting exons of the human GR gene were designed and produced. Table 2 below indicates the target sequences cleaved by each TALE-nuclease. The GR TALE-nuclease comprises two independent entities (called half TALE-nucleases), each containing a repeat sequence engineered to bind to and cleave GR target sequences consisting of two 17-bp long sequences (called half targets) separated by a 15-bp spacer.

[0286]

[0287] Table 2: Sequences of TALE-nuclease target sites within the human GR gene and description of GR TALE-nucleases.

[0288] The amino acid sequences of the repeat and N-terminal and C-terminal domains are based on AvrBs3 TALE (Ref.: GenBank: X16130.1). The C-terminal and N-terminal domains are separated by two BsmBI restriction sites. Repeat arrays (Sequence Nos. 7 to 18) targeting the desired sequences (Sequence Nos. 1 to 6) were synthesized using a solid-supported method comprising consecutive restriction / ligation / washing steps (International PCT application WO2013 / 017950). Simply put, the first block (coding for a di-repeat) was immobilized on a solid support via biotin / streptavidin interactions, the second block (a tri-repeat) was then ligated to the first, and the third block (a tri-repeat) was coupled after SfaNI digestion. The process was repeated using tri- or di-repeat blocks to obtain the desired repeat array. The product was then cloned and sequenced within a traditional pAPG10 cloning plasmid for amplification in E. coli. Therefore, the obtained repeat array sequences were subcloned into a yeast expression TALE vector using the type IIS restriction enzymes BsmBI to receive the plasmid and BbvI and SfaNI for the inserted repeat sequences. DNA encoding a half TALE-nuclease, containing a TALE-derived DNA binding domain fused to the catalytic domain of the FokI restriction enzyme, was amplified in E. coli, recovered by standard miniprep techniques, and sequenced to evaluate the integrity of the insert.

[0289] Activity of TALE-nucleases in yeast :

[0290] The nuclease activities of six GR-TALE-nucleases were tested at 37 °C and 30 °C in our aforementioned yeast SSA assay on targets comprising two opposing TALE target sequences on DNA strands separated by a 15 bp spacer causing SEQ NOs: 1 to 6 (International PCT Applications WO 2004 / 067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006). All yeast target reporter plasmids containing TALE-nuclease DNA target sequences were constructed as previously described (International PCT Applications WO 2004 / 067736 and in (Epinat, Arnould et al. 2003; Chames, Epinat et al. 2005; Arnould, Chames et al. 2006; Smith, Grizot et al. 2006). The TALE-nuclease cleavage activity levels of individual clones on the targets in yeast are shown in Table 3.

[0291]

[0292] The values ​​are between 0 and 1. The maximum value is 1.

[0293] Table 3: Cleavage activity of GR TALE-nucleases in yeast.

[0294] In HEK293 cells GR TALE-nuclease activity :

[0295] Each The TALE-nuclease construct is pEF1 alphaIt was subcloned in mammalian expression vectors using restriction enzyme digestion under the control of a length promoter.

[0296] One million HEK293 cells were seeded one day prior to transfection. Following the manufacturer's instructions, they were co-transfected with 2.5 μg each of two plasmids encoding the left and right halves of GRex2, GRex3T2, GRex3T4, GRex5T1, GRex5T2, or GRex5T3 TALE-nucleases, which recognize the genomic sequences of the two half targets of interest within the GR gene under the control of the EF1 alpha promoter, using 25 μL of lipofectamine (Invitrogen). As a control group, cells were co-transfected with 2.5 μg each of two plasmids encoding the left and right halves of TALE-nucleases targeting the T-cell receptor alpha constant chain region (TRAC_T01) under the control of the EF1 alpha promoter (TRAC_T01-L and -R TALE-nucleases (SEQ No. 41 and SEQ No. 42, TRAC_T01 target site (SEQ No. 37)). Double-strand breaks caused by TALE-nucleases within the GR coding sequence induce non-homologous end joining (NHEJ), which is an error-prone mechanism. The activity of TALE-nucleases is measured by the frequency of deletions or insertions at targeted genomic locations.

[0297] Two or seven days after transfection, cells were harvested, and locus-specific PCRs were performed on extracted genomic DNA using the following primers: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-3' (forward adapter sequence)-10N (TAG)- locus-specific forward sequence for GR exon 2: 5'-GGTTCATTTAACAAGCTGCC-3' (SEQ No. 31), for GR exon 3: 5'-GCATTCTGACTATGAAGTGA-3' (SEQ No. 32), and for GR exon 5: 5'-TCAGCAGGCCACTACAGGAGTCTCACAAG-3' (SEQ No. 33), and reverse primer 5'-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-3' (Reverse adapter sequence) - Locus-specific reverse sequences for GR exon 2: 5'-AGCCAGTGAGGGTGAAGACG-3' (SEQ No.: 34), for GR exon 3: 5'-GGGCTTTGCATATAATGGAA-3' (SEQ No.: 35) and for GR exon 5: 5'-CTGACTCTCCCCTTCATAGTCCCCAGAAC-3' (SEQ No.: 36).

[0298] PCR products were sequenced using a 454 sequencing system (454 Life Sciences). Nearly 10,000 sequences were obtained per PCR product, and the presence of site-specific insertion or deletion events was analyzed. Table 4 shows the percentage of sequences exhibiting insertions or deletions at TALE-nuclease target sites out of the total number of sequences in the sample. GRex2, GRex3T2, and GRex3T4, the results of representative experiments, are listed in Table 4.

[0299] In all tested cases, the percentage of mutagenesis at 7 days was similar to that of the samples 2 days after transfection. The characteristics of mutagenic events were also analyzed, showing that deletions were the most frequent in all cases compared to insertions.

[0300]

[0301] Table 4: Percentage of targeted mutagenesis at endogenous TALE-nuclease target sites in HEK293 cells.

[0302] Activity of GR TALE-nucleases in primary T lymphocytes:

[0303] Each TALE-nuclease construct was subcloned in an expression vector under the control of a T7 promoter using restriction enzyme digestion.

[0304] mRNA encoding TALE-nucleases that cleave GR genome sequences was synthesized from each plasmid carrying downstream coding sequences from the T7 promoter. T lymphocytes isolated from peripheral blood were activated for 5 days using anti-CD3 / CD28 activator beads (Life Technologies), and 5 million cells were transfected by electroporation with 10 μg each of 2 mRNAs encoding both half TALE-nucleases using the CytoLVT-P instrument (BTX-Harvard apparatus). T cells transfected with 10 μg each of two mRNAs encoding both half TALE-nucleases targeting the CD52 gene (CD52_T02-L and -R TALEN (sequence numbers: 55 and 56), target sequence CD52_T02 sequence number: 40) are used as a control.

[0305] Three and seven days after transfection, genomic DNA was isolated from the transfected cells, and locus-specific PCRs were performed using the aforementioned primers. The PCR products were sequenced using a 454 sequencing system (454 Life Sciences). Nearly 10,000 sequences were obtained per PCR product, and the presence of site-specific insertion or deletion events was analyzed; the results are shown in Table 5.

[0306]

[0307] Table 5: Percentage of targeted mutagenesis at endogenous TALE-nuclease target sites of primary T lymphocytes.

[0308] Example 2: TALE-nucleases that cleave the human CD52 gene, the human T-cell receptor alpha constant chain (TRAC), and the human T-cell receptor beta constant chains 1 and 2 (TRBC).

[0309] As described in Example 1, heterodimeric TALE-nucleases targeting the CD52, TRAC, and TRBC genes, respectively, were designed and produced. The targeted genomic sequences consist of two 17-bp sequences (referred to as half targets) separated by an 11 or 15-bp spacer. Each half target is recognized by repeats of the half TALE-nucleases listed in Table 6. The human genome contains two functional T-cell receptor beta chains (TRBC1 and TRBC2). During the development of alpha / beta T lymphocytes, one of these two constant chains is selected in each cell to splice to the variable region of the TCR-beta and form a functional full-length beta chain. 2 TRBC targets were selected from conserved sequences between TRBC1 and TRBC2, so the corresponding TALE-nuclease will cleave both TRBC1 and TRBC2 simultaneously.

[0310]

[0311] Table 6: Sequences of TALE-nuclease target sites in corresponding human genes and description of CD52, TRAC, and TRBC TALE-nucleases.

[0312] Other target sequences within the TRAC and CD52 genes were designed, as shown in Table 7.

[0313]

[0314] Table 7: Additional target sequences for TRAC and CD52 TALE-nucleases.

[0315] Activity of CD52-TALE-nuclease, TRAC-TALE-nuclease, and TRBC-TALE-nuclease in HEK293 cells

[0316] Each TALE-nuclease construct was subcloned in a mammalian expression vector under the control of a pEF1-alpha long promoter using restriction enzyme digestion. One million HEK293 cells were seeded one day prior to transfection. Cells were co-transfected according to the manufacturer's instructions using 25 μl of lipofectamine (Invitrogen) with 5 μg of a control pUC vector (pCLS0003) or 2.5 μg each of two plasmids encoding TALE-nucleases that recognize two half targets within the genomic sequence of interest in the T-cell receptor beta constant chain region (TRBC), T-cell receptor alpha constant chain region (TRAC), or CD52 gene under the control of an EF1-alpha promoter. Double-strand cleavages produced by TALE-nucleases within CD52 or TRAC coding sequences are repaired in living cells by non-homologous end joining (NHEJ), which is an error-prone mechanism. In living cells, the activity of TALE-nucleases is measured by the frequency of insertions or deletions at targeted genomic loci.48 hours after transfection, genomic DNA was isolated from these transfected cells, and locus-specific PCRs were performed using the following primers: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG (forward adapter sequence)-10N (TAG)- locus-specific forward sequence, for CD52: 5'-CAGATCTGCAGAAAGGAAGC-3' (SEQ No. 66), for TRAC: 5'-ATCACTGGCATCTGGACTCCA-3' (SEQ No. 67), for TRBC1: 5'-AGAGCCCCTACCAGAACCAGAC-3' (SEQ No. 68), or for TRBC2: 5'-GGACCTAGTAACATAATTGTGC-3' (SEQ No. 69), and reverse primers 5'-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG (reverse adapter sequence) - endogenous locus-specific reverse sequence, for CD52: 5'-CCTGTTGGAGTCCATCTGCTG-3' (Sequence No. 70), for TRAC: 5'-CCTCATGTCTAGCACAGTTT-3' (Sequence No. 71), for TRBC1 and TRBC2: 5'-ACCAGCTCAGCTCCACGTGGT-3' (Sequence No. 72). PCR products were sequenced by the 454 Sequencing System (454 Life Sciences). Nearly 10,000 sequences were obtained per PCR product and the presence of site-specific insertion or deletion events was analyzed; the results are in Table 8.

[0317]

[0318] Table 8: Percentages of insertions and deletions for TALE nucleases targeting CD52_T02, TRAC_T01, TRBC_T01, and TRBC_T02 targets.

[0319] Activity of CD52-TALE-nuclease, TRBC-TALE-nuclease, and TRAC-TALE-nuclease in primary T lymphocytes

[0320] Each TALE-nuclease construct was subcloned in a mammalian expression vector under the control of a T7 promoter using restriction enzyme digestion.

[0321] mRNA encoding TALE-nucleases that cleave CD52 TRAC and TRBC genomic sequences was synthesized from a plasmid carrying downstream coding sequences from the T7 promoter. T lymphocytes isolated from peripheral blood were activated for 5 days using anti-CD3 / CD28 activator beads (Life Technologies), and 5 million cells were then transfected by electroporation with 10 μg each of two mRNAs encoding both half TALE-nucleases (or RNA not encoding as controls) using the CytoLVT-P instrument. As a result of insertions and deletions induced by NHEJ, the coding sequences for CD52 and / or TRAC will be out of frame in the fraction of cells causing non-functional genes. Five days after electroporation, cells were labeled with fluorochrome-conjugated anti-CD52 or anti-TCR antibodies by flow cytometry for the presence of CD52 or TCR on their cell surfaces. Since all expanded T lymphocytes from peripheral blood normally expressed CD52 and TCR, the proportion of CD52-negative or TCR-negative cells is a direct measure of TALE-nuclease activity. The results of representative experiments are listed in Table 9. Table 10 shows the results of representative experiments testing the efficiency of TRBC TALE-nucleases.

[0322]

[0323] Table 9: Percentages of CD52-negative, TCR-negative, and CD52 / TCR-double-negative T lymphocytes after transfection with corresponding TALE-nuclease-expressing polynucleotides.

[0324]

[0325] Table 10: Percentages of TCR-negative T lymphocytes after transfection with TRBC TALE-nuclease-expressing polynucleotides.

[0326] Functional analysis of T cells with the targeted CD52 gene

[0327] The goal of CD52 gene inactivation is to make T lymphocytes resistant to anti-CD52 antibody-mediated immunosuppression. As described in the previous paragraph, T lymphocytes were transfected with mRNA encoding a TALE-nuclease that cleaves CD52. Seven days after transfection, cells were treated with 50 μg / ml anti-CD52 monoclonal antibody (or rat IgG as a control) with or without 30% rabbit complement (Cedarlane). After incubation at 37°C for 2 hours, cells were labeled with a fluorochrome-conjugated anti-CD52 antibody along with a fluorescent viability dye (eBioscience) and analyzed by flow cytometry to determine the frequency of CD52-positive and CD52-negative cells among the living cells. Figure 6 shows the results of a representative experiment, which indicates that CD52-negative cells are completely resistant to complement-mediated anti-CD52 antibody toxicity.

[0328] Functional analysis of T cells with targeted TRAC gene

[0329] The goal of TRAC gene inactivation is to make T lymphocytes unresponsive to T-cell receptor stimulation. As described in the previous paragraph, T lymphocytes were transfected with mRNA encoding a TALE-nuclease that cleaves TRAC or CD52. Sixteen days after transfection, the cells were treated with up to 5 μg / ml of phytohemagglutinin (PHA, Sigma-Aldrich), a T-cell mitogen that acts through T-cell receptors. Cells possessing functional T-cell receptors should increase in size after PHA treatment. Three days after culture, the cells were labeled with fluorochrome-conjugated anti-CD52 or anti-TCR antibodies and analyzed by flow cytometry to compare cell size distributions between TCR-positive and TCR-negative cells, or between CD52-positive and CD52-negative cells. Figure 7 shows that TCR-positive cells increased in size significantly after PHA treatment, whereas TCR-negative cells had the same size as untreated cells, indicating that TRAC inactivation made them unresponsive to TCR signaling. In contrast, CD52-positive and CD52-negative cells increased in size to the same degree.

[0330] Functional analysis of T cells with targeted CD52 and TRAC genes

[0331] To demonstrate that genomic modification does not affect the ability of T cells to exhibit anti-tumor activity when provided to a chimeric antigen receptor (CAR), we transfected T cells treated with TRAC-TALE-nuclease and CD52-TALE-nuclease with 10 μg of RNA encoding the anti-CD19 CAR (SEQ ID: 73). After 24 hours, the T cells were cultured with CD19-expressing Daudi cells for 4 hours. Cell surface upregulation of CD107a, a marker of cytotoxic granule release (called degranulation) by T lymphocytes, was measured by flow cytometry analysis (Betts, Brenchley et al. 2003). The results are included in Figure 8 and show that CD52-negative / TCRαβ-negative cells and CD52-positive / TCRαβ-positive cells have the same degranulating ability in response to CD19+ Daudi cells or PMA / ionomycin (positive control). CD107 upregulation is dependent on the presence of CD19+. These data suggest that genomic manipulation does not have a negative effect on the ability of T cells to mount a controlled anti-tumor response.

[0332] Genomic safety of CD52-TALE-nuclease and TRAC-TALE-nuclease in primary T lymphocytes

[0333] Since our constructs contain nuclease subunits, an important question is whether multiple TALE-nuclease transfections can lead to off-target cleavage and genotoxicity due to mispairing of half-TALE-nucleases or 'close matches' of target sequences. To estimate the effects of TRAC-TALE-nuclease and CD52-TALE-nuclease on the integrity of cellular genomes, we listed sequences in the human genome that show potential for off-site cleavage. To create this list, we examined all sequences within the genome with up to 4 substitutions compared to the original half targets and identified potential half target pairs in a head-to-head orientation with a spacer of 9 to 30 bp from each other. This analysis includes sites potentially targeted by heterodimers formed by one TRAC half-TALE-nuclease and one CD52 half-TALE-nuclease, or by homodimers of one half-TALE-nuclease molecule. We scored the potential of off-site targets based on specificity data, considering the position of the substitutions (where mismatches tolerate bases better at the 3' end of the half target) and the values ​​of each substitution. We obtained 173 unique sequences with scores reflecting the estimation that they would be cleavage.We selected 15 high-scoring cells and simultaneously analyzed the frequencies of mutations found at these loci by deep sequencing in T cells transfected with CD52 and TRAC TALE nucleases and purified by magnetic separation to be CD52-negative and TCRαβ-negative. The results are shown in Figure 9. The highest frequency of insertions / deletions was 7x10. -4 These results suggest an estimated offsite target that is at least 600 times less mutagenic than the intended targets. Therefore, the TALE-nuclease reagents used in this study appear to be extremely specific.

[0334] Example 3: TALE-nucleases cleaving the human PDCD1 gene and the human CTLA4 gene

[0335] As described in Example 1, heterodimeric TALE-nucleases targeting the PDCD1 and CTLA4 genes, respectively, were designed and produced. The targeted genomic sequences consist of two 17-bp long sequences (referred to as half targets) separated by an 11 or 15-bp spacer. Each half target is recognized by repeats of the half TALE-nucleases listed in Table 11.

[0336]

[0337] Table 11: Sequences of TALE-nuclease target sites in corresponding human genes and description of CTLA4 and PDCD1 TALE-nucleases.

[0338] Activity of CTLA4-TALE-nuclease and PDCD1-TALE-nuclease in HEK293 cells

[0339] Each TALE-nuclease construct was subcloned in a mammalian expression vector under the control of a pEF1-alpha long promoter using restriction enzyme digestion. One million HEK293 cells were seeded one day prior to transfection. Cells were co-transfected with 5 μg of a control pUC vector (pCLS0003) or 2.5 μg each of two plasmids encoding TALE-nucleases that recognize two half targets within the genomic sequences of interest in the PDCD1 and CTLA-4 genes under the control of an EF1-alpha promoter, using 25 μl of lipofectamine (Invitrogen) as per the manufacturer's instructions.

[0340] Double-strand cleavage by TALE-nucleases within PDCD1 or CTLA-4 coding sequences is repaired in living cells by non-homologous end joining (NHEJ), which is an error-prone mechanism. The activity of TALE-nucleases in living cells is measured by the frequency of insertions or deletions at targeted genomic loci.48 hours after transfection, genomic DNA was isolated from the transfected cells, and locus-specific PCRs were performed using the following primers: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG (forward adapter sequence)-10N (TAG)- locus-specific forward sequence, for CTLA4_T01: 5'-CTCTACTTCCTGAAGACCTG-3' (SEQ No. 99), for CTLA4_T03 / T04: 5'-ACAGTTGAGAGATGGAGGGG-3' (SEQ No. 100), for PDCD1_T01: 5'-CCACAGAGGTAGGTGCCGC-3' (SEQ No. 101), or for PDCD1_T03: 5'-GACAGAGATGCCGGTCACCA-3' (SEQ ID NO: 102) and reverse primer 5'-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG (reverse adapter sequence)- endogenous locus-specific reverse sequence for CTLA4_T01: 5'-TGGAATACAGAGCCAGCCAA-3' (SEQ ID NO: 103), for CTLA4_T03 / T04: 5'-GGTGCCCGTGCAGATGGAAT-3' (SEQ ID NO: 104), for PDCD1_T01: 5'-GGCTCTGCAGTGGAGGCCAG-3' (SEQ ID NO: 105) or for PDCD1_T03: 5'-GGACAACGCCACCTTCACCT-3' (SEQ ID NO: 106).

[0341] PCR products were analyzed by T7-endonuclease assay: Briefly, after denaturation and reannealing of the PCR products, T7 endonuclease will specifically digest mismatched DNA containing wild-type and mutated strands. The digested products are then resolved by polyacrylamide gel electrophoresis. The presence of digested products indicates mutated sequences induced by TALE-nuclease activity. The results are shown in Figure 10, where arrows indicate the digested PCR products. They demonstrate that PDCD1_T1, PDCD1_T3, CTLA4_T1, CTLA4_T3, and CTLA4_T4 TALE-nucleases all exhibit mutagenic nuclease activity at their target sites.

[0342] CTLA4 Inactivation in Primary T Cells:

[0343] Human primary T cells were activated with CD3 / 28 beads. After 5 days, 5x10 6 Cells were electroporated with 20 μg of RNA encoding one of three TALENTMs (T1, T2, and T3) designed for the CTLA4 gene, or without RNA as a control. Three days after electroporation, CTLA4 expression was measured by intracellular staining using fluorescent antibodies and flow cytometry analysis (Figs. 27 and 28).

[0344] Three TALENs TM All of them induced downregulation of CTLA4 expression in HEK293 cell lines in a manner related to their efficiency (T1 was more effective than T3 and T4).

[0345] Deep sequencing analysis of genomic DNA isolated from transfected cells using 454 technology (Roche) revealed that 96% of CTLA4 alleles were mutated in TALEN T1-treated cells compared to 0.1% in control samples without TALEN.

[0346] PD1 Inactivation in Primary T Cells:

[0347] Human primary T cells were activated with CD3 / 28 beads. 5x10 after 10 days 6 Cells were electroporated without RNA as a control or with 20 μg of RNA encoding one of two TALENs specific to the human PD1 gene. After 10 days, the cells were reactivated, and 3 days after reactivation, PD1 expression was measured by surface staining using fluorescent antibodies and flow cytometry analysis (Fig. 29).

[0348] Both TALENs induced significant downregulation of PD1 expression. Deep sequencing analysis of genomic DNA isolated from cells transfected with TALEN T1 and TALEN T03, respectively, using 454 technology (Roche) revealed that 34% and 39% of PD1 alleles were mutated, respectively (results shown in Fig. 30).

[0349] Enhanced anti-tumor active PD1-TALEN treated cells:

[0350] Human primary T cells were activated with CD3 / 28 beads. 5x10 after 10 days 6Cells were electroporated with 20 μg of RNA encoding a TALEN specific to the human PD1 gene or without RNA as a control. After one week, cells were electroporated with mRNA encoding a chimeric antigen receptor specific to human CD19 or without RNA as a negative control. The following day, their antitumor activity was measured in a cellular cytotoxicity assay using HCT116 cells expressing PD1 ligand 1 (PDL1) transduced with a CD19 expression vector (vs. parental HCT116 cells as a control) or CD19+ Daudi cells (vs. K562 as a control). Cytotoxic activity was determined by comparing the viability of the control cells and the target cells. The results are shown in the diagrams of Figure 31. PD1 TALEN transfection restored cytotoxic activity against PDL1-expressing HCT116 cells and improved cytotoxic activity against Daudi cells.

[0351] Example 4: pTalpha enables CD3 surface expression in inactivated TCR alpha T lymphocytes:

[0352] Description in other preTalpha versions:

[0353] The human pTalpha gene codes for a transmembrane glycoprotein containing an extracellular Ig-like domain, a hydrophobic transmembrane domain, and a large C-terminal intracytoplasmic tail. Other versions derived from the human pTalpha glycoprotein have been designed and are listed in Table 12 and shown in Figure 11.

[0354]

[0355]

[0356] Table 12: Description of a subset of pTalpha structures

[0357] Other tested preTalpha constructs include the following:

[0358] 1) pTalpha deletion mutants: Different deletions are made in the intracellular cytoplasmic tail of the human pTalpha protein (SEQ No. 107) (containing 114 amino acids). The constructs tested include mutants and a full-length version of the protein (FL) in which 18, 48, 62, 78, 92, 110, and 114 amino acids are deleted from the C-terminus of the protein (SEQ Nos. 108 to 114).

[0359] 2) pTalpha mutants containing intracellular activation domains: FL and △48 variants fused to CD8, CD28, or 41BB intracellular activation domains at their C-terminus (SEQ ID NOs: 115 to 120).

[0360] 3) pTalpha / TCRα chimeric mutants: In one of the constructs, the intracellular domain (IC) of TCRα is fused to a tailless version (△114) of pTalpha (SEQN: 121). A second construct was also created by fusing the extracellular domain of pTalpha to the IC domains and transmembrane (TM) from TCRα (SEQN: 122).

[0361] 4) pTalpha dimerization mutants: Several mutations have been described in the literature as capable of altering the oligomerization / dimerization ability of the preTCR complex. These mutants are proposed to enable preTCR expression at the cell surface without inducing the constitutive signaling pathways (presumed to induce preTCR oligomerization). The mutants have been introduced into the pTalpha△48 variant and are as follows:

[0362] 1xMUT: W46R (Sequence No.: 123)

[0363] - 4x MUT: D22A, K24A, R102A, R117A (Sequence No.: 124)

[0364] Activation of other preTalpha constructs in TRAC-inactivated Jurkat cells:

[0365] To screen other pTalpha variants for their ability to restore CD3 surface expression in TCR-alpha inactivated cells, TCR-alpha gene-disrupted cell lines were created using TALENs targeting TRAC. Jurkat cells (T-cell leukemia cell lines) and KO cells (TCR α / β NEG ; CD3 NEGThe cells were transfected with plasmids encoding TALEN that cleave TRAC using ) and CytoPulse electroporation, and then purified by negative selection using CD3 magnetic beads. The KO population (JKT_KOx3 cells) was amplified and used for screening other pTalpha variants. Screening was performed by transfecting one million JKT_KOx3 cells with 15 μg of plasmids encoding other pTalpha variants under the control of the EF1α promoter, followed by analysis of CD3 cell surface expression by flow cytometry 48 hours (h) after transfection. Figure 12 is a representative example of the activity and transfection efficiencies (%) of BFP+ cells of FL, △18, and △48 pTalpha constructs of JKT_KOx3 cells, based on the % of CD3+ cells determined by flow cytometry. Results from other constructs are grouped in Table 13.

[0366]

[0367] Table 13: Activity of other pT-alpha constructs in Jurkat TCR-alpha inactivated cells.

[0368] CD3 expression activity in Jurkat TCR alpha-inactivated cells transfected with other preTalpha constructs was measured by flow cytometry analysis.

[0369] Activation of pTalpha-FL and pTalpha-△48 in TCR alpha-inactivated primary T lymphocytes:

[0370] Inducing CD3 surface expression in TCR alpha-inactivated T lymphocytes To test the activity of the pTalpha-FL and pTalpha-△48 versions, the pTalpha-FL and pTalpha-△48 coding sequences were cloned into a self-inactivating pLV-SFFV-BFP-2A-PCTRA lentiviral vector encoding a blue fluorescent protein (BFP) under a self-cleaved T2A peptide following the SFFV promoter (Fig. 13).

[0371] T lymphocytes isolated from peripheral blood were activated for 72 hours using anti-CD3 / CD28 activator beads (Life Technologies), and 4.5 million cells were transfected by electroporation with 10 μg of mRNA encoding a TALE-nuclease targeting the TCR alpha constant chain region (TRAC) using the CytoLVT-S instrument (BTX-Harvard Harbour). Two days after electroporation, T cells were transduced with LV-SFFV-BFP-2A-pTalpha-△48 or LV-SFFV-BFP-2A-control lentiviral vectors. CD3-negative and CD3low T cells were then purified using anti-CD3 magnetic beads (Miltenyi Biotech). The experimental protocol is shown in Figure 14A.

[0372] Figure 14B shows BFP expression, CD3 cell surface expression, and flow cytometry analysis of TCR-alpha / beta on TCR-alpha inactivated T cells (KO) transduced with a control BFP lentiviral vector (KO / BFP) or BFP-2A-pTalpha△48 (KO / △48) before and after purification with CD3 beads. TCR-alpha inactivated cells transduced with the BFP-T2A-pTalpha-△48 vector (BFP+ cells) show higher levels of CD3 compared to untransduced cells (BFP- cells). No difference was observed between cells transduced with the control BFP vector. These results indicate that pT-alpha mediates the restoration of CD3 expression on the cell surface of TCR-alpha inactivated cells. In contrast, TCR alpha / beta staining remained unchanged in cells without the pTalpha-△48 expression vector or in the transduced cells, as expected.

[0373] pTalpha-mediated CD3 expression supports activation in TCR-deficient T-cells:

[0374] To determine the ability of pTalpha to transduce cell activation signals, the expression of early and later activation markers was analyzed in TCR alpha-inactivated T cells transduced with pTalpha-△48 and pTalpha-△48.41BB. TCR alpha-inactivated T cells transduced with pTalpha-△48.41BB and pTalpha-△48 were generated from primary human T cells as described in Figure 14A and the preceding section.

[0375] To detect CD3-via signaling, cells were reactivated using anti-CD3 / CD28-coated beads 3 days after purification of TCR alpha-inactivated T cells with CD3 beads (Fig. 14A). Cells were stained with fluorochrome-conjugated anti-CD69 (early activation marker) and anti-CD25 (late activation marker) and analyzed by flow cytometry 24 and 48 hours after reactivation, respectively (Figs. 15A-B). As shown in Figures 15A-B, TCR alpha-inactivated cells expressing pTalpha-△48 (KO / pTα-△48) or pTalpha-△48.41BB (KO / pTα-△48.BB) showed upregulation of activation markers at levels similar to those observed in TCR alpha / beta-expressing cells (NEP: non-electroporated cells).

[0376] Another indicator of T cell activation is an increase in cell size, sometimes referred to as "blasting." The ability of preTCR complexes to induce "blasting" was measured by flow cytometry analysis of cell size 72 hours after reactivation using anti-CD3 / CD28 beads (Fig. 15C). Stimulation with anti-CD3 / CD28 beads induced a comparable increase in cell size in cells expressing pTalpha-△48 or pTalpha-△48.41BB versus cells expressing TCR alpha / beta complexes. Taken together, these results suggest that preTCR complexes have the ability to transduce signals that effectively couple to mechanisms mediating the upregulation of activation markers.

[0377] pTalpha-mediated CD3 expression supports the expansion of TCR-deficient primary T-cells using stimulating anti-CD3 / CD28 antibodies.

[0378] To evaluate the ability of preTCR complexes to support long-term cell proliferation, the proliferation of cells produced as described above was measured. Ten days after initial activation, cells were maintained in IL2 (non-re-act) or in IL2 with anti-CD3 / CD28 beads (re-act). For each condition, cells were counted and analyzed by flow cytometry at different time points to estimate the number of BFP+ cells. The growth of TCR-alpha inactivated cells (KO) transduced with BFP-T2A-preTCRα-△48 vectors or BFP was compared, and the fold induction of these cells was estimated against values ​​obtained two days after reactivation. Figure 16 shows the results obtained from two independent donors. In both cases, TCR-alpha inactivated cells expressing pTalpha-△48 showed greater expansion than TCR-alpha inactivated cells expressing only the BFP control vector. For the second donor, TCR-alpha inactivated cells expressing full-length pTalpha (pTalpha) or pTalpha-△48.41BB were also included and showed greater expansion than TCR-alpha inactivated cells expressing only the BFP control vector.

[0379] Example 5: Optimization of mRNA transfection in T cells using Cytopulse technology.

[0380] Determination of the optimized cytopulse program

[0381] The first set of experiments was performed on inactivated PBMCs to determine the voltage range in which cells could be transfected. Five different programs were tested as listed in Table 14.

[0382]

[0383] Table 14: Different cytopulse programs were used to determine the minimum voltage required for electroporation in PBMC-derived T-cells.

[0384] 3 or 6 million cells using different Cytopulse programs with 20 μg of control plasmids pUC and plasmids encoding GFP in a 0.4 cm gap cuvette (30 or 15x10⁻⁶ 6 Cells ( / ml) were electroporated. 24 hours after electroporation, GFP expression was analyzed in the electroporated cells by flow cytometry to determine the efficiency of transfection. The data shown in Figure 17 indicate the minimum voltage required for plasmid electroporation in PBMC-derived T cells. These results demonstrate that cytopulse programs 3 and 4 enable efficient transformation of T cells (EP#3 and #4).

[0385] mRNA electroporation of activated purified T cells

[0386] After determining the optimal cytopulse program that enables efficient DNA electroporation of T cells, we tested whether this method was also applicable to mRNA electroporation.

[0387] 5x10 preactivated for 6 days with PHA / IL2 6Purified T cells were resupplying in cytoporation buffer T (BTX-Harvard apparatus) and electroporated in 0.4 cm cuvettes with 20 μg of plasmids encoding pUC or GFP or 10 μg of mRNA encoding GFP using the preferred cytopulse program as determined in the previous section (Table 15).

[0388]

[0389] Table 15: Cytopulse program used for electroporating purified T-cells.

[0390] 48 hours (h) after transfection, cells were stained with a viability dye (eFluor-450), and cell viability and the percentage of viable GFP+ cells were determined by flow cytometry analysis (Fig. 18).

[0391] The data shown in Figure 18 indicate that, under the optimal conditions determined here, RNA electroporation is non-toxic and enables transfection of more than 95% of viable cells.

[0392] In synthesis, the entire dataset demonstrates that T-cells can be efficiently transfected with DNA or RNA. In particular, RNA transfection has no effect on cell viability and enables uniform expression levels of the transfected gene of interest within the cell population.

[0393] Efficient transfection can be achieved early after cell activation, independently of the activation method used (PHA / IL-2 or CD3 / CD28-coated beads). The inventors succeeded in transfecting cells from 72 hours (h) after activation with efficiencies of >95%. Furthermore, efficient transfection of T cells after thawing and activation can also be obtained using the same electroporation protocol.

[0394] mRNA electroporation in primary human T cells for TALE-nuclease functional expression

[0395] After demonstrating that mRNA electroporation enables the efficient expression of GFP in primary human T cells, we tested whether this method could be applied to the expression of other proteins of interest. Transcription activator-like effector nucleases (TALE-nucleases) are site-specific nucleases created by the fusion of a TAL DNA binding domain to a DNA cleavage domain. They are powerful genome editing tools because they induce double-strand breaks in virtually any desired DNA sequence. These double-strand breaks activate non-homologous end-joining (NHEJ) and error-front DNA repair mechanisms, potentially leading to the inactivation of any desired gene of interest. Alternatively, if a suitable repair template is simultaneously introduced into the cells, TALE-nuclease-induced DNA damage can be repaired by homologous recombination, thus providing the possibility to modify the gene sequence at will.

[0396] We used mRNA electroporation to express a TALE-nuclease designed to specifically cleave the sequence within the human gene encoding the alpha chain of the T cell antigen receptor (TRAC). Mutations induced in this sequence are expected to cause the loss of the TCRαβ complex from the cell surface and gene inactivation. As a control, non-coding RNA or TRAC TALE-nuclease RNA was transfected into activated primary human T lymphocytes using Cytopulse technology. The electroporation sequence consisted of two pulses of 1200 V, followed by four pulses of 130 V as described in Table 15.

[0397] By flow cytometry analysis of TCR surface expression 7 days after electroporation (Fig. 19, top panel), we observed that 44% of T cells had lost TCRαβ expression. We analyzed the genomic DNA of transfected cells by PCR amplification of the TRAC locus, followed by 454 high-throughput sequencing. Of the sequenced alleles, 33% (727 out of 2153) contained insertions or deletions at the TALE-nuclease cleavage site. Fig. 19 (bottom panel) shows examples of the mutated alleles.

[0398] These data are based on cytopulse technology m This indicates that the electroporation of RNA causes the functional expression of TRAC TALE-nuclease.

[0399] Electroporation of T cells with monocistronic mRNA encoding the anti-CD19 single-strand chimeric antigen receptor (CAR):

[0400] 5X10 preactivated for several days (3-5) with IL2 and anti-CD3 / CD28 coated beads 6 T cells were resuspended in cytoporation buffer T and electroporated in 0.4 cm cuvettes with or without mRNA 10 μg of single-strand CAR (SEQ No. 73) using the program described in Table 15.

[0401] After 24 hours of electroporation, cells were stained with a PE-conjugated goat anti-mouse IgG F(ab')2 fragment and the fixable viability dye eFluor-780, which are specific for evaluating cell surface expression of CAR on live cells. The data are shown in Fig. 20. A indicates that the overwhelming majority of live T cells electroporated with the previously described monocistronic mRNA express CAR on their surface. After 24 hours of electroporation, T cells were cocultured with Daudi (CD19+) cells for 6 hours and analyzed by flow cytometry to detect the expression of the degranulation marker CD107a on their surface (Betts, Brenchley et al. 2003).

[0402] The data shown in Figure 20 indicates that a majority of the cells electroporated with the previously described monocistronic mRNA degranulate in the presence of target cells expressing CD19. These results clearly demonstrate that the CAR expressed on the surface of the electroporated T cells is active.

[0403] Electroporation of T cells with polycistronic mRNA encoding anti-CD19 multisubunit chimeric antigen receptor (CAR):

[0404] 5 x 10⁶ preactivated for several days (3-5) with IL2 and anti-CD3 / CD28 coated beads 6 T cells were electroporated in cytoporation buffer T and, using a program as described in Table 15 (coded by sequence number 125, sequence number 126, FIG. 21A and FIG. 4B (csm4)), were electroporated in 0.4 cm cuvettes with 45 μg of mRNA encoding a multi-chain CAR or without mRNA.

[0405] 24 hours after electroporation, cells were stained with a PE-conjugated goat anti-mouse IgG F(ab')2 fragment and a fixable viability dye eFluor-780, which are specific for evaluating cell surface expression of CAR on live cells. The data shown in Figure 21 indicate that the overwhelming majority of live T cells electroporated with the previously described polycistronic mRNA express CAR on their surface.

[0406] After 24 hours of electroporation, T cells were co-cultured with Daudi (CD19+) for 6 hours and analyzed by flow cytometry to detect the expression of the degranulation marker CD107a on their surface. The data shown in Figure 21 indicate that a majority of the cells electroporated with the previously described polycistronic mRNA degranulate in the presence of target cells expressing CD19. These results clearly demonstrate that the CAR expressed on the surface of the electroporated T cells is active.

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Claims

Claim 1 An in vitro method for preparing T-cells for immunotherapy comprising the following: (a) a step of inactivating at least first and second immune checkpoint proteins in said T-cells, comprising introducing into said T-cells at least first and second rare-cutting endonucleases capable of selectively inactivating first and second genes by DNA cleavage, wherein said first and second rare-cutting endonucleases target said first and second genes encoding said first and second immune checkpoint proteins, said first rare-cutting endonuclease is directed toward PD1, and said second rare-cutting endonuclease is directed toward CTLA-4, LAG3, or TIGIT; and (b) a step of expanding the T-cells, each of which expresses an endogenous T-cell receptor (TCR). Claim 2 In claim 1, the first and second rare-cleavage nucleases are TALE-nucleases, method. Claim 3 In claim 2, each of the first and second rare-cleavage endonucleases is encoded by RNA, method. Claim 4 In claim 3, a method wherein each of the first and second rare-cleavage endonucleases is introduced into the T-cell by RNA electroporation. Claim 5 In claim 4, the first TALE-nuclease is directed toward one of the gene target sequences of PD-1 selected from the group consisting of SEQ ID NO: 77 and SEQ ID NO:

78. Claim 6 A method according to claim 5, wherein (a) the first TALE-nuclease comprises amino acid sequences selected from the group consisting of (i) sequence numbers: 85 and 86, and (ii) sequence numbers: 87 and 88; and / or (b) the first TALE-nuclease comprises amino acid sequences encoded by nucleic acid sequences selected from the group consisting of (i) sequence numbers: 95 and 96, and (ii) sequence numbers: 97 and 98. Claim 7 A method according to claim 1, wherein the second TALE-nuclease is directed toward one of the gene target sequences of CTLA-4 selected from the group consisting of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO:

76. Claim 8 In claim 7, the method comprises: (a) the second TALE-nuclease comprising amino acid sequences selected from the group consisting of (i) sequence numbers: 79 and 80, (ii) sequence numbers: 81 and 82, and (iii) sequence numbers: 83 and 84; and / or (b) the second TALE-nuclease comprising amino acids encoded by nucleic acid sequences selected from the group consisting of (i) sequence numbers: 89 and 90, (ii) sequence numbers: 91 and 92, and (iii) sequence numbers: 93 and 94. Claim 9 A method according to any one of claims 1 to 8, wherein introducing the first and second rare-cleavage endonucleases into the T-cell comprises: (1) contacting the T-cell with RNA encoding the first and second rare-cleavage endonucleases; and (2) applying an agile pulse sequence composed of: (i) one electrical pulse having a pulse interval of 0.2 to 10 ms, a pulse width of 0.1 ms, and a voltage range of 2250 to 3000 V per centimeter between the electrical pulses of steps (i) and (ii); (ii) one electrical pulse having a voltage range of 2250 to 3000 V, a pulse interval of 100 ms, and a pulse width of 100 ms between the electrical pulse of step (ii) and the first electrical pulse of step (iii); and (iii) four electrical pulses of voltage of 325 V with a pulse interval of 2 ms and a pulse width of 0.2 ms between each of the four electrical pulses. Claim 10 A method according to any one of claims 1 to 8, wherein the T-cells are tumor infiltrating lymphocytes (TILs). Claim 11 T-cells obtainable by a method according to any one of claims 1 to 8. Claim 12 A population of T-cells produced by a method according to any one of claims 1 to 8 for use as a medicine for cancer treatment.

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