Immunotherapeutic cells engineered to target antigens present on both immune and pathological cells

Abstract

Methods for developing genetically engineered immune cells for immunotherapy, by inactivating in said immune cells by rare-cutting endonucleases such as TALEN, Cas9 or argonaute the genes encoding antigenic markers common to both pathological cells and said immune cells (e.g.: CD38, CS1 or CD70), said cells being capable of having chimeric antigen receptors targeting said markers.

Description

Technical Field

[0001] The present invention relates to methods for developing genetically engineered, preferably non-alloreactive, immune cells for immunotherapy having chimeric antigen receptors targeting antigenic markers shared by both pathological cells and immune cells (eg, CD38).

[0002] The method includes expressing a CAR for the antigen marker and inactivating the gene in the immune cell to facilitate the presence of the antigen marker on the surface of the immune cell. This inactivation is usually implemented by using a transgene encoding an RNA-guided endonuclease (e.g., Cas9 / CRISPR), a meganuclease, a zinc finger nuclease, or a TAL nuclease. The engineered immune cells, preferably T-cells, direct their immune activity to malignant, infected cells or defective immune cells while avoiding mutual destruction, autostimulation or aggregation. The present invention opens up a way to use standard and affordable adoptive immunotherapy strategies (adoptive immunotherapy strategy) of immune cells for the treatment of cancer, infection and autoimmune diseases. Background Art

[0003] Adoptive immunotherapy, which involves the transfer of autologous antigen-specific immune cells generated in vitro, is a promising strategy for treating viral infections and cancer. For example, T-cells used in adoptive immunotherapy can be generated by expansion of antigen-specific T-cells or by genetically engineered T-cell redirection (Park, Rosenberg et al. 2011).

[0004] New specificity in T-cells is successfully generated by genetic transfer of transgenic T-cell receptors or chimeric antigen receptors (CAR) (Jena, Dotti et al. 2010). CAR is a synthetic receptor consisting of a targeting portion associated with one or more signaling domains in a single fusion molecule. In general, the binding portion of CAR is composed of the antigen binding domain of a single-chain antibody (scFv), including the light fragment and variable fragment of a monoclonal antibody connected by a flexible linker. Binding portions based on receptor or ligand domains have also been successfully used. The signaling domain of the first generation of CAR is derived from the cytoplasmic region of the CD3ζ or the Fc receptor γ chain. The first generation of CAR has been shown to successfully redirect T-cell cytotoxicity, however, they have failed to provide prolonged amplification and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules, including CD28, OX-40 (CD134) and 4-1BB (CD137), have been added alone (second generation) or in combination (third generation) to enhance survival and increase the proliferation of CAR-modified T cells. CARs have successfully redirected T cells against antigens expressed on the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0005] The current scheme of using adoptive immunotherapy to treat patients is based on autologous cell transfer. In this method, T lymphocytes are recovered from patients, genetically modified or selected in vitro, cultured in vitro to expand the number of cells when necessary, and finally infused into patients. In addition to lymphocyte infusion, the host can be manipulated in other ways that support the implantation of the T-cell or its participation in the immune response, such as pre-conditioning (with radiation or chemotherapy) and administration of lymphocyte growth factors (such as IL-2). Each patient receives a treatment made separately, using the patient's own lymphocytes (i.e., autologous therapy). Autologous therapy faces a large number of technical logistical obstacles for practical application, and their offspring require expensive special equipment and expert personnel, they must be produced in a very short time after the patient's diagnosis, and in many cases, the patient's pre-treatment has caused a degraded immune function, making the patient's lymphocytes possibly dysfunctional and present in very low quantities. Because of these obstacles, the autologous cell preparation of each patient is effectively a new product, thereby causing efficacy and safety to change significantly.

[0006] Ideally, it would be desirable to use a standardized therapy in which allogeneic therapeutic cells could be pre-made, characterized in detail, and administered to patients immediately. Allogeneic refers to cells obtained from individuals belonging to the same species but genetically different. However, there are currently many disadvantages to the use of allogeneic cells. Allogeneic cells are rapidly rejected in immunocompetent hosts, a process known as host versus transplant rejection (HVG), which significantly limits the efficacy of the transferred cells. In immunocompetent hosts, allogeneic cells can be transplanted, but their endogenous T-cell receptor (TCR) specificity can recognize the host tissue as foreign, leading to graft versus host disease (GVHD), which can cause severe tissue damage and death.

[0007] To provide allogeneic T-cells, the present inventors have previously disclosed methods of genetically engineering T-cells in which different effector genes, specifically those encoding T-cell receptors, are expressed by using specific TAL-nucleases, under the trademark TALEN. TM (Cellectis, 8, rue de la Croix Jarry, 75013 PARIS), is better known for inactivation. This approach has been shown to be highly efficient in primary cells when using RAN transfection as part of a platform allowing large-scale production of allogeneic T-cells (WO 2013 / 176915).

[0008] CD38 (cluster of differentiation 38), also known as cyclic ADP ribose hydrolase, is a glycoprotein found on the surface of a variety of immune cells (white blood cells), specifically T-cells, including CD4+, CD8+, B lymphocytes and natural killer cells. CD38 plays a role in cell adhesion, signal transduction and calcium signaling. Structural information about this protein can be found in the UniProtKB / SWISS-PROT database under document P28907. In humans, the CD38 protein is encoded by the CD38 gene located on chromosome set 4. CD38 is a multifunctional extracellular enzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose (cADPR) from NAD+ to ADP-ribose. These reaction products are believed to be responsible for the intracellular Ca 2+ In addition, the loss of CD38 function is associated with impaired immune response and metabolic disorders (Malavasi F., et al. (2008). "Evolution and function of the ADP ribosyl cyclase / CD38 gene family in physiology and pathology". Physiol. Rev. 88(3): 841-86).

[0009] In another aspect, CD38 protein is a marker for HIV infection, leukemia, myeloma, solid tumors, type II diabetes and bone metabolism and some other genetically determined disorders. Specifically, it is used as a prognostic marker in leukemia (Ibrahim, S. et al. (2001) CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 98: 181-186).

[0010] Although cells expressing CD38 and many other tumor antigen markers indicated in Table 1, such as CD70 and CS1, can be attractive targets for CAR, the fact that this antigen marker is also expressed on the surface of most T-cells has significantly hampered the selection of these markers for the implementation of immunotherapy.

[0011] The inventors herein provide a strategy for immunotherapy involving pathological cells expressing specific antigenic markers also present on the surface of T-cells, such as, for example, malignant CD38-positive B-cells that cause leukemia, CD70 and CS1. Summary of the invention

[0012] The methods disclosed herein are used to engineer T-cells that are intended to target pathological cells that express one or more antigenic markers that are also present on the surface of T-cells. Examples of such antigenic markers can be found in Table 1. An example of such an antigenic marker is CD38. Other examples are CD70 and CS1. Antigenic markers refer to the entire protein of its immunologically active fragments.

[0013] According to the invention, the T-cells are engineered to inactivate the expression of a gene encoding such an antigenic marker, or to participate in the presentation of such an antigenic marker on the cell surface.

[0014] This inactivation is preferably carried out by genome modification, more specifically by expression in T-cells of specific rare-cutting endonucleases that are able to target gene sites directly or indirectly involved in the generation or presentation of the antigenic marker on the surface of the T-cells. Different types of rare-cutting endonucleases can be used, such as meganucleases, TAL-nucleases, zinc finger nucleases (ZFNs), or RNA / DNA-guided endonucleases such as Cas9 / CRISPR or Argonaute.

[0015] According to a preferred embodiment, said T-cells are endowed with at least one chimeric antigen receptor (CAR) allowing specific binding of said cells carrying said targeted antigen marker.

[0016] According to another embodiment, the T-cells can be further engineered to become allogeneic, in particular by deleting genes involved in self-recognition, such as, for example, those encoding components of the T-cell receptor (TCR) or the HLA complex.

[0017] The present invention encompasses isolated cells or cell lines comprising the genetic modifications set forth in the detailed description, examples and figures, as well as any protein, polypeptide or vector for engineering the T-cells.

[0018] Thanks to the present invention, the engineered T-cells can be used as therapeutic products in methods of treating or preventing cancer, infection or autoimmune disease, ideally as "off-the-shelf" products.

[0019] Preferred immune cells according to the present invention are those that produce the phenotype described below:

[0020] ·[CAR targeting the antigen markers in Table 1] + [Antigen markers in Table 1] -

[0021] Such as the following:

[0022] ·[CAR CD38] + [CD38] - , and preferably [TCR] negative;

[0023] ·[CAR CD70] + [CD70] - , and preferably [TCR] negative;

[0024] ·[CAR CS1] + [CS1] - , and preferably [TCR] negative;

[0025] They are used as therapeutic products, preferably allogeneic ones. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Brief description of the figures and tables

[0027] Figure 1 : Schematic representation of engineered T cells that have CD38 destruction and are endowed with a chimeric antigen receptor (represented as a single-chain CAR) that targets malignant cells carrying the antigen marker CD38 according to the present invention.

[0028] Figure 2 : Schematic representation of a multi-subunit chimeric antigen receptor.

[0029] Figure 3: Schematic representation of a therapeutic strategy for binding T-cells endowed with multi-subunit CAR and circulating bispecific antibodies according to the present invention. In this specific aspect, the receptor present on the extracellular chain of the multi-subunit CAR is composed of an epitope recognized by a bispecific antibody. The bispecific antibody is intended to bind to the epitope on the one hand and to bind to the antigen marker on the other hand to facilitate the binding of the T-cell to the pathological cell.

[0030] Figure 4 : A schematic illustration of a therapeutic strategy for combining T-cells endowed with multi-subunit CARs and circulating monoclonal antibodies according to the present invention. In this specific aspect, the receptor present on the extracellular chain of the multi-subunit CAR is, for example, composed of an Fc receptor intended to bind a monoclonal antibody directed against the antigen marker. The monoclonal antibody increases the chance of T-cells binding to pathological cells.

[0031] Figure 5 : A schematic illustration of a therapeutic strategy according to the present invention combining a T-cell endowed with a multi-subunit CAR containing two extracellular cell domain structures and a circulating bispecific antibody. In this specific aspect, the extracellular cell domains are located on different subunits. These domains are composed of an epitope recognized by a bispecific antibody and a receptor for a targeted antigen, respectively. The receptor is directed to a first antigen marker, and the bispecific antibody is intended to bind an epitope and a second antigen marker. The display is intended to selectively target pathological cells carrying the first and second antigen markers on their surface.

[0032] Figure 6 : Shows something like Figure 5 , but the stimulatory and co-stimulatory domains (4-1BB and CD3ζ protein domains, respectively) have been exchanged to modulate the intensity of activation of the T-cells caused by the binding of the chimeric antigen receptor to the pathological cells.

[0033] Figure 7 : Shows something like Figure 5 , but the stimulatory and co-stimulatory domains (4-1BB and CD3ζ protein domains, respectively) have been exchanged and a CD3ζ domain has been added to increase the strength of activation of the T-cells caused by the binding of the chimeric antigen receptor to the pathological cells.

[0034] Figure 8: A schematic illustration of a therapeutic strategy according to the present invention combining a T-cell endowed with a multi-subunit CAR containing two extracellular cell domains and a circulating monoclonal antibody. In this specific aspect, the extracellular cell domains are located on different subunits. These domains are respectively composed of an antigen binding domain targeting an antigen marker and an Fc receptor intended to bind a monoclonal antibody to a second antigen marker. The display is intended to selectively target pathological cells carrying the first and second antigen markers on their surface.

[0035] Fig. 9 : CD38 expression by activated T-cells. A. CD38 expression by T-cells at day 6 after activation with CD3 / CD28 coated beads + IL2. B. Longitudinal analysis of CD38 expression by T-cells over a 17 day period after activation.

[0036] Figure 10: Knockout (KO) on the CD38 gene: A. Location on the CD38 exon 1 sequence of the three different TALENs (T2, T4 and T5) designed to knock out Cd38 in T-cells. B. Expression of CD38 in T-cells after transfection with TALEN CD38ex1_T2. C. Controlled CD38 staining for purification of CD38 KO T-cells.

[0037] Fig.11 : CD38CAR: A. Schematic representation of the three versions of CAR designed. B. CD38 expression levels by the target cell lines.

[0038] Fig.12 : Timing experiments for engineering of the CAR CS1+ and KO CS1T- cells and their subsequent testing;

[0039] Fig.13 : constructs of T01, T02 and T03 with the described TAL repeats for KO of CS1 gene;

[0040] Figure 14: Targeting of the TALs T01, T02 and T03 within the CS1 (SLAMF7) gene. T01 and T02 target exon 1 ( Fig.14A ), while T03 targets exon 2 ( Fig. 14B ).

[0041] Fig.15 A: Percentage measurement of target cell viability for TALEn or non-TALEn transfection combined with CAR+ or non-transduced cells: CS1(+) cells showed reduced cell viability when they were co-cultured with CAR+T- cells, while there was no effect on CS1(-) cell viability.

[0042] Fig.15B: Determination of the percentage of specific cell lysis (CS1+) calculated using flow cytometry data. It was shown that specific cell lysis was 2-fold higher when transfected with TALEn targeting the CS1 gene prior to T-cell CAR transduction.

[0043] Fig.16 : Results from FACS analysis of cytotoxic activity experiments, which show that transduction efficiency is higher in mock-transfected cells than in cells that have been transfected with TALEn targeting the CS1 gene (NTD: not transduced).

[0044] Fig.17 : Results from FACS analysis when the different samples were reactivated with CD3 / CD28 beads at D11 after transduction, showing the transduction efficiency and CD8 / CS1 expression levels in each sample. Increased CS1 levels were observed in mock-transfected cells upon reactivation, while a small number of cells were able to express CS1 in the TALEn-transfected population.

[0045] Table 1: Different cytopulse programs used for T-cell electroporation.

[0046] Table 2: Suitable target sequences for the guide RNAs using Cas9 in T-cells.

[0047] Table 3: List of genes encoding immune checkpoint proteins.

[0048] Table 4: Cluster of differentiation (CD) antigen markers found to be expressed on the surface of T-cells and characteristic of different tumor types.

[0049] Tables 5 to 13: Major surface antigen markers expressed in T-cells and overexpressed in solid tumor cells from different cancer types. The listed antigen markers were identified as described in Example 1.

[0050] Table 5: Colon tumor cells;

[0051] Table 6: Breast tumor cells;

[0052] Table 7: Digestive tract tumor cells;

[0053] Table 8: Renal tumor cells;

[0054] Table 9: Liver tumor cells;

[0055] Table 10: Lung tumor cells;

[0056] Table 11: Ovarian tumor cells;

[0057] Table 12: Pancreatic tumor cells;

[0058] Table 13: Prostate tumor cells;

[0059] Table 14: Major surface antigen markers expressed in T-cells and overexpressed in liquid tumor cells from various cancers (ALL, AML, CML, MDS, CLL, CTRL). The listed antigen markers were identified as described in Example 1.

[0060] Table 15: Sequences of the tested CD38 targets and TALENs for inactivating the CD38 antigen;

[0061] Table 16: Sequences of two other CD38 targets and the corresponding TALENs used for their inactivation;

[0062] Table 17: Sequences of the VH and VL chains and specific CDRs for the VH and VL chains of the scFv anti-CD38 antibodies daratumumab and MOR202;

[0063] Table 18: Three different structures of scFv daratumumab-like anti-CD38 CAR and the polypeptide sequences of the individual components used;

[0064] Table 19: Sequences of the VH and VL chains of the scFv anti-CS1 antibodies;

[0065] Table 20: Based on Fig.11 The polypeptide sequences of the anti-CS1CAR versions V1, V2 and V3 in A;

[0066] Table 21: Sequences of the CS1 targets and the TALENs used for their inactivation;

[0067] Table 22: Sequences of the CD70 target and the TALENs used for its inactivation;

[0068] Table 23: Polynucleotide and nucleic acid sequences of the VH and VL chains of the scFv anti-CD70AB4, AB8 and 1F6 antibodies;

[0069] Table 24: Based on Fig.11 The polypeptide sequences of the V1, V2 and V3 versions of the anti-CD70CAR in A. DETAILED DESCRIPTION

[0070] Unless specifically defined herein, all technical and scientific terms used have the same meanings as commonly understood by those skilled in the fields of gene therapy, biochemistry, genetics and molecular biology.

[0071] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention with the appropriate methods and materials described herein. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, shall prevail. Furthermore, unless otherwise specified, the materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0072] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art and are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al. al.USPat.No.4,683,195; Nucleic AcidHybridization (BD Harries & S.J. Higgins eds.1984); Transcription And Translation (BDHames&S.J.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); Series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially, Volumes 154 and 155 (Wu et al. eds.) and Volume 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 (DMWeir and CCBlackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0073] In a general aspect, the present invention relates to methods for novel adoptive immunotherapy strategies for treating diseases associated with the development of pathological cells, such as cancer, infection, and autoimmune diseases.

[0074] As a main object of the present invention is the possibility to target pathological cells that carry specific antigenic markers like T-cells.Pathological cells are any type of cells present in a patient that are believed to contribute to a deterioration of the health condition.

[0075] Generally speaking, pathological cells are malignant or infected cells that need to be reduced or eliminated to obtain remission from the patient.

[0076] In a first embodiment, the method of the present invention relates to a method for preparing suitable immune cells, preferably T-cells, for immunotherapy, comprising the following steps:

[0077] (a) genetically inactivated or mutated genes in immune cells that are involved in the expression or presentation of antigenic markers known to be present on the surface of the T cells and on the surface of the pathological cells;

[0078] (b) expressing a transgene encoding a chimeric antigen receptor targeting the antigen marker present on the surface of the pathological cell into the immune cell.

[0079] The immune cells according to the present invention are endowed with chimeric antigen receptors for antigen markers that are usually expressed by pathological cells and immune cells, or known to be present on the surface of the T-cells. The phrase "known to exist" means that the antigen markers have been reported to be found on the surface of the immune cells grown under natural conditions in vivo, particularly in the blood, but are not necessary when they are cultured in vitro. In any case, the method of the present invention results in the absence of antigen markers on the surface of the immune cells, thereby preventing the chimeric antigen receptor from reacting with the surface of the engineered T-cells. In this regard, the method may include a further step of purifying the resulting T-cells by excluding the cells that present the labeled antigen on their surface.

[0080] As shown in Table 4, the present invention relates to a number of antigen marker candidates reportedly expressed by tumor cells and also expressed by T- cells. Some of them, such as CD38, have been used as specific markers for some time in diagnostic methods, especially for leukemia pathological cells, but have not been used in treatment. In fact, although these markers are recognized as very specific markers in the art, they cannot be used as targets for immunotherapy because antibodies against these markers will destroy or interfere with the patient's T- cells. The inventors have confirmed that CS1 and CD70 are also present on the surface of T- cells, and expressing CARs targeting CS1 and CD70 in such T- cells will lead to their depletion (see Example 2).

[0081] According to a preferred embodiment of the present invention, the gene mutation or inactivation in step a) of the above method is performed using a rare-cutting endonuclease.

[0082] Inactivation gene refers to that the gene of interest will not be expressed in the form of functional protein. In a specific embodiment, the genetic modification of the method depends on the expression of rare-cutting nuclease in the cell provided for engineering, so that it also catalyzes the cleavage in a targeted gene to inactivate the targeted gene. The nucleic acid chain break caused by the nuclease is usually repaired by different mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process, which often causes the DNA sequence of the cleavage site to change. These mechanisms involve rejoining of the two DNA ends (either by direct rejoining (Critchlow and Jackson 1998) or via so-called microhomology-mediated end joining (Betts, Brenchley et al. 2003; Ma, Kim et al. 2003)). Repair by non-homologous end joining (NHEJ) usually results in small insertions or deletions and can be used to generate specific gene knockouts. The modification can be a substitution, deletion, or addition of at least one nucleotide. Cells in which cleavage-induced mutagenesis events, i.e., mutagenesis events following NHEJ events, occur can be identified and / or selected by methods well known in the art.

[0083] The term "rare-cutting endonuclease" refers to a wild-type or variant enzyme that can catalyze the hydrolysis (cutting) of bonds between nucleic acids in a DNA or RNA molecule, preferably a DNA molecule. Specifically, the nuclease can be an endonuclease that recognizes a nucleic acid target site with a length range of 10 to 45 base pairs (bp), usually 10 to 35 base pairs, more usually 12 to 20 base pairs, more preferably a highly specific rare-cutting endonuclease. The endonuclease according to the present invention recognizes at a specific polynucleotide sequence, further referred to as a "target sequence", and cuts nucleic acids within these target sequences or enters into sequences adjacent thereto, depending on the molecular structure of the endonuclease. The rare-cutting endonuclease can recognize and produce single or double-strand breaks at a specific nucleotide sequence.

[0084] In a specific embodiment, the rare-cutting endonuclease according to the present invention is an RNA-guided endonuclease such as the Cas9 / CRISPR complex. RNA-guided endonucleases constitute a new generation of genetic engineering tools when endonucleases are associated with RNA molecules. In this system, the nucleotide sequence of the RNA molecule determines the target specificity and activates the endonuclease ((Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ranet al. 2013; Mali, Yang et al. 2013).

[0085] Cas9

[0086] Cas9, also known as Csn1 (COG3513), is a giant protein involved in crRNA biogenesis and destruction of invading DNA. Cas9 has been described in different bacterial species such as Streptococcus thermophilus, Listeria innocua ((Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and Streptococcus pyogenes ((Deltcheva, Chylinski et al. 2011). The giant Cas9 protein (>1200 amino acids) contains two predicted nuclease domains, i.e., the HNH (McrA-like) nuclease domain located in the middle of the protein and the split RuvC-like nuclease domain (RNAase H fold) (Makarova, Grishin et al. (2006)).

[0087] "Cas9" refers to an engineered endonuclease or a homologue of Cas9 that can process a target nucleic acid sequence. In a specific embodiment, Cas9 can induce cutting in the nucleic acid target sequence (which may correspond to a double-strand break or a single-strand break). Cas9 variants can be Cas9 endonucleases that are not naturally present in nature and are obtained by protein engineering or by random mutagenesis. According to the present invention, Cas9 variants, for example, can be obtained by mutation, i.e., the deletion of at least one residue in the amino acid sequence of Streptococcus pyogenes Cas9 endonuclease (COG3513), or insertion or substitution. In the framework of the present invention, this Cas9 variant retains function, i.e., they retain the ability to process target nucleic acid sequences. Cas9 variants can also be homologous to the deletion of at least one residue in the amino acid sequence that can contain Streptococcus pyogenes Cas9, or insertion or substitution of Streptococcus pyogenes Cas9. Any combination of deletion, insertion and substitution can also be made to obtain the final construct, provided that the final construct has the desired activity, specifically the ability to bind to guide RNA or nucleic acid target sequence.

[0088] The RuvC / RNase H motif includes proteins that show a broad spectrum of nucleic acid hydrolysis functions and can act on both RNA and DNA (RNaseH, RuvC, DNA transposases and retroviral integrases and the PIWI domain of Argonaut proteins). The RuvC catalytic domain of the Cas9 protein described in the present invention can be characterized by the sequence motif: D-[I / L]-GXXSXGWA, wherein X represents any one of the 20 natural amino acids, and [I / L] represents isoleucine or leucine. In other aspects, the Cas9 variants involved in the present invention include at least the D-[I / L]-GXXSXGWA sequence, wherein X represents any one of the 20 natural amino acids, and [I / L] represents isoleucine or leucine.

[0089] The HNH motif is characteristic of many nucleases acting on double-stranded DNA including colicins, restriction enzymes and homing endonucleases. The domain HNH (SMART ID: SM00507, SCOP name: HNH family) is associated with a range of DNA binding proteins, performing various binding and cleavage functions. Those with known functions are involved in a range of cellular processes including bacterial toxicity, homing functions in group I and II introns and inteins, recombination, developmentally controlled DNA rearrangements, phage packaging and restriction endonuclease activity (Dalgaard, Klar et al. 1997). These proteins are found in viruses, archaea, eubacteria and eukaryotes. Interestingly, with respect to the LAGLI-DADG and GIY-YIG motifs, the HNH motif is often associated with self-propagating elements such as inteins, endonuclease domains of group I and II introns (Dalgaard, Klar et al. 1997). The HNH domain can be characterized by the presence of conserved Asp / His residues flanked by conserved His (amino terminus) and His / Asp / Glu (carboxyl terminus) residues at a certain distance. A considerable number of these proteins can also have a CX2C motif on either side of the central Asp / His residue. Structurally, the HNH motif appears as a central hairpin of twisted β strands flanked on each side by α helices (Kleanthous, Kuhlmann et al. 1999). The giant HNH domain of Cas9 is represented by SEQ ID NO.5. In the present invention, the HNH motif can be characterized by the sequence motif: YXXDHXXPXSXXXDXS, wherein X represents any one of the 20 natural amino acids. The present invention relates to a Cas9 variant comprising at least a YXXDHXXPXSXXXDXS sequence, wherein X represents any one of the 20 natural amino acids.

[0090] The present invention may be particularly interesting in that targeted multiple gene modifications and the generation of inducible nuclease systems are indeed readily performed by introducing the guide RNA into the Cas9 cells. For the purposes of the present invention, the inventors have demonstrated that the Cas9 protein can be divided into two separate split Cas9RuvC and HNH domains, which can process target nucleic acid sequences together with or alone the guide RNA.

[0091] Moreover, the RuvC and HNH domains from different RNA-guided endonucleases or Cas homologs can be assembled to improve nuclease efficiency or specificity. Domains from different species can be split into two proteins or fused to each other to form variant Cas proteins. The Cas9 split system is considered to be particularly suitable for inducible methods of genome targeting and avoids the potential toxic effects of overexpression of Cas9 in the cell. In fact, the first split Cas9 domain can be preferably introduced into the cell by stably transforming the cell with a transgenic encoding the split domain. Then, the complementary split part of the Cas9 can be introduced into the cell, so that the two split parts are reassembled into the cell to reconstruct the functional Cas9 protein at the desired time point.

[0092] The reduced size of the split Cas9 relative to wild-type Cas9 facilitates vectorization and delivery to the cells, for example, by using cell-penetrating peptides. Rearrangement of domains from different Cas proteins allows, for example, modulation of specificity and nuclease activity through slightly different targeting PAM motifs than the Streptococcus pyogenes Cas9.

[0093] Split Cas9 system

[0094] The previous characterization of the RuvC and HNH domains has prompted the inventors to engineer Cas9 proteins to produce split Cas9 proteins. Surprisingly, the inventors have demonstrated that the two split Cas9s can process the nucleic acid target together or alone. This observation allows the development of new Cas9 systems using split Cas9 proteins. Each split Cas9 domain can be prepared and used separately. Therefore, this split system has multiple advantages for the vectorization and delivery of the RNA-guided nuclease in T-cells, allowing the delivery of shorter and / or inactivated proteins, and is particularly suitable for inducing genome engineering in T-cells at the desired time, thereby limiting the potential toxicity of the integrated Cas9 nuclease.

[0095] "Split Cas9" herein refers to a reduced or truncated form of a Cas9 protein or Cas9 variant that contains either a RuvC or HNH domain, but not both of these domains. Such a "split Cas9" can be used independently or in a complementary manner with a guide RNA, such as, for example, one split Cas9 provides a RuvC domain and another provides a HNH domain. Different split RNA-guided endonucleases can be used together, with RuvC and / or NHN domains.

[0096] Each Cas9 split domain can be derived from a different or the same Cas9 homolog. Many homologs of Cas9 have been identified in genome databases.

[0097] The Cas9 split domain (RuvC and HNH domain) can be introduced into the cell simultaneously or sequentially so that the split Cas9 domain processes the target nucleic acid sequence in the cell. The Cas9 split domain and guide RNA can be introduced into the cell by using cell penetrating peptides or other transfection methods as described elsewhere.

[0098] In another aspect of the present invention, only one split Cas9 domain, called compact Cas9, is introduced into the cell. In fact, surprisingly, the inventors have demonstrated that the split Cas9 domain comprising the RuvC motif as described above can independently cut the target nucleic acid sequence of the split domain comprising the HNH motif. Therefore, they can prove that the guide RNA does not require the presence of the HNH domain to bind to the target nucleic acid sequence and is fully and stably bound by the RuvC split domain. In a preferred embodiment, the split Cas9 domain alone can cut off the target nucleic acid sequence.

[0099] Each split domain can be fused to at least one active domain in the N-terminus and / or C-terminus, and the active domain can be selected from the group consisting of: nuclease (e.g., endonuclease or exonuclease), polymerase, kinase, phosphatase, methylase, demethylase, acetylase, deacetylase, topoisomerase, integrase, transposase, ligase, helicase, recombinase, transcription activator (e.g., VP64, VP16), transcription repressor (e.g., KRAB), DNA end processing enzyme (e.g., Trex2, Tdt), reporter (e.g., fluorescent protein, lacZ, luciferase).

[0100] The HNH domain is responsible for nicking one strand of the target double-stranded DNA, while the RuvC-like RNaseH fold domain is involved in nicking the other strand of the double-stranded nucleic acid target (including the PAM motif) (Jinek, Chylinski et al. 2012). However, in wild-type Cas9, these two domains lead to blunt cleavage of the invasive DNA within the same target sequence (original spacer) in the immediate vicinity of the PAM ((Jinek, Chylinski et al. 2012). Cas 9 can be a nickase and induce nicking events in different target sequences.

[0101] As a non-limiting example, Cas9 or split Cas9 can include mutations in the catalytic residues of the HNH or RuvC-like domains to induce nicking events in different target sequences. As a non-limiting example, the catalytic residues of the Cas9 protein are those corresponding to amino acids D10, D31, H840, H868, N882 and N891 on homologs of Cas family members or alignment positions using the CLUSTALW method. Any of these residues can be replaced by any other amino acid, preferably by alanine residues. Mutations in catalytic residues refer to substitutions by another amino acid, or deletions or additions of amino acids that induce inactivation of at least one of the catalytic domains of cas9. (See, in one embodiment, Cas9 or split Cas9 may comprise one or more of the above mutations. In another embodiment, the split Cas9 comprises only one of the two RuvC and HNH catalytic domains. In the present invention, Cas9 from different species, Cas9 homologs, Cas9 engineered and functional variants thereof can be used. The present invention contemplates the use of any RNA-guided endonuclease or split RNA-guided endonuclease variant to perform nucleic acid cleavage in a genetic sequence of interest.

[0102] Preferably, the Cas9 variant according to the present invention has an amino acid sequence that shares at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably 95% identity with Cas9 of Streptococcus pyogenes (COG3513).

[0103] Meganuclease

[0104] Rare-cutting endonucleases can also be homing endonucleases, also known as megabase meganucleases. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases are highly specific and recognize DNA target sites of 12 to 45 base pairs (bp) in length, typically in the range of 14 to 40 bp in length. The homing endonuclease according to the present invention can correspond, for example, to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease. A preferred homing endonuclease according to the present invention can be an I-Crel variant. A "variant" endonuclease, i.e., an endonuclease that is naturally not present in nature and that is obtained by engineering or by random mutation, can bind to a DNA sequence different from that recognized by a wild-type endonuclease (see International Application WO2006 / 097854).

[0105] The rare-cutting endonuclease can be a modular DNA binding nuclease. A modular DNA binding nuclease refers to any fusion protein or protein of a specified nucleic acid target sequence comprising at least one catalytic domain of an endonuclease and at least one DNA binding domain. The DNA binding domain is generally an RNA or DNA binding domain formed by an independently folded polypeptide or protein domain comprising at least one motif that recognizes a double or single-stranded polynucleotide. Many such polypeptides have been described in the art and have the ability to bind to specific nucleic acid sequences. Such binding domains generally include, as non-limiting examples, a helix-turn helix domain, a leucine zipper domain, a winged helix domain, a helix-loop-helix domain, an HMG-box domain, an immunoglobulin domain, a B3 domain, or an engineered zinc finger domain.

[0106] Zinc finger nuclease

[0107] Initially developed for in vitro cleavage of DNA, "zinc finger nucleases" (ZFNs) are fusions between the IIS type restriction enzyme cleavage domain, FokI, and a DNA recognition domain containing three or more C2H2 type zinc finger motifs. Heterologous dimers of two separate ZFNs at specific locations in DNA in precise orientations and spacings result in double strand breaks (DSBs) in the DNA. The use of such chimeric endonucleases has been widely reported in the art, as reviewed by Urnov et al. (Genome editing with engineered zinc finger nucleases (2010) Nature reviews Genetics 11:636-646).

[0108] Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. To allow the two cleavage domains to dimerize and cleave DNA, the two separate ZFNs bind opposite strands of DNA to their C-termini at a distance. The most commonly used linker sequence between the zinc finger domain and the cleavage domain requires that the 5' edge of each binding site be separated by 5-7 bp.

[0109] The most straightforward approach to generating new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The most common modular assembly process involves combining three individual zinc fingers that can each recognize a 3 base pair DNA sequence to produce a 3-finger array that can recognize a 9 base pair target site. Many selection methods have been used to generate zinc finger arrays that can target desired sequences. Initial selection work utilized phage display to select proteins that bind to a given DNA target from a larger collection of partially randomized zinc finger arrays. More recent work has utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0110] TAL-nuclease

[0111] "TALE-nuclease" or "MBBBD-nuclease" refers to an engineered protein produced by the fusion of a DNA binding domain, typically derived from a transcription activator-like effector protein (TALE) or a modular base-per-base binding domain (MBBBD), containing a catalytic domain with endonuclease activity. Such catalytic domains are typically derived from enzymes, such as, for example, I-TevI, ColE7, NUCA, and Fok-I. TALE-nucleases can be formed in monomeric or dimer form depending on the selected catalytic domain (WO2012138927). This engineered TALE-nuclease can be marketed under the trade name TALEN TM Commercially available (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

[0112] According to a preferred embodiment of the present invention, the DNA binding domain is derived from a transcription activator-like effector (TALE), in which the sequence specificity is driven by a series of 33 to 35 amino acid repeats derived from Xanthomonas or Ralstonia solanacearum proteins AvrBs3, PthXo1, AvrHah1, PthA, Tal1c as non-limiting examples.

[0113] These repeats differ essentially in the position of the two amino acids that specify the interaction with the base pairs ((Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). Each base pair in the DNA target is contacted by a single repeat, while the specificity is due to the two variant amino acids of the repeat (the so-called repeat variable dipeptide, RVD). The TALE binding domain may further comprise an N-terminal translocation domain responsible for the requirement of the first thymine base (T0) of the targeting sequence and a C-terminal domain containing a nuclear localization signal (NLS). The TALE nucleic acid binding domain specifically corresponds to an engineered core TALE scaffold comprising a plurality of TALE repeat sequences, each repeat comprising an RVD specific for each nucleotide base of the TALE recognition site. In the present invention, each TALE repeat sequence of the core scaffold is made of 30 to 42 amino acids, more preferably 33 or 34 amino acids, wherein two key amino acids located at positions 12 and 13 (the so-called repeat variable dipeptide, RVD) mediate the binding of one nucleotide of the TALE binding site sequence. Recognition; equivalent to two key amino acids being able to be specifically located at positions other than positions 12 and 13 in the TALE repeat sequence with a height exceeding 33 or 34 amino acids in length. Preferably, the RVD associated with the recognition of the different nucleotides is HD that recognizes C, NG that recognizes T, NI that recognizes A, and NN that recognizes G or A. In another embodiment, key amino acids 12 and 13 can mutate toward other amino acid residues to adjust their specificity for nucleotides A, T, C and G and especially enhance this specificity. The TALE nucleic acid binding domain generally comprises 8 to 30 TALE repeat sequences. More preferably, the core scaffold of the present invention comprises 8 to 20 TALE repeat sequences, and more preferably 15 TALE repeat sequences. It can also comprise an additional single truncated TALE repeat sequence consisting of 20 amino acids located at the C-terminus of the TALE repeat sequence group, i.e., another C-terminal half-TALE repeat sequence.

[0114] Other engineered DNA binding domains can be used as replacement sequences to form so-called modular base-per-base specific nucleic acid binding domains (MBBBDs), as described in WO2014 / 018601. The MBBBDs can, for example, be engineered from recently identified proteins, namely EAV36_BURRH, E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH proteins from the recently sequenced genome of the endosymbiotic fungus Burkholderia Rhizoxinica (Lackner, Moebius et al. 2011). These nucleic acid binding polypeptides contain modules of about 31 to 33 base-specific amino acids. These modules exhibit less than 40% sequence identity with Xanthomonas TALE common repeats and exhibit more polypeptide sequence variability. The different domains of the above proteins (modules, N and C-termini) from Burkholderia and Xanthomonas are suitable for engineering new proteins or scaffolds with binding properties to specific nucleic acid sequences and can be combined to form chimeric TALE-MBBBD proteins.

[0115] As an example, the present invention encompasses methods of engineering T-cells in order to inactivate the expression of genes encoding antigenic markers such as CD38, CS1 and CD70 by using specific TALE-nucleases.

[0116] Particularly suitable for use in the practice of the present invention are TALE-nucleases such as those in SEQ ID NOs: 2-3; 5-6; 8-9, SEQ ID NOs: 64-65; 67-68; 70-71 and SEQ ID NOs: 73-74; 76-77; 79-80, respectively, for the CD38, CS1 and CD70 genes. These specific TALE-nucleases, their sequence targets and the protocols used are more thoroughly described in Examples 1-3 below.

[0117] Delivery Method

[0118] The inventors have considered any method known in the art to allow delivery of polynucleotides expressing the endonuclease, its possible co-effectors (e.g., guide RNA or DNA associated with Cas9 or argonaute nuclease) and the chimeric antigen receptor within the cell or subcellular compartment of the cell. These include, as non-limiting examples, viral transduction, electroporation, and also liposome delivery, polymer carriers, chemical carriers, lipid complexes (lipoplex), polymer complexes (polyplex), dendrimers, nanoparticles, emulsions, natural endocytosis or phagocytosis pathways.

[0119] As a preferred embodiment of the present invention, the polynucleotide encoding the endonuclease of the present invention is transfected in the form of mRNA so that, for example, transient expression is obtained by electroporation and chromosome integration of exogenous DNA is avoided. The inventors have determined the different optimal conditions for mRNA electroporation in the T-cells shown in Table 1. The inventors have used the cell pulse (cytoPulse) technology, which allows the instantaneous penetration of living cells by using pulsed electric fields to deliver substances into the cells (US Pat. No. 6,010,613 and WO 2004 / 083379). The pulse duration, intensity, and interval between pulses can be modified to achieve the optimal conditions of high transfection efficiency and lowest mortality. Basically, the first high electric field pulse allows pore formation, and the subsequent low electric field pulse allows the polynucleotide to be migrated into the cell. In one aspect of the present invention, the inventors describe the steps of causing mRNA transfection efficiency>95% in T-cells, and transiently expressing different types of proteins in T-cells using electroporation schemes. In particular, the present invention relates to a method of transforming a T-cell, comprising contacting the T-cell with RNA and applying to the T-cell an agile pulse sequence consisting of:

[0120] (a) an electric pulse with a voltage range of 2250 to 3000 V / cm, a pulse width of 0.1 ms and a pulse interval of 0.2 to 10 ms between the electric pulses in steps (a) and (b);

[0121] (b) an electric pulse with a voltage ranging from 2250 to 3000 V, a pulse width of 100 ms, and a pulse interval of 100 ms between the electric pulse of step (b) and the first electric pulse of step (c); and

[0122] (c) 4 electric pulses, voltage 325V, pulse width 0.2ms and pulse interval 2ms between every 4 electric pulses.

[0123] In a specific embodiment, the method of transforming a T-cell comprises contacting the T-cell with RNA and applying to the T-cell a flexible pulse sequence consisting of:

[0124] (a) an electric pulse with a voltage of 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000 V / cm, 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 electric pulses of steps (a) and (b);

[0125] (b) an electric pulse having a voltage ranging from 2250, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000 V, a pulse width of 100 ms and a pulse interval of 100 ms between the electric pulse of step (b) and the first electric pulse of step (c); and

[0126] (c) 4 electric pulses, voltage 325V, pulse width 0.2ms and pulse interval 2ms between every 4 electric pulses.

[0127] Any value included in the above numerical range is disclosed in the present application. The electroporation medium can be any suitable medium known in the art. Preferably, the electroporation medium has a conductivity range of 0.01 to 1.0 millisiemens.

[0128] Table 1: Different cell pulsing programs used to determine the minimum voltage required for electroporation in PBMC-derived T cells

[0129]

[0130] Viral transduction

[0131] According to the present invention, the use of retroviral vectors and more preferably lentiviral vectors are particularly suitable for expressing the chimeric antigen receptor into the T-cell. Methods for viral transduction are well known in the art (Walther et al. (2000) Viral Vectors for Gene Transfer. Drugs. 60 (2): 249-271). Integrative viral vectors allow stable integration of the polynucleotide in the T-cell genome and allow expression of the chimeric antigen receptor for a longer period of time.

[0132] Non-alloreactive T cells

[0133] Although the method of the present invention can be used as part of gene therapy, for example, by using a viral vector targeting T-cells in the blood circulation for in vivo implementation, it will include a genetic sequence expressing a specific rare-cutting endonuclease together with other genetic sequences expressing CAR, but the method of the present invention is more generally intended to practice in vitro the cultured T-cells that can be obtained from patients or donors. The engineered T-cells engineered in vitro can be reimplanted into the patient from which they are derived as part of autologous treatment, or used as part of allogeneic treatment. In the latter case, it is preferred that the cells are further engineered to make them non-alloreactive to ensure their correct engraftment. Therefore, the method of the present invention may include an additional step of obtaining the T-cells from the donor and inactivating the genes involved in MHC recognition and or the target of immunosuppressive drugs, as described in, for example, WO 2013 / 176915.

[0134] T-cell receptor (TCR) is a cell surface receptor that participates in T-cell activation in response to the appearance of antigen. The TCR is generally composed of two chains, α and β, which are assembled to form a heterodimer and associated with the CD3 transduction subunit to form the T-cell receptor complex present on the cell surface. Each α and β chain of the TCR is composed of immunoglobulin-like N-terminal variable (V) and constant (C) regions, hydrophobic transmembrane domains and short cytoplasmic regions. For immunoglobulin molecules, the variable regions of the α and β chains are produced by V (D) J recombination, producing a large number of different antigen specificities in T-cell populations. However, compared to immunoglobulins that recognize complete antigens, T-cells are activated by processed peptide fragments associated with MHC molecules, introducing an additional dimension (dimension) into antigen recognition by T-cells, also known as MHC restriction. The recognition of the MHC differences between the donor and the recipient by the T cell receptor leads to the potential development of T-cell proliferation and GVHD. It has been shown that normal surface expression of the TCR depends on the coordinated synthesis and assembly of all seven components of the complex (Ashwell and Klusner 1990). Inactivation of TCRα or TCRβ may lead to the elimination of the TCR from the surface of T-cells to prevent recognition of alloantigens, thereby preventing recognition of GVHD.

[0135] Therefore, still according to the invention, the engraftment of the T-cells can be improved by inactivating at least one gene encoding a TCR component. By inactivating the TCR alpha gene and / or the TCR beta gene the TCR will be rendered non-functional in the cells.

[0136] Relative to the use of Cas9 / CRISPR system, the inventors have determined suitable target sequences within the 3 exons encoding TCR, allowing significantly reduced toxicity in living cells while retaining cutting efficiency. The preferred target sequences are indicated in Table 2 (+ for lower ratios of TCR negative cells, ++ for intermediate ratios, +++ for higher ratios).

[0137] Table 2: Suitable target sequences for the guide RNAs using Cas9 in T-cells

[0138]

[0139]

[0140] MHC antigens are also proteins that play an important role in transplantation reactions. Rejection is mediated by T-cells that react to tissue compatibility antigens on the surface of the implanted tissue, and the largest group of these antigens is the major histocompatibility antigens (MHC). These proteins are expressed on the surface of all higher vertebrates and are called HLA antigens (human leukocyte antigens) in human cells. Like TCR, the MHC protein plays a vital role in T-cell stimulation. Antigen presenting cells (usually dendritic cells) display peptides that are degradation products of foreign proteins on the cell surface on the MHC. In the presence of a co-stimulatory signal, the T-cell is activated and will act on target cells that also display the same peptide / MHC complex. For example, stimulated T helper cells will target macrophages that bind to their MHC display antigens, or cytotoxic T-cells (CTLs) will act on virus-infected cells that display foreign viral peptides.

[0141] Therefore, in order to provide less alloreactive T-cells, the method of the present invention can further comprise the step of inactivating or mutating a HL gene.

[0142] The class I HL gene cluster in the human body includes three major loci, B, C and A, and multiple minor loci. The class II HLA cluster also includes three major loci, DP, DQ and DR, and both class I and class II gene clusters are polymorphic, because both class I and II genes have multiple different alleles in the population. There are also multiple auxiliary proteins that also play a role in HLA function. The Tap1 and Tap2 subunits are parts of the TAP transporter complex that are crucial for loading peptide antigens on class I HLA complexes, and the LMP2 and LMP7 proteomic subunits play a role in the proteolytic degradation of antigens into peptides to display on the HLA. LMP7 reduction has been shown to reduce the amount of MHC class I on the cell surface, perhaps by lack of stability (Fehling et al. (1999) Science 265: 1234-1237). In addition to TAP and LMP, there is also the tapasin gene, whose product forms a bridge between the TAP complex and the HLA class I chain and enhances peptide loading. The reduction of tapasin can lead to impaired MHC class I assembly of cells, reduced cell surface expression of the MHC class I and impaired immune response (Grandea et al. (2000) Immunity 13: 213-222 and Garbi et al. (2000) Nat. Immunol. 1: 234-238). Any of the above genes can be used as part of the present invention, for example, as disclosed in WO2012 / 012667, inactivated.

[0143] Methods for engineering drug-resistant T-cells:

[0144] To improve cancer treatment and selective implantation of allogeneic T-cells, drug resistance can be imparted to engineered T-cells to protect them from the toxic side effects of chemotherapy or immunosuppressants. In fact, the inventors have observed that most patients are treated with chemotherapy and immune depleting agents as standard of care before receiving T-cell immunotherapy. In addition, they have found that they can use these treatments to help select the engineered T-cells (by adding chemotherapy drugs to the culture medium to expand the cells in vitro before treatment, or by obtaining selective expansion of the engineered T-cells in vivo in patients under chemotherapy or immunosuppressive treatment.

[0145] Moreover, the drug resistance of T-cells also allows their enrichment in vivo or in vitro, because T-cells expressing the drug resistance gene will survive and multiply relative to drug-sensitive cells. Specifically, the present invention relates to a method for engineering allogeneic and drug-resistant T-cells that tolerate immunotherapy, comprising:

[0146] (a) providing T-cells;

[0147] (b) selecting at least one drug;

[0148] (c) modifying T-cells to confer drug resistance to said T-cells;

[0149] (d) expanding said engineered T-cells in the presence of said drug, and optionally

[0150] The aforementioned steps may be combined with the steps of the method described previously.

[0151] T-cell drug resistance can be conferred by inactivating one or more genes responsible for the sensitivity of the cell to drugs (drug sensitivity genes), such as the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (Gene Bank: M26434.1). Specifically, HPRT can be inactivated in engineered T-cells to confer resistance to the cell growth inhibitory metabolite 6-thioguanine (6TG), which is converted by HPRT into cytotoxic thioguanine nucleotides and is currently used to treat cancer patients, specifically leukemia patients (Hacke, Treger et al. 2013). Another example is that the inactivation of CD3 normally expressed on the surface of the T-cell can confer resistance to anti-CD3 antibodies such as teplizumab.

[0152] It is also possible to confer drug resistance to T-cells by expressing drug resistance genes. The drug resistance gene refers to a nucleic acid sequence that encodes "tolerance" to an agent such as a chemotherapeutic agent (e.g., methotrexate). In other words, expression of a drug resistance gene in a cell allows the cell to proliferate to a greater extent in the presence of the drug than a corresponding cell without the drug resistance gene. The drug resistance gene of the present invention can encode resistance to antimetabolites, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, analogs or derivatives thereof, and the like.

[0153] Variant alleles of multiple genes such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanyl transferase (MGMT) have been identified as being able to confer drug resistance to cells. The drug resistance gene can be expressed in the cell by introducing a transgene encoding the gene into the cell, or integrating the drug resistance gene into the genome of the cell by homologous recombination. A variety of other drug resistance genes have been identified as being potentially useful for conferring drug resistance to targeted cells (Takebe, Zhao et al. 2001; Sugimoto, Tsukahara et al. 2003; Zielske, Reese et al. 2003; Nivens, Felder et al. 2004; Bardenheuer, Lehmberg et al. 2005; Kushman, Kabler et al. 2007).

[0154] DHFR is an enzyme involved in the regulation of the amount of tetrahydrofolate in the cell and is important for DNA synthesis. Folic acid analogs such as methotrexate (MTX) inhibit DHFR and are therefore used as clinical anti-tumor agents. Different mutant forms of DHFR that have improved tolerance to the inhibitory effects of antifolates used in therapy have been described. In a specific embodiment, the drug resistance gene according to the present invention can be a nucleic acid sequence encoding a mutant form of human wild-type DHFR (Gene Bank: AAH71996.1), which comprises at least one mutation that provides tolerance to anti-folate therapy, such as methotrexate. In a specific embodiment, the mutant form of DHFR comprises at least one mutated amino acid at position G15, L22, F31 or F34, preferably at position L22 or F31 ((Schweitzer, Dicker et al. 1990); International Application WO 94 / 24277; US Patent US 6,642,043).

[0155] As used herein, "antifolate agent" or "folate analog" refers to a molecule that interferes with the folate metabolic pathway to some extent. Examples of antifolate agents include, for example, methotrexate (MTX); aminopterin; trimetrexate (Neutrexin TM); edatrexate; N10-propargyl-5,8-dideazafolate (CB3717); ZD1694 (Tumodex); 5,8-dideazaisofolate (IAHQ); 5,10-dideazatetrahydrofolate (DDATHF); 5-deazafolate; PT523 (Nα-(4-amino-4-deoxypteroyl)-Nδ-semiphthaloyl-L-ornithine); 10-ethyl-10-deazaaminopterin (DDATHF, lometrexol); pyrexine; 10-EDAM; ZD1694; GW1843; pemetrexed and PDX (10-propargyl-10-deazaaminopterin).

[0156] Another example of a drug resistance gene can also be inosine-5'-monophosphate dehydrogenase II (IMPDH2), a mutant or modified form of the rate-limiting enzyme for de novo synthesis of guanosine nucleotides. The mutant or modified form of IMPDH2 is an IMPDH inhibitor resistance gene. The IMPDH inhibitor can be mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF). The mutant IMPDH2 can contain at least one, preferably two mutations in the MAP binding site (NP_000875.2) of wild-type human IMPDH2 that leads to significantly increased resistance to IMPDH inhibitors. The mutation is preferably at position T333 and / or S351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al. 2007; Jonnalagadda, Brown et al. 2013). In a specific embodiment, the threonine residue at position 333 is replaced by an isoleucine residue and the serine residue at position 351 is replaced by a tyrosine residue.

[0157] Another resistance gene is a mutant form of calcineurin. Calcineurin (PP2B) is a ubiquitously expressed serine / threonine protein phosphatase that participates in many biological processes and is a T cell activation center. Calcineurin is a heterodimer composed of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). After the T-cell receptor binds, calcineurin dephosphorylates the transcription factor NFAT, allowing it to translocate to the nucleus and active key target genes such as IL2. FK506 in a complex with FKBP12, or cyclosporine A (CSA) in a complex with CyPA-blocked NFAT will approach the active site of calcineurin, preventing it from dephosphorylating, and thereby inhibiting T-cell activation (Brewin, Mancao et al. 2009). The resistance gene of the present invention can be a nucleic acid sequence (such as FK506 and / or CsA) encoding a mutant form of calcineurin that is resistant to calcineurin inhibitors. In a specific embodiment, the mutant form can comprise at least one mutant amino acid of the wild-type calcineurin heterodimer a at the following positions: V314, Y341, M347, T351, W352, L354, K360, preferably double mutations at positions T351 and L354 or V314 and Y341. The correspondence of the amino acid positions described herein is often expressed according to the amino acid positions of the form of the wild-type human calcineurin heterodimer (GenBank: ACX34092.1).

[0158] In another specific embodiment, the mutant form can comprise at least one mutant amino acid of the wild-type calcineurin heterodimer b at the following positions: V120, N123, L124 or K125, preferably a double mutation at positions L124 and K125. The correspondence of the amino acid positions described herein is often expressed according to the positions of the amino acids of the form of the wild-type human calcineurin heterodimer b polypeptide (GenBank: ACX34095.1).

[0159] Another drug resistance gene is 0(6)-methylguanine methyltransferase (MGMT) encoding human alkylguanyltransferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents such as nitrosoureas and temozolomide (TMZ). 6-Benzylguanine (6-BG) is an inhibitor of AGT that enhances the toxicity of nitrosoureas and is co-administered with TMZ to enhance the cytotoxic effects of this agent. Various mutant forms of MGMT encoding AGT variants are highly resistant to inactivation by 6-BG, but retain their ability to repair DNA damage (Maze, Kurpad et al. 1999). In a specific embodiment, the AGT mutant form can comprise a mutant amino acid at position P140 of wild-type AGT (UniProtKB: P16455).

[0160] Another drug resistance gene can be the multidrug resistance protein 1 (MDR1) gene. This gene encodes a membrane glycoprotein, called the P-glycoprotein (P-GP) that participates in the transmission of metabolic byproducts across the cell membrane. The P-Gp protein shows a wide range of specificity for a variety of structurally unrelated chemotherapeutic drugs. Therefore, it is possible to confer cell resistance by expressing a nucleic acid sequence (NP_000918) encoding MDR-1.

[0161] Resistance genes can also be cytotoxic antibiotics, such as the ble gene or the mcr gene. Ectopic expression of the ble gene or mcr in immune cells provides a selective advantage when exposed to chemotherapeutic agents, bleomycin or mitomycin C, respectively.

[0162] The T-cells can also be made tolerant to immunosuppressants. Immunosuppressants are agents that suppress immune function through one of a variety of mechanisms of action. In other words, immunosuppressants act by compounds that show the ability to eliminate the degree and / or voracity of immune response. As non-limiting examples, immunosuppressants can be calcineurin inhibitors, targets of rapamycin, interleukin-2α-chain blockers, inosine monophosphate dehydrogenase inhibitors, dihydrofolate reductase inhibitors, corticosteroids or immunosuppressive antimetabolites. Classical cytotoxic immunosuppressants work by inhibiting the synthesis of DNA. Others can work by the activation of T-cells or by inhibiting the activation of helper cells. The method according to the present invention allows the immunosuppressive tolerance of T-cells to immunotherapy by inactivating the target of the immunosuppressant in T-cells. As non-limiting examples, the target of the immunosuppressant can be a receptor of an immunosuppressant, such as: CD52, glucocorticoid receptor (GR), FKBP family gene members and cyclophilin family gene members.

[0163] In an immunocompetent host, allogeneic cells are usually quickly rejected by the host's immune system. It has been shown that allogeneic leukocytes present in non-irradiated blood products will last no more than 5-6 days. Therefore, in order to prevent the rejection of allogeneic cells, the host's immune system must be effectively suppressed. Glucocorticoid steroids are widely used in therapy for immunosuppression. This type of steroid hormone binds to the glucocorticoid receptor (GR) present in the cytosol of T-cells, resulting in translocation into the nucleus and binding to specific DNA motifs that regulate the expression of many genes involved in immune processes. T-cells treated with glucocorticoid steroids produce reduced levels of cytokine production that lead to T-cell anergy and interfere with T-cell activation. Alemtuzumab, also known as CAMPATH1-H, is a humanized monoclonal antibody that targets CD52, a 12-amino acid glucosylphosphatidyl-inositol-(GPI)-linked glycoprotein (Waldmann and Hale, 2005). CD52 is expressed at high levels on T and B lymphocytes and at low levels on monocytes, while being absent on granulocytes and myeloid precursors. Treatment with alemtuzumab, a humanized monoclonal antibody against CD52, has been shown to induce rapid depletion of circulating lymphocytes and monocytes. It is frequently used in the treatment of T-cell lymphomas and, in some cases, as part of the conditioning regimen for transplantation. However, in the case of adoptive immunotherapy, the use of immunosuppressive drugs can also have an adverse effect on the introduced therapeutic T-cells. Therefore, in order to effectively use adoptive immunotherapy in these conditions, the introduced cells will need to be resistant to the immunosuppressive therapy.

[0164] In a preferred embodiment of the above steps, the gene (specific for immunosuppressive therapy) of step (b) is CD52, and the immunosuppressive therapy of step (d) comprises a humanized antibody targeting the CD52 antigen. As another embodiment, the gene (specific for immunosuppressive therapy) of step (b) is a glucocorticoid receptor (GR), and the immunosuppressive therapy of step (d) comprises a corticosteroid such as dexamethasone. As another embodiment, the target gene (specific for immunosuppressive therapy) of step (b) is a member of the FKBP family gene or a variant thereof, and the immunosuppressive therapy of step (d) comprises FK506, also known as tacrolimus or fujimycin. As another embodiment, the FKBP family gene member is FKBP12 or a variant thereof. As another embodiment, the gene (specific for immunosuppressive therapy) of step (b) is a member of the cyclophilin family gene or a variant thereof, and the immunosuppressive therapy of step (d) comprises cyclosporine.

[0165] In a specific embodiment of the present invention, the genetic modification step of the method depends on the inactivation of two genes selected from the group consisting of CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ. In another embodiment, the genetic modification step of the method depends on the inactivation of more than two genes. The genetic modification is preferably performed in vitro using at least two RNA guides targeting different genes.

[0166] Inactive genes refer to genes that are not expressed in the form of functional proteins.

[0167] Engineering highly active T-cells for immunotherapy

[0168] According to the present invention, the T-cell can be selected from the group consisting of the following: inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. In another embodiment, the cell can be derived from the group consisting of CD4+T-lymphocytes and CD8+T-lymphocytes. They can be extracted from blood or derived from stem cells. The stem cell can be an adult stem cell, an embryonic stem cell, more specifically a non-human stem cell, an umbilical cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell or a hematopoietic stem cell. Representative human cells are CD34+ cells. Before amplification and genetic modification of the cell of the present invention, the cell source can be obtained from a subject by various non-limiting methods. T-cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the infection site, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments of the present invention, any number of T-cell lines available and known to those skilled in the art can be used. In another embodiment, the cells can be derived from a healthy donor, from a patient diagnosed with cancer or obtained from a patient diagnosed with infection. In another embodiment, the cells are part of a mixed population of cells with different phenotypic properties. Also encompassed within the scope of the present invention are cell lines obtained from transformed T-cells according to the methods previously described.

[0169] As a further aspect of the invention, the T-cells according to the invention may be further engineered, preferably genetically engineered, to enhance their activity and / or activation, in particular by modulating the expression of proteins involved in overall T-cell regulation (also called "immune checkpoints").

[0170] Immune checkpoints

[0171] It should be understood by those of ordinary skill in the art that the term "immune checkpoint" refers to a group of molecules expressed by T-cells. These molecules effectively act as a "brake" to downregulate or inhibit immune responses. Molecules of immune checkpoints include, but are not limited to, programmed death (PD-1, also known as PDCD1 or CD279, accession number: NM_005018), cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152, gene bank accession number AF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, gene bank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM_18 1780.3), BY55 (also known as CD160, GenBank accession number: CR541888.1), TIGIT (also known as lVSTM3, accession number: NM_173799), LAIR1 (also known as CD305, GenBank accession number: CR542051.1, {Meyaard, 1997 #122}), SIGLEC10 (GenBank accession number: AY358337.1), 2B4 (also known as CD244, accession number: NM_00116 6664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7{Nicoll, 1999#123}, SIGLEC9{Zhang, 2000 #124;Ikehara, 2004#125}, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII , TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF {Quigley, 2010 #121}, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, which directly inhibit immune cells. For example, CTLA-4 is a cell-surface protein expressed on certain CD4 and CD8 T-cells; when bound by its ligands (B7-1 and B7-2) on antigen-presenting cells, T-cell activation and effector function are inhibited. The present invention thus relates to an engineered T-cell, in particular for use in immunotherapy, comprising genetically modifying the T-cell by inactivating at least one protein involved in an immune checkpoint, in particular PD1 and / or CTLA-4 or any immune checkpoint protein referred to in Table 3.

[0172] Table 3: List of genes encoding immune checkpoint proteins

[0173]

[0174] Engineered T-cells expressing chimeric antigen receptors for pathological cells

[0175] The chimeric antigen receptor introduced into the T-cell according to the present invention can adopt different designs, such as single-chain or multi-chain CAR. These different designs allow various strategies to improve the specificity and binding efficiency of the targeted pathological cells. Some of these strategies are illustrated in the drawings of the present application. Single-chain CAR is the most classic version in the art. The multi-chain CAR structure was developed by the applicant as a method that allows the activity of T-cells to be regulated according to specificity and intensity. The multiple subunits can mask other costimulatory domains or keep such domains at a certain distance, as well as other types of receptors, while traditional single-chain structures may sometimes be considered to be too sensitive and too little allowed for multi-specific interactions.

[0176] Single-chain CAR

[0177] Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T-cells generated in vitro, is a promising strategy for treating viral infections and cancer. T-cells used for adoptive immunotherapy can be generated by amplifying antigen-specific T-cells or by redirecting T-cells through genetic engineering (Park, Rosenberg et al. 2011). The transfer of viral antigen-specific T-cells is a well-established procedure for the treatment of transplant-related viral infections and rare virus-related malignancies. Similarly, the separation and transfer of tumor-specific T-cells has been shown to be successful in the treatment of melanoma.

[0178] New specificities of T-cells have been successfully generated by genetically transferring transgenic T-cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. al.2010). CAR is a synthetic receptor consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule. In general, the binding portion of the CAR is composed of an antigen-binding domain of a single-chain antibody (scFv), including a light fragment and a variable fragment of a monoclonal antibody connected by a flexible linker. Binding moieties based on receptor domains or ligand domains have also been successfully used. The signaling domain of the first generation of CARs is derived from the cytoplasmic region of the CD3ζ or Fc receptor γ chain. The first generation of CARs has been shown to successfully redirect T-cell cytotoxicity. However, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134) and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to improve survival and increase proliferation of CAR-modified T-cells. CAR has successfully allowed T-cells to redirect antigens expressed on the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dottiet al.2010).

[0179] In addition to CARs targeting antigenic markers shared by pathological cells and T-cells, such as CD38, it is conceivable to express other CARs directed against other antigenic markers that are not necessarily expressed by said T-cells, thereby enhancing the specificity of the T-cells.

[0180] Examples of chimeric antigen receptors that can be further expressed by the T-cells to generate multispecific cells are antigen receptors for multiple myeloma or lymphoblastic leukemia antigen markers, such as TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708) and FCRL5 (UNIPROT Q68SN8).

[0181] As a further example, the target antigen can be from any cluster of differentiation molecules (e.g., CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138), tumor-associated surface antigens such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigens, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate enzyme-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGF1 receptor, mesothelin, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin and A1 domain of tenascin-C (TNC A1) and fibroblast-associated protein (fap); lineage-specific or tissue-specific antigens such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF17), or virus-specific surface antigens such as HIV-specific antigens (such as HIV gp120); EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lassa virus-specific antigens, influenza virus-specific antigens, and any derivatives or variants of these surface markers. The antigen is not necessarily a surface marker antigen, but can also be an endogenous small antigen presented by HLA-I class on the cell surface.

[0182] As an example, the present invention encompasses single-chain CARs that specifically target cell surface markers (such as CD38, CS1 and / or CD70, as described in the Examples), together with inactivation of genes encoding CD38, CS1 and / or CD70, respectively, in cells expressing said CARs.

[0183] As a specific example, the VH and VL chains of the scFv anti-CD38 share at least 80%, preferably 90% and more preferably 95% identity with SEQ ID NOs: 10 and 12 and SEQ ID NOs: 11 and 13, respectively.

[0184] As a specific example, an antibody or epitope binding on the CD38 antigen is characterized in that the antibody or its epitope binding fragment comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three consecutive complementarity determining regions having an amino acid sequence represented by SEQ ID NOS: 14-17, and wherein the light chain comprises three consecutive complementarity determining regions having an amino acid sequence represented by SEQ ID NOS: 21-23.

[0185] As another specific example, an antibody or epitope binding on the CD38 antigen is characterized in that the antibody or epitope binding fragment comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three consecutive complementarity determining regions having an amino acid sequence represented by SEQ ID NOS: 18-20, and wherein the light chain comprises three consecutive complementarity determining regions having an amino acid sequence represented by SEQ ID NOS: 24-26.

[0186] As another specific example, the VH and VL chains of the scFv anti-CS1 share at least 80%, preferably 90%, and more preferably 95% identity with SEQ ID NO: 38-40-42-44-46 and SEQ ID NO: 39-41-42-45-46, respectively.

[0187] As yet another specific example, the VH and VL chains of the scFv anti-CD70 share at least 80%, preferably 90%, and more preferably 95% identity at the polynucleotide or nucleic acid level with SEQ ID NOs: 81-82; 85-86; 89-91 and SEQ ID NOs: 83-84; 87-88; 91-92, respectively.

[0188] In one embodiment, the present invention encompasses polynucleotides encoding single CAR anti-CD38, which share at least 80%, preferably 90%, and more preferably 95% identity with SEQ ID NOs: 35-37. In another embodiment, the present invention encompasses polynucleotides encoding single CAR anti-CS1, which share at least 80%, preferably 90%, and more preferably 95% identity with SEQ ID NOs: 48-62.

[0189] In yet another embodiment, the present invention encompasses polynucleotides encoding single CAR anti-CD70 that share at least 80%, preferably 90%, and more preferably 95% identity with SEQ ID NOs: 93-101.

[0190] The present invention more specifically focuses on CARs that have some consistency with those described in the present application and will carry rare-cutting endonuclease-induced mutations in genes encoding the cell markers targeted by the CAR. Immune cells (i.e., the CAR exhibits affinity for the product of the inactivated gene). Consistency refers to at least 70%, preferably 80%, more preferably 90%, and even more preferably 95% polynucleotide or polypeptide consistency determined by software such as FASTA, or BLAST, which can be used as part of the GCG sequence analysis software package (University of Wisconsin, Madison, Wis.). BLASTP "consistency" indicates the number and score of total residues in the consistent high-scoring sequence pair. Amino acid sequences with these degrees of consistency or similarity or any intermediate degree of consistency or similarity with the amino acid sequences disclosed herein have been envisioned and are covered by the disclosure of the present invention. This is also applicable to the use of BLASTN polynucleotide sequences.

[0191] Multimeric CAR

[0192] The chimeric antigen receptors of the prior art introduced into T-cells consist of single-chain polypeptides that require continuous attachment of signaling domains. However, moving the signaling domain from its natural proximal membrane position may interfere with its function. In order to overcome this shortcoming, the applicant has recently designed a multi-chain CAR derived from FcεRI to allow normal proximal membrane positions for all relevant signaling domains. In this new architecture, the high-affinity IgE binding domain of the FcεRIα chain is replaced by an extracellular ligand binding domain such as scFv to redirect the specificity of T-cells to cellular targets, and the N- and / or C-terminal tails of the FcεRIβ chain are used to place costimulatory signals at normal proximal membrane positions.

[0193] Therefore, the CAR expressed by the engineered T-cell according to the present invention can be a multi-chain chimeric antigen receptor (CAR) that is particularly suitable for the production and amplification of the engineered T-cell of the present invention. This multi-chain CAR comprises at least two of the following components:

[0194] a) a polypeptide comprising the transmembrane domain and the extracellular ligand binding domain of the FcεRIα chain,

[0195] b) a polypeptide comprising a portion of the N- and C-terminal cytoplasmic tails and the transmembrane domain of the FcεRIβ chain and / or

[0196] c) at least two polypeptides each comprising a portion of the intracytoplasmic tail and the transmembrane domain of the FcεRIγ chain, whereby the different polypeptides spontaneously multimerize together to form a dimeric, trimeric or tetrameric CAR.

[0197] According to this structure, the ligand binding domain and the signaling domain are produced on a separate polypeptide. The different polypeptides are anchored on the membrane of the close neighbors to allow interaction with each other. In such a structure, the signal and the costimulatory domain can be in a near-membrane position (i.e., adjacent to the cell membrane on its inner side), which is considered to allow the function of the costimulatory domain to be improved. The multi-subunit architecture also provides more flexibility and possibilities for designing CARs with more control over T-cell activation. For example, it is possible to include a variety of extracellular antigen recognition domains with different specificities to obtain a multi-specific CAR structure. It is also possible to control the relative ratio between the different subunits to become a multi-chain CAR. This type of structure has recently been introduced by the applicant in PCT / US2013 / 058005 (WO2014 / 039523).

[0198] For example, by using the different α, β and γ chains of the high affinity receptor for IgE (FcεRI) (Metzger, Alcarazet al. 1986) fused with the signal and co-stimulatory domains, the different chains can be assembled as part of a single multi-chain CAR. The γ chain contains a transmembrane region and a cytoplasmic tail containing an immunoreceptor tyrosine-based activation motif (ITAM) (Cambier 1995).

[0199] The multi-chain CAR can include multiple extracellular ligand binding domains to simultaneously bind to different components in the target to increase the activation and function of immune cells. In one embodiment, the extracellular ligand binding domain can be placed in series on the same transmembrane polypeptide, and can optionally be separated by a linker. In another embodiment, the different extracellular ligand binding domains can be placed on different transmembrane polypeptides constituting the multi-chain CAR. In another embodiment, the present invention relates to a multi-chain CAR group each comprising a different extracellular ligand binding domain. In a specific embodiment, the present invention relates to a method for engineering immune cells, including providing immune cells, and expressing a population of multi-chain CARs each comprising a different extracellular ligand binding domain on the cell surface. In another specific embodiment, the present invention relates to a method for engineering immune cells, including providing immune cells, and introducing into the cell a polynucleotide encoding a polypeptide consisting of a population of multi-chain CARs each comprising a different extracellular ligand binding domain. In a specific embodiment, the method for engineering immune cells includes expressing at least a portion of FcεRIβ and / or γ chains fused to a signal transduction domain and multiple portions fused to FcεRIα chains of different extracellular ligand binding domains on the surface of the cell. In a more specific embodiment, the method includes introducing into the cell at least one polynucleotide encoding a portion of FcεRIβ and / or γ chain fused to a signal transduction domain and a plurality of FcεRIα chains fused to different extracellular ligand binding domains. For a multi-chain CAR group, it refers to at least two, three, four, five, six or more multi-chain CARs each comprising a different extracellular ligand binding domain. The different extracellular ligand binding domains according to the present invention can preferably bind to different components in the target simultaneously to increase the activation and function of immune cells.

[0200] The present invention also relates to an isolated immune cell comprising a population of multi-chain CARs, each comprising a different extracellular ligand binding domain.

[0201] The signal transduction domain or intracellular signaling domain of the multi-chain CAR of the present invention is responsible for intracellular signaling after the extracellular ligand binding domain binds to the target to cause the immune cell activation and immune response. In other words, the signal transduction domain is responsible for the activation of at least one normal effector function of the immune cell expressing the multi-chain CAR. For example, the effector function of T-cells may be cytolytic activity or auxiliary activity including cytokine secretion.

[0202] In the present application, the term "signal transduction domain" refers to the portion of a protein that transduces the effector signaling function signal and directs the cell to perform a specialized function.

[0203] Preferred examples of signal transduction domains for single or multi-chain CARs can be the cytoplasmic sequences of the co-receptors of the Fc receptor or T-cell receptor and the co-receptors that act in concert to start signal transduction after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences of the same functional capabilities. The signal transduction domain includes two unique types of cytoplasmic signal sequences, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide secondary or costimulatory signals. The primary cytoplasmic signal sequence can include a signal motif of an activation motif of the immune receptor tyrosine-like called ITAM. ITAM is a well-defined signal motif found in the cytoplasmic tail of various receptors that act as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention can include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d as non-limiting examples. In a preferred embodiment, the signal transduction domain of the multi-chain CAR can include the CD3ζ signaling domain, or the intracytoplasmic domain of the FcεRIβ or γ chain.

[0204] In a specific embodiment, the signal transduction domain of the multi-chain CAR of the present invention comprises a co-stimulatory signal molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or ligands required for their effective immune response.

[0205] The ligand binding domain can be any previously used and mentioned antigen receptor for single-chain CARs referred to in the literature, specifically scFv from a monoclonal antibody. As described in WO2014 / 4011988, bispecific or multispecific CARs are incorporated herein by reference.

[0206] Similar to what has been described previously about single-chain CARs, the present invention encompasses immune cells endowed with multi-chain CARs that specifically target cell surface markers such as CD38, CS1, or CD70. According to a preferred embodiment of the present invention, the above-mentioned CARs are expressed in immune cells, and the inactivation of the endogenous gene encoding the surface marker is induced by the expression of a rare-cutting endonuclease.

[0207] T-cell activation and expansion

[0208] The method according to the invention generally comprises a further step of activating and / or expanding the T-cells. This can be accomplished before or after genetic modification of the T-cells using methods such as, for example, those described in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. According to these methods, the T-cells of the present invention can be expanded by contacting with an agent that stimulates a signal associated with the CD3TCR complex and a surface to which a ligand that stimulates a co-stimulatory molecule on the surface of the T-cell is attached.

[0209] Specifically, the T-cell population can be stimulated in vitro, such as by contacting with anti-CD3 antibodies, or antigen-binding fragments thereof, or anti-CD2 antibodies fixed on the surface, or by contacting with protein kinase C activators (e.g., bryostatin) in combination with calcium ion carriers. For the co-stimulatory effect of auxiliary molecules on the surface of the T-cells, ligands that bind auxiliary molecules are used. For example, the T-cell population can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating the proliferation of the T-cells. In order to stimulate the proliferation of CD4+T-cells or CD8+T-cells, anti-CD3 antibodies and anti-CD28 antibodies. For example, the reagent providing each signal can be in a solution or coupled to a surface. Because it is easy to understand for those of ordinary skill in the art, the ratio of particles to cells can depend on the particle size relative to the target cell. In a further embodiment of the present invention, the cells, such as T-cells, are combined with beads coated with reagents, the beads and cells are subsequently separated, and the cells are then cultured. In an alternative embodiment, before culture, the beads and cells coated with the reagents are not separated but cultured together. Cell surface proteins can be linked by allowing paramagnetic beads (3×28 beads) to which anti-CD3 and anti-CD28 are attached to contact the T-cells. In one embodiment, the cells (e.g., 4 to 10 T-cells) and beads (e.g., in a ratio of 1:1) are M-450CD3 / CD28T paramagnetic beads) are combined with a buffer, preferably PBS (without divalent cations such as calcium and magnesium). Moreover, it is readily understood by those of ordinary skill in the art that any cell concentration can be used. The mixture can be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value therebetween. In another embodiment, the mixture can be cultured for 21 days. Conditions suitable for T- cell culture include suitable media (e.g., Minimal Essential Media or RPMI culture medium 1640 or X-vivo5, (Lonza)) that can contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, -10, -2, IL-15, TGFp and TNF- or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, human plasma proteins, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The medium can include RPMI 1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-Vivo 1 and X-Vivo20, Optimizer, and additional amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokines sufficient for T-cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures to be injected into a subject. The target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37° C.) and atmosphere (e.g., air + 5% CO). 2 ). T-cells that have been exposed to varying stimulation times can exhibit different properties.

[0210] In another specific embodiment, the cells can be expanded by co-culturing with tissues or cells. The cells can also be expanded in vivo after administering the cells to the subject, for example, in the blood of the subject.

[0211] Therapeutic applications

[0212] The T-cells obtainable by the various methods described above are intended to be used as a drug for treating cancer, infection or immune disease, etc. in a patient in need thereof.

[0213] The treatment can be palliative, therapeutic or preventive. It can be part of an allogeneic immunotherapy or part of an autologous immunotherapy treatment. By autologous, it is meant that the cells, cell line or cell population used to treat the patient is derived from the patient or a human leukocyte antigen (HLA) compatible donor. By allogeneic, it is meant that the cells or cell population used to treat the patient is not derived from the patient, but from a donor.

[0214] The T-cells engineered according to one of the previous methods can be pooled, frozen, and administered to one or more patients. When they are made non-alloreactive, they can be used as "off-the-shelf" therapeutic products, meaning that they can be universally infused into patients in need thereof.

[0215] The treatment is mainly used to diagnose patients with cancer, viral infection, autoimmune disease or graft-versus-host disease (GVHD). Cancer is preferably leukemia and lymphoma, which are liquid tumors, but solid tumors may also be involved. The types of cancer treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma and sarcoma, as well as certain leukemia or lymphoma, benign and malignant tumors, and malignant tumors, for example, sarcoma, carcinoma and melanoma. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0216] The present invention provides in Tables 4 to 14 examples of antigenic markers that can be targeted using the engineered cells of the present invention for treating different cancer types.

[0217] Preferred antigenic markers for use in the immunotherapy of the present invention are more specifically CD38, CD319 (CS1) and CD70.

[0218] The T-cells of the present invention, when equipped with specific CARs for the patient's own immune cells, particularly T-cells, allow the cells to be inhibited or regulated, which is a key step for treating autoimmune diseases such as rheumatoid polyarthritis, systemic lupus erythematosus, Sjögren's syndrome, scleroderma, fibromyalgia, myositis, ankylosing spondylitis, type I insulin-dependent diabetes, Hashimoto's thyroiditis, Addison's disease, Crohn's disease, celiac's disease, amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). Therefore, the present invention covers methods for treating immune diseases by targeting the previously described engineered T-cells to the patient's own T-cells.

[0219] The above-mentioned treatment can be combined with one or more therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy and radiotherapy.

[0220] The engineered T-cells as previously described are particularly suitable for treating various forms of cancer when they are made resistant to chemotherapeutic and immunosuppressive drugs used as standard of care, particularly methotrexate and fludarabine and cyclophosphamide combinations. In fact, the present invention preferably relies on cells or cell populations, in which respect it is expected that the chemotherapeutic and / or immunosuppressive treatment should facilitate the in vivo selection and expansion of the engineered T-cells.

[0221] In certain embodiments of the invention, the cells are administered to a patient in conjunction with any number of related treatment modalities (e.g., before, simultaneously, or after), including, but not limited to, treatment with agents such as antiviral therapy, 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 further embodiments, the T-cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin) which is important for growth factor-induced signaling (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 a further embodiment, the cell compositions of the invention are administered to a patient in conjunction with bone marrow transplantation, T-cell ablative therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH (e.g., before, simultaneously or after), and in another embodiment, the cell compositions of the invention are administered after B-cell ablative therapy such as an agent reactive with CD20, e.g., rituximab (Rituxan). For example, in one embodiment, the subject can undergo peripheral blood stem cell transplantation after standard treatment with high-dose chemotherapy. In certain embodiments, after transplantation, the subject receives an injection of the amplified immune cells of the present invention. In other embodiments, the amplified cells are administered before or after surgery. The modified cells obtained by any of the methods described herein can be used in specific aspects of the present invention to treat patients in need thereof against host-versus-graft (HVG) rejection and graft-versus-host disease (GVHD); therefore, within the scope of the present invention is a method for treating patients in need thereof against host-versus-graft (HVG) rejection and graft-versus-host disease (GVHD), including treating the patient by administering to the patient an effective amount of modified cells comprising inactivated TCRα and / or TCRβ genes.

[0222] According to one embodiment, the T-cells of the present invention, once administered to a patient, may undergo robust in vivo T-cell expansion and be present in body fluids for an extended period of time, preferably 1 week, more preferably 2 weeks, and even more preferably at least one month. Although the T-cells according to the present invention are expected to persist during this period, their lifespan into the patient's body is expected to be no more than one year, preferably 6 months, more preferably 2 months, and even more preferably one month.

[0223] Administration of the cells or cell populations according to the present invention can be implemented in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intramedullaryly, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

[0224] Administration of the cell or cell population may consist of administering 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6 Cells / kg body weight, including all integer values ​​of cell numbers within those ranges. The cells or cell groups can be administered in one or more doses. In another embodiment, the cells of the effective amount are administered as a single dose. In another embodiment, the cells of the effective amount are administered as more than one dose for a period of time. The timing plan for administration is within the judgment of the managing physician and depends on the clinical condition of the patient. The cells or cell groups can be obtained from any source, such as a blood bank or a donor. Although individual needs change, the determination of the optimal range of the effective amount of a given cell type for a specific disease or condition is within the technology of the art. An effective amount refers to an amount that provides a therapeutic or preventive benefit. The dosage of the administration will depend on the age, health status and weight of the recipient, the type of concurrent treatment if present, the frequency of treatment and the nature of the desired therapeutic effect.

[0225] In another embodiment, the effective amount of cells or a composition comprising these cells is administered parenterally. The administration can be intravenous. The administration can be done directly by injection in the tumor.

[0226] Identification of surface antigen markers expressed on the surface of T-cells that are overexpressed in cells involved in different cancer types (Tables 5 to 13) in solid tumors

[0227] We used BioGPS microarray data from a small set of normal tissue (Human U133A / GNF1H Gene Atlas) cancer microarray data that can also be downloaded from BioGPS (Human Primary Tumor (U95)) UniProt data that contain the subcellular localization described.

[0228] We derived the distribution of values ​​from normal tissue and determined a threshold of 5 for the relative expression.

[0229] We browsed all genes analyzed by microarray (44.000 probes representing about 13000 genes) and checked their localization in the membrane (proteins that were not indicated as membrane proteins were discarded). Expression in CD8+T-cells was checked by the BioGPS database. The genes are listed according to cancer type, where the corresponding expression is the highest (Tables 5 to 13).

[0230] Identification of surface antigen markers expressed on the surface of T-cells and overexpressed in different liquid hematological tumors (Table 14)

[0231] For this study, no RNA-seq data were available, so we used microarray data from a large study by the MILE consortium (Microarray Innovations in Leukemia), involving 11 laboratories (http: / / www.ngrl.org.uk / wessex / downloads / tm08 / TM08-S4-1_KenMills.pdf-Haferlach et al. 2010, http: / / www.ncbi.nlm.nih.gov / pubmed / 20406941). These raw data include results for ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), CLL (chronic lymphoblastic leukemia), CML (chronic myeloid leukemia), and MDS (myelodysplastic syndrome). We also used UniProt data for general subcellular localization.

[0232] We first derived the overall distribution of the values ​​of all genes on all studied tissues. Then, in order to understand the necessary level of expression, we selected a gene list (CD52, CD 20, CD33, CD19, CD25, CD44, CD47, CD96, CD116, CD117, CD135, TIM-3) that is expressed in some liquid tumors and therapeutic antibodies are available. For each gene, we studied the value obtained in the tumor of its expression. Then, we calculated the mean value of each tumor and gene pair that seemed to provide cell membrane proteins (cell membrane localization+description of at least one transmembrane domain in protein) for the gene. We discarded genes that were expressed below the threshold of 0.15 in all tissues. We listed those genes that were relatively expressed higher than 0.2 in T-cells in Table 14 and sorted. Therefore, Table 4 provides targeted liquid tumor cells according to the present invention, specifically, hypothetical antigen marker candidates for the treatment of ALL, AML, CLL, CML and MDS.

[0233] Examples of procedures for engineering T-cells for immunotherapy according to the invention

[0234] For a better understanding of the present invention, an example of the steps followed to generate T-cells against leukemic CD38-positive cells is provided below:

[0235] 1. Provide T-cells from cell culture or blood samples from an individual patient or blood bank and use anti-CD3 / C28 activator beads Activating the T-cells. The beads provide the primary and co-stimulatory signals required for T-cell activation and expansion.

[0236] 2. Transduce the cells with a retroviral vector comprising a transgene encoding a chimeric antigen receptor consisting of a fusion of a CD3ζ activation domain, a 4-1BB co-stimulatory domain, a transmembrane domain, and a hinge from CD28 fused to a sequence encoding an anti-CD38 antibody variable chain. In order to safely improve the transformed T-cells, a suicide gene sensitive to rituximab can be further introduced into the lentiviral vector separated by the T2A split sequence as described in WO2013 / 153391.

[0237] 3. (Optionally) Engineering non-alloreactive and / or resistant T-cells:

[0238] a) It is possible to inactivate TCRα in the cells to eliminate the TCR from the cell surface and prevent allogeneic TCR from recognizing host tissue as foreign and thereby avoid GvHD by following the approach proposed in WO2013 / 176915.

[0239] b) It is also possible to inactivate a gene encoding a target for an immunosuppressant or chemotherapeutic drug to render the cells tolerant to immunosuppressive or chemotherapeutic treatment to prevent transplant 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 (e.g., alemtuzumab), as described in WO2013 / 176915.

[0240] 4. Gene inactivation by using TAL-encoding specific endonucleases (TALEN TM -Cellectis, 8 rue de la Croix Jarry, France) by electroporating T-cells with mRNA. Inactivated T-cells are sorted using magnetic beads. For example, T-cells that still express the targeted gene (e.g., CD38, CD70 and CD70) can be removed by fixation on a solid surface, while the inactivated cells are not exposed to the stress of passing through the column. This flexible approach increases the concentration of correctly engineered T-cells.

[0241] 5. Expansion of engineered T-cells in vitro prior to administration to the patient or in vivo following administration to the patient via CD3 complex stimulation. Prior to the administration step, the patient can undergo immunosuppressive therapy such as CAMPATH1-H, a humanized monoclonal anti-CD52 antibody.

[0242] 6. Optionally, the engineered cells are brought into proximity with the target antigen by exposing the cells to the bispecific antibody in vitro prior to administration to the patient or in vivo after administration to the patient.

[0243] Monocistronic mRNA encoding anti-CD38 single-chain chimeric antigen receptor (CAR CD38) Functional analysis of electroporated engineered T-cells:

[0244] In order to verify that genome engineering does not affect the ability of engineered T-cells to exhibit anti-tumor activity, especially when a chimeric antigen receptor (CAR CD38) is provided. The engineered T-cells were incubated for 4 hours with Daudi cells expressing CD38 on their surface. The cell surface upregulation of CD107a, marked by the release of cytotoxic granules of T lymphocytes (called degranulation), was determined by flow cytometry analysis (Betts, Brenchley et al. 2003).

[0245] 24h after electroporation, cells were stained with a fixable viability dye eFluor-780 and a PE-conjugated goat anti-mouse IgG F(ab')2 fragment for specific evaluation of cell surface expression of CAR on the living cells. Most of the live T-cells with genetic disruption of CD38 expressed the CAR on their surface. T-cells were co-cultured with Daudi (CD38+) cells for 6h and analyzed by flow cytometry to detect the expression of the degranulation marker CD107a on its surface (Betts, Brenchleyet al.2003).

[0246] The results show that CD38-destroyed T-cells retain the same ability to degranulate in response to PMA / ionomycin (positive control) or CD38+Daudi cells. CD107 upregulation is dependent on the presence of CD38+. These data indicate that the genome engineering of the T-cells of the present invention has no negative impact on the ability of the T-cells to mount a controlled anti-tumor response.

[0247] Table 4: Clusters of differentiation (CD) antigen markers found to be expressed on the surface of T-cells for various cancers

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257] Table 5: Antigen markers expressed on the surface of colon tumor cells and T-cells

[0258]

[0259]

[0260] Table 6: Antigen markers expressed on the surface of breast tumor cells and T-cells

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269] Table 7: Antigen markers expressed on the surface of digested tumor cells and T-cells

[0270]

[0271]

[0272]

[0273] Table 8: Antigen markers expressed on the surface of renal tumor cells and T-cells

[0274]

[0275]

[0276]

[0277] Table 9: Antigen markers expressed on the surface of liver tumor cells and T-cells

[0278]

[0279] Table 10: Antigen markers expressed on the surface of lung tumor cells and T-cells

[0280]

[0281]

[0282]

[0283]

[0284]

[0285] Table 11: Antigen markers expressed on the surface of ovarian tumor cells and T-cells

[0286]

[0287]

[0288] Table 12: Antigen markers expressed on the surface of pancreatic tumor cells and T-cells

[0289]

[0290]

[0291] Table 13: Antigen markers expressed on the surface of prostate tumor cells and T-cells

[0292]

[0293]

[0294] Table 14: Antigen markers expressed on the surface of T-cells and overexpressed in liquid tumor cells (ALL, AML, CML, MDS, CLL, CTRL)

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312] Example 1-Knockout (KO) of CD38 gene & expression of anti-CD38 CAR

[0313] Description of the CD38 target - cyclic ADP ribose hydrolase

[0314] CD38 is a glycoprotein found on many immune cells, including multiple myeloma (MM) cells that express high levels of CD38 in most patients. CD38 is a confirmed target of MM, because many studies have shown that anti-CD38 mAb can effectively kill CD38+MM cells and CD38+MM cell lines from patients by CDC and ADCC (Ellis, JHKetal, Journal of Immunology, 1995, 155 (2), 925-937). Daratumumab is a therapeutic human CD38 monoclonal antibody that induces killing of multiple myeloma and other hematological tumors (De Weers, M.et al, J Immunol 2011 186: 1840-1848). In some studies, it has been shown that CD38 is also highly expressed by activated T- cells (Sandoval-Montes CJ et a:, 2005, Leukoc Biol.77 (4): 513-21).

[0315] CD38 expression by T-cells

[0316] CD38 expression by T-cells after CD3 / CD28 beads and IL-2 stimulation was analyzed by FACS every 3-4 days over 17 days. It was observed that more than 90% of T-cells expressed ( Fig. 10B ).

[0317] Therefore, in order to avoid killing activated T-cells by anti-CD38CAR+T-cells, it is necessary to prevent the surface expression of CD38 in T-cells. This can be done by inactivating the CD38 gene using TALE-nuclease. TALEN is a trademark owned by the applicant (Cellectis, 8 rue de la Croix Jarry, 75013 PARIS), specifying a custom form of TAL nuclease.

[0318] The CD38 knockout (KO) strategy

[0319] Heterodimeric TALE-nucleases targeting two 17-pb long sequences separated by a 13-pb spacer in the CD38 gene were designed and produced. Each half of the target was expressed as listed in Tables 15 and Fig. 10A Repeat recognition of the half TALE-nuclease in.

[0320] The repeat sequence of the left TALEN of the CD38ex1_T2 target is NN-NI-NN-NN-NG-NN-NN-NN-NG-NG-NN-NN-HD-NN-NI-NG, while the repeat sequence of the right TALEN is NN-NG-HD-HD-HD-HD-NN-HD-NI-NN-NG-NN-HD-HD-NG.

[0321] Table 15: Sequences of tested CD38 targets and TALENs used for activation of the CD38 antigen

[0322]

[0323] Each TALE-nuclease construct was subcloned using restriction enzyme digestion in a mammalian expression vector under the control of a T7 promoter. mRNA encoding a TALE-nuclease cleaving CD38 was synthesized from a plasmid carrying the coding sequence downstream from the T7 promoter.

[0324] Purified T-cells activated by anti-CD3 / CD28 coated beads and recombinant IL-2 were transfected by electroporation (Cytopulse) with each 2mRNA (10 μg each) encoding two and a half CD38ex1_T2TALE-nucleases. For the study, the percentage of CD38 KO, CD38 negative T-cells was evaluated by flow cytometry at days 3, 6, 10 and 13 after TALEN mRNA gene transfection. It was observed that 15% of the transfected T-cells were CD38 deficient (Figure 10) and this deficiency was stable within 13 days after transfection.

[0325] In addition, two alternative TALE-nucleases targeting the CD38 gene were designed. Each half-target was identified by the following Table 16 and Fig. 10A The repeats of the half TALE-nucleases listed in are recognized. The repeat sequence of the left TALEN of the CD38ex1_T4 target is NG-NN-HD-NN-NI-NN-NG-NG-HD-NI-NN-HD-HD-HD-NN-NN-NG, while the repeat sequence of the right TALEN is NG-NN-HD-NG-NN-HD-NN-NN-NN-HD-NG-HD-NG-HD-NI. The repeat sequence of the left TALEN of the CD38ex1_T5 target is NG-NN-NI-NG-HD-HD-NG-HD-NN-NG-HD-NN-NG-NN-NN-NG, while the repeat sequence of the right TALEN is HD-NN-NI-NN-NN-NG-NN-NN-HD-NN-HD-NI-NN-HD-NI.

[0326] Table 16: Sequences of two other CD38 targets and corresponding TALENs for their activation

[0327]

[0328]

[0329]

[0330] Expression strategy of the CAR anti-CD38

[0331] Structure and composition of CAR anti-CD38

[0332] The VH and VL chains of scFv anti-CD38 are provided in Table 17. SEQ ID NOs: 10-11 correspond to the humanized anti-CD38 antibody daratumumab (Genmab) and SEQ ID NOs: 12-13 correspond to the MOR202 (or MOR03087), as described in US 8,263,746B patent.

[0333] SEQ ID NOs: 14-20 and SEQ ID NOs: 21-26 correspond to the CDR sequences of the VH chain (HCDR) and the VL chain (LCDR), respectively, as described in the WO2008 / 047242 application.

[0334] Table 17: Sequences of the VH and VL chains of the scFv anti-CD38 antibodies daratumumab, MOR202 and sequences of specific CDRs of the VH and VL chains.

[0335]

[0336]

[0337] For the daratumumbab scFv3, different CAR constructs (GMB005-V1&V2&V3) were designed, such as Fig.11 A, and its sequence is listed in Table 18 below. All three constructs share the same components in terms of signal clause peptide (CD8α), GS linker (between the scFvVH and VL chains), transmembrane domain (TM), 4-1BB costimulatory domain and CD3ζ activation domain (sequences listed in Table 18 below). The difference comes from the choice of the hinge (Table 18):

[0338] V1: FcRIIa hinge

[0339] -V2: CD8a hinge

[0340] -V3: IgG1 hinge

[0341] Table 18: Three different structures of anti-CD38 CAR of the scFv daratumumab class and the peptide sequences of each component used

[0342]

[0343] filter

[0344] CD38 TALEN will be transfected on day 4 after activation. After 3 days, the CD38-deficient cells will be sorted by negative selection and transfected with anti-CD38 CAR mRNA 3 days later. Once the CAR mRNA temporary transfection is completed, the generated CAR molecules will then be screened for expression and degranulation activity towards the target cell line expressing CD38. Different expression levels of CD38 expression ( Fig.11 B) Target cell lines to be used for activity testing:

[0345] -U266 CD38+ and U266 CD38- obtained by magnetic separation using anti-CD38 microbeads

[0346] -L363, a multiple myeloma cell line expressing moderate levels of CD38

[0347] -Daudi, a cell line derived from Burkitt's lymphoma that expresses high levels of CD38

[0348] - K562, a cell line derived from chronic myeloid leukemia, is a CD38-negative cell line.

[0349] This first screen will be followed by a second screening step, in which many of the selected candidates will be tested for their ability to induce degranulation, IFNγ release, and specific cytotoxic activity against the selected target cell line. The selection of candidates will then be narrowed, and some of the selected candidates for lentiviral vector production and CAR activity will be evaluated in CD38 KO T-cells stably expressing the CAR.

[0350] Example 2 Activity of anti-CS1 CAR in the context of CS1 KO

[0351] Introduction of CS1 target

[0352] Multiple myeloma (MM) is a B-cell malignancy characterized by abnormal clonal expansion of plasma cells (PCs) within the bone marrow, with an estimated 21,700 new cases of MM and 10,710 deaths identified in the United States in 2012 (Siegel R, et al. Cancer J Clin 2012 62: 10–29). In 2013, it is estimated that 22,350 individuals will be newly diagnosed with MM and 10,710 will die from the disease in the United States, accounting for 20% of all deaths from hematological malignancies. Despite the use of proteasome inhibitors and immunomodulatory drugs, which have improved overall survival (Palumbo A, et al. Leukemia 2009 23: 449–456), MM remains an incurable malignancy (Podar K, et al. Leukemia 2009 23: 10–24), for which new treatments are urgently needed.

[0353] The cell surface glycoprotein CS1 (also referred to in the literature as SLAMF7, CD319 or CRACC-NCBI reference sequence: NP_067004.3) is highly and widely expressed on the surface of the myeloma cells (Hsi ED, et al. Clin Cancer Res 2008 14: 2775–84). CS1 is expressed at very low levels on most immune effector cells, including natural killer (NK) cells, certain subsets of T-cells and normal B cells, and is almost undetectable on myeloid cells (Hsi ED, et al. Clin Cancer Res 2008 14: 2775–84). It is noteworthy that the expression of CS1 in human hematopoietic stem cells is negligible (Hsi ED, et al. Clin Cancer Res 2008 14: 2775–84), which can be used for stem cell transplantation to treat hematological malignancies, including MM. The function of CS1 in MM is still not fully understood, and it has been documented that CS1 may play a role in myeloma cell adhesion, growth of colony formation, and tumorigenesis (Benson DM Jr, et al. J Clin Oncol 2012 30:2013–5; Tai YT, et al. Blood 2009 113:4309–18).

[0354] The structure of the CAR anti-CS1

[0355] As in Example 1, for the anti-CD38 antigen target single-chain CAR, a hinge, a transmembrane domain, a co-activation domain and a transduction domain (such as Fig.11 A and the sequences shown in Table 18) with respect to the same components and the same structures V1, V2 and V3.

[0356] The VH and VL chains of the scFv anti-CS1 are given in Table 19. SEQ ID NOs: 38-40-42-44-46 and SEQ ID NOs: 39-41-43-45-47 correspond to the VH and VL chains of the murine scFvs Luc63, Luc90, Luc34, LucX1 and LucX2, respectively.

[0357] Table 20 shows anti-CS1 CARs with the above scFvs; these CARs are based on Fig.11 The versions V1, V2 and V3 of A, in which a short FcERγ hinge, a medium hinge CD8α hinge and a long IgG1 hinge are used, respectively. The underlined parts correspond to the scFv VH and VL chains joined by the linker.

[0358] Table 19: Sequences of the VH and VL chains of scFv anti-CS1 antibodies

[0359]

[0360] Table 20: Based on Fig.11 The polypeptide sequences of the anti-CS1 CAR of the V1, V2 and V3 versions described in A

[0361]

[0362]

[0363]

[0364] Strategies for CAR CS1+ and KO CS1 Engineering

[0365] CS1 is expressed at high levels in plasmacytoid cells from patients with multiple myeloma, making them an interesting target for CAR development. T-cells, especially the CD8 subset, express low levels of CS1, which is a disadvantage for T-cell CAR development because they may be killed when expressing anti-CS1 CAR.

[0366] In this example, we evaluated the activity of Luc90-V2CAR (sequence shown in Table 20) in mock transfected or human T-cells transfected with TALEN targeting the CS1 (SLAMF7) gene to observe whether the CAR activity is enhanced when the CS1 gene is disrupted in CAR+T-cells. The process of the experiment is as follows Fig.12 as shown in .

[0367] T-cells were purified from buffy coat samples and activated using CD3 / CD28 coated beads. Cells were co-transfected with 10 μg of mRNA encoding the T01_left TAL and 10 μg of mRNA encoding the T01_right TAL 72 h after activation. The sequences of the TALs are shown in Table 21 below and the plasmid constructs (T01, T02 and T03) with the TAL repeats are as follows: Fig.13 as shown in .

[0368] FIG. 14 shows the target locations of the TAL T01, T02 and T03 within the CS1 (SLAMF7) gene: T01 and T02 target exon 1 ( Fig.14A ), while T03 targets exon 2 ( Fig. 14B ).

[0369] Table 21: Sequences of the targets of CS1 and the TALENs used for its inactivation

[0370]

[0371] Three days after TALEn transfection, cells were transduced with a recombinant lentiviral vector that drove the L90-V2CAR to leave the EF1A promoter for expression. The lentiviral vector was constructed in such a way that CAR expression was combined with BFP expression (blue fluorescent protein) by ribosomal skippeptide. The L90-V2CAR was constructed by recognizing the extracellular binding domain of the CS1 target (scFv L90), followed by a hinge and a transmembrane region derived from the hCD8α protein. The intracellular portion of the molecule contains a 41BB-derived costimulatory domain, followed by a CD3γ signaling domain (sequences shown in Tables 18-19-20 above for individual components, scFv, and CAR sequences, respectively).

[0372] Transduction efficiency was assessed by flow cytometry following BFP expression 6 days after transduction. Cells were also stained with anti-CD8 and anti-CS1 antibodies.

[0373] result

[0374] CAR CS1+ expression

[0375] Fig.16The results showed that the transduction efficiency was higher in mock-transfected cells than in cells transfected with TALEn targeting the CS1 gene. This may be due to specific cell killing of non-transduced CS1-expressing T-cells, while this population was not affected when the cells no longer expressed CS1 due to TALEN-driven gene disruption.

[0376] No significant difference in CS1 levels was observed between TALEN or mock transfected (negative control-no plasmid transfection) cells at this time point, because CS1 levels decreased over time after initial activation of T- cells. On the other hand, a significant decrease in CD8+ cells was observed in mock transfected CAR-expressing cells compared to TALEN-transfected CAR+ cells, indicating that a high proportion of CD8+ cells have been eliminated by the CAR+T- cells.

[0377] Evaluation of cytotoxic activity

[0378] By co-culturing the same amount of T-cells with the cell line (L363 cells) expressing CS1 or the negative control cell line (MOLM13) lacking CS1 expression, the cytotoxicity of these cells was evaluated 8 days after CAR transduction. The viability of the target cell line was measured by flow cytometry 4h after starting cell co-culture. Fig.15 The results shown in A indicate that the cell viability of CS1(+) cells was reduced when they were co-cultured with CAR+T-cells, while no effect on the viability of CS1(-) cells was observed. The specific cell breakdown was calculated using the flow cytometry data and it was 2-fold higher when the T-cells were transfected with TALEn targeting the CS1 gene prior to CAR transduction. It should be considered that the effect could be even higher because the amount of CAR+T-cells present in the co-culture was higher when the cells were mock-transfected (see Fig.16 The experimental results are as follows:

[0379] - For the sham / NTD samples, the % of BFP+ cells was 0.1% and the amount of CD8+ cells was 53.9%;

[0380] - For the TALEn / NTD sample, the % of BFP+ cells was 0.2% and the amount of CD8+ cells was 49.5%;

[0381] - For the sham / L90-2 sample, the % of BFP+ cells was 94% and the amount of CD8+ cells was 1.8%

[0382] - For the TALEn / L90-2 sample, the % of BFP+ cells was 61% and the amount of CD8+ cells was 8.3%.

[0383] The transduction efficiency was higher in mock-transfected cells than in cells transfected with TALEn targeting the CS1 gene (NTD: not transduced).

[0384] Reactivation after transduction

[0385] To confirm that the CS1 gene had been disrupted in TALEn-transfected T-cells, the different samples were reactivated with CD3 / CD28 beads at D11 after transduction. 72 h after reactivation, cells were stained with anti-CD8 and anti-CS1 antibodies and expression was analyzed by flow cytometry.

[0386] Fig.17 Transduction efficiency and CD8 / CS1 expression levels in each sample are shown. As shown in the lower panel, an increase in CS1 levels was observed in mock-transfected cells upon reactivation, whereas the number of cells capable of expressing CS1 was low in the TALEn-transfected population.

[0387] The results of the experiment are as follows:

[0388] - For the sham / NTD samples, the % of BFP+ cells was 0.01%, CS1 was expressed in 65.2% of cells, and the amount of CD8+ cells was 80.7%;

[0389] - For the TALEn / NTD sample, the % of BFP+ cells was 0.2%, CS1 was expressed in 9.7% of cells, and the amount of CD8+ cells was 78.8%;

[0390] - For the sham / L90-2 sample, the % of BFP+ cells was 94%, CS1 was expressed in 37.5% of the cells, and the amount of CD8+ cells was 16%;

[0391] - For the TALEn / L90-2 sample, the BFP intensity was 61%, the CS1 expression was 8.5%, and the CD8 expression was 68.5%;

[0392] Increased CS1 levels were observed in mock-transfected cells upon reactivation, whereas the number of cells capable of expressing CS1 was lower in the TALEn-transfected population.

[0393] Together, these results indicate that the CS1 gene was disrupted in TALEn-transfected T-cells and that this enhanced the cytotoxic activity of anti-CS1CAR+ cells, primarily by preserving the cytotoxic CD8+ T-cells.

[0394] Example 3: CD70 Target

[0395] Introduction of CD70 target

[0396] The CD70 is a cytokine that binds to CD27 and is part of the TNF family (Goodwin R.G. et al, 1993, Cell 73: 447-456). This protein plays a role in adaptive T-cell responses, induces co-stimulated T-cell proliferation, and enhances the production of cytolytic T-cells. Its accession number is P32970 (Uniprot). As shown in some studies in the literature Schürch, C. et al. (J. Clin. Invest., 2012; doi: 10.1172 / JCI45977), blocking the CD27-CD70 interaction may help treat chronic myeloid leukemia (CML).

[0397] Strategy of CD70 KO

[0398] The same strategy for CD70 gene KO was performed as in Examples 1 and 2. A heterodimeric TALE-nuclease targeting two 49pb long sequences separated by a 15pb spacer in the CD70 gene and a TALE nuclease targeting a 57-pb long sequence separated by a 23bp spacer were designed and produced. Each half target was recognized by a repeat of the half TALE nuclease listed in Table 2 below.

[0399] Table 22: Sequences of the CD70 targets and TALENs used for their inactivation

[0400]

[0401] Anti-CD70 CAR expression strategy

[0402] The same strategy for expressing CAR anti-CD70 was performed as described in Examples 1 and 2.

[0403] As in Example 1-2, the same components are used in terms of signal peptide, linker between the VH and VL chains, transmembrane domain, co-activation and transduction domains ( Fig.11 The same structures V1, V2 and V3 were designed based on the general structure shown in A and the sequences of the individual components shown in Table 18. Only the hinges differed between the three versions V1, V2 and V3, where a short FcERγ hinge, a medium hinge CD8α hinge and a long IgG1 hinge were used, respectively.

[0404] The VH and VL chains of scFv anti-CD70 are given in Table 23. SEQ ID NOs: 81-82, 85-86, 89-90 and SEQ ID NOs: 83-84, 87-88, 91-92 correspond to the VH and VL chains of scFv Ab4, Ab8 from AMGEN and 1F6 from Seattle Genetics, respectively.

[0405] Table 24 shows the anti-CD70 CARs with the above scFvs; these CARs are based on Fig.11 Versions of A, V1, V2, and V3, in which a short FcEγ hinge, a medium hinge CD8, and a long IgG1 hinge were used, respectively.

[0406] Table 23: Polynucleotides and nucleic acid sequences of the VH and VL chains of the scFv anti-CD70Ab4, Ab8 and 1F6 antibodies

[0407]

[0408]

[0409] Table 24: Based on Fig.11 The polypeptide sequences of the anti-CD70CAR versions of V1, V2 and V3 of A

[0410]

[0411]

[0412] References

[0413] Bardenheuer, W., K.Lehmberg, et al. (2005). "Resistance to cytarabine andgemcitabine and in vitro selection of transduced cells after retroviralexpression of cytidine deaminase in human hematopoietic progenitor cells." Leukemia 19(12):2281-8.

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Claims

1. A method for preparing T-cells for immunotherapy of pathological cells, comprising the following steps: a) genetically inactivating a gene encoding an antigen marker in a T-cell, the antigen marker being present on the surface of both the T-cell and the pathological cell, wherein the antigen marker is selected from the group consisting of CD38, CS1 and CD70; b) expressing in said T-cells a transgene encoding a chimeric antigen receptor directed against said antigen marker present on the surface of said pathological cells.

2. The method of claim 1, wherein the antigen marker is CD38.

3. The method according to any one of claims 1 to 2, wherein the method comprises the further step of activating and expanding the T-cells.

4. The method according to any one of claims 1 to 2, wherein the method comprises the further step of purifying the obtained T-cells by excluding cells presenting the antigen marker on their surface.

5. The method according to any one of claims 1 to 2, wherein the method comprises a preliminary step of obtaining the T-cells from a donor.

6. The method according to any one of claims 1 to 2, wherein the method comprises a preliminary step of obtaining the T-cells from a patient affected by the development of the pathological cells.

7. The method according to any one of claims 1 to 2, wherein the T-cells are derived from primary stem cells, iPS cells or hES cells.

8. The method according to claim 7, wherein the T-cells are derived from iPS cells obtained from a patient affected by the development of the pathological cells.

9. The method according to any one of claims 1 to 2, wherein step a) is performed using a rare-cutting endonuclease.

10. The method according to claim 9, wherein step a) is performed using TAL-nuclease.

11. The method of claim 9, wherein step a) is performed using an RNA-guided endonuclease.

12. The method of claim 11, wherein the RNA-guided endonuclease is Cas9.

13. The method of claim 11, wherein the RNA-guided endonuclease cleaves into at least two polypeptides, one comprising RuvC and the other comprising HNH.

14. The method of claim 9, wherein the endonuclease is expressed from transfected mRNA.

15. The method according to any one of claims 1 to 2, wherein the method comprises the further step of inactivating genes encoding components of the T-cell receptor.

16. The method of claim 15, wherein the component of the T-cell receptor is TCRα.

17. The method according to any one of claims 1 to 2, wherein the method comprises the further step of inactivating genes encoding components of HLA.

18. The method according to any one of claims 1 to 2, wherein the method comprises the further step of inactivating the gene encoding β2m.

19. The method according to any one of claims 1 to 2, wherein the method comprises inactivating a gene encoding a protein selected from the group consisting of CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, C A further step is the genes of immune checkpoint proteins ASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.

20. The method according to any one of claims 1 to 2, wherein the method comprises the further step of inactivating the gene encoding PDCD1 and / or the gene encoding CTLA-4.

21. The method according to any one of claims 1 to 2, wherein the method comprises the further step of inactivating a gene that provides sensitivity of the T-cells to chemotherapeutic or immunosuppressive drugs.

22. The method of claim 21, wherein the gene in said further step encodes CD52.

23. The method of claim 21, wherein the gene in the further step is hypoxanthine-guanine phosphoribosyltransferase.

24. The method of claim 21, wherein the gene in the further step encodes a glucocorticoid receptor.

25. The method of claim 21, wherein the genes in said further step are involved in the DCK regulatory pathway.

26. The method of claim 25, wherein the gene in said further step is involved in DCK expression.

27. The method according to any one of claims 1 to 2, wherein the T-cells in step a) are derived from inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes.

28. The method according to claim 27, wherein the T-cells are derived from CD4+ T-lymphocytes and / or CD8+ T-lymphocytes.

29. The method according to any one of claims 1 to 2, wherein the transformed T-cells are expanded in vitro.

30. The method according to any one of claims 1 to 2, wherein the transformed T-cells are expanded in vivo.

31. The method according to any one of claims 1 to 2, wherein the pathological cells are selected from malignant cells or infected cells.

32. The method according to any one of claims 1 to 2, wherein the pathological cells are B-cells.

33. The method according to any one of claims 1 to 2, wherein the pathological cells are solid tumor cells.

34. The method according to any one of claims 1 to 2, for preparing T-cells to be used as a medicament.

35. Use of T-cells prepared according to the method of claim 32 in the preparation of a medicament for treating lymphoma.

36. Use of the T-cell prepared according to the method of claim 32 in the preparation of a medicament for treating leukemia.

37. Use of T-cells prepared according to the method of claim 34 in the preparation of a medicament for treating chronic lymphocytic leukemia.

38. An engineered T-cell obtainable by the method according to any one of claims 1 to 34, characterized in that: The gene encoding the antigen marker CD38, CS1 or CD70 present on the surface of both the T-cell and the pathological cell is genetically inactivated, resulting in the lack of the antigen marker on the surface of the T-cell; and the T-cell expresses a chimeric antigen receptor for the antigen marker present on the surface of the pathological cell.

39. The engineered T-cell according to claim 38, producing the phenotype [CARCD38] + [CD38] - .

40. The engineered T-cell according to claim 38, producing the phenotype [CARCD70] + [CD70] - .

41. The engineered T-cell of claim 38, producing the phenotype [CAR CS1] + [CS1] - .

42. Use of a population of engineered T-cells as claimed in any one of claims 38 to 41 in the preparation of a medicament for treating a patient diagnosed with the presence of pathological cells presenting the specific antigen markers CD38, CD70 or CS1 common to T-cells.

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