MANIPULATED CELLS FOR IMMUNE THERAPY TARGETTING ANTIGEN IN IMMUNE CELLS AS WELL AS PATHOGEN CELLS

Description

Field of the invention

[0001] The present invention relates to genetically engineered, preferably non-alloreactive, T cells for immunotherapy, which are endowed with Chimeric Antigen Receptors targeting an antigen marker, characterized in that a gene encoding said antigen marker selected from CD38, CS1 and CD70 is genetically inactivated through the expression in the T cell of a transcription activator-like effector (TALE)-nuclease able to target said gene.

[0002] The engineered T-cells direct their immune activity towards malignant, infected cells or defective immune cells, while avoiding their mutual destruction, auto-stimulation or aggregation. The invention opens the way to standard and affordable adoptive immunotherapy strategies using immune cells for treating cancer, infections and auto-immune diseases.Background of the invention

[0003] Adoptive immunotherapy, which involves the transfer of autologous antigen-specific immune cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy, for instance, can be generated either by expansion of antigen-specific T-cells or redirection of T-cells through genetic engineering (Park, Rosenberg et al. 2011).

[0004] Novel specificities in T-cells have been successfully generated through the genetic transfer of transgenic T-cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have 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 enhance survival and increase proliferation of CAR modified T cells. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0005] US 2013 / 315884 and Schiffer-Mannioui et al (Journal for Immunotherapy of Cancer, 2013, 1(Suppl.1): 34) disclose the use of TALENs to delete antigen markers expressed on T-cells.

[0006] The current protocol for treatment of patients using adoptive immunotherapy is based on autologous cell transfer. In this approach, T lymphocytes are recovered from patients, genetically modified or selected ex vivo, cultivated in vitro in order to amplify the number of cells if necessary and finally infused into the patient. In addition to lymphocyte infusion, the host may be manipulated in other ways that support the engraftment of the T cells or their participation in an immune response, for example pre-conditioning (with radiation or chemotherapy) and administration of lymphocyte growth factors (such as IL-2). Each patient receives an individually fabricated treatment, using the patient's own lymphocytes (i.e. an autologous therapy). Autologous therapies face substantial technical and logistic hurdles to practical application, their generation requires expensive dedicated facilities and expert personnel, they must be generated in a short time following a patient's diagnosis, and in many cases, pretreatment of the patient has resulted in degraded immune function, such that the patient's lymphocytes may be poorly functional and present in very low numbers. Because of these hurdles, each patient's autologous cell preparation is effectively a new product, resulting in substantial variations in efficacy and safety.

[0007] Ideally, one would like to use a standardized therapy in which allogeneic therapeutic cells could be pre-manufactured, characterized in detail, and available for immediate administration to patients. By allogeneic it is meant that the cells are obtained from individuals belonging to the same species but are genetically dissimilar. However, the use of allogeneic cells presently has many drawbacks. In immune-competent hosts allogeneic cells are rapidly rejected, a process termed host versus graft rejection (HvG), and this substantially limits the efficacy of the transferred cells. In immune-incompetent hosts, allogeneic cells are able to engraft, but their endogenous T-cell receptors (TCR) specificities may recognize the host tissue as foreign, resulting in graft versus host disease (GvHD), which can lead to serious tissue damage and death.

[0008] In order to provide allogeneic T-cells, the inventors previously disclosed a method to genetically engineer T-Cells, in which different effector genes, in particular those encoding T-cell receptors, were inactivated by using specific TAL-nucleases, better known under the trade mark TALEN ™< (Cellectis, 8, rue de la Croix Jarry, 75013 PARIS). This method has proven to be highly efficiency in primary cells using RNA transfection as part of a platform allowing the mass production of allogeneic T-cells (WO 2013 / 176915).

[0009] CD38 (cluster of differentiation 38), also known as cyclic ADP ribose hydrolase is a glycoprotein found on the surface of many immune cells (white blood cells),in particular T-cells, including CD4+, CD8+, B lymphocytes and natural killer cells. CD38 also functions in cell adhesion, signal transduction and calcium signaling. Structural information about this protein can be found in the UniProtKB / Swiss-Prot database under reference P28907.In humans, the CD38 protein is encoded by the CD38 gene which located on chromosome 4. CD38 is a multifunctional ectoenzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose (cADPR) from NAD+ to ADP-ribose. These reaction products are deemed essential for the regulation of intracellular Ca2+. Also, loss of CD38 function was associated with impaired immune responses and metabolic disturbances (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).

[0010] On another hand, CD38 protein is a marker of HIV infection, leukemias, myelomas, solid tumors, type II diabetes mellitus and bone metabolism, as well as some other genetically determined conditions. In particular, it has been 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).

[0011] Although, cells expressing CD38, as well as many other tumor antigen markers referred to in Table 1, such as CD70 and CS1 could be regarded as attractive targets for CARs, the fact that such antigen markers are also expressed at the surface of most T-cells, has hampered significantly the selection of these markers to perform immunotherapy.

[0012] The inventors here provide strategies for immunotherapy involving pathological cells expressing specific antigen markers also present at the surface of T-cells, i.e. CD38, CD70 and CS1.Summary of the invention

[0013] The present invention is set out in the appended set of claims.

[0014] Particularly, in one aspect the present invention provides the use of a transcription activator-like effector (TALE)-nuclease specific for a target sequence within a gene encoding an antigen marker selected from CD38, CD70 and CS1 for inactivating the expression of said gene in a T-cell expressing a chimeric antigen receptor (CAR) specific for said antigen marker. By antigen marker is meant the whole protein or an immune-reactive fragment thereof.

[0015] In another aspect, the present invention provides an engineered T cell expressing a chimeric antigen receptor (CAR) specific for an antigen marker characterized in that a gene encoding said antigen marker selected from CD38, CS1 and CD70 is genetically inactivated through the expression in the T cell of a transcription activator-like effector (TALE)-nuclease able to target said gene.

[0016] In another aspect, the present invention provides said engineered T cell for use as a medicament, and more specifically for use in the treatment of cancer, infections or autoimmune disease.

[0017] According to the invention, the T-cells are engineered in order to inactivate the expression of the genes encoding such antigen markers.

[0018] This inactivation is performed by a genome modification, more particularly through the expression in the T-cell of a TALE-nuclease able to target a genetic locus encoding said antigen marker.

[0019] According to one embodiment, the T-cells can be further engineered to make them allogeneic, especially by deleting genes involved into self-recognition, such as those, for instance, encoding components of T-cell receptors (TCR) or HLA complex.

[0020] As a result of the invention, the engineered T-cells can be used as therapeutic products, ideally as an "off the shelf" product, in methods for treating or preventing cancer, infections or auto-immune disease.

[0021] Preferred T cells according to the present invention are those resulting into the phenotypes: [CAR CD38] +< [CD38] -< , preferably also [TCR] negative; [CAR CD70] +< [CD70] -< , preferably also [TCR] negative; [CAR CS1] +< [CS1] -< , preferably also [TCR] negative; for their use as therapeutic products, preferably allogeneic ones.Brief description of the figures and tables

[0022] Figure 1: Schematic representation of an engineered T-cell according to the present invention disrupted for CD38 and endowed with a chimeric antigen receptor (represented as a single-chain CAR) targeting a malignant cell bearing the antigen marker CD38. Figure 2: Schematic representation of a multi-subunit chimeric antigen receptor. Figure 3: Schematic representation of a therapeutic strategy according to the invention combining T-cells endowed with a multi-subunit CAR and circulating bi-specific antibody. In this particular aspect, the receptor present on the extracellular chain of the multi-subunit CAR is composed of an epitope which is recognized by a bi-specific antibody. The bi-specific antibody is intended to bind said epitope one the one hand and the antigen marker on the other hand to facilitate the binding of the T-cell to the pathological cell. Figure 4: Schematic representation of a therapeutic strategy according to the invention combining T-cells endowed with a multi-subunit CAR and circulating monoclonal antibody. In this particular aspect, the receptor present on the extracellular chain of the multi-subunit CAR is composed, for instance, of a Fc receptor intended to bind a monoclonal antibody that is directed against the antigen marker. The monoclonal antibody increases the chance of T-cells binding the pathological cells. Figure 5: Schematic representation of a therapeutic strategy according to the invention combining T-cells endowed with a multi-subunit CAR that comprises two extracellular cellular domains and one circulating bi-specific antibody. In this particular aspect, the extracellular cellular domains are located on distinct sub-units. These domains are respectively composed of an epitope that is recognized by a bi-specific antibody and of a receptor targeting an antigen. The receptor is directed against a first antigen marker, whereas the bi-specific antibody is intended to bind the epitope and a second antigen marker. This display aims to selectively target pathological cells bearing at their surface both the first and second antigen markers. Figure 6: display is similar to Figure 5, but stimulation and co-stimulation domains (respectively 4-1BB and CD3zeta protein domains) have been exchanged to modulate the intensity of the activation of the T-cell resulting from the binding of the chimeric antigen receptor with the pathological cell. Figure 7: display is similar to Figure 5, but stimulation and co-stimulation domains (respectively 4-1BB and CD3zeta protein domains) have been exchanged and one CD3zeta domain has been added to increase the intensity of the activation of the T-cell resulting from the binding of the chimeric antigen receptor with the pathological cell. Figure 8: Schematic representation of a therapeutic strategy according to the invention combining T-cells endowed with a multi-subunit CAR that comprises two extracellular cellular domains and one circulating monoclonal antibody. In this particular aspect, the extracellular cellular domains are located on distinct sub-units. These domains are respectively composed of an antigen binding domain targeting an antigen marker and a Fc receptor intended to bind a monoclonal antibody that is directed against a second antigen marker. This display aims to selectively target pathological cells bearing at their surface both the first and second antigen markers. Figure 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 during 17 days after activation. Figure 10 Knock-out (KO) on CD38 gene: A. Position on CD38 exon 1 sequence of the 3 differents TALEN (T2, T4 and T5) designed to knock out Cd38 in T cell. B. Expression of CD38 in T cells after transfection with the TALEN CD38ex1_T2. C. CD38 staining to control for the purification of CD38 KO T cells. Figure 11: CD38 CAR : A. Representation of the 3 versions of CARs designed. B. CD38 expression level by the target cell lines. Figure 12: Timing experiment for the engineering of the CAR CS1+ and KO CS1 T-cells and their subsequent testing; Figure 13: Constructs of T01, T02 and T03 with the TAL repeats used for the KO of CS1 gene; Figure 14: Target location for the TALs T01, T02 and T03 within the CS1 (SLAMF7) gene. T01 and T02 target the exon 1 (Figure 14A), whereas T03 targets the exon 2 (Figure 14B). Figure 15A : Measurement of percentage of target cell viability for TALEn or not TALEn transfected combined with CAR+ or not transduced cells: a reduced cell viability of CS1(+) cells shown when they were co-cultured with CAR+ T-cells, while no impact on CS1(-) cell viability was observed. Figure 15B: Measurement of percentage of specific cell lysis (CS1+) calculated using the flow cytometry data. It is shown that specific cell lysis is 2-times higher when T-cells have been transfected with TALEn targeting the CS1 gene prior to CAR transduction. Figure 16: Results of FACS analysis from cytoxic activity experiment, which show that transduction efficiencies are higher in mock transfected cells than in cells that have been transfected with TALEn targeting the CS1 gene (NTD: not transduced). Figure 17: Results from FACS analysis when the different samples are reactivated with CD3 / CD28 beads at D11 after transduction, showing the transduction efficiencies and CD8 / CS1 expression levels in each sample. An increase in CS1 levels upon re-activation is observed in mock transfected cells, while a low amount of cells are able to express CS1 in the TALEn transfected populations. Table 1: Different cytopulse programs used for T-cells electroporation. Table 2: appropriate target sequences for the guide RNA using Cas9 in T-cells Table 3: List of genes encoding immune checkpoint proteins Table 4: Cluster of differentiation (CD) antigen markers found to be expressed on the surface of T-cells, while being characteristic of different types of tumors. Table 5 to 13: Main surface antigen markers expressed in T-cells, while being over-expressed in solid tumor cells from various types of cancer. The listed antigen markers were identified as explained in Example 1. Table 5: colon tumor cells; Table 6: breast tumor cells; Table 7: digestive track tumor cells; Table 8: kidney tumor cells; Table 9: liver tumor cells; Table 10: lung tumor cells; Table 11: ovary tumor cells; Table 12: pancreas tumor cells; Table 13: prostate tumor cells; Table 14: Main surface antigen markers expressed in T-cells, while being over-expressed in liquid tumor cells from various types of cancer (ALL, AML, CML, MDS, CLL, CTRL). The listed antigen markers were identified as explained in Example 1. Table 15: Sequences of the tested CD38 target and TALENs for inactivation of the CD38 antigen; Table 16: Sequences of two other CD38 targets and the corresponding TALENs for their inactivation; Table 17: Sequences of VH and VL chains of the scFv anti-CD38 antibodies daratumumab and MOR202 and of specific CDRs for VH and VL chains Table 18: Polypeptide sequence of the 3 different structures of scFv daratumumab-based anti-CD38 CARs and of the individual components used; Table 19: Sequences of VH and VL chains of the scFv anti-CS1 antibodies; Table 20: Polypeptide sequence of anti-CS1 CARs based on the V1, V2 and V3 versions in Figure 11A; Table 21: Sequences of the CS1 target and TALENs for its inactivation; Table 22: Sequences of the CD70 target and TALENs for its inactivation; Table 23: Polynucleotide and nucleic acid sequences of VH and VL chains of the scFv anti-CD70 Ab4, Ab8 and 1F6 antibodies; Table 24: Polypeptide sequence of anti-CD70 CARs based on the V1, V2 and V3 versions in Figure 11A Detailed description of the invention

[0023] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0024] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

[0025] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully 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 (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0026] As a main objective of the invention is the possibility to target pathological cells that bear specific antigen markers in common with T-cells. By pathological cell is meant any types of cells present in a patient, which are deemed causing health deterioration.

[0027] In general, pathological cells are malignant or infected cells that need to be reduced or eliminated to obtain remission of a patient.

[0028] The T cells according to the invention are endowed with a chimeric antigen receptor directed to an antigen marker selected from CD38, CD70 and CS1 that is commonly expressed by the pathological cells and immune cells, or known to be present on the surface of said T Cells. The expression "known to be present" means that the antigen marker is reported to be found on the surface of the immune cells grown in natural conditions in-vivo, especially in the blood, but not necessarily when they are cultured in-vitro. In any event, the method of the invention results into the absence of the antigen marker on the surface of the immune cell, thereby preventing the chimeric antigen receptor from reacting with the engineered T-cell surface. In this respect, the method may include a further step of purifying the resulting T-cells by excluding the cells presenting said marker antigen on their surface.

[0029] As shown in Table 4, an important number of antigen marker candidates have been reported to be expressed by tumor cells, but also by T-cells. Some of them, like CD38, have been used as specific markers in diagnostic methods for a while, especially with respect to Leukemia pathological cells, but not in therapy. Indeed, although these markers were identified in the art as quite specific markers, they could not be used as targets for immunotherapy because antibodies directed against these markers would have destroyed or interfered with patients' T-cells. The present inventors have established that CS1 and CD70 are also present on the surface of T-cells and that expressing CARs targeting CS1 and CD70 in such T cells leads to their depletion (see example 2).

[0030] According to the invention, the inactivation of the gene encoding said antigen is performed using a rare-cutting endonuclease that is a TALE-nuclease.

[0031] By inactivating a gene it is intended that the gene of interest is not expressed in a functional protein form. In particular embodiments, the genetic modification of the method relies on the expression, in provided cells to engineer, of a rare-cutting endonuclease such that same catalyzes cleavage in one targeted gene thereby inactivating said targeted gene. The nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. Mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson 1998) or via the so-called microhomology-mediated end joining (Betts, Brenchley et al. 2003; Ma, Kim et al. 2003). Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions and can be used for the creation of specific gene knockouts. Said modification may be a substitution, deletion, or addition of at least one nucleotide. Cells in which a cleavage-induced mutagenesis event, i.e. a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and / or selected by well-known method in the art.

[0032] The term "rare-cutting endonuclease" refers to a wild type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. Particularly, said nuclease can be an endonuclease, more preferably a rare-cutting endonuclease which is highly specific, recognizing nucleic acid target sites ranging from 10 to 45 base pairs (bp) in length, usually ranging from 10 to 35 base pairs in length, more usually from 12 to 20 base pairs. The endonuclease used according to the present invention recognizes at specific polynucleotide sequences, further referred to as "target sequence" and cleaves nucleic acid inside these target sequences or into sequences adjacent thereto, depending on the molecular structure of said endonuclease. The rare-cutting endonuclease can recognize and generate a single- or double-strand break at specific polynucleotides sequences.TAL-nucleases

[0033] "TALE-nuclease" or "MBBBD-nuclease" refers to engineered proteins resulting from the fusion of a DNA binding domain typically derived from Transcription Activator Like Effector proteins (TALE) or Modular Base-per-Base Binding domain (MBBBD), with a catalytic domain having endonuclease activity. Such catalytic domain usually comes from enzymes, such as for instance I-Tevl, ColE7, NucA and Fok-I. TALE-nuclease can be formed under monomeric or dimeric forms depending of the selected catalytic domain (WO2012138927). Such engineered TALE-nucleases are commercially available under the trade name TALEN ™< (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

[0034] According to a preferred embodiment of the invention, the DNA binding domain is derived from a Transcription Activator like Effector (TALE), wherein sequence specificity is driven by a series of 33-35 amino acids repeats originating from Xanthomonas or Ralstonia bacterial proteins AvrBs3, PthXo1, AvrHah1, PthA, Tallc as non-limiting examples.

[0035] These repeats differ essentially by two amino acids positions that specify an interaction with a base pair (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009). Each base pair in the DNA target is contacted by a single repeat, with the specificity resulting from the two variant amino acids of the repeat (the so-called repeat variable dipeptide, RVD). TALE binding domains may further comprise an N-terminal translocation domain responsible for the requirement of a first thymine base (T0) of the targeted sequence and a C-terminal domain that containing a nuclear localization signals (NLS). A TALE nucleic acid binding domain generally corresponds to an engineered core TALE scaffold comprising a plurality of TALE repeat sequences, each repeat comprising a RVD specific to each nucleotides base of a TALE recognition site. In the present invention, each TALE repeat sequence of said core scaffold is made of 30 to 42 amino acids, more preferably 33 or 34 wherein two critical amino acids (the so-called repeat variable dipeptide, RVD) located at positions 12 and 13 mediates the recognition of one nucleotide of said TALE binding site sequence; equivalent two critical amino acids can be located at positions other than 12 and 13 specially in TALE repeat sequence taller than 33 or 34 amino acids long. Preferably, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A. In another embodiment, critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. A TALE nucleic acid binding domain usually comprises between 8 and 30 TALE repeat sequences. More preferably, said core scaffold of the present invention comprises between 8 and 20 TALE repeat sequences; again more preferably 15 TALE repeat sequences. It can also comprise an additional single truncated TALE repeat sequence made of 20 amino acids located at the C-terminus of said set of TALE repeat sequences, i.e. an additional C-terminal half- TALE repeat sequence.

[0036] Other engineered DNA binding domains can be used as alternative sequences to form so-called modular base-per-base specific nucleic acid binding domains (MBBBD) as described in WO 2014 / 018601. Said MBBBD can be engineered, for instance, from newly identified proteins, namely EAV36_BURRH, E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH proteins from the recently sequenced genome of the endosymbiont fungi Burkholderia Rhizoxinica (Lackner, Moebius et al. 2011). These nucleic acid binding polypeptides comprise modules of about 31 to 33 amino acids that are base specific. These modules display less than 40 % sequence identity with Xanthomonas TALE common repeats and present more polypeptides sequence variability. The different domains from the above proteins (modules, N and C-terminals) from Burkholderia and Xanthomonas are useful to engineer new proteins or scaffolds having binding properties to specific nucleic acid sequences and may be combined to form chimeric TALE-MBBBD proteins.

[0037] As examples, the present invention encompasses the use of a TALE-nuclease specific for a target sequence within a gene encoding an antigen marker selected from CD38, CD70 and CS1 for inactivating the expression of said gene in a T-cell expressing a chimeric antigen receptor (CAR) specific for said antigen marker.

[0038] Particularly suitable for the realization of the invention, TALE-nucleases such as the ones in SEQ ID NO: 2-3; 5-6; 8-9, SEQ ID NO: 64-65; 67-68; 70-71 and SEQ ID NO: 73-74; 76-77; 79-80, for respectively CD38, CS1 and CD70 genes. These specific TALE-nucleases, their sequence target and the protocol used are presented more thoroughly in the following Examples 1-3.Delivery methods

[0039] The inventors have considered any means known in the art to allow delivery inside cells or subcellular compartments of said cells the polynucleotides expressing the endonucleases, their possible co-effectors (e.g. guide RNA or DNA associated with Cas9 or Argonaute nucleases) as well as the chimeric antigen receptors. These means include viral transduction, electroporation and also liposomal delivery means, polymeric carriers, chemical carriers, lipoplexes, polyplexes, dendrimers, nanoparticles, emulsion, natural endocytosis or phagocytose pathway as non-limiting examples.

[0040] Polynucleotides encoding the endonucleases used in the present invention may be transfected under mRNA form in order to obtain transient expression and avoid chromosomal integration of foreign DNA, for example by electroporation. The inventors have determined different optimal conditions for mRNA electroporation in T-cell displayed in Table 1. The inventor used the cytoPulse technology which allows, by the use of pulsed electric fields, to transiently permeabilize living cells for delivery of material into the cells (U.S. patent 6,010,613 and WO 2004 / 083379). Pulse duration, intensity as well as the interval between pulses can be modified in order to reach the best conditions for high transfection efficiency with minimal mortality. Basically, the first high electric field pulses allow pore formation, while subsequent lower electric field pulses allow to moving the polynucleotide into the cell. Described herewith are steps that led to achievement of >95% transfection efficiency of mRNA in T cells, and the use of the electroporation protocol to transiently express different kind of proteins in T cells. In particular, disclosed but not claimed is a method of transforming T cell comprising contacting said T cell with RNA and applying to T cell an agile pulse sequence consisting of: (a) one electrical pulse with a voltage range from 2250 to 3000 V per centimeter, a pulse width of 0.1 ms and a pulse interval of 0.2 to 10 ms between the electrical pulses of step (a) and (b); (b) one electrical pulse with a voltage range from 2250 to 3000 V with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c) ; and (c) 4 electrical pulses with a voltage of 325 V with a pulse width of 0.2 ms and a pulse interval of 2 ms between each of 4 electrical pulses. Disclosed but not claimed is the method of transforming T cell comprising contacting said T cell with RNA and applying to T cell an agile pulse sequence consisting of: (a) one electrical pulse with a voltage of 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V per centimeter, a pulse width of 0.1 ms and a pulse interval of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ms between the electrical pulses of step (a) and (b); (b) one electrical pulse with a voltage range from 2250, of 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and (c) 4 electrical pulses with a voltage of 325 V with a pulse width of 0.2 ms and a pulse interval of 2 ms between each of 4 electrical pulses. Any values included in the value range described above are disclosed herewith. Electroporation medium can be any suitable medium known in the art. Preferably, the electroporation medium has conductivity in a range spanning 0.01 to 1.0 milliSiemens. Table 1 : Different cytopulse programs used to determine the minimal voltage required for electroporation in PBMC derived T-cells. Group 1 Group 2 Group 3 Cyto-pulse programPulsesVduration (ms)Interval (ms)PulsesVduration (ms)Interval (ms)PulsesVduration (ms)Interval (ms)116000.10.216000.110041300.22219000.10.219000.110041300.223112000.10.2112000.110041300.224112000.11019000.110041300.22519000.12016000.110041300.22 Viral transduction

[0041] According to the present invention, the use of retroviral vectors and more preferably of lentiviral vectors is particularly suited for expressing the chimeric antigen receptors into the T-cells. 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 the stable integration of the polynucleotides in the T-cells genome and to expressing the chimeric antigen receptors over a longer period of time.Non alloreactive T cells

[0042] The use of the present invention is intended to be practiced ex-vivo on cultured T-cells obtainable from patients or donors. The engineered T-cells engineered ex-vivo can be either re-implanted into a patient from where they originate, as part of an autologous treatment, or to be used as part of an allogeneic treatment. In this later case, it is preferable to further engineer the cells to make them non-alloreactive to ensure their proper engraftment. T-cells from a donor may be modified to inactivate genes thereof involved in MHC recognition and or being targets of immunosuppressive drugs such as described for instance in WO 2013 / 176915.

[0043] T cell receptors (TCR) are cell surface receptors that participate in the activation of T cells in response to the presentation of antigen. The TCR is generally made from two chains, alpha and beta, which assemble to form a heterodimer and associates with the CD3-transducing subunits to form the T-cell receptor complex present on the cell surface. Each alpha and beta chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. As for immunoglobulin molecules, the variable region of the alpha and beta chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells. However, in contrast to immunoglobulins that recognize intact antigen, T cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T cells, known as MHC restriction. Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of 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). The inactivation of TCRalpha or TCRbeta can result in the elimination of the TCR from the surface of T cells preventing recognition of alloantigen and thus GVHD.

[0044] Thus, engraftment of the T-cells of the present invention may be improved by inactivating at least one gene encoding a TCR component. TCR is rendered not functional in the cells by inactivating TCR alpha gene and / or TCR beta gene(s).

[0045] With respect to the use of Cas9 / CRISPR system, the inventors have determined appropriate target sequences within the 3 exons encoding TCR, allowing a significant reduction of toxicity in living cells, while retaining cleavage efficiency. The preferred target sequences are noted in Table 2 (+ for lower ratio of TCR negative cells, ++ for intermediate ratio, +++ for higher ratio). Table 2: appropriate target sequences for the guide RNA using Cas9 in T-cellsExon TCRPositionStrandTarget genomic sequenceSEQ IDefficiencyEx178-1GAGAATCAAAATCGGTGAATAGG102+++Ex3261TTCAAAACCTGTCAGTGATTGGG103+++Ex11531TGTGCTAGACATGAGGTCTATGG104+++Ex374-1CGTCATGAGCAGATTAAACCCGG105+++Ex14-1TCAGGGTTCTGGATATCTGTGGG106+++Ex15-1GTCAGGGTTCTGGATATCTGTGG107+++Ex333-1TTCGGAACCCAATCACTGACAGG108+++Ex360-1TAAACCCGGCCACTTTCAGGAGG109+++Ex1200-1AAAGTCAGATTTGTTGCTCCAGG110++Ex11021AACAAATGTGTCACAAAGTAAGG111++Ex139-1TGGATTTAGAGTCTCTCAGCTGG112++Ex159-1TAGGCAGACAGACTTGTCACTGG113++Ex122-1AGCTGGTACACGGCAGGGTCAGG114++Ex121-1GCTGGTACACGGCAGGGTCAGGG115++Ex128-1TCTCTCAGCTGGTACACGGCAGG116++Ex3251TTTCAAAACCTGTCAGTGATTGG117++Ex363-1GATTAAACCCGGCCACTTTCAGG118++Ex217-1CTCGACCAGCTTGACATCACAGG119++Ex132-1AGAGTCTCTCAGCTGGTACACGG120++Ex127-1CTCTCAGCTGGTACACGGCAGGG121++Ex2121AAGTTCCTGTGATGTCAAGCTGG122++Ex3551ATCCTCCTCCTGAAAGTGGCCGG123++Ex3861TGCTCATGACGCTGCGGCTGTGG124++Ex11461ACAAAACTGTGCTAGACATGAGG125+Ex186-1ATTTGTTTGAGAATCAAAATCGG126+Ex23-1CATCACAGGAACTTTCTAAAAGG127+Ex2341GTCGAGAAAAGCTTTGAAACAGG128+Ex351-1CCACTTTCAGGAGGAGGATTCGG129+Ex318-1CTGACAGGTTTTGAAAGTTTAGG130+Ex2431AGCTTTGAAACAGGTAAGACAGG131+Ex1236-1TGGAATAATGCTGTTGTTGAAGG132+Ex11821AGAGCAACAGTGCTGTGGCCTGG133+Ex31031CTGTGGTCCAGCTGAGGTGAGGG134+Ex3971CTGCGGCTGTGGTCCAGCTGAGG135+Ex31041TGTGGTCCAGCTGAGGTGAGGGG136+Ex12671CTTCTTCCCCAGCCCAGGTAAGG137+Ex115-1ACACGGCAGGGTCAGGGTTCTGG138+Ex11771CTTCAAGAGCAACAGTGCTGTGG139+Ex1256-1CTGGGGAAGAAGGTGTCTTCTGG140+Ex3561TCCTCCTCCTGAAAGTGGCCGGG141+Ex3801TTAATCTGCTCATGACGCTGCGG142+Ex357-1ACCCGGCCACTTTCAGGAGGAGG143+Ex12681TTCTTCCCCAGCCCAGGTAAGGG144+Ex1266-1CTTACCTGGGCTGGGGAAGAAGG145+Ex12621GACACCTTCTTCCCCAGCCCAGG146+Ex31021GCTGTGGTCCAGCTGAGGTGAGG147+Ex3511CCGAATCCTCCTCCTGAAAGTGG148+

[0046] MHC antigens are also proteins that played a major role in transplantation reactions. Rejection is mediated by T cells reacting to the histocompatibility antigens on the surface of implanted tissues, 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 (for human leukocyte antigens) in human cells. Like TCR, the MHC proteins serve a vital role in T cell stimulation. Antigen presenting cells (often dendritic cells) display peptides that are the degradation products of foreign proteins on the cell surface on the MHC. In the presence of a co-stimulatory signal, the T cell becomes activated, and will act on a target cell that also displays that same peptide / MHC complex. For example, a stimulated T helper cell will target a macrophage displaying an antigen in conjunction with its MHC, or a cytotoxic T cell (CTL) will act on a virally infected cell displaying foreign viral peptides.

[0047] Thus, in order to provide less alloreactive T-cells, one HLA gene can be inactivated or mutated.

[0048] The class I HLA gene cluster in humans comprises three major loci, B, C and A, as well as several minor loci. The class II HLA cluster also comprises three major loci, DP, DQ and DR, and both the class I and class II gene clusters are polymorphic, in that there are several different alleles of both the class I and II genes within the population. There are also several accessory proteins that play a role in HLA functioning as well. The Tapl and Tap2 subunits are parts of the TAP transporter complex that is essential in loading peptide antigens on to the class I HLA complexes, and the LMP2 and LMP7 proteosome subunits play roles in the proteolytic degradation of antigens into peptides for display on the HLA. Reduction in LMP7 has been shown to reduce the amount of MHC class I at the cell surface, perhaps through a lack of stabilization (Fehling et al. (1999) Science 265:1234-1237). In addition to TAP and LMP, there is the tapasin gene, whose product forms a bridge between the TAP complex and the HLA class I chains and enhances peptide loading. Reduction in tapasin results in cells with impaired MHC class I assembly, reduced cell surface expression of the MHC class I and impaired immune responses (Grandea et al. (2000) Immunity 13:213-222 and Garbi et al. (2000) Nat. Immunol. 1:234-238). Any of the above genes may be inactivated as part of the present invention as disclosed, for instance in WO 2012 / 012667.Method of engineering drug-resistant T-cells:

[0049] To improve cancer therapy and selective engraftment of allogeneic T-cells, drug resistance can be conferred to the engineered T-cells to protect them from the toxic side effects of chemotherapy or immunosuppressive agents. Indeed, the inventors have observed that most patients were treated with chemotherapy and immune depleting agents as a standard of care, prior to receiving T-cell immunotherapy. Also they found that they could take advantage of these treatments to help the selection of the engineered T-cells, either by adding chemotherapy drugs in culture media for expansion of the cells ex-vivo prior to treatment, or by obtaining a selective expansion of the engineered T-cells in-vivo in patients under chemotherapy or immunosuppressive treatments.

[0050] Also the drug resistance of T-cells also permits their enrichment in or ex vivo, as T-cells which express the drug resistance gene, will survive and multiply relative to drug sensitive cells. Disclosed herewith, but not part of the invention, is a method of engineering allogeneic and drug resistance T-cells resistant for immunotherapy comprising: (a) Providing a T-cell; (b) Selecting at least one drug; (c) Modifying T-cell to confer drug resistance to said T-cell; (d) Expanding said engineered T-cell in the presence of said drug, and optionally the preceding steps may be combined with the steps of the methods as previously described.

[0051] Drug resistance can be conferred to a T-cell by inactivating one or more gene(s) responsible for the cell's sensitivity to the drug (drug sensitizing gene(s)), such as the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (Genbank: M26434.1). In particular HPRT can be inactivated in engineered T-cells to confer resistance to a cytostatic metabolite, the 6-thioguanine (6TG) which is converted by HPRT to cytotoxic thioguanine nucleotide and which is currently used to treat patients with cancer, in particular leukemias (Hacke, Treger et al. 2013). Another example if the inactivation of the CD3 normally expressed at the surface of the T-cell can confer resistance to anti-CD3 antibodies such as teplizumab.

[0052] Drug resistance can also be conferred to a T-cell by expressing a drug resistance gene. Said drug resistance gene refers to a nucleic acid sequence that encodes "resistance" to an agent, such as a chemotherapeutic agent (e.g. methotrexate). In other words, the expression of the drug resistance gene in a cell permits proliferation of the cells in the presence of the agent to a greater extent than the proliferation of a corresponding cell without the drug resistance gene. A drug resistance gene can encode resistance to anti-metabolite, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, their analogs or derivatives, and the like.

[0053] Variant alleles of several genes such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT) have been identified to confer drug resistance to a cell. Said drug resistance gene can be expressed in the cell either by introducing a transgene encoding said gene into the cell or by integrating said drug resistance gene into the genome of the cell by homologous recombination. Several other drug resistance genes have been identified that can potentially be used to confer 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).

[0054] DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in the cell and is essential to DNA synthesis. Folate analogs such as methotrexate (MTX) inhibit DHFR and are thus used as anti-neoplastic agents in clinic. Different mutant forms of DHFR which have increased resistance to inhibition by anti-folates used in therapy have been described. For example, the drug resistance gene can be a nucleic acid sequence encoding a mutant form of human wild type DHFR (GenBank: AAH71996.1) which comprises at least one mutation conferring resistance to an anti-folate treatment, such as methotrexate. For example, mutant form of DHFR comprises at least one mutated amino acid at position G15, L22, F31 or F34, preferably at positions L22 or F31 ((Schweitzer, Dicker et al. 1990); International application WO 94 / 24277; US patent US 6,642,043).

[0055] As used herein, "antifolate agent" or "folate analogs" refers to a molecule directed to interfere with the folate metabolic pathway at some level. Examples of antifolate agents include, e.g., methotrexate (MTX); aminopterin; trimetrexate (Neutrexin ™< ); edatrexate; N10-propargyl-5,8-dideazafolic acid (CB3717); ZD1694 (Tumodex), 5,8-dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid (DDATHF); 5-deazafolic acid; PT523 (N alpha-(4-amino-4- deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine); 10-ethyl-10-deazaaminopterin (DDATHF, lomatrexol); piritrexim; 10-EDAM; ZD1694; GW1843; Pemetrexate and PDX (10-propargyl-10- deazaaminopterin).

[0056] Another example of drug resistance gene can also be a mutant or modified form of ionisine-5'- monophosphate dehydrogenase II (IMPDH2), a rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. The mutant or modified form of IMPDH2 is a IMPDH inhibitor resistance gene. IMPDH inhibitors can be mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF). The mutant IMPDH2 can comprises at least one, preferably two mutations in the MAP binding site of the wild type human IMPDH2 (NP_000875.2) that lead to a significantly increased resistance to IMPDH inhibitor. The mutations are preferably at positions T333 and / or S351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al. 2007; Jonnalagadda, Brown et al. 2013). In one example, the threonine residue at position 333 is replaced with an isoleucine residue and the serine residue at position 351 is replaced with a tyrosine residue.

[0057] Another drug resistance gene is the mutant form of calcineurin. Calcineurin (PP2B) is an ubiquitously expressed serine / threonine protein phosphatase that is involved in many biological processes and which is central to T-cell activation. Calcineurin is a heterodimer composed of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). After engagement of the T-cell receptor, calcineurin dephosphorylates the transcription factor NFAT, allowing it to translocate to the nucleus and active key target gene such as IL2. FK506 in complex with FKBP12, or cyclosporine A (CsA) in complex with CyPA block NFAT access to calcineurin's active site, preventing its dephosphorylation and thereby inhibiting T-cell activation (Brewin, Mancao et al. 2009). A drug resistance gene can be a nucleic acid sequence encoding a mutant form of calcineurin resistant to calcineurin inhibitor such as FK506 and / or CsA. For example, said mutant form can comprise at least one mutated amino acid of the wild type calcineurin heterodimer a at positions: V314, Y341, M347, T351, W352, L354, K360, preferably double mutations at positions T351 and L354 or V314 and Y341. Correspondence of amino acid positions described herein is frequently expressed in terms of the positions of the amino acids of the form of wild-type human calcineurin heterodimer (GenBank: ACX34092.1).

[0058] In another example, said mutant form can comprise at least one mutated amino acid of the wild type calcineurin heterodimer b at positions: V120, N123, L124 or K125, preferably double mutations at positions L124 and K125. Correspondence of amino acid positions described herein is frequently expressed in terms of the positions of the amino acids of the form of wild-type human calcineurin heterodimer b polypeptide (GenBank: ACX34095.1).

[0059] Another drug resistance gene is 0(6)-methylguanine methyltransferase (MGMT) encoding human alkyl guanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents, such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is an inhibitor of AGT that potentiates nitrosourea toxicity and is co-administered with TMZ to potentiate the cytotoxic effects of this agent. Several mutant forms of MGMT that encode variants of AGT are highly resistant to inactivation by 6-BG, but retain their ability to repair DNA damage (Maze, Kurpad et al. 1999). In one example, AGT mutant form can comprise a mutated amino acid of the wild type AGT position P140 (UniProtKB: P16455).

[0060] Another drug resistance gene can be multidrug resistance protein 1 (MDR1) gene. This gene encodes a membrane glycoprotein, known as P-glycoprotein (P-GP) involved in the transport of metabolic byproducts across the cell membrane. The P-Gp protein displays broad specificity towards several structurally unrelated chemotherapy agents. Thus, drug resistance can be conferred to cells by the expression of nucleic acid sequence that encodes MDR-1 (NP_000918).

[0061] Drug resistance gene can also be cytotoxic antibiotics, such as ble gene or mcrA gene. Ectopic expression of ble gene or mcrA in an immune cell gives a selective advantage when exposed to the chemotherapeutic agent, respectively the bleomycine or the mitomycin C.

[0062] The T-cells can also be made resistant to immunosuppressive agents. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. In other words, an immunosuppressive agent is a role played by a compound which is exhibited by a capability to diminish the extent and / or voracity of an immune response. As non-limiting example, an immunosuppressive agent can be a calcineurin inhibitor, a target of rapamycin, an interleukin-2 α-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite. Classical cytotoxic immunosuppressants act by inhibiting DNA synthesis. Others may act through activation of T-cells or by inhibiting the activation of helper cells. The method according to the invention allows conferring immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent in T cells. As non-limiting examples, targets for immunosuppressive agent can be a receptor for an immunosuppressive agent such as: CD52, glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member.

[0063] In immunocompetent hosts, allogeneic cells are normally rapidly rejected by the host immune system. It has been demonstrated that, allogeneic leukocytes present in non-irradiated blood products will persist for no more than 5 to 6 days. Thus, to prevent rejection of allogeneic cells, the host's immune system must be effectively suppressed. Glucocorticoidsteroids are widely used therapeutically for immunosuppression. This class of steroid hormones binds to the glucocorticoid receptor (GR) present in the cytosol of T cells resulting in the translocation into the nucleus and the binding of specific DNA motifs that regulate the expression of a number of genes involved in the immunologic process. Treatment of T cells with glucocorticoid steroids results in reduced levels of cytokine production leading to T cell anergy and interfering in T cell activation. Alemtuzumab, also known as CAMPATH1-H, is a humanized monoclonal antibody targeting CD52, a 12 amino acid glycosylphosphatidyl-inositol- (GPI) linked glycoprotein (Waldmann and Hale, 2005). CD52 is expressed at high levels on T and B lymphocytes and lower levels on monocytes while being absent on granulocytes and bone marrow precursors. Treatment with Alemtuzumab, a humanized monoclonal antibody directed against CD52, has been shown to induce a rapid depletion of circulating lymphocytes and monocytes. It is frequently used in the treatment of T cell lymphomas and in certain cases as part of a conditioning regimen for transplantation. However, in the case of adoptive immunotherapy the use of immunosuppressive drugs will also have a detrimental effect on the introduced therapeutic T cells. Therefore, to effectively use an adoptive immunotherapy approach in these conditions, the introduced cells would need to be resistant to the immunosuppressive treatment.

[0064] As an example of the above steps, said gene of step (b), specific for an immunosuppressive treatment, is CD52, and the immunosuppressive treatment of step (d) comprises a humanized antibody targeting CD52 antigen. As another embodiment, said gene of step (b), specific for an immunosuppressive treatment, is a glucocorticoid receptor (GR) and the immunosuppressive treatment of step d) comprises a corticosteroid such as dexamethasone. As another embodiment, said target gene of step (b), specific for an immunosuppressive treatment, is a FKBP family gene member or a variant thereof and the immunosuppressive treatment of step (d) comprises FK506 also known as Tacrolimus or fujimycin. As another example, said FKBP family gene member is FKBP12 or a variant thereof. As another embodiment, said gene of step (b), specific for an immunosuppressive treatment, is a cyclophilin family gene member or a variant thereof and the immunosuppressive treatment of step (d) comprises cyclosporine.

[0065] For example, the genetic modification step of the method relies on the inactivation of two genes selected from the group consisting of CD52 and GR, CD52 and TCR alpha, CDR52 and TCR beta, GR and TCR alpha, GR and TCR beta, TCR alpha and TCR beta. In another embodiment, the genetic modification step of the method relies on the inactivation of more than two genes. The genetic modification is preferably operated ex-vivo using at least two RNA guides targeting the different genes.

[0066] By inactivating a gene it is intended that the gene of interest is not expressed in a functional protein form.Engineering highly active T cells for immunotherapy

[0067] According to the present invention, the T-cells can be selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes. In another embodiment, said 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 cells can be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, or hematopoietic stem cells. Representative human cells are CD34+ cells. Prior to expansion and genetic modification of the cells of the invention, a source of cells can be obtained from a subject through a variety of non-limiting methods. T-cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, 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, may be used. In another embodiment, said cell can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In another embodiment, said cell is part of a mixed population of cells which present different phenotypic characteristics. In the scope of the present invention is also encompassed a cell line obtained from a transformed T- cell according to the method previously described.

[0068] 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, especially by modulating the expression of proteins involved in overall T-cell regulation, referred to as "immune-checkpoints".Immune check points

[0069] It will be understood by those of ordinary skill in the art, that the term "immune checkpoints" means a group of molecules expressed by T cells. These molecules effectively serve as "brakes" to down-modulate or inhibit an immune response. Immune checkpoint molecules include, but are not limited to Programmed Death 1 (PD-1, also known as PDCD1 or CD279, accession number: NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, GenBank accession number AF414120.1), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM_181780.3), BY55 (also known as CD160, GenBank accession number: CR541888.1), TIGIT (also known as IVSTM3, accession number: NM_173799), LAIR1 (also known as CD305, GenBank accession number: CR542051.1, {Meyaard, 1997 #122}), SIGLEC10 (GeneBank accession number: AY358337.1), 2B4 (also known as CD244, accession number: NM_001166664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7 {Nicoll, 1999 #123}, SIGLEC9 {Zhang, 2000 #124;lkehara, 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 engaged by its ligands (B7-1 and B7-2) on antigen presenting cells, T-cell activation and effector function are inhibited. Thus, disclosed herewith, but not part of the invention, is a method of engineering T-cells, especially for immunotherapy, comprising genetically modifying T-cells by inactivating at least one protein involved in the immune check-point, in particular PD1 and / or CTLA-4 or any immune-checkpoint proteins referred to in Table 3. Table 3: List of genes encoding immune checkpoint proteins.PathwayGenes that can be inactivated In the pathwayCo-inhibitory receptorsCTLA4 (CD152)CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22PDCD1 (PD-1, CD279)PDCD1CD223 (lag3)LAG3HAVCR2 (tim3)HAVCR2BTLA(cd272)BTLACD160(by55)CD160IgSF familyTIGITCD96CRTAMLAIR1(cd305)LAIR1SIGLECsSIGLEC7SIGLEC9CD244(2b4)CD244Death receptorsTRAILTNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7FASFADD, FASCytokine signallingTGF-beta signalingTGFBRII, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1IL10 signallingIL10RA, IL10RB, HMOX2IL6 signallingIL6R, IL6STArginine / tryptopha n starvationEIF2AK4Prevention of TCR signallingCSK, PAG1SIT1Induced Treginduced TregFOXP3Transcription factors controlling exhaustiontranscription factors controlling exhaustionPRDM1 (=blimp1, heterozygotes mice control chronic viral infection better than wt or conditional KO)BATFHypoxia mediated toleranceiNOS induced guanylated cyclaseGUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3 Engineered T-cells expressing chimeric antigen receptors against pathological cells

[0070] The chimeric antigen receptors introduced into the T-cells according to the invention can adopt different design such as single-chain or multi-chain CARs. These different designs allow various strategies for improving specificity and binding efficiency towards the targeted pathological cells. Some of these strategies are illustrated in the figures of the present application. Single-chain CARs are the most classical version in the art. Multi-chain CAR architectures were developed by the applicant as allowing modulation of the activity of T-cells in terms of specificity and intensity. The multiple subunits can shelter additional co-stimulation domains or keep such domains at a distance, as well as other types of receptors, whereas classical single chain architecture can sometimes be regarded as too much sensitive and less permissive to multispecific interactions.Single-Chain CAR

[0071] Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.

[0072] Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have 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 enhance survival and increase proliferation of CAR modified T cells. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).

[0073] In addition to the CAR targeting the antigen marker, which is common to the pathological cells and the T-cells, such as CD38, it is envisioned to express further CARs directed towards other antigen markers not necessarily expressed by the T-cells, so as to enhancing T-cells specificity.

[0074] Examples of chimeric antigen receptor that can be further expressed by the T-cells to create multi-specific cells, are antigen receptors directed against 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).

[0075] As further examples, the antigen of the target can be from any cluster of differentiation molecules (e.g. CD16, CD64, CD78, CD96,CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138), a tumor-associated surface antigen, 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 mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), or a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen as well as any derivate or variant of these surface markers. Antigens are not necessarily surface marker antigens but can be also endogenous small antigens presented by HLA class I at the surface of the cells.

[0076] As examples, the present invention encompasses single-chain CARs which target specifically a cell surface marker that is CD38, CS1 or CD70, as described in the examples, together with an inactivation of the gene encoding respectively CD38, CS1 or CD70 in the cells expressing said CARs.

[0077] As a specific example, the VH and VL chains of the scFv anti-CD38 share at least 80%, preferably 90% and more preferably 95% of identity with respectively SEQ ID NO:10 and 12 and SEQ ID NO: 11 and 13.

[0078] As a specific example, the antibody or epitope-binding on CD38 antigen,characterized in that said antibody or epitope-binding fragment thereof comprises at least one heavy chain and at least one light chain, wherein said heavy chain comprises three sequential complementarity-determining regions having amino acid sequences represented by SEQ ID NOS: 14-17, and wherein said light chain comprises three sequential complementarity-determining regions having amino acid sequences represented by SEQ ID NOS: 21-23.

[0079] As a another specific example, the antibody or epitope-binding on CD38 antigen,characterized in that said antibody or epitope-binding fragment thereof comprises at least one heavy chain and at least one light chain, wherein said heavy chain comprises three sequential complementarity-determining regions having amino acid sequences represented by SEQ ID NOS: 18-20, and wherein said light chain comprises three sequential complementarity-determining regions having amino acid sequences represented by SEQ ID NOS: 24-26.

[0080] As another specific example, the VH and VL chains of the scFv anti-CS1 share at least 80%, preferably 90% and more preferably 95% of identity with respectively SEQ ID NO:38-40-42-44-46 and SEQ ID NO: 39-41-42-45-46.

[0081] As still another specific example, the VH and VL chains of the scFv anti-CD70 share at least 80%, preferably 90% and more preferably 95% of identity at the polynucleotide or nucleic acid level with respectively SEQ ID NO:81-82; 85-86; 89-91 and SEQ ID NO: 83-84; 87-88; 91-92.

[0082] In an embodiment, the invention encompasses a polynucleotide encoding a single CAR anti-CD38 which shares at least 80%, preferably 90% and more preferably 95% of identity with SEQ ID NO: 35-37.In another embodiment, the invention encompassed a polynucleotide encoding a single CAR anti-CS1 which shares at least 80%, preferably 90% and more preferably 95% of identity with SEQ ID NO: 48-62.

[0083] In still another embodiment, the invention encompasses a polynucleotide encoding a single CAR anti-CD70 which shares at least 80%, preferably 90% and more preferably 95% of identity with SEQ ID NO: 93-101.

[0084] The present invention is more particularly drawn to immune cells that are endowed with a CAR presenting some identity with those described in the present application and that would bear rare-cutting endonuclease induced mutations in a gene encoding the cell marker targeted by said CAR ( i.e. the CAR displays affinity with the product of said inactivated gene). By identity is meant at least 70%, preferably 80%, more preferably 90% and even more preferably 95% polynucleotide or polypeptide identity as determined by the software such as FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.). BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The same applies with respect to polynucleotide sequences using BLASTN.Multi-subunit CAR

[0085] Chimeric antigen receptors from the prior art introduced in T-cells have been formed of single chain polypeptides that necessitate serial appending of signaling domains. However, by moving signaling domains from their natural juxtamembrane position may interfere with their function. To overcome this drawback, the applicant recently designed a multi-chain CAR derived from FcεRI to allow normal juxtamembrane position of all relevant signaling domains. In this new architecture, the high affinity IgE binding domain of FcεRI alpha chain is replaced by an extracellular ligand-binding domain such as scFv to redirect T-cell specificity against cell targets and the N and / or C-termini tails of FcεRI beta chain are used to place costimulatory signals in normal juxtamembrane positions.

[0086] Accordingly, the CAR expressed by the engineered T-cell according to the invention can be a multi-chain chimeric antigen receptor (CAR) particularly adapted to the production and expansion of engineered T-cells of the present invention. Such multi-chain CARs comprise at least two of the following components: a) one polypeptide comprising the transmembrembrane domain of FcεRI alpha chain and an extracellular ligand-binding domain, b) one polypeptide comprising a part of N- and C- terminal cytoplasmic tail and the transmembrane domain of FcεRI beta chain and / or c) at least two polypeptides comprising each a part of intracytoplasmic tail and the transmembrane domain of FcεRI gamma chain, whereby different polypeptides multimerize together spontaneously to form dimeric, trimeric or tetrameric CAR.

[0087] According to such architectures, ligands binding domains and signaling domains are born on separate polypeptides. The different polypeptides are anchored into the membrane in a close proximity allowing interactions with each other. In such architectures, the signaling and co-stimulatory domains can be in juxtamembrane positions (i.e. adjacent to the cell membrane on the internal side of it), which is deemed to allow improved function of co-stimulatory domains. The multi-subunit architecture also offers more flexibility and possibilities of designing CARs with more control on T-cell activation. For instance, it is possible to include several extracellular antigen recognition domains having different specificity to obtain a multi-specific CAR architecture. It is also possible to control the relative ratio between the different subunits into the multi-chain CAR. This type of architecture has been recently described by the applicant in PCT / US2013 / 058005 (WO2014 / 039523).

[0088] The assembly of the different chains as part of a single multi-chain CAR is made possible, for instance, by using the different alpha, beta and gamma chains of the high affinity receptor for IgE (FcεRI) (Metzger, Alcaraz et al. 1986) to which are fused the signaling and co-stimulatory domains. The gamma chain comprises a transmembrane region and cytoplasmic tail containing one immunoreceptor tyrosine-based activation motif (ITAM) (Cambier 1995).

[0089] The multi-chain CAR can comprise several extracellular ligand-binding domains, to simultaneously bind different elements in target thereby augmenting immune cell activation and function. In one embodiment, the extracellular ligand-binding domains can be placed in tandem on the same transmembrane polypeptide, and optionally can be separated by a linker. In another embodiment, said different extracellular ligand-binding domains can be placed on different transmembrane polypeptides composing the multi-chain CAR. In another embodiment, the present invention relates to a population of multi-chain CARs comprising each one different extracellular ligand binding domains. In a particular, the present invention relates to a method of engineering immune cells comprising providing an immune cell and expressing at the surface of said cell a population of multi-chain CAR each one comprising different extracellular ligand binding domains. In another particular embodiment, the present invention relates to a method of engineering an immune cell comprising providing an immune cell and introducing into said cell polynucleotides encoding polypeptides composing a population of multi-chain CAR each one comprising different extracellular ligand binding domains. In a particular embodiment the method of engineering an immune cell comprises expressing at the surface of the cell at least a part of FcεRI beta and / or gamma chain fused to a signal-transducing domain and several part of FcεRI alpha chains fused to different extracellular ligand binding domains. In a more particular embodiment, said method comprises introducing into said cell at least one polynucleotide which encodes a part of FcεRI beta and / or gamma chain fused to a signal-transducing domain and several FcεRI alpha chains fused to different extracellular ligand biniding domains. By population of multi-chain CARs, it is meant at least two, three, four, five, six or more multi-chain CARs each one comprising different extracellular ligand binding domains. The different extracellular ligand binding domains according to the present invention can preferably simultaneously bind different elements in target thereby augmenting immune cell activation and function.

[0090] The present invention also relates to an isolated immune cell which comprises a population of multi-chain CARs each one comprising different extracellular ligand binding domains.

[0091] The signal transducing domain or intracellular signaling domain of the multi-chain CAR of the invention is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response. In other words, the signal transducing domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the multi-chain CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines.

[0092] In the present application, the term "signal transducing domain" refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.

[0093] Preferred examples of signal transducing domain for use in single or multi-chain CAR can be the cytoplasmic sequences of the Fc receptor or T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivate or variant of these sequences and any synthetic sequence that as the same functional capability. Signal transduction domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAM used in the invention can include as non-limiting examples those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the signaling transducing domain of the multi-chain CAR can comprise the CD3zeta signaling domain, or the intracytoplasmic domain of the FcεRI beta or gamma chains.

[0094] In particular embodiment the signal transduction domain of the multi-chain CAR of the present invention comprises a co-stimulatory signal molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient immune response.

[0095] Ligand binding-domains can be any antigen receptor previously used, and referred to, with respect to single- chain CAR referred to in the literature, in particular scFv from monoclonal antibodies. Bispecific or multi-specific CARs are described in WO 2014 / 4011988.

[0096] Similarly as described before with respect to single-chain CARs, the present invention encompasses T cells endowed with multi-chain CARs which target specifically a cell surface marker that is CD38, CS1 or CD70. According to a preferred embodiment of the invention the CARs described above are expressed in immune cells, whereas inactivation of the endogenous genes encoding said surface marker(s) is induced by expression of a rare-cutting endonuclease.Activation and expansion of T cells

[0097] A step of activating and / or expanding the T-cells can be done prior to or after genetic modification of the T cells, using the methods as described, for example, 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 invention can be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells.

[0098] In particular, T cell populations may be stimulated in vitro such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. For example, the agents providing each signal may be in solution or coupled to a surface. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. In further embodiments of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. Cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells. In one embodiment the cells (for example, 4 to 10 T cells) and beads (for example, DYNABEADS ®< M-450 CD3 / CD28 T paramagnetic beads at a ratio of 1:1) are combined in a buffer, preferably PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. The mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g , 1L-4, 1L-7, GM-CSF, -10, - 2, 1L-15, TGFp, and TNF- or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 , and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% C02). T-cells that have been exposed to varied stimulation times may exhibit different characteristics.Therapeutic applications

[0099] The T-cells of the present invention are intended for use as a medicament for treating, among others, cancer, infections or immune diseases in a patient in need thereof.

[0100] Said treatment can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment. By autologous, it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor. By allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.

[0101] The T-cells may be pooled, frozen, and administrated to one or several patients. When they are made non-alloreactive, they are available as an "off the shelf" therapeutic product, which means that they can be universally infused to patients in need thereof.

[0102] Said treatments are primarily intended to patients diagnosed with cancer, viral infection, autoimmune disorders or Graft versus Host Disease (GvHD). Cancers are preferably leukemias and lymphomas, which have liquid tumors, but may also concern solid tumors. Types of cancers to be treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0103] Antigen markers used for the immunotherapy of the present invention are CD38, CD319 (CS1) and CD70.

[0104] The present T-cells, when armed with specific CARs directed against patient's own immune cells, especially T-cells, allow the inhibition or regulation of said cells, which is a key step for treating auto-immune disease, such as rheumatoid polyarthritis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, fibromyalgia, myositis, ankylosing spondylitis, insulin dependent diabetes of type I, Hashimoto's thyroiditis, Addison's disease, Crohn's disease, Celiac's disease, amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). Accordingly, the present invention encompasses a method for treating an immune disease by directing engineered T-cells as previously described against patient's own T-cells.

[0105] The above treatments can take place in combination with one or more therapies selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.

[0106] The engineered T-cells as previously described, when they are made resistant to chemotherapy drugs and immunosuppressive drugs that are used as standards of care, especially methotrexate and the combination of fludarabine and Cyclophosphamide, are particularly suited for treating various forms of cancer. Indeed, the present invention preferably relies on cells or population of cells. In this aspect, it is expected that the chemotherapy and / or immunosuppressive treatment should help the selection and expansion of the engineered T-cells in-vivo.

[0107] In certain embodiments of the present invention, cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, 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 may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1 1; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Citrr. Opin. mm n. 5:763-773, 93). In a further embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgeiy. Said modified cells obtained by any one of the methods described here can be used in a particular aspect of the invention for treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD); therefore in the scope of the present invention is a method of treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating said patient by administering to said patient an effective amount of modified cells comprising inactivated TCR alpha and / or TCR beta genes.

[0108] T cells of the invention can undergo robust in vivo T cell expansion upon administration to a patient, and can persist in the body fluids for an extended amount of time, preferably for a week, more preferably for 2 weeks, even more preferably for at least one month. Although the T-cells according to the invention are expected to persist during these periods, their life span into the patient's body are intended not to exceed a year, preferably 6 months, more preferably 2 months, and even more preferably one month.

[0109] The administration of the cells or population of cells according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermaliy, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0110] The administration of the cells or population of cells can consist of the administration of 10 4< -10 9< cells per kg body weight, preferably 10 5< to 10 6< cells / kg body weight including all integer values of cell numbers within those ranges. The cells or population of cells can be administrated in one or more doses. In another embodiment, said effective amount of cells are administrated as a single dose. In another embodiment, said effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.

[0111] In another embodiment, said effective amount of cells or composition comprising those cells are administrated parenterally. Said administration can be an intravenous administration. Said administration can be directly done by injection within a tumor.Identification of surface antigen marker expressed on the surface of T-cells, while being overexpressed in solid tumors involved into different types of cancer (Tables 5 to 13)

[0112] We used BioGPS microarray data from a panel of normal tissues (Human U133A / GNF1H Gene Atlas) cancer microarray data that also can be downloaded from BioGPS (Human Primary Tumors (U95)) uniprot data that contains the subcellular localization.

[0113] We drew the distribution of values coming from normal tissues and determined a threshold value of 5 for the relative expression.

[0114] We browsed all the genes assayed with microarrays (44.000 probes representing about 13 000 genes) and checked their localization in the membrane (protein not referred to as being a membrane protein were discarded). Expression in CD8+ T-cells was checked from the BioGPS database. The genes were listed according to the type of cancer where the corresponding expression was the highest (Tables 5 to 13).Identification of surface antigen marker expressed on the surface of T-cells, while being overexpressed in different liquid blood tumors (Table 14)

[0115] For that study, no RNA-seq data were available and thus we used microarray data that were obtained from a large study from 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). This raw data include results for ALL (acute lymphoblastic leukemia), AML (acute myelogenous leukemia), CLL (chronic lymphoblastic leukemia) and CML (chronic myelogenous leukemia) and MDS (myelodysplastic syndrome). We also used uniprot data for subcellular localization as usual.

[0116] We first drew the overall distribution of values from all genes on all studied tissues. Then, to have an idea of the level necessary for expression, we took a list of genes which are expressed in some liquid tumors and for which therapeutic antibodies are available (CD52, CD 20, CD33, CD19, CD25, CD44, CD47, CD96, CD116, CD117, CD135, TIM-3). For each gene, we looked at the value obtained in the tumor in which it is expressed. Then, we computed the average for each tumor and gene pair for which the gene seems to give a cell membrane protein (cell membrane localization + description of at least one transmembrane domain in the protein). We discarded genes for which the expression in all the tissues was below this threshold of 0.15. We listed and ranked in Table 14, those genes which relative expression in T-cells was above 0.2. Thus, Table 4 provides putative antigen marker candidates for targeting liquid tumor cells as per the invention, in particular for treating ALL, AML, CLL, CML and MDS.Example of steps to engineer T-cells according to the invention for immunotherapy

[0117] For a better understanding of the invention, it is provided below an example of the steps to follow to produce T-cells directed against leukemia CD38 positive cells: 1. Providing T-cells from a cell culture or from a blood sample from one individual patient or from blood bank and activating said T cells using anti-CD3 / C28 activator beads (Dynabeads ®< ). The beads provide both the primary and co-stimulatory signals that are required for activation and expansion of T cells. 2. Transducing said cells with a retroviral vector comprising a transgene encoding a Chimeric antigen receptor consisting of the fusion of CD3zeta activation domain, 4-1BB co-stimulation domain, a transmembrane domain and a hinge from CD28 fused to a sequence encoding the variable chain of an anti-CD38 antibody. For security improvement of the transformed T-cell, a suicide gene sensitive to rituximab may further be introduced as described in WO 2013 / 153391 into the lentiviral vector separated by T2A splitting sequences. 3. (Optionally) Engineering non alloreactive and / or resistant T cells: a) It is possible to Inactivate TCR alpha in said cells to eliminate the TCR from the surface of the cell and prevent recognition of host tissue as foreign by TCR of allogenic and thus to avoid GvHD by following the protocols set forth in WO 2013 / 176915. b) It is also possible to inactive one gene encoding target for an immunosuppressive agent or a chemotherapy drug to render said cells resistant to immunosuppressive or chemotherapy treatment to prevent graft rejection without affecting transplanted T cells. In this example, target of immunosuppressive agents is CD52 and immunosuppressive agent is a humanized monoclonal anti-CD52 antibody (ex: Alemtuzumab) as described in WO 2013 / 176915. 4. Gene Inactivation is performed by electoporating T-cells with mRNA encoding specific TAL-endonuclease (TALEN ™< - Cellectis, 8 rue de la Croix Jarry, France). Inactivated T cells are sorted using magnetic beads. For example, T cells still expressing the targeted gene (e.g. CD38, CD70 and CD70) can be removed by fixation on a solid surface, and inactivated cells are not exposed of the stress of being passed through a column. This gentle method increases the concentration of properly engineered T-cells. 5. Expansion in vitro of engineered T-cells prior to administration to a patient or in vivo following administration to a patient through stimulation of CD3 complex. Before administration step, patients can be subjected to an immunosuppressive treatment such as CAMPATH1-H, a humanized monoclonal anti-CD52 antibody. 6. Optionally exposed said cells with bispecific antibodies ex vivo prior to administration to a patient or in vivo following administration to a patient to bring the engineered cells into proximity to a target antigen. Functional analysis of the engineered T-cells electroporated with a monocistronic mRNA encoding for an anti-CD38 single chain chimeric antigen receptor (CAR CD38):

[0118] To verify that genome engineering did not affect the ability of the engineered T-cells to present anti-tumor activity, especially when provided with a chimeric antigen receptor (CAR CD38), The engineered T-cells were incubated for 4 hours with Daudi cells expressing CD38 on their surface. The cell surface upregulation of CD107a, a marker of cytotoxic granule release by T lymphocytes (called degranulation) was measured by flow cytometry analysis (Betts, Brenchley et al. 2003).

[0119] 24 hours post electroporation, cells were stained with a fixable viability dye eFluor-780 and a PE-conjugated goat anti mouse IgG F(ab')2 fragment specific to assess the cell surface expression of the CAR on the live cells. The vast majority of the live T-cells genetically disrupted for CD38, express the CAR on their surface. T cells were co-cultured with Daudi (CD38 +< ) cells for 6 hours and analyzed by flow cytometry to detect the expression of the degranulation marker CD107a at their surface (Betts, Brenchley et al. 2003).

[0120] The results showed that CD38 disrupted T-cells kept the same ability to degranulate in response to PMA / ionomycin (positive control) or CD38+ Daudi cells. CD107 upregulation is dependent on the presence of a CD38+. These data suggest that the genome engineering of the present T-cells had no negative impact on the ability of T cells to mount a controlled anti-tumor response. Table 4: Cluster of differentiation (CD) antigen markers of various cancers found to be expressed on the surface of T-cellsAntigen Other Names Structure main Distribution Function CD1aT6IgSF, MHC-likecortical thymocytes, Langerhans cells, DCantigen presentation, with beta2mCD1bT6IgSF, MHC-likecortical thymocytes, Langerhans cells, DCantigen presentation, with beta2mCD1cT6IgSF, MHC-likecortical thymocytes, Langerhans cells, DC, B subsetantigen presentation, with beta2mCD1dIgSF, MHC-likeintestinal epith, B subset, monolow, DCantigen presentation, with beta2mCD3 gamma, CD3 deltaT3IgSFT, thymocyte subsetwith TCR, TCR surface expression / signal transductionCD3 epsilonT3IgSFT, thymocyte subsetwith TCR, TCR surface expression / signal transductionCD4T4IgSFthymocyte subset, T subset, mono, macMHC class II coreceptor, HIV receptor, T cell differentiation / activationCD5T1, Tp67Scavenger R SFthymocytes, T, B subset, B-CLLCD72 receptor, TCR or BCR signaling, T-B interactionCD7IgSFhematopoietic progenitors, thymocytes, T, NKT costimulationCD8aT8, Leu-2IgSFthymocyte subset, T subset, NKMHC class I coreceptor, receptor for some mutated HIV-1, T cell differentiation / activationCD8bIgSFthymocyte subset, T subsetCD9p24, MRP-1TM4SFpre-B, eosinophils, basophils, platelets, Tactcellular adhesion and migrationCD10CALLA, NEP, gp100type II TMB precursors, T precursors, neutrophilszinc-binding metalloproteinase, B cell developmentCD11aLFA-1, integrin alphaLIntegrin familylymph, gran, mono, macCD11a / CD18 receptor for ICAM-1, -2,-3, intercellular adhesion, T costimulationCD11bMac-1, integrin alphaMIntegrin familymyeloid cells, NKbinds CD54, ECM, iC3bCD11cp150, 95, CR4, integrin alphaXIntegrin familyDC, myeloid cells, NK, B, T subsetbinds CD54, fibrinogen and iC3bCD13Aminopeptidase N, APNtype II TMmyeloid cellszinc-binding metalloproteinase, antigen processing, receptor for corona virus strainsCD14LPS-RGPI-linkedmono, mac, Langerhans cells, granlowreceptor for LPS / LBP, LPS recognitionCD15Lewis-x, LexCHOneutrophils, eosinophils, monoadhesionCD16aFcgammaRllIAIgSFneutrophils, mac, NKcomponent of low affinity Fc receptor, phagocytosis and ADCCCD16bFcgammaRIIIBIgSFneutrophilscomponent of low affinity Fc receptor, phagocytosis and ADCCCD20B1, Bp35TM4SFB, T subsetB cell activationCD21C3DR, CR2, EBV-RCCRSFB, FDC, T subsetcomplement C3d and EBV receptor, complex with CD19 and CD81, BCR coreceptorCD22BL-CAM, Siqlec-2IgSF, sialoadhesinsBadhesion, B-mono, B-T interactionsCD23FcepsilonRIIC-type lectinB, activated mac, eosinophils, FDC, plateletsCD19-CD21-CD81 receptor, IgE low affinity receptor, signal transductionCD24BA-1GPI-linkedthymocytes, erythrocytes, peripheral lymph, myeloidbinds P-selectinCD25Tac, p55type I TMTact, Bact, lymph progenitorsIL-2Ralpha, with IL-2Rbeta and gamma to form high affinity complexCD31PECAM-1IgSFmono, platelets, gran, endoth, lymph subsetCD38 receptor, adhesionCD33p67, Siglec-3IgSF, sialoadhesinsmyeloid progenitors, mono, gran, DC, mast cells, TactadhesionCD37TM4SFB, Tlow, granlowsignal transductionCD38T10variable levels on majority of hematopoietic cells, high expression on plasma cells, B and Tactecto-ADP-ribosyl cyclase, cell activationCD40TNFRSFB, mono, mac, FDC, endoth, T subsetCD154 receptor, B differentiation / costimulation, isotype-switching, rescues B cells from apoptosisCD43Leukosialin, sialophorinSialomucin, type I TMleukocytes, except resting B, plateletslowinhibition of T cell interaction, CD54R, adhesionCD44H-CAM, Pgp-1hyaladherin familyhematopoietic and non-hematopoietic cells, except platelets, hepatocytes, testisbinds hyaluronic acid, adhesionCD45LCA, T200, B220hematopoietic cells, multiple isoforms from alternative splicingtyrosine phosphatase, enhanced TCR & BCR signalsCD45RAB, T subset(naive), monoexon A isoforms of CD45CD45RBT subset, B, mono, mac, granexon B isoforms of CD45CD45ROTact, memory T, B subset, mono, mac, granisoform of CD45 lacking A, B, C exonsCD46MCPCCRSFnucleated cellsmembrane cofactor protein, binds C3b & C4b allowing degradation by Factor I, measles virus receptorCD47IAPIgSFhematopoietic cells, epith, endoth, fibroblasts, other tissuesleukocyte adhesion, migration, activationCD48Blast-1IgSFbroad, all leukocytescell adhesionCD52CAMPATH-1thymocytes, T, B (not plasma cells), mono, macCD53TM4SFleukocytes, DC, osteoblasts, osteoclastssignal transductionCD55DAFGPI-linkedhematopoietic, endothbinds C3b, complement regulationCD56NCAMIgSFNK, T subset, neurons, some large granular lymphocyte leukemias, myeloid leukemiasadhesionCD57HNK-1, Leu-7NK subset, T subsetCD58LFA-3IgSFhematopoietic, non-hematopoietic cellsCD2 receptor, adhesionCD59Protectin, MAC-inhibitorGPI-linkedhematopoietic, non-hematopoietic cellsbinds complement C8 and C9, blocks assembly of membrane attack complexCD60aGD3CHOT subset, platelets, thymic epith, astrocytescostimulationCD63LIMP, LAMP-3TM4SFactivated platelets, mono, maclysosomal membrane protein, moves to cell surface after activationCD68Macrosialin, gp110Sialomucinintracellularly in mono, mac, neutrophils, basophils, large lymph, mast cells, DC, myeloid progenitors, liverCD69AIMC-type lectinTact, B, NK and gran, thymocytes, platelets, Langerhans cellssignal transductionCD70Ki-24TNFSFBact and TactCD27 ligand, T and B cell costimulationCD74li, invariant chainB, mac, mono, Langerhans cells, DC, TactMHC class II traffic and functionCD79aIgaIgSFBcomponent of BCR, BCR surface expression and signal transductionCD79bIgbIgSFBcomponent of BCR, BCR surface expression and signal transductionCD81TAPA-1TM4SFT, B, NK, thymocytes, DC, endoth, fibroblast, neuroblastomas, melanomascomplex with CD19 & CD21, signaling, T costimulationCD82R2TM4SFleukocytessignal transductionCD83HB15IgSFBact and Tact, DC, Langerhans cellsCDw84mono, platelets, B, T subset, mac subsetCD86B70, B7-2IgSFmono, DC, Bact and Tactbinds to CD28, CD152, T costimulationCD87UPA-RGPI-linkedgran, mono, NK, Tact, endoth, fibroblastsurokinase plasminogen activator receptor, inflammatory cell invasion, metastasisCD90Thy-1IgSF, GPI-linkedCD34+ hematopoietic subset, neuronshematopoietic stem cell and neuron differentiationCD94KP43C-type lectinNK, T subsetcomplex with NKG2, inhibits NK functionCD95Apo-1, FasTNFRSFlymph (high upon activation), mono, neutrophilsFasL (CD178) receptor, apoptosisCD96TACTILEIgSFNK, Tactadhesion of activated T and NKCD97TM7SFBact and Tact, mono, granCD984F2T, B, NK, gran, all human cell linescellular activationCD99MIC2, E2leukocytesT cell activation, adhesionCD100hematopoietic cells except immature bone marrow cells, RBC and plateletscell adhesion, cellular activationCD103HML-1, alpha6, integrin alphaEIntegrin familyintraepithelial lymph, lymph subset, activated lymphwith integrin beta7, binds E-cadherin, lymph homing / retentionCD107aLAMP-1activated platelets, T, endoth, metastatic tumorsa lysosomal membrane proteinCD107bLAMP-2activated platelets, T, endoth, metastatic tumorsa lysosomal membrane proteinCD109Tact and platelets, CD34+ subset, endothCD123IL-3RCRSFlymph subset, basophils, hematopoietic progenitors, mac, DC, megakaryocytesIL-3Ralpha, with CDw131CD146MUC18, S-endoIgSFendoth, melanomas, FDC, TactadhesionCD154CD40L, gp39, TRAPTNFSFTactCD40 ligand, B and DC costimulationCD158ap58.1IgSF, KIR familyNK subset, T subsetinhibition of NK cell cytolytic activity, MHC class-I specific NK receptorCD158bp58.2IgSF, KIR familyNK subset, T subsetinhibition of NK cell cytolytic activity, MHC class-I specific NK receptorCD163130kDScavenger receptor SFmono, macCD164MGC-24epith, mono, lymphlow, bone marrow stromal cells, CD34+ erythroid progenitorshematopoietic progenitor cell-stromal cell interactionCD168RHAMMmono, T subset, thymocyte subset, intracellularly in breast cancer cellsadhesion, tumor migration, metastasisCD171L1IgSFCNS, PNS, glial cells, mono, T subset, B, DC, several human tumor cellskidney morphogenesis, lymph node architecture, T costimulation, neurohistogenesis, homotypic interaction, binds CD9, CD24, CD56, CD142, CD166, integrinsCD177NB1neutrophil subsetCD178FasL, CD95LTNFSFTact, testisCD95 ligand, apoptosis, immune privilege, soluble form in serumCD180RP-105LRRF, TLR familyB subset, mono, DCB cell activation, LPS signaling, with MD-1CD182CXCR2, IL-8RBGPCR1 familyneutrophils, basophils, NK, T subset, monobinding of IL-8 induces chemotaxis of neutrophilsCD185CXCR5, BLR1GPCR1 familymature B and Burkitt Lymphoma cellswith chemokine BLC, possible regulatory function in Burkitt Lymphomagenesis and / or B differentiation, activation of mature BCD191CCR1, MIP-1alphaR, RANTES-RGPCR1 familyT, mono, stem cell subsetbinds C-C type chemokines and transduces signal by increasing intracellular calcium ion levelsCD193CCR3, CKR3GPCR1 familyeosinophils, lower expression in neutrophils and mono, T subsetbinds eotaxin, eotaxin-3, MCP-3, MCP-4, RANTES & MIP-1delta, alternative coreceptor with CD4 for HIV-1 infectionggCD196CCR6, LARC receptor, DRY6GPCR1 familyT subset, B, DC subsetbinds MIP-3alpha / LARCCD197CCR7T subset, DC Subset6Ckine and MIP-2beta receptorCD200OX-2thymocytes, endoth, B, Tactinhibition of immune responseCD209DC-SIGNDC subsetICAM-3 receptor, HIV-1 binding proteinCD227MUC1, EMAMucin family, type I TMepith, stem cell subset, FDC, mono, B subset, some myelomasadhesion, signaling, binds CD169, CD54, & selectinsCD231TALLA-1, A15TM4SFT leukemias, neuroblastomas, brain neuronsmarker for T cell acute lymphoblastic leukemiaCD246ALK, Ki-1anaplastic T cell leukemias, small intestine, testis, brain, not on normal lymphbrain development, implicated in ALK lymphomasCD254TRANCE, RANKL, OPGLTNFSFlymph node & BM stroma Tactbinds OPG and RANK, osteoclast differentiation, enhances DC to stimulate naïve-T proliferationCD263TRAIL-R3, DcR1, LITperipheral blood lymphocytesreceptor for TRAIL but lacks death domainCD272BTLAIgSFTact, B , remains on Th1HVEM receptor, inhibitory responseCD273B7DC, PD-L2, PDCD1L2IgSFDC subset, mono, macPD-1 receptor, costimulation or suppression of T proliferationCD276B7-H3B7 Family, ASVin vitro cultured DC and mono, Tact, mammary tissuecostimulation, T activationCD277BT3.1, butyrophilin SF3 A1, BTF5B7 / BT family, ASVT, B, NK, mono, DC, endoth, CD34+ cells, tumor cell linesT activationCD279PD1, SLEB2Tact and BactB7-H1 & B7-DC receptor, autoimmune disease and peripheral toleranceCD298Na+ / K+-ATPase beta3 subunitbroadtransport sodium & potassium ions across membraneCD300aCMRF35H, IRC1, IRp60IgSF, ASVNK, mono, neutrophils, T and B subset and lymphocytic cell lines, AMLunknownCD300cCMRF35A, LIRIgSFmono, neutrophils, monocytic cell lines, B & T subsetsunknownCD304BDCA4, neuropilin 1semaphorin familyneurons, CD4+ / CD25+ Treg, DC, endothelial and tumor cellsinteracts with VEGF165 & semaphorins, coreceptor with plexin, axonal guidance, angiogenesis, cell survival, migrationCD305LAIR1IgSF, ASVNK, B, T, monoinhibitory receptor on NK and T cellsCD314NKG2D, KLRType II lectin-like receptorNK, CD8+ activated, NK1.1+ T, some myeloid cellsbinds MHC class I, MICA, MICB, Rae1 & ULBP4, activates cytolysis and cytokine production, costimulationCD317BST2, HM1.24Type IIB, T, NK, mono, DC, fibroblast cell line, myelomapre-B cell growth, overexpressed in multiple myelomaCD319CS1, CRACC, SLAM F7SLAM receptor familyB Cells, Dendritic Cells, NK, NKTmultiple myeloma Table 5: antigen markers expressed on the surface of both colon tumor cells and T-cells Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells EPCAMEpithelial cell adhesion molecule2,9713,99IFITM1Interferon-induced transmembrane protein 110,5513,06CLDN4Claudin-42,8711,62CDH17Cadherin-171,8511,52CEACAM1Carcinoembryonic antigen-related cell adhesion molecule 13,3310,84SLC26A3Chloride anion exchanger2,5710,59ATP1A1Sodium / potassium-transporting ATPase subunit alpha-19,2810,51SIIsomaltase2,8610,46ABCB1Multidrug resistance protein 16,0910,24KCNQ1Potassium voltage-gated channel subfamily KQT member 13,369,99FCGRTIgG receptor FcRn large subunit p514,89,98EPHB3Ephrin type-B receptor 35,239,74DSG2Desmoqlein-23,048,5EPHB4Ephrin type-B receptor 46,58,44GUCY2CHeat-stable enterotoxin receptor2,238,05EPHA2Ephrin type-A receptor 22,87,95LY6G6DLymphocyte antigen 6 complex locus protein G6f2,027,91CD97CD97 antigen subunit beta7,77,87SIGMAR1Sigma non-opioid intracellular receptor 14,587,85EREGEpiregulin2,936,9FAIM2Protein lifequard 22,946,82PIGRSecretory component4,26,8SLC7A6Y+L amino acid transporter 28,066,55SCNN1DAmiloride-sensitive sodium channel subunit delta1,775,74GPR35G-protein coupled receptor 351,985,5ABCG2ATP-binding cassette sub-family G member 21,795,35LPAR4Lysophosphatidic acid receptor 42,935,05GPR161G-protein coupled receptor 1612,714,96CD1CT-cell surface glycoprotein CD1c2,734,89SGCAAlpha-sarcoglycan2,324,84CD22B-cell receptor CD224,124,75CD22B-cell receptor CD223,584,75CD22B-cell receptor CD222,734,75CD22B-cell receptor CD222,144,75SLC22A18Solute carrier family 22 member 182,324,62HTR75-hydroxytryptamine receptor 73,024,46LCTPhlorizin hydrolase2,324,24CD33Myeloid cell surface antigen CD333,424,14PVRPoliovirus receptor5,074,07PLXDC1Plexin domain-containing protein 15,853,99P2RY2P2Y purinoceptor 22,153,97CHRNB2Neuronal acetylcholine receptor subunit beta-26,313,88PTGDRProstaglandin D2 receptor4,083,65NCR1Natural cytotoxicity triggering receptor 12,633,33GYPAGlycophorin-A3,183,31TNFRSF8Tumor necrosis factor receptor superfamily member 822,75KELKell blood group glycoprotein1,932,48EDAEctodysplasin-A, secreted form2,72,42ACEAngiotensin-converting enzyme, soluble form2,392,19DRD2D(2) dopamine receptor2,491,97CXCR3C-X-C chemokine receptor type 34,191,66MC2RAdrenocorticotropic hormone receptor1,941,43 Table 6: antigen markers expressed on the surface of both breast tumor cells and T-cells Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells ABCA8ATP-binding cassette sub-family A member 83,157,73ABCC10Multidrug resistance-associated protein 76,485,29ABCC6Multidrug resistance-associated protein 62,672,17ACCN2Acid-sensing ion channel 13,622,49ADAM 12Disintegrin and metalloproteinase domain-containing protein 124,967,72ADCYAP1R1Pituitary adenylate cyclase-activating polypeptide type I receptor2,172,88ADRA1AAlpha-1A adrenergic receptor3,314,85ADRA1BAlpha-1B adrenergic receptor1,491,6ADRA1DAlpha-1D adrenergic receptor2,393,38ADRA2AAlpha-2A adrenergic receptor2,641,79ADRB3Beta-3 adrenergic receptor2,362,16AGERAdvanced glycosylation end product-specific receptor2,852,38AGTR2Type-2 angiotensin II receptor3,083,7ALKALK tyrosine kinase receptor4,974,27ANO3Anoctamin-32,393,69ANPEPAminopeptidase N3,2610,78APLNRApelin receptor2,472,06AQP2Aquaporin-22,121,43ATP10AProbable phospholipid-transporting ATPase VA3,966,02ATP2B2Plasma membrane calcium-transporting ATPase 42,754,81ATP2B3Plasma membrane calcium-transporting ATPase 33,74,14ATP4APotassium-transporting ATPase alpha chain 11,5611,49ATP4BPotassium-transporting ATPase subunit beta2,4913,56ATP6V0A2V-type proton ATPase 116 kDa subunit a isoform 22,512,57ATRNAttractin4,099,44AVPR1AVasopressin V1a receptor2,524,03AVPR1BVasopressin V1b receptor2,973,32AVPR2Vasopressin V2 receptor2,682,93BAI1Brain-specific angiogenesis inhibitor 12,730,33BAI2Brain-specific angiogenesis inhibitor 22,344,14BAI3Brain-specific angiogenesis inhibitor 32,734,76BDKRB1B1 bradykinin receptor2,073,28BRS3Bombesin receptor subtype-32,744,12BTF3Butyrophilin subfamily 3 member A211,2913,02C18orf1Low-density lipoprotein receptor class A domain-containing protein 43,188,45C3AR1C3a anaphylatoxin chemotactic receptor3,045,15C6orf105Androgen-dependent TFPI-regulating protein2,343,84CASRExtracellular calcium-sensing receptor2,525CCBP2Atypical chemokine receptor 21,723,29CCKARCholecystokinin receptor type A2,463CCKBRGastrin / cholecystokinin type B receptor2,255,66CCR2C-C chemokine receptor type 25,943,56CCR3C-C chemokine receptor type 31,894,17CCR6C-C chemokine receptor-like 23,335,23CCR8C-C chemokine receptor type 82,283,93CCR9C-C chemokine receptor type 91,681,98CD1AT-cell surface glycoprotein CD1 a1,984,88CD1BT-cell surface glycoprotein CD1 b2,354,94CD1DAntigen-presenting glycoprotein CD1d2,824,96CD300CCMRF35-like molecule 62,045,04CD4T-cell surface glycoprotein CD42,846,17CD40LGCD40 ligand, soluble form2,13,49CD5T-cell surface glycoprotein CD53,141,01CD63CD63 antigen8,613,18CD84SLAM family member 54,73,17CDH15Cadherin-152,073,55CDH19Protocadherin-162,828,4CDH22Cadherin-2234,9CDH8Cadherin-83,635,87CDONCell adhesion molecule-related / down-regulated by oncogenes2,353,61CHRNA4Neuronal acetylcholine receptor subunit alpha-42,143,33CHRNA5Neuronal acetylcholine receptor subunit alpha-52,24,88CHRNA6Neuronal acetylcholine receptor subunit alpha-62,264,93CHRNB3Neuronal acetylcholine receptor subunit beta-31,853,91CHRNEAcetylcholine receptor subunit epsilon2,562,83CLDN3Claudin-32,9113,56CLDN7Claudin-71,8912,87CLDN8Claudin-82,4610,67CLDN9Claudin-91,741,69CLEC4MC-type lectin domain family 4 member M2,73,32CMKLR1Chemokine-like receptor 12,625CNNM2Metal transporter CNNM22,475,32CNR2Cannabinoid receptor 22,383,66CRHR1Corticotropin-releasing factor receptor 12,1510,71CRHR2Corticotropin-releasing factor receptor 22,326,44CSF1Processed macrophage colony-stimulating factor 15,637,61CSF1RMacrophage colony-stimulating factor 1 receptor2,24,02CSF3RGranulocyte colony-stimulating factor receptor1,852,8CX3CL1Processed fractalkine2,359,31CXCR5C-X-C chemokine receptor type 52,076,06DAGLASn1-specific diacylglycerol lipase alpha2,62,11DRD1D(1A) dopamine receptor2,675,71DRD3D(3) dopamine receptor2,724,99DRD4D(4) dopamine receptor1,490,89DRD5D(1B) dopamine receptor2,264,91DSC2Desmocollin-22,2611,12DSCAMDown syndrome cell adhesion molecule2,543,76DSG1Desmoqlein-12,627,71EMR2EGF-like module-containing mucin-like hormone receptor-like 22,253,38EPHA5Ephrin type-A receptor 52,427,48EPHA7Ephrin type-A receptor 72,614,87ERBB3Receptor tyrosine-protein kinase erbB-32,3912,76F2RL2Proteinase-activated receptor 33,25,16FAM168BMyelin-associated neurite-outgrowth inhibitor8,3411,16FAPSeprase1,8710,15FASTumor necrosis factor receptor superfamily member 65,687,24FASLGFasL intracellular domain2,232,66FCARImmunoglobulin alpha Fc receptor2,83,85FCER1AHigh affinity immunoglobulin epsilon receptor subunit alpha2,544,59FCGR2ALow affinity immunoglobulin gamma Fc region receptor II-a2,778,81FCGR2BLow affinity immunoglobulin gamma Fc region receptor II-b2,465,35FGFR2Fibroblast growth factor receptor 24,019,83FGFR4Fibroblast growth factor receptor 42,567,42FLT3LGFms-related tyrosine kinase 3 ligand7,864,37FPR1fMet-Leu-Phe receptor3,385,92FPR3N-formyl peptide receptor 31,912,61FSHRFollicle-stimulating hormone receptor1,893,78FZD5Frizzled-52,825,2FZD5Frizzled-51,815,2FZD9Frizzled-92,663,16GABRA1Gamma-aminobutyric acid receptor subunit alpha-12,26,26GABRA5Gamma-aminobutyric acid receptor subunit alpha-52,493,24GABRA6Gamma-aminobutyric acid receptor subunit alpha-62,542,98GABRB1Gamma-aminobutyric acid receptor subunit beta-11,892,37GABRB2Gamma-aminobutyric acid receptor subunit beta-22,263,89GABRG3Gamma-aminobutyric acid receptor subunit gamma-32,232,85GABRPGamma-aminobutyric acid receptor subunit pi2,9312,34GABRR1Gamma-aminobutyric acid receptor subunit rho-12,353,47GABRR2Gamma-aminobutyric acid receptor subunit rho-24,165,43GALR2Galanin receptor type 21,850,46GALR3Galanin receptor type 30,680,48GCGRGlucagon receptor1,383,4GHRHRGrowth hormone-releasing hormone receptor1,613,49GJA5Gap junction alpha-5 protein1,722,05GJA8Gap junction alpha-8 protein2,396,51GJC1Gap junction delta-3 protein1,943,89GLP1RGlucagon-like peptide 1 receptor5,723,41GLRA1Glycine receptor subunit alpha-12,153,87GLRA3Glycine receptor subunit alpha-33,193,1GNRHRGonadotropin-releasing hormone receptor2,724,1GPNMBTransmembrane glycoprotein NMB2,1413,94GPR1G-protein coupled receptor 13,834,1GPR135Probable G-protein coupled receptor 1354,151,91GPR143G-protein coupled receptor 1431,933,65GPR15G-protein coupled receptor 151,814,41GPR17Uracil nucleotide / cysteinyl leukotriene receptor1,931,74GPR171Probable G-protein coupled receptor 1717,736,32GPR18N-arachidonyl glycine receptor7,053,52GPR182G-protein coupled receptor 1821,661,29GPR19Probable G-protein coupled receptor 191,895,26GPR20G-protein coupled receptor 202,022,53GPR3G-protein coupled receptor 33,015,36GPR3112-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid receptor1,631,64GPR37L1Prosaposin receptor GPR37L12,234GPR39G-protein coupled receptor 391,811,36GPR44Prostaglandin D2 receptor 222,32GPR45Probable G-protein coupled receptor 452,785,31GPR6G-protein coupled receptor 62,563,38GPR65Psychosine receptor6,594,5GPR68Ovarian cancer G-protein coupled receptor 12,121,09GPR98G-protein coupled receptor 981,894,7GRIA1Glutamate receptor 14,174,77GRIA3Glutamate receptor 32,516,83GRIK2Glutamate receptor ionotropic, kainate 52,564,94GRIK3Glutamate receptor ionotropic, kainate 32,053,58GRIN1Glutamate receptor ionotropic, NMDA 14,521,49GRIN2BGlutamate receptor ionotropic, NMDA 2B2,223,56GRIN2CGlutamate receptor ionotropic, NMDA 2C2,563,37GRM1Metabotropic glutamate receptor 13,213,69GRM2Metabotropic glutamate receptor 22,040,44GRM3Metabotropic glutamate receptor 32,393,41GRM4Metabotropic glutamate receptor 45,23,78GRM5Metabotropic glutamate receptor 52,265,28GRM7Metabotropic glutamate receptor 72,863,07GYPBGlycophorin-B2,434,02HBP1Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 17,329,27HCRTR2Orexin receptor type 22,322,42HTR1B5-hydroxytryptamine receptor 1B2,823,51HTR1D5-hydroxytryptamine receptor 1D2,292,33HTR1E5-hydroxytryptamine receptor 1E1,722,4HTR2A5-hydroxytryptamine receptor 2A2,13,67HTR2C5-hydroxytryptamine receptor 2C2,495,18HTR45-hydroxytryptamine receptor 43,864,25ICAM4Intercellular adhesion molecule 42,512,16ICOSInducible T-cell costimulator3,913,86IL6RInterleukin-6 receptor subunit alpha4,243,08IL6RInterleukin-6 receptor subunit alpha2,643,08IL6STInterleukin-6 receptor subunit beta9,4312,67IL9RInterleukin-9 receptor2,712, 86ITGB3Integrin beta-34,163,69KCNA3Potassium voltage-gated channel subfamily A member 32,094,9KCND2Potassium voltage-gated channel subfamily D member 22,674,25KCNH1Potassium voltage-gated channel subfamily H member 12,314,48KCNJ4Inward rectifier potassium channel 42,433,49KCNMA1Calcium-activated potassium channel subunit alpha-12,357,17KCNS1Potassium voltage-gated channel subfamily S member 15,666,49KCNV2Potassium voltage-gated channel subfamily V member 22,384,06KIR2DL4Killer cell immunoglobulin-like receptor 2DL41,683,31KIR3DL1Killer cell immunoglobulin-like receptor 3DL12,562,73KIR3DL3Killer cell immunoglobulin-like receptor 3DL31,73,06KLRG1Killer cell lectin-like receptor subfamily G member 18,35,76LAMP1I ysosome-associated membrane glycoprotein 110,913,6LHCGRLutropin-choriogonadotropic hormone receptor2,234,92LNPEPLeucyl-cystinyl aminopeptidase, pregnancy serum form2,685,05LPAR2Lysophosphatidic acid receptor 25,54,23LRIG2Leucine-rich repeats and immunoglobulin-like domains protein 23,355,48LRRTM2Leucine-rich repeat transmembrane neuronal protein 22,424,24LTB4RLeukotriene B4 receptor 14,962,26MAS1Proto-oncogene Mas1,913,11MC1RMelanocyte-stimulating hormone receptor2,940,96MC5RMelanocortin receptor 52,281,63MEP1BMeprin A subunit beta2,613,87MFSD5Molybdate-anion transporter1,984,72MOGMyelin-oligodendrocyte glycoprotein3,084,74MTNR1BMelatonin receptor type 1B1,611,67MUC1Mucin-1 subunit beta2,7313,68MUSKMuscle, skeletal receptor tyrosine-protein kinase2,394,75NCAM2Neural cell adhesion molecule 22,124,49NCR2Natural cytotoxicity triggering receptor 24,797,09NCR3Natural cytotoxicity triggering receptor 34,552,74NIPA2Magnesium transporter NIPA26,773,9NLGN1Neuroligin-12,627,71NLGN4YNeuroliqin-4, Y-linked2,525,26NMBRNeuromedin-B receptor1,682,47NPHS1Nephrin2,744,33NPY2RNeuropeptide Y receptor type 22,684,43NPY5RNeuropeptide Y receptor type 52,385,05NTSR2Neurotensin receptor type 21,723OPRD1Delta-type opioid receptor2,262,14OPRL1Nociceptin receptor2,311,51OPRM1Mu-type opioid receptor3,184,01OR10H3Olfactory receptor 10H31,634,02OR1E1Olfactory receptor 1E13,044,77OR2F1Olfactory receptor 2F12,645,73OR2F2Olfactory receptor 2F22,192,3OR2H1Olfactory receptor 2H13,393,82OR2H2Olfactory receptor 2H23,796,37OR2J2Olfactory receptor 2J22,412,16OR2J2Olfactory receptor 2J21,932,16OR511Olfactory receptor 5I11,852,8OR7E24Olfactory receptor 7E242,53,47P2RX7P2X purinoceptor 72,362,15PANX1Pannexin-12,144,38PCDHA9Protocadherin alpha-92,823,56PCDHB11Protocadherin beta-111,915,23PCDHGA8Protocadherin qamma-A83,134,48PLA2R1Soluble secretory phospholipase A2 receptor2,915,16PLXNA3Plexin-A32,423,25POP1Blood vessel epicardial substance1,742,59PPYR1Neuropeptide Y receptor type 42,22,75PTGER1Prostaglandin E2 receptor EP1 subtype1,960,94PTGFRProstaglandin F2-alpha receptor2,754,89PTGIRProstacyclin receptor2,782,12PTPRJReceptor-type tyrosine-protein phosphatase eta2,634,6PTPRRReceptor-type tyrosine-protein phosphatase R2,479,99PVRL1Poliovirus receptor-related protein 12,524,51PVRL2Poliovirus receptor-related protein 23,8410,05ROS1Proto-oncogene tyrosine-protein kinase ROS2,933,38S1PR2Sphingosine 1-phosphate receptor 21,741,17S1PR4Sphingosine 1-phosphate receptor 440,21SCNN1BAmiloride-sensitive sodium channel subunit beta1,893,16SCNN1GAmiloride-sensitive sodium channel subunit gamma2,232,61SEMA4DSemaphorin-4D10,661,56SEMA6ASemaphorin-6A4,557,81SEMA6CSemaphorin-6C5,023,73SGCBBeta-sarcoglycan2,693,45SGCBBeta-sarcoglycan2,043,45SLC12A3Solute carrier family 12 member 32,263,36SLC14A1Urea transporter 12,976,21SLC14A2Urea transporter 22,854,4SLC16A1Monocarboxylate transporter 13,468,84SLC16A2Monocarboxylate transporter 81,775,17SLC16A6Monocarboxylate transporter 72,4111,66SLC22A1Solute carrier family 22 member 12,9511,61SLC22A6Solute carrier family 22 member 62,262,53SLC5A12Sodium-coupled monocarboxylate transporter 22,984,45SLC6A1Sodium- and chloride-dependent GABA transporter 12,454,3SLC6A4Sodium-dependent serotonin transporter2,172,66SLC6A6Sodium- and chloride-dependent taurine transporter2,544,13SLC7A7Y+L amino acid transporter 12,229,78SLC8A1Sodium / calcium exchanger 12,072,36SLC9A1Sodium / hydrogen exchanger 13,155,54SLC9A3Sodium / hydrogen exchanger 32,123,15SLCO1A2Solute carrier organic anion transporter family member 1A23,874,98SLCO2B1Solute carrier organic anion transporter family member 2B14,438,92SORT1Sortilin2,934,6SSTR2Somatostatin receptor type 23,084,47SSTR3Somatostatin receptor type 32,231,5SSTR4Somatostatin receptor type 41,831,53SSTR5Somatostatin receptor type 52,571,47TACR1Substance-P receptor2,663,2TACR3Neuromedin-K receptor2,325,7TLR6Toll-like receptor 62,24,58TMPRSS6Transmembrane protease serine 64,023,69TNFSF11Tumor necrosis factor ligand superfamily member 11,2,575,18TNFSF14Tumor necrosis factor ligand superfamily member 14, soluble form3,342,83TPOThyroid peroxidase1,961,89TRAT1T-cell receptor-associated transmembrane adapter 17,515,29TRHRThyrotropin-releasing hormone receptor24,18TRPM1Transient receptor potential cation channel subfamily M member 12,435,22TSHRThyrotropin receptor2,94,87TSHRThyrotropin receptor2,124,87UNC93AProtein unc-93 homolog A2,644,94VIPR2Vasoactive intestinal polypeptide receptor 22,583,37ZP2Processed zona pellucida sperm-binding protein 21,943,55 Table 7: antigen markers expressed on the surface of both digestive tumor cells and T-cells Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells ACVR1BActivin receptor type-1B5,1610,48AMIGO2Amphoterin-induced protein 26,738,2ATP1B1Sodium / potassium-transporting ATPase subunit beta-12,6412,31ATP8B1Probable phospholipid-transporting ATPase IC8,222,17CCR7C-C chemokine receptor type 710,2511,52CD164Sialomucin core protein 2410,2712,12CD180CD180 antigen2,56,47CD40Tumor necrosis factor receptor superfamily member 55,026CD53Leukocyte surface antigen CD5310,7911,3CD79AB-cell antigen receptor complex-associated protein alpha chain3,749,17CD79BB-cell antigen receptor complex-associated protein beta chain3,66,66CD8BT-cell surface glycoprotein CD8 beta chain8,432,62CELSR1Cadherin EGF LAG seven-pass G-type receptor 12,728,68CLCN5H(+) / Cl(-) exchange transporter 52,714,97CLDN18Claudin-183,0514,51CLIC1Chloride intracellular channel protein 19,9413,83COL13A1Collagen alpha-1(XIII) chain2,966,24DIO3Type III iodothyronine deiodinase2,042,9EDNRAEndothelin-1 receptor2,98,96EMR1EGF-like module-containing mucin-like hormone receptor-like 11,837,29ENPP1Nucleotide pyrophosphatase2,579,66EPHB1Ephrin type-B receptor 12,026,33EPHB1Ephrin type-B receptor 11,816,33F2RProteinase-activated receptor 13,049,78F2RL1Proteinase-activated receptor 2, alternate cleaved 23,319,47FCER2Low affinity immunoglobulin epsilon Fc receptor soluble form2,498,77GABBR1Gamma-aminobutyric acid type B receptor subunit 15,18,52GABRA3Gamma-aminobutyric acid receptor subunit alpha-32,123,84GPR183G-protein coupled receptor 1834,7910,22GPR37Prosaposin receptor GPR373,18,23GPRC5ARetinoic acid-induced protein 31,8713,69GRPRGastrin-releasing peptide receptor2,043,35GYPCGlycophorin-C9,227,58IL1R2Interleukin-1 receptor type 2, soluble form2,8212,83KIAA0319Dyslexia-associated protein KIAA03192,435,61LAMP2I ysosome-associated membrane glycoprotein 24,0511,29LRP8Low-density lipoprotein receptor-related protein 84,248,84LSRLipolysis-stimulated lipoprotein receptor4,9911,48MICBMHC class I polypeptide-related sequence B5,279,89MMP16Matrix metalloproteinase-163,196,18MS4A1B-lymphocyte antigen CD202,158,02MYOFMyoferlin2,4111,56NAT1Sodium-coupled neutral amino acid transporter 33,4912,09NFASCNeurofascin3,788,28NPY1RNeuropeptide Y receptor type 12,326,93OR2B6Olfactory receptor 2B62,784,24P2RY10Putative P2Y purinoceptor 103,396,62PCDH1Protocadherin-14,4510,07PROM 1Prominin-12,5211,77PSEN1Presenilin-1 CTF122,948,83PTGER2Prostaglandin E2 receptor EP2 subtype6,336,74PTGER4Prostaglandin E2 receptor EP4 subtype8,625,12PTPRKReceptor-type tyrosine-protein phosphatase kappa2,1410,9RETExtracellular cell-membrane anchored RET cadherin 120 kDa fragment2,3812,3SERINC3Serine incorporator 37,9312,01SIT1Sodium- and chloride-dependent transporter XTRP35,924,82SLAMF1Signaling lymphocytic activation molecule4,49,03SLC29A1Equilibrative nucleoside transporter 12,076,12SLC39A6Zinc transporter ZIP66,6915,23SLC7A5Large neutral amino acids transporter small subunit 13,7910,98STX4Syntaxin-45,687,67TGFBR3Transforming growth factor beta receptor type 37,557,29TGOLN2Trans-Golgi network integral membrane protein 29,5911,3TLR1Toll-like receptor 12,344,57TMED10Transmembrane emp24 domain-containing protein 109,3412,24TMEM97Transmembrane protein 972,759,02TNFTumor necrosis factor, soluble form1,633,18TNFRSF17Tumor necrosis factor receptor superfamily member 171,8910,47TNFRSF1BTumor necrosis factor-binding protein 25,519,4VDAC1Voltage-dependent anion-selective channel protein 16,5211,5 Table 8: antigen markers expressed on the surface of both kidney tumor cells and T-cells Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells ADORA3Adenosine receptor A31,894,56ATP11AProbable phospholipid-transporting ATPase IH3,628,8BSGBasiqin4,7711,34BTN3A2Butyrophilin subfamily 3 member A210,868,19C10orf72V-set and transmembrane domain-containing protein 42,046,85CADM3Cell adhesion molecule 33,576,39CD8AT-cell surface glycoprotein CD8 alpha chain10,356,6CDH16Cadherin-162,177,09CDH4Cadherin-42,153,6CDH5Cadherin-52,59,55CHL1Processed neural cell adhesion molecule L1-like protein2,6910,43CHRNB1Acetylcholine receptor subunit beta2,123,6CLIC4Chloride intracellular channel protein 43,3413,12CNR1Cannabinoid receptor 12,265,64CRIM1Processed cysteine-rich motor neuron 1 protein3,5712,39CSPG4Chondroitin sulfate proteoglycan 43,336,59CYBBCytochrome b-245 heavy chain2,868,07EDNRBEndothelin B receptor3,048,97FLT1Vascular endothelial growth factor receptor 12,758,5FZD1Frizzled-12,727,59GJC2Gap junction gamma-2 protein2,092,94GLRBGlycine receptor subunit beta2,517,15GPERG-protein coupled estrogen receptor 12,348,64GPM6ANeuronal membrane glycoprotein M6-a2,956,88GPR162Probable G-protein coupled receptor 1622,752,81GPR4G-protein coupled receptor 42,938,09GRM8Metabotropic glutamate receptor 83,438,25HLA-DPB1HLA class II histocompatibility antigen, DP beta 1 chain9,9313,99HTR65-hydroxytryptamine receptor 64,8310,07INSRInsulin receptor subunit beta3,448,95ITM2BBri23 peptide11,1612,19KCNJ1ATP-sensitive inward rectifier potassium channel 12,54,17KDRVascular endothelial growth factor receptor 22,999,95KLKlotho peptide2,837,59LAIR1Leukocyte-associated immunoglobulin-like receptor 15,644,25MFAP3Microfibril-associated glycoprotein 33,77,3MFAP3LMicrofibrillar-associated protein 3-like3,448,7MICAMHC class I polypeptide-related sequence A4,072,01NCAM1Neural cell adhesion molecule 12,457,31NOTCH3Notch 3 intracellular domain3,2112,41NOTCH4Notch 4 intracellular domain5,898,84OLR1Oxidized low-density lipoprotein receptor 1, soluble form2,848,41P2RY14P2Y purinoceptor 142,634,63PCDH17Protocadherin-171,77,36PDGFRBPlatelet-derived growth factor receptor beta2,6810,48PECAM1Platelet endothelial cell adhesion molecule7,710,85PLXND1Plexin-D15,0211,68PPAP2BLipid phosphate phosphohydrolase 34,1712,46PTAFRPlatelet-activating factor receptor3,014,81PTGER3Prostaglandin E2 receptor EP3 subtype4,7610,26PTH1RParathyroid hormone / parathyroid hormone-related peptide receptor2,357,31RAM P3Receptor activity-modifying protein 31,798,84ROR2Tyrosine-protein kinase transmembrane receptor ROR23,25,98S1PR1Sphingosine 1-phosphate receptor 15,176,51SCARB1Scavenger receptor class B member 13,0110,4SLC13A3Solute carrier family 13 member 33,327,89SLC16A4Monocarboxylate transporter 52,8812,54SLC17A3Sodium-dependent phosphate transport protein 41,5811,55SLC28A1Sodium / nucleoside cotransporter 14,766,3SLC2A5Solute carrier family 2, facilitated glucose transporter member 52,748,5SLC39A14Zinc transporter ZIP142,6611,63SLC6A13Sodium- and chloride-dependent GABA transporter 22,757,44SLC7A8Large neutral amino acids transporter small subunit 25,0310,46SLCO2A1Solute carrier organic anion transporter family member 2A13,468,06TBXA2RThromboxane A2 receptor4,013,64TGFBR2TGF-beta receptor type-210,4110,94THSD7AThrombospondin type-1domain-containing protein 7A3,058TIE1Tyrosine-protein kinase receptor Tie-12,044,41TNFRSF1ATumor necrosis factor-binding protein 16,8410,52TNFSF12Tumor necrosis factor ligand superfamily member 12, secreted form4,354,1VAMP5Vesicle-associated membrane protein 53,496,18 Table 9: antigen markers expressed on the surface of both liver tumor cells and T-cells Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells ABCB4Multidrug resistance protein 32,023,7ADAM 10Disintegrin and metalloproteinase domain-containing protein 109,429,41ATRAnthrax toxin receptor 16,989,9BST2Bone marrow stromal antigen 27,3812,45BTN3A3Butyrophilin subfamily 3 member A39,727,48C9Complement component C9b2,4110,52CHRNDAcetylcholine receptor subunit delta2,434,05CLDN14Claudin-142,792,4EPORErythropoietin receptor4,6710,55ERBB2Receptor tyrosine-protein kinase erbB-22,3614,12F2RL3Proteinase-activated receptor 42,172,61GJB1Gap junction beta-1 protein2,969,4GPR126G-protein coupled receptor 1262,2311,32IL1R1Interleukin-1 receptor type 1, soluble form2,8812,57ITGB1Integrin beta-18,7613,48NAALADL1N-acetylated-alpha-linked acidic dipeptidase-like protein3,031,46OR7A5Olfactory receptor 7A51,513,83SGCDDelta-sarcoglycan3,997,21SIGLEC6Sialic acid-binding lg-like lectin 63,573,49SLC38A3Sodium-coupled neutral amino acid transporter 31,898,91TFR2Transferrin receptor protein 22,7410,47 Table 10: antigen markers expressed on the surface of both lung tumor cells and T-cells Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells ABCB6ATP-binding cassette sub-family B member 6, mitochondrial2,889,82ABCC1Multidrug resistance-associated protein 17,058,16ACCN1Acid-sensing ion channel 22,250,8ADAM23Disintegrin and metalloproteinase domain-containing protein 232,514,73ADORA1Adenosine receptor A14,498,22ADORA2BAdenosine receptor A2b1,667,5AJAP1Adherens junction-associated protein 11,856,24APLP1C302,226,02AQP3Aquaporin-38,3813,88ATP10DProbable phospholipid-transporting ATPase VD2,437,4ATP1A3Sodium / potassium-transporting ATPase subunit alpha-33,013,13ATP1B2Sodium / potassium-transporting ATPase subunit beta-23,213,8ATP1B3Sodium / potassium-transporting ATPase subunit beta-38,614,26AXLTyrosine-protein kinase receptor UFO2,519,58BEST1Bestrophin-12,494,44BTCBetacellulin2,864,59BTN3A1Butyrophilin subfamily 3 member A110,6611,63CALCRCalcitonin receptor2,958,62CALCRLCalcitonin gene-related peptide type 1 receptor2,127,67CCR1C-C chemokine receptor type 12,639,77CD163Soluble CD1632,668,76CD300ACMRF35-like molecule 87,964,23CD300ACMRF35-like molecule 82,294,23CD68Macrosialin4,028,92CD74HLA class II histocompatibility antigen gamma chain9,113,44CD86T-lymphocyte activation antigen CD862,935,04CHRNA3Neuronal acetylcholine receptor subunit alpha-32,544,62CHRNA3Neuronal acetylcholine receptor subunit alpha-324,62CKAP4Cytoskeleton-associated protein 46,1511,94CLCA2Calcium-activated chloride channel regulator 2, 35 kDa form2,999,81CLDN5Claudin-53,667,73CLSTN1CTF1-alpha8,2612,51CNIH3Protein cornichon homolog 32,76,09COMTCatechol O-methyltransferase7,7812,13CSPG5Chondroitin sulfate proteoglycan 52,845,69CXCR6C-X-C chemokine receptor type 63,163,91CXCR7Atypical chemokine receptor 32,58,95DCHS1Protocadherin-164,292,28DSC3Desmocollin-22,828,95DSG3Desmoglein-32,2310,73EGFREpidermal growth factor receptor3,810,92FAT2Protocadherin Fat 22,259,29FCER1GHigh affinity immunoglobulin epsilon receptor subunit gamma3,138,96FCGR1AHigh affinity immunoglobulin gamma Fc receptor I2,099,65FLT4Vascular endothelial growth factor receptor 33,193,36FPR2N-formyl peptide receptor 22,97,14FURINFurin6,427,5FZD6Frizzled-62,6410,45GABBR2Gamma-aminobutyric acid type B receptor subunit 23,799,19GABRB3Gamma-aminobutyric acid receptor subunit beta-32,468,83GABRDGamma-aminobutyric acid receptor subunit delta1,721,67GABREGamma-aminobutyric acid receptor subunit epsilon1,859,18GIPRGastric inhibitory polypeptide receptor3,435,37GJA1Gap junction alpha-1 protein2,8412,65GJB3Gap junction beta-3 protein3,723,79GJB5Gap junction beta-5 protein1,776,69GLRA2Glycine receptor subunit alpha-22,266,15GPR109BHydroxycarboxylic acid receptor 31,772,91GPR12G-protein coupled receptor 1221,76GPR176Probable G-protein coupled receptor 1762,053,86GPR50Melatonin-related receptor2,263,16GRIK1Glutamate receptor ionotropic, kainate 14,665,65GRIN2DGlutamate receptor ionotropic, NMDA 2D2,172,32HCRTR1Orexin receptor type 12,343,56HLA-DPA1HLA class II histocompatibility antigen, DP alpha 1 chain8,3112,86HLA-DQA1HLA class II histocompatibility antigen, DQ alpha 1 chain2,3511,44HLA-DQB1HLA class II histocompatibility antigen, DQ beta 1 chain7,412,71HLA-DRAHLA class II histocompatibility antigen, DR alpha chain6,4214,18HLA-DRB4HLA class II histocompatibility antigen, DR beta 4 chain2,7211,24IGSF9BProtein turtle homolog B3,922,81IL1RAPInterleukin-1 receptor accessory protein3,9911,4IL1RL1Interleukin-1 receptor-like 12,555,15IL4RSoluble interleukin-4 receptor subunit alpha4,159,56IL7RInterleukin-7 receptor subunit alpha11,6211,26ITGA6Integrin alpha-6 light chain7,9912,76JPH3Junctophilin-32,342,5KCNS3Potassium voltage-gated channel subfamily S member 32,458,91KITMast / stem cell growth factor receptor Kit2,858,67KITLGSoluble KIT ligand2,587,27LILRB3Leukocyte immunoglobulin-like receptor subfamily B member 35,658,03LILRB4Leukocyte immunoglobulin-like receptor subfamily B member 43,1210,44LPAR1Lysophosphatidic acid receptor 14,125,47LPHN3Latrophilin-32,856,43MMP24Processed matrix metalloproteinase-245,195,73MPZMyelin protein P02,563,63MUC4Mucin-4 beta chain3,0410,34NCKAP1LNck-associated protein 1-like6,697,51NKG7Protein NKG710,923,66NOTCH2Notch 2 intracellular domain6,626,22NRCAMNeuronal cell adhesion molecule2,788,16NRG2Neurequlin-23,559,22NRXN1Neurexin-12,565,33NTRK2BDNF / NT-3 growth factors receptor2,5610,7NTSR1Neurotensin receptor type 11,749,74P2RY1P2Y purinoceptor 12,347,62P2RY6P2Y purinoceptor 64,275,79PCDH8Protocadherin-82,679,29PCDHA3Protocadherin alpha-32,143,54PIK3IP1Phosphoinositide-3-kinase-interacting protein 18,683,47PLXNA2Plexin-A22,887,3PRR4Processed poliovirus receptor-related protein 43,248,02PTPREReceptor-type tyrosine-protein phosphatase epsilon6,037,92PTPROReceptor-type tyrosine-protein phosphatase U10,469,01PTPRUReceptor-type tyrosine-protein phosphatase U3,726,18RABAC1Prenylated Rab acceptor protein 17,548,82SCTRSecretin receptor2,22,48SECTM1Secreted and transmembrane protein 12,426,9SGCEEpsilon-sarcoglycan2,159,65SGCGGamma-sarcoglycan2,565,74SLC16A3Monocarboxylate transporter 45,8912,72SLC16A7Monocarboxylate transporter 25,396,97SLC20A2Sodium-dependent phosphate transporter 22,5112,69SLC26A4Pendrin3,579,39SLC2A1Solute carrier family 2, facilitated glucose transporter member 15,15,83SLC4A7Sodium bicarbonate cotransporter 34,898,7SLCO3A1Solute carrier organic anion transporter family member 3A14,877,91SYNE2Nesprin-29,4310,43TACR2Substance-K receptor2,236,68TFRCTransferrin receptor protein 1, serum form7,3214,31TMEFF1Tomorequlin-13,225,05TMPRSS11DTransmembrane protease serine 11D catalytic chain2,358,32 Table 11: antigen markers expressed on the surface of both ovary tumor cells and T-cells; Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells ACVR2BActivin receptor type-2B2,14,26ADAM28Disintegrin and metalloproteinase domain-containing protein 282,839,22ADRA2CAlpha-2C adrenergic receptor4,65,13ATP2B1Plasma membrane calcium-transporting ATPase 15,311,49ATP2B4Plasma membrane calcium-transporting ATPase 48,2110,1ATP7ACopper-transporting ATPase 13,917,31CD200OX-2 membrane glycoprotein2,8310,51CD47Leukocyte surface antigen CD479,8810,42CDH12Cadherin-122,315,91CDH18Cadherin-182,284,79CDH2Cadherin-23,7211,97CDH6Cadherin-62,778,68CDIPTCDP-diacylglycerol--inositol 3-phosphatidyltransferase8,8810,73CELSR2Cadherin EGF LAG seven-pass G-type receptor 22,668,38CHRNA1Acetylcholine receptor subunit alpha2,425,71CLSTN3Calsyntenin-33,874,54CX3CR1CX3C chemokine receptor 1911,42DDR1Epithelial discoidin domain-containing receptor 13,8312,36EPHA1Ephrin type-A receptor 12,025,96EPHA4Ephrin type-A receptor 42,398,56ERBB4ERBB4 intracellular domain2,299,76FGFR1Fibroblast growth factor receptor 15,4211,4FGFR3Fibroblast growth factor receptor 32,9511,35FZD2Frizzled-21,918,06FZD7Frizzled-72,5510,24GJA4Gap junction alpha-4 protein2,046,7GPR125Probable G-protein coupled receptor 1252,357,88GPR56GPR56 C-terminal fragment8,611,27GPR64G-protein coupled receptor 642,048,57GPRC5BG-protein coupled receptor family C group 5 member B1,9610,29GRIA2Glutamate receptor 21,9611,78GRIK5Glutamate receptor ionotropic, kainate 55,793,36GRIN2AGlutamate receptor ionotropic, NMDA 2A1,682,96HEG1Protein HEG homolog 14,810,1HRH1Histamine H1 receptor2,316,26HTR3A5-hydroxytryptamine receptor 3A2,19,35IFITM2Interferon-induced transmembrane protein 210,2711,36IFITM3Interferon-induced transmembrane protein 38,5513,48KCNH2Potassium voltage-gated channel subfamily H member 22,095,36KCNJ12ATP-sensitive inward rectifier potassium channel 122,292,21L1CAMNeural cell adhesion molecule L12,618,73LGR5Leucine-rich repeat-containing G-protein coupled receptor 52,4512,12LPHN1Latrophilin-14,55,56LPHN1Latrophilin-11,635,56LPHN2Latrophilin-21,937,14MGAGlucoamylase5,155,65NEO1Neogenin1,8510,31NPTNNeuroplastin8,4613,14NRG1Neurequlin-12,616,53NTRK1High affinity nerve growth factor receptor2,092,49PCDH7Protocadherin-72,898,52PCDH9Protocadherin-92,996,15PDGFRAPlatelet-derived growth factor receptor alpha3,698,44PDGFRAPlatelet-derived growth factor receptor alpha2,268,44PLXNB1Plexin-B12,266,71PLXNB2Plexin-B23,110,68PODXLPodocalyxin2,7311,41PRSS8Prostasin heavy chain2,0710,77PTH2RParathyroid hormone 2 receptor1,858,67PVRL3Poliovirus receptor-related protein 32,5610,15SCNN1AAmiloride-sensitive sodium channel subunit alpha5,9710,63SLC29A2Equilibrative nucleoside transporter 22,931,89SSPNSarcospan3,499,16STARHeat-stable enterotoxin receptor2,367,13TGFATransforming growth factor alpha2,641,71TMED1Transmembrane emp24 domain-containing protein 14,799,3TMEM59Transmembrane protein 598,8312,74TNFRSF25Tumor necrosis factor receptor superfamily member 257,534,27TYRO3Tyrosine-protein kinase receptor TYRO34,1110,27UPK2Uroplakin-22,297,49 Table 12: antigen markers expressed on the surface of both pancreas tumor cells and T-cells Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells ADAM9Disintegrin and metalloproteinase domain-containing protein 93,4910,99B4GALT1Processed beta-1,4-galactosyltransferase 17,448,99BDKRB2B2 bradykinin receptor2,524,44CA9Carbonic anhydrase 93,3411,9CACNA1CVoltage-dependent L-type calcium channel subunit alpha-1C2,364,54CD58Lymphocyte function-associated antigen 36,518,16CDH11Cadherin-112,8510,38CDH3Cadherin-31,9610,91CFTRCystic fibrosis transmembrane conductance regulator3,1211,45CHRNB4Neuronal acetylcholine receptor subunit beta-42,380,66CLDN10Claudin-102,3611,5CXCR4C-X-C chemokine receptor type 411,7410,98DAG1Beta-dystroglycan5,6510,98DDR2Discoidin domain-containing receptor 22,348DMPKMyotonin-protein kinase3,74,21FAT1Protocadherin Fat 1, nuclear form3,312,45HTR2B5-hydroxytryptamine receptor 2B2,227,73LDLRLow-density lipoprotein receptor2,9312,14NCKAP1Nck-associated protein 13,3411,99PM P22Peripheral myelin protein 222,0910,66PNPLA2Patatin-like phospholipase domain-containing protein 25,463,45PNPLA2Patatin-like phospholipase domain-containing protein 22,353,45TEKAnqiopoietin-1 receptor3,878,52TGFBR1TGF-beta receptor type-12,174,3 Table 13: antigen markers expressed on the surface of both prostate tumor cells and T-cells Antigen Protein Name Relative expression in T-Cell Relative Expression in colon cancer cells ACCN3Acid-sensing ion channel 32,472,03ADRB1Beta-1 adrenergic receptor2,855,09ADRB2Beta-2 adrenergic receptor5,749,43AGTR1Type-1 angiotensin II receptor2,8111,62APLP2Amyloid-like protein 27,0613,06ATP1A2Sodium / potassium-transporting ATPase subunit alpha-23,077,55ATP8A1Probable phospholipid-transporting ATPase IA7,239,16CADM1Cell adhesion molecule 14,4212,28CHRM3Muscarinic acetylcholine receptor M31,859,23CHRNA2Neuronal acetylcholine receptor subunit alpha-22,835,34CXADRCoxsackievirus and adenovirus receptor3,3112,74DPP4Dipeptidyl peptidase 4 soluble form6,4211,22ECE1Endothelin-converting enzyme 17,144,7ENPP4Bis(5'-adenosyl)-triphosphatase ENPP46,577,49EPHA3Ephrin type-A receptor 32,847,85ERGPotassium voltage-gated channel subfamily H member 22,7211,3FAM38APiezo-type mechanosensitive ion channel component 18,49,57FOLH1Glutamate carboxypeptidase 22,9613,18GABRA2Gamma-aminobutyric acid receptor subunit alpha-236,42GHRGrowth hormone-binding protein2,526,84GPM6BNeuronal membrane glycoprotein M6-b3,226,56GPR116Probable G-protein coupled receptor 1163,6910,09HBEGFHeparin-binding EGF-like growth factor2,878,12JAM3Junctional adhesion molecule C4,297,26KCND3Potassium voltage-gated channel subfamily D member 33,099,77LIFRLeukemia inhibitory factor receptor2,716,8LRBALipopolysaccharide-responsive and beige-like anchor protein5,359,26MMENeprilysin2,628,05NOVPlexin-A12,4310,41NRP1Neuropilin-13,177,85OPRK1Kappa-type opioid receptor2,074,92PLXNB3Plexin-B32,573,59PPAP2ALipid phosphate phosphohydrolase 13,611,55SCAM P5Secretory carrier-associated membrane protein 53,038,43SLC23A2Solute carrier family 23 member 23,557,04SLC2A4Solute carrier family 2, facilitated glucose transporter member 42,675,96SLC36A1Proton-coupled amino acid transporter 13,389,28SLC4A4Electrogenic sodium bicarbonate cotransporter 13,1411,29STIM1Stromal interaction molecule 13,686,51TMPRSS2Transmembrane protease serine 2 catalytic chain2,679,63TRPV6Transient receptor potential cation channel subfamily V member 64,848,09VIPR1Vasoactive intestinal polypeptide receptor 14,417,73IYIPF3Protein YIPF3, 36 kDa form III44,3 Table 14: antigen markers expressed on the surface of T-cells and overexpressed in liquid tumor cells (ALL, AML, CML, MDS, CLL, CTRL) Antigen Protein Name Relative expression on T cell CD63CD63 antigen0.83CXCR4C-X-C chemokine receptor type 40.82IFITM2Interferon-induced transmembrane protein 20.82ITM2BBri23 peptide0.81BTF3Butyrophilin subfamily 3 member A20.8HLA-DRB1HLA class II histocompatibility antigen, DRB1-12 beta chain0.79HLA-DRAHLA class II histocompatibility antigen, DR alpha chain0.78IFITM3Interferon-induced transmembrane protein 30.78NKG7Protein NKG70.78FCER1GHigh affinity immunoglobulin epsilon receptor subunit gamma0.78IFITM1Interferon-induced transmembrane protein 10.76NPTNNeuroplastin0.76GYPCGlycophorin-C0.76GPR160Probable G-protein coupled receptor 1600.76HLA-DPB1HLA class II histocompatibility antigen, DP beta 1 chain0.75BRI3CT-BRI30.75SLC38A2Sodium-coupled neutral amino acid transporter 20.74C5AR1C5a anaphylatoxin chemotactic receptor 10.74CDIPTCDP-diacylglycerol--inositol 3-phosphatidyltransferase0.73TNFSF13BTumor necrosis factor ligand superfamily member 13b, soluble form0.73CSF3RGranulocyte colony-stimulating factor receptor0.73HLA-DPA1HLA class II histocompatibility antigen, DP alpha 1 chain0.71CD164Sialomucin core protein 240.71CD97CD97 antigen subunit beta0.7C3AR1C3a anaphylatoxin chemotactic receptor0.69P2RY8P2Y purinoceptor 80.68BSGBasigin0.68APLP2Amyloid-like protein 20.67TFRCTransferrin receptor protein 1, serum form0.67MGAMGlucoamylase0.67GYPAGlycophorin-A0.67TMED10Transmembrane emp24 domain-containing protein 100.66FCGRTIgG receptor FcRn large subunit p510.66CKAP4Cytoskeleton-associated protein 40.66DYSFDysferlin0.66SPPL2ASignal peptide peptidase-like 2A0.65LAMP2Lysosome-associated membrane glycoprotein 20.65SLC7A5Large neutral amino acids transporter small subunit 10.65TNFRSF1BTumor necrosis factor-binding protein 20.64TREM1Triggering receptor expressed on myeloid cells 10.64GPR183G-protein coupled receptor 1830.63SERINC3Serine incorporator 30.63CD58Lymphocyte function-associated antigen 30.63GYPBGlycophorin-B0.63RABAC1Prenylated Rab acceptor protein 10.62KCNH2Potassium voltage-gated channel subfamily H member 20.62FPR1fMet-Leu-Phe receptor0.62P2RY13P2Y purinoceptor 130.62CLEC5AC-type lectin domain family 5 member A0.62SLC7A7Y+L amino acid transporter 10.61MICBMHC class I polypeptide-related sequence B0.61CD300LFCMRF35-like molecule 10.61GJB6Gap junction beta-6 protein0.61ATP1A1Sodium / potassium-transporting ATPase subunit alpha-10.6PTGER4Prostaglandin E2 receptor EP4 subtype0.6CD8AT-cell surface glycoprotein CD8 alpha chain0.6PTGER2Prostaglandin E2 receptor EP2 subtype0.6GPR97Probable G-protein coupled receptor 970.6IMP3Signal peptide peptidase-like 2A0.59LAMP1Lysosome-associated membrane glycoprotein 10.59LILRB3Leukocyte immunoglobulin-like receptor subfamily B member 30.59GPR109BHydroxycarboxylic acid receptor 30.59SAT2Sodium-coupled neutral amino acid transporter 20.58GPR65Psychosine receptor0.58AMICA1Junctional adhesion molecule-like0.58PAG1Phosphoprotein associated with glycosphingolipid-enriched microdomains 10.58ENPP4Bis(5'-adenosyl)-triphosphatase ENPP40.57SLC40A1Solute carrier family 40 member 10.57OLR1Oxidized low-density lipoprotein receptor 1, soluble form0.57LRRC33Negative regulator of reactive oxygen species0.56IL7RInterleukin-7 receptor subunit alpha0.56LAIR1Leukocyte-associated immunoglobulin-like receptor 10.56ITM2CCT-BRI30.56GPR84G-protein coupled receptor 840.56SLC12A7Solute carrier family 12 member 70.55PTAFRPlatelet-activating factor receptor0.55CD33Myeloid cell surface antigen CD330.55SLC22A16Solute carrier family 22 member 160.55CCR7C-C chemokine receptor type 70.54TLR1Toll-like receptor 10.54TGOLN2Trans-Golgi network integral membrane protein 20.54YIPF3Protein YIPF3, 36 kDa form III0.54BST2Bone marrow stromal antigen 20.54MAGT1Magnesium transporter protein 10.54TMEM173Stimulator of interferon genes protein0.54ERMAPErythroid membrane-associated protein0.54CEACAM1Carcinoembryonic antigen-related cell adhesion molecule 10.54NIPA2Magnesium transporter NIPA20.53PECAM1Platelet endothelial cell adhesion molecule0.53CD1DAntigen-presenting glycoprotein CD1d0.53TMEM59Transmembrane protein 590.53NCKAP1LNck-associated protein 1-like0.53FASTumor necrosis factor receptor superfamily member 60.53IL6RInterleukin-6 receptor subunit alpha0.53TNFRSF1ATumor necrosis factor-binding protein 10.53KELKell blood group glycoprotein0.53TMEM149IGF-like family receptor 10.52SLC3A24F2 cell-surface antigen heavy chain0.52ORAI1Calcium release-activated calcium channel protein 10.52XKR8XK-related protein 8, processed form0.52C9orf46Plasminogen receptor (KT)0.52TMEM127Transmembrane protein 1270.52SLC2A1Solute carrier family 2, facilitated glucose transporter member 10.52FCGR1BHigh affinity immunoglobulin gamma Fc receptor IB0.52CXCR2C-X-C chemokine receptor type 20.52IL4RSoluble interleukin-4 receptor subunit alpha0.51HSD17B73-keto-steroid reductase0.51SEMA4DSemaphorin-4D0.51ZDHHC5Palmitoyltransferase ZDHHC50.51ADRB2Beta-2 adrenergic receptor0.51S1PR4Sphingosine 1-phosphate receptor 40.51PILRAPaired immunoglobulin-like type 2 receptor alpha0.51LTB4RLeukotriene B4 receptor 10.51SORT1Sortilin0.51SLCO4C1Solute carrier organic anion transporter family member 4C10.51ANO10Anoctamin-100.51CLSTN1CTF1-alpha0.5RHBDF2Inactive rhomboid protein 20.5CCR1C-C chemokine receptor type 10.5EPCAMEpithelial cell adhesion molecule0.5PNPLA2Patatin-like phospholipase domain-containing protein 20.49SLC12A6Solute carrier family 12 member 60.49SLC30A1Zinc transporter 10.49GPR27Probable G-protein coupled receptor 270.49EPORErythropoietin receptor0.49CD79AB-cell antigen receptor complex-associated protein alpha chain0.48HLA-DQB1HLA class II histocompatibility antigen, DQ beta 1 chain0.48HBP1Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 10.48ABCA7ATP-binding cassette sub-family A member 70.48RAG1AP1Sugar transporter SWEET10.48CD47Leukocyte surface antigen CD470.48CXCL16C-X-C motif chemokine 160.48SLC14A1Urea transporter 10.48TGFBR2TGF-beta receptor type-20.47LRBALipopolysaccharide-responsive and beige-like anchor protein0.47MFSD5Molybdate-anion transporter0.47RELTTumor necrosis factor receptor superfamily member 19L0.47ATP2B4Plasma membrane calcium-transporting ATPase 40.47FURINFurin0.47GAPTProtein GAPT0.47NFAM1NFAT activation molecule 10.47ATP2B1Plasma membrane calcium-transporting ATPase 10.46SLC26A11Sodium-independent sulfate anion transporter0.46STX4Syntaxin-40.46NAT1Sodium-coupled neutral amino acid transporter 30.46STIM1Stromal interaction molecule 10.46SLC39A4Zinc transporter ZIP40.46ESYT2Extended synaptotagmin-20.46TM7SF3Transmembrane 7 superfamily member 30.46SEMA4ASemaphorin-4A0.46CYBBCytochrome b-245 heavy chain0.46FCARImmunoglobulin alpha Fc receptor0.46GABBR1Gamma-aminobutyric acid type B receptor subunit 10.45CD53Leukocyte surface antigen CD530.45SIGLEC10Sialic acid-binding Ig-like lectin 100.45S1PR1Sphingosine 1-phosphate receptor 10.45BTN3A2Butyrophilin subfamily 3 member A20.45NOTCH2Notch 2 intracellular domain0.45PIK3IP1Phosphoinositide-3-kinase-interacting protein 10.45FAM168BMyelin-associated neurite-outgrowth inhibitor0.45LPAR2Lysophosphatidic acid receptor 20.45ATP1B3Sodium / potassium-transporting ATPase subunit beta-30.45FLVCR1Feline leukemia virus subgroup C receptor-related protein 10.45SECTM1Secreted and transmembrane protein 10.45SLC38A5Sodium-coupled neutral amino acid transporter 50.45GPR18N-arachidonyl glycine receptor0.44LMBR1LProtein LMBR1L0.44ABCC1Multidrug resistance-associated protein 10.44SLC22A18Solute carrier family 22 member 180.44CSF1RMacrophage colony-stimulating factor 1 receptor0.44EMR1EGF-like module-containing mucin-like hormone receptor-like 10.44FPR2N-formyl peptide receptor 20.44KITMast / stem cell growth factor receptor Kit0.44MS4A1B-lymphocyte antigen CD200.43MICAMHC class I polypeptide-related sequence A0.43GPR172ASolute carrier family 52, riboflavin transporter, member 20.43F11RJunctional adhesion molecule A0.43ADAM10Disintegrin and metalloproteinase domain-containing protein 100.43FAM38APiezo-type mechanosensitive ion channel component 10.43CD68Macrosialin0.43SLC26A6Solute carrier family 26 member 60.43MCOLN1Mucolipin-10.43SLCO3A1Solute carrier organic anion transporter family member 3A10.43PPAP2BLipid phosphate phosphohydrolase 30.43ICAM4Intercellular adhesion molecule 40.43CXCR1C-X-C chemokine receptor type 10.43CD300ACMRF35-like molecule 80.43RELL1RELT-like protein 10.43TAPBPLTapasin-related protein0.42FCGR2CLow affinity immunoglobulin gamma Fc region receptor II-c0.42SLC16A6Monocarboxylate transporter 70.42TMED1Transmembrane emp24 domain-containing protein 10.42CD86T-lymphocyte activation antigen CD860.42SLC16A3Monocarboxylate transporter 40.42SLC2A5Solute carrier family 2, facilitated glucose transporter member 50.42SLC29A1Equilibrative nucleoside transporter 10.42SLC16A14Monocarboxylate transporter 140.42P2RY2P2Y purinoceptor 20.42SUCNR1Succinate receptor 10.42BTN3A1Butyrophilin subfamily 3 member A10.41LAT2Linker for activation of T-cells family member 20.41PLXND1Plexin-D10.41ECE1Endothelin-converting enzyme 10.41TGFBR1TGF-beta receptor type-10.41CCRL2C-C chemokine receptor-like 20.41TFR2Transferrin receptor protein 20.41SLC44A1Choline transporter-like protein 10.41ITGA6Integrin alpha-6 light chain0.41PMP22Peripheral myelin protein 220.41LAX1Lymphocyte transmembrane adapter 10.4AMIGO2Amphoterin-induced protein 20.4SLC38A1Sodium-coupled neutral amino acid transporter 10.4SLC41A1Solute carrier family 41 member 10.4C2orf89Metalloprotease TIKI10.4ABCC10Multidrug resistance-associated protein 70.4CLDN15Claudin-150.4SLC39A6Zinc transporter ZIP60.4SLC16A5Monocarboxylate transporter 60.4TTYH3Protein tweety homolog 30.4ATP7ACopper-transporting ATPase 10.4COMTCatechol O-methyltransferase0.4SLC17A5Sialin0.4TMIGD2Transmembrane and immunoglobulin domain-containing protein 20.4CLEC7AC-type lectin domain family 7 member A0.4SLC31A1High affinity copper uptake protein 10.4LRRC4Leucine-rich repeat-containing protein 40.4P2RY10Putative P2Y purinoceptor 100.39ATP10DProbable phospholipid-transporting ATPase VD0.39BTN3A3Butyrophilin subfamily 3 member A30.39LIME1Lck-interacting transmembrane adapter 10.39TNFTumor necrosis factor, soluble form0.39PAQR8Membrane progestin receptor beta0.39OXER1Oxoeicosanoid receptor 10.39TRAT1T-cell receptor-associated transmembrane adapter 10.39GPBAR1G-protein coupled bile acid receptor 10.39SLC36A1Proton-coupled amino acid transporter 10.39PTPREReceptor-type tyrosine-protein phosphatase epsilon0.39PROM1Prominin-10.39CD74HLA class II histocompatibility antigen gamma chain0.38CNSTConsortin0.38TMEM49Vacuole membrane protein 10.38CLIC4Chloride intracellular channel protein 40.38NAALADL1N-acetylated-alpha-linked acidic dipeptidase-like protein0.38ANTXR2Anthrax toxin receptor 20.38FGFR1Fibroblast growth factor receptor 10.38IL1RAPInterleukin-1 receptor accessory protein0.38ATP1B2Sodium / potassium-transporting ATPase subunit beta-20.38ABCG2ATP-binding cassette sub-family G member 20.38CLEC12AC-type lectin domain family 12 member A0.38HLA-DQA1HLA class II histocompatibility antigen, DQ alpha 1 chain0.37B4GALT1Processed beta-1,4-galactosyltransferase 10.37CNNM3Metal transporter CNNM30.37ATP1B1Sodium / potassium-transporting ATPase subunit beta-10.37SLC39A1Zinc transporter ZIP10.37ATRNAttractin0.37CYSLTR1Cysteinyl leukotriene receptor 10.37TRPV2Transient receptor potential cation channel subfamily V member 20.37SLC27A1Long-chain fatty acid transport protein 10.37GPR171Probable G-protein coupled receptor 1710.37DAGLBSn1-specific diacylglycerol lipase beta0.37KCNQ1Potassium voltage-gated channel subfamily KQT member 10.37FZD6Frizzled-60.37CSF2RAGranulocyte-macrophage colony-stimulating factor receptor subunit alpha0.37PTH2RParathyroid hormone 2 receptor0.37MARCH1E3 ubiquitin-protein ligase MARCH10.36BACE2Beta-secretase 20.36CD5T-cell surface glycoprotein CD50.36TMEM219Insulin-like growth factor-binding protein 3 receptor0.36XPR1Xenotropic and polytropic retrovirus receptor 10.36CD1CT-cell surface glycoprotein CD1c0.36CNNM2Metal transporter CNNM20.36TMEM88Transmembrane protein 880.36ICOSInducible T-cell costimulator0.36KLRG1Killer cell lectin-like receptor subfamily G member 10.36LRP8Low-density lipoprotein receptor-related protein 80.36F2RProteinase-activated receptor 10.36HM13Minor histocompatibility antigen H130.36EMR2EGF-like module-containing mucin-like hormone receptor-like 20.36TREML1Trem-like transcript 1 protein0.36C17orf60Allergin-10.36GPR146Probable G-protein coupled receptor 1460.36SLAM F6SLAM family member 60.35SLC7A6Y+L amino acid transporter 20.35RELL2RELT-like protein 20.35LGR6Leucine-rich repeat-containing G-protein coupled receptor 60.35PANX1Pannexin-10.35C18orf1Low-density lipoprotein receptor class A domain-containing protein 40.35SLMAPSarcolemmal membrane-associated protein0.35CCR5C-C chemokine receptor type 50.35MUC1Mucin-1 subunit beta0.35EMR3EGF-like module-containing mucin-like hormone receptor-like 3 subunit beta0.35COL23A1Collagen alpha-1(XXIII) chain0.35OR2W3Olfactory receptor 2W30.35LNPEPLeucyl-cystinyl aminopeptidase, pregnancy serum form0.34PRR7Proline-rich protein 70.34NOTCH1Notch 1 intracellular domain0.34RFT1Solute carrier family 52, riboflavin transporter, member 10.34TNFRSF25Tumor necrosis factor receptor superfamily member 250.34ANO6Anoctamin-60.34AQP3Aquaporin-30.34ADAM9Disintegrin and metalloproteinase domain-containing protein 90.34INSRInsulin receptor subunit beta0.34FZD5Frizzled-50.34ERGPotassium voltage-gated channel subfamily H member 20.34MMENeprilysin0.34FCGR2BLow affinity immunoglobulin gamma Fc region receptor II-b0.33LSRLipolysis-stimulated lipoprotein receptor0.33DDR1Epithelial discoidin domain-containing receptor 10.33CNR2Cannabinoid receptor 20.33ATRAnthrax toxin receptor 10.33P2RY14P2Y purinoceptor 140.33VEZTVezatin0.33ALG10BPutative Dol-P-Glc:Glc(2)Man(9)GlcNAc(2)-PP-Dol alpha-1,2-glucosyltransferase0.33PAQR7Membrane progestin receptor alpha0.33FLT3LGFms-related tyrosine kinase 3 ligand0.33CD40LGCD40 ligand, soluble form0.33FCGR2ALow affinity immunoglobulin gamma Fc region receptor II-a0.33CLDN12Claudin-120.33GP6Platelet glycoprotein VI0.33EPHB4Ephrin type-B receptor 40.33SEMA4CSemaphorin-4C0.33CD300CCMRF35-like molecule 60.33PEAR1Platelet endothelial aggregation receptor 10.33FFAR2Free fatty acid receptor 20.33SLC2A6Solute carrier family 2, facilitated glucose transporter member 60.32TMEM150ATransmembrane protein 150A0.32ANO8Anoctamin-80.32CD200R1Cell surface glycoprotein CD200 receptor 10.32FCER1AHigh affinity immunoglobulin epsilon receptor subunit alpha0.32BEST1Bestrophin-10.32CLDN5Claudin-50.32SLC47A1Multidrug and toxin extrusion protein 10.32SLC5A10Sodium / glucose cotransporter 50.32CD40Tumor necrosis factor receptor superfamily member 50.31ANO9Anoctamin-90.31CLEC2DC-type lectin domain family 2 member D0.31VIPR1Vasoactive intestinal polypeptide receptor 10.31SLC16A7Monocarboxylate transporter 20.31UTS2RUrotensin-2 receptor0.31CLSTN3Calsyntenin-30.31GPR35G-protein coupled receptor 350.31SYT15Synaptotagmin-150.31FAM57AProtein FAM57A0.31CD8BT-cell surface glycoprotein CD8 beta chain0.31IL17RCInterleukin-17 receptor C0.31GLDNGliomedin0.31FZD2Frizzled-20.31KCNA3Potassium voltage-gated channel subfamily A member 30.3MGAGlucoamylase0.3GPR1G-protein coupled receptor 10.3IL6STInterleukin-6 receptor subunit beta0.3PCDHGB5Protocadherin gamma-B50.3OR1I1Olfactory receptor 1I10.3PTH1RParathyroid hormone / parathyroid hormone-related peptide receptor0.3NLGN2Neuroligin-20.3MMP24Processed matrix metalloproteinase-240.3CDH22Cadherin-220.3TNFRSF8Tumor necrosis factor receptor superfamily member 80.3CHRNGAcetylcholine receptor subunit gamma0.3PSEN1Presenilin-1 CTF120.3GPR114Probable G-protein coupled receptor 1140.3PLXNB2Plexin-B20.3CHRNA2Neuronal acetylcholine receptor subunit alpha-20.3GPR34Probable G-protein coupled receptor 340.3LPAR6Lysophosphatidic acid receptor 60.3ATP8A1Probable phospholipid-transporting ATPase IA0.3FZD1Frizzled-10.3CCR2C-C chemokine receptor type 20.3P2RY1P2Y purinoceptor 10.3SLC16A9Monocarboxylate transporter 90.3C20orf103Lysosome-associated membrane glycoprotein 50.3ADORA2BAdenosine receptor A2b0.3CLEC12BC-type lectin domain family 12 member B0.3FCRL3Fc receptor-like protein 30.29CD180CD180 antigen0.29TIGITT-cell immunoreceptor with Ig and ITIM domains0.29PPAP2ALipid phosphate phosphohydrolase 10.29ATP11CProbable phospholipid-transporting ATPase IG0.29TNFRSF17Tumor necrosis factor receptor superfamily member 170.29TNFSF12Tumor necrosis factor ligand superfamily member 12, secreted form0.29TBXA2RThromboxane A2 receptor0.29OR3A3Olfactory receptor 3A30.29GPR153Probable G-protein coupled receptor 1530.29ATP11AProbable phospholipid-transporting ATPase IH0.29LRFN1Leucine-rich repeat and fibronectin type III domain-containing protein 10.29OR51B2Olfactory receptor 51B20.29KCNS1Potassium voltage-gated channel subfamily S member 10.29OR12D2Olfactory receptor 12D20.29GRM4Metabotropic glutamate receptor 40.29NEO1Neogenin0.29DRD5D(1B) dopamine receptor0.29PLXDC1Plexin domain-containing protein 10.29GPR157Probable G-protein coupled receptor 1570.29CD300LBCMRF35-like molecule 70.29MARVELD1MARVEL domain-containing protein 10.29MFAP3Microfibril-associated glycoprotein 30.29CHRNB1Acetylcholine receptor subunit beta0.29PVRL2Poliovirus receptor-related protein 20.29F2RL1Proteinase-activated receptor 2, alternate cleaved 20.29GPR124G-protein coupled receptor 1240.29BACE1Beta-secretase 10.29C6orf105Androgen-dependent TFPI-regulating protein0.28CXCR3C-X-C chemokine receptor type 30.28IGSF8Immunoglobulin superfamily member 80.28ATP8B1Probable phospholipid-transporting ATPase IC0.28TP53I13Tumor protein p53-inducible protein 130.28MC1RMelanocyte-stimulating hormone receptor0.28CD84SLAM family member 50.28CALHM1Calcium homeostasis modulator protein 10.28CHRNA6Neuronal acetylcholine receptor subunit alpha-60.28CDH10Cadherin-100.28SLC16A1Monocarboxylate transporter 10.28GPRC5DG-protein coupled receptor family C group 5 member D0.28AGERAdvanced glycosylation end product-specific receptor0.28FASLGFasL intracellular domain0.28GPR56GPR56 C-terminal fragment0.28SIGLEC1Sialoadhesin0.28KIR2DL5AKiller cell immunoglobulin-like receptor 2DL5A0.28PLB1Lysophospholipase0.28CD200OX-2 membrane glycoprotein0.27ADAM28Disintegrin and metalloproteinase domain-containing protein 280.27SIT1Sodium- and chloride-dependent transporter XTRP30.27SLC23A2Solute carrier family 23 member 20.27CCR10C-C chemokine receptor type 100.27PRR4Processed poliovirus receptor-related protein 40.27GJD2Gap junction delta-2 protein0.27SLC2A8Solute carrier family 2, facilitated glucose transporter member 80.27CD209CD209 antigen0.27CD274Programmed cell death 1 ligand 10.27PROM2Prominin-20.27ATP6V0A2V-type proton ATPase 116 kDa subunit a isoform 20.27MPZMyelin protein P00.27TNFRSF18Tumor necrosis factor receptor superfamily member 180.27MFSD2AMajor facilitator superfamily domain-containing protein 2A0.27HEG1Protein HEG homolog 10.27OXTROxytocin receptor0.27CD99L2CD99 antigen-like protein 20.27LILRB4Leukocyte immunoglobulin-like receptor subfamily B member 40.27SMAGPSmall cell adhesion glycoprotein0.27OR51I2Olfactory receptor 51I20.27LY6G6DLymphocyte antigen 6 complex locus protein G6f0.27KCNQ4Potassium voltage-gated channel subfamily KQT member 40.27HRH2Histamine H2 receptor0.27SLC39A2Zinc transporter ZIP20.27CLDN10Claudin-100.27GPM6BNeuronal membrane glycoprotein M6-b0.27STEAP4Metalloreductase STEAP40.27APOLD1Apolipoprotein L domain-containing protein 10.27S1PR3Sphingosine 1-phosphate receptor 30.27SGMS2Phosphatidylcholine:ceramide cholinephosphotransferase 20.27KIR2DS5Killer cell immunoglobulin-like receptor 2DS50.27STARHeat-stable enterotoxin receptor0.27NIPA1Magnesium transporter NIPA10.26CNNM4Metal transporter CNNM40.26SLAMF1Signaling lymphocytic activation molecule0.26KIAA1919Sodium-dependent glucose transporter 10.26TLR6Toll-like receptor 60.26CRB3Protein crumbs homolog 30.26SLC12A9Solute carrier family 12 member 90.26GPR68Ovarian cancer G-protein coupled receptor 10.26OR51J1Olfactory receptor 51J10.26TREML2Trem-like transcript 2 protein0.26GPR176Probable G-protein coupled receptor 1760.26FLVCR2Feline leukemia virus subgroup C receptor-related protein 20.26LPAR1Lysophosphatidic acid receptor 10.26PANX2Pannexin-20.26SLC6A6Sodium- and chloride-dependent taurine transporter0.26PROKR2Prokineticin receptor 20.26CLDN9Claudin-90.26MYOFMyoferlin0.26LY6G6FLymphocyte antigen 6 complex locus protein G6f0.26ESAMEndothelial cell-selective adhesion molecule0.26NCR3Natural cytotoxicity triggering receptor 30.25HLA-DQB2HLA class II histocompatibility antigen, DQ beta 2 chain0.25SLC4A5Electrogenic sodium bicarbonate cotransporter 40.25P2RY4P2Y purinoceptor 40.25ABCB1Multidrug resistance protein 10.25SLC9A1Sodium / hydrogen exchanger 10.25CELSR2Cadherin EGF LAG seven-pass G-type receptor 20.25SYT8Synaptotagmin-80.25PCDHA9Protocadherin alpha-90.25TMEM204Transmembrane protein 2040.25PTPRJReceptor-type tyrosine-protein phosphatase eta0.25GRPRGastrin-releasing peptide receptor0.25SEMA6BSemaphorin-6B0.25CLCN5H(+) / Cl(-) exchange transporter 50.25GLRA2Glycine receptor subunit alpha-20.25PLVAPPlasmalemma vesicle-associated protein0.25ACVR1BActivin receptor type-1B0.25JAM3Junctional adhesion molecule C0.25LDLRAD3Low-density lipoprotein receptor class A domain-containing protein 30.25XGGlycoprotein Xg0.25SLC2A11Solute carrier family 2, facilitated glucose transporter member 110.24PCDH9Protocadherin-90.24VAMP5Vesicle-associated membrane protein 50.24CDHR2Cadherin-related family member 20.24DRD2D(2) dopamine receptor0.24LRIG2Leucine-rich repeats and immunoglobulin-like domains protein 20.24RAM P3Receptor activity-modifying protein 30.24SLC39A14Zinc transporter ZIP140.24STRA6Stimulated by retinoic acid gene 6 protein homolog0.24ADRA2CAlpha-2C adrenergic receptor0.24CLDN19Claudin-190.24CX3CR1CX3C chemokine receptor 10.24CD79BB-cell antigen receptor complex-associated protein beta chain0.24KIR2DL2Killer cell immunoglobulin-like receptor 2DL20.24CXCR7Atypical chemokine receptor 30.24OR5L2Olfactory receptor 5L20.24LRRC52Leucine-rich repeat-containing protein 520.24JPH1Junctophilin-10.24ADORA1Adenosine receptor A10.24GPRC5CG-protein coupled receptor family C group 5 member C0.24RETExtracellular cell-membrane anchored RET cadherin 120 kDa fragment0.24PVRPoliovirus receptor0.24ITGB3Integrin beta-30.24PTGIRProstacyclin receptor0.24LPHN1Latrophilin-10.24OR10J1Olfactory receptor 10J10.24MFAP3LMicrofibrillar-associated protein 3-like0.24GPNMBTransmembrane glycoprotein NMB0.24CELSR3Cadherin EGF LAG seven-pass G-type receptor 30.23CCR6C-C chemokine receptor-like 20.23DMPKMyotonin-protein kinase0.23UPK3BUroplakin-3b0.23OR1D2Olfactory receptor 1D20.23OR7D2Olfactory receptor 7D20.23ITGB1Integrin beta-10.23HRH3Histamine H3 receptor0.23GRIN2CGlutamate receptor ionotropic, NMDA 2C0.23KIR3DL1Killer cell immunoglobulin-like receptor 3DL10.23EPHB2Ephrin type-B receptor 20.23OR2S2Olfactory receptor 2S20.23KIR2DL4Killer cell immunoglobulin-like receptor 2DL40.23CNNM1Metal transporter CNNM10.23MARVELD2MARVEL domain-containing protein 20.23CXCR6C-X-C chemokine receptor type 60.23NOVPlexin-A10.23ABCB6ATP-binding cassette sub-family B member 6, mitochondrial0.23PVRL1Poliovirus receptor-related protein 10.23SLC46A2Thymic stromal cotransporter homolog0.23ADORA3Adenosine receptor A30.23GPR125Probable G-protein coupled receptor 1250.23CD22B-cell receptor CD220.22FZD3Frizzled-30.22LPAR5Lysophosphatidic acid receptor 50.22TMEM8BTransmembrane protein 8B0.22PLXNA1Plexin-A10.22NPFFR1Neuropeptide FF receptor 10.22SEZ6L2Seizure 6-like protein 20.22LRRTM2Leucine-rich repeat transmembrane neuronal protein 20.22SLC16A11Monocarboxylate transporter 110.22GRIK5Glutamate receptor ionotropic, kainate 50.22SYT6Synaptotagmin-60.22TMEM102Transmembrane protein 1020.22OR8B8Olfactory receptor 8B80.22GJB1Gap junction beta-1 protein0.22GRM6Metabotropic glutamate receptor 60.22C20orf54Solute carrier family 52, riboflavin transporter, member 30.22OR52D1Olfactory receptor 52D10.22SLC46A1Proton-coupled folate transporter0.22DSC2Desmocollin-20.22FAT1Protocadherin Fat 1, nuclear form0.22GCGRGlucagon receptor0.22POP1Blood vessel epicardial substance0.22CXADRCoxsackievirus and adenovirus receptor0.22ABCC6Multidrug resistance-associated protein 60.22GJA1Gap junction alpha-1 protein0.22CXCR5C-X-C chemokine receptor type 50.21ABCB4Multidrug resistance protein 30.21CTLA4Cytotoxic T-lymphocyte protein 40.21TRPV1Transient receptor potential cation channel subfamily V member 10.21MRGPRX4Mas-related G-protein coupled receptor member X40.21SIGLEC6Sialic acid-binding Ig-like lectin 60.21IL9RInterleukin-9 receptor0.21CHRNB2Neuronal acetylcholine receptor subunit beta-20.21PDGFRBPlatelet-derived growth factor receptor beta0.21TMPRSS11DTransmembrane protease serine 11D catalytic chain0.21CDH24Cadherin-240.21PRRT2Proline-rich transmembrane protein 20.21GALR3Galanin receptor type 30.21OR51I1Olfactory receptor 51I10.21PTPRUReceptor-type tyrosine-protein phosphatase U0.21LPAR4Lysophosphatidic acid receptor 40.21ZNRF3E3 ubiquitin-protein ligase ZNRF30.21P2RY6P2Y purinoceptor 60.21AGTR1Type-1 angiotensin II receptor0.21GPR182G-protein coupled receptor 1820.21PODXLPodocalyxin0.21BDKRB1B1 bradykinin receptor0.21DCHS1Protocadherin-160.21GRIN3BGlutamate receptor ionotropic, NMDA 3B0.21PTGDRProstaglandin D2 receptor0.21PVRL4Processed poliovirus receptor-related protein 40.21GPR77C5a anaphylatoxin chemotactic receptor 20.21PARM1Prostate androgen-regulated mucin-like protein 10.21OR10H1Olfactory receptor 10H10.21OR10D3Putative olfactory receptor 10D30.21TNFSF14Tumor necrosis factor ligand superfamily member 14, soluble form0.21FCRL5Fc receptor-like protein 50.2RNF43E3 ubiquitin-protein ligase RNF430.2AMIGO1Amphoterin-induced protein 10.2OR1F1Olfactory receptor 1F10.2SLCO4A1Solute carrier organic anion transporter family member 4A10.2TTYH2Protein tweety homolog 20.2GABRR2Gamma-aminobutyric acid receptor subunit rho-20.2GJD3Gap junction delta-3 protein0.2GRID1Glutamate receptor ionotropic, delta-10.2CLDN1Claudin-10.2SLC6A13Sodium- and chloride-dependent GABA transporter 20.2SLC30A8Zinc transporter 80.2KIR2DL3Killer cell immunoglobulin-like receptor 2DL30.2GPR78G-protein coupled receptor 780.2UPK2Uroplakin-20.2CLDN14Claudin-140.2EDAEctodysplasin-A, secreted form0.2PTGER1Prostaglandin E2 receptor EP1 subtype0.2TRPV5Transient receptor potential cation channel subfamily V member 50.2PRIMA1Proline-rich membrane anchor 10.2GJA9Gap junction alpha-9 protein0.2SLC7A3Cationic amino acid transporter 30.2SSTR2Somatostatin receptor type 20.2CD1AT-cell surface glycoprotein CD1a0.2SLC7A8Large neutral amino acids transporter small subunit 20.2CLIC6Chloride intracellular channel protein 60.2EPHA8Ephrin type-A receptor 80.2SLC20A2Sodium-dependent phosphate transporter 20.2SCNN1AAmiloride-sensitive sodium channel subunit alpha0.2OR51B6Olfactory receptor 51B60.2OR14J1Olfactory receptor 14J10.2OR10C1Olfactory receptor 10C10.2OPRL1Nociceptin receptor0.2CCR9C-C chemokine receptor type 90.2JPH4Junctophilin-40.2HTR1E5-hydroxytryptamine receptor 1E0.2MC3RMelanocortin receptor 30.2CD163L1Scavenger receptor cysteine-rich type 1 protein M1600.2SEZ6Seizure protein 6 homolog0.2PRSS8Prostasin heavy chain0.2CDH26Cadherin-like protein 260.2ODZ1Teneurin C-terminal-associated peptide0.2FGFR3Fibroblast growth factor receptor 30.2 EXAMPLE 1 - KNOCK OUT (KO) ON CD38 GENE & EXPRESSION OF ANTI-CD38 CARPresentation of the CD38 target -cyclic ADP ribose hydrolase

[0121] CD38 is a glycoprotein found on the surface of many immune cells, including multiple myeloma (MM) cells that express a high level of CD38 in a large majority of patients. CD38 is a validated target for MM as many studies have shown efficient killing of CD38+ MM cells from patients and CD38+ MM cell lines using anti-CD38 mAbs by CDC and ADCC (Ellis, J. H. K. et al, Journal of Immunology, 1995, 155 (2), 925-937). Daratumumab is a therapeutic human CD38 monoclonal antibody which 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).Expression of CD38 by T-cells

[0122] The CD38 expression by T cells after CD3 / CD28 beads and IL-2 stimulation was analyzed by FACS every 3-4 days during 17 days. It was observed that more than 90% T cells express between day 6 and day 17 after activation (Figure 10B).

[0123] Thus, in order to avoid killing of activated T cells by anti-CD38 CAR+ T cells CD38 surface expression in T cells needs to be prevented. This may be accomplished by the inactivation of the CD38 gene using TALE-nucleases. TALEN is a trademark owned by the applicant (Cellectis, 8 rue de la Croix Jarry, 75013 PARIS) designating customized format of TAL nucleases.Strategy for the CD38 knock-out (KO)

[0124] Heterodimeric TALE-nuclease targeting two 17-pb long sequences separated by a 13-pb spacer within the CD38 gene were designed and produced. Each half target is recognized by repeats of the half TALE-nucleases listed in the following Table 15 and Figure 10A.

[0125] The repeats sequence of the left TALEN for the CD38ex1_T2 target was NN-NI-NN-NN-NG-NN-NN-NN-NG-NG-NN-NN-HD-NN-NI-NG, and the one for the right TALEN was NN-NG-HD-HD-HD-HD-NN-HD-NI-NN-NG-NN-HD-HD-HD-NG. Table 15: Sequences of the tested CD38 target and TALENs for inactivation of the CD38 antigenNameTALEN L / RSEQ ID #Nucleic acid sequence or polypeptide sequenceCD38 targetN / A1TGAGGTGGGTTGGCGAC taaggcgcaccgg TGGGCACTGCGGGGACACD38e x1_T2-L1 TALENL2CD38e x1_T2-R1 TALENR3

[0126] Each TALE-nuclease construct was subcloned using restriction enzyme digestion in a mammalian expression vector under the control of the T7 promoter. mRNA encoding TALE-nuclease cleaving CD38 were synthesized from plasmids carrying the coding sequence downstream from the T7 promoter.

[0127] Purified T cells activated during 72 hours with anti CD3 / CD28 coated beads and recombinant IL-2 were transfected by electroporation (Cytopulse) with each of the 2 mRNAs (10µg each) encoding both half CD38ex1_T2 TALE-nucleases. To investigate, the CD38 KO, the percentage of CD38 negative T cells was assessed by flow cytometry at day 3, 6, 10 and 13 after TALEN mRNA transfection. It was observed that 15% of transfected T cells were CD38 deficient (Figure 10 C) and this deficiency was stable during 13 days after transfection.

[0128] In addition two alternative TALE-nucleases targeting the CD38 gene have been designed. Each half target is recognized by repeats of the half TALE-nucleases listed in the following Table 16 and Figure 10A. The repeats sequence of the left TALEN for the CD38ex1_T4 target was NG-NN-HD-NN-NI-NN-NG-NG-HD-NI-NN-HD-HD-HD-NN-NN-NG, and the one for the right TALEN was NG-NN-HD-NG-NN-HD-HD-NN-NN-HD-NG-HD-NG-HD-NG-Ni. The repeats sequence of the left TALEN for the CD38ex1_T5 target was NG-NN-NI-NG-HD-HD-NG-HD-NN-NG-HD-NN-NG-NN-NN-NG, and the one for the right TALEN was HD-NN-NI-NN-NN-NG-NN-NN-HD-NN-HD-HD-NI-NN-HD-Ni. Table 16: Sequences of two other CD38 targets and the corresponding TALENs for their inactivationNameTALEN L / RSEQ ID #Nucleic acid sequence or repeats sequenceCD38ex 1_T4 targetN / A4TGCGAGTTCAGCCCGGtgtccggggacaaacccTGCTGCCGGCTCTCTACD38ex 1_T4-L TALENL5 CD38ex 1_T4-R TALENR6CD38ex 1_T5 targetN / A7TGATCCTCGTCGTGGTgctcgcggtggtcgtccCGAGGTGGCGCCAGCACD38ex 1_T5-L TALENL8 CD38ex 1_T5-R TALENR9 Strategy for the expression of the CAR anti-CD38 Structure and composition of CARs anti-CD38

[0129] In Table 17 are presented VH and VL chain of scFv anti-CD38. SEQ ID NO:10-11 correspond to the humanized anti-CD38 antibody daratumumab (Genmab) and SEQ ID NO: 12-13 to the MOR202 (or MOR03087) such as described in the US 8,263,746B patent.

[0130] SEQ ID NO:14-20 and SEQ ID NO:21-26 correspond to the CDR sequence for respectively the VH chain (HCDR) and the VL chain (LCDR) such as described in the WO 2008 / 047242 application.

[0131] Table 17: Sequences of VH and VL chains of the scFv anti-CD38 antibodies daratumumab, MOR202 and of specific CDRs for VH and VL chains.

[0132] For the daratumumbab scFv 3 different CARs constructs (GMB005-V1&V2&V3) have been NameVH or VL chainSEQ ID #Polypeptide or nucleic acid sequenceDaratumumabVH10VL11MOR202 (or MOR03087)VH12VL13HCDR1-1VH14GFTFSSYYMNHCDR1-2VH15SYYMNHCDR2VH16GISGDPSNTYYADSVKGHCDR3VH17DLPLVYTGFAYHCDR4VH18DYWMQHCDR5VH19TIYPGDGDTGYAQKFKHCDR6VH20GDYYGSNSLDYLCDR1VL21SGDNLRHYYVYLCDR2VL22GDSKRPSLCDR3VL23QTYTGGASLLCDR4VL24KASQDVSTVVALCDR5VL25SASYRYILCDR6VL26QQHSPPYT designed such as presented in Figure 11A and their sequence displayed in the following Table 18. All three constructs share the same components, in terms of signal peptide (CD8α), GS linker (between the scFv VH and VL chains), transmembrane domain (TM), 4-1BB costimulatory domain, and CD3ζ activation domain (sequences displayed in the following Table 18). Their differences come from the choice of the hinge (Table 18): V1 : FcRlla hinge V2 : CD8a hinge V3 : IgG1 hinge Table 18: Polypeptide sequence of the 3 different structures of scFv daratumumab-based anti-CD38 CARs and of the individual components used Name of CARSEQ ID #CD8α-Signal peptide (SP)27MALPVTALLLPLALLLHAARPGS linker28GGGGSGGGGSGGGGSFCRIIα hinge29GLAVSTISSFFPPGYQCD8α hinge30IgG1 hinge31TM domain32IYIWAPLAGTCGVLLLSLVITLYC4-1 BB costimulatory domain33KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCD3ζ activation domain34GMB005-V1 CAR35GMB005-V2 CAR36GMB005-V3 CAR37 Screening

[0133] CD38 TALENs will be transfected at day 4 after activation. 3 days after the CD38 deficient cells will be sorted by negative selection and transfected 3 days after with anti-CD38 CAR mRNAs. The CAR molecules generated will then be screened for expression and degranulation activity toward target cell lines expression CD38 upon CAR mRNA transient transfection. Target cell lines expressing different expression levels of CD38 (Figure 11B) will be used for activity testing: U266 CD38+ and U266 CD38- obtained by magnetic separation using anti-CD38 microbeads L363, a multiple myeloma cell line expressing intermediate levels of CD38 Daudi, a cell line derived from Burkitt lymphoma expressing high levels of CD38 K562, a cell line CD38 negative cell line derived from chronic myelogenous leukemia.

[0134] This first screening will be followed by a second screening step in which a number of selected candidates will be tested for their ability to induce degranulation, IFNy release and specific cytotoxic activity towards the selected target cell lines. Candidate selection will then be narrowed and some candidates selected for lentivirus vector production and CAR activity will be assessed in CD38 KO T-cells stably expressing the CARs.EXAMPLE 2 ACTIVITY OF ANTI-CS1 CAR IN THE CONTEXT OF CS1 KOPresentation of CS1 target

[0135] Multiple myeloma (MM) is a B-cell malignancy characterized by the aberrant clonal expansion of plasma cells (PCs) within the bone marrow, with an estimated 21,700 new cases and 10,710 deaths from MM identified in the United States in 2012 (Siegel R, et al. Cancer J Clin 2012 62:10-29). In 2013, it has been estimated that 22,350 individuals will be newly diagnosed with MM in the United States and 10,710 people will die from it, accounting for 20% of the deaths from all hematologic malignancies. Despite the use of proteasome inhibitors and immune-modulating 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 novel therapeutic approaches are urgently needed.

[0136] The cell surface glycoprotein CS1 (also referred in the literature as SLAMF7, CD319 or CRACC - NCBI Reference Sequence: NP_067004.3) is highly and ubiquitously expressed on the surface of myeloma cells (Hsi ED, et al. Clin Cancer Res 2008 14:2775-84). CS1 is expressed at very low levels in the majority of immune effector cells, including natural killer (NK) cells, some 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). Notably, CS1 is negligibly expressed in human hematopoietic stem cells (Hsi ED, et al. Clin Cancer Res 2008 14:2775-84), which can be used for stem cell transplantation to treat hematologic malignancies, including MM. The functions of CS1 in MM remain incompletely understood, and it has been documented that CS1 may play a role in myeloma cell adhesion, clonogenic growth, and tumorigenicity (Benson DM Jr, et al. J Clin Oncol 2012 30:2013-5; Tai YT, et al. Blood 2009 113:4309-18).Structure of the CAR anti-CS1

[0137] The same structures V1, V2 and V3 are designed such as in the Example 1 for the anti-CD38 antigen target single-chain CAR, with the same components in terms of hinge, transmembrane domain, co-activation and transduction domains (such as depicted in the Figure 11A and sequences shown in Table 18).

[0138] In Table 19 are presented the VH and VL chains of scFv anti-CS1. SEQ ID NO:38-40-42-44-46 and SEQ ID NO:39-41-43-45-47 correspond to respectively the VH chain and the VL chain of the murine scFv Luc63, Luc90, Luc34, LucX1 and LucX2 .

[0139] In Table 20 are presented anti-CS1 CARs with the above scFv; these CARs being based on the versions V1, V2 and V3 of Figure 11A, wherein respectively the short FcERy hinge, the medium hinge CD8α hinge and the long IgG1 hinge are used. The underlined parts correspond to the scFv VH and VL chains bound by a linker. Table 19: Sequences of VH and VL chains of the scFv anti-CS1 antibodiesNameVH or VL chainSEQ ID NO :Polypeptide sequenceLuc63VH38VL39Luc90VH40VL41Luc34VH42VL43LucX1VH44VL45LucX2VH46VL47 Table 20: Polypeptide sequence of anti-CS1 CARs based on the V1, V2 and V3 versions in Figure 11A Name of CARSEQ ID #Polypeptide sequenceLuc63-V1 CAR48Luc63-V2 CAR49Luc63-V3 CAR50Luc90-V1 CAR51Luc90-V2 CAR52Luc90-V3 CAR53Luc34-V1 CAR54Luc34-V2 CAR55Luc34-V3 CAR56LucX1-V1 CAR57LucX1-V2 CAR58LucX1-V3 CAR59LucX2-V1 CAR60LucX2-V2 CAR61LucX2-V3 CAR62 Strategy for CAR CS1+ and KO CS1 engineering

[0140] CS1 is expressed at high levels in plasmacytoid cells from patients with Multiple Myeloma, making this an interesting target for CAR development. T-cells, especially the CD8 subset, express low levels of CS1, which is a drawback for T-cell CAR development, since they could be killed when expressing an anti-CS1 CAR.

[0141] In this example we assessed the activity of the Luc90-v2 CAR (sequence shown in Table 20) in human T-cells that were either mock transfected, or transfected with a TALEN targeting the CS1 (SLAMF7) gene, to see if the CAR activity was enhanced when the CS1 gene was disrupted in CAR+ T-cells. The course of the experiment is shown in the Figure 12.

[0142] T-cells were purified from buffy-coat samples and activated using CD3 / CD28-coated beads. Cells were co-transfected 72h after activation with 10 µg of mRNA encoding the T01_left TAL and 10 µg of the mRNA encoding the T01_right TAL. Sequences of the TALs are shown in the following Table 21 and the plasmid constructs (T01, T02 and T03) with the TAL repeats shown in Figure 13.

[0143] Figure 14 shows the target location for the TALs T01, T02 and T03 within the CS1 (SLAMF7) gene: T01 and T02 target the exon 1 (Figure 14A), whereas T03 targets the exon 2 (Figure 14B). Table 21: Sequences of the CS1 target and TALENs for its inactivationNameTALEN L / RSEQ ID #Nucleic acid sequenceTarget of T0163L64TGACTTCCAGAGAGCAAR65AACATGCCTCACCCTCATarget of T0266L67TTCCAGAGAGCAATATGR68TGCCTCACCCTCATCTATarget of T0369L70TTGACTCTATTGTCTGGR71CCTCTTGTCACCATACA

[0144] 3 days after TALEn transfection, cells were transduced with a recombinant lentiviral vector driving expression of the L90-v2 CAR off an EF1a promoter. The lentiviral vector is built in a way that CAR expression is coupled with BFP expression (Blue Fluorescent Protein) through a ribosomal skip peptide. The L90-v2 CAR is constituted by an extracellular binding domain recognizing the CS1 target (scFv L90) followed by hinge and transmembrane regions derived from the hCD8α protein. The intracellular portion of the molecule contains a 41BB-derived costimulatory domain, followed by the CD3γ signaling domain (sequences displayed in previous Table 18-19-20 for individual components, scFv and CAR sequences respectively).

[0145] Transduction efficiency was assessed 6 days after transduction by flow cytometry, by following BFP expression. Cells were also stained with anti-CD8 and anti-CS1 antibodies.Results CAR CS1+ expression

[0146] The results from Figure 16 show that the transduction efficiencies are higher in mock transfected cells than in cells that have been transfected with TALEn targeting the CS1 gene. This is probably due to specific cell killing of non-transduced CS1-expressing T-cells, while this population is not affected when the cells no longer express CS1 as a consequence of TALEN-driven gene disruption.

[0147] No significant differences in CS1 levels are observed at this timepoint between TALEN or mock transfected cells (negative control- transfection without plasmid), since CS1 levels decrease over time after initial activation of T-cells. On the other hand, a significant decrease in the % of CD8+ cells is observed in mock transfected CAR expressing cells compared to TALEN transfected CAR+ cells, indicating that a high proportion of CD8+ cells has been eliminated by the CAR+ T-cells.Cytotoxic activity assessment

[0148] The cytotoxic activity of these cells was evaluated 8 days after CAR transduction, by co-culturing the same amount of T-cells either with a cell line expressing CS1 (L363 cells) or a negative control cell line lacking expression of CS1 (MOLM13). The viability of the target cell lines was measured by flow cytometry 4h after starting cell co-cultures. The results shown in Figure 15A show reduced cell viability of CS1(+) cells when they were co-cultured with CAR+ T-cells, while no impact on CS1(-) cell viability was observed. The specific cell lysis was calculated using the flow cytometry data, and it was 2-times higher when T-cells have been transfected with TALEn targeting the CS1 gene prior to CAR transduction (Figure 15B). It should be considered that the impact might be even higher, since the amount of CAR+ T-cells present in the co-cultures is higher when the cells were mock transfected (see flow cytometry data from Figure 16). The results from the experiment are the following: for the Mock / NTD sample, the % of BFP+ cells is 0.1% and the amount of CD8+ cells is 53.9%; for the TALEn / NTD sample, the % of BFP+ cells is 0.2% and the amount of CD8+ cells is 49.5%; for the Mock / L90-2 sample, the % of BFP+ cells is 94% and the amount of CD8+ cells is 1.8%; for the TALEn / L90-2 sample, the % of BFP+ cells is 61% and the amount of CD8+ cells is 8.3%.

[0149] Transduction efficiencies are higher in mock transfected cells than in cells that have been transfected with TALEn targeting the CS1 gene (NTD: not transduced).Reactivation after transduction

[0150] In order to confirm that the CS1 gene has been disrupted in TALEn transfected T-cells, the different samples were reactivated with CD3 / CD28 beads at D11 after transduction. 72h after reactivation cells were stained with anti-CD8 and anti-CS1 antibodies and expression analyzed by flow cytometry.

[0151] Figure 17 shows the transduction efficiencies and CD8 / CS1 expression levels in each sample. As shown in the lower panel, an increase in CS1 levels upon re-activation is observed in mock transfected cells, while a low amount of cells are able to express CS1 in the TALEn transfected populations.

[0152] The results from the experiment are the following: for the Mock / NTD sample, the % of BFP+ cells is 0.01%, CS1 is expressed in 65.2% of cells, and the amount of CD8+ cells is 80.7%; for the TALEn / NTD sample, the % of BFP+ cells is 0.2%, the CS1 is expressed in 9.7% of cells and the amount of CD8+ cells is 78.8%; for the Mock / L90-2 sample, the % of BFP+ cells is 94%, the CS1 is expressed in 37.5% of cells and the amount of CD8+ cells is 16%. for the TALEn / L90-2 sample, the BFP intensity is 61%, the CS1 expression is 8.5% and the CD8 expression is 68.5%.

[0153] An increase in CS1 levels upon re-activation is observed in mock transfected cells, while a low amount of cells are able to express CS1 in the TALEn transfected populations.

[0154] Altogether, these results indicate that the CS1 gene is disrupted in TALEn transfected T-cells, and that this enhances the cytotoxic activity of anti-CS1 CAR+ cells, mainly by preserving the cytotoxic CD8+ T-cells.EXAMPLE 3: CD70 TARGETPresentation of CD70 target

[0155] 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 has a role in adaptive T cell responses, induces the proliferation of costimulated T-cells and enhances the generation of cytolytic T-cells. Its accession number is P32970 (Uniprot). Some studies such as in Schürch, C. et al. (J. Clin. Invest., 2012; doi:10.1172 / JCI45977) suggest that blocking CD27-CD70 interactions could help treat chronic myelogenous leukemia (CML).Strategy for CD70 KO

[0156] The same strategy for the KO of CD70 gene will be performed such as in Example 1 and Example 2. Heterodimeric TALE-nuclease targeting two 49-pb long sequences separated by a 15pb spacer within the CD70 gene and one TALE-nuclease targeting a 57-pb long sequence separated by a 23pb spacer were designed and produced. Each half target is recognized by repeats of the half TALE-nucleases listed in the following Table 22. Table 22: Sequences of the CD70 target and TALENs for its inactivationNameTALEN L / RSEQ ID #Nucleic acid sequence72TGGTCTTTTCTTCCAGTgggacgtagctgagcTGCAGCTGAATCACACATarget 1L73TGGTCTTTTCTTCCAGTTALEN 1R74TGCAGCTGAATCACACATarget 275TGGTGATCTGCCTCGTGgtgtgcatccagcgcTTCGCACAGGCTCAGCATALEN 2L76TGGTGATCTGCCTCGTGR77TTCGCACAGGCTCAGCATarget 378TALEN 3L79TGCGGGCTGCTTTGGTCR80CTGCCTCGTGGTGTGCA Strategy for the expression of anti-CD70 CAR

[0157] The same strategy for expressing a CAR anti-CD70 will be performed such as in Example 1 and in Example 2.

[0158] The same structures V1, V2 and V3 are designed such as in the Example 1-2 with the same components in terms of signal peptide, linker between the VH and VL chains, transmembrane domain, co-activation and transduction domains (general architectures shown in Figure 11A, and sequences for individual components shown in Table 18). Only the hinge differs between the 3 versions V1, V2 and V3, wherein respectively the short FcERy hinge, the medium hinge CD8α hinge and the long IgG1 hinge are used.

[0159] In Table 23 are presented VH and VL chain of scFv anti-CD70. SEQ ID NO:81-82, 85-86, 89-90 and SEQ ID NO:83-84,87-88,91-92 correspond to respectively the VH chain and the VL chain of the scFv Ab4, Ab8 from AMGEN and 1F6 from Seattle Genetics.

[0160] In Table 24 are presented the anti-CD70 CARs with the above scFv; these CARs being based on the versions V1, V2 and V3 according to Figure 11A, wherein respectively a short FcEy hinge, a medium hinge CD8 and a long IgG1 hinge are used. Table 23: Polynucleotide and nucleic acid sequences of VH and VL chains for the scFv anti-CD70 Ab4, Ab8 and 1F6 antibodiesNameVH or VL chainSEQ ID #Polypeptide and nucleic acid sequenceAb4VH8182VL8384Ab8VH8586VL87881F6VH8990VL9192 Table 24: Polypeptide sequences of anti-CD70 CARs based on the V1, V2 and V3 versions according to Figure 11A Name of CARSEQ ID NO:Polypeptide sequenceAb4-V1 CAR93Ab4-V2 CAR94Ab4-V3 CAR95Ab8-V1 CAR96Ab8-V2 CAR97Ab8-V3 CAR981F6 V1 CAR991F6 V2 CAR1001F6 V3 CAR101 REFERENCES

[0161] Bardenheuer, W., K. Lehmberg, et al. (2005). "Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells." Leukemia 19(12): 2281-8. Betts, M. R., J. M. Brenchley, et al. (2003). "Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation." J Immunol Methods 281(1-2): 65-78. Boch, J., H. Scholze, et al. (2009). "Breaking the code of DNA binding specificity of TAL-type III effectors." Science 326(5959): 1509-12. Brewin, J., C. Mancao, et al. (2009). "Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease." Blood 114(23): 4792-803. Cambier, J.C. (1995) "Antigen and Fc Receptor Signaling:The Awesome Power of the Immunoreceptor Tyrosine- I Based Activation Motif (ITAM)" The Journal of Immunology 155 (7) 3281-3285. Cong, L., F. A. Ran, et al. (2013). "Multiplex genome engineering using CRISPR / Cas systems." Science 339(6121): 819-23. Critchlow, S. E. and S. P. Jackson (1998). "DNA end-joining: from yeast to man." Trends Biochem Sci 23(10): 394-8. Dalgaard, J. Z., A. J. Klar, et al. (1997). "Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family." Nucleic Acids Res 25(22): 4626-38. Deltcheva, E., K. Chylinski, et al. (2011). "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Nature 471(7340): 602-7. Garneau, J. E., M. E. Dupuis, et al. (2010). "The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA." Nature 468(7320): 67-71. Gasiunas, G., R. Barrangou, et al. (2012). "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria." Proc Natl Acad Sci U S A 109(39): E2579-86. Hacke, K., J. A. Treger, et al. (2013). "Genetic modification of mouse bone marrow by lentiviral vector-mediated delivery of hypoxanthine-Guanine phosphoribosyltransferase short hairpin RNA confers chemoprotection against 6-thioguanine cytotoxicity." Transplant Proc 45(5): 2040-4. Jena, B., G. Dotti, et al. (2010). "Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor." Blood 116(7): 1035-44. Jinek, M., K. Chylinski, et al. (2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Science 337(6096): 816-21. Jonnalagadda, M., C. E. Brown, et al. (2013). "Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy." PLoS One 8(6): e65519. Kushman, M. E., S. L. Kabler, et al. (2007). "Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in stably transfected V79MZ cells co-expressing hCYP1A1." Carcinogenesis 28(1): 207-14. Lackner, G., N. Moebius, et al. (2011). "Complete genome sequence of Burkholderia rhizoxinica, an Endosymbiont of Rhizopus microsporus." J Bacteriol 193(3): 783-4. Ma, J. L., E. M. Kim, et al. (2003). "Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences." Mol Cell Biol 23(23): 8820-8. Mak, A. N., P. Bradley, et al. (2012). "The crystal structure of TAL effector PthXo1 bound to its DNA target." Science 335(6069): 716-9. Mali, P., L. Yang, et al. (2013). "RNA-guided human genome engineering via Cas9." Science 339(6121): 823-6. Metzger, H. et al. (1986) "The Receptor with High Affinity for Immunoglobulin E" Annual Review of Immunology. 4: 419-470 Moscou, M. J. and A. J. Bogdanove (2009). "A simple cipher governs DNA recognition by TAL effectors." Science 326(5959): 1501. Nivens, M. C., T. Felder, et al. (2004). "Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase." Cancer Chemother Pharmacol 53(2): 107-15. Park, T. S., S. A. Rosenberg, et al. (2011). "Treating cancer with genetically engineered T cells." Trends Biotechnol 29(11): 550-7. Sangiolo, D., M. Lesnikova, et al. (2007). "Lentiviral vector conferring resistance to mycophenolate mofetil and sensitivity to ganciclovir for in vivo T-cell selection." Gene Ther 14(21): 1549-54. Schweitzer, B. I., A. P. Dicker, et al. (1990). "Dihydrofolate reductase as a therapeutic target." Faseb J 4(8): 2441-52. Sugimoto, Y., S. Tsukahara, et al. (2003). "Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91." J Gene Med 5(5): 366-76. Takebe, N., S. C. Zhao, et al. (2001). "Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of the human aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reductase gene." Mol Ther 3(1): 88-96. Waldmann H. and Hale G. (2005) "CAMPATH: from concept to clinic". Phil. Trans. R. Soc. B 360: 1707-1711. Yam, P., M. Jensen, et al. (2006). "Ex vivo selection and expansion of cells based on expression of a mutated inosine monophosphate dehydrogenase 2 after HIV vector transduction: effects on lymphocytes, monocytes, and CD34+ stem cells." Mol Ther 14(2): 236-44. Zielske, S. P., J. S. Reese, et al. (2003). "In vivo selection of MGMT(P140K) lentivirus-transduced human NOD / SCID repopulating cells without pretransplant irradiation conditioning." J Clin Invest 112(10): 1561-70.

Claims

1. Use of a transcription activator-like effector (TALE)-nuclease specific for a target sequence within a gene encoding an antigen marker selected from CD38, CD70 and CS1 for inactivating the expression of said gene in a T-cell expressing a chimeric antigen receptor (CAR) specific for said antigen marker.

2. Use according to claim 1, wherein said TALE nuclease is specific for a target sequence within the gene encoding CD38 selected from SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7.

3. Use according to claim 1, wherein said TALE nuclease is specific for a target sequence within exon 1 or exon 2 of the gene encoding CS1 or which is specific for a target sequence within the gene encoding CS1 selected from SEQ ID NO: 63, SEQ ID NO: 66 and SEQ ID NO: 69.

4. Use according to claim 1, wherein said TALE nuclease is specific for a target sequence within the gene encoding CD70 selected from SEQ ID NO: 72, SEQ ID NO: 75 and SEQ ID NO: 78.

5. Use according to claim 1, wherein said TALE nuclease is specific for the gene encoding CD38 and comprises the sequences of SEQ ID NO: 2 and SEQ ID NO: 3, the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, or the sequences of SEQ ID NO: 8 and SEQ ID NO: 9.

6. An engineered T cell expressing a chimeric antigen receptor (CAR) specific for an antigen marker characterized in that a gene encoding said antigen marker selected from CD38, CS1 and CD70 is genetically inactivated through the expression in the T cell of a transcription activator-like effector (TALE)-nuclease able to target said gene.

7. The engineered T cell according to claim 6, wherein the TALE nuclease is specific for a target sequence within the gene encoding CD38 selected from SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7.

8. The engineered T cell according to claim 6, wherein the TALE nuclease is specific for a target sequence within the gene encoding CS1 selected from SEQ ID NO: 63, SEQ ID NO: 66 and SEQ ID NO: 69.

9. The engineered T cell according to claim 6, wherein the TALE nuclease is specific for a target sequence within the gene encoding CD70 selected from SEQ ID NO: 72, SEQ ID NO: 75 and SEQ ID NO: 78.

10. The engineered T cell according to claim 6, characterized in that a gene encoding CD38 is genetically inactivated through the expression in the T cell of a transcription activator-like effector (TALE)-nuclease able to target said gene and comprising the sequences of SEQ ID NO: 2 and SEQ ID NO: 3, the sequences of SEQ ID NO: 5 and SEQ ID NO: 6, or the sequences of SEQ ID NO: 8 and SEQ ID NO: 9.

11. The engineered T cell according to claim 6, characterized in that a gene encoding CS1 is genetically inactivated through the expression in the T cell of a transcription activator-like effector (TALE)-nuclease able to target said gene and comprising the sequences of SEQ ID NO: 64 and SEQ ID NO: 65, the sequences of SEQ ID NO: 67 and SEQ ID NO: 68, or the sequences of SEQ ID NO: 70 and 71, or characterized in that a gene encoding CD70 is genetically inactivated through the expression in the T cell of a transcription activator-like effector (TALE)-nuclease able to target said gene and comprising the sequences of SEQ ID NO: 73 and SEQ ID NO: 74, the sequences of SEQ ID NO: 76 and 77, or the sequences of SEQ ID NO: 79 and 80.

12. The T cell according to any one of claims 6 to 11, wherein said T cell further expresses a CAR which is directed towards any of the following antigens: CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, 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 mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1) and fibroblast associated protein (fap); or CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD38, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF17), an HIV-specific antigen (such as HIV gp120), an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, or an Influenza Virus-specific antigen.

13. The T cell according to any one of claims 6 to 12 further engineered by deleting genes encoding components of T-cell receptors (TCR) and / or HLA complex.

14. The T cell according to any one of claims 6 to 13 for use as a medicament.

15. The T cell according to any one of claims 6 to 14 for use in the treatment of cancer, infections or autoimmune disease.