Universal anti-CD22 chimeric antigen receptor engineered immune cells

Abstract

To provide efficient and safe treatments for their aggressive or refractory / relapsed forms of hematological cancers.SOLUTION: The present invention relates to an engineered immune cell endowed with a new CD22 Chimeric Antigen Receptor (CD22 CAR) with a deletion in the TRAC gene that is able to redirect the immune cell specificity and reactivity toward selected tumor cells. The engineered immune cells endowed with such CARs are particularly suited for treating relapsed refractory CD22 expressing cancers.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates generally to the field of immunotherapy, and more specifically to universal chimeric antigen receptor T cells specific for CD22 (UCART22) and chimeric antigen receptor (CAR) specific for cluster of differentiation 22 (CD22) (CAR CD22), which are engineered human primary immune cells containing at least one edited gene (preferably a gene encoding a TCR subunit or the CD52 gene), and methods for engineering said cells. The present invention further relates to UCART 22 for use in patients who may or may not be the original donors of cells ("allogeneic" or "autologous" CD22 CAR engineered primary human immune cells) as a treatment for relapsed and refractory hematological cancers. Cells expressing CD22 according to the present invention are particularly efficient and safe for immunotherapy, especially for aggressive or recurrent cancers. [Background technology]

[0002] 2. Background of the Invention Over 45,000 deaths were expected from blood cancers (non-Hodgkin's lymphoma, leukemia) in the United States in 2000 (Greenlee et al., CA Cancer J. Clin., 50:7-33 (2000)). The numbers published in 2014 were similar, and despite advances in treatments such as chemotherapy, the prognosis for such cancers remains essentially unchanged. (EK Mai, U Bertsch, J Durig, C Kunz, M Haenel, IW Blau, M Munder, A Jauch, B Schurich, T Hielscher, M Merz, B Huegle-Doerr, A Seckinger, D Hose, J Hillengass, MS Raab, K Neben, HW Lindemann, M Zeis, C Gerecke, IGH Schmidt-Wolf, K Weisel, C Scheid, H Salwender and H Goldschmidt. Phase III trial of bortezomib, cyclophosphamide and dexamethasone (VCD) versus bortezomib, doxorubicin and dexamethasone (PAd) in newly diagnosed myeloma. Leukemia (19 March 2015) | doi:10.1038 / leu.2015.80.

[0003] Unique among these novel investigational therapies for hematological cancers is the genetic modification of cells with cytolytic capabilities, such as T cells, by gene transfer of chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010).

[0004] CAR is a synthetic receptor that consists of a targeting moiety that is associated with one or more signaling intracellular domains in a single molecule or in multiple transmembrane domains that form multimers.In certain CARs, the binding moiety consists of the antigen-binding domain derived from a single chain antibody (scFv), which comprises the variable fragment of a monoclonal antibody linked by a linker.Binding moieties based on receptor or ligand domains have also been successfully used to create CARs.

[0005] Signaling domains from costimulatory molecules of the T cell receptor (TCR), as well as specific transmembrane and hinge domains, have been added to form second- and third-generation CARs, which have been successful in human clinical trials. In these studies, T cells from patients with blood ("liquid") cancers were redirected against malignant cells expressing, for example, CD19 or CD22 (June et al., 2011, Haso et al., 2013) and reinfused into the same patients. (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood. 2013 Feb 14;121(7):1165-74. doi: 10.1182 / blood-2012-06-438002. Epub 2012 Dec 14).

[0006] Methods that allow immune cells from one individual to be manipulated before reinfusion into the same individual (usually afflicted with cancer) are not well suited, particularly for aggressive forms of cancer where time may be a race. Moreover, such methods may be problematic or uncertain in patients with altered immune systems.

[0007] To mitigate this problem, immunotherapy using so-called “allogeneic” T cells expressing CARs (also known as universal or “off-the-shelf” T cells) has recently been implemented, and the first two patients treated with such cells are still in remission almost two years after treatment.

[0008] Nevertheless, there are still aspects of such therapy that can be improved, such as the efficiency of anti-T cell drug presence, the efficiency against escaping cancer cells, persistence, control measures, etc.In fact, it seems that cancer cells can escape from treatment and survive despite the persistence of redirected immunity in treated patients by downregulating the expression of the surface antigen recognized by CAR.In addition, one major adverse, sometimes fatal, effect observed in patients treated with either autologous or allogeneic T cells is cytokine release syndrome.

[0009] Therefore, there remains a need to develop efficient and safe treatments for these pathologies, especially for aggressive or refractory / recurrent forms of hematological cancers. Summary of the Invention

[0010] The inventors have now developed a novel, efficient therapy ("UCART22") comprising engineered primary human T cells carrying a deletion of at least the TRAC gene, endowed with a chimeric antigen receptor targeting CD22 and a safety marker that allows the number and, consequently, the activity of the cells to be controlled in vivo.

[0011] In a novel, efficient therapy comprising engineered primary human T cells having a deletion of at least the TRAC gene ("UCART22"), at least one additional deletion in the CD52, dCK, beta 2 microglobulin genes is contemplated.

[0012] These novel UCART22s are particularly efficient for adoptive transfer in patients suffering from CD22-mediated pathologies, regardless of whether the patient is the initial donor of immune cells and whether the patient is already under immune-affecting therapy.

[0013] The UCART 22 cells of the invention are resistant to at least one chemical or antibody drug commonly used to treat CD22-mediated pathologies, such as Campath and / or purine nucleotide analogs (PNAs). The UCART 22 cells of the invention can survive and be viable in the presence of said drugs used at doses that kill greater than 80% of the cells.

[0014] Significant and unexpected clinical benefits of engineered isolated primary immune cells UCART 22 are observed, including low cytokine release, no or minimal graft-versus-host disease, and still significant activity against refractory and relapsed hematologic cancer cells.

[0015] Other advantages are disclosed in this study. [The present invention 1001] A chimeric antigen receptor (CAR) specific for CD22, preferably expressed on the cell surface, of a safety-switched engineered human T cell receptor knockout (TCR KO) cell (UCART 22), wherein the anti-CD22 CAR (CD22 CAR) (i) the hinge domain from CD8 alpha; An antigen-binding domain specific for CD22, optionally with a leader sequence at least one extracellular domain comprising (ii) a transmembrane domain derived from CD8 alpha, and (iii) Intracellular signaling domain Including, the CD22 CAR has a polypeptide sequence having at least 80% identity to SEQ ID NO:15; The safety switch, - RQR8 transiently linked to the CD22 CAR by peptide 2A, or - at least two Rituximab mAb specific epitopes, or three Rituximab mAb specific epitopes, or three Rituximab mAb specific epitopes and a QBEN-10 mAb specific epitope, linked to the CD22 CAR, preferably located between the VH and hinge domains; including engineered human T cell receptor knockout (TCR KO) cells (UCART 22). [The present invention 1002] A polynucleotide sequence having at least 80% identity to SEQ ID NO:11 is inserted into the genome, Further included are inactivated TRAC genes having insertions, deletions, or mutations within SEQ ID NO: 18, wherein the inactivated TRAC genes have undetectable levels of T cell receptor (TCR) at the cell surface as measured by flow cytometry, and undetectable levels of off-site events as measured by guide sequence technology. UCART 22 of the present invention 1001. [The present invention 1003] UCART22 of the present invention 1002, wherein said anti-CD22 CAR is preferably inserted into the TRAC gene, preferably having SEQ ID NO:18. [The present invention 1004] UCART22 of any of 1001 to 1002, comprising another inactivated gene selected from a dCK gene, a B2M gene, and a CD52 gene, preferably a CD52 gene. [The present invention 1005] UCART22 of any of claims 1001 to 1004, wherein at least one additional gene is inactivated, and the gene is selected from genes encoding aryl hydrocarbon receptor (AHR), transforming growth factor beta receptor (TGF beta receptor), interleukin 10 receptor (IL-10 R), programmed cell death protein 1, and combinations thereof. [The present invention 1006] UCART22 of any of claims 1001 to 1005, comprising an additional scfv specific for any one of tumor associated surface antigens selected from CD19, CD20, CD30, a major histocompatibility complex (MHC) molecule, an immunoglobulin (Ig), CD3, CD5, CD34, CD79, preferably CD79b, CD138, B7-1 (CD80), BCMA (CD269, TNFRSF 17), FLT-3, or PAX5, preferably CD19. [The present invention 1007] The UCART22 of any of claims 1001 to 1006, wherein the CD22CAR further comprises an antigen-binding domain specific for CD19, or the UCART 22 further comprises a CD19CAR, preferably a CD19 CAR having at least 80% identity with SEQ ID NO:25 or SEQ ID NO:26. [The present invention 1008] UCART22 of any of claims 1001 to 1007, wherein the anti-CD22 CAR is a single-chain CAR or a multi-chain CAR. [The present invention 1009] UCART22 of any of the inventions 1006 or 1007, wherein the anti-CD19 CAR is a single-chain CAR or a multi-chain CAR. [The present invention 1010] A population of UCART cells comprising UCART22 of any one of 1001 to 1009 of the present invention. [The present invention 1011] A population of UCART cells of the present invention 1010, comprising UCART 19, preferably UCART19 expressing an anti-CD19 CAR comprising the sequence of SEQ ID NO:25 or SEQ ID NO:26 on the cell surface. [The present invention 1012] A kit comprising UCART 22 and UCART19 for sequential administration (at least once), or simultaneous administration, or sequential administration (at least once) followed by simultaneous administration, in a patient in need thereof. [The present invention 1013] The kit of the present invention 1012, wherein UCART 19 is first used at least once, twice, three times, four times, or several times, and then UCART 22 is used alone or together with UCART19. [The present invention 1014] The kit of the present invention 1012 or 1013, wherein UCART 22 is first used at least once, twice, three times, four times, or several times, and then UCART 19 is used alone or together with UCART 22. [The present invention 1015] The kit of any of claims 1012 to 1014, further comprising a lymphodepletion treatment administered prior to UCART. [The present invention 1016] Lymphocyte depletion is with fludarabine and cyclophosphamide, preferably fludarabine 25 mg / m on days -6 to -2. 2 Any of the kits of the present invention 1012-1015, achieved using iv x 5 doses and 1 dose of cyclophosphamide 60 mg / kg iv on day -5. [The present invention 1017] Any of the kits of 1012 to 1016, comprising at least one other UCART cell directed against a cancer antigen selected from CD79a, CD79b, CD20, CD30, CD52, CD40, CD80, CD86, CD74, and VEGF. [The present invention 1018] A pharmaceutical composition comprising a population of cells containing the UCART22 of any one of 1001 to 1009 or the UCART 22 of 1010 or 1011 of the present invention, and a pharma- ceutically acceptable excipient. [The present invention 1019] The pharmaceutical composition of the present invention 1018, further comprising a bryostatin, preferably bryostatin-1. [The present invention 1020] The pharmaceutical composition of the present invention 1018 or 1019 or the kit of any of the present inventions 1012 to 1017 for use thereof as a medicament for the prevention or treatment of a patient suffering from a CD22+ mediated cancer or a CD22+ mediated inflammatory disease. [The present invention 1021] A pharmaceutical composition according to any of claims 1018 or 1019 or a kit according to any of claims 1012 to 1017 for use thereof as a medicament for the prevention or treatment of a patient suffering from a CD19+ mediated cancer or a CD19+ mediated inflammatory disease. [The present invention 1022] A pharmaceutical composition of any of 1018 or 1019 or a kit of any of 1012 to 1017 for use thereof as a medicament for the prevention or treatment of a patient suffering from a CD19+CD22+ mediated cancer or a CD19+CD22+ mediated inflammatory disease. [The present invention 1023] A pharmaceutical composition of any of the present inventions 1018 or 1019 or a kit of any of the present inventions 1012 to 1017, for use thereof as in any of the present inventions 1020 or 1022, wherein the treatment of the patient includes a step of administering (re-dosing) the pharmaceutical composition at least twice to avoid recurrence / refractory development of the cancer. [The present invention 1024] CD22-mediated hematological cancers selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphoid carcinoma, acute myeloid leukemia, preferably lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia, A pharmaceutical composition of any of the present inventions 1018 or 1019 or a kit of any of the present inventions 1012 to 1017 for use thereof of any of the present inventions 1020 or 1022 for the treatment of CD22-expressing blood cancer selected from acute lymphoblastic carcinoma, acute myeloid leukemia, and Burkitt's lymphoma, acute lymphocytic carcinoma, acute myeloid leukemia, more preferably a relapsed or refractory CD22-expressing blood cancer, even more preferably an aggressive form of said CD22-related blood cancer. [The present invention 1025] CD19-mediated hematological cancers selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphoid carcinoma, acute myeloid leukemia, preferably lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic ... A pharmaceutical composition of any of the present inventions 1018 or 1019 or a kit of any of the present inventions 1012 to 1017 for use thereof of any of the present inventions 1021 or 1022 for the treatment of CD19-expressing blood cancer selected from acute lymphoblastic carcinoma, acute myeloid leukemia, and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, more preferably a relapsed or refractory CD19-expressing blood cancer, even more preferably an aggressive form of said CD19-related blood cancer. [The present invention 1026] A pharmaceutical composition according to any of the inventions 1018 or 1019 or a kit according to any of the inventions 1012 to 1017 for use thereof according to any of the inventions 1020 to 1023 for the treatment of relapsed or refractory onset B ALL, preferably as a pediatric indication. [The present invention 1027] The pharmaceutical composition of any of the inventions 1018 or 1019 or the kit of any of the inventions 1012 to 1017, for use thereof, of any of the inventions 1020 to 1026, wherein the treatment of the patient comprises administering at least one monoclonal antibody (mAb), preferably QBEN 10 and or rituximab, in the patient in a dose that allows contact of said UCART22 with at least one specific mAb.

[0016] This study provides the following: 1. A chimeric antigen receptor (CAR) specific for CD22, preferably expressed on the cell surface, safety-switched, engineered human T cell receptor knockout (TCR KO) cells (UCART 22), comprising: (i) the hinge domain from CD8 alpha; An antigen-binding domain specific for CD22, optionally with a leader sequence at least one extracellular domain comprising (ii) a transmembrane domain derived from CD8 alpha, and (iii) Intracellular signaling domain Including, the CD22 CAR has a polypeptide sequence having at least 80% identity to SEQ ID NO:15; The safety switch, - RQR8 transiently linked to the CD22 CAR by peptide 2A, or - at least two Rituximab mAb specific epitopes, or three Rituximab mAb specific epitopes, or three Rituximab mAb specific epitopes and a QBEN-10 mAb specific epitope, linked to the CD22 CAR, preferably located between the VH and hinge domains; including engineered human T cell receptor knockout (TCR KO) cells (UCART 22).

[0017] 2. A polynucleotide sequence having at least 80% identity to SEQ ID NO:11 is inserted into the genome; Further included are inactivated TRAC genes having insertions, deletions, or mutations within SEQ ID NO: 18, wherein the inactivated TRAC genes have undetectable levels of T cell receptor (TCR) at the cell surface as measured by flow cytometry, and undetectable levels of off-site events as measured by guide sequence technology. UCART 22 of embodiment 1.

[0018] UCART22 according to 2, wherein the anti-CD22 CAR is preferably inserted into the TRAC gene, preferably having SEQ ID NO:18.

[0019] 3. UCART22 according to any one of aspects 1-2, comprising another inactivated gene selected from the dCK gene, the B2M gene, the CD52 gene, preferably the CD52 gene.

[0020] 4. The UCART22 of any one of aspects 1-3, wherein at least one additional gene is inactivated, and the gene is selected from genes encoding aryl hydrocarbon receptor (AHR), transforming growth factor beta receptor (TGF beta receptor), interleukin 10 receptor (IL-10 R), programmed cell death protein 1, or a combination thereof.

[0021] 5. UCART22 according to any one of aspects 1 to 4, wherein the gene encoding beta 2 microglobulin (B2M) is inactivated.

[0022] 6. UCART22 according to any one of aspects 1 to 5, wherein the gene encoding the aryl hydrocarbon receptor (AHR) is inactivated.

[0023] 7. UCART22 according to any one of aspects 1 to 6, wherein the gene encoding transforming growth factor beta receptor) (TGF beta receptor) is inactivated.

[0024] 8. UCART22 according to any one of aspects 1 to 7, wherein the gene encoding interleukin-10 receptor (IL-10 R) is inactivated.

[0025] 9. UCART22 according to any one of aspects 1 to 8, wherein the gene encoding programmed cell death protein 1 (PD1) is inactivated.

[0026] 5. UCART22 according to any one of aspects 1-4, comprising an additional scfv specific for any one of a tumor associated surface antigen selected from CD19, CD20, CD30, a major histocompatibility complex (MHC) molecule, an immunoglobulin (Ig), CD3, CD5, CD34, CD79, preferably CD79b, CD138, B7-1 (CD80), BCMA (CD269, TNFRSF 17), FLT-3, or PAX5, preferably CD19.

[0027] 6. The UCART22 of any one of aspects 1-5, wherein the CD22CAR further comprises an antigen-binding domain specific for CD19, or the UCART 22 further comprises a CD19CAR, preferably a CD19 CAR having at least 80% identity to SEQ ID NO:25 or SEQ ID NO:26.

[0028] 7. UCART22 according to any one of aspects 1 to 5, wherein the anti-CD22 CAR is a single-chain CAR or a multi-chain CAR.

[0029] 8. UCART22 according to any one of aspects 5-6, wherein the anti-CD19 CAR is a single-chain CAR or a multi-chain CAR.

[0030] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD19.

[0031] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD20.

[0032] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD30.

[0033] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for a major histocompatibility complex (MHC) molecule.

[0034] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for immunoglobulin (Ig).

[0035] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD3.

[0036] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD5.

[0037] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD34.

[0038] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD79, preferably CD79b.

[0039] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD138.

[0040] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD80.

[0041] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for BCMA (CD269).

[0042] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for TNFRSF 17.

[0043] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for FLT-3.

[0044] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD19.

[0045] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD79a or CD79b.

[0046] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD20.

[0047] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD30.

[0048] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for a major histocompatibility complex (MHC) molecule.

[0049] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for immunoglobulin (Ig).

[0050] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD3.

[0051] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD5.

[0052] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD34.

[0053] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD138.

[0054] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD80.

[0055] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for BCMA (CD269).

[0056] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for TNFRSF 17.

[0057] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for FLT-3.

[0058] 9. A population of UCART cells, comprising UCART22 of any one of aspects 1 to 8.

[0059] 10. The population of UCART cells of embodiment 9, comprising UCART 19, preferably UCART19 expressing an anti-CD19 CAR comprising the sequence of SEQ ID NO:25 or SEQ ID NO:26 on the cell surface.

[0060] 11. A kit comprising UCART 22 and UCART19 for sequential administration (at least once), or simultaneous administration, or sequential administration (at least once) followed by simultaneous administration, in a patient in need thereof.

[0061] 12. The kit of embodiment 11, wherein UCART 19 is first used at least once, twice, three times, four times, or several times, and then UCART 22 is used alone or together with UCART19.

[0062] 13. The kit of embodiment 11 or 12, wherein UCART 22 is first used at least once, twice, three times, four times, or several times, and then UCART 19 is used alone or together with UCART22.

[0063] 14. The kit according to any one of aspects 11 to 13, further comprising a lymphodepletion treatment administered prior to UCART.

[0064] 15. Lymphocyte depletion is treated with fludarabine and cyclophosphamide, preferably fludarabine 25 mg / m on days -6 to -2. 2 iv x 5 doses and one dose of cyclophosphamide 60 mg / kg iv on day -5.

[0065] 16. The kit according to any one of aspects 11 to 15, comprising at least one other UCART cell directed against a cancer antigen selected from CD79a, CD79b, CD20, CD30, CD52, CD40, CD80, CD86, CD74, VEGF.

[0066] 17. A pharmaceutical composition comprising a population of cells comprising UCART22 according to any one of aspects 1 to 8, or said UCART 22 according to aspect 9 or 10, and a pharma- ceutically acceptable excipient.

[0067] 18. The pharmaceutical composition according to embodiment 17, further comprising a bryostatin, preferably bryostatin-1.

[0068] 19. A pharmaceutical composition according to embodiment 17 or 18 or a kit according to any one of embodiments 11 to 16, for its use as a medicament for the prevention or treatment of a patient suffering from a CD22+ mediated cancer or a CD22+ mediated inflammatory disease.

[0069] 20. A pharmaceutical composition according to any one of aspects 17 or 18 or a kit according to any one of aspects 11 to 16 for its use as a medicament for the prevention or treatment of a patient suffering from a CD19+ mediated cancer or a CD19+ mediated inflammatory disease.

[0070] 21. A pharmaceutical composition according to any one of aspects 17 or 18 or a kit according to any one of aspects 11 to 16 for its use as a medicament for the prevention or treatment of a patient suffering from a CD19+CD22+ mediated cancer or a CD19+CD22+ mediated inflammatory disease.

[0071] 22. The pharmaceutical composition according to any one of aspects 17 or 18 or the kit according to any one of aspects 11 to 16, for its use according to any one of aspects 19 or 21, wherein the treatment of the patient comprises a step of administering (re-dosing) the pharmaceutical composition at least twice to avoid recurrence / refractory occurrence of the cancer.

[0072] 23. A pharmaceutical composition according to any one of aspects 17 or 18 or a kit according to any one of aspects 11 to 16 for its use according to any one of aspects 19 or 21 for the treatment of a CD22-mediated hematological cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphoid carcinoma, acute myeloid leukemia, preferably a CD22-expressing hematological cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphoid carcinoma, acute myeloid leukemia, more preferably a relapsed refractory CD22-expressing hematological cancer, even more preferably an aggressive form of said CD22-related hematological cancer.

[0073] 24. A pharmaceutical composition according to any one of aspects 17 or 18 or a kit according to any one of aspects 11 to 16 for its use according to any one of aspects 20 or 21 for the treatment of a CD19-mediated hematological cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphoid carcinoma, acute myeloid leukemia, preferably a CD19-expressing hematological cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphoid carcinoma, acute myeloid leukemia, more preferably a relapsed refractory CD19-expressing hematological cancer, even more preferably an aggressive form of said CD19-related hematological cancer.

[0074] 25. A pharmaceutical composition according to any one of aspects 17 or 18 or a kit according to any one of aspects 11 to 16 for its use according to any one of aspects 19 to 22 for the treatment of relapsed or refractory manifestations of B ALL, preferably as a pediatric indication.

[0075] 26. A pharmaceutical composition according to any one of aspects 17 or 18 or a kit according to any one of aspects 11 to 16, for its use according to any one of aspects 19 to 25, wherein treatment of a patient comprises administering in the patient at least one monoclonal antibody (mAb), preferably QBEN 10 and or rituximab, in a dose allowing contact of said UCART22 with the at least one specific mAb.

[0076] Aspects of CD22 CAR The present invention provides an anti-CD22 CAR comprising a hinge domain selected from FcRIIIα, CD8 alpha, IgG1, IgG4, and PD1, preferably CD8 alpha or IgG4.

[0077] An anti-CD22 CAR as described above, wherein the CD22-specific scfv comprises a VH and a VL linked to each other by a linker L1, preferably a linker comprising 1 to 3 "GGGGS" motifs, more preferably 1 GGGGS" motif.

[0078] An anti-CD22 CAR according to any one of the above, wherein said scfv specific for CD22 is linked to the transmembrane domain by a hinge selected from FcRIIIα, CD8 alpha, IgG1 derived, preferably CD8 alpha derived hinge.

[0079] The anti-CD22 CAR of any one of the preceding claims, comprising an intracellular domain, wherein the intracellular domain comprises a CD3 zeta signaling domain and a 4-1BB signaling domain.

[0080] An anti-CD22 CAR as described in any one of the above, wherein the anti-CD22 CAR comprises at least one, preferably two, three or four, monoclonal antibody (mAb) specific epitopes, preferably two, inserted into the linker L and / or into the hinge of a CD22 specific scfv.

[0081] The mAb specific epitope is The anti-CD22 CAR described in any one of the above, which is a polypeptide selected from TIFF0007634045000001.tif35168.

[0082] The present invention provides a polynucleotide encoding any one of the anti-CD22 CARs described above, or a polynucleotide having at least 80% identity with the polynucleotide encoding the anti-CD22 CAR, or a vector comprising a polynucleotide according to any one of the above polynucleotides.As disclosed herein below, in said UCART22, other genes are edited, in particular dCK gene, B2M gene, CD52 gene.

[0083] UCART provided Provided is UCART22 as described above, wherein at least one additional gene has been edited or engineered, the gene being selected from genes encoding aryl hydrocarbon receptor (AHR), transforming growth factor beta receptor (TGF beta receptor), interleukin 10 receptor (IL-10 R), programmed cell death protein 1, or combinations thereof.

[0084] UCART22 as described above, wherein at least one additional gene contains a mutation, deletion or insertion that inactivates its activity and / or expression, said gene being selected from genes encoding aryl hydrocarbon receptor (AHR), transforming growth factor beta receptor (TGF beta receptor), interleukin 10 receptor (IL-10 R), programmed cell death protein 1, or combinations thereof.

[0085] UCART22 as described above, wherein at least one additional gene contains a mutation, deletion or insertion that inactivates its activity and / or expression, said gene being selected from genes encoding aryl hydrocarbon receptor (AHR), transforming growth factor beta receptor) (TGF beta receptor), interleukin 10 receptor (IL-10 R), or combinations thereof.

[0086] UCART22 as described above, wherein at least one additional gene contains a mutation, deletion or insertion that inactivates its activity and / or expression, and the gene is selected from genes encoding transforming growth factor beta receptor (TGF beta receptor), interleukin 10 receptor (IL-10 R), programmed cell death protein 1, or combinations thereof.

[0087] UCART22 as described above, wherein at least one additional gene contains a mutation, deletion or insertion that inactivates its activity and / or expression, and the gene is selected from genes encoding aryl hydrocarbon receptor (AHR), interleukin 10 receptor (IL-10 R), programmed cell death protein 1, or combinations thereof.

[0088] UCART22 as described above, wherein at least one additional gene contains a mutation, deletion or insertion that inactivates its activity and / or expression, said gene being selected from genes encoding aryl hydrocarbon receptor (AHR), transforming growth factor beta receptor (TGF beta receptor), programmed cell death protein 1, or combinations thereof.

[0089] The genes into which the CAR can be inserted are shown in Table 9 below.

[0090] Table 9: List of genes encoding immune checkpoint proteins that can be inactivated according to the present invention in the CD22 CAR engineered T cells of the present invention. TIFF0007634045000002.tif156140 The bolded characters are preferred genes to be inactivated in the UCART of the present invention.

[0091] An anti-CD22 CAR (UCART22) as described above, comprising any one of the above, and a polynucleotide encoding said anti-CD22 CAR (UCART22) inserted into the genome, into the CD25 gene.

[0092] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR or a multi-chain CAR.

[0093] UCART22 of any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for any one of tumor associated surface antigens selected from CD19, CD20, CD30, a major histocompatibility complex (MHC) molecule, an immunoglobulin (Ig), CD3, CD5, CD34, CD79, preferably CD79b, CD138, B7-1 (CD80), BCMA (CD269, TNFRSF 17), FLT-3, or PAX5, preferably CD19.

[0094] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD19.

[0095] UCART22 according to any one of the above, expressing an anti-CD22 CAR and an anti-CD19 CAR, preferably of SEQ ID NO:94 or of SEQ ID NO:95.

[0096] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD20.

[0097] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD30.

[0098] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for a major histocompatibility complex (MHC) molecule.

[0099] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for immunoglobulin (Ig).

[0100] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD3.

[0101] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD5.

[0102] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD34.

[0103] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD79, preferably CD79b.

[0104] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD138.

[0105] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for CD80.

[0106] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for BCMA (CD269).

[0107] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for TNFRSF 17.

[0108] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scfv specific for FLT-3.

[0109] UCART22 according to any one of the preceding claims, wherein the anti-CD22 CAR is a multi-chain CAR.

[0110] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD19.

[0111] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD79a or CD79b.

[0112] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain (mc) CAR comprising an additional scfv specific for CD19.

[0113] UCART22 according to any one of the above, expressing an anti-CD22 mcCAR and an anti-CD19 CAR, preferably of SEQ ID NO:94 or of SEQ ID NO:95.

[0114] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD20.

[0115] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD30.

[0116] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for a major histocompatibility complex (MHC) molecule.

[0117] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for immunoglobulin (Ig).

[0118] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD3.

[0119] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD5.

[0120] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD34.

[0121] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD138.

[0122] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for CD80.

[0123] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for BCMA (CD269).

[0124] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for TNFRSF 17.

[0125] UCART22 according to any one of the above, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scfv specific for FLT-3.

[0126] A population of cells comprising UCART22 of any one of the above.

[0127] A population of cells comprising UCART22 as described in any one of the above, wherein the cells expressing the anti-CD22 CAR further express an anti-CD19 CAR, preferably wherein the anti-CD19 CAR comprises the sequence of SEQ ID NO:25 or SEQ ID NO:26 at the cell surface.

[0128] The CD22 CAR T cells (UCART22), optionally in combination with a protein kinase C inhibitor such as bryostatin 1, as a pharmaceutical composition of the present invention, are particularly useful for the treatment of CLL, ALL, multiple myeloma (MM), blastic plasmacytoid dendritic cell neoplasm (BPDCN), in particular refractory / relapsed ALL, refractory / relapsed CLL, and or aggressive forms of these diseases, more preferably refractory or relapsed B-ALL.

[0129] The CD22 CAR T cells (UCART22), derived from m971, optionally in combination with a protein kinase C inhibitor such as bryostatin 1, as a pharmaceutical composition of the present invention, are particularly useful for the treatment of CLL, ALL, multiple myeloma (MM), blastic plasmacytoid dendritic cell neoplasm (BPDCN), in particular refractory / relapsed ALL, refractory / relapsed CLL, and or aggressive forms of these diseases, more preferably refractory or relapsed B-ALL.

[0130] The engineered immune cells of the present invention not only exhibit high levels of in vivo activity against malignant cells, less cytokine release, but also their numbers and activity are controlled, conferring safety and efficacy for immunotherapy.

[0131] The present invention provides engineered human T cell receptor knockout (TCR KO) cells (UCART 22) endowed with a chimeric antigen receptor specific for CD22 (CD22 CAR), preferably expressed on the cell surface, wherein the CD22 CAR comprises: (i) the hinge domain from CD8 alpha; An antigen-binding domain specific for CD22, optionally with a leader sequence at least one extracellular domain comprising (ii) a transmembrane domain derived from CD8 alpha, and (iii) Intracellular signaling domain Including, The UCART22 contains a deletion in at least one additional edited gene, preferably the CD52 gene.

[0132] The present invention also provides a UCART CD22 as described above, wherein the antigen binding domain comprises a scfv specific for CD22 derived from the m917 antibody (m971), preferably said scfv comprises a sequence selected from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, more preferably a sequence from SEQ ID NO:15.

[0133] In one aspect, the present invention also provides an engineered human T cell receptor knockout (TCR KO) cell (UCART 22) endowed with a chimeric antigen receptor specific for CD22 (CD22 CAR), preferably expressed on the cell surface, wherein the CD22 CAR comprises: (i) the hinge domain from CD8 alpha; An antigen-binding domain specific for CD22, optionally with a leader sequence at least one extracellular domain comprising (ii) a transmembrane domain derived from CD8 alpha, and (iii) Intracellular signaling domain Including, The UCART22 comprises a deletion in at least one additional edited gene, preferably the CD52 gene; and wherein the antigen-binding domain comprises an scfv specific for the distal portion of CD22, and the CAR has SEQ ID NO:20.

[0134] The following array: The UCART CD22 described above, comprising TIFF0007634045000003.tif27152 or having 80-99% homology with each of said sequences.

[0135] The present invention also provides a UCART 22 as described above comprising a polypeptide of SEQ ID NO15 and a safety switch, the safety switch comprising: RQR8 transiently linked to the CD22 CAR by peptide 2A, or At least two Rituximab mAb specific epitopes, preferably three Rituximab mAb specific epitopes, more preferably three Rituximab mAb specific epitopes and a QBEN-10 mAb specific epitope linked to the CD22 CAR. [The CD22 CAR allows for both selection and / or depletion of immunocytes.]

[0136] The present invention also provides UCART 22 as described above, which comprises at least one additional edited gene, preferably a deletion in the CD52 gene or in the dCK gene or a deletion in the beta 2 microglobulin gene or in the CTIIA gene, even more preferably a deletion in the CD52 gene, and / or an insertion in the HIF-1 alpha gene, which confers resistance to hypoxia.

[0137] The present invention provides UCART 22, as described above, which contains a deletion in the CD52 gene, in combination with alemtuzumab.

[0138] The present invention also provides UCART 22 as described above, wherein said CD22 CAR is expressed under hypoxia (low O2 concentration less than 5% O2, preferably less than 1% O2).

[0139] The present invention also provides UCART 22 as described above, comprising a polynucleotide encoding a polypeptide comprising a CD22 CAR and a safety switch. In a preferred embodiment, the present invention provides UCART 22 as described above, comprising the polynucleotides of SEQ ID NO:22 and SEQ ID NO:18 in the same gene, preferably in the TRAC gene.

[0140] The present invention also provides UCART 22 as described above, comprising a polynucleotide encoding a polypeptide comprising a CD22 CAR and a safety switch. In a preferred embodiment, the present invention provides UCART 22 as described above, comprising the polynucleotides of SEQ ID NO:20 and SEQ ID NO:18 in the same gene, preferably in the TRAC gene.

[0141] The present invention provides a population of cells comprising UCART 22 as described above.

[0142] The present invention provides a pharmaceutical composition comprising any of UCART 22 as described above or any of the above populations of cells comprising said UCART 22, and a pharma- ceutically acceptable excipient.

[0143] In certain embodiments, a pharmaceutical composition is provided that includes UCART 22 according to the invention or a population of cells comprising said UCART 22, in combination with UCART CD19, and a pharma- ceutically acceptable excipient. UCART 19 and UCART 22 may be given simultaneously, contemporaneously, or sequentially, beginning with UCART 19 or UCART 22, as a function of CD19 and CD22 expression levels in the patient's cancer cells.

[0144] In certain embodiments, the present invention provides a pharmaceutical composition comprising UCART 22 / 19 or UCART 19 / 22 (bispecific CAR single chain or multi-chain CAR or cells expressing both CD19CAR and CD22CAR) and a pharma- ceutical acceptable excipient.The present invention provides a pharmaceutical composition as described above, further comprising a compound of bryostatin family, preferably bryostatin 1.

[0145] The present invention provides a therapeutically effective amount of UCART 22 as described above, or a pharmaceutical composition comprising said UCART 22 as described above, for use as a medicament for treating a patient.

[0146] The invention provides a therapeutically effective amount of UCART 22 as described above, or a pharmaceutical composition as described above, for use as a medicament for treating a patient, wherein treatment of the patient comprises administering (redosing) said therapeutically effective amount of UCART CD22 or said pharmaceutical composition at least twice to avoid relapse.

[0147] In certain embodiments, the invention provides UCART CD22 as described above, or a pharmaceutical composition as described above, for use as a medicament for treating a patient, wherein treatment of the patient comprises administering at least two doses of UCART 22 as described above, or a pharmaceutical composition as described above, to avoid relapse.

[0148] The present invention provides a therapeutically effective amount of UCART 22 as described above, or a pharmaceutical composition as described above, for use as a medicament for treating a patient as described above, for the treatment of a CD22-associated pathology, preferably a CD22-associated B-cell malignancy (e.g., a CD22-associated hematological cancer).

[0149] In certain embodiments, the present invention provides: A therapeutically effective amount of UCART 22 as described above or a pharmaceutical composition as described above for use as a medicament for the treatment of hematological cancer, such as hematological cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphocytic carcinoma, acute myeloid leukemia, preferably a CD22-expressing hematological cancer, more preferably a relapsed refractory CD22-expressing hematological cancer, and even more preferably an aggressive form of said CD22-related hematological cancer.

[0150] Preferably, UCART22 is used to treat patients with relapsed / refractory B ALL.

[0151] The present invention provides a therapeutically effective amount of UCART 22 as described above or a pharmaceutical composition as described above for use as a medicament to treat a patient, wherein the patient is suffering from a cancer selected from the following: alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, Esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, mast cell tumor, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer.

[0152] The present invention provides: 1. An engineered human T cell receptor knockout (TCR KO) cell (UCART 22) endowed with a chimeric antigen receptor (CAR) specific for CD22, preferably expressed on the cell surface, wherein the anti-CD22 CAR comprises: (i) the hinge domain from CD8 alpha; An antigen-binding domain specific for CD22, optionally with a leader sequence at least one extracellular domain comprising (ii) a transmembrane domain derived from CD8 alpha, and (iii) Intracellular signaling domain Including, An engineered human T cell receptor knockout (TCR KO) cell (UCART 22), wherein the UCART22 comprises at least one additional edited gene, preferably a CD52 inactivation gene, a dCK inactivation gene, a beta 2 microglobulin inactivation gene, or an inserted HIF-1 alpha gene.

[0153] 2. A UCART CD22 according to 1, wherein the antigen binding domain comprises a scfv specific for CD22, said scfv being derived from the m917 antibody (m971), preferably said scfv comprising a sequence selected from SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or from SEQ ID NO:15, more preferably the sequence from SEQ ID NO:15.

[0154] 3. A polypeptide of SEQ ID NO15 and a safety switch, the safety switch comprising: - RQR8 transiently linked to CD22 CAR by peptide 2A; - at least two rituximab mAb specific epitopes, preferably three rituximab mAb specific epitopes, more preferably three rituximab mAb specific epitopes and a QBEN-10 mAb specific epitope linked to the CD22 CAR [The CD22 CAR allows for both selection and / or depletion of loaded immune cells] UCART 22 according to any one of 1 to 2, comprising:

[0155] 4. A UCART 22 according to any one of 1 to 3, comprising a CD52 inactivation gene, even more preferably a CD52 inactivation gene and a HIF-1 alpha insertion gene that confers resistance to alemtuzumab and hypoxia.

[0156] 5. A UCART 22 described in any one of 1 to 4, wherein the anti-CD22 CAR is expressed on the cell surface under low O2 concentration (<5% O2).

[0157] 6. A UCART 22 according to any one of 1 to 5, comprising a polynucleotide encoding a polypeptide comprising an anti-CD22 CAR as in 1 to 5.

[0158] UCART 22 according to any one of the above, comprising an anti-CD22 CAR on the cell surface, wherein a polynucleotide encoding said anti-CD22 CAR is inserted into the TRAC gene, and an exogenous polynucleotide sequence encoding IL-12 is inserted into the CD25, B2M or PD1 genomic sequence.

[0159] UCART 22 of any one of the above, comprising an anti-CD22 CAR at the cell surface, wherein a polynucleotide encoding said anti-CD22 CAR is inserted into the TRAC gene, and an exogenous polynucleotide sequence encoding IL-12 is inserted into the CD25 genomic sequence or into the PD1 genomic sequence, and further comprising a genomic KO gene selected from IL-10 receptor, TGF beta receptor, TIM-3, LAG-3 (see Table 9 above).

[0160] 7. UCART 22 according to any one of 1 to 6, further comprising a polynucleotide of SEQ ID NO: 22, preferably comprising the sequences of SEQ ID NO: 18 and 22 in the same TRAC gene.

[0161] 8. A population of cells comprising the UCART 22 according to any one of 1 to 7.

[0162] 9. A pharmaceutical composition comprising a population of cells comprising UCART 22 according to any one of 1 to 7 or said UCART 22 according to 8, and a pharma- ceutically acceptable excipient.

[0163] 10. The pharmaceutical composition according to claim 9, further comprising a bryostatin, preferably bryostatin-1.

[0164] 11. A therapeutically effective amount of UCART according to any one of 1 to 7, or a pharmaceutical composition according to 9 or 10, for use as a medicament for treating a patient.

[0165] 12. A therapeutically effective amount of UCART CD22 according to any one of 1-7 or the pharmaceutical composition according to 9 or 10, for use as a medicament for treating a patient according to 11, wherein treatment of the patient comprises administering (redosing) said therapeutically effective amount of UCART CD22 or said pharmaceutical composition at least twice to avoid relapse.

[0166] 13. A therapeutically effective amount of UCART 22 according to any one of 1 to 7 or the pharmaceutical composition according to 9 or 10 for use as a medicament for treating a patient according to 11 or 12, for the treatment of a CD22-associated pathology, preferably a CD22-associated B-cell malignancy.

[0167] 14. A therapeutically effective amount of UCART 22 or the pharmaceutical composition according to any one of 1 to 7 for use as a medicament for treating a patient according to any one of 11 to 13 (for use in the treatment of a blood cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphocytic carcinoma, acute myeloid leukemia, preferably a CD22-expressing blood cancer, more preferably a relapsed or refractory CD22-expressing blood cancer, even more preferably an aggressive form of said CD22-related blood cancer).

[0168] 15. A therapeutically effective amount of UCART according to any one of 1 to 7 or the pharmaceutical composition according to 9 or 10 for use as a medicament for treating a patient according to any one of 11 to 13 for use in the treatment of relapsed and / or refractory CD22-positive B-ALL.

[0169] 16. The patient is diagnosed with alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, 16. A therapeutically effective amount of UCART 22 according to any one of 1 to 7 or the pharmaceutical composition according to 9 or 10, for use as a medicament for treating a patient according to any one of 11 to 13 suffering from a cancer, preferably mediated by CD22+ cells, selected from: malignant mesothelioma, mast cell tumor, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, preferably liver cancer and lung cancer. [Brief description of the drawings]

[0170] [Figure 1] Schematic diagram of engineered immune cells according to the present invention. The engineered immune cells shown in FIG. 1 can be T cells that have been given a polynucleotide encoding the CAR of the present invention. The T cells are further engineered to allow for better and safer engraftment into patients. X or Y are edited genes that can be mutated, deleted, and / or have an insertion. For example, genes expressing components of the T cell receptor (TCR), such as TCR alpha or TCR beta, can be deleted or contain an insertion, and Y can be a gene involved in the sensitivity of T cells to immunosuppressive drugs, such as dCK (for resistance to purine nucleotide analogs) or CD52 (for Campath) or HPRT (for 6-thioguanine). [Diagram 2]Diagram of single and multi-chain CD22 CARs. Figure 2 shows an example of a CD22 CAR according to the invention, comprising an scfv specific for CD22, and optionally comprising a safety switch, hinge and transmembrane domains from CD8 alpha, intracellular domains from 4-1BB and CD3 zeta, and optionally a domain that confers resistance to hypoxia. [Diagram 3] An example of a CD22 CAR construct of the present invention comprising a safety switch. At least a Rituximab (R) mAb specific epitope (black box), preferably three Rituximab mAb specific epitopes, more preferably three Rituximab mAb specific epitopes and a QBEN-10 (Q) mAb specific epitope (gray box) are inserted into the CD22 CAR. R can be inserted into the scfv between the VH and VL domains (or between the VL and VH domains) and / or into the hinge. [Figure 4] Examples of polypeptides encoded by polynucleotides. Figure 4 shows an example of a polypeptide that is cleaved so that the safety switch (RQR8) and CD22 CAR are expressed on the cell surface. R = CD20 mimotope (bound by rituximab), Q = CD34 epitope (bound by QBEnd10). [Diagram 5] Degranulation activity of UCART22. Degranulation activity of UCART22 (scfv-V1 against the proximal domain of CD22) compared to that of non-transduced (NT) or T cells transduced with a CAR targeting the distal part of CD22 (scfv-V2) in the presence of CD22-positive NALM-16 cells compared to CD22-negative SUP-T1 cells. [Figure 6] Cytotoxic activity of UCART22. Cytotoxic activity of UCART22 (scfv-V1 against the proximal domain of CD22) compared to the cytotoxic activity of non-transduced (NT) or T cells transduced with a CAR targeting the distal part of CD22 (scfv-V2) cells in the presence of CD22-positive NALM-16 cells compared to CD22-negative SUP-T1 cells. [Figure 7]Interferon gamma production in UCART22. [Figure 8] Mice survival in the presence of control cells, UCART22 (scfv-V1 against the proximal domain of CD22) or CART22 (no TRAC deletion). [Figure 9] General strategy for inserting genes into the TRAC gene using TALENs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0171] Detailed Description of the Invention Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics, immunology, and molecular biology.

[0172] Suitable methods and materials are described herein, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise specified.

[0173] 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 that are within the skill of the art, and such techniques are fully described in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harries & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D.Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0174] CD22-specific chimeric antigen receptor The present invention relates to a novel design of a chimeric antigen receptor, an anti-CD22 chimeric antigen receptor (CAR or CD22 CAR or anti-CD22 CAR), which is capable of binding to CD22, in particular to the proximal domain of CD22.

[0175] The CD22-specific chimeric antigen receptor of the present invention comprises an extracellular domain comprising an extracellular ligand-binding domain and a hinge, optionally a suicide domain, a transmembrane domain, and an intracellular domain comprising a signaling transduction domain.

[0176] The CD22 CAR according to the present invention, which is expressed on the cell surface, comprises an extracellular domain that includes an extracellular ligand-binding domain.

[0177] The term "extracellular ligand-binding domain" as used herein is defined as an oligo- or polypeptide capable of binding to at least one epitope of CD22. Preferably, the extracellular ligand-binding domain is capable of at least partially interacting with CD22 and a cell surface molecule that interacts with another cell surface antigen or another membrane-bound antigen, or interacting directly with CD22, or interacting with human CD22, more precisely with the proximal region of human CD22 (from amino acid 243 to amino acid 687).

[0178] In one embodiment, a CD22 CAR according to the invention comprises an extracellular domain comprising an extracellular ligand-binding domain capable of interacting with the proximal region of CD22 (from amino acid 243 to amino acid 687) and the distal part of CD22 (from aa 20 to aa 425).

[0179] In the present invention, the full-length extracellular domain of CD22 is from amino acid (aa) 20 to aa 687, the membrane proximal domain of CD22 is from aa 243 to aa 687, and the membrane distal domain of CD22 is from aa 20 to aa 425.

[0180] In one embodiment, the extracellular ligand-binding domain can be selected to recognize a particular form (glycosylated) of CD22 that acts as a cell surface marker on target cells associated with a particular disease state.

[0181] In a preferred embodiment, the extracellular ligand-binding domain comprises a light (V) domain of a target antigen-specific monoclonal anti-CD22 antibody linked by a linker, in particular a flexible linker. L ) and heavy (V H At least one single chain antibody fragment (scFv) containing a V L and V H are preferably derived from antibody m971 as in Table 2 below. They are preferably linked together by a flexible linker, for example comprising the sequence SEQ ID NO:10.

[0182] For purposes of the present invention, certain portions of the m971 antibody (m971), a fully human anti-CD22 antibody previously identified and characterized using phage display technology (Xiao X, Ho M, Zhu Z, Pastan I, Dimitrov DS. Identification and characterization of fully human anti-CD22 monoclonal antibodies. mAbs. 2009;1(3):297-303), were combined with specific sequences in accordance with the present invention to generate novel CD22 CARs. See also WO 2014065961, which is incorporated by reference.

[0183] A preferred embodiment of the present invention provides a CAR comprising an antigen-binding domain that comprises, consists of, or consists essentially of a single chain variable fragment (scFv) of the antigen-binding domain of m971 with the following moieties as in Table 1:

[0184] Table 1. Sequences of various domains in CD22 CAR TIFF0007634045000004.tif211167

[0185] Table 2: Sequences of various CD22 scfv VH and VL TIFF0007634045000005.tif43168

[0186] Table 3: CD22 CAR of structure V-1 TIFF0007634045000006.tif49160

[0187] Table 4: CAR of structure V-3 TIFF0007634045000007.tif49159

[0188] In a preferred embodiment, the CD22 CAR of the present invention comprises the sequence of SEQ ID NO:15.

[0189] In a preferred embodiment, the CD22 CAR of the present invention comprises the sequence of SEQ ID NO:23.

[0190] The sequence of the CD22 CAR of the present invention comprises a peptide signal, a TM domain derived from CD8 alpha, and a linker between the VH and VL domains derived from m971.

[0191] SCFV In the present invention, the scfv comprises a heavy chain (V) of an immunoglobulin or part of an immunoglobulin specific for CD22 linked by a short linker peptide of 10 to 25 amino acids, preferably of SEQ ID NO: 10. Hドメイン ) and light chain (V Lドメイン ) variable region fusion protein.

[0192] In a preferred embodiment, the CAR preferentially comprises an extracellular ligand-binding domain comprising a polypeptide sequence exhibiting at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO:12.

[0193] In a preferred embodiment, the CAR preferentially comprises an extracellular ligand-binding domain comprising a polypeptide sequence exhibiting at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO:13.

[0194] In a preferred embodiment, the CAR preferentially comprises an extracellular ligand-binding domain comprising a polypeptide sequence exhibiting at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID NO:12 and a polypeptide sequence exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:13.

[0195] Hinge The extracellular domain may further comprise a hinge region between the extracellular ligand-binding domain and the transmembrane domain. The term "hinge region" as used herein generally refers to any oligo- or polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the hinge region is used to provide additional flexibility and accessibility to the extracellular ligand-binding domain. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids, most preferably 10-50 amino acids. The hinge region may be derived from all or part of a naturally occurring molecule, for example, all or part of the extracellular region of CD8, or CD4, or all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence. In preferred embodiments, the hinge domain comprises a portion of the human CD8 alpha chain, FcRIIIα receptor or IgG1, designated herein as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or in a more preferred embodiment, the hinge polypeptide exhibits at least 80%, preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to SEQ ID NO:4, and even more preferably 100% sequence identity to SEQ ID NO:4.

[0196] IgG4-derived or PD1-derived hinges are part of the present invention and are disclosed in WO2016120216 and may be used in the construction of a CD22 CAR according to the present invention.

[0197] The CD22 CAR according to the present invention is anchored in the cell membrane. Thus, such CD22 CAR further comprises a transmembrane domain. The distinguishing feature of a suitable transmembrane domain includes the ability to be expressed on the surface of a cell, preferably herein an immune cell, in particular a lymphocytic cell or a natural killer (NK) cell, and to interact together to direct the cellular response of the immune cell to a given target cell. The transmembrane domain may be derived from natural or synthetic origin. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. As a non-limiting example, the transmembrane polypeptide may be a subunit of the T cell receptor, such as α, β, or δ, a polypeptide constituting the CD3 complex, the p55 (α chain), p75 (β chain), or β chain of the IL2 receptor, an Fc receptor, in particular a subunit chain of the Fc receptor III or a CD protein. Alternatively, the transmembrane domain may be synthetic and may mainly comprise hydrophobic residues such as leucine and valine.

[0198] In a preferred embodiment, said transmembrane domain is derived from the human CD8 alpha chain (e.g. NP_001139345.1).

[0199] A CD22 CAR according to the invention generally further comprises a transmembrane domain (TM), more particularly from CD8α, that exhibits identity to the polypeptide of SEQ ID NO:6 or SEQ ID NO:7. Preferably, a CAR according to the invention comprises a TM that exhibits at least 70%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polypeptide of SEQ ID NO:6.

[0200] A CD22 CAR according to the invention generally further comprises a transmembrane domain (TM) from CD8α that exhibits identity to the polypeptide of SEQ ID NO: 6. Preferably, a CAR according to the invention comprises a TM that exhibits at least 70%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polypeptide of SEQ ID NO: 6.

[0201] The CD22 CAR according to the present invention comprises an intracellular domain that comprises a signal transduction domain or an intracellular signaling domain.

[0202] The signal transduction domain or intracellular signaling domain of the CD22 CAR according to the present invention is responsible for intracellular signaling after the binding of the extracellular ligand binding domain to the target, which leads to the activation of immune cells and immune response (cytolytic activity against target cells). In other words, the signal transduction domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "signal transduction domain" refers to the part of a protein that transmits effector signal function signals and instructs cells to perform specialized functions.

[0203] A preferred example of the signaling domain in the CD22 CAR of the present invention can be the cytoplasmic sequences of T cell receptors and co-receptors that act in concert to initiate signal transduction after antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional ability. Signaling domains include two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation, and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can include signaling motifs known as ITAM immunoreceptor tyrosine-based activation motifs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that serve as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention may include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the signaling transduction domain of the CAR may comprise a CD3 zeta signaling domain having an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of (SEQ ID NO:9). Optionally, the CD3 zeta signaling domain comprises a polypeptide sequence exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:9.

[0204] In a more preferred embodiment, the signaling transduction domain of the CD22 CAR is composed of the CD3 zeta signaling domain of SEQ ID NO:9, excluding any sequence derived from the human CD28 signaling domain. In a particular embodiment, the signaling domain of the CAR of the present invention comprises a costimulatory signal molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient immune response. A "costimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including, but not limited to, proliferation activation, differentiation, etc., in addition to the primary signal provided by the engagement of the TCR / CD3 complex with, for example, a peptide-loaded MHC molecule. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, toll ligand receptor, and ligands that specifically bind to B7-H3. Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, CD83, among others.

[0205] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0206] In a preferred embodiment, the signaling domain of the CAR of the present invention comprises a portion of a costimulatory signal molecule consisting of a fragment of 4-1BB (GenBank: AAA53133.) In particular, the signaling domain of the CAR of the present invention comprises an amino acid sequence that comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:8.

[0207] In a more preferred embodiment, the signaling domain of the CAR of the present invention does not comprise a sequence derived from CD28 (NP_006130.1).

[0208] In an even more preferred embodiment, all aspects of the invention do not include sequences derived from CD28 (NP_006130.1).

[0209] In an even more preferred embodiment, the signaling domain of the CD22 CAR of the present invention comprises a portion of the costimulatory signal molecule 4-1BB (GenBank: AAA53133) and does not contain any sequence derived from CD28 (NP_006130.1).

[0210] The present invention provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising: The extracellular domain contains: - a binding domain specific for CD22, preferably a binding domain specific for human CD22, more preferably said binding domain specific for human CD22 is a single chain variable fragment (scFv), - hinge, preferably from CD8 alpha; A transmembrane domain, preferably from CD8 alpha; An intracellular domain that contains: - a costimulatory signal molecule derived from human 4-1BB, and - an intracellular signaling domain comprising the human CD3 zeta signaling domain.

[0211] In a preferred embodiment, the CD22 CAR of the present invention has no sequence derived from CD28.

[0212] In a preferred embodiment, the CD22 CAR of the present invention does not contain any sequence from CD28 and comprises a sequence leader, a TM domain and a hinge from CD8α, preferably does not contain any sequence from CD28 and comprises a sequence leader of SEQ ID NO:1, a TM domain of SEQ ID NO:6 and a hinge of SEQ ID NO:4 from CD8α.

[0213] In one embodiment, the CD22 CAR of the present invention comprises a leader sequence from human CD8α (SEQ ID NO:1) or a leader sequence having at least 95% identity to SEQ ID NO:1.

[0214] In another embodiment, the CD22 CAR of the present invention comprises a leader sequence of SEQ ID NO:2 or a leader sequence having at least 95% identity to SEQ ID NO:2.

[0215] In one embodiment, the present invention provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising: a binding domain specific for CD22, preferably a binding domain specific for human CD22, more preferably said domain specific for human CD22 is a single chain variable fragment (scFv), even more preferably comprising a VH and a VL from m971, - hinge from human CD8 alpha (CD8α), - the transmembrane domain from human CD8 alpha (α), - a costimulatory signal molecule derived from human 4-1BB, - an intracellular signaling domain comprising the human CD3 zeta signaling domain.

[0216] In one embodiment, the present invention provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising: a binding domain specific for CD22, preferably a binding domain specific for human CD22, more preferably said domain specific for human CD22 is a single chain variable fragment (scFv), even more preferably comprising a VH and a VL from m971, - a hinge from human FcRIIIα, - the transmembrane domain from human CD8 alpha (α), - a costimulatory signal molecule derived from human 4-1BB, - an intracellular signaling domain comprising the human CD3 zeta signaling domain.

[0217] The present invention also provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising: a binding domain specific for CD22, preferably a binding domain specific for human CD22, more preferably said domain specific for human CD22 is a single chain variable fragment (scFv), even more preferably comprising a VH and a VL from m971, - a hinge from human IgG1, - the transmembrane domain from human CD8 alpha (α), - a costimulatory signal molecule derived from human 4-1BB, - an intracellular signaling domain comprising the human CD3 zeta signaling domain.

[0218] These last three embodiments encompass a CD22 CAR having a signal peptide of SEQ ID NO:1 or SEQ ID NO:2, preferably SEQ ID NO:1.

[0219] More preferably, said CD22 CAR has no sequence derived from CD28.

[0220] The scfv of the present invention is derived from an antibody specific for CD22, which comprises a VH domain separated from a VL domain by a linker, said VH and / or VL domain contributing to binding to CD22.

[0221] In a preferred embodiment, the UCART 22 of the present invention comprises an scfv derived from the m971 antibody, with one GS4 linker between the VH and VL and 2R between the VL and the CD8 alpha hinge.

[0222] In the present invention, the scfv comprises the heavy chain (V) of SEQ ID NO:12: of an immunoglobulin specific for CD22 (m971) linked with a linker peptide, preferably the linker of SEQ ID NO:10. Hドメイン ) and the light chain (V Lドメイン ) variable region fusion protein.

[0223] In one embodiment, said scfv of the invention further comprises a leader sequence (or signal peptide), preferably said leader sequence linked to the VH domain.

[0224] The embodiment in which said leader sequence is linked to the VL domain is part of the present invention.

[0225] In one embodiment the VH domain is linked to the hinge, in another embodiment the VL domain is linked to the hinge.

[0226] The invention provides scfv linked to hinges of various lengths, preferably derived from CD8α, IgG1 or FcRIIIα (see FIG. 2), more preferably derived from CD8α.

[0227] Preferably, the present invention provides a CD22 CAR comprising: a signal peptide, for example the signal peptide from CD8 alpha of SEQ ID NO:2 or of SEQ ID NO:1. - comprising a VH domain separated from a VL domain by a linker (scFv), said VH and VL and said linker contributing to binding to CD22; a hinge derived from the human CD8 alpha chain, or a hinge derived from human IgG1, or a hinge derived from FcRIIIα, preferably from CD8 alpha. - Transmembrane domain (TM) from CD8 alpha (α) - A costimulatory signal molecule derived from human 4-1BB - an intracellular signaling domain that comprises a CD3 zeta signaling domain.

[0228] One CD22 CAR of the invention is comprised of: - a leader sequence (e.g., the CD8α leader sequence or the CD8α signal peptide) - an anti-CD22 scfv comprising a VH, a linker and a VL, or a VL, a linker and a VH, said VH and VL being derived from m971 CD8α hinge CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0229] One CD22 CAR of the invention comprises, consecutively: - CD8α signal peptide, which can be removed upon expression on the cell surface; - an epitope recognized by QBEN10 and an epitope recognized by rituximab, - an anti-CD22 scfv comprising a VH, a linker and a VL, said VH and VL being derived from m971; Two consecutive epitopes recognized by rituximab CD8α hinge CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0230] One CD22 CAR of the invention comprises, consecutively: - CD8α signal peptide, which can be removed upon expression on the cell surface; - an epitope recognized by QBEN10 and an epitope recognized by rituximab, - an anti-CD22 scfv comprising a VH, a linker and a VL, said VH and VL being derived from m971; Two consecutive epitopes recognized by rituximab CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0231] One CD22 CAR of the invention comprises, consecutively: - CD8α signal peptide, which can be removed upon expression on the cell surface; - an anti-CD22 scfv comprising a VH, a linker and a VL, said VH and VL being derived from m971 Two consecutive epitopes recognized by rituximab CD8α hinge CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0232] One CD22 CAR of the invention comprises, consecutively: - CD8α signal peptide, which can be removed upon expression on the cell surface; - an anti-CD22 scfv comprising a VH, a linker and a VL, said VH and VL being derived from m971 Two consecutive epitopes recognized by rituximab CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0233] One CD22 CAR of the invention comprises, consecutively: - CD8α signal peptide, which can be removed upon expression on the cell surface; - an anti-CD22 scfv comprising a VH, a linker and a VL, said VH and VL being derived from m971; an anti-CD19 scfv Two consecutive epitopes recognized by rituximab CD8α hinge CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0234] One CD22 CAR of the invention comprises, consecutively: - CD8α signal peptide, which can be removed upon expression on the cell surface; - an anti-CD22 scfv comprising a VH, a linker and a VL, said VH and VL being derived from m971; an anti-CD19 scfv Two consecutive epitopes recognized by rituximab CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0235] One CD22 CAR of the invention comprises, consecutively: - CD8α signal peptide, which can be removed upon expression on the cell surface; - an anti-CD19 scfv and an anti-CD22 scfv comprising a VH, a linker and a VL, said VH and VL being derived from m971; Two consecutive epitopes recognized by rituximab CD8α hinge CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0236] One CD22 CAR of the invention comprises, consecutively: - CD8α signal peptide, which can be removed upon expression on the cell surface; - an anti-CD19 scfv and an anti-CD22 scfv comprising a VH, a linker and a VL, said VH and VL being derived from m971; Two consecutive epitopes recognized by rituximab CD8αTM ·Costimulatory signal molecule derived from 4-1BB · Intracellular CD3 zeta signaling domain.

[0237] Linker-SCFV Linkers according to the invention are, for example, multimers of pentapeptides, (GGGGS)n or (G4S)n or (Gly4Ser)n (where n=1 to 4, preferably n=3), 18mers TIFF0007634045000008.tif3128 (Andris-Widhopf et al., 2011) and 20-mer (G4S)4 (Schaefer et al., 2010). Linkers according to the invention may comprise sequences with added functionality, such as epitope tags (Sblattero & Bradbury, 2000 Nature Biotechnology 18, 75-80), at least one sequence (preferably two) of SEQ ID NO:20 separated by a linker, often a sequence that improves the properties of the scFv of the invention in the context of a particular antibody sequence.

[0238] Among other linkers, preferred within the present invention are 15-mer peptide linkers TIFF0007634045000009.tif4128 (Zhihong Shen, Heping Yan, Ying Zhang, Raymond L. Mernaugh, and Xiangqun Zeng (2008), Anal Chem. 80(6): 1910-1917).

[0239] In a preferred embodiment, the linker connecting the VH to VL sequence (VL to VH sequence) of m971 is a linker of the formula (G4S)n, where n is 1 to 3; preferably, n=3 of the sequence (G4S)3 (SEQ ID NO:10).

[0240] In one aspect, the present invention provides: CD22 CAR including: - the human CD8α leader sequence of SEQ ID NO:1 (CD8α leader or CD8α signal peptide) - an anti-CD22 scfv antibody comprising a VH of SEQ ID NO:12, a linker of SEQ ID NO:10, and a VL of SEQ ID NO:13; human CD8α hinge of SEQ ID NO:4, Human CD8αTM of SEQ ID NO:6 A costimulatory signaling molecule derived from 4-1BB of SEQ ID NO:8 The intracellular CD3 zeta signaling domain of SEQ ID NO:9.

[0241] In one aspect, the present invention also provides: CD22 CAR including: - the human CD8α leader sequence of SEQ ID NO:1 (CD8α leader or CD8α signal peptide) - an anti-CD22 scfv antibody comprising a VH of SEQ ID NO:12, a linker of SEQ ID NO:10, and a VL of SEQ ID NO:13; human FcγRIIIα hinge of SEQ ID NO:3, Human CD8αTM of SEQ ID NO:6 A costimulatory signaling molecule derived from 4-1BB of SEQ ID NO:8 The intracellular CD3 zeta signaling domain of SEQ ID NO:9.

[0242] In one aspect, the present invention provides: CD22 CAR including: - the human CD8α leader sequence of SEQ ID NO:1 (CD8α leader or CD8α signal peptide) an anti-CD22 scfv comprising a VL of SEQ ID NO: 13, a linker of SEQ ID NO: 10, and a VH of SEQ ID NO: 12; human FcγRIIIα hinge of SEQ ID NO:3, Human CD8αTM of SEQ ID NO:6 A costimulatory signaling molecule derived from 4-1BB of SEQ ID NO:8 The intracellular CD3 zeta signaling domain of SEQ ID NO:9.

[0243] In one aspect, the present invention provides: CD22 CAR including: - the human CD8α leader sequence of SEQ ID NO:1 (CD8α leader or CD8α signal peptide) an anti-CD22 scfv comprising a VL of SEQ ID NO: 13, a linker of SEQ ID NO: 10, and a VH of SEQ ID NO: 12; human CDA alpha hinge of SEQ ID NO:4; Human CD8αTM of SEQ ID NO:6 A costimulatory signaling molecule derived from 4-1BB of SEQ ID NO:8 The intracellular CD3 zeta signaling domain of SEQ ID NO:9.

[0244] In one embodiment, the present invention provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising: - a signal peptide having an amino acid sequence that has at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the polypeptide of SEQ ID NO:1 or 2; preferably, the signal peptide has an amino acid sequence that has at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the polypeptide of SEQ ID NO:1.

[0245] a VH domain separated from a VL domain by a linker, said VH and VL contributing to binding to CD22; said linker having at least 90%, 95%, 97%, 99% or 100% sequence identity with the polypeptide of SEQ ID NO:10.

[0246] The VH domain has at least 90%, 95%, 97%, 99% or 100% sequence identity to the polypeptide of SEQ ID NO:12.

[0247] The VL domain has at least 90%, 95%, 97%, 99% or 100% sequence identity to the polypeptide of SEQ ID NO:13.

[0248] - a hinge derived from the human CD8 alpha chain having an amino acid sequence having at least 80%, more preferably at least 80%, 90%, 95%, 97%, 99% or 100% sequence identity to the polypeptide of SEQ ID NO:4. - a transmembrane domain derived from CD8 alpha (α) having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the polypeptide of SEQ ID NO:6; - a costimulatory signal molecule derived from human 4-1BB (or the 4-1BB intracellular domain) having an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:8; - an intracellular signaling domain comprising a CD3 zeta signaling domain having an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:9.

[0249] In a preferred embodiment, the CD22-specific chimeric antigen receptor (CD22 CAR) of the invention does not contain any sequence from human CD28, in particular from the human CD28 intracellular signaling domain. In a more preferred embodiment, the CD22-specific chimeric antigen receptor (CD22 CAR) of the invention does not contain any sequence from human CD28, in particular from the human CD28 intracellular signaling domain, and further contains a signal peptide from CD8α, preferably fused to the VH domain of a scfv specific for CD22.

[0250] In one embodiment, the present invention provides a CD22 CAR of SEQ ID NO:15.

[0251] In one aspect, the present invention provides a CD22 CAR having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the polypeptide of SEQ ID NO:15.

[0252] In one embodiment, the present invention provides a CD22 CAR having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the polynucleotide of SEQ ID NO:22.

[0253] The CD22 CAR of the present invention comprises the following sequence: TIFF0007634045000010.tif140159

[0254] In a preferred embodiment, the CD22 CAR of the present invention comprises the following sequence: TIFF0007634045000011.tif58159

[0255] The sequence of CD22 CAR with a peptide signal from SEQ ID NO:2, a TM domain from CD8α, and a linker between the VH and VL domains: TIFF0007634045000012.tif92153TIFF0007634045000013.tif204153

[0256] In one embodiment, the present invention provides the following sequence: TIFF0007634045000014.tif58152There is no signal peptide in this CD22CAR.

[0257] In one embodiment, the UCART 22 of the present invention comprises the sequence of SEQ ID NO:20: TIFF0007634045000015.tif58152

[0258] In one embodiment, the UCART 22 of the present invention comprises the sequence of SEQ ID NO:22.

[0259] In a preferred embodiment, the UCART 22 of the present invention comprises the sequence of SEQ ID NO:22 inserted into the human TRAC gene (e.g., into the human TRAC gene chromosome 14 - NC_000014.9) and expresses an anti-CD22 CAR specific for the proximal part of CD22 on the cell surface.

[0260] In a more preferred embodiment, the UCART 22 of the present invention comprises the sequence of SEQ ID NO:20 inserted into the human TRAC gene (e.g., into the human TRAC gene chromosome 14 - NC_000014.9) and expresses an anti-CD22 CAR specific for the distal part of CD22 on the cell surface.

[0261] In one aspect, the anti-CD22 binding domain of the CD22 CAR of the invention is an anti-CD22 binding domain specific for the distal portion of CD22.

[0262] HA22-CAR Thus, the present invention provides a CD22 CAR having the following sequence: TIFF0007634045000016.tif45152

[0263] Preferably, the UCART 22 of the present invention has the sequence: Contains TIFF0007634045000017.tif118153.

[0264] An anti-CD22 binding domain specific for the distal portion of CD22 can be expressed in the UCART22 of the present invention alone or together with an anti-CD22 binding domain specific for the proximal portion of CD22.

[0265] In one aspect, the anti-CD22 binding domain of the CD22 CAR of the present invention is an optimized anti-CD22 binding domain.

[0266] As used herein, an "optimized" antibody (or scfv) refers to a form of an antibody (or scfv) that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from an immunoglobulin. Preferably, the antibody is a human immunoglobulin (recipient antibody) in which residues from the complementarity determining regions (CDRs) have been replaced by residues from the CDRs to achieve the desired specificity, affinity, and capacity.

[0267] The donor CDRs may undergo minor amino acid alterations that may significantly affect or alter the binding characteristics of the CD22 CAR of the present invention. Indeed, one of the inventions provided herein is a CD22 CAR that maintains binding (and cytolytic activity) to CD22-expressing cells, but has modified affinity to reduce the intensity of the response (cytokine release).

[0268] Amino acid modifications are typically conservative modifications, including amino acid substitutions, additions, and deletions in the antibody fragment in the CAR and / or in other parts of the CAR molecule. Modifications can be introduced into the antibody, antibody fragment, or other parts of the CAR molecule of the invention by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis, or by using optimized germline sequences.

[0269] Generally, an optimized CAR will comprise substantially all of at least one, and typically two, variable domains, where all or substantially all of the CDR regions correspond to the original human immunoglobulin.

[0270] Conservative amino acid substitution refers to a substitution in which an amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CAR of the present invention can be replaced with other amino acid residues of the same side chain family, and the modified CAR can be tested using the functional assays described herein.

[0271] In a preferred embodiment, the present invention provides a CD22 CAR having conservative sequence modifications (or amino acid sequence changes) compared to the amino acid sequence of the polypeptide of SEQ ID NO:15.

[0272] In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence that has two amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO:15.

[0273] In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence having three amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO:15.

[0274] In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence which has four amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO:15.

[0275] In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence having five amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO:15, optionally comprising at least one sequence of SEQ ID NO:20.

[0276] In a more preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence having five amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO:15, wherein at least one CDR in SEQ ID NO:15 is conserved.

[0277] In a more preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence having 1 to 15 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO:15, wherein at least one CDR in SEQ ID NO:15 is conserved.

[0278] In a preferred embodiment, the sequence of the CD22 CAR of the present invention is modified by changing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids compared to the wt CD22 CAR in order to reduce HAMA (human anti-mouse response) without affecting the ability of the CAR to bind to its target (CD22).

[0279] In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence with at least one amino acid alteration compared to the amino acid sequence of wt (which is m971), wherein said at least one amino acid alteration has no effect on or improves the binding and / or activity of said CD22 CAR in primary T cells.

[0280] The binding characteristics can be modified using adaptation techniques first described in Mitchell Ho, Satoshi Nagata, and Ira Pastan. Isolation of anti-CD22 Fv with high affinity by Fv display on human cells PNAS 2006 103 (25) 9637-9642; published ahead of publication June 8, 2006, doi:10.1073 / pnas.0603653103, which is incorporated herein by reference.

[0281] These optimized scfvs also have at least one mutation equivalent to the mutations Pro-91-Thr-92 (PT) Gly-91-Ala-92 and Val-91-Phe-92.

[0282] In one embodiment, the present invention provides an anti-CD22 CAR comprising: - An extracellular domain that contains: Signal peptide an optionally optimized ligand binding domain comprising a VH domain and a VL domain derived from a monoclonal anti-CD22 antibody having one of the following: Pro-91-Thr-92 (PT), Gly-91-Ala-92, Val-91-Phe-92, mutations or equivalents; Hinge, including CD8 alpha (α) hinge - CD8 alpha transmembrane domain, and - a cytoplasmic domain containing the CD3 zeta signaling domain and the costimulatory domain derived from 4-1BB.

[0283] In a preferred embodiment, the invention provides an anti-CD22 CAR as described above, wherein said monoclonal anti-CD22 antibody is derived from the m971 antibody with a hotspot in the CDR, preferably one of the following mutations: Pro-91-Thr-92 (PT), Gly-91-Ala-92, Val-91-Phe-92.

[0284] In a more preferred embodiment, the present invention provides isolated engineered (TCR and dCK KO) immune T cells comprising an anti-CD22 CAR comprising: An extracellular domain comprising: A ligand-binding domain comprising a VH domain and a VL domain derived from a monoclonal anti-CD22 antibody having a signal peptide and at least one of the following mutations: Pro-91-Thr-92, Gly-91-Ala-92; Val-91-Phe-92; Hinge, including CD8 alpha (α) hinge - CD8 alpha transmembrane domain, and - a cytoplasmic domain containing the CD3 zeta signaling domain and the costimulatory domain derived from 4-1BB.

[0285] In a more preferred embodiment, the present invention provides isolated engineered (TCR and CD52 KO) immune T cells comprising an anti-CD22 CAR comprising: An extracellular domain comprising: A ligand-binding domain comprising a VH domain and a VL domain derived from a monoclonal anti-CD22 antibody having a signal peptide and at least one of the following mutations: Pro-91-Thr-92, Gly-91-Ala-92; Val-91-Phe-92; Hinge, including CD8 alpha (α) hinge - CD8 alpha transmembrane domain, and - a cytoplasmic domain containing the CD3 zeta signaling domain and the costimulatory domain derived from 4-1BB.

[0286] In a more preferred embodiment, the present invention provides isolated engineered (TCR, CD52 and dCK KO) immune T cells comprising an anti-CD22 CAR comprising: An extracellular domain comprising: A ligand-binding domain comprising a VH domain and a VL domain derived from a monoclonal anti-CD22 antibody having a signal peptide and at least one of the following mutations: Pro-91-Thr-92, Gly-91-Ala-92; Val-91-Phe-92; Hinge, including CD8 alpha (α) hinge - CD8 alpha transmembrane domain, and - a cytoplasmic domain containing the CD3 zeta signaling domain and the costimulatory domain derived from 4-1BB.

[0287] In one aspect, CAR 22 may be co-expressed on the cell surface with at least one, preferably two, more preferably three, monoclonal antibody (mAb) specific epitopes, which may be fused to the transmembrane domain of CD8. In one embodiment, the mAb specific epitope is an epitope recognized by rituximab and / or by QBEN10, and the peptide co-expressed with cD22 CAR is RQR8.

[0288] In another embodiment, at least one, preferably two, more preferably three, monoclonal antibody (mAb) specific epitopes can be inserted into the linker L (which joins the VH to the VL) of a CD22 specific scfv and / or into the hinge of the CD22 CAR.

[0289] The monoclonal antibody (mAb) specific epitopes are: ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemcitabine, It may be one of the following mAb specific epitopes specifically recognized by a monoclonal antibody selected from tuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and ustekinumab, preferably from rituximab (R) and / or from QBEN10 (Q).

[0290] Epitope-specific mAbs can be used for in vitro cell sorting and / or in vivo cell depletion of immune cells expressing CD22.

[0291] In certain embodiments, the extracellular binding domain of the CD22 CAR of the invention may comprise one of the following sequences: V1-L1-V2-(L) x -Epitope 1-(L) x -; V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -; V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x - V1-L1-V2; (L) x -Epitope 1-(L) x -Epitope 2-(L) x - V1-L1-V2; Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x - V1-L1-V2; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x ; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x -; (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -; V1-(L) x -Epitope 1-(L) x -V2; V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x ; V1-(L) x -Epitope 1-(L) x -V2-(L) x-Epitope 2-(L) x -Epitope 3-(L) x ; V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x ; (L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2; or, (L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2-(L) x -Epitope 3-(L) x ; Where: V1 is a VL, preferably of SEQ ID NO:12, and V2 is a VH, preferably of SEQ ID NO:13, or V1 is a VH and V2 is a VL; L1 is a linker suitable for linking a VH chain to a VL chain, preferably of SEQ ID NO:10; L is a linker comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain may be identical to or different from other occurrences of L in the same extracellular binding domain, and x is 0 or 1, and each occurrence of x is selected independently of the others; and Epitope 1, epitope 2 and epitope 3 are mAb-specific epitopes and may be the same or different.

[0292] In one embodiment, the extracellular binding domain comprises one of the following sequences: V1-L1-V2-L-epitope 1;V1-L1-V2-L-epitope 1-L;V1-L1-V2-L-epitope 1-L-epitope 2;V1-L1-V2-L-epitope 1-L-epitope 2-L;V1-L1-V2-L-epitope 1-L-epitope 2-L-epitope 3;V1-L1-V2-L-epitope 1-L-epitope 2-L-epitope 3-L;V1-L1-V2-epitope 1;V1-L1-V2-epitope 1-L;V1-L1-V2-epitope 1-L-epitope 2;V1-L1-V2-epitope 1- L-epitope 2-L;V1-L1-V2-epitope 1-L-epitope 2-L-epitope 3;V1-L1-V2-epitope 1-L-epitope 2-L-epitope 3-L;epitope 1-V1-L1-V2;epitope 1-L-V1-L1-V2;L-epitope 1-V1-L1-V2;L-epitope 1-L-V1-L1-V2;epitope 1-L-epitope 2-V1-L1-V2;epitope 1-L-epitope 2-L-V1-L1-V2;epitope 1-L-epitope 2-L-V1-L1-V2;L-epitope 1-L-epitope 2-V1-L1-V2;L-epitope 1-L-epitope epitope 2-L-V1-L1-V2; epitope 1-L-epitope 2-L-epitope 3-V1-L1-V2; epitope 1-L-epitope 2-L-epitope 3-L-V1-L1-V2; L-epitope 1-L-epitope 2-L-epitope 3-V1-L1-V2; L-epitope 1-L-epitope 2-L-epitope 3-L-V1-L1-V2; V1-L-epitope 1-L-V2; L-epitope 1-L-V1-L-epitope 2-L-V2; V1-L-epitope 1-L-V2-L-epitope 2-L; V1-L-epitope 1-L-V2-L-epitope 2-L; V1-L-epitope 1-L-V2-L-epitope 2-L-epitope 3;V1-L-epitope 1-L-V2-L-epitope 2-epitope 3;V1-L-epitope 1-L-V2-L-epitope 2-L-epitope 3-epitope 4;L-epitope 1-L-V1-L-epitope 2-L-V2-L-epitope 3-L;epitope 1-L-V1-L-epitope 2-L-V2-L-epitope 3-L;L-epitope 1-L-V1-L-epitope 2-L-V2-L-epitope 3-L;L-epitope 1-L-V1-L-epitope 2-L-V2-L-epitope 3-L;L-epitope 1-L-V1-L-epitope 2-L-V2-L-epitope 3;L-epitope 1-L-V1-L1-V2-L-epitope 2-L;L-epitope1-L-V1-L1-V2-L-epitope2-L-epitope3; L-epitope1-L-V1-L1-V2-L-epitope2-epitope3, or epitope1-L-V1-L1-V2-L-epitope2-L-epitope3-epitope4, where; V1 is V L And V2 is V H or V1 is V H And V2 is V L and the VH, and VL are the optimized VH and VL from m971; L1 is V L Chain to V H any linker suitable for linking chains; L is a linker comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain may be identical to or different from other occurrences of L in the same extracellular binding domain, and Epitope 1, epitope 2 and epitope 3 are mAb-specific epitopes and may be the same or different.

[0293] L1 is a linker containing glycine and / or serine, preferably L1 has the amino acid sequence (Gly-Gly-Gly-Ser) n or (Gly-Gly-Gly-Gly-Ser) n (wherein n is 1, 2, 3, 4 or 5), or a linker comprising the amino acid sequence (Gly4Ser)4 or (Gly4Ser)3.

[0294] Similarly, L is a linker comprising glycine and / or serine, preferably L is TIFF0007634045000018.tif19153, and preferably L is SGGGG, GGGGS or SGGGGS.

[0295] In a preferred embodiment, epitope 1, epitope 2, epitope 3 and epitope 4 are selected from the group consisting of ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab , ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and ustekinumab, and preferably epitope 1, epitope 2, epitope 3 and epitope 4 are recognized by rituximab or QBEND-10.

[0296] Epitope 1, Epitope 2, Epitope 3 and Epitope 4 are mAb-specific epitopes specifically recognized by: TIFF0007634045000019.tif171145TIFF0007634045000020.tif240145TIFF0007634045000021.tif192145

[0297] The mAb specific epitope is therefore The polypeptide may comprise one polypeptide selected from TIFF0007634045000022.tif118165.

[0298] In a preferred embodiment, the mAb specific epitope is This is a mAb-specific epitope having the amino acid sequence of TIFF0007634045000023.tif4128.

[0299] In a more preferred embodiment, the CD22 CAR of the present invention comprises three mAb-specific epitopes having the amino acid sequence of CPYSNPSLC®, and TIFF0007634045000024.tif4128.

[0300] In an even more preferred embodiment, the CD22 CAR of the present invention comprises two mAb-specific epitopes having the amino acid sequence CPYSNPSLC® located in the NT part of the hinge, immediately following the VL.

[0301] In one aspect, at least one sequence (R) to which rituximab is bound and / or the sequence (Q) to which QBEN10 is bound may be linked with a linker, as previously described in (WO2016120216). TIFF0007634045000025.tif3128 and / or into the hinge.

[0302] In certain embodiments, the CD22 CAR of the present invention is a single-chain CAR (scCAR).

[0303] In a particular embodiment, the single chain anti-CD22 CAR of the present invention comprises an scfv from m971 and at least one other binding domain, preferably specific for the distal part of CD22 or for another B cell antigen, particularly when expressed by B cell malignancies, e.g., CD34, CD10, CD79a, CD20, IgD, CD5, CD23, CD19, STAT5, CD3, CD30, BCMA. In a particular embodiment, the CD22 CAR of the present invention is a multi-chain CAR (mcCAR). Multi-chain CD22 CARs are part of the present invention and can be produced as described in detail in WO2014039523, which is incorporated herein by reference. In a particular embodiment, the V of an immunoglobulin specific for CD22 is Hドメイン and V Lドメイン Alternatively, the immunoglobulin portion can be carried by two distinct and sequestered (non-covalently linked) chains of a multi-chain CAR.

[0304] In its multi-chain form, the CD22 CAR of the present invention comprises at least two, preferably three, transmembrane domains (non-covalently linked to each other), at least one of the transmembrane domains comprising a CD22-specific scfv of the present invention.

[0305] In a particular embodiment, the V of an immunoglobulin specific for CD22, preferably from m971, Hドメイン and V Lドメイン can be carried by one strand of a multi-stranded CAR.

[0306] Examples of sc CD22 CARs and mc CD22 CARs of the present invention are provided in FIG.

[0307] In a particular embodiment, the V of an immunoglobulin specific for CD22, preferably from m971, Hドメイン and V Lドメイン can be carried by one chain of a multi-chain CAR and can be coupled to the V of another immunoglobulin specific for CD22. Hドメイン and V Lドメイン can be carried by another strand of the mc CAR.

[0308] Downregulation or mutation of target antigen is commonly observed in cancer cells, which creates antigen loss escape variants.Therefore, in order to counteract tumor escape and make immune cells more specific to targets, the CD22-specific CAR according to the present invention can also contain other extracellular ligand binding domains to simultaneously bind different elements in targets, thereby enhancing the activation and function of immune cells.In one embodiment, the extracellular ligand binding domains can be placed in tandem on the same transmembrane polypeptide, and can be optionally separated by linkers.

[0309] In another embodiment, the different extracellular ligand binding domains can be located on different transmembrane polypeptides that make up the multi-chain CAR. In another embodiment, the present invention relates to a population of CARs that comprise different extracellular ligand binding domains, one of each being specific for CD22.

[0310] In one embodiment, the extracellular binding domain specific for CD22 and the second extracellular binding domain are on the same scCAR.

[0311] In another embodiment, the extracellular binding domain specific for CD22 and the second extracellular binding domain are on the same mc CAR and belong to the same or two different and non-covalently linked transmembrane domains of the mc CAR.

[0312] Other second extracellular binding domains can be any extracellular binding domain that is binding specific for an antigen associated (even temporarily co-expressed) with CD22 on a pathological cell, such as CD34, CD10, CD79a, CD20, IgD, CD5, CD23, CD19, STAT5, CD3, CD30, BCMA, PAX5, CD19, CD20, CD30, glycosphingolipids, major histocompatibility complex (MHC) molecules, Ig, CD3, CD34, CD79, preferably CD79a, CD138, B7-1 (CD80), B7-2 (CD86), major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF 17) or FLT-3, Pax5.

[0313] The present invention also provides engineered TCR KO cells, preferably T cells, comprising at least another edited gene endowed with a CD22 CAR, associated CD22 CAR nucleic acid, CD22 CAR recombinant expression vectors, populations of said TCR KO cells endowed with a CD22 CAR, and pharmaceutical compositions, proteins, expression vectors relating to the CD22 CAR, engineered TCR KO CD52 KO cells expressing said CD22CAR of the present invention.

[0314] The present invention provides: engineered TCR KO cells comprising at least another edited gene selected from a gene conferring resistance to hypoxia, a gene conferring resistance to alemtuzumab, a gene conferring resistance to a protease inhibitor (e.g., bortezomib), a gene conferring resistance to PNA(dCK) and conferred with a CD22 CAR, and related nucleic acids, populations of engineered TCR KO cells comprising at least another edited gene as described below and conferred with said CD22 CAR, and pharmaceutical compositions comprising the same as medicaments.

[0315] Polynucleotides, Vectors: The present invention relates to polynucleotides, vectors encoding the above-mentioned CD22 CAR according to the present invention.

[0316] The polynucleotide may be present in an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector, such as a baculovirus vector, for transfection of an insect host cell, or a plasmid or viral vector, such as a lentivirus or adeno-associated virus, for introduction into a mammalian, preferably human host cell).

[0317] In certain embodiments, different nucleic acid sequences can be included in one polynucleotide or vector that includes a nucleic acid sequence encoding a ribosomal skip sequence, such as a sequence encoding a 2A peptide. The 2A peptide, identified in the Aphthovirus subgroup of picornaviruses, causes a ribosomal "skip" from one codon to the next, without a peptide bond being formed between the two amino acids encoded by the codons (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)).

[0318] Thus, the present invention provides a vector as in FIG. 4 encoding RQR8 and an anti-CD22 CAR linked by peptide 2A.

[0319] "Codon" refers to three nucleotides on an mRNA (or the sense strand of a DNA molecule) that are translated into one amino acid residue by the ribosome. Thus, two polypeptides can be synthesized from a single continuous open reading frame in an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosome skipping mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.

[0320] Another object of the present invention is a vector that allows the CD22 CAR of the present invention to be expressed in a cell. In a preferred embodiment, said vector allows the transient expression of the CD22 CAR of the present invention. In a more preferred embodiment, said vector allows the constitutive and stable expression of the CD22 CAR of the present invention by inserting the sequence encoding said CD22 CAR into the genome of the cell.

[0321] Expression of the CD22 CAR of the present invention and / or survival of cells expressing the CD22 CAR of the present invention can be controlled using a gene under the control of an inducible promoter, as described in (R. Kuhn, F. Schwenk, M. Aguet, K. Rajewsky. Inducible gene targeting in mice. Science 8 September 1995: Vol. 269 no. 5229 pp. 1427-1429 DOI:10.1126 / science.7660125, and references cited therein.

[0322] In one embodiment, a CD22 CAR is provided, the extracellular domain of which comprises at least two CD20 mimotopes of SEQ ID NO:19 (CPYSNPSLC) located between the m971 scfv domain and the hinge from human CD8 alpha. Patent document WO2016120216A1 discloses a method for preparing such a construct and is incorporated herein by reference.

[0323] In one embodiment, the present invention provides a vector comprising a sequence encoding a CD22 CAR selected from SEQ ID NO:22.

[0324] To direct a transmembrane polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence, or pre sequence) is provided in the polynucleotide sequence or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence. That is, the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are generally located 5' of the nucleic acid sequence encoding the polypeptide of interest, although some secretory signal sequences can be located elsewhere in the nucleic acid sequence of interest (see, for example, Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In a preferred embodiment, the signal peptide comprises the amino acid sequence SEQ ID NO: 1 and 2.

[0325] In a more preferred embodiment, the signal peptide of the CAR of the present invention comprises the amino acid sequence of SEQ ID NO:1 derived from human CD8 alpha.

[0326] Those skilled in the art will recognize that, given the degeneracy of the genetic code, considerable sequence variation is possible in these polynucleotide molecules.Preferably, the nucleic acid sequences of the present invention are codon-optimized for expression in mammalian cells, preferably for expression in human cells.Codon optimization refers to the exchange of codons in a sequence of interest that are usually rare in highly expressed genes of a given species with codons that are generally frequent in highly expressed genes of such species.Such codons code for the amino acids as the exchanged codons.

[0327] How to engineer immune cells with CD22 CAR: The present invention encompasses a method for preparing immune cells for immunotherapy, comprising the step of introducing ex vivo into said immune cells a polynucleotide or vector encoding one of the CD22 CARs of the present invention, preferably SEQ ID NO:15, as previously described.

[0328] In a preferred embodiment, the polynucleotide is contained in a vector allowing for stable expression in immune cells.

[0329] According to a further embodiment, the method further comprises the step of genetically modifying the cells to make them more suitable for adoptive transfer and / or for use with drugs that affect the immune cell survival, in particular for transplantation (also called allogeneic, or allogeneic, transplantation) alone or in combination with a drug to which the immune cells have been made tolerant.

[0330] In this latter case, the engineered cells may be first isolated from a donor and used for re-infusion into the same donor in combination with the drug to which the engineered cells have been made tolerant.

[0331] For gene editing, the methods described in PA201670503 are incorporated herein by reference and illustrated in the Examples below, where gene editing, as used herein, means modifying a gene or inactivating a gene, e.g., mutating a gene, deleting a gene, inserting a sequence into a gene, modifying the methylation of the gene (including the gene's promoter), etc.

[0332] The method described in MacLeod et al., Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells, Molecular Therapy (2017), http: / / dx.doi.org / 10.1016 / j.ymthe.2017.02.005, which is incorporated herein by reference, is also a potential alternative to the method used in the present invention to prepare TCR KO CD22 CARs, or cells equipped with CD22 CARs that are resistant to hypoxia by overexpressing HIF-1a.

[0333] The method of the present invention is based on the cellular homology directed repair (HDR) mechanism to "knock in" the CD22 CAR into the TRAC gene (encoding the TCR alpha subunit of the TCR), resulting in a more efficient product.

[0334] HDR with exogenous DNA sequences has been previously described in T cells using short oligonucleotides paired with CRISPR / Cas9. Others have shown that adeno-associated virus (AAV) vectors can be used as templates in conjunction with site-specific nucleases Crispr / Cas9 or MegaTAL to achieve gene insertion by HDR. In the present invention, a novel method is provided that combines adeno-associated virus (AAV) vectors and TALENs to insert CAR into TCR genes.

[0335] In UCART22, AAV6 vectors can be used following gene editing using specific TALENs of a gene such as the TRAC gene (the gene encoding the alpha subunit of the TCR) or any of the genes disclosed in PCT / EP2017 / 076798.

[0336] Since TALENs are specific for DNA sequences and allow the integration of sequences into a gene, preferably the TRAC gene, the present invention also provides engineered immune cells comprising a sequence encoding a CAR, preferably a CD22 CAR as described above, located in a precise region of the TRAC gene determined by the TALEN used, the sequence in the TRAC gene of said engineered immune cells being unique due to the TALEN specificity.

[0337] Accordingly, the present invention provides an engineered immune cell comprising the following sequence: (YYY) n -ZZZ-(XXX) m where n = 1 to at least 10, and m is = 1 to 100, preferably m is > 100, and represents the number of base pairs of the sequence to be integrated; wherein ZZZ encodes a self-cleaving peptide, such as a 2A peptide, in frame with a sequence encoding TRAC; Y is A or T or G or C, and at least TIFF0007634045000026.tif4128 preferably and (XXX)n is A or T or G or C and is a portion of the exogenous sequence to be inserted into the TRAC gene, preferably a sequence encoding a CAR, more preferably a sequence encoding a CD22 CAR.

[0338] In one embodiment, the TRAC gene is deleted and the inserted gene is expressed under the control of the TRAC promoter.

[0339] Additional or alternative sequences, such as an IRES internal ribosome entry site, can be inserted between the TALEN target and the XXX.

[0340] In the present invention, the TALEN target is SEQ ID NO:21 and the CAR CD22 is SEQ ID NO:22.

[0341] In one embodiment, the sequence cleaved by the TALEN is TIFF0007634045000028.tif3128. Array TIFF0007634045000029.tif3128 may be at least partially conserved in engineered cells of the invention depending on the insert sequence (see FIG. 9).

[0342] The present invention provides engineered immune cells that contain a TRAC gene comprising SEQ ID NO:22.

[0343] The present invention provides a method for the preparation of a nucleic acid sequence for a nucleic acid sequence encoding a nucleic acid fragment of a human TRAC gene, preferably at the locus: The present invention provides an engineered immune cell comprising a sequence encoding a CD22-specific CAR or a sequence encoding a CD19-specific CAR inserted anywhere in TIFF0007634045000030.tif3128.

[0344] Thus, in certain embodiments, the engineered immune cells of the invention comprise two different sequences encoding CARs and express the two different CARs. Undetectable levels of TCR at the cell surface.

[0345] Thus, in certain embodiments, the engineered immune cells of the invention comprise two different sequences encoding CARs and express the two different CARs: undetectable levels of TCR and MHC class I at the cell surface.

[0346] The engineered immune cells of the present invention comprise in their genomic TRAC gene a sequence encoding CD22, preferably SEQ ID NO:22, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD19 has been inserted.

[0347] The engineered immune cells of the present invention comprise, in their genomic TRAC gene, a sequence encoding CD22, preferably SEQ ID NO:22, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD34 has been inserted.

[0348] The engineered immune cells of the present invention comprise a sequence encoding CD22, preferably SEQ ID NO:22, in their genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD79a has been inserted.

[0349] The engineered immune cells of the present invention comprise a sequence encoding CD22, preferably of SEQ ID NO:22, in their genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD79b has been inserted.

[0350] The engineered immune cells of the present invention comprise in their genomic TRAC gene a sequence encoding CD22, preferably SEQ ID NO:22, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD10 has been inserted.

[0351] The engineered immune cells of the present invention comprise a sequence encoding CD22, preferably of SEQ ID NO:22, in their genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for IgD has been inserted.

[0352] The engineered immune cells of the present invention comprise in their genomic TRAC gene a sequence encoding CD22, preferably SEQ ID NO:22, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD5 has been inserted.

[0353] The engineered immune cells of the present invention comprise in their genomic TRAC gene a sequence encoding CD22, preferably SEQ ID NO:22, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD23 has been inserted.

[0354] The engineered immune cells of the present invention comprise in their genomic TRAC gene a sequence encoding CD22, preferably SEQ ID NO:22, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD30 has been inserted.

[0355] The engineered immune cells of the present invention comprise a sequence encoding CD22, preferably SEQ ID NO:22, in their genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for BCMA has been inserted.

[0356] The engineered immune cells of the present invention comprise a sequence encoding CD22, preferably SEQ ID NO:22, in their genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for FLT3 has been inserted.

[0357] The engineered immune cells of the present invention comprise a sequence encoding CD22, preferably of SEQ ID NO:22, in their genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD138 has been inserted.

[0358] The engineered immune cells of the present invention comprise a sequence encoding CD22, preferably SEQ ID NO:22, in their genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD80 has been inserted.

[0359] The engineered immune cells of the present invention comprise a sequence encoding CD22, preferably of SEQ ID NO:22, in their genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD86 has been inserted.

[0360] The other genomic sequence into which another exogenous gene encoding a second CAR is inserted can be any one of the genes disclosed in PCT / EP2017 / 076798 or WO2017069958A2.

[0361] Other exogenous genes encoding CARs can be inserted into the TRAC gene in frame with the self-cleaving peptide and the CD22 CAR sequence (preferably SEQ ID NO:), or into another gene encoding a protein such as those disclosed in PCT / EP2017 / 076798 or selected from among the following: TIFF0007634045000031.tif112153

[0362] Preferred edited (KO) genes in UCART 22 are: The file is TIFF0007634045000032.tif24160.

[0363] Preferably, the gene into which the second CAR is inserted is a genomic gene active during T cell activation selected from any one of those listed in Table A: (Table A) TIFF0007634045000033.tif153128 or in the safe harbor locus or Table AA as disclosed in PCT / EP2017 / 076798.

[0364] (Table AA) TIFF0007634045000034.tif142158

[0365] The other gene into which the second CAR can be inserted can be a gene encoding any one of the proteins in Table B selected from the following:

[0366] (Table B) TIFF0007634045000035.tif43128TIFF0007634045000036.tif242111TIFF0007634045000037.tif246111 TIFF0007634045000038.tif250111TIFF0007634045000039.tif238111TIFF0007634045000040.tif150128

[0367] UCART22 can be prepared using an AAV vector, preferably an AAV6 vector, and even more preferably an AAV6 / 2 vector, to insert CD22 CAR or another gene as disclosed herein. The AAV vector can be used after gene editing, such as the TRAC gene (the gene encoding the TCR or alpha subunit) or any gene disclosed in PCT / EP2017 / 076798, using a specific endonuclease.

[0368] Adoptive cell transfer is the transfer of cells into a patient. The cells may originate from the patient themselves, then be modified and then transferred back (syngeneic transfer), or they may originate from another individual. The cells are most commonly derived from the immune system, with the aim of transferring improved immune functionality and characteristics back to the patient along with the cells. Transferring autologous cells, or cells from the patient, minimizes graft-versus-host disease (GVHD) or tissue or organ rejection. Similarly, transferring TCR-deficient T-cell CD22 CART cells minimizes GVHD. Transferring TCR-deficient MHCI-deficient CD22 CART minimizes GVHD and HVGD.

[0369] In one embodiment, the step of genetically modifying (manipulating) the immune cells is carried out before the step of introducing into the cells a polynucleotide or vector encoding one of the CD22 CARs of the present invention.According to the first aspect, the immune cells can be made less allogeneic by inactivating at least one gene expressing one or more components of T cell receptor (TCR), as described in WO 2013 / 176915, which may be combined with the inactivation of a gene encoding or controlling HLA expression, such as the β2m gene, as described in WO2008102199 or WO2015136001 or WO2016201047 (all of which are incorporated herein by reference).Therefore, the risk of graft-versus-host syndrome and the risk of graft rejection are significantly reduced.

[0370] According to another aspect, the immune cells of the present invention can be further genetically engineered to improve the resistance of the engineered immune cells to immunosuppressive drugs or chemotherapy treatments used as standard medical care to treat CD22-positive malignant cells, as disclosed in WO201575195, which is incorporated herein by reference.

[0371] Resistance to Campath (alemtuzumab) In one preferred embodiment, the genes that can be inactivated to confer drug resistance to T cells are glucocorticoid receptor (GR) and CD52. The genes are inactivated to make the cells resistant to these treatments and give the cells a competitive advantage over the patient's own T cells that have not been given a specific CD22 CAR.

[0372] Inactivation of the CD52 and TRAC genes in engineered immune cells according to the present invention is carried out using TALE nucleases or the CRISPR CAS9 system.

[0373] In one more preferred embodiment, the gene that can be inactivated to confer drug resistance to T cells is CD52 in TCR KO immune T cells loaded with a CD22 CAR.

[0374] In one preferred embodiment, the gene that can be inactivated to confer drug resistance to T cells is the glucocorticoid receptor (GR).

[0375] Expression of the CD3 gene may also be suppressed or reduced to confer resistance to another immunosuppressant, teplizumab. Expression of HPRT may also be suppressed or reduced in accordance with the present invention to confer resistance to 6-thioguanine, a cytostatic drug commonly used in chemotherapy, particularly for the treatment of acute lymphoblastic leukemia.

[0376] Deletion of the human deoxycytidine kinase (dCK) gene confers resistance to purine nucleotide analogues. In one preferred embodiment, the gene that can be inactivated to confer drug resistance to T cells is the human deoxycytidine kinase (dCK) gene. This enzyme is required for the phosphorylation of the deoxyribonucleosides deoxycytidine (dC), deoxyguanosine (dG) and deoxyadenosine (dA). Purine nucleotide analogs (PNAs) are metabolized by dCK to PNA monophosphate, PNA diphosphate, and PNA triphosphate. Their triphosphate forms, especially clofarabine triphosphate, compete with ATP for DNA synthesis, act as proapoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production.

[0377] Inactivation of dCK gene in engineered immune cells according to the present invention is mediated by TALE nuclease or CRISPR CAS9 system.To achieve this goal, several pairs of dCK TALE nucleases are designed at polynucleotide level, assembled and verified by sequencing.Examples of TALE nuclease pairs that can be used according to the present invention are described in PCT / EP2014 / 075317.

[0378] This dCK inactivation in the engineered immune cells of the present invention confers resistance to purine nucleoside analogs (PNAs) such as clofarabine and fludarabine.

[0379] In another preferred embodiment, dCK inactivation in the engineered immune cells of the present invention, combined with inactivation of the TRAC gene, renders these double knockout (KO) (TCR or TRAC KO and dCK KO) cells resistant to drugs such as clofarabine and less allogenic.

[0380] In another preferred embodiment, CD52 inactivation in the engineered immune cells of the present invention, combined with inactivation of the TRAC gene, renders these double knockout (KO) (TCR or TRAC KO and CD52 KO) cells resistant to drugs such as CAMPATH (alemtuzumab) and less allogeneic.

[0381] This dual feature is particularly useful for therapeutic purposes, allowing the use of immunotherapy "off-the-shelf" allogeneic cells (UCART22) in combination with chemotherapy to treat patients with cancer in need of treatment. This dual KO inactivation of dCK / TRAC or CD52 / TRAC can be performed simultaneously or sequentially. One example of a successful TALE nuclease dCK / TRAC pair in the present invention is described in PCT / EP2014 / 075317, in particular the target sequences of two loci (dCK and TRAC). The document PCT / EP2014 / 075317 is incorporated herein in its entirety.

[0382] The present invention provides primary T cells expressing a CD22 CAR of SEQ ID NO:15, wherein the CD52 and TRAC genes have been inactivated by deletion for their use in the treatment of CLL, ALL, preferably aggressive, relapsed and refractory forms thereof, more preferably relapsed and refractory forms of B-ALL, optionally in lymphodepleted patients.

[0383] According to a further aspect of the invention, immune cells can be further engineered to be more active or to limit attrition by inactivating genes that code for proteins that act as "immune checkpoints" that act as regulators of T cell activation, such as PDCD1 or CTLA-4. Examples of genes whose expression can be reduced or suppressed are shown in Table 9.

[0384] The present invention also provides primary T cells expressing the CD22 CAR of SEQ ID NO:15, in which the CD52, TRAC and dCK genes have been deleted.

[0385] In another embodiment, the present invention also provides primary T cells expressing a CD22 CAR that are made tolerant to hypoxia.

[0386] The present invention also provides primary T cells expressing the CD22 CAR of SEQ ID NO:15 in which the CD52, TRAC and dCK genes have been deleted and a sequence for HIF-1a has been inserted, rendering the cells resistant to hypoxia.

[0387] Engineered cell tolerance to hypoxia In certain embodiments, under low oxygen conditions (hypoxia) (1-5% O2 compared to normal oxygen conditions 20% O2), expression and cytolytic activity of the CD22 CAR T cells of the invention is maintained, or expression of the CD22 CAR T cells is induced and activity is maintained, and the cells still target and destroy tumor cells when implanted into tissue.

[0388] Examples of hypoxia-inducible CARs in T cells are described in (WO2013123061 or Juillerat, A. et al. An oxygen sensitive self-decision making engineered CAR T-cellSci. Rep. 7, 39833; doi: 10.1038 / srep39833 (2017); both incorporated by reference): A synthetic promoter specific for OxiTF was constructed that drives expression of the CD22 CAR. OxiTF is designed to activate a synthetic genetic element that encodes the CD22 CAR. Upon encountering a tumor (making hypoxia), the engineered T cells can "detect" the decrease in oxygen levels (when compared to the average level of O2 in the blood) and trigger expression of the CD22 CAR. Cell surface exposure of the CD22 CAR allows for recognition of tumor antigens under hypoxia, which ultimately triggers T cell activation and proliferation via activation and co-stimulatory domains present within the CD22 CAR. Finally, the tumor antigen-expressing cells are lysed by UCART22 of the present invention.

[0389] In the present invention, immune cells can also be engineered to remain efficient under low O2 conditions (low oxygen concentration average 1-5%) by overexpressing at least one, and preferably all, of the following factors: Oct3, Oct4, Sox2, Klf4 and c-Myc, or by editing the HIF-1a factor.

[0390] In the present invention, oxygen-sensitive CD22 single-chain CAR-expressing engineered cells and hypoxia-resistant CD22 CAR-expressing engineered cells were successfully constructed and tested.

[0391] Since CD22 is mainly expressed on CD22-expressing B-cell malignancies, which are "liquid" tumors and therefore not supposed to create hypoxia in contrast to solid tumors, it was not expected that CD22 CAR-engineered immune cells that are resistant to hypoxia would be more efficient against patient-derived B-ALL than CD22 CAR-engineered immune cells that are not resistant to hypoxia. Indeed, the UCART22 of the present invention, which reaches nested cancer cells clustered or living in tissues, can lyse these cells.

[0392] Other genes, such as those in Table 5 above, may be edited in the UCART22 of the present invention.

[0393] Table 5: List of genes encoding immune checkpoint proteins that can be inactivated according to the present invention in the CD22 CAR engineered T cells of the present invention. TIFF0007634045000041.tif201140

[0394] The present invention provides isolated engineered immune T cells expressing CD22 CAR of SEQ ID NO:15, for their use in the treatment of CLL, ALL, preferably aggressive, relapsed, refractory forms thereof, in lymphodepleted patients, wherein the dCK and / or CD52 and TRAC genes have been edited, i.e., inactivated by deletion.

[0395] The present invention provides isolated engineered immune T cells expressing CD22 CAR of SEQ ID NO:15, wherein the b2m and TRAC genes have been edited, i.e., inactivated by deletion, for their use in the treatment of CLL, ALL, preferably aggressive, relapsed and refractory forms thereof, in lymphodepleted patients.

[0396] The present invention provides isolated engineered immune T cells expressing CD22 CAR of SEQ ID NO:15, in which the TRAC and IL-10R genes have been edited, i.e., inactivated by deletion, for their use in the treatment of CLL, ALL, preferably aggressive, relapsed, refractory forms thereof, in lymphodepleted patients. The present invention provides engineered immune T cells expressing CD22 CAR (UCART22) that contain a defective TCR alpha gene (resulting in impaired cell surface expression of TCR), a defective B2M gene (resulting in impaired expression of MHC class I molecules at the cell surface), and additionally a TGF beta receptor KO gene.

[0397] The present invention provides engineered immune T cells expressing a CD22 CAR (UCART22) that contain a defective TCR alpha gene (resulting in impaired cell surface expression of the TCR), a defective B2M gene (resulting in impaired expression of MHC class I molecules at the cell surface), and additionally an IL-10 receptor KO gene.

[0398] The present invention provides engineered immune T cells expressing a CD22 CAR (UCART22) that contain a defective TCR alpha gene (resulting in impaired cell surface expression of the TCR), a defective B2M gene (resulting in impaired expression of MHC class I molecules at the cell surface), and additionally an AHR KO gene.

[0399] The present invention provides engineered immune T cells expressing a CD22 CAR (UCART22) that contain a defective TCR alpha gene (resulting in impaired cell surface expression of the TCR), a defective B2M gene (resulting in impaired expression of MHC class I molecules at the cell surface), and additionally a PD1 KO gene.

[0400] The present invention provides engineered immune T cells expressing a CD22 CAR (UCART22) that contain a defective TCR alpha gene (resulting in impaired cell surface expression of the TCR), a defective B2M gene (resulting in impaired expression of MHC class I molecules at the cell surface), and additionally a LAG-3 KO gene.

[0401] The present invention provides engineered immune T cells expressing a CD22 CAR (UCART22) that contain a defective TCR alpha gene (resulting in impaired cell surface expression of the TCR), a defective B2M gene (resulting in impaired expression of MHC class I molecules at the cell surface), and additionally a TIM-3 KO gene.

[0402] The present invention provides isolated engineered immune T cells expressing CD52 and the CD22 CAR of SEQ ID NO:15, in which the TRAC gene has been inactivated by deletion for their use in the treatment of relapsed refractory forms of BALL in lymphodepleted patients.

[0403] In a preferred embodiment, the method of engineering immune cells involves the introduction into said T cells of a polynucleotide, in particular an mRNA, encoding a specific rare-cutting endonuclease that selectively inactivates genes, such as those mentioned above, by DNA cleavage.

[0404] In a more preferred embodiment, the rare-cutting endonuclease is a TALE nuclease or a Cas9 endonuclease. TAL nucleases have been previously demonstrated to have higher specificity and cleavage efficiency than other types of rare-cutting endonucleases, and are therefore the endonuclease of choice for large-scale production of engineered immune cells with constant turnover.

[0405] Delivery method The above-mentioned various methods involve the introduction of the CD22 CAR of the present invention into cells. As a non-limiting example, the CD22 CAR can be introduced as a transgene encoded by one of the plasmid vectors of the present invention. The plasmid vector encoding the CD22 CAR of the present invention can also contain a selection marker that provides for the identification and / or selection of cells that have received the vector.

[0406] Methods by which the CD22 CAR according to the present invention can be introduced into isolated immune cells and then expressed are described elsewhere, for example in WO2013126720 or WO2015121454, which are incorporated herein by reference in their entirety.

[0407] The polypeptide corresponding to the CD22 CAR of the present invention can be synthesized in situ in a cell as a result of introducing a polynucleotide encoding the polypeptide into the cell. Alternatively, the polypeptide can be produced outside the cell and then introduced into the cell. Methods for introducing a polynucleotide construct into a cell are known in the art, and include, as non-limiting examples, stable transformation methods in which the polynucleotide construct is integrated into the genome of the cell, transient transformation methods in which the polynucleotide construct is not integrated into the genome of the cell, and methods mediated by viruses. The polynucleotide can be introduced into a cell, for example, by recombinant viral vectors (e.g., retroviruses, e.g., lentiviruses, adenoviruses, adeno-associated viruses), liposomes, etc. For example, transient transformation methods include, for example, microinjection, electroporation, or particle bombardment. The polynucleotide can be included in a vector, more specifically, a plasmid or a virus, for expression in the cell.

[0408] In a preferred embodiment, the polynucleotide encoding the CD22 CAR is inserted into an AAV6 vector and introduced into a predetermined gene. Methods for preparing vectors that allow the CD22 CAR of the present invention to be introduced into isolated T cells and then expressed are described elsewhere, for example in WO2013126720, which is incorporated herein by reference.

[0409] Manipulated immune cells (UCART) Engineered immune cells loaded with CD22 CAR (UCART 22) are another object of the present invention.

[0410] Preferably, said immune cells are isolated immune cells, more preferably isolated immune T cells, more preferably isolated primary immune T cells.

[0411] UCART 22 is provided as a medicament, and thus a therapeutically effective amount of UCART 22 is provided as a medicament.

[0412] By "primary immune cells" according to the present invention is meant cells derived from tissues, e.g. blood samples, or peripheral blood mononuclear cells (PBMCs), which may be cultured for several passages and eventually frozen before use, said primary immune cells having a limited division potential compared to transformed or cancerous cells (Raulf-Heimsoth M. T cell - primary culture from peripheral blood. Methods Mol Med. 2008;138:17-30. doi: 10.1007 / 978-1-59745-366-0).

[0413] The immune cells according to the invention are preferably immune T or NK cells. Thus, the engineered immune cells according to the invention are isolated from a blood sample, are primary cells, are derived from immune T cells selected from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes, natural killer T cells, preferably cytotoxic T lymphocytes, and are further engineered.

[0414] By engineered it is meant that the primary immune cells have been modified such that they are loaded with a CD22 CAR and at least one gene is edited, preferably the cells have been loaded with a CD22 CAR and modified such that they do not express a TCR and do not die in the presence of purine nucleotide analogues.

[0415] In other words, an engineered immune cell refers to a TCR KO isolated immune T cell that expresses a CD22 CAR and contains at least one other edited gene.

[0416] In certain embodiments, engineered means that the primary immune cells have been modified to have a CD22 CAR attached to them, preferably the cells have been modified to have a CD22 CAR attached to them and not die in the presence of purine nucleotide analogs (1-5 micromoles / L) or in the presence of alemtuzumab (50 micrograms / mL). Valton et al., Molecular Therapy vol. 23 no. 9, 1507-1518 sep. 2015).

[0417] Preferably, the T cells are armed with a CD22 CAR of SEQ ID NO:15.

[0418] More preferably, the T cells are armed with a CD22 CAR of SEQ ID NO:15 and comprise at least one sequence of SEQ ID NO:22.

[0419] More preferably, the T cells are armed with a CD22 CAR of SEQ ID NO:15 and comprise at least one sequence and at least a portion of the sequence of SEQ ID NO:22.

[0420] More preferably, the T cells are armed with a CD22 CAR of SEQ ID NO:18, which comprises at least one sequence of SEQ ID NO:20.

[0421] The present invention provides primary immune T cells that express the CD22 CAR of the present invention and exhibit CTL and / or degranulation activity against CD22-expressing cells.

[0422] The present invention also provides primary T cells expressing the CD22 CAR of the present invention for lysing CD22-expressing cells, particularly CD22-expressing cancerous cells.

[0423] Preferably, cells targeted by T cells equipped with the CD22 CAR of SEQ ID NO:16 of the present invention are preferably efficient in the treatment of relapsed / refractory / aggressive ALL or CLL.

[0424] The present invention also relates to an isolated cell or cell line obtainable by said method for engineering cells.Specifically, said isolated cell comprises at least one of the above-mentioned CD22 CARs of the present invention.In another embodiment, said isolated cell comprises a population of CARs, each of which comprises a different extracellular ligand-binding domain.Specifically, said isolated cell comprises an exogenous polynucleotide sequence encoding a CAR.The genetically modified immune cell of the present invention is activated and can proliferate independently of antigen binding mechanism.

[0425] Also included within the scope of the present invention is an isolated immune cell, preferably an isolated immune T cell (T-cell), more preferably an engineered isolated immune T cell, obtained by any one of the methods already described. Said immune cell refers to a cell of hematopoietic origin functionally involved in the initiation and / or execution of an innate and / or adaptive immune response. Said immune cell according to the present invention may be derived from a stem cell. Stem cells may be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. A representative human cell is a CD34+ cell. Said isolated cell may be a dendritic cell, a killer dendritic cell, a mast cell, a NK cell, a B cell, or a T cell selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another embodiment, said cell may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes.

[0426] Prior to the expansion and genetic modification of the cells of the present invention, the source of cells can be obtained from a subject through various non-limiting methods. Cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue, ascites, pleural effusion, splenic tissue, and tumor. In certain embodiments of the present invention, a number of T cell lines available and known to those skilled in the art can be used. In another embodiment, the cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with infection. In another embodiment, the cells are part of a mixed population of cells that present different phenotypic characteristics. Cell lines obtained from engineered T cells according to the described method are also within the scope of the present invention. Modified cells that are resistant to immunosuppressive treatment and are obtainable by previous methods are also within the scope of the present invention.

[0427] In a preferred embodiment, the present invention provides a T cell or population of T cells bearing the above-mentioned CD22 CAR of the present invention, which does not express a functional TCR and is reactive against CD22 positive cells, for their adoptive transfer into patients.

[0428] In a more preferred embodiment, the present invention provides a T cell or population of T cells armed with the above-mentioned CD22 CAR that does not express a functional TCR and is rendered resistant to chemotherapy, in particular to purine nucleotide analogues (PNAs).

[0429] According to a preferred embodiment of the present invention, immune cells equipped with CD22 CAR are engineered to be resistant to chemotherapeutic drugs, in particular to purine nucleotide analogs (PNAs), and are therefore suitable for cancer treatment combining adoptive immunotherapy and chemotherapy.

[0430] Purine nucleotide analogs are included in many chemotherapy compositions for the treatment of cancer. They are used as standard treatment for leukemia or lymphoma. The most widely used PNAs, alone or in combination, are clofarabine, fludarabine and cytarabine. PNAs are metabolized to PNA monophosphate, PNA diphosphate and PNA triphosphate by enzymes with deoxycytidine kinase (dCK) activity [EC 2.7.1.74]. Their triphosphate forms, especially chlorofarabine triphosphate, compete with ATP for DNA synthesis, acting as proapoptotic agents and are potent inhibitors of ribonucleotide reductase (RNR) involved in trinucleotide production.

[0431] The present invention therefore includes a method for producing ex vivo immune cells, preferably primary T cells, that do not express a TCR, that are resistant to purine analog drugs and that are capable of targeting CD22-positive malignant cells.

[0432] The method for preparing UCART22 according to the present invention may also be that disclosed in WO 2013176915 or WO 2014191128, the entireties of which are incorporated herein by reference.

[0433] The method for preparing UCART22 comprises the steps of: (a) providing immune cells from a donor, preferably an isolated T cell or a population of isolated T cells; (b) introducing into said immune cells (preferably by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets - a gene expressing an enzyme with deoxycytidine kinase activity (dcK - EC 2.7.1.74), in particular the human deoxycytidine kinase gene (NCBI Gene ID: 1633), and / or - a gene encoding one of the TCR subunits alpha and / or beta, preferably alpha, and / or - The gene encoding human CD52 (c) expressing the endonuclease in the immune cells to obtain targeted inactivation of the gene; (d) expanding the engineered immune cells obtained in step c), optionally in the presence of a purine analog drug; (e) introducing the CD22 CAR of the present invention, preferably of SEQ ID SEQ ID NO:15, into said immune cells.

[0434] The method for preparing UCART22 comprises the steps of: (a) providing immune cells from a donor, preferably an isolated T cell or a population of isolated T cells; (b) introducing into said immune cells (preferably by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets - a gene expressing an enzyme with deoxycytidine kinase activity (dcK - EC 2.7.1.74), in particular the human deoxycytidine kinase gene (NCBI Gene ID: 1633), and / or - a gene encoding one of the TCR subunits alpha and / or beta, preferably alpha, and / or - The gene encoding human beta 2 microglobulin (B2M) (c) expressing the endonuclease in the immune cells to obtain targeted inactivation of the gene; (d) expanding the engineered immune cells obtained in step c), optionally in the presence of a purine analog drug; (e) introducing a CAR of the present invention, preferably a CD22 CAR, alone or in combination with a CD19 CAR, even more preferably a CD22 CAR of SEQ ID NO:15, alone or in combination with a CD19 CAR, into the immune cells.

[0435] In a preferred embodiment, the present invention comprises a method for producing ex vivo immune cells, preferably primary T cells, that do not express TCR, that are resistant to purine analog drugs and capable of targeting CD22-positive malignant cells, comprising the steps of: Providing immune cells from a donor, preferably isolated T cells (or a population of isolated T cells), introducing into said immune cells a CD22 CAR of the invention, preferably of a sequence ID selected from SEQ ID NO:15; - expanding the engineered immune cells obtained in step b) (d) introducing into said immune cells (by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets a gene expressing an enzyme with deoxycytidine kinase activity (dcK - EC 2.7.1.74), in particular the human deoxycytidine kinase gene (NCBI Gene ID: 1633), and / or a gene expressing one of the TCR subunits alpha or beta, (e) expressing the endonuclease in the immune cells to obtain targeted inactivation of the gene; (f) Expanding the engineered immune cells obtained in step e), optionally in the presence of a purine analog drug.

[0436] The method for producing ex vivo immune cells, preferably primary T cells, that do not express a TCR, that are resistant to purine analog drugs and that are capable of targeting CD22-positive malignant cells, optionally comprises a further step of introducing (by transfection or transduction) into said immune cells a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets one of the genes cited in Table 9, preferably PD-1, CD279, more preferably PDCD1 (PD-1, CD279) or CTLA4 (CD152).

[0437] The inventors have successfully generated CD22 CAR-expressing primary T cells that are resistant to purine nucleotide analogs (dCK-KO), more specifically to chlorofarabine and / or fludarabine, by mediating inactivation (deletion) of dCK gene expression in the cells by using specifically nucleases, in particular TAL-nucleases.

[0438] Transfection of T cells with mRNA encoding a specific TAL nuclease directed against the dCK gene, preferably by using electroporation as described in WO2013176915, induced significant resistance to the drug while maintaining T cell cytotoxic activity against CD22-bearing cells.

[0439] The present application also provides TCR-KO, CD22 CAR (preferably of SEQ ID NO:15) primary T cells with silenced or inactivated expression of deoxycytidine kinase (dCK-KO) for the treatment of leukemia or lymphoma, preferably aggressive, resistant or relapsed forms thereof, more preferably B-ALL.

[0440] According to a preferred embodiment of the present invention, immune cells equipped with CD22 CAR are engineered to be resistant to chemotherapeutic drugs, in particular to alemtuzumab (CAMPATH), and are therefore suitable for cancer treatment combining adoptive immunotherapy and chemotherapy.

[0441] Alemtuzumab is used for many cancer treatments. It is used as a standard of care for leukemia or lymphoma, especially in the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma. It is known under the trademarks Campath, MabCampath and Campath-1H. It is also used in some conditioning regimens for bone marrow transplants, kidney transplants and islet cell transplants.

[0442] It is a monoclonal antibody that binds to CD52, a protein present on the surface of mature lymphocytes but not on the stem cells from which these lymphocytes originate. Following treatment with alemtuzumab, these CD52-bearing lymphocytes are targeted for destruction.

[0443] Alemtuzumab is also used as a second-line therapy for CLL. It has been approved by the U.S. Food and Drug Administration for CLL patients who have been treated with alkylating agents and who have failed fludarabine therapy.

[0444] The present invention therefore includes a method of producing UCART22 ex vivo that is resistant to alemtuzumab and thus does not express TCR.

[0445] The method for preparing UCART22 CD52 KO comprises the following steps: (a) providing immune cells from a donor, preferably an isolated T cell or a population of isolated T cells; (b) introducing into said immune cells (preferably by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets - a gene encoding one of the TCR subunits alpha and / or beta, preferably alpha, and / or - the gene encoding CD52, (c) expressing the endonuclease in the immune cells to obtain targeted inactivation of the gene; (d) expanding the engineered immune cells obtained in step c), optionally in the presence of alemtuzumab; (e) introducing the CD22 CAR of the present invention, preferably of SEQ ID NO:15, into said immune cells.

[0446] In a preferred embodiment, the present invention comprises a method for producing ex vivo immune cells, preferably primary T cells, that do not express TCR and are resistant to alemtuzumab and capable of targeting CD22-positive malignant cells, comprising the steps of: Providing immune cells from a donor, preferably isolated T cells (or a population of isolated T cells), - introducing the CD22 CAR of the invention, preferably of SEQ ID NO:15, into said immune cells. - expanding the engineered immune cells obtained in step b) (d) introducing into said immune cells (by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets a gene expressing CD52 and a gene expressing one of the TCR subunits alpha or beta; (e) expressing the endonuclease in the immune cells to obtain targeted inactivation of the gene; (f) Expanding the engineered immune cells obtained in step e), optionally in the presence of a purine analog drug.

[0447] In one embodiment, the method for preparing UCART22 comprises the steps of: (a) providing immune cells from a donor, preferably an isolated T cell or a population of isolated T cells; (b) introducing into said immune cells (preferably by transfection or transduction): - a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets a gene encoding one of the TCR subunits alpha and / or beta, preferably alpha; and - a nucleic acid encoding the sequence to be inserted, preferably encoding HIF-1 alpha, (c) expressing the endonuclease in the immune cells to obtain targeted insertion of the sequence to be inserted; (d) expanding the engineered immune cells obtained in step c), optionally in the presence of low O2 concentration (5% O2, preferably 1% O2); (e) introducing the CD22 CAR of the present invention, preferably of SEQ ID SEQ ID NO:15, into said immune cells.

[0448] In a preferred embodiment, the present invention comprises a method for producing ex vivo immune cells, preferably primary T cells, that do not express TCR, that are resistant to hypoxia and capable of targeting CD22-positive malignant cells, comprising the steps of: Providing immune cells from a donor, preferably isolated T cells (or a population of isolated T cells), - introducing into said immune cells the CD22 CAR of the invention, preferably of SEQ ID NO:15, - expanding the engineered immune cells obtained in step b) (d) introducing into said immune cells (preferably by transfection or transduction): - a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets a gene encoding one of the TCR subunits alpha and / or beta, preferably alpha; and - a nucleic acid encoding an inserted sequence encoding HIF-1 alpha, (e) expressing the endonuclease in the immune cells to obtain targeted insertion of the gene; (f) Expanding the engineered immune cells obtained in step e), optionally in the presence of low O2 concentration (5% O2, preferably 1% O2).

[0449] The method of ex vivo production of immune cells, preferably primary T cells, that do not express a TCR, that are resistant to either purine analog drugs, alemtuzumab or hypoxia, and that are capable of targeting CD22-positive malignant cells, optionally comprises a further step of introducing (by transfection or transduction) into said immune cells a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets one of the genes cited in Table 9, preferably (PD-1, CD279), more preferably PDCD1 (PD-1, CD279) and CTLA4 (CD152).

[0450] The inventors have successfully generated CD22 CAR-expressing primary T cells that are resistant to purine nucleotide analogs (dCK-KO), more specifically to chlorofarabine and / or fludarabine, by mediating inactivation (deletion) of dCK gene expression in the cells by using specifically nucleases, in particular TAL-nucleases.

[0451] Transfection of T cells with mRNA encoding a specific TAL nuclease directed against the dCK gene, preferably by using electroporation as described in WO2013176915, induced significant resistance to the drug while maintaining T cell cytotoxic activity against CD22-bearing cells.

[0452] The same method is applied to the deletion of human CD52 using the specific TALENs described by the inventors.

[0453] The present application also provides TCR-KO, CD22 CAR (preferably of SEQ ID NO:15) primary T cells with silenced or inactivated expression of deoxycytidine kinase (dCK-KO) for the treatment of leukemia or lymphoma, preferably aggressive, resistant, relapsed forms thereof; more preferably relapsed B-ALL.

[0454] Such cells are "universal" T cells (or UCARTs).

[0455] In one aspect, the application provides CD22 CAR primary T cells (preferably of SEQ ID NO:15) with suppressed or inactivated expression of deoxycytidine kinase (dCK-KO) for the treatment of leukemia or lymphoma, preferably aggressive, resistant, relapsed forms thereof; more preferably relapsed B-ALL after autologous transfer.

[0456] In one aspect, the application provides CD22 CAR primary T cells (preferably of SEQ ID NO:15) with suppressed or inactivated expression of CD52 (CD52-KO) for the treatment of leukemia or lymphoma, preferably aggressive, resistant, relapsed forms thereof; more preferably relapsed B-ALL after autologous transfer.

[0457] In one aspect, the present application relates to a method for the treatment of leukemia or lymphoma, preferably aggressive, resistant, relapsed forms thereof; more preferably relapsed B-ALL after autologous transfer. Provided are CD22 CAR primary T cells (preferably of SEQ ID NO:15) in which expression of HIF-1 alpha is increased by inserting a coding sequence into the TRAC sequence without knocking out the TCR.

[0458] In one aspect, the application provides CD22 CAR primary T cells (preferably of SEQ ID NO:15) with suppressed or inactivated expression of deoxycytidine kinase and CD52 (dCK- and CD52KO) for the treatment of leukemia or lymphoma, preferably aggressive, resistant, relapsed forms thereof; more preferably relapsed B-ALL after autologous transfer.

[0459] In one aspect, the application provides CD22 CAR primary T cells (preferably of SEQ ID NO:15) in which expression of deoxycytidine kinase and CD52 has been suppressed or inactivated (dCK- and CD52KO) and expression of HIF-1 alpha has been increased by inserting HIF1 alpha coding sequence into the TRAC sequence without knocking out the TCR, for the treatment of leukemia or lymphoma, preferably aggressive, resistant, relapsed forms thereof; more preferably relapsed B-ALL after autologous transfer.

[0460] The method for producing ex vivo immune cells, preferably primary T cells, that do not express a TCR, that are resistant to purine analog drugs and / or alemtuzumab and / or hypoxia and that are capable of targeting CD22-positive malignant cells, optionally comprises a further step of introducing (by transfection or transduction) into said immune cells a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets one of the genes cited in Table 9, preferably PD-1, CD279, more preferably PDCD1 (PD-1, CD279) or CTLA4 (CD152).

[0461] The inventors have successfully generated CD22 CAR-expressing primary T cells that are resistant to purine nucleotide analogs (dCK-KO), more specifically to chlorofarabine and / or fludarabine, by mediating inactivation (deletion) of dCK gene expression in the cells by using specifically nucleases, in particular TAL-nucleases.

[0462] The inventors have successfully generated CD22 CAR-expressing primary T cells that are resistant to hypoxia by targeted insertion of the HIF-1 alpha gene into the cells, specifically by using nucleases, in particular TAL-nucleases.

[0463] Transfection of T cells with mRNA encoding a specific TAL nuclease directed against the dCK gene, preferably by using electroporation as described in WO2013176915, induced significant resistance to the drug while maintaining T cell cytotoxic activity against CD22-bearing cells.

[0464] The present application also provides TCR-KO, CD22 CAR (preferably of SEQ ID NO:15) primary T cells, resistant to hypoxia, for the treatment of leukemia or lymphoma, preferably aggressive, resistant, relapsed forms thereof; more preferably B-ALL.

[0465] T cell activation and expansion Although the genetically modified immune cells of the present invention are activated and proliferate independent of antigen binding mechanisms, the immune cells of the present invention, specifically T cells, can be activated and proliferate independent of antigen binding mechanisms, either before or after genetic modification of the T cells, as described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,6 T cells can be further activated and expanded generally using methods such as those described in U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

[0466] Generally, the T cells of the present invention are expanded by contact with an agent that stimulates the CD3 TCR complex and costimulatory molecules on the surface of the T cells to generate an activation signal for the T cells.For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate an activation signal for the T cells.

[0467] As a non-limiting example, a population of T cells can be stimulated in vitro, for example, by contacting with a surface-immobilized anti-CD3 antibody or an antigen-binding fragment thereof or an anti-CD2 antibody, or by contacting with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under appropriate conditions to stimulate proliferation of T cells. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 5 (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human fetal serum), interleukin 2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2, 1L-15, TGFp, and TNF-, or other additives for proliferation of cells known to those skilled in the art. Other additives for the growth of cells include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for the growth and expansion of T cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% C02). T cells exposed to various stimulation times may exhibit different characteristics.

[0468] In another specific embodiment, the cells can be expanded by co-culture with tissue or cells. The cells can be expanded in vivo, for example in the blood of a subject, after administration of the cells to the subject.

[0469] Pharmaceutical Compositions A pharmaceutical composition comprising engineered (TCR and dCK KO) or (TRAC and CD52 KO) immune T cells expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) and a pharma- ceutically acceptable vehicle is another object of the present invention.

[0470] In another embodiment, the present invention provides a composition comprising a UCART22 of the present invention (as in any one of the above embodiments) together with a compound of the bryostatin family of compounds, preferably bryostatin-1.

[0471] Bryostatins are a group of macrolide lactones derived from the bryozoan Bugula neritina. The structure of bryostatin 1 was determined in the 1980s. To date, 20 different bryostatins have been isolated; furthermore, certain bryostatin analogues are called "bryologs". Bryostatin is a potent modulator of protein kinase C (Wender, Paul A., Jeremy L. Baryza,Chad E. Bennett,F. Christopher Bi,Stacey E. Brenner,Michael O. Clarke,Joshua C. Horan,Cindy Kan,Emmanuel Lacote,Blaise Lippa,Peter G. Nell, and, and Tim M. Turner. The Practical Synthesis of a Novel and Highly Potent Analogue of Bryostatin. Journal of the American Chemical Society 2002 124 (46), 13648-13649 DOI: 10.1021 / ja027509+).

[0472] Examples of bryostatin compounds suitable for compibed with the UCART22 of the present invention and methods for preparing these compounds are described in WO2001040214A1 or EP2737904A2, WO1997034598, which are incorporated herein by reference.

[0473] Examples of doses of bryostatin-1 that may be used in combination with UCART22 of the present invention are as previously described in Varterasian ML1, Mohammad RM, Shurafa MS, Hulburd K, Pemberton PA, Rodriguez DH, Spadoni V, Eilender DS, Murgo A, Wall N, Dan M, Al-Katib AM. Phase II trial of bryostatin 1 in patients with relapsed low-grade non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Clin Cancer Res. 2000 Mar;6(3):825-8.

[0474] Engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) [UCART22] or a pharmaceutical composition comprising said UCART22 are provided as medicaments.

[0475] Engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) [UCART22] for use in treating cancer or reducing inflammation is another object of the present invention.

[0476] Engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) [UCART22] for use in the treatment of ALL, CLL, relapsed refractory aggressive forms of CLL or ALL are another object of the present invention.

[0477] Provided are engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) [UCART22] for use in the treatment of CD19 recurrent cancer, preferably CD19 recurrent B-ALL.

[0478] In another embodiment, hypoxia-tolerant, engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) [UCART22] or a pharmaceutical composition comprising said UCART22 is provided as a medicament.

[0479] In another embodiment, hypoxia-tolerant, engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) [UCART22] for use in treating cancer or reducing inflammation is another object of the present invention.

[0480] In another embodiment, hypoxia-tolerant, engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) [UCART22] for use in the treatment of ALL, CLL, relapsed refractory aggressive forms of CLL or ALL is another object of the present invention.

[0481] In another embodiment, there is provided a hypoxia-tolerant, engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cell [UCART22] expressing the CD22 CAR of the present invention (preferably of SEQ ID NO:15) for use in the treatment of CD19 recurrent cancer, preferably CD19 recurrent B-ALL.

[0482] In another embodiment, the isolated cell obtained by the various methods of the present invention, or the cell line derived from said isolated cell, can be used as a medicament.In another embodiment, said medicament can be used to treat cancer in patients who need treatment, particularly for the treatment of B-cell lymphoma and leukemia.In another embodiment, said isolated cell according to the present invention or the cell line derived from said isolated cell can be used in the manufacture of medicament for the treatment of cancer in patients who need treatment.

[0483] The present invention encompasses autologous transfer of engineered cells, where cells are isolated from one donor, a human donor, engineered, and then transferred back to the original donor in need thereof.

[0484] In this particular embodiment, the cells can be engineered to be resistant to drugs such as, for example, alemtuzumab (campath) and / or pna, and optionally, to be resistant to hypoxia.

[0485] Therapeutic applications The term "cancer" refers to a disease characterized by the uncontrolled growth of one or more cells.

[0486] Examples of cancer are described herein and include, but are not limited to, liquid tumor or blood cancers.

[0487] Hematological cancers according to the present invention may be selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphoid carcinoma, acute myeloid leukemia, preferably CD22-expressing hematological cancers, more preferably relapsed or refractory CD22-expressing hematological cancers, and even more preferably aggressive forms of said CD22-associated hematological cancers.

[0488] In a preferred embodiment, the relapsed or refractory CD22-expressing hematological cancer is relapsed and / or refractory CD22 expressing or positive B-ALL.

[0489] Thus, a therapeutically effective amount of UCART 22 according to any one of the above-mentioned embodiments, or a therapeutically effective amount of the above-mentioned pharmaceutical composition, is provided for use as a medicament for treating patients suffering from relapsed and / or refractory CD22-expressing or positive B-ALL.

[0490] In another embodiment, a therapeutically effective amount of UCART 22 according to any one of the above-mentioned embodiments, or a therapeutically effective amount of the above-mentioned pharmaceutical composition is provided for use as a medicament for treating a patient suffering from a CD22-positive blood cancer selected from leukemia and lymphoma, hairy cell leukemia, any acute lymphoid cancer, acute lymphoid leukemia (ALL), acute myeloid leukemia, chronic lymphoid leukemia, B-chronic lymphoid leukemia, chronic myeloid cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Burkitt's lymphoma, multiple myeloma.

[0491] In another embodiment, a therapeutically effective amount of UCART 22 according to any one of the above-mentioned embodiments, or a therapeutically effective amount of the above-mentioned pharmaceutical composition is administered to treat or prevent the development of a cancer, such as alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cavity cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., The present invention provides a method for treating a patient suffering from a CD22-positive cancer selected from the group consisting of head and neck squamous cell carcinoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer), malignant mesothelioma, mast cell tumor, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0492] Other examples of CD22-mediated cancers are described herein and include, but are not limited to, liver cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, pancreatic cancer, thyroid cancer, and urothelial cancer.

[0493] In a particular embodiment, the objects of the present invention are provided for the treatment of aggressive forms of these cancers, defined as those that grow at least twice as fast as the general average growth rate of such cancers in the population.

[0494] In one embodiment, the subject invention is used in the treatment of leukemia during the accelerated phase of treatment.

[0495] In a particular embodiment, the object of the present invention is provided for the treatment of triple cancer consisting of refractory / recurrent diffuse large B-cell non-Hodgkin's lymphoma - breast metastasis in lung - chronic lymphocytic leukemia with bladder cancer and prostate cancer.

[0496] Preferably, the cancer is a hematological malignancy (e.g., a leukemia or lymphoma, including but not limited to Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphocytic carcinoma, acute myeloid leukemia, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma).

[0497] More preferably, the cancer is characterized by expression of CD22, more preferably, the cancer is characterized by expression of CD22 by cancerous cells, even more preferably, the cancer is characterized by overexpression of CD22 by cancer cells.

[0498] In one embodiment, the cancer cells are relapsed / refractory CD19-negative cancer cells.

[0499] In one embodiment, the cancer cells are relapsed / refractory CD22-expressing cancer cells.

[0500] In a preferred embodiment, the cancer cells are relapsed refractory CD19 negative CD22 positive expressing B-ALL.

[0501] B-cell ALL is: Early precursor B (early pre-B) ALL (also called pro-B ALL) ·Common ALL ·Pre-B ALL Mature B-cell ALL, also called Burkitt leukemia or non-Hodgkin lymphoma, in children Includes.

[0502] The term "disease associated with expression of CD22" as used herein includes, but is not limited to, a disease associated with expression of CD22 or a condition associated with the activity of cells expressing CD22, including, for example, tumor cells of various cancers such as CD22-expressing B-ALL.

[0503] Lytic cell destruction is one of the mechanisms by which the CD22 CAR T cells of the invention act on CD22-expressing cells to reduce or eliminate tumors, promote infiltration of host immune cells to tumor sites, and enhance / expand anti-tumor responses.

[0504] In another aspect, the present invention relies on a method for treating a patient in need of treatment, comprising at least one of the following steps: Providing the UCART22 of the present invention administering said transformed immune cells to said patient.

[0505] In one embodiment, the UCART22 cells of the present invention are capable of robust in vivo T cell expansion and persist in the host for an extended period of time.

[0506] In another aspect, the present invention provides a method for treating a patient in need thereof, the method comprising at least one of the following steps: A process for conditioning patients suffering from cancer Providing the UCART22 of the present invention administering said transformed immune cells to said patient.

[0507] The conditioning may include lymphodepletion or any suitable conditioning that a person skilled in the art, and preferably a physician, recognizes as a determinant for curing said patient.

[0508] In a preferred embodiment, the method further comprises the step of bone marrow transplantation.

[0509] In one embodiment, the UCART22 cells of the present invention are capable of robust in vivo T cell expansion and persist in the host for an extended period of time.

[0510] Said treatment may be an ameliorative treatment, a therapeutic treatment or a prophylactic treatment. It may be either a part of an autologous immunotherapy treatment or a part of an allogeneic immunotherapy treatment. Autologous means that the cells, cell lines or cell populations used to treat a patient originate from the patient or from a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cells or cell populations used to treat a patient originate from a donor, not from the patient.

[0511] In one embodiment, survival of said T cells of the present invention in a host is controlled using anti-CD20 (rituximab) and / or QBEN10 treatment.

[0512] subject The compositions and methods of the present invention can be used to treat subjects who have been characterized as having pathological cells or tissues that express CD22, or who are suspected of having pathological cells or tissues that express CD22. For example, subjects who would benefit from treatment according to the present invention include subjects with B-ALL or CLL, refractory BALL, and relapsed B-ALL.

[0513] In a preferred embodiment, the patient is a child with BALL, relapsed BALL, refractory BALL (pediatric indications).

[0514] Treatment with engineered immune cells according to the present invention may be combined with one or more treatments for cancer selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, and radiation therapy.

[0515] Thus, the present invention provides a pharmaceutical composition comprising a therapeutically active amount of UCART22 for the treatment of children with BALL, relapsed BALL, or refractory BALL.

[0516] The present invention also provides a pharmaceutical composition comprising UCART22 and a pharma- ceutical acceptable excipient for the treatment of children with BALL, relapsed BALL, or refractory BALL.

[0517] In a preferred embodiment, the pharmaceutical composition comprises UCART22 of the present invention and a bryostatin family compound, preferably bryostatin-1, and a pharmaceutically acceptable excipient for the treatment of children with BALL, relapsed BALL, or refractory BALL.

[0518] According to a preferred embodiment of the present invention, said treatment can be administered to a patient undergoing immunosuppressive treatment, (conditioning treatment), more preferably lymphodepletion.Indeed, the present invention preferably relies on a cell or a population of cells that is made resistant to at least one immunosuppressive agent due to the inactivation of the gene that codes for the receptor of such immunosuppressive agent.In this aspect, the immunosuppressive treatment or lymphodepletion should support the selection and expansion of T cells according to the present invention in the patient, and the destruction of CD22 cancer cells.

[0519] Administration of the cells or cell populations according to the present invention can be performed in any convenient manner, including aerosol inhalation, injection, oral, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, 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.

[0520] Administration of cells or populations of cells is at least 10 per kg of body weight, including all integer values ​​of cell number within these ranges. 4 ~10 9 cells, preferably 10 per kg of body weight 5 ~10 6 In another embodiment, the effective amount of cells is administered in a single dose or in multiple doses. The cells or population of cells may be administered once or multiple times, preferably in several consecutive doses (remedied) to avoid escape (relapsed cells). In another embodiment, the effective amount of cells is administered as a single dose or in divided doses. In another embodiment, the effective amount of cells is administered multiple times over a period of time.

[0521] The timing of administration is at the discretion of the managing physician and will depend on the clinical status of the patient.

[0522] The cell or cell population can be obtained from any source, such as a blood bank or a donor. Although individual needs vary, the determination of the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. By effective amount is meant an amount that provides therapeutic or prophylactic benefit. The dosage administered will depend on the age, health, and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the effect desired.

[0523] The UCART22 of the present invention is designed to limit cytokine storm, being efficient but not active. In the case of over-reacting patients, the present invention may be combined with suitable medicines for preventing or blocking cytokine storm, such as anti-IL-6 drugs.

[0524] In another embodiment, the effective amount of cells or a composition comprising the cells is administered parenterally. The administration can be intravenous. The administration can be by injection directly into a tumor.

[0525] In certain embodiments of the invention, the cells are administered to the patient in conjunction with (e.g., before, simultaneously, or after) a number of relevant treatment modalities, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir, and interleukin 2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efalizumab 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, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, alemtuzumab, anti-CD3 antibodies, or other antibody therapies, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993).

[0526] In further embodiments, the cell composition of the present invention is administered to the patient in conjunction with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or T cell depletion therapy using either antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after B cell depletion therapy, such as agents that react with CD20, e.g., Rituxan, or QBEN10. For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0527] In another embodiment, after transplant, the subject receives an agent that reacts with CD20, such as Rituxan or Ritiximab, preferably an agent that reacts with CD22 and CD20.

[0528] Other definitions

[0529] Amino acid residues within a polypeptide sequence are represented herein by single-letter codes, e.g., Q stands for Gln, a glutamine residue, R stands for Arg, an arginine residue, and D stands for Asp, an aspartic acid residue.

[0530] An amino acid substitution refers to the replacement of one amino acid residue with another; for example, the replacement of an arginine residue with a glutamine residue in a peptide sequence is an amino acid substitution.

[0531] Nucleotides are represented as follows: one-letter codes are used to represent the nucleoside bases: a is adenine, t is thymine, c is cytosine, and g is guanine. For degenerate nucleotides, r stands for g or a (purine nucleotides), k stands for g or t, s stands for g or c, w stands for a or t, m stands for a or c, y stands for t or c (pyrimidine nucleotides), d stands for g, a, or t, v stands for g, a, or c, b stands for g, t, or c, h stands for a, t, or c, and n stands for g, a, t, or c.

[0532] As used herein, "nucleic acid" or "polynucleotide" refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by either ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have changes in the sugar moiety and / or the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications at the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Nucleic acids can be either single-stranded or double-stranded.

[0533] Chimeric antigen receptor (CAR) refers to a molecule that combines a binding domain for a component present on a target cell, e.g., an antibody-based specificity for a desired antigen (e.g., a tumor antigen), with an intracellular domain that activates the T cell receptor to generate a chimeric protein that exhibits specific anti-target cell immune activity. CARs usually consist of an extracellular single-chain antibody (scFvFc) fused to an intracellular signaling domain of the T cell antigen receptor complex ζ chain (scFvFc:ζ), and when expressed in T cells, have the ability to redirect antigen recognition based on the specificity of the monoclonal antibody. One example of a CAR used in the present invention is a CAR for the CD22 antigen, which may include, as a non-limiting example, the amino acid sequence: SEQ ID NO: 15-18, preferably SEQ ID NO: 16 or 18, more preferably SEQ ID NO: 16.

[0534] The term "endonuclease" refers to a wild-type or variant enzyme that can catalyze the hydrolysis (cleavage) of bonds between nucleic acids of DNA or RNA molecules, preferably DNA molecules. Endonucleases do not cleave DNA or RNA molecules regardless of their sequence, but rather recognize and cleave DNA or RNA molecules at specific polynucleotide sequences, further referred to as "target sequences" or "target sites". Endonucleases can be classified as rare-cutting endonucleases when they have polynucleotide recognition sites that are typically longer than 12 base pairs (bp), more preferably 14-55 bp. Rare-cutting endonucleases significantly increase HR by inducing DNA double-strand breaks (DSBs) at defined locations (Perrin, Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007). Rare-cutting endonucleases can be, for example, homing endonucleases (Paques and Duchateau 2007), chimeric zinc finger nucleases (ZFNs) resulting from the fusion of an engineered zinc finger domain with the catalytic domain of a restriction enzyme such as FokI (Porteus and Carroll 2005), Cas9 endonucleases derived from CRISPR systems (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013), or chemical endonucleases (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). In chemical endonucleases, a chemical or peptidic cleaving agent is conjugated to either a polymer of nucleic acid or other DNA that recognizes a specific target sequence, thereby targeting the cleavage activity to that specific sequence.Chemical endonucleases also include synthetic nucleases such as conjugates of orthophenanthroline, DNA cleavage molecules, and triplex-forming oligonucleotides (TFOs), which are known to bind to specific DNA sequences (Kalish and Glazer 2005). Such chemical endonucleases are included in the term "endonuclease" according to the present invention.

[0535] "TALE nuclease" (TALEN) typically refers to a fusion protein consisting of a nucleic acid binding domain from a Transcription Activator Like Effector (TALE) and one nuclease catalytic domain that cleaves a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain, more preferably a domain with endonuclease activity, such as I-TevI, ColE7, NucA, Fok-I. In a specific embodiment, the TALE domain can be fused to a meganuclease, such as I-CreI and I-OnuI or functional variants thereof. In a more preferred embodiment, said nuclease is a monomeric TALE nuclease. A monomeric TALE nuclease is a TALE nuclease that does not require dimerization for specific recognition and cleavage, such as the fusion of engineered TAL repeats with the catalytic domain of I-TevI ​​described in WO2012138927. Transcription activator-like effectors (TALEs) are proteins from the bacterial species Xanthomonas that contain multiple repeats, each of which contains two residues (RVDs) at positions 12 and 13 specific for each nucleotide base of a nucleic acid target sequence. A binding domain (MBBBD) with similar modular base-per-base nucleic acid binding properties can also be derived from a new modular protein recently discovered by the applicant in a different bacterial species. The new modular protein has the advantage of exhibiting more sequence variability than the TAL repeats.Preferably, the RVDs involved in the recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A, YG for recognizing T, TL for recognizing A, VT for recognizing A or G, and SW for recognizing A. In another embodiment, the critical amino acids 12 and 13 may be mutated to other amino acid residues to modulate the specificity for the nucleotides A, T, C and G, in particular to enhance this specificity. TALE nucleases have been described and used to stimulate gene targeting and modification (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011). Custom TAL nucleases are commercially available under the trademark TALEN™ (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

[0536] The rare-cutting endonuclease according to the present invention can also be Cas9 endonuclease, zinc finger endonuclease, MegaTAL endonuclease. From the type II prokaryotic CRISPR (Clustered Regularly Interspaced Short palindromic Repeats) adaptive immune system (see (Sorek, Lawrence et al. 2013) for a review), genome engineering tools have been developed based on RNA-guided Cas9 nuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013). CRISPR-associated (Cas) systems were first discovered in bacteria and function as defense against foreign DNA, viruses or plasmids. CRISPR-mediated genome engineering first proceeds by the selection of a target sequence, which is often flanked by short sequence motifs called protospacer adjacent motifs (PAMs). After target sequence selection, a specific crRNA complementary to this target sequence is engineered. The trans-activating crRNA (tracrRNA), required in CRISPR type II systems, pairs with the crRNA and binds to the supplied Cas9 protein. Cas9 acts as a molecular anchor that facilitates base pairing of the tracrRNA with the cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the double tracrRNA:crRNA structure acts as a guide RNA that directs the endonuclease Cas9 to the cognate target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence for homology between the target sequence and the crRNA. In addition to target sequence-crRNA complementarity, DNA targeting requires the presence of a short motif adjacent to the protospacer (protospacer adjacent motif-PAM).Then, following pairing between the duplex RNA and the target sequence, Cas9 introduces a blunt-ended double-stranded break three bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).

[0537] The rare-cutting endonuclease may be a homing endonuclease, also known by the name meganuclease. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and generate single- or double-stranded breaks. Homing endonucleases are highly specific and recognize DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually 14 to 40 bp in length. A homing endonuclease according to the invention may correspond, for example, to a LAGLIDADG endonuclease, a HNH endonuclease, or a GIY-YIG endonuclease. A preferred homing endonuclease according to the invention may be an I-CreI variant.

[0538] "Delivery vector" refers to a delivery vector that can be used in the present invention to contact (i.e., "contact") a cell or to deliver (i.e., "introduce") a required drug / chemical and molecule (protein or nucleic acid) to a cell or intracellular compartment. It includes, but is not limited to, liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymer carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions, or other suitable transfer vectors. These delivery vectors allow the delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as plasmids, peptides developed by Diatos. In these cases, the delivery vector is a molecular carrier. "Delivery vector" also refers to a delivery method for performing transfection.

[0539] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In the present invention, "vector" includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vectors) and / or expression (expression vectors) of the nucleic acids to which they are linked. Many suitable vectors are known to those of skill in the art and are commercially available.

[0540] Viral vectors include negative-stranded RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses, particularly aav6), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma, mammalian type C, B, and D viruses, the HTLV-BLV complex, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0541] "Lentiviral vector" refers to HIV-based lentiviral vectors, which are highly promising for gene delivery due to their relatively large packaging capacity, reduced immunogenicity, and ability to stably transduce a wide range of different cell types with high efficiency. Lentiviral vectors are usually generated after transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. After entry, viral RNA undergoes reverse transcription mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA that serves as a substrate for viral integration into the DNA of infected cells. "Integrated lentiviral vector (or LV)" refers, by way of non-limiting example, to such vectors that can be integrated into the genome of target cells. Conversely, "non-integrated lentiviral vector (or NILV)" refers to efficient gene delivery vectors that do not integrate into the genome of target cells through the action of viral integrase.

[0542] Delivery vectors and vectors may be associated with or combined with cell permeabilization techniques such as sonoporation or electroporation, or variations of these techniques.

[0543] Cells refers to viable cells of eukaryotic organisms, primary cells, and cell lines derived from these organisms for in vitro culture.

[0544] "Primary cells" refer to cells taken directly from viable tissue (i.e., biopsy material) and established for growth in vitro that have undergone few population doublings and therefore better represent the main functional components and characteristics of the tissue from which they were derived, as compared to tumorigenic continuous cell lines or artificially immortalized cell lines.

[0545] As non-limiting examples, cell lines may be selected from the group consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.

[0546] All of these cell lines can be modified by the methods of the present invention to provide cell line models for making, expressing, quantifying, detecting, and studying genes or proteins of interest; these models can also be used to research, make, and screen bioactive molecules of interest in various areas such as chemicals, biofuels, therapeutics, and agriculture, as non-limiting examples.

[0547] "Mutation" refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50 or more nucleotide / amino acid substitutions, deletions, insertions in a polynucleotide (cDNA, gene) or polypeptide sequence. Mutations may affect the coding sequence of a gene or its regulatory sequences. They may also affect the structure of the genomic sequence or the structure / stability of the encoded mRNA.

[0548] "Variant" refers to a repeat variant, variant, DNA binding variant, TALE nuclease variant, polypeptide variant obtained by mutation or replacement of at least one residue in the amino acid sequence of the parent molecule.

[0549] "Functional variant" refers to a catalytically active variant of a protein or protein domain; such a variant may have the same activity, or additional properties, or higher or lower activity, compared to its parent protein or protein domain.

[0550] "Identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions of each sequence that can be aligned for comparison purposes. When a position of the compared sequences is occupied by the same base, the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used, for example, with default settings. For example, polypeptides and polynucleotides encoding such polypeptides that have at least 70%, 85%, 90%, 95%, 98%, or 99% identity with the specific polypeptides described herein and preferably exhibit substantially the same function are contemplated. Unless otherwise indicated, the similarity score will be based on the use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positive score, and the percent sequence identity is based on the BLASTP identity score. BLASTP "identity" indicates the number and fraction of total residues in a high-scoring sequence pair that are identical; BLASTP "positive" indicates the number and fraction of residues that have a positive alignment score and are similar to each other. Amino acid sequences having these degrees of identity or similarity with the amino acid sequences disclosed herein, or intermediate degrees of identity or similarity, are contemplated and encompassed by the present disclosure. Polynucleotide sequences of similar polypeptides are predicted using the genetic code and can be obtained by conventional means, in particular by back-translating the amino acid sequence using the genetic code.

[0551] "Signaling domain" or "costimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including, but not limited to, proliferation activation, differentiation, etc., in addition to the primary signal provided by, for example, engagement of the TCR / CD3 complex with a peptide-loaded MHC molecule. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to the Toll ligand receptor, and antibodies that specifically bind B7-H3. Co-stimulatory ligands may include, but are not limited to, ligands that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83, among others.

[0552] "Costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0553] A "costimulatory signal," as used herein, refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0554] The term "extracellular ligand-binding domain" as used herein is defined as an oligopeptide or polypeptide capable of binding to a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as ligands include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0555] The term "subject" or "patient" as used herein includes all members of the animal kingdom, including non-human primates and humans, preferably humans. In one embodiment, the patient is a patient with aggressive, refractory or relapsed ALL, or aggressive, refractory, relapsed CLL.

[0556] A mammal is any warm-blooded vertebrate of the class Mammalia, preferably a human.

[0557] By "suicide domain or switch", or "safety on-and-off switch", is meant a domain, usually a cell surface domain, that is recognized by molecules, proteins, chemicals, antibodies to immunoselect expressing cells and ultimately control their function and survival.

[0558] The foregoing description of the invention provides manners and processes for making and using the invention to enable any person skilled in the art to make and use the invention, this capability being provided with particularity for the subject matter of the claims annexed hereto and forming a part of the original specification.

[0559] When a numerical limit or range is stated herein, the endpoints are included. All values ​​and subranges included in the numerical limit or range are specifically included, as if expressly written.

[0560] The above description is presented to enable any person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, the present invention is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0561] general law Screening and selection of CARs ·Primary T cell culture T cells were purified from buffy coat samples provided by EFS (Etablissement Francais du Sang, Paris, France) using Ficoll gradient density medium. The PBMC layer was harvested. T cells were activated in X-Vivo™-15 medium (Lonza) supplemented with 20 ng / mL human IL-2, 5% human, and Dynabeads Human T activator CD3 / CD28 (Life Technologies) at a bead:cell ratio of 1:1.

[0562] CAR mRNA transfection After purification and activation of T cells, transfection of CAR mRNA encoding each CAR construct was performed on day 4 or day 11. Cells were immediately diluted with X-Vivo™-15 medium and incubated at 37° C. with 5% CO2. IL-2 was added at 20 ng / mL 2 hours after electroporation.

[0563] T cell transduction A vector encoding the CD22 CAR is introduced into T cells as previously described. CAR detection on the T-cell surface is performed using a recombinant protein consisting of a fusion of the extracellular domain of the human CD22 protein (whole protein, CD22 distal part or CD22 proximal part) with a mouse IgG1 Fc fragment. Binding of this protein to the CAR molecule is detected with a PE-conjugated secondary antibody (Jackson Immunoresearch) targeting the mouse Fc part of the protein and analyzed by flow cytometry.

[0564] Inactivation of specific genes in primary T cells Inactivation of specific genes in primary T cells can be performed before, or preferably after, CD22 CAR is introduced into cells using appropriately designed endonucleases, such as TAL endonucleases, optionally Crispr Cas 9 endonucleases. At least one gene is inactivated, and one, two or three genes may be inactivated in one step or in several consecutive steps. In a preferred embodiment, two genes are inactivated, preferably the TCR alpha gene and a gene whose deletion confers resistance to a drug selected from purine nucleotide analogs, alemtuzumab, platins (cisplatin or carboplatin), antitopoisomerase I (irinotecan), antitopoisomerase II (etoposide), methotrexate (folate analogs), preferably purine nucleotide analogs, alemtuzumab.

[0565] Generally, heterodimeric nucleases, in particular TALE nucleases, are designed and manufactured that target two long sequences (called half targets) separated by a spacer within a target gene.

[0566] Each TALE nuclease construct can be cloned into a suitable mammalian expression vector. The mRNA encoding the TALE nuclease that cleaves the targeted genomic sequence can be synthesized from a plasmid carrying the coding sequence downstream of a promoter. Purified T cells preactivated by anti-CD3 / CD28 coated beads are used and transfected with each of the two mRNAs encoding both half TALE nucleases. The cells can be reactivated by soluble anti-CD28 and cell proliferation measured for various times and the activation marker CD25 detected to assess the activation state of the cells.

[0567] Degranulation assay (CD107a mobilization) Cells were incubated with equal amounts of cells expressing various levels of the targeted protein (CD22) in 96-well plates. Co-cultures were maintained at 37°C with 5% CO2 for 6 hours. CD107a staining was performed during cell stimulation by adding fluorescent anti-CD107a antibody at the beginning of co-culture, along with anti-CD49d, anti-CD28, and 1x monensin solution as controls. After a 6-hour incubation period, cells were stained with fixable viability dye and fluorochrome-conjugated anti-CD8 and analyzed by flow cytometry. Degranulation activity was determined as % of CD8+ / CD107a+ cells and by determining the mean fluorescence intensity signal (MFI) for CD107a staining in CD8+ cells. Degranulation assays were performed 24 hours after mRNA transfection.

[0568] IFNγ release assay 24 hours after mRNA transfection, CD22 CAR-expressing T cells were incubated with cell lines expressing various levels of the targeted proteins for 24 hours at 37° C. Supernatants were collected and IFNγ detection in cell culture supernatants was performed by ELISA assay.

[0569] Cytotoxicity Assay Cells were incubated with target cells (expressing different levels of CD22) or (negative control). Target and control cells were labeled with fluorescent intracellular dyes (CFSE or Cell Trace Violet) before co-culture with CAR+T cells. Co-cultures were incubated for 4 hours at 37°C. After this incubation period, cells were labeled with a fixable viability dye and analyzed by flow cytometry. The viability of each cell population (target or negative control cells) was determined and the % of specific cell lysis was calculated. Cytotoxicity assays were performed 48 hours after mRNA transfection.

[0570] Antitumor Mouse Model Immunodeficient mice are implanted with tumor cells (patient-derived CD22 BALL) or targeted protein expressing luciferase cells in the flank. The cells are then implanted into the mouse brain. Serial transplantation into further generations of mice continues the maintenance of the xenograft cell line in vivo. Optionally, mice received anti-cancer treatment (alemtuzumab and / or flu) before / or together with the injection of CAR+T cells. Different doses of the tested CAR+T cells or T cells that do not express CD22CAR are then injected iv into the mice (either 2 or 7 days after the injection of the tumor cell line). To follow tumor progression in different animals, bioluminescence signals are determined on the day of T cell injection (D0), 7 days, 14 days, 21 days, 28 days, and 40 days after T cell injection.

[0571] A Phase I Dose-Escalation Study to Evaluate the Safety, Expansion, and Durability of Allogeneic CD22 CART (UCART22) in Patients with Relapsed or Refractory or MRD+ CD22+ B-Cell Acute Lymphoblastic Leukemia (B-ALL)

[0572] Background and Rationale With current multiagent chemotherapy regimens, long-term survival is seen in >80% of childhood acute lymphoblastic leukemia (ALL) and approximately 40% of adult ALL (1). Further intensification of chemotherapy has not proven effective (2). In recent years, there have been significant advances in our understanding of the biology of ALL, which provides opportunities for "targeted therapy" (3, 4).

[0573] Relapsed / refractory ALL remains a challenging disease. Post-relapse therapy results in a second CR (CR2) in 30-40% of patients, with a 5-year OS of only about 10%. In the largest report of relapsed adult ALL patients to date, Fielding and coworkers analyzed the outcome of relapsed adult ALL patients treated in the MRC UKALLXII / ECOG E2993 trial (5). Of 1508 evaluable patients, 1372 (91%) achieved CR1, of which 609 (44% of CR1 patients) relapsed at a median of 11 months. 5-year OS was only 7% for patients who relapsed. Median OS for relapsed patients was 5.5 months. Tavernier and coworkers reported the outcome of 421 ALL patients who experienced a first relapse treated in the French LALA-94 trial (6). CR2 was achieved in 44% of patients, with a median DFS of 5.2 months and a median OS of 6.3 months. Oriol and colleagues reported the outcome of 263 ALL patients in first relapse treated in four consecutive PETHEMA trials (7). CR2 was achieved in 45% of patients, a rate similar to that in the French LALA trial. Median OS after relapse was 4.5 months and 5-year OS was 10%.

[0574] CD22 expression occurs in >90% of patients with ALL and is a legitimate therapeutic target. Cellular therapies, such as chimeric antigen receptor (CAR) T-cell therapy, are increasingly being used to treat patients with hematological malignancies (8-16). In patients with relapsed acute lymphoblastic leukemia (ALL), very high complete response rates (80-90%) have been reported with autologous CD19-CAR T cells (12). Similarly, response rates of 40-50% have been seen in patients with relapsed chronic lymphocytic leukemia (CLL) receiving autologous CD19 CAR T therapy (9).

[0575] This study evaluates allogeneic CART cells directed against CD22 in patients with relapsed and / or refractory CD22 B-ALL.

[0576] the purpose Main purpose To evaluate the safety and tolerability of allogeneic CD22 CART and to determine the maximum tolerated dose (MTD)

[0577] Secondary Objectives To determine the efficacy of allogeneic CD22 CART To determine the incidence of GVHD

[0578] exploratory purpose To determine the expansion, phenotype, trafficking and persistence of infused CART cells

[0579] Inclusion criteria 1. Relapsed or refractory CD22-positive ALL (for expansion phase: patients with MRD+ disease are allowed) 2. Patients aged ≥ 2 years 3.ECOG performance status ≤ 2 4. Normal organ function including bilirubin ≤ 2 mg / dl, ALT / AST < 3x ULN, and creatinine ≤ 2 mg / dl 5.Left ventricular ejection fraction (LVEF)≧40%

[0580] Exclusion criteria 1. The patient is pregnant or breastfeeding 2.Patients with uncontrolled active infection 3. Isolated extramedullary recurrence (i.e., testis, CNS) 4. Known active CNS leukemia. Note: Patients with a history of effectively treated CNS disease are eligible if they were in CNS remission >4 weeks prior to enrollment 5. Active hepatitis B or C 6.HIV infection 7. Active GVHD requiring systemic steroid therapy. Steroid therapy for physiological replacement is permitted. 8. Received DLI within 4 weeks of CD22 CART infusion 9. Allo-SCT within 60 days of CD22 CART infusion

[0581] Exam description This is a Phase 1 trial. There are two phases to this trial: dose escalation, and dose expansion. Patients will receive lymphodepleting chemotherapy followed by CD22 allogeneic CART. Dose Escalation: Four dose levels will be studied in a standard 3x3 design. A total of 9-18 patients will be enrolled.

[0582] TIFF0007634045000042.tif34128

[0583] Once the R2PD level is identified, dose escalation will commence. A total of 20 patients will then be enrolled (10 R / R ALL; 10 MRD+ post-SCT). Total sample size: 29–38 patients. TIFF0007634045000043.tif128165

[0584] The second study will compare allogeneic CART cells directed against CD22 with allogeneic CART cells directed against CD22 and CD19 (two forms) in patients with CD19+CD22+ B-ALL.

[0585] the purpose Main purpose To evaluate the safety and tolerability of allogeneic CD19+CD22 CART and to determine the maximum tolerated dose (MTD)

[0586] Secondary Objectives To determine the efficacy of allogeneic CD22 CART To determine the incidence of GVHD

[0587] exploratory purpose To determine the expansion, phenotype, trafficking and persistence of infused CART cells

[0588] Inclusion criteria 1. Relapsed or refractory CD22-positive ALL (for expansion phase: patients with MRD+ disease are allowed) 2. Patients aged ≥ 2 years 3.ECOG performance status ≤ 2 4. Normal organ function including bilirubin ≤ 2 mg / dl, ALT / AST < 3x ULN, and creatinine ≤ 2 mg / dl 5.Left ventricular ejection fraction (LVEF)≧40%

[0589] Exclusion criteria 1. The patient is pregnant or breastfeeding 2.Patients with uncontrolled active infection 3. Isolated extramedullary recurrence (i.e., testis, CNS) 4. Known active CNS leukemia. Note: Patients with a history of effectively treated CNS disease are eligible if they were in CNS remission >4 weeks prior to enrollment 5. Active hepatitis B or C 6.HIV infection 7. Active GVHD requiring systemic steroid therapy. Steroid therapy for physiological replacement is permitted. 8. Received DLI within 4 weeks of CD22 CART infusion 9. Allo-SCT within 60 days of CD22 CART infusion

[0590] Exam description This is a Phase 1 trial. There are two phases to this trial: dose escalation, and dose expansion. Patients will receive lymphodepleting chemotherapy followed by CD22 allogeneic CART. Dose Escalation: Four dose levels will be studied in a standard 3x3 design. A total of 9-18 patients will be enrolled.

[0591] TIFF0007634045000044.tif34128

[0592] Once the R2PD level is identified, dose escalation will commence. A total of 20 patients will then be enrolled (10 R / R ALL; 10 MRD+ post-SCT). Total sample size: 29–38 patients. TIFF0007634045000045.tif128165

[0593] References TIFF0007634045000046.tif235161TIFF0007634045000047.tif39160 EXAMPLES

[0594] Example 1: Expansion of TCR alpha inactivated cells expressing CD22-CAR We designed and engineered heterodimeric TALE nucleases that target two 17-bp long sequences (called half targets) separated by a 15-bp spacer in the T-cell receptor alpha constant chain region (TRAC) gene. Each half target is recognized by a repeat of the half TALE nuclease listed in Table 6.

[0595] Table 6. TAL nucleases targeting the TCRα gene TIFF0007634045000048.tif63163

[0596] Each TALE nuclease construct was subcloned into a mammalian expression vector using restriction enzyme digestion so that it was under the control of a T7 promoter. mRNA encoding the TALE nuclease cleaving the TRAC genomic sequence was synthesized from a plasmid carrying the coding sequence downstream of the T7 promoter.

[0597] Purified T cells preactivated for 72 hours by anti-CD3 / CD28 coated beads were transfected with each of the two mRNAs encoding both half-TRAC_T01 TALE nucleases. 48 hours after transfection, different groups of T cells from the same donor were transduced with vectors encoding one of the CD22 CARs of the present invention. Two days after transduction, CD3 陰性 Cells were purified using anti-CD3 magnetic beads, and 5 days after transduction, cells were reactivated with soluble anti-CD28 (5 μg / ml).

[0598] Cell proliferation was followed up to 30 days after reactivation by counting cells twice a week. Proliferation of TCRα-inactivated cells expressing the CD22 CAR was comparable to that of non-inactivated cells and was increased especially when reactivated with anti-CD28.

[0599] Multiplex transfection of TALEN mRNA Activated T cells were transfected with mRNA encoding the TRAC TALENs (left and right) and a TALEN pair specific for the B2M gene, followed by transduction with exogenous polynucleotides encoding a CD22-specific CAR or a CD19-specific CAR or both.

[0600] Double KO TRAC and B2M or CD56 genes expressed undetectable levels of TCR and MHCI or TCR and CD56. After purification, cells were re-transduced.

[0601] Example 2: CD22 CAR-T Development of engineered CAR T cells targeting CD22 for the treatment of refractory, relapsed or aggressive ALL or CLL CD22 CAR: (Figure 2) Constructs as m971 and m971 variants with reduced affinity and high selectivity for CD22 Different scfv were used to design and prepare the CD22 CAR. The m971 scfv is derived from the 971 antibody (Haso W 1 , Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood. 2013 Feb 14;121(7):1165-74. doi: 10.1182 / blood-2012-06-438002. Epub 2012 Dec 14).

[0602] The CAR construct (Figure 2) was constructed using a 41BB costimulatory domain, a CD3ζ activation domain, a CD8α transmembrane domain and a hinge, a CD8α hinge (SEQ ID NO: 15). The construct containing the FcγRIIIα-hinge corresponds to (SEQ ID NO: 14).

[0603] The constructs were inserted into vectors for stable expression and screening of the designed CARs.

[0604] The CD22 CAR was m971-V3 CAR (SEQ ID NO:22); and SEQ ID NO:24.

[0605] The sequences may also be optimized for CD22 binding and treatment of ALL and CLL, preferably refractory, relapsed and aggressive forms thereof.

[0606] CAR expression CD22 CAR was introduced into primary KO TCR T cells 5 days after activation with anti-CD3CD28 coated beads and IL-2. CAR expression was assessed by flow cytometry. All CARs were expressed on the cell surface. Activity against CD22+ transformed cell lines and patient-derived refractory or relapsed CD22+ B ALL.

[0607] To test the functionality of the anti-CD22 CAR, B cells expressing CD22 (ALL lines REH, SEM, NALM6-GL, KOPN8, Daudi, Raji, and K562 were used (see Haso et al., 2013, 2013; Blood: 121 (7): 1165 - 1174 for experimental details) obtained from patients with refractory or relapsed CD22+ B ALL.

[0608] As expected, all cells expressing CD22 were positively stained and targeted by the m971-derived CD22 CAR of the present invention, as was the CD22 CAR (scfv2-derived HA22).

[0609] Degranulation assay To validate the CD22 CAR construct, a degranulation assay was performed on target cells using T cells expressing the CD22 CAR of the present invention. CART degranulation was assessed by flow cytometry. The readout is CD107a expression at the T cell plasma membrane after 5 hours of incubation with target cells. The results showed that degranulation from CARm971- (scfv-1) was more significant than that for CD22 CAR T cells against the distal part of CD22 (SEQ ID 20) scfv-2 (Figure 5).

[0610] Cytotoxicity Assay Cytotoxicity assays were performed on these same target cells using T cells expressing the CD22 CAR of the present invention. The CD22 CAR showed strong specific lysis of the CD22 cells of the present invention UCART 22 when compared to cells expressing scfv (scfv2) against the distal part of CD22 (Figure 6).

[0611] Interferon gamma assay Interferon gamma production of UCART22 (scfv-V1 against the proximal domain of CD22) compared to that of non-transduced (NT) or T cells transduced with a CAR targeting the distal part of CD22 (scfv-V2) cells in the presence of CD22-positive NALM-16 cells compared to CD22-negative SUP-T1 cells (Figure 7).

[0612] Mouse survival Compared to control cells, mouse survival was improved in the presence of UCART22 (scfv-V1 against the proximal domain of CD22) or CART22 (without TRAC inactivation) (Figure 8).

[0613] Tolerance to hypoxia and / or drugs The engineered cells of the present invention, UCART22, were not significantly affected (viability and CTL activity) in the presence of alemtuzumab (50 micrograms / mL) or PNA(flu) when compared to non-engineered sensitive cells, which died 48 hours following the addition of the drugs in cell culture or following culture conditions under hypoxia (less than 5%, preferably less than 1% O2).

[0614] Studies performed under low O2 conditions (<5% or <1%) produced similar results, confirming that UCART22 with increased HIF-1a expression can survive, express the CD22 CAR, and be active under hypoxia.

[0615] Similar results (survival, CTL activity) were obtained in mice treated with Campath (50 micrograms / mL), confirming the resistance of UCART22 to the drug. It is suggested that the UCART 22 cells of the present invention may reach cancer cells nested in tissues or clustered in vivo, because the amount of cancer cells that "recover" or "escape" from treatment with UCART22 was much smaller (about 15% reduction) than in mice treated with UCART22 that is not resistant to O2. Thus, it seems that the local hypoxia created by liquid tumors prevents immune cells from fighting them.

[0616] Experiments were performed to determine whether UCART22 cells could be eliminated if desired (Figures 3 and 4). In mice, rituximab induced a significant reduction in UCART, as indicated by an increase in the fluorescence intensity of tumor cells in mice treated with rituximab and UCART, compared to cells alone.

[0617] Initial clinical data obtained show that UCAR T cells significantly reduce relapsed and refractory ALL in vivo and in vitro, with absent or minimally mild (grade 1) GVHD and mild to absent uncontrolled cytokine storm.

[0618] Such treatment is less "toxic" compared to autologous CD22 CAR T and can be controlled in patients using rituximab and / or QBEN10.

[0619] The cells persist in humans long enough to be active (over a month) and can be depleted using QBEN10.

[0620] Examples of CD22 CAR polypeptide sequences to be prepared The framing sequences correspond to the preferred VH and VL sequences, which can be exchanged (modifications in hot spots) to improve CAR efficiency, as described above. TIFF0007634045000049.tif97159TIFF0007634045000050.tif160159

[0621] result The CAR construct selected for preparation in UCART22 contains the m971 anti-human CD22 scFv (described in Haso et al., 2013), a CD8α hinge (optional) and transmembrane domain, and a cytoplasmic tail composed of the 4-1BB costimulatory domain and the CD3ζ signaling domain (Figure 3).

[0622] Another CAR construct contains, consecutively, the sequence m971 anti-human CD22 scFv, two binding domains for rituximab (CD20), the CD8α hinge and transmembrane domains, and a cytoplasmic tail composed of the 4-1BB costimulatory domain and the CD3ζ signaling domain (Figure 3).

[0623] The alternative CAR construct consecutively contained a binding domain for rituximab (CD20), a binding domain for Q-BEN10 (CD34), the m971 anti-human CD22 scFv, two binding domains for rituximab (CD20), a CD8α hinge and transmembrane domain, and a cytoplasmic tail composed of the 4-1BB costimulatory domain and the CD3ζ signaling domain (Figure 3).

[0624] In UCART22, recombinant third generation self-inactivating (SIN) lentiviral vectors (rLVs) are used for the generation of T cells expressing anti-CD22 CAR and RQR8 depletion mechanism under the control of human EF1α promoter (Figure 4). Recombinant lentiviral vectors derived from HIV are genetically stable and have not yet shown evidence of vector-attributable pathological outcomes (Chang and Sadelain, 2007; Wang et al., 2009). In addition, the conditions for transduction of T cells with rLVs are known and compatible with preservation of T cell functional properties.

[0625] In UCART22, AAV6 vectors can be used after gene editing of a gene such as the TRAC gene (the gene encoding the TCR or alpha subunit) or any of the genes disclosed in PCT / EP2017 / 076798 (Figure 9).

[0626] Variants of the anti-CD22 CAR construct of the invention were further prepared, including an additional CD19 scfv (before or after the CD22 scfv), as well as a multi-chain CD22 CAR, a multi-chain CD22, CD19 CAR (Figure 2) were also constructed and inserted into a lentiviral vector, an adeno-associated virus 6 (AAV6) vector, which allows the CD22 CAR construct or the CD19 CAR construct described below to be inserted into the TRAC gene or into the CD25 gene or into the beta2 microglobulin gene, making these with the CAR sequences flanking the sequence 5' and 3', allowing homologous recombination into the TRAC gene, into the CD25 gene or into the beta2 microglobulin gene after specific cleavage of the gene using a specific TAL-protein-Fok-1 (TALEN).

[0627] UCART22 was generated, which further contains a TGF beta receptor KO gene, an IL-10 receptor KO gene, an AHR KO gene, a PD1 KO gene, a LAG-3 KO gene, a TIM-3 KO gene, or a combination thereof.

[0628] Examples of each UCART22 were generated that further contained either a TGF beta receptor KO gene, or an IL-10 receptor KO gene, or an aryl hydrocarbon receptor (AHR) KO gene, or a PD1 KO gene, or a LAG-3 KO gene, or a TIM-3 KO gene.

[0629] To test the effect of depleting CD19+ CD22+ cancer cells, an anti-CD19 construct was also prepared, previously described in “Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Waseem Qasim, et al., 2017. Science Translational Medicine25 Jan 2017), and further containing a suicide gene different from that carried by the CD22 CAR (as described in WO2016120216A1). In this study, 1) The combination of UCART19 + UCART 22 (two single-chain CARs) compared to each individual cell alone (all at 5x106 cells), 2) TCR-negative cells expressing the bispecific anti-CD22-anti-CD19 CAR (bispecific single chain CAR) compared to the individual cells alone (all at 5x106 cells); 3) or a multi-chain CAR comprising both CD22 and CD19 scfv or CD19 CD22scfv, The cytolytic activity of each was measured in comparison to the individual cells alone (all at 5x106 cells).

[0630] Expression of RQR8, R2CD22CAR or QR3CD22CAR to confer sensitivity to rituximab / rituximab and QBEN10 The lentiviral vector cassette driving the expression of CD22CAR was designed to co-express RQR8 (via a 2A peptide linker). RQR8 is a depletion ligand that can potentially be activated in the event of unmanageable adverse events related to the administration of UCART22. RQR8 is a 136 amino acid artificial cell surface protein that combines an antibody binding epitope from human CD34 and human CD20, recognized by the QBEnd-10 antibody (Philip et al., 2014). The CD20 epitope present within the construct is recognized by rituximab, thus allowing deletion of RQR8-expressing cells through complement-mediated cell killing (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of rituximab and / or QBEN10.

[0631] In other experiments, constructs corresponding to anti-CD22 CARs containing two epitopes recognized by rituximab (R) (R2 CD22 CAR: two epitopes recognized by rituximab inserted immediately after scfv and immediately before the hinge, or inserted in place of the hinge to shorten the extracellular domain compared to the entire construct with a hinge), or "QR3" QR3 CD22 CAR (Q: epitopes recognized by QBEN 10, QR - mid-N-terminus before VH -linker-VL RR hinge TM -41BB- CD3 zeta) were prepared and tested against cells expressing various levels of CD22.

[0632] TRAC gene knockout to limit alloreactivity of UCART22 A potential limitation of allogeneic adoptive immunotherapy approaches is that recognition of donor-recipient MHC differences by the TCRαβ complex of donor cells can lead to donor T cell activation / proliferation and the development of graft-versus-host disease (GvHD).TCRαβ is composed of α and β subunits, with TCRα encoded by a single gene and TCRβ encoded by two homologous genes.

[0633] UCART22 cells are genetically modified to specifically disrupt the T cell receptor alpha constant (TRAC) gene. Inactivation of the TRAC gene prevents cell surface expression of the TCRαβ complex, eliminating TCR-mediated recognition of histocompatibility antigens that can lead to GvHD. At the end of the UCART22 manufacturing process, remaining TCRαβ+ cells are depleted to ensure a residual level of ≦3.0% of TCRαβ+ cells in the final product, in accordance with release criteria.

[0634] CD52 gene knockout to confer resistance to alemtuzumab UCART22 is CD52 - and CD52 + The cells have been engineered to be a mixture of cells, allowing for the potential use of alemtuzumab in lymphodepletion regimens prior to administration of UCART22. Alemtuzumab is a monoclonal antibody that binds to human CD52. Thus, the CD52 - A fraction is resistant to alemtuzumab.

[0635] TALEN® technology is used to disrupt both TRAC and CD52 genes. In the UCART22 manufacturing process, TRAC and CD52 TALEN® are introduced into cells as messenger ribonucleic acid (mRNA) using an electroporation system. This transient expression allows TALEN® to perform efficient targeted gene knockout before being degraded by the cell. This approach avoids the potential risks associated with long-term expression of nucleases in cells injected into patients.

[0636] B2M gene knockout to limit UCART22 alloreactivity TALEN® technology is used to disrupt both TRAC and B2M genes. In the UCART22 manufacturing process, TRAC and B2M TALEN® are introduced into cells as messenger ribonucleic acid (mRNA) using an electroporation system. This transient expression allows TALEN® to perform efficient targeted gene knockout before being degraded by the cell.

[0637] Working Example Source of T cells used in non-clinical trials (pharmacology and toxicology) A description of the cell products utilized in non-clinical trials is summarized in the Table.

[0638] Table C. Engineered T cells used in in vitro and in vivo preclinical studies TIFF0007634045000051.tif152168DKO: double KO, KO: knockout, rLV: recombinant lentiviral vector MHCI: Major histocompatibility complex class I B2m: beta 2 microglobulin

[0639] overview UCART22 activation is driven by contact of anti-CD22 chimeric antigen receptor (CD22CAR) with the CD22 antigen, leading to the destruction of CD22+ B-ALL cells through T cell-mediated cytotoxicity and potentially proinflammatory cytokine production.

[0640] Proof-of-concept studies conducted during the development of other UCART products (UCART19 and UCART123) demonstrated that UCART cells (TCRαβ-deficient CAR T cells) are as active as non-gene-edited CAR T cells (TCRαβ-positive CAR T cells) in vitro and in an in vivo tumor xenograft model in NSG mice (Poirot et al., 2015). This was confirmed for TCRαβ-deficient CD22 CAR T cells and TCRαβ-deficient, MHC I-deficient CD22 CAR T cells compared to TCRαβ-positive CD22 CAR T cells. In vitro pharmacology studies performed during UCART22 nonclinical development are summarized in the Table. The studies demonstrated the following: - Antitumor activity of UCART22 by CD22 antigen-dependent cytotoxicity and cytokine secretion assays. Activity assays were performed on B-ALL cell lines and primary B-ALL samples. - Efficient TALEN®-mediated inactivation of the TRAC and CD52 genes. - Efficient TALEN®-mediated inactivation of the TRAC and beta2 microglobulin genes. - Their CD52- T cells are resistant to alemtuzumab. - Efficient elimination of RQR8+ or R2 CD22CAR+ cells using rituximab.

[0641] Table D. List of in vitro pharmacology studies TIFF0007634045000052.tif173170CDC = complement-dependent cytotoxicity; PHA = phytohemagglutinin.

[0642] In vivo pharmacology studies (summarized in the table) demonstrated the following: -Antitumor activity of UCART22 in vivo against tumor xenografts in immunodeficient mice using B-ALL cell lines. In vivo activity of UCART22 manufactured under validated GMP conditions against B-ALL cell lines. - Improved antitumor activity of UCART22 / 19 in vivo against tumor xenografts in immunodeficient mice using B-ALL cell lines. In vivo activity of UCART22 / 19 and 19 / 22 manufactured under GMP conditions against B-ALL cell lines. - The ability of rituximab to eliminate RQR8+ cells in an immunocompetent mouse model.

[0643] Table E. List of in vivo studies TIFF0007634045000053.tif144170

[0644] To demonstrate the activity of UCART22 cells, CAR-driven cytotoxicity and cytokine secretion assays were performed on tumor cell lines and primary B-ALL cells.

[0645] UCART22 activity assessed by cytotoxicity assay Cytotoxic activity against B-ALL cell lines The potential cytotoxicity of UCART22 was evaluated against several tumor cell lines derived from pediatric or adult B-ALL patients (Table F). The results show that MHH-CALL-4, MUTZ-5, SEMK2, PALL-2, LAX2, BALL-1, NALM-6 and RS4;11 are all CD22+ cell lines expressing different levels of CD22. Two acute myeloid leukemia (AML) cell lines (OCI-AML2 and MOLM13) that do not express CD22 were used as negative controls.

[0646] Table F. Cell line characteristics TIFF0007634045000054.tif86168

[0647] The specific cytotoxicity of UCART22 against CD22+ cells is assessed by measuring the viability of CD22+ target cells after co-culture with UCART22 cells. Non-transduced (CAR-) TRAC and CD52 double KO T cells are used as controls to calculate the percentage of specific cytolysis of target CD22+ B-ALL cells by UCART22. Error! Reference source not found shows the cytotoxic activity of UCART22 cells against a panel of CD22+ B-ALL tumor cell lines. UCART22 shows specific cytolytic activity against all cell lines expressing CD22, even at minimal levels.

[0648] Cytotoxic activity against primary B-ALL samples The cytotoxic activity of UCART22 was confirmed against several primary B-ALL samples. The characteristics of the primary samples are shown in Table G. Only patient samples with more than 50% blast cells were used for the co-incubation studies, which accounted for 14 of the 19 samples.

[0649] (Table G) Primary ALL samples evaluated in preclinical studies. ND: not determined TIFF0007634045000055.tif213170

[0650] CD22 and CD19 expression levels were also assessed in all primary B-ALL samples except Pt1. Eleven had CD22 surface expression above CD22-controls, and four B-ALL patient samples showed CD22 surface expression within the range seen in B-ALL cell lines (>1000 CD22 molecules / cell). High CD22 expressers (>60%) also expressed CD19, except for two relapse samples.

[0651] Using the same approach as for the cell lines, CTL activity (>10% specific cell lysis) was confirmed against eight B-ALL patient samples.

[0652] Cytokine secretion assays for B-ALL cell lines and primary B-ALL samples The release of interferon-gamma (IFNγ) and other cytokines by UCART22 cells co-incubated with several cell lines or B-ALL patient samples was assessed using the BioLegend Legend PLEX 13-cytokine assay. IFNγ was found to have the highest concentration of all detected cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, and tumor necrosis factor alpha (TNF-α)) secreted into the medium after 25 hours of co-culture of B-ALL cells with UCART22 or control T cells (NTD DKO).

[0653] When UCART22 was co-incubated with both B-ALL cell lines and primary B-ALL samples, high levels of IFNγ secretion were observed.

[0654] In vitro study 2 TALEN®-induced inactivation of TRAC, B2M and CD52 genes The ability to use UCART22 in an allogeneic setting depends on its ability to prevent cell surface expression of TCRαβ, MHC CI, and eliminate TCR-mediated recognition of histocompatibility antigens that can lead to GvHD and attack by host CD8+ cells. To effectively eliminate TCRαβ and MHC CI from the cell surface, TALEN®-mediated gene editing was used to inactivate TRAC and beta 2 microglobulin gene (B2M), genes. In addition, another TALEN® was used during the manufacturing process of UCART22 to inactivate the CD52 gene, allowing the use of alemtuzumab in lymphodepletion conditioning regimens. In a specific setting, TALEN® was used during the manufacturing process of UCART22 to inactivate beta 2 microglobulin gene (B2M), and alter major histocompatibility complex class I (MHC class I) molecule expression, preventing rejection by host T cells.

[0655] Molecular analysis of TRAC, CD52, and B2M gene disruptions To demonstrate targeted modification of TRAC and CD52 genes at the molecular level, genomic deoxyribonucleic acid (DNA) was isolated from T cells electroporated with GMP-grade TRAC and CD52 TALEN® mRNA or TRAC and B2M TALEN® mRNA and purified for TCRαβ- cells MHCCI-. Regions around the TRAC, B2M and CD52 TALEN® cleavage sites were amplified by polymerase chain reaction (PCR) and analyzed by Next Generation Sequencing (NGS, Illumina). In control samples, the same analysis was performed with cells that were removed before electroporation of TALEN® mRNA (on day 6). This analysis shows that control cells at day 6 did not show any modification at the TRAC and CD52 loci, while high frequency modifications were detected in TALEN®-treated T cells (Table).

[0656] (Table H) Percentage of non-homologous end joining (NHEJ) at TALEN® cleavage in the TRAC, CD52 and B2M loci.

[0657] The percentages are determined by NGS in TALEN®-treated T cells from three different batches. Control cells were sampled before TALEN® mRNA electroporation for all batches.

[0658] (Table H) TIFF0007634045000056.tif24170

[0659] Further analysis of the sequences indicates that the TALEN®-induced modifications are located in small regions surrounding the target site, and that 93-97% of these modifications are deletions. These deletions are generally small, with an average of 97% smaller than 150 bp and 81% smaller than 50 bp.

[0660] Functional assays demonstrating TRAC gene disruption in UCART22 To demonstrate functional inactivation of TCRαβ, UCART22 cells were assayed for their ability to express activation markers such as CD25 and CD69 following phytohemagglutinin (PHA)-mediated TCR stimulation. For UCART22, all made with GMP raw materials, T cells were harvested either before (TRAC) or after (UCART22) depletion of TCRαβ+ cells. Cells were reactivated with 0.5 μg / mL PHA for 24 h. Expression of activation markers (CD25 and CD69) was measured by flow cytometry. While control TRAC cells (65.2% TCRαβ-) upregulate CD25 and CD69, no upregulation was observed in UCART22 cells (98.9% TCRαβ-), confirming the absence of TCRαβ receptors in UCART22.

[0661] Resistance of CD52-UCART22 cells to alemtuzumab The ability to use alemtuzumab, an anti-CD52 monoclonal antibody, in lymphodepletion regimens relies on effectively eliminating the CD52 glycoprotein from the cell surface of UCART22 cells, preventing their elimination by alemtuzumab. UCART22 is therefore engineered using CD52 TALENs to be a mixture of CD52+ and CD52- cells. Flow cytometry analysis performed on development batches of UCART22 at the end of the manufacturing process indicates that on average, 72% of T cells (CD45+ / CD4+ or CD8+ cells) are CD52- (range 62.3-76.5%, N=6).

[0662] To demonstrate that CD52- cells are resistant to alemtuzumab, a complement-dependent cytotoxicity (CDC) assay was performed. Human T cells containing TALEN®-mediated CD52 gene inactivation were treated with 50 μg / mL of the rat anti-CD52 antibody from which therapeutic antibody alemtuzumab is derived or rat immunoglobulin G (IgG) as a control, with or without rabbit complement, at 37° C. for 2 hours, and then analyzed for viability and CD52 expression by flow cytometry.

[0663] The results show that cells that do not contain CD52 gene inactivation are specifically eliminated through complement-dependent cytotoxicity in the presence of alemtuzumab.

[0664] Resistance to host CD8+ T cells Interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression and promoted CD8+ T cell-mediated killing of control UCART22, whereas MHC class I-deficient UCART22 cells were resistant to CD8+ T cell-mediated destruction.

[0665] Demonstration of effective depletion of UCART22 following treatment with rituximab UCART22 is an allogeneic T cell and therefore should be eliminated with the recovery of the patient's immune system. In addition, UCART22 has been engineered to co-express CD22CAR and RQR8, a short membrane protein that provides two epitopes that bind to rituximab (RTX), a therapeutic monoclonal antibody specific for human CD20. RQR8 therefore offers the possibility of depleting UCART22 through the administration of rituximab.

[0666] The ability to eliminate UCART22 cells by complement-dependent cytotoxicity (CDC) after treatment with RTX was examined. Frozen UCART22 (from five different development batches) were thawed and co-cultured with Raji CD22+ cells at a 1:0.25 ratio for 3 days. After this reactivation period, a CDC assay was performed (cells were incubated with RTX (100 μg / ml) for 2 hours in the presence or absence of baby rabbit complement (BRC)). The results demonstrate that approximately 85% of CAR+ cells are efficiently eliminated in vitro in the presence of both RTX and complement.

[0667] Another mechanism of action for RTX, by which depletion of RQR8+ cells can be achieved, is antibody-dependent cell-mediated cytotoxicity (ADCC) (Seidel et al., 2013), in which effector cells of the immune system (mainly natural killer - NK cells) can actively lyse target cells whose membrane surface antigens are bound by specific antibodies.

[0668] Complementary experiments have been published by Martin Pule at UCL (Philip et al., 2014) demonstrating effective in vitro mediated elimination of RQR8+ T cells by CDC and ADCC (primary human T cells were transduced with a bicistronic retroviral vector encoding RQR8 and GFP (SFG.RQR8.IRES.eGFP) and selected with Miltenyi QBEnd10 beads). Time course and RTX dose titration demonstrate that CDC is highly effective at RTX concentrations of 25 μg / mL and above, with killing occurring within 30 minutes. Similarly, ADCC mediated sensitivity was demonstrated for RQR8+ T cells.

[0669] In vivo studies Rationale for species / model selection Due to the human specificity of UCART22, studies in standard immunocompetent animal models are not applicable due to rapid targeting and elimination of human UCART22 cells by the xenogeneic immune response.

[0670] Therefore, two approaches can be envisioned for in vivo modeling of UCART22 activity: syngeneic animal models and human tumor xenograft models in immunodeficient animals.

[0671] A syngeneic animal model approach would involve re-engineering / redeveloping a species-specific form of UCART22 for a model organism, followed by evaluation of the functional properties of this species-specific UCART22 surrogate product when engrafted into a syngeneic host. This approach is not considered valid due to the fact that the properties of individual CARs vary considerably, and species-specific differences in immune function, physiology, and genetics severely impair the estimation of the activity of UCART22 in humans.

[0672] We chose immunodeficient mouse xenograft models because they allow the engraftment of both UCART22 and human tumor CD22+ cells (B-ALL cell lines or primary samples). This animal model is widely used for the evaluation of the in vivo activity of CAR T cell therapy. Although these models have the same limitations as the immunocompetent models described above in terms of on-target / off-tumor cytotoxicity and off-target cytotoxicity, and completely exclude any role of MHC mismatch, they have proven to be extremely effective for evaluating the in vivo antitumor efficacy of CAR T cells. They have emerged as the de facto standard for the evaluation of the in vivo activity of CAR T cell products (see, for example, Carpenito et al., 2009; Gade et al., 2005; Gill et al., 2014; Hudecek et al., 2010; Kenderian et al., 2015; Mardiros et al., 2013; Zhou et al., 2013, among many published articles). They proved capable of discriminating the relative antitumor activity of different CARs, highlighting their ability to capture and semi-quantitatively read out useful human in vivo T cell functions (see, e.g., Milone et al., 2009). Thus, human CD22+ tumor cells were engrafted into immunodeficient mice (NSG mice) followed by administration of UCART22, and tumor burden and survival time were serially assessed as indicators of antitumor activity.

[0673] Selected tumor models For tumor xenografts, UCART22 activity in vivo was evaluated against Daudi cells (CCL-213, ATCC), a CD22+ B-ALL cell line. To track tumor burden by in vivo imaging, the cell line was modified to express firefly luciferase (and GFP) by transduction with a lentiviral vector (amsbio LVP438-PBS) (Daudi-Luc-GFP cells).

[0674] Selected mouse strains Mouse strains used in in vivo preclinical studies were: 1 / To demonstrate anti-tumor activity, highly immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ; The Jackson Laboratory line #5557) lacking mature T cells, B cells and functional NK cells. Immunocompetent mouse model (C57BL / 6 x Balb / c (F1) mice) to demonstrate efficacy of 2 / RQR8-depleted or R2 CD22CAR mechanisms.

[0675] Analysis method The activity of UCART22 in vivo was assessed using: - Bioluminescence measurements of treated mice versus control groups (for xenograft models with luciferase expressing cell lines), - clinical signs, - Overall survival of the mice.

[0676] Route of administration The route of administration of UCART22 in animals is intravenous injection (single, undivided dose). In humans, UCART 19 and UCART 22 were also intravenously injected once, twice, or three or four times (undivided doses) either alone or alternating according to the following sequence: 19. 22 19 22 19 or 22 19 22 19 or 19 / 22 19 / 22.

[0677] Demonstration of antitumor activity of UCART22 in vivo UCART22 activity was demonstrated in immunodeficient mice engrafted with Daudi-Luc-GFP human tumor cells using several developmental batches of UCART22. An example study is shown in Figure 8. In addition, validation studies are ongoing to evaluate the in vivo activity of GMP batches of UCART22.

[0678] Briefly, NSG mice were intravenously injected with 0.5x106 Daudi-Luc-GFP cells on day -7 and treated with UCART22 on day 0 (intravenous injection, 2 doses: 3x106 and 10x106 UCART22 cells / mouse, 5 mice / group) or left untreated (vehicle injection). Non-transduced T cells double KO for TRAC and CD52 (NTD DKO) were injected as controls. The administered UCART22 dose was determined according to the dose of CAR+ T cells used in the literature to demonstrate the in vivo antitumor activity of other CD22CAR T cells (Haso et al., 2013). UCART22 treatment led to the elimination of tumor cells and extended survival, as shown by in vivo imaging, and all treated mice were alive at the end of the study (80 days after UCART22 treatment). The results demonstrated the antitumor activity of UCART22.

[0679] CD52- cells are resistant to alemtuzumab in vivo, and CD52- CAR+ T-cells are active in vivo in the presence of alemtuzumab Studies conducted during the development of UCART19, another UCART product containing the same TRAC / CD52 double knockout, demonstrated that CD52- T cells were resistant to alemtuzumab in vivo ( Poirot et al., 2015 ).

[0680] In addition, the activity of UCART19 cells in the presence of alemtuzumab was demonstrated in a tumor xenograft model in NSG mice. All mice that received tumor cells but not UCART19 cells showed tumor progression and were sacrificed by 13 days after injection. In five of the seven mice that received tumor and UCART19 cells, the tumor was completely controlled by day 13, and in the remaining two mice, partial responses were observed. In contrast, alemtuzumab treatment was observed to only slow tumor progression without UCART19 cell injection (6 / 6 mice). In mice treated with alemtuzumab in combination with UCART19 cells, tumor cells were cleared from the bone marrow, as assessed by luminometry or flow cytometry of cell suspensions obtained from bone marrow isolated 13 days after injection.

[0681] In conclusion, mice that received alemtuzumab therapy 2 days prior to injection of UCART19 cells demonstrate the antitumor efficacy of UCART19 cells in the presence of alemtuzumab in vivo. In addition, spleen engraftment data at day 13 indicates that CD52- T cells are resistant to alemtuzumab in vivo.

[0682] Demonstration of effective rituximab-induced depletion of RQR8+ cells in vivo The sensitivity of RQR8+ UCART22 to RTX depletion was previously shown in vitro. Furthermore, Martin Pule's group showed that RTX can eliminate RQR8+ cells in an immunocompetent mouse model using re-engineered RTX against mouse IgG2a (mRtx-IgG2a), the functional equivalent of human IgG1 (Philip et al., 2014). This antibody was adapted for use in the mouse system by transfer of the binding portion of RTX to mouse Fc, allowing binding to mouse Fc receptors with affinity close to that with which RTX binds to human Fc receptors. Studies were performed using an immunocompetent haploidentical adoptive transfer model with RQR8-transduced C57BL / 6 splenocytes transferred into non-lethally irradiated C57BL / 6 x Balb / c cross (F1) recipients. This model allows good engraftment levels in all lymphoid tissues maintained by allogeneic stimulation, but also preserves endogenous lymphocytes.

[0683] On day 1, 1.5x106 RQR8+ donor splenocytes from C57BL / 6 mice (splenocytes transduced with a retrovirus encoding the RQR8-2A-GD2CAR construct and purified using Miltenyi CD34 beads) were injected intravenously into F1 mice (C57BL / 6 x Balb / c) 4 hours after 5Gy X-ray irradiation preconditioning. On day 7, donor cell engraftment was confirmed by flow cytometry in peripheral blood. Mice were treated three times with murine RTX (ritux-mIgG2a 150μg, injected intravenously via tail vein on days 7, 10 and 12) or PBS (untreated). Each cohort had 5 mice. Animals were sacrificed on day 14 for flow cytometric analysis of spleen, bone marrow, blood and lymph nodes. In RTX-treated mice, 50, 60 and 70% depletion of RQR8+ cells was observed in the spleen, bone marrow and blood, respectively, within 6 hours after ritux-mIgG2a therapy of mice.

[0684] The same study was also performed with the anti-CD19 CAR used in the UCART19 product, an allogeneic engineered CAR T cell product that contains the same RQR8 construct as UCART22.

[0685] Remedy This study evaluated the antitumor activity of single or multiple injections of UCART22. CD22 expressing B-ALL tumor cell line (Daudi expressing high levels of CD22). Cells were transduced with lentiviral vectors to express GFP and firefly luciferase. Tumor cells (0.5x106 Daudi-Luc-GFP) were injected intravenously via the tail vein into NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, strain 005557, The Jackson Laboratory) on day -7. On day 0, mice were randomized into 11 groups of 6 mice according to bioluminescence signal and body weight measured on day -1.

[0686] One, two or three treatments of UCART22 (at 1 million or 3 million CAR+ cells / mouse) were administered. UCART22 was injected intravenously into mice on days 0 + / - 10 + / - 20. The antitumor activity of UCART22 was assessed by tumor burden tracked by bioluminescence imaging on days -1, 7, 14, 21, 28 and 35, observation of clinical signs, and survival of mice.

[0687] Daudi model, treatment with 1 x 106 UCART22 cells / mouse Mice treated two or three times with 1 x 10 UCART22 show better control of tumor progression and longer survival compared to mice treated once with 1 x 10 UCART22 (80% survival at day 60 for 3 doses versus 0% for 1 dose).

[0688] In one experiment, UCART19 was injected intravenously into mice on day 30. In those mice that were still alive, cancer cell levels were undetectable at day 90.

[0689] summary The activity of UCART22 was demonstrated in vitro and in vivo against B-ALL cell lines and in vitro against primary B-ALL samples.

[0690] UCART22 are allogeneic T cells and therefore should be eliminated with the recovery of the patient's immune system. In addition, UCART22 has been engineered to co-express RQR8, a short membrane protein that provides two epitopes that bind the monoclonal antibody, rituximab. RQR8 thus provides a means to deplete RQR8+ UCART22 by administration of rituximab in the event of unmanageable UCART22-associated toxicity, such as cytokine release syndrome (CRS) or GvHD, or prior to allo-HSCT. The efficacy of rituximab to eliminate RQR8+ cells in vitro was demonstrated using CDC and ADCC assays. Furthermore, the efficiency of rituximab-induced depletion of RQR8+ cells was demonstrated in vivo in blood, spleen, bone marrow and lymph nodes using an immunocompetent mouse model and a murine version of rituximab.

[0691] Similar results were obtained using the R2 or QR3 anti-CD22 CARs expressed in TCR-deficient MHC CI-deficient T cells.

[0692] Resistance of CD52- cells to alemtuzumab was demonstrated in vitro and in vivo, allowing for the potential use of alemtuzumab in lymphodepleting regimens.

[0693] Finally, resistance to CD8+ T cell-mediated destruction was observed using the R2 − anti-CD22 CAR expressed in TCR-deficient MHC CI-deficient T cells.

[0694] Additional studies conducted to evaluate various risks are summarized in the Tables and and detailed in the sections below.

[0695] Table I: List of in vitro toxicology studies performed TIFF0007634045000057.tif231170TIFF0007634045000058.tif88170

[0696] Table J: List of in vivo toxicology studies TIFF0007634045000059.tif81170

[0697] The potential risk of GvHD was evaluated in immunodeficient mice during the development of previous UCART products: this animal model allows efficient engraftment of human T cells and has previously been shown to consistently develop xenogeneic GvHD following infusion of human PBMCs or T cells ( Ali et al., 2012 ; Schroeder and DiPersio, 2011 ).

[0698] Off-target activity of CD22 CAR The CD22 specificity of CD22CAR has been evaluated by GLP-compliant tissue cross-reactivity studies that showed no significant unexpected off-target binding of the scFv component of CD22CAR with human tissues, and Retrogenix screens are ongoing. Furthermore, published clinical data on patients treated with m971-derived CAR T cells showed no off-target toxicity (Fry et al., 2017).

[0699] Because CD22 is expressed on normal B cells, a risk of B cell hypoplasia due to the on-target / off-tumor activity of UCART22 is expected as long as UCART22 is persisted.

[0700] GVHD of UCART 22 CAR An essential safety feature of the UCART22 product is whether the potential of UCART22 to mediate GvH reactions was abolished by disruption of the TRAC gene and purification of TCRαβ-cells. This risk was evaluated during the development of previous UCART products (UCART19 and UCART123) in in vivo preclinical studies. GvHD signs were observed in all NSG mice injected with unmodified T cells, with more severe findings at the highest dose, whereas no treatment-related changes were observed in mice injected with UCART. In the hospital, of 14 patients treated with UCART19 so far, 4 of them developed cutaneous GvHD that was mild to moderate (3 grade 1 and 1 grade 2 (Qasim et al., 2017) and data presented at the ASH meeting 2017 by R. Benjamin et W. Qasim) and was manageable without systemic treatment.

[0701] CRS Other potential safety risks associated with the administration of the immunotherapy product and the underlying treated disease were identified: infusion-related reactions, cytokine release syndrome (CRS), tumor lysis syndrome, infections and neurotoxicity. CRS is a frequent adverse event in immunotherapy, especially in CAR T cell trials. In an autologous anti-CD19 CAR T cell clinical trial, the intensity of the observed cytokine release was the result of a combination of several parameters, such as the dose of infused CAR T cells, the signaling domain of the CAR (4-1BB / CD28), the level of CAR T cell proliferation and the patient's tumor burden on the day of administration. These risks will be assessed in vivo in a model similar to a recently published one (Taraseviciute, A., Kean, L., & Jensen, MC (2016). Creation of the First Non-Human Primate Model That Faithfully Recapitulates Chimeric Antigen Receptor (CAR) T Cell-Mediated Cytokine Release Syndrome (CRS) and Neurologic Toxicity Following B Cell-Directed CAR-T Cell Therapy. Blood, 128(22), 651. Accessed March 28, 2018. Retrieved from http: / / www.bloodjournal.org / content / 128 / 22 / 651).

[0702] Starting dose recommendations for FIH trials Standard methods for clinical dosing are not transposable to CAR T cell-based immunotherapy due to the limited validity of animal models, the biomechanical properties of the product, and the adaptogenic mechanism of action.

[0703] Therefore, the selection of the starting clinical dose was mainly based on: (i) previous experience obtained with CAR T cells administered to humans (literature on autologous CAR T cells); (ii) compassionate cases and patients treated in ongoing clinical studies with UCART19 at University College London (UCL) and King's College London (KCL) (see (Qasim et al., 2017) and the presentation by R. Benjamin and W. Qasim at the ASH meeting 2017), and (iii) doses currently studied or approved in other B-ALL autologous CAR studies. The approach is described in the clinical section.

[0704] First results of the clinical study show a significant reduction in tumor burden in both assays (>80%) in patients treated with UCART22. Furthermore, the data demonstrate that a short but efficient treatment (from two doses) can block and evade malignant B cells (73% remission at 150 days), especially after re-dosing and / or use of UCART22 and 19.

[0705] Sequence information SEQUENCE LISTING <110> Cellectis SA <120> Universal anti-CD22 CHIMERIC ANTIGEN RECEPTOR ENGINEERED IMMUNE CELLS <150> DK PA201770239 <151> 2017-03-31 <150> DK PA201770240 <151> 2017-03-31 <150> DK PA201770542 <151> 2017-06-30 <150> PCT / EP2017 / 076800 <151> 2017-10-19 <160> 26 <210> 1 <211> 21 <212> PRT <213> Homo sapiens <220> <223> CD8alpha signal peptide <400> 1 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 2 <211> 20 <212> PRT <213> Homo sapiens <220> <223> signal peptides <400> 2 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly 20 <210> 3 <211> 16 <212> PRT <213> Homo sapiens <220> <223> FcgRIIIa hinge <400> 3 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 1 5 10 15 <210> 4 <211> 45 <212> PRT <213> homo sapiens <220> <223> CD8alpha hinge <400> 4 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 5 <211> 231 <212> PRT <213> homo sapiens <220> <223> IgG1 hinge <400> 5 Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 6 <211> 24 <212> PRT <213> Homo sapiens <220> <223> CD8alpha transmembrane domain <400> 6 I Tyr I Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 7 <211> 27 <212> PRT <213> Homo sapiens <220> <223> 41BB transmembrane domain <400> 7 Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu 1 5 10 15 Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val 20 25 <210> 8 <211> 42 <212> PRT <213> Homo sapiens <220> <223> Fragment of 4-1BB (residues 214-255) <400> 8 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 9 <211> 112 <212> PRT <213> homo sapiens <220> <223> fragment of T-cell surface glycoprotein CD3 zeta chain <400> 9 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 10 <211> 15 <212> PRT <213> artificial sequence <220> <223> G4Sx3 linker sequence <400> 10 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 11 <211> 1532 <212> DNA <213> homo sapiens <220> <223> UCART22 <400> 11 tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca gctgagagaa 60 tggctctgcc cgtcaccgct ctgctgctgc cactggccct gctgctgcac gcagcaagac 120 cacaggtgca gctgcagcag agcggccctg gcctggtgaa gccaagccag acactgtccc 180 tgacctgcgc catcagcggc gattccgtga gctccaactc cgccgcctgg aattggatca 240 ggcagtcccc ttctcggggc ctggagtggc tgggaaggac atactatcgg tctaagtggt 300 acaacgatta tgccgtgtct gtgaagagca gaatcacaat caaccctgac acctccaaga 360 atcagttctc tctgcagctg aatagcgtga caccagagga caccgccgtg tactattgcg 420 ccagggaggt gaccggcgac ctggaggatg cctttgacat ctggggccag ggcacaatgg 480 tgaccgtgtc tagcggagga ggaggatccg gaggaggagg atctggcggc ggcggcagcg 540 atatccagat gacacagtcc ccatcctctc tgagcgcctc cgtgggcgac agagtgacaa 600 tcacctgtag ggcctcccag accatctggt cttacctgaa ctggtatcag cagaggcccg 660 gcaaggcccc taatctgctg atctacgcag caagctccct gcagagcgga gtgccatcca 720 gattctctgg caggggctcc ggcacagact tcaccctgac catctctagc ctgcaggccg 780 aggacttcgc cacctactat tgccagcagt cttatagcat cccccagaca tttggccagg 840 gcaccaagct ggagatcaag accacaaccc cagcaccaag gccacctaca cctgcaccaa 900 ccatcgcctc tcagcccctg agcctgagac ctgaggcatg taggccagca gcaggaggag 960 1020. aaggggtctg gattttgcat gcgacatcta catctgggca cctctggcag gaacatgtgg cgtgctcctg ctcagcctgg tcatcaccct gtactgcaag agaggcagga agaagctgct gtatatcttc aagcagccct tcatgcgccc cgtgcagaca acccaggagg aggatggctg ctcctgtagg ttcccagaag aggaggg aggatgtgag ctgcgcgtga 1200 agttttcccg gtctgccgac gcacctgcat accagcaggg ccagaaccag ctgtataacg agctgaatct gggccggaga gaggagtacg atgtgctgga caagaggcgc ggcagagatc cagagatggg cggcaagccc cggagaaga accctcagga gggcctgtac aatgagctgc agaaggataa gatggccgag gcctattctg agatcggcat gaagggagag aggcgccggg gcaagggaca cgacggactg taccagggac tgagcacagc caccaaggat acctatgacg ccctgcatat gcaggcactg cctccaaggt ga <210> 12 <211> 124 <212> PRT <213> artificial sequence <220> <223> 971- heavy chain <400> 12 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu Glu Asp Ala Phe Asp 100 105 110 Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 13 <211> 107 <212> PRT <213> artificial sequence <220> <223> 971- light chain <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 461 <212> PRT <213> artificial sequence <220> <223> 971-v1 polypeptide CAR sequence <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp 35 40 45 Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro 50 55 60 Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp 65 70 75 80 Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro 85 90 95 Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu 115 120 125 Glu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 165 170 175 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr 180 185 190 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile 195 200 205 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 210 215 220 Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 225 230 235 240 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln 245 250 255 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Leu Ala Val Ser 260 265 270 Thr Ile Ser Ser Phe Phe Pro Gly Tyr Gln Ile Tyr Ile Trp Ala 275 280 285 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 290 295 300 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 305 310 315 320 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 325 330 335 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 340 345 350 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 355 360 365 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 370 375 380 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 385 390 395 400 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 405 410 415 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 420 425 430 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 435 440 445 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 450 455 460 <210> 15 <211> 490 <212> PRT <213> artificial sequence <220> <223> 971-v3 polypeptide CAR sequence <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu 20 25 30 Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp 35 40 45 Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro 50 55 60 Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp 65 70 75 80 Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro 85 90 95 Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro 100 105 110 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Val Thr Gly Asp Leu 115 120 125 Glu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 165 170 175 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr 180 185 190 Leu Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile 195 200 205 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 210 215 220 Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 225 230 235 240 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln 245 250 255 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala 260 265 270 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 325 330 335 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 340 345 350 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 355 360 365 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 370 375 380 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 385 390 395 400 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 405 410 415 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 420 425 430 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 435 440 445 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 450 455 460 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 465 470 475 480 Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 16 <211> 530 <212> PRT <213> artificial sequence <220> <223> TAL binding domain TRAC_T01-L <400> 16 Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn 65 70 75 80 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 100 105 110 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 170 175 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 275 280 285 Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala 290 295 300 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 340 345 350 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala 370 375 380 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 385 390 395 400 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 405 410 415 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val 435 440 445 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 465 470 475 480 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala 515 520 525 Leu Glu 530 <210> 17 <211> 530 <212> PRT <213> artificial sequence <220> <223> TAL binding domain TRAC_T01-R <400> 17 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His 65 70 75 80 Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 100 105 110 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu 115 120 125 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala 130 135 140 Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 165 170 175 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 275 280 285 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 290 295 300 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn 340 345 350 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 370 375 380 Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 385 390 395 400 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 405 410 415 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val 435 440 445 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln 465 470 475 480 Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala 515 520 525 Leu Glu 530 <210> 18 <211> 49 <212> DNA <213> artificial sequence <220> <223> target TALEN TRAC_T01 <400> 18 ttgtcccaca gatatccaga accctgaccc tgccgtgtac cagctgaga 49 <210> 19 <211> 9 <212> PRT <213> artificial sequence <220> <223> Mimotope of CD20 antigen <400> 19 Cys Pro Tyr Ser Asn Pro Ser Leu Cys 1 5 <210> 20 <211> 1473 <212> DNA <213> artificial sequence <220> <223> CAR 11 dis 16 <400> 20 atggctctgc ccgtcaccgc tctgctgctg ccactggccc tgctgctgca cgcagcaaga 60 ccacaggtgc agctgcagca gagcggccct ggcctggtga agccaagcca gacactgtcc 120 ctgacctgcg ccatcagcgg cgattccgtg agctccaact ccgccgcctg gaattggatc 180 aggcagtccc cttctcgggg cctggagtgg ctgggaagga catactatcg gtctaagtgg 240 tacaacgatt atgccgtgtc tgtgaagagc agaatcacaa tcaaccctga cacctccaag 300 aatcagttct ctctgcagct gaatagcgtg acaccagagg acaccgccgt gtactattgc 360 gccagggagg tgaccggcga cctggaggat gcctttgaca tctggggcca gggcacaatg 420 gtgaccgtgt ctagcggagg aggaggatcc ggaggaggag gatctggcgg cggcggcagc 480 gatatccaga tgacacagtc cccatcctct ctgagcgcct ccgtgggcga cagagtgaca 540 atcacctgta gggcctccca gaccatctgg tcttacctga actggtatca gcagaggccc 600 ggcaaggccc ctaatctgct gatctacgca gcaagctccc tgcagagcgg agtgccatcc 660 agattctctg gcaggggctc cggcacagac ttcaccctga ccatctctag cctgcaggcc 720 gaggacttcg ccacctacta ttgccagcag tcttatagca tcccccagac atttggccag 780 ggcaccaagc tggagatcaa gaccacaacc ccagcaccaa ggccacctac acctgcacca 840 accatcgcct ctcagcccct gagcctgaga cctgaggcat gtaggccagc agcaggagga 900 gcagtccata caaggggtct ggattttgca tgcgacatct acatctgggc acctctggca 960 ggaacatgtg gcgtgctcct gctcagcctg gtcatcaccc tgtactgcaa gagaggcagg 1020 aagaagctgc tgtatatctt caagcagccc ttcatgcgcc ccgtgcagac aacccaggag 1080 gaggatggct gctcctgtag gttcccagaa gaggaggagg gaggatgtga gctgcgcgtg 1140 aagttttccc ggtctgccga cgcacctgca taccagcagg gccagaacca gctgtataac 1200 gagctgaatc tgggccggag agaggagtac gatgtgctgg acaagaggcg cggcagagat 1260 ccagagatgg gcggcaagcc ccggagaaag aaccctcagg agggcctgta caatgagctg 1320 cagaaggata agatggccga ggcctattct gagatcggca tgaagggaga gaggcgccgg 1380 ggcaagggac acgacggact gtaccaggga ctgagcacag ccaccaagga tacctatgac 1440 gccctgcata tgcaggcact gcctccaagg tga 1473 <210> 21 <211> 59 <212> DNA <213> artificial sequence <220> <223> TARGET TALEN(R) <400> 21 tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca gctgagaga 59 <210> 22 <211> 1473 <212> DNA <213> Artificial sequence <220> <223> 971-v3 polynucleotide CAR sequence <400> 22 atggctctgc ccgtcaccgc tctgctgctg ccactggccc tgctgctgca cgcagcaaga 60 ccacaggtgc agctgcagca gagcggccct ggcctggtga agccaagcca gacactgtcc 120 ctgacctgcg ccatcagcgg cgattccgtg agctccaact ccgccgcctg gaattggatc 180 aggcagtccc cttctcgggg cctggagtgg ctgggaagga catactatcg gtctaagtgg 240 tacaacgatt atgccgtgtc tgtgaagagc agaatcacaa tcaaccctga cacctccaag 300 aatcagttct ctctgcagct gaatagcgtg acaccagagg acaccgccgt gtactattgc 360 gccagggagg tgaccggcga cctggaggat gcctttgaca tctggggcca gggcacaatg 420 gtgaccgtgt ctagcggagg aggaggatcc ggaggaggag gatctggcgg cggcggcagc 480 gatatccaga tgacacagtc cccatcctct ctgagcgcct ccgtgggcga cagagtgaca 540 atcacctgta gggcctccca gaccatctgg tcttacctga actggtatca gcagaggccc 600 ggcaaggccc ctaatctgct gatctacgca gcaagctccc tgcagagcgg agtgccatcc 660 agattctctg gcaggggctc cggcacagac ttcaccctga ccatctctag cctgcaggcc 720 gaggacttcg ccacctacta ttgccagcag tcttatagca tcccccagac atttggccag 780 ggcaccaagc tggagatcaa gaccacaacc ccagcaccaa ggccacctac acctgcacca 840 accatcgcct ctcagcccct gagcctgaga cctgaggcat gtaggccagc agcaggagga 900 gcagtccata caaggggtct ggattttgca tgcgacatct acatctgggc acctctggca 960 ggaacatgtg gcgtgctcct gctcagcctg gtcatcaccc tgtactgcaa gagaggcagg 1020 aagaagctgc tgtatatctt caagcagccc ttcatgcgcc ccgtgcagac aacccaggag 1080 gaggatggct gctcctgtag gttcccagaa gaggaggagg gaggatgtga gctgcgcgtg 1140 aagttttccc ggtctgccga cgcacctgca taccagcagg gccagaacca gctgtataac 1200 gagctgaatc tgggccggag agaggagtac gatgtgctgg acaagaggcg cggcagagat 1260 ccagagatgg gcggcaagcc ccggagaaag aaccctcagg agggcctgta caatgagctg 1320 cagaaggata agatggccga ggcctattct gagatcggca tgaagggaga gaggcgccgg 1380 ggcaagggac acgacggact gtaccaggga ctgagcacag ccaccaagga tacctatgac 1440 gcctgcata tgcaggcact gcctccaagg is 1473 <210> 23 <211> 489 <212> PRT <213> artificial sequence <220> <223> CAR distal CD22 <400> 23 Put Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Wing Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Ala Phe Ser Ile Tyr Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys 50 55 60 Arg Leu Glu Trp Val Ala Tyr Ile Ser Ser Gly Gly Gly Thr Tyr Tyr 65 70 75 80 Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala 100 105 110 Met Tyr Tyr Cys Ala Arg His Ser Gly Tyr Gly Thr His Trp Gly Val 115 120 125 Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 145 150 155 160 Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg 165 170 175 Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 180 185 190 Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr ...

Claims

1. A polynucleotide encoding: A) A chimeric antigen receptor specific for CD22 (CD22 CAR), comprising: (i) at least one extracellular domain comprising: - an antigen-binding domain specific for CD22, comprising a single chain variable fragment (scFv) comprising a heavy chain variable fragment (VH) of SEQ ID NO: 12 and a light chain variable fragment (VL) of SEQ ID NO: 13, and - a hinge derived from human CD8α having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4, (ii) a transmembrane domain derived from human CD8α having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6; and (iii) an intracellular signaling domain comprising a human CD3 zeta signaling domain having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and a costimulatory signal molecule derived from human 4-1BB having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8; B) Safety switches, including RQR8, as well as C) Peptide 2A linking the safety switch to the CD22 CAR. (a) the hinge of the CD22 CAR has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:4; (b) the transmembrane domain of the CD22 CAR has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6; (c) the CD3 zeta signaling domain of the CD22 CAR has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9; and (d) the human 4-1BB derived costimulatory signal molecule of the CD22 CAR has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8; The polynucleotide of claim 1. (a) the hinge of the CD22 CAR has the amino acid sequence of SEQ ID NO: 4; (b) the transmembrane domain of the CD22 CAR has the amino acid sequence of SEQ ID NO: 6; (c) the CD3 zeta signaling domain of the CD22 CAR has the amino acid sequence of SEQ ID NO: 9; and (d) the human 4-1BB derived costimulatory signal molecule of the CD22 CAR has the amino acid sequence of SEQ ID NO: 8; The polynucleotide of claim 1.

4. A polynucleotide described in any one of claims 1 to 3, wherein the VH and VL of the CD22 CAR are separated by a linker.

5. A polynucleotide described in any one of claims 1 to 4, wherein the CD22 CAR further comprises a leader sequence.

6. 1. Use of an engineered human immune cell, or a population of cells comprising said engineered human immune cell, that expresses a chimeric antigen receptor (CAR) specific for CD22 (CD22 CAR) on its cell surface, in the manufacture of a medicament for use in the prophylaxis or treatment of a patient suffering from a CD22+ mediated cancer, comprising The CD22 CAR comprises: (i) at least one extracellular domain comprising: - an antigen-binding domain specific for CD22, comprising a single chain variable fragment (scFv) comprising a heavy chain variable fragment (VH) of SEQ ID NO: 12 and a light chain variable fragment (VL) of SEQ ID NO: 13, and - a hinge derived from human CD8α having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4, (ii) a transmembrane domain derived from human CD8α having an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6; and (iii) an intracellular signaling domain comprising a human CD3 zeta signaling domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9, and a costimulatory signal molecule derived from human 4-1BB having an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

8. and The CAR further comprises a safety switch, the safety switch comprising RQR8 transiently linked to the CD22 CAR by peptide 2A. The above uses. (a) the hinge of the CD22 CAR has an amino acid sequence having at least 95% sequence identity to SEQ ID NO:4; (b) the transmembrane domain of the CD22 CAR has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6; (c) the CD3 zeta signaling domain of the CD22 CAR has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 9; and (d) the human 4-1BB derived costimulatory signal molecule of the CD22 CAR has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8; 7. The use according to claim 6. (a) the hinge of the CD22 CAR has the amino acid sequence of SEQ ID NO: 4; (b) the transmembrane domain of the CD22 CAR has the amino acid sequence of SEQ ID NO: 6; (c) the CD3 zeta signaling domain of the CD22 CAR has the amino acid sequence of SEQ ID NO: 9; and (d) the human 4-1BB derived costimulatory signal molecule of the CD22 CAR has the amino acid sequence of SEQ ID NO: 8; 7. The use according to claim 6.

9. The use described in any one of claims 6 to 8, wherein the VH and VL of the CD22 CAR are separated by a linker.

10. The use described in any one of claims 6 to 9, wherein the CD22 CAR further comprises a leader sequence.

11. The use according to any one of claims 6 to 10, wherein the engineered human immune cells are T cells (UCART 22) characterized in that the genes encoding TCR subunits are inactivated, or natural killer (NK) cells.

12. The use described in claim 11, wherein the TCR subunit is TCR alpha.

13. The use according to any one of claims 6 to 12, wherein the gene encoding CD52 is inactivated in the engineered human immune cells.

14. The use according to any one of claims 6 to 13, wherein in the engineered human immune cells, a gene selected from the group consisting of the dCK gene and the B2M gene is inactivated.

15. The use according to any one of claims 6 to 14, wherein the polynucleotide according to any one of claims 1 to 5 is inserted into the genome of the engineered human immune cell.

16. 16. The use according to any one of claims 6 to 15, wherein the cancer is a blood cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphocytic carcinoma, acute myeloid leukemia.

17. The use according to any one of claims 6 to 15, wherein the CD22+ mediated cancer is acute lymphoblastic leukemia (ALL).

18. The use of any one of claims 6 to 15, wherein the CD22+ mediated cancer is relapsed or refractory B-ALL.

19. 19. The use according to claim 18, wherein relapsed or refractory B-ALL is as a pediatric indication.

20. An ex vivo method for preparing immune cells for immunotherapy, comprising the step of introducing the polynucleotide according to any one of claims 1 to 5 into immune cells.

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