Universal anti-CD22 chimeric antigen receptor engineered immune cells

Abstract

The present application relates to an engineered immune cell with a novel CD22 chimeric antigen receptor (CD22 CAR) having a deletion in the TRAC gene that can redirect the specificity and responsiveness of the immune cell to selected tumor cells. Engineered immune cells with such CARs are particularly suitable for treating relapsed, refractory, CD22-expressing cancers.

Description

Technical Field

[0001] The present invention generally relates to the field of immunotherapy, more specifically, to a universal chimeric antigen receptor T cell (UCART22) specific to CD22 and a method for engineering the cell, the universal chimeric antigen receptor T cell (UCART22) specific to CD22 is an engineered human primary immune cell comprising at least one edited gene and a chimeric antigen receptor (CAR) (CAR CD22) specific to differentiation cluster 22 (CD22), and the edited gene is preferably a gene encoding a TCR subunit or a CD52 gene. The present invention further relates to UCART22 in patients who may or may not be cell initial donors ("allogeneic (allogenic)" or "autologous" CD22 CAR engineered human primary immune cells) as a treatment for relapsed refractory hematological cancers. The cells expressing CD22 according to the present invention are particularly effective and safe for immunotherapy, particularly for aggressive or recurrent cancers. Background Art

[0002] In 2000, blood cancers (non-Hodgkin lymphoma, leukemia) were estimated to kill more than 45,000 people in the United States (Greenlee et al., CA Cancer J. Clin., 50:7-33 (2000)). Similar figures were reported in 2014, and despite advances in treatment, such as chemotherapy, the prognosis for this type of cancer remains essentially unchanged. (EK Mai, U Bertsch, J Dürig, C Kunz, M Haenel, IW Blau, M Munder, A Jauch, B Schurich, T Hielscher, M Merz, BHuegle-Doerr, A Seckinger, D Hose, J Hillengass, MS Raab, K Neben, HW Lindemann, MZeis, C Gerecke, IGH Schmidt-Wolf, K Weisel, C Scheid, H Salwender and HGoldschmidt.Phase III trial of bortezomib,cyclophosphamide and dexamethasone(VCD)versus bortezomib,doxorubicin and dexamethasone(PAd)in newly A phase III trial of bortezomib, cyclophosphamide, and dexamethasone (VCD) versus bortezomib, doxorubicin, and dexamethasone (PAd) in newly diagnosed myeloma. Leukemia (March 19, 2015) | doi: 10.1038 / leu.2015.80.)

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

[0004] CAR is a synthetic receptor composed of a targeting portion associated with one or more intracellular signaling domains in a single molecule or in multiple transmembrane domains forming a multimer. In a specific CAR, the binding portion consists of an antigen binding domain from a single-chain antibody (scFV), comprising a variable fragment of a monoclonal antibody connected by a linker. Binding portions based on receptor or ligand domains have also been successfully used to prepare CARs.

[0005] The addition of signal transduction domains from co-stimulatory molecules of the T cell receptor (TCR), as well as specific transmembrane and hinge domains, was used to form second and third generation CARs, resulting in successful therapeutic trials in humans. 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 re-injected into the same patient. (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 antigenreceptors targeting B-cell precursor acute lymphoblasticleukemia. Blood. 2013Feb 14;121(7):1165-74.doi:10.1182 / blood-2012-06-438002.Epub 2012Dec 14).

[0006] The approach, which allows immune cells from one individual to be engineered before being reinjected into the same individual (usually with cancer), is not well suited for use with aggressive forms of tumors that may be racing against time. In addition, the approach can be problematic or inconclusive in patients with altered immune systems.

[0007] To alleviate this problem, immunotherapy using so-called “allogeneic” T cells expressing CARs (also called universal or “off-the-shelf” T cells) has recently been implemented, and the first two patients who received such cell therapy remain in remission approximately two years after treatment.

[0008] Nevertheless, there are still some aspects of this therapy that need to be improved, such as effectiveness in the presence of anti-T cell drugs, effectiveness against escaped tumor cells, persistence, means of control, etc. In fact, it seems that by downregulating the expression of surface antigens recognized by CAR, cancer cells may escape treatment and survive, although redirected immunity still exists in treated patients. In addition, the main adverse reaction observed in patients treated with autologous or allogeneic T cells, which is sometimes fatal, is the cytokine release syndrome.

[0009] Therefore, there remains a need to develop effective and safe treatments for these diseases, particularly for aggressive or refractory / relapsed forms of hematological cancers. Summary of the invention

[0010] Here, the inventors have developed a new effective therapy ("UCART22") comprising engineered primary human T cells with a deletion of at least the TRAC gene, endowed with a chimeric antigen receptor targeting CD22 and a safety marker, allowing the control of the number and thus the activity of said cells in vivo.

[0011] In a new effective therapy comprising engineered primary human T cells with a deletion of at least the TRAC gene ("UCART22"), at least one additional deletion in the CD52, dCK, β2 microglobulin genes is contemplated.

[0012] These new UCART22s are particularly effective for adoptive transfer in patients with CD22-mediated pathologies, whether or not the patient is a naive donor of the immune cells, and whether or not the patient has received immune-affecting treatments.

[0013] The UCART22 cells of the present invention are resistant to at least one chemical or antibody drug such as CAMPATH and / or purine nucleotide analogs (PNA) commonly used to treat CD22-mediated pathology. The UCART22 cells of the present invention are able to survive and be active in the presence of the drug used at a dose that kills more than 80% of the cells.

[0014] Significant and unexpected clinical advantages of engineered isolated primary immune cells UCART22 have been observed, including low cytokine release, no or very mild graft-versus-host disease, and significant activity against refractory relapsed forms of blood cancer cells.

[0015] Other advantages will be revealed in this study.

[0016] This study provides:

[0017] 1. An engineered human T cell receptor knockout (TCR KO) cell (UCART22) having a chimeric antigen receptor (CAR) specific for CD22, a safety switch, preferably expressed on the cell surface, the anti-CD22 CAR (CD22 CAR) comprising:

[0018] i) at least one extracellular domain comprising:

[0019] - the hinge domain from CD8α,

[0020] - an antigen binding domain specific for CD22, optionally a leader sequence,

[0021] ii) the transmembrane domain from CD8α, and

[0022] iii) an intracellular signaling domain, the CD22 CAR having a polypeptide sequence having at least 80% identity to SEQ ID N°15,

[0023] The safety switch comprises:

[0024] - Peptide 2A is transiently linked to RQR8 of CD22 CAR, or

[0025] - At least two rituximab mAb specific epitopes linked to the CD22 CAR, preferably located between the VH and hinge domains, or 3 rituximab mAb specific epitopes or 3 rituximab mAb specific epitopes and a QBEN-10 mAb specific epitope.

[0026] 2. UCART22 according to embodiment 1, wherein a polynucleotide sequence having at least 80% identity with SEQ ID N°11 is inserted into the genome, and further comprises an inactivated TRAC gene having an insertion, deletion or mutation within SEQ ID N°18, and has an undetectable level of T cell receptor (TCR) on the cell surface as measured by flow cytometry, and an undetectable level of off site events as measured by guide sequence technology.

[0027] According to UCART22 of embodiment 2, the anti-CD22 CAR is preferably inserted into the TRAC gene, preferably having SEQ ID N°18.

[0028] 3. The UCART22 according to any one of embodiments 1 to 2 comprises another inactivated gene selected from the group consisting of dCK gene, B2M gene, and CD52 gene, preferably CD52 gene.

[0029] 4. The UCART22 of any one of embodiments 1 to 3, wherein at least one additional gene is inactivated, the gene being selected from genes encoding aryl hydrocarbon receptor (AHR), transforming growth factor β receptor (TGFβ receptor), interleukin 10 receptor (IL-10R), programmed cell death protein 1, and combinations thereof.

[0030] 5. The UCART22 of any one of embodiments 1 to 4, wherein the gene encoding β2 microglobulin (B2M) is inactivated.

[0031] 6. The UCART22 of any one of embodiments 1 to 5, wherein the gene encoding the aryl hydrocarbon receptor (AHR) is inactivated.

[0032] 7. The UCART22 of any one of embodiments 1 to 6, wherein the gene encoding transforming growth factor beta receptor (TGFβ receptor) is inactivated.

[0033] 8. The UCART22 of any one of embodiments 1 to 7, wherein the gene encoding interleukin 10 receptor (IL-10R) is inactivated.

[0034] 9. The UCART22 of any one of embodiments 1 to 8, wherein the gene encoding programmed cell death protein 1 (PD1) is inactivated.

[0035] 5. The UCART22 according to any one of embodiments 1 to 4, comprising an additional scfv specific for any one of the following tumor-associated surface antigens selected from the group consisting of CD19, CD20, CD30, major histocompatibility complex (MHC) molecules, immunoglobulins (Ig), CD3, CD5, CD34, CD79, preferably CD79b, CD138, B7-1 (CD80), BCMA (CD269, TNFRSF 17), FLT-3 or PAX5, preferably CD19.

[0036] 6. According to any one of embodiments 1 to 5, the UCART22, wherein the CD22 CAR further comprises an antigen binding domain specific for CD19, or wherein the UCART22 further comprises a CD19 CAR, preferably a CD19 CAR having at least 80% identity with SEQ ID N°25 or SEQID N°26.

[0037] 7. According to any one of embodiments 1 to 5, the UCART22, wherein the anti-CD22 CAR is a single-chain CAR or a multi-chain CAR.

[0038] 8. UCART22 according to any one of embodiments 5 or 6, wherein the anti-CD19 CAR is a single-chain CAR or a multi-chain CAR.

[0039] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD19.

[0040] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD20.

[0041] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD30.

[0042] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for a major histocompatibility complex (MHC) molecule.

[0043] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for immunoglobulin (Ig).

[0044] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD3.

[0045] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD5.

[0046] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD34.

[0047] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD79, preferably CD79b.

[0048] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD138.

[0049] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD80.

[0050] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for BCMA (CD269).

[0051] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for TNFRSF 17.

[0052] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scFv specific for FLT-3.

[0053] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD19.

[0054] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD79a or CD79b.

[0055] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD20.

[0056] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD30.

[0057] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for a major histocompatibility complex (MHC) molecule.

[0058] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for immunoglobulin (Ig).

[0059] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD3.

[0060] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD5.

[0061] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD34.

[0062] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD138.

[0063] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD80.

[0064] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for BCMA (CD269).

[0065] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for TNFRSF 17.

[0066] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for FLT-3.

[0067] 9. A population of UCART cells comprising UCART22 according to any one of embodiments 1 to 8.

[0068] 10. A population of UCART cells according to embodiment 9, comprising UCART19, preferably UCART19 expressing an anti-CD19 CAR comprising a sequence of SEQ ID N°25 or SEQ ID N°26 on the cell surface.

[0069] 11. A kit comprising UCART22 and UCART19 for administration in a patient in need thereof, either consecutively (at least once), or concomitantly, or consecutively (at least once) followed by concomitantly.

[0070] 12. The kit according to embodiment 11, wherein UCART19 is used first at least once, twice, three times, four times or several times, followed by the use of UCART22 alone or together with UCART19.

[0071] 13. The kit according to embodiment 11 or 12, wherein UCART22 is used first at least once, twice, three times, four times or several times, followed by the use of UCART19 alone or together with UCART22.

[0072] 14. The kit according to any one of embodiments 11 to 13, further comprising a lymphodepletion treatment administered prior to UCART.

[0073] 15. The kit according to any one of embodiments 11 to 14, wherein lymphocyte depletion is achieved with fludarabine and cyclophosphamide, preferably 25 mg / m on day -6 to day -2. 2 iv×5 doses of fludarabine and 1 dose of cyclophosphamide 60 mg / kg iv on day -5.

[0074] 16. The kit according to any one of embodiments 11 to 15 comprises at least one other UCART cell directed against a cancer antigen selected from CD79a, CD79b, CD20, CD30, CD52, CD40, CD80, CD86, CD74, and VEGF.

[0075] 17. A pharmaceutical composition comprising the UCART22 according to any one of embodiments 1 to 8 or a cell population comprising the UCART22 according to embodiment 9 or 10 and a pharmaceutically acceptable excipient.

[0076] 18. The pharmaceutical composition according to embodiment 17, further comprising bryostatin, preferably bryostatin 1.

[0077] 19. The pharmaceutical composition according to embodiment 17 or 18 or the kit according to any one of embodiments 11 to 16, for use as a medicament for preventing or treating patients suffering from CD22+-mediated cancer or CD22+-mediated inflammatory diseases.

[0078] 20. A pharmaceutical composition according to any one of embodiments 17 or 18 or a kit according to any one of embodiments 11 to 16, for use as a medicament for preventing or treating a patient suffering from a CD19+-mediated cancer or a CD19+-mediated inflammatory disease.

[0079] 21. A pharmaceutical composition according to any one of embodiments 17 or 18 or a kit according to any one of embodiments 11 to 16, for use as a medicament for preventing or treating patients suffering from CD19+CD22+ mediated cancer or CD19+CD22+ mediated inflammatory diseases.

[0080] 22. A pharmaceutical composition according to any one of embodiments 17 or 18 or a kit according to any one of embodiments 11 to 16, for use according to any one of embodiments 19 or 21, wherein treating the patient comprises a step of administering the pharmaceutical composition at least twice (re-administration) to avoid recurrent / refractory development of cancer.

[0081] 23. A pharmaceutical composition according to any one of embodiments 17 to 18 or a kit according to any one of embodiments 11 to 16, for use according to any one of embodiments 19 or 21, for treating a CD22-mediated blood cancer selected from the group consisting of lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic leukemia (ALL), Cancer, acute myeloid leukemia, preferably a blood cancer expressing CD22 selected from the group consisting of lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, more preferably a relapsed refractory blood cancer expressing CD22, even more preferably an aggressive form of said CD22-associated blood cancer.

[0082] 24. A pharmaceutical composition according to any one of embodiments 17 to 18 or a kit according to any one of embodiments 11 to 16, for use according to any one of embodiments 20 or 21, for treating a CD19-mediated blood cancer selected from the group consisting of lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic leukemia (ALL), Cancer, acute myeloid leukemia, preferably a blood cancer expressing CD19 selected from the group consisting of lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, more preferably a relapsed refractory blood cancer expressing CD19, even more preferably an aggressive form of the CD19-associated blood cancer.

[0083] 25. The pharmaceutical composition according to any one of embodiments 17 to 18 or the kit according to any one of embodiments 11 to 16, for use according to any one of embodiments 19 to 22, for the treatment of relapsed or refractory B-expressing ALL, preferably as a pediatric indication.

[0084] 26. A pharmaceutical composition according to any one of embodiments 17 to 18 or a kit according to any one of embodiments 11 to 16, for use according to any one of embodiments 19 to 25, wherein treating the patient comprises administering to the patient at least one monoclonal antibody (mAb), preferably QBEN 10 and / or rituximab, at a dose that allows contact of the UCART22 with at least one specific mAb.

[0085] Implementation of CD22 CAR

[0086] The present invention provides an anti-CD22 CAR comprising a hinge domain selected from the group consisting of FcRIIIα, CD8α, IgG1, IgG4 and PD1, preferably selected from CD8α or IgG4.

[0087] The above-mentioned anti-CD22 CAR, wherein the scfv specific for CD22 comprises VH and VL connected to each other by a linker L1, preferably comprising 1-3 "GGGGS" motifs, more preferably one "GGGGS" motif.

[0088] An anti-CD22 CAR according to any of the above items, wherein the scfv specific for CD22 is connected to the transmembrane domain via a hinge selected from a hinge from FcRIIIα, CD8α, IgG1, preferably a hinge from CD8α.

[0089] The anti-CD22 CAR according to any one of the above items, comprises an intracellular domain, wherein the intracellular domain comprises a CD3ζ signaling domain and a 4-1BB signaling domain.

[0090] An anti-CD22 CAR as described 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 hinge of a scFv specific for CD22.

[0091] An anti-CD22 CAR as described in any one of the above, wherein the mAb specific epitope is a polypeptide selected from the following: CPYSNPSLC, NSELLSLINDMPITNDQKKLMSNN, CQFDLSTRRLKC, CQYNLSSRALKC, CVWQRWQKSYVC, CVWQRWQKSYVC, SFVLNWYRMSPSNQTDKLAAFPEDR, SGTYLCGAISLAPKAQIKE, ELPTQGTFSNVSTNVSPAKPTTTA, ELPTQGTFSNVSTNVSPAKPTTTA, GQNDTSQTSSPS, preferably ELPTQGTFSNVSTNVSPAKPTTTA and / or CPYSNPSLC (SEQ ID N°19).

[0092] The present invention provides a polynucleotide encoding an anti-CD22 CAR according to any one of the above items or a polynucleotide having at least 80% identity with the polynucleotide encoding an anti-CD22 CAR, and a vector comprising any one of the above polynucleotides.

[0093] As disclosed herein below, other genes in the UCART22, especially the dCK gene, B2M gene, and CD52 gene, are edited.

[0094] Provided by UCART

[0095] Wherein provided is UCART22 as described above, and at least one additional gene is edited or engineered, wherein the gene is selected from genes encoding: aryl hydrocarbon receptor (AHR), transforming growth factor β receptor (TGFβ receptor), interleukin 10 receptor (IL-10R), programmed cell death protein 1, and combinations thereof.

[0096] Wherein UCART22 as described above and at least one additional gene contain mutations, deletions or insertions that inactivate their activity and / or expression, and the genes are selected from genes encoding the following: aryl hydrocarbon receptor (AHR), transforming growth factor β receptor (TGFβ receptor), interleukin 10 receptor (IL-10R), programmed cell death protein 1, and combinations thereof.

[0097] Wherein UCART22 as described above and at least one additional gene contain mutations, deletions or insertions that inactivate their activity and / or expression, and the genes are selected from genes encoding the following: aryl hydrocarbon receptor (AHR), transforming growth factor β receptor (TGFβ receptor), interleukin 10 receptor (IL-10R), and combinations thereof.

[0098] Wherein UCART22 as described above and at least one additional gene contain mutations, deletions or insertions that inactivate their activity and / or expression, and the genes are selected from genes encoding the following: transforming growth factor β receptor (TGFβ receptor), interleukin 10 receptor (IL-10R), programmed cell death protein 1, and combinations thereof.

[0099] Wherein UCART22 as described above and at least one additional gene comprise a mutation, deletion or insertion that inactivates their activity and / or expression, and the gene is selected from genes encoding: aryl hydrocarbon receptor (AHR), interleukin 10 receptor (IL-10R), programmed cell death protein 1, and combinations thereof.

[0100] Wherein UCART22 as described above and at least one additional gene contain mutations, deletions or insertions that inactivate their activity and / or expression, and the genes are selected from genes encoding: aryl hydrocarbon receptor (AHR), transforming growth factor β receptor (TGFβ receptor), programmed cell death protein 1, and combinations thereof.

[0101] Table 9 describes the genes into which the CAR can be inserted.

[0102] 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

[0103]

[0104] The genes shown in bold are preferably inactivated in the UCART of the present invention.

[0105] UCART22 as described above comprises an anti-CD22 CAR (UCART22) according to any one of the above items and a polynucleotide encoding the anti-CD22 CAR (UCART22) inserted into the genome (inserted into the CD25 gene).

[0106] The UCART22 according to any one of the above items, wherein the anti-CD22 CAR is a single-chain CAR or a multi-chain CAR.

[0107] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for any one of the following tumor-associated surface antigens selected from: CD19, CD20, CD30, major histocompatibility complex (MHC) molecules, immunoglobulins (Ig), CD3, CD5, CD34, CD79, preferably CD79b, CD138, B7-1 (CD80), BCMA (CD269, TNFRSF 17), FLT-3 or PAX5, preferably CD19.

[0108] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD19.

[0109] The UCART22 according to any one of the above items expresses anti-CD22 CAR and anti-CD19 CAR, preferably anti-CD22 CAR and anti-CD19 CAR of SEQ ID N°94 or SEQ ID N°95.

[0110] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD20.

[0111] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD30.

[0112] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for a major histocompatibility complex (MHC) molecule.

[0113] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for immunoglobulin (Ig).

[0114] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD3.

[0115] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD5.

[0116] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD34.

[0117] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD79, preferably CD79b.

[0118] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD138.

[0119] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for CD80.

[0120] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single-chain CAR comprising an additional scFv specific for BCMA (CD269).

[0121] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single chain CAR A comprising an additional scFv specific for TNFRSF 17.

[0122] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a single chain CAR comprising an additional scFv specific for FLT-3.

[0123] The UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR.

[0124] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD19.

[0125] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD79a or CD79b.

[0126] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain (mc) CAR comprising an additional scFv specific for CD19.

[0127] The UCART22 according to any of the above items expresses anti-CD22 mcCAR and anti-CD19 CAR, preferably anti-CD22 mcCAR and anti-CD19 CAR of SEQ ID N°94 or SEQ ID N°95.

[0128] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD20.

[0129] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD30.

[0130] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for a major histocompatibility complex (MHC) molecule.

[0131] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for immunoglobulin (Ig).

[0132] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD3.

[0133] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD5.

[0134] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD34.

[0135] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD138.

[0136] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for CD80.

[0137] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for BCMA (CD269).

[0138] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for TNFRSF 17.

[0139] UCART22 according to any of the above items, wherein the anti-CD22 CAR is a multi-chain CAR comprising an additional scFv specific for FLT-3.

[0140] A cell population comprising UCART22 according to any of the above items.

[0141] A cell population comprising UCART22 according to any one of the above items, wherein the cells expressing the anti-CD22 CAR also express anti-CD19 CAR on the cell surface, preferably the anti-CD19 CAR comprises the sequence of SEQ ID N°25 or SEQ ID N°26.

[0142] A CD22 CAR T cell (UCART22), optionally in combination with a protein kinase C inhibitor such as bryostatin 1 as a pharmaceutical composition of the present invention, is particularly useful in treating CLL, ALL, multiple myeloma (MM), blastic plasmacytoid dendritic cell neoplasm (BPDCN), especially refractory / relapsed ALL, refractory / relapsed CLL and / or aggressive forms of these diseases, more preferably refractory or relapsed B-ALL.

[0143] CD22 CAR T cells derived from m971 (UCART22) are optionally combined with a protein kinase C inhibitor such as bryostatin 1 as a pharmaceutical composition of the present invention in the treatment of CLL, ALL, multiple myeloma (MM), blastic plasmacytoid dendritic cell neoplasm (BPDCN), especially refractory / relapsed ALL, refractory / relapsed CLL and / or aggressive forms of these diseases, more preferably refractory or relapsed B-ALL.

[0144] The engineered immune cells of the present invention not only show a high level of in vivo activity against malignant cells and less cytokine release, but also their number and activity are controlled, thereby providing a safe and effective method for immunotherapy.

[0145] The present invention provides engineered human T cell receptor knockout (TCR KO) cells having a chimeric antigen receptor specific for CD22 (CD22 CAR) (UCART22), wherein the chimeric antigen receptor specific for CD22 (CD22 CAR) is preferably expressed on the cell surface,

[0146] Wherein the CD22 CAR comprises:

[0147] i) at least one extracellular domain comprising:

[0148] - the hinge domain from CD8α,

[0149] - an antigen binding domain specific for CD22, optionally a leader sequence,

[0150] ii) the transmembrane domain from CD8α, and

[0151] iii) Intracellular signal transduction domain.

[0152] The UCART22 comprises at least one additional edited gene, preferably a deletion in the CD52 gene.

[0153] The present invention also provides UCART CD22 as described above, wherein the antigen binding domain comprises a Antibody The scfv specific for CD22, preferably the scfv comprises a sequence selected from SEQ ID N°12, SEQ ID N°13, SEQ ID N°14 and SEQ ID N°15, more preferably a sequence from SEQ ID N°15.

[0154] In one embodiment, the present invention also provides an engineered human T cell receptor knockout (TCR KO) cell (UCART22) having a chimeric antigen receptor specific for CD22 (CD22 CAR), preferably a chimeric antigen receptor specific for CD22 (CD22 CAR) expressed on the cell surface,

[0155] Wherein the CD22 CAR comprises:

[0156] i) at least one extracellular domain comprising:

[0157] - the hinge domain from CD8α,

[0158] - an antigen binding domain specific for CD22, optionally a leader sequence,

[0159] ii) the transmembrane domain from CD8α, and

[0160] iii) Intracellular signal transduction domain.

[0161] The UCART22 comprises at least one additional edited gene, preferably a deletion in the CD52 gene.

[0162] And wherein the antigen binding domain comprises a scfv specific to the distal portion of CD22, and the CAR has SEQ ID N°20.

[0163] According to the above UCART CD22 contains the following sequence:

[0164] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSANDDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK or has 80 to 99% homology with each of the sequences.

[0165] The present invention also provides UCART22 as described above comprising the polypeptide of SEQ ID N°15 and a safety switch, wherein the safety switch comprises:

[0166] - Peptide 2A is transiently linked to RQR8 of CD22 CAR, or

[0167] - At least two rituximab mAb specific epitopes linked to the CD22 CAR, preferably 3 rituximab mAb specific epitopes, and more preferably 3 rituximab mAb specific epitopes and a QBEN-10 mAb specific epitope.

[0168] [Enables the sorting and / or depletion of immune cells having the CD22 CAR]

[0169] The present invention also provides UCART22 as described above, comprising at least one additional edited gene, preferably a deletion in the CD52 gene or the dCK gene, or a deletion in the β2 microglobulin gene or the CTIIA gene, even more preferably a deletion in the CD52 gene, and / or an insertion in the HIF-1α gene, resulting in resistance to hypoxia.

[0170] The present invention provides UCART22 as described above comprising a deletion in the CD52 gene, in combination with alemtuzumab.

[0171] The present invention also provides UCART22 as described above, wherein the CD22 CAR is in hypoxia (less than 5% O 2 , preferably less than 1% O 2 Low O 2 concentration).

[0172] The present invention also provides UCART22 comprising a polynucleotide as described above, the polynucleotide encoding a polypeptide comprising a CD22CAR and a safety switch. In a preferred embodiment, the present invention provides UCART22 as described above, which comprises a polynucleotide of SEQ ID N°22 and SEQ ID N°18 in the same gene, preferably in the TRAC gene.

[0173] The present invention also provides UCART22 comprising a polynucleotide as described above, the polynucleotide encoding a polypeptide comprising CD22CAR and a safety switch. In a preferred embodiment, the present invention provides UCART22 as described above, which comprises a polynucleotide of SEQ ID N°20 and SEQ ID N°18 in the same gene, preferably in the TRAC gene.

[0174] The present invention provides a cell population comprising UCART22 as described above.

[0175] The present invention provides a pharmaceutical composition comprising the UCART22 or cell population as described above and a pharmaceutically acceptable excipient, wherein the cell population comprises any one of the UCART22 as described above.

[0176] In a specific embodiment, a pharmaceutical composition is provided, comprising a UCART22 according to the present invention or a cell population comprising the UCART22 in combination with UCART CD19 and a pharmaceutically acceptable excipient. UCART19 and UCART22 can be administered at the same time, concomitantly or sequentially with UCART19 or UCART22 first, as a function of the expression levels of CD19 and CD22 in the patient's cancer cells.

[0177] In a specific embodiment, a pharmaceutical composition is provided, comprising UCART22 / 19 or UCART19 / 22 (bispecific CAR single chain or multi-chain CAR or cells expressing both CD19 CAR and CD22 CAR) and a pharmaceutically acceptable excipient. The present invention provides a pharmaceutical composition as described above, further comprising a compound of the bryostatin family, preferably bryostatin 1.

[0178] The present invention provides a therapeutically effective amount of UCART22 as described above or a pharmaceutical composition containing UCART22 as described above, for use as a drug for treating patients.

[0179] The present invention provides a therapeutically effective amount of UCART22 as described above or a pharmaceutical composition as described above, for use as a drug for treating a patient, wherein treating the patient comprises administering at least twice (re-administering) the therapeutically effective amount of UCARTCD22 or the pharmaceutical composition to avoid recurrence.

[0180] In a specific embodiment, the present invention provides any one of the UCART CD22 described above or any one of the pharmaceutical compositions described above for use as a drug for treating a patient, wherein treating the patient comprises administering the UCART CD22 described above or the pharmaceutical composition described above at least twice to avoid recurrence.

[0181] The present invention provides a therapeutically effective amount of UCART22 as described above or a pharmaceutical composition as described above, for use as a drug for treating a patient as described above, for treating a CD22-related disease, preferably a CD22-related B-cell malignancy (e.g., a CD22-related blood cancer).

[0182] In certain embodiments, the present invention provides:

[0183] A therapeutically effective amount of UCART22 as described above or a pharmaceutical composition as described above, for use as a medicament for treating a blood cancer, such as a blood cancer selected from the following: lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, preferably a blood cancer expressing CD22, more preferably a relapsed refractory blood cancer expressing CD22, and even more preferably an invasive form of the CD22-related blood cancer.

[0184] Preferably, UCART22 is used to treat patients with relapsed refractory B-ALL.

[0185] The present invention provides a therapeutically effective amount of UCART22 as described above or a pharmaceutical composition as described above, for use as a medicament for treating a patient, wherein the patient suffers from a cancer selected from the following: alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder tumor), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cancer, middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney 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 tumors, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer.

[0186] The present invention provides:

[0187] 1. An engineered human T cell receptor knockout (TCR KO) cell (UCART22) having a chimeric antigen receptor (CAR) specific for CD22 that is preferentially expressed on the cell surface,

[0188] Wherein the anti-CD22 CAR comprises:

[0189] i) at least one extracellular domain comprising:

[0190] - the hinge domain from CD8α,

[0191] - an antigen binding domain specific for CD22, optionally a leader sequence,

[0192] ii) the transmembrane domain from CD8α, and

[0193] iii) Intracellular signal transduction domain.

[0194] The UCART22 comprises at least one additional edited gene, preferably an inactivated CD52 gene, an inactivated dCK gene, an inactivated β2 microglobulin gene or an inserted HIF-1α gene.

[0195] 2. According to UCART CD22 described in 1, wherein the antigen binding domain comprises a scFv specific for CD22, and the scFv is derived from the m971 antibody (m971), and preferably the scFv comprises a sequence selected from SEQ ID N°12, SEQ ID N°13, SEQ ID N°14 or SEQ ID N°15, more preferably a sequence from SEQ ID N°15.

[0196] 3. The UCART22 according to any one of 1 to 2, comprising a polypeptide of SEQ ID N°15 and a safety switch, wherein the safety switch comprises:

[0197] - Peptide 2A is temporarily linked to RQR8 of CD22 CAR,

[0198] - At least two rituximab mAb specific epitopes, preferably 3 rituximab mAb specific epitopes, more preferably 3 rituximab mAb specific epitopes and a QBEN-10 mAb specific epitope attached to the CD22 CAR

[0199] [Enabling the sorting and / or depletion of immune cells having the CD22 CAR].

[0200] 4. The UCART22 according to any one of 1 to 3, comprising a CD52 inactivated gene, even more preferably a CD52 inactivated gene and a HIF-1α inserted gene, conferring resistance to alemtuzumab and hypoxia.

[0201] 5. UCART22 according to any one of 1 to 4, wherein the anti-CD22 CAR is at low O 2 Concentration (<5% O 2 ) are expressed on the cell surface.

[0202] 6. The UCART22 according to any one of 1 to 5, comprising a polynucleotide encoding a polypeptide comprising the anti-CD22 CAR in 1 to 5.

[0203] The UCART22 of any one of the above-described items comprises an anti-CD22 CAR on the cell surface, a polynucleotide encoding the anti-CD22 CAR inserted into the TRAC gene, and an exogenous polynucleotide sequence encoding IL-12 inserted into the CD25 genomic sequence, the B2M genomic sequence, or the PD1 genomic sequence.

[0204] Any of the UCART22 described above comprises an anti-CD22 CAR on the cell surface, a polynucleotide encoding the anti-CD22 CAR inserted into the TRAC gene, an exogenous polynucleotide sequence encoding IL-12 inserted into the CD25 genomic sequence or the PD1 genomic sequence, and further comprises a genomic KO gene selected from IL-10R receptor, TGFβ receptor, TIM-3, and LAG-3 (see Table 9 above).

[0205] 7. UCART22 according to any one of 1 to 6, further comprising the polynucleotide of SEQ ID N°22, preferably comprising the sequences of SEQ ID N°18 and SEQ ID N°22 in the same TRAC gene.

[0206] 8. A cell population comprising the UCART22 according to any one of 1 to 7.

[0207] 9. A pharmaceutical composition comprising the UCART22 according to any one of 1 to 7 or the cell population comprising the UCART22 according to 8 and a pharmaceutically acceptable excipient.

[0208] 10. The pharmaceutical composition according to 9, further comprising bryostatin, preferably bryostatin 1.

[0209] 11. A therapeutically effective amount of UCART22 according to any one of 1 to 7 or the pharmaceutical composition according to 9 or 10, for use as a drug for treating a patient.

[0210] 12. A therapeutically effective amount of UCART22 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 according to 11, wherein treating the patient comprises administering at least twice (re-administering) the therapeutically effective amount of UCART CD22 or the pharmaceutical composition to avoid relapse.

[0211] 13. A therapeutically effective amount of UCART22 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 according to 11 or 12, for treating a CD22-related disease, preferably a CD22-related B-cell malignancy.

[0212] 14. A therapeutically effective amount of UCART22 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 according to any one of 11 to 13, for treating a blood cancer selected from the following: lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, preferably a blood cancer expressing CD22, more preferably a relapsed refractory blood cancer expressing CD22, and even more preferably an invasive form of the CD22-related blood cancer.

[0213] 15. A therapeutically effective amount of UCART22 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 according to any one of 11 to 13, for treating relapsed and / or refractory CD22-positive B-ALL.

[0214] 16. A therapeutically effective amount of UCART22 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 according to any one of 11 to 13, wherein the patient suffers from cancer, preferably a CD22+ cell-mediated cancer, selected from the following: alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer, pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal tumors, 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, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, preferably liver cancer and lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0215] Figure 1 : Schematic diagram of engineered immune cells according to the present invention

[0216] Figure 1 The engineered immune cells shown in can be T cells with polynucleotides encoding CAR of the present invention. The T cells are further engineered to be better and more safely implanted into the patient. X or Y is an edited gene that can be mutated, deleted and / or have an insertion. For example, a gene expressing a T cell receptor (TCR) component (e.g., TCRα or TCRβ) can be deleted or can include an insertion, and Y can be a gene related to the sensitivity of T cells to immunosuppressive drugs, such as dCK (about tolerance to purine nucleotide analogs) or CD52 (about Campas (Campath)) or HPRT (about 6-thioguanine).

[0217] Figure 2 : Schematic diagram of single-chain and multi-chain CD22 CAR

[0218] Figure 2 The CD22 CAR of the present invention is illustrated, comprising a scFv specific for CD22, optionally comprising a safety switch, hinge and transmembrane domains from CD8α, intracellular domains from 4-1BB and CD3ζ, and optionally a domain conferring resistance to hypoxia.

[0219] Figure 3 : Examples of CD22 CAR constructs of the invention comprising a safety switch

[0220] At least one rituximab (R) mAb specific epitope (black box), preferably 3 rituximab mAb specific epitopes, and more preferably 3 rituximab mAb specific epitopes and a QBEN-10 (Q) mAb specific epitope (gray box) are inserted into the CD22CAR.

[0221] R may be inserted into the scFv between the VH and VL domains (or the VL and VH domains), and / or into the hinge.

[0222] Figure 4 Examples of polypeptides encoded by polynucleotides

[0223] Figure 4 represents an example of a polypeptide that will be cleaved such that a safety switch (RQR8) and CD22 CAR will be expressed on the cell surface.

[0224] R = CD20 mimotope (bound by rituximab), Q = CD34 epitope (bound by QBEnd10)

[0225] Figure 5 :Degranulation activity of UCART22

[0226] Degranulation activity of UCART22 (scFv-V1 directed against the proximal domain of CD22) compared to that of T cells not transduced (NT) or transduced with a CAR targeting the distal portion of CD22 (scFv-V2) in the presence of CD22-positive NALM-16 cells compared to CD22-negative SUP-T1 cells.

[0227] Figure 6 :Cytotoxic activity of UCART22

[0228] Cytotoxic activity of UCART22 (scFv-V1 directed against the proximal domain of CD22) compared to that of T cells not transduced (NT) or transduced with a CAR targeting the distal end of CD22 (scFv-V2) in the presence of CD22-positive NALM-16 cells compared to CD22-negative SUP-T1 cells.

[0229] Figure 7 : Interferon-γ production by UCART22

[0230] Figure 8 : Survival of mice in the presence of control cells, UCART22 (scfv-V1 targeting the proximal domain of CD22), or CART22 (without TRAC deletion).

[0231] Fig. 9: General strategy for inserting genes into TRAC using TALEN. DETAILED DESCRIPTION

[0232] Unless specifically defined herein, all technical and scientific terms used have the same meanings as commonly understood by those skilled in the fields of gene therapy, biochemistry, genetics, immunology, and molecular biology.

[0233] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, wherein suitable methods and materials are described herein. All publications, patent applications, patents and other references are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. In addition, unless otherwise specified, materials, methods and examples are illustrative only and are not intended to be limiting.

[0234] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art and are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gaited., 1984); Mullis et al.USPat.No.4,683,195; Nucleic Acid Hybridization (BD Harries&S.J.Higgins eds.1984); Transcription And Translation (BDHames&S.J.Higgins eds.1984); Culture Of Animal Cells (RIFreshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes(IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelsonand M.Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols.154and 155 (Wu et al.eds.) and Vol.185, "Gene Expression Technology"(D.Goeddel,ed.); Gene Transfer Vectors For Mammalian Cells(JHMiller andM.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods InCell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DMWeir and CCBlackwell, eds., 1986); Harbor, NY, 1986). .

[0235] CD22-specific chimeric antigen receptor

[0236] The present invention relates to a new design of anti-CD22 chimeric antigen receptor (CAR or CD22 CAR or anti-CD22 CAR), which is a chimeric antigen receptor capable of binding to CD22, particularly the proximal domain of CD22.

[0237] The CD22-specific chimeric antigen receptor of the present invention comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises an extracellular ligand binding domain and a hinge, and optionally a suicide domain, and the intracellular domain comprises a signal transduction domain.

[0238] The CD22 of the present invention expressed on the cell surface comprises an extracellular domain, and the extracellular domain comprises an extracellular ligand-binding domain.

[0239] As used herein, the term "extracellular ligand binding domain" is defined as an oligopeptide or polypeptide capable of binding to at least one epitope of CD22. Preferably, the extracellular ligand binding domain will be capable of interacting at least partially with a cell surface molecule (which interacts with CD22) and with another cell surface antigen or another membrane-bound antigen, or directly with CD22, or with human CD22, more precisely directly with the proximal region of human CD22 (from amino acid 243 to amino acid 687).

[0240] In one embodiment, the CD22 CAR of the present invention comprises an extracellular domain comprising an extracellular ligand binding domain capable of interacting with the proximal region of CD22 (from amino acid position 243 to amino acid position 687) and the distal portion of CD22 (from amino acid position 20 to amino acid position 425).

[0241] In the present invention, the full-length extracellular domain of CD22 is from amino acid position 20 to amino acid position 687, the membrane proximal domain of CD22 is from amino acid position 243 to amino acid position 687, and the membrane distal domain of CD22 is from amino acid position 20 to amino acid position 425.

[0242] In one embodiment, the extracellular ligand binding domain can be selected to recognize a specific form (glycosylated) of CD22 that serves as a cell surface marker associated with a particular disease state.

[0243] In a preferred embodiment, the extracellular ligand binding domain comprises at least one single-chain antibody fragment (scFv) comprising a light (V) fragment of a target antigen-specific monoclonal anti-CD22 antibody connected by a linker, in particular a flexible linker. L ) and weight (V H ) variable fragment. L and V H Preferred is antibody m971 from the following Table 2. They are preferably linked together by a flexible linker comprising, for example, the sequence SEQ ID NO.10.

[0244] For the purpose of the present invention, according to the present invention, a complete human anti-CD22 antibody, a specific portion of the m971 antibody (m971) previously identified and characterized using a phage display method (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) is combined into a specific sequence to generate a new CD22 CAR. See also WO2014065961, which is incorporated herein by reference.

[0245] A preferred embodiment of the present invention provides a CAR comprising an antigen binding domain, which antigen binding domain comprises a single-chain variable fragment (scFv) of an m971 antigen binding domain having the following parts shown in Table 1 below, consists of a single-chain variable fragment (scFv) of an m971 antigen binding domain having the following parts shown in Table 1 below, or basically consists of a single-chain variable fragment (scFv) of an m971 antigen binding domain having the following parts shown in Table 1 below.

[0246] Table 1: Sequences of different domains in CD22 CAR

[0247]

[0248] Table 2: Sequences of different CD22 scfv VH and VL

[0249]

[0250] Table 3: CD22 of structure V-1

[0251]

[0252] Table 4: CAR of structure V-3

[0253]

[0254] In a preferred embodiment, the CD22 CAR of the present invention comprises the sequence of SEQ ID N°15.

[0255] In a preferred embodiment, the CD22 CAR of the present invention comprises the sequence of SEQ ID N°23.

[0256] The sequence of the CD22 CAR of the present invention comprises a signal peptide, a TM domain from CD8α, and a linker between the VH and VL domains from m971.

[0257] SCFV

[0258] In the present invention, scFv is a heavy chain (V H结构域 ) and light chain (V L结构域 ) is a fusion protein connected to a short linker peptide of 10-25 amino acids (preferably SEQ ID N°10).

[0259] In a preferred embodiment, the CAR preferably comprises an extracellular ligand binding domain, which comprises a polypeptide sequence that exhibits at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID N°12.

[0260] In a preferred embodiment, the CAR preferably comprises an extracellular ligand binding domain, which comprises a polypeptide sequence that exhibits at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence SEQ ID N°13.

[0261] In a preferred embodiment, the CAR preferably comprises an extracellular ligand binding domain, which comprises a polypeptide sequence exhibiting at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID N°12 and a polypeptide sequence exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence selected from the group consisting of SEQ ID No.13.

[0262] Hinge

[0263] The extracellular domain may further comprise a hinge region between the extracellular ligand binding domain and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide for connecting a transmembrane domain to an extracellular ligand binding domain. In particular, the hinge region is used to provide more flexibility and accessibility for the extracellular ligand binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids, more preferably 10 to 50 amino acids. The hinge region may be derived from all or part of a naturally occurring molecule, such as all or part of an extracellular region derived from CD8 or CD4, or from 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 fully synthetic hinge sequence. In a preferred embodiment, the hinge domain comprises a portion of a human CD8α chain, an FcRIIIα receptor or an IgG1, see SEQ ID N°4, SEQ ID N°5 and SEQ ID N°6 of the present specification, respectively. In a more preferred embodiment, the hinge polypeptide exhibits at least 90%, 95%, 97&, 99% or 100% sequence identity to SEQ ID N°4, and even more preferably exhibits 100% sequence identity to SEQ ID N°4.

[0264] A hinge from IgG4 or from PD1 is part of the present invention and disclosed in WO2016120216 and can be used to construct the CD22 CAR of the present invention.

[0265] The CD22 CAR of the present invention is anchored to the membrane of the cell. Therefore, this CD22 CAR also includes a transmembrane domain. The distinguishing features of a suitable transmembrane domain include the ability to express on the surface of the cell and the interaction to guide the cellular response of immune cells to predetermined target cells, and the above-mentioned cells are preferably immune cells in the present invention, especially lymphocytes or natural killer (NK) cells. The transmembrane domain can be derived from a natural or synthetic source. The transmembrane domain can be derived from any membrane-bound protein or transmembrane protein. As a non-limiting example, a transmembrane polypeptide can be a subunit of a T cell receptor such as α, β or δ, a polypeptide constituting a CD3 complex, an IL2 receptor p55 (α chain), p75 (β chain) or chain, an Fc receptor, in particular a subunit chain of Fc receptor III or CD protein. Alternatively, the transmembrane domain can be synthetic and can include mainly hydrophobic residues such as leucine and valine.

[0266] In a preferred embodiment, the transmembrane domain is derived from human CD8 α chain (eg, NP_001139345.1).

[0267] The CD22 CAR of the present invention generally further comprises a transmembrane domain (TM), in particular a transmembrane domain from CD8α showing identity to the polypeptide of SEQ ID N°6 or SEQ ID N°7. Preferably, the CAR of the present invention comprises a TM showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the polypeptide of SEQ ID N°6.

[0268] The CD22 CAR of the present invention generally further comprises a transmembrane domain (TM) from CD8α showing identity to the polypeptide of SEQ ID N° 6. Preferably, the CAR of the present invention comprises a TM showing 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 N° 6.

[0269] The CD22 CAR of the present invention comprises an intracellular domain, which comprises a signal transduction domain or an intracellular signaling domain.

[0270] The signal transduction domain or intracellular signaling domain of the CD22 CAR of the present invention is responsible for intracellular signal transduction after the extracellular ligand binding domain binds to the target, causing immune cell activation and immune response (cell lysis activity to target cells). In other words, the signal transduction domain is responsible for the activation of at least one normal effector function of the immune cells expressing CAR. For example, the effector function of T cells can be cell lysis activity or auxiliary activity including the secretion of cytokines. Therefore, the term "signal transduction domain" refers to the part of the protein that transduces the effector signal function signal and instructs the cell to perform a specific function.

[0271] Preferred examples of signal transduction domains in the CD22 CAR of the present invention can be cytoplasmic sequences of auxiliary receptors that cooperate with T cell receptors and synergistically initiate signal transduction and then antigen receptor coordination, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities. The signal transduction domain contains two different types of cytoplasmic signal transduction sequences, one of which is a sequence that initiates antigen-dependent primary activation, and the other is a sequence that acts in an antigen-independent manner to provide a secondary or co-stimulatory signal. The primary cytoplasmic signal transduction sequence may include a signal transduction motif known as an immunoreceptor tyrosine-based activation motif of ITAM. ITAM is a well-defined signal transduction motif found in the cytoplasmic tail of a variety of receptors used as a syk / zap70 class tyrosine kinase binding site. Examples of ITAM used in the present invention may include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d as non-limiting examples. In a preferred embodiment, the signal transduction domain of CAR may comprise a CD3 ζ signal transduction 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 N ° 9. Alternatively, the CD3 ζ signal transduction domain comprises a polypeptide sequence showing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID N ° 9.

[0272] In a more preferred embodiment, the signal transduction domain of CD22 CAR is composed of the CD3ζ signal transduction domain of SEQ ID N°9, and does not include any sequence from the human CD28 signal transduction domain. In a specific embodiment, the signal transduction domain of the CAR of the present invention comprises a co-stimulatory signal molecule. Co-stimulatory signal molecules are cell surface molecules other than ligands required for antigen receptors or their effective immune responses. "Co-stimulatory ligands" refer to molecules on antigen presenting cells that specifically bind to associated public stimulatory molecules on T cells to provide signals, in addition to the primary signal provided by, for example, the TCR / CD3 complex binds to the peptide-loaded MHC molecule, which mediates T cell responses, including but not limited to proliferation activation, differentiation, etc. Costimulatory ligands may 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 molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, among others, antibodies that specifically bind to costimulatory molecules presented on T cells, such as, but not limited to, CD27, 4-1BB, OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0273] "Costimulatory molecules" refer to associative binding partners on T cells that specifically bind to co-stimulatory ligands to mediate co-stimulatory responses of cells (such as but not limited to proliferation). Co-stimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors. Examples of co-stimulatory molecules include CD27, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3 and ligands that specifically bind to CD83.

[0274] In a preferred embodiment, the signal transduction 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 signal transduction domain of the CAR of the present invention comprises an amino acid sequence comprising 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 N°8.

[0275] In a more preferred embodiment, the signal transduction domain of the CAR of the present invention does not contain a sequence from CD28 (NP_006130.1).

[0276] In an even more preferred embodiment, all embodiments of the invention do not comprise sequences from CD28 (NP_006130.1).

[0277] In a more preferred embodiment, the signal transduction domain of the CD22 CAR of the present invention comprises a portion of the co-stimulatory signal molecule 4-1BB (GenBank: AAA53133) and does not comprise a sequence from CD28 (NP_006130.1).

[0278] The present invention provides a CD22-specific chimeric antigen receptor (CD22 CAR), comprising:

[0279] The extracellular domain contains:

[0280] 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),

[0281] - Hinge, preferably from CD8α

[0282] - transmembrane domain, preferably from CD8α

[0283] -Intracellular domain, comprising:

[0284] Co-stimulatory signaling molecules from human 4-1BB, and

[0285] An intracellular signaling domain comprising the human CD3ζ signaling domain.

[0286] In a preferred embodiment, the CD22 CAR of the present invention lacks sequences from CD28.

[0287] In a preferred embodiment, the CD22 CAR of the present invention does not contain any sequence from CD28 and comprises a leader sequence, a TM domain and a hinge from CD8α, preferably does not contain a sequence from CD28 and comprises a leader sequence of SEQ ID N°1, a TM domain of SEQ ID N°6 and a hinge of SEQ ID N°4 from CD8α.

[0288] In one embodiment, the CD22 CAR of the present invention comprises a leader sequence from human CD8α (SEQ ID N°1) or a leader sequence having at least 95% identity to SEQ ID N°1.

[0289] In another embodiment, the CD22 CAR of the present invention comprises a leader sequence of SEQ ID N°2 or a leader sequence having at least 95% identity to SEQ ID N°2.

[0290] In one embodiment, the present invention provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising:

[0291] - 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), even more preferably comprises VH and VL from m971,

[0292] - Hinge from human CD8α

[0293] - Transmembrane domain from human CD8α

[0294] -Co-stimulatory signaling molecule from human 4-1BB

[0295] - an intracellular signaling domain comprising a human CD3ζ signaling domain.

[0296] In one embodiment, the present invention provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising:

[0297] - 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), even more preferably comprises VH and VL from m971,

[0298] - Hinge from human FcRIIIα

[0299] - Transmembrane domain from human CD8α

[0300] -Co-stimulatory signaling molecule from human 4-1BB

[0301] - an intracellular signaling domain comprising a human CD3ζ signaling domain.

[0302] The present invention also provides a CD22-specific chimeric antigen receptor (CD22 CAR), comprising:

[0303] - 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), even more preferably comprises VH and VL from m971,

[0304] - Hinge from human IgG1

[0305] - Transmembrane domain from human CD8α

[0306] -Co-stimulatory signaling molecule from human 4-1BB

[0307] - an intracellular signaling domain comprising a human CD3ζ signaling domain.

[0308] The last three embodiments comprise a CD22 CAR having a signal peptide of SEQ ID N°1 or SEQ ID N°2, preferably SEQ ID N°1.

[0309] More preferably, the CD22 CAR lacks sequences from CD28.

[0310] The scFv of the present invention is derived from an antibody specific for CD22, and comprises a VH domain separated from a VL domain by a linker, wherein the VH and / or VL domain contribute to binding to CD22.

[0311] In a preferred embodiment, UCART22 of the present invention comprises an scfv from the m971 antibody, wherein there is a GS4 linker between VH and VL, and there is a 2R between VL and the CD8α hinge.

[0312] In the present invention, scFv is the heavy chain (V H结构域 ) and SEQ ID N° 13 light chain (V L结构域 ) with a linker peptide, preferably a fusion protein connected to the variable region of SEQ ID N°10.

[0313] In one embodiment, the scFv of the present invention further comprises a leader sequence (or signal peptide), preferably the leader sequence is connected to the VH domain.

[0314] Embodiments wherein the leader sequence is linked to the VL domain are part of the present invention.

[0315] In one embodiment, the VH domain is attached to the hinge, and in another embodiment, the VL domain is attached to the hinge.

[0316] The present invention provides scFvs connected to hinges of different lengths, preferably from CD8α, IgG1 or FcRIIIα (see Figure 2 ), more preferably a hinge from CD8α.

[0317] Preferably, the present invention provides a CD22 CAR comprising:

[0318] - a signal peptide, for example the signal peptide of SEQ ID N°2 or the signal peptide from CD8α SEQ ID N°1.

[0319] - (scFv), comprising a VH domain separated from a VL domain by a linker, said VH and VL and the linker facilitating binding to CD22,

[0320] - A hinge from the human CD8 alpha chain or a hinge from human IgG1 or a hinge from human FcRIII alpha, preferably from CD8 alpha.

[0321] - Transmembrane domain (TM) from CD8α

[0322] -Co-stimulatory signaling molecule from human 4-1BB

[0323] - an intracellular signaling domain comprising a CD3 zeta signaling domain.

[0324] One of the CD22 CARs of the present invention consists of:

[0325] - Leader sequence (e.g., CD8α leader sequence or CD8α signal peptide)

[0326] -Anti-CD22 scFv comprising VH, linker and VL, or VL, linker and VH, wherein the VH and VL are derived from m971

[0327] CD8α hinge

[0328] CD8αTM

[0329] Co-stimulatory signaling molecules from 4-1BB

[0330] Intracellular CD3ζ signaling domain.

[0331] One of the CD22 CARs of the present invention comprises in succession:

[0332] -CD8α signal peptide, which can be removed after being expressed on the cell surface,

[0333] - an epitope recognized by QBEN 10 and an epitope recognized by rituximab,

[0334] - an anti-CD22 scFv comprising a VH, a linker and a VL, said VH and VL being derived from two consecutive epitopes of m971 recognized by rituximab,

[0335] CD8α hinge

[0336] CD8αTM

[0337] Co-stimulatory signaling molecules from 4-1BB

[0338] Intracellular CD3ζ signaling domain

[0339] One of the CD22 CARs of the present invention comprises in succession:

[0340] -CD8α signal peptide, which can be removed after being expressed on the cell surface,

[0341] - an epitope recognized by QBEN 10 and an epitope recognized by rituximab,

[0342] - an anti-CD22 scFv comprising a VH, a linker and a VL, said VH and VL being derived from two consecutive epitopes of m971 recognized by rituximab,

[0343] CD8αTM

[0344] Co-stimulatory signaling molecules from 4-1BB

[0345] Intracellular CD3ζ signaling domain

[0346] One of the CD22 CARs of the present invention comprises in succession:

[0347] -CD8α signal peptide, which can be removed after being expressed on the cell surface,

[0348] -Anti-CD22 scFv comprising VH, linker and VL, wherein the VH and VL are from m971

[0349] Two consecutive epitopes recognized by rituximab,

[0350] CD8α hinge

[0351] CD8αTM

[0352] Co-stimulatory signaling molecules from 4-1BB

[0353] Intracellular CD3ζ signaling domain

[0354] One of the CD22 CARs of the present invention comprises in succession:

[0355] -CD8α signal peptide, which can be removed after being expressed on the cell surface,

[0356] -Anti-CD22 scFv comprising VH, linker and VL, wherein the VH and VL are from m971

[0357] Two consecutive epitopes recognized by rituximab,

[0358] CD8αTM

[0359] Co-stimulatory signaling molecules from 4-1BB

[0360] Intracellular CD3ζ signaling domain

[0361] One of the CD22 CARs of the present invention comprises in succession:

[0362] -CD8α signal peptide, which can be removed after being expressed on the cell surface,

[0363] -Anti-CD22 scFv, comprising VH, linker and VL, said VH and VL being derived from m971, anti-CD19 scFv

[0364] Two consecutive epitopes recognized by rituximab,

[0365] CD8α hinge

[0366] CD8αTM

[0367] Co-stimulatory signaling molecules from 4-1BB

[0368] Intracellular CD3ζ signaling domain

[0369] One of the CD22 CARs of the present invention comprises in succession:

[0370] -CD8α signal peptide, which can be removed after being expressed on the cell surface,

[0371] -Anti-CD22 scFv, comprising VH, linker and VL, said VH and VL being derived from m971, anti-CD19 scFv

[0372] Two consecutive epitopes recognized by rituximab,

[0373] CD8αTM

[0374] Co-stimulatory signaling molecules from 4-1BB

[0375] Intracellular CD3ζ signaling domain

[0376] One of the CD22 CARs of the present invention comprises in succession:

[0377] -CD8α signal peptide, which can be removed after being expressed on the cell surface,

[0378] -Anti-CD19 scFv and anti-CD22 scFv, comprising VH, linker and VL, wherein the VH and VL are from m971

[0379] Two consecutive epitopes recognized by rituximab,

[0380] CD8α hinge

[0381] CD8αTM

[0382] Co-stimulatory signaling molecules from 4-1BB

[0383] Intracellular CD3ζ signaling domain

[0384] One of the CD22 CARs of the present invention comprises in succession:

[0385] -CD8α signal peptide, which can be removed after being expressed on the cell surface,

[0386] -Anti-CD19 scFv and anti-CD22 scFv, comprising VH, linker and VL, wherein the VH and VL are from m971,

[0387] Two consecutive epitopes recognized by rituximab,

[0388] CD8αTM

[0389] Co-stimulatory signaling molecules from 4-1BB

[0390] Intracellular CD3ζ signaling domain.

[0391] Connector – SCFV

[0392] The linker of the present invention can be, for example, a pentapeptide (GGGGS) n or (G4S) n or (Gly4Ser) n wherein n=1 to 4, preferably n=3, 18-mer GGSSRSSSSGGGGSGGGG (Andris-Widhopf et al., 2011) and 20-mer (G4S) 4(Schaefer et al., 2010). The linker of the present invention may include sequences with additional functions, such as epitope tags (Sblattero & Bradbury, 2000 Nature Biotechnology 18, 75-80), at least on the sequence of SEQ ID NO 20, preferably two sequences separated by a linker that usually improve the performance of the scFv of the present invention in the context of a specific antibody sequence.

[0393] Among other linkers, suitable for the present invention is a 15-mer peptide linker (RGRGRGRGRSRGGGS) (Zhihong Shen, Heping Yan, Ying Zhang, Raymond L. Mernaugh, and Xiangqun Zeng (2008), Anal Chem. 80(6): 1910-1917).

[0394] In a preferred embodiment, the linker connecting the VH of m971 to the VL sequence (or connecting the VL to the VH sequence) is a linker of the formula (G4S)n, wherein n is 1-3, preferably the sequence (G4S)3 (SEQ ID N°10) with n=3.

[0395] In one embodiment, the present invention provides:

[0396] CD22 CAR, including:

[0397] - Human CD8α leader sequence (CD8α leader sequence or CD8α signal peptide) of SEQ ID N°1

[0398] -Anti-CD22 scfv comprising the VH of SEQ ID N°12, the linker of SEQ ID N°10 and the VL of SEQ ID N°13

[0399] Human CD8α hinge of SEQ ID N°4,

[0400] Human CD8αTM of SEQ ID N°6

[0401] · SEQ ID N°8 co-stimulatory signal molecule from 4-1BB

[0402] The intracellular CD3ζ signaling domain of SEQ ID N°9.

[0403] In one embodiment, the present invention also provides:

[0404] CD22 CAR, including:

[0405] - Human CD8α leader sequence (CD8α leader sequence or CD8α signal peptide) of SEQ ID N°1

[0406] -Anti-CD22 scfv comprising the VH of SEQ ID N°12, the linker of SEQ ID N°10 and the VL of SEQ ID N°13

[0407] Human FcγRIIIα hinge of SEQ ID N°3,

[0408] Human CD8αTM of SEQ ID N°6

[0409] · SEQ ID N°8 co-stimulatory signal molecule from 4-1BB

[0410] The intracellular CD3ζ signaling domain of SEQ ID N°9.

[0411] In one embodiment, the present invention provides:

[0412] CD22 CAR, including:

[0413] - Human CD8α leader sequence (CD8α leader sequence or CD8α signal peptide) of SEQ ID N°1

[0414] -Anti-CD22 scfv comprising the VL of SEQ ID N°13, the linker of SEQ ID N°10 and the VH of SEQ ID N°12

[0415] Human FcγRIIIα hinge of SEQ ID N°3,

[0416] Human CD8αTM of SEQ ID N°6

[0417] · SEQ ID N°8 co-stimulatory signal molecule from 4-1BB

[0418] The intracellular CD3ζ signaling domain of SEQ ID N°9.

[0419] In one embodiment, the present invention provides:

[0420] CD22 CAR, including:

[0421] - Human CD8α leader sequence (CD8α leader sequence or CD8α signal peptide) of SEQ ID N°1

[0422] -Anti-CD22 scfv comprising the VL of SEQ ID N°13, the linker of SEQ ID N°10 and the VH of SEQ ID N°12

[0423] Human CDA alpha hinge of SEQ ID N° 4,

[0424] Human CD8αTM of SEQ ID N°6

[0425] · SEQ ID N°8 co-stimulatory signal molecule from 4-1BB

[0426] The intracellular CD3ζ signaling domain of SEQ ID N°9.

[0427] In one embodiment, the present invention provides a CD22-specific chimeric antigen receptor (CD22CAR), comprising:

[0428] - a signal peptide having at least 80%, preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the polypeptide of SEQ ID N°1 or SEQ ID N°2; preferably the signal peptide has at least 80%, preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the polypeptide of SEQ ID N°1.

[0429] - 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 N°10.

[0430] The VH domain has at least 90%, 95%, 97%, 99% or 100% sequence identity with the polypeptide of SEQ ID N°12.

[0431] The VL domain has at least 90%, 95%, 97%, 99% or 100% sequence identity with the polypeptide of SEQ ID N°13.

[0432] - a hinge derived from 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 with the polypeptide of SEQ ID N°4.

[0433] - a transmembrane domain derived from human CD8α, having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID N°6.

[0434] - A co-stimulatory signal molecule derived from human 4-1BB (or a 4-1BB intracellular domain), which has 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 the amino acid sequence selected from the group consisting of SEQ ID N°8.

[0435] - an intracellular signaling domain comprising a CD3ζ signaling domain, which has 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 N°9.

[0436] In a preferred embodiment, the CD22-specific chimeric antigen receptor (CD22CAR) of the present invention does not include any sequence from human CD28, particularly from the internal signal transduction domain of human CD28. In a more preferred embodiment, the CD22-specific chimeric antigen receptor (CD22 CAR) of the present invention does not include any sequence from human CD28, particularly from the internal signal transduction domain of human CD28, and further includes a signal peptide from CD8α, preferably fused to the VH domain of a scfv specific for CD22.

[0437] In one embodiment, the present invention provides a CD22 CAR of SEQ ID N°15.

[0438] In one embodiment, the present invention provides a CD22 CAR having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID N°15.

[0439] In one embodiment, the present invention provides a CD22 CAR having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide of SEQ ID N°22.

[0440] The CD22 CAR of the present invention comprises the following sequence:

[0441] v1-m971(FcγRIIIα-CD8αTM-41BB.IC-CD3ζ.IC)

[0442]

[0443] V3-m971(CD8α-CD8αTM-41BB.IC-CD3ζ.IC)

[0444]

[0445] In a preferred embodiment, the CD22 CAR of the present invention comprises the following sequence.

[0446]

[0447] Sequence of CD22 CAR with signal peptide from SEQ ID N°2, TM domain from CD8α and linker between VH and VL domains:

[0448]

[0449] M971 V1

[0450]

[0451] M971 V3

[0452]

[0453] In one embodiment, the invention provides the following sequence:

[0454] In this CD22 CAR, the signal peptide is missing.

[0455] In one embodiment, UCART22 of the present invention comprises the sequence of SEQ ID N°20:

[0456] In one embodiment, UCART22 of the present invention comprises the sequence of SEQ ID N°22.

[0457] In a preferred embodiment, the UCART22 of the present invention comprises a sequence of SEQ ID N°22, which is inserted into the human TRAC gene (such as the TRAC gene of human chromosome 14—NC_000014.9) and expresses an anti-CD22 CAR specific to the proximal portion of CD22 on the cell surface.

[0458] In a more preferred embodiment, the UCART22 of the present invention comprises a sequence of SEQ ID N°20, which is inserted into the human TRAC gene (such as the TRAC gene of human chromosome 14—NC_000014.9) and expresses an anti-CD22 CAR specific to the distal portion of CD22 on the cell surface.

[0459] In one aspect, the anti-CD22 binding domain of the CD22 CAR of the present invention is an anti-CD22 binding domain specific for the distal portion of CD22.

[0460] HA22-CAR

[0461] Therefore, the present invention provides a CD22 CAR having the following sequence:

[0462]

[0463] Preferably, the UCART22 of the present invention comprises the sequence:

[0464]

[0465] In the UCART22 of the present invention, the anti-CD22 binding domain specific to the distal portion of CD22 can be expressed alone or together with the anti-CD22 binding domain specific to the proximal portion of CD22.

[0466] In one aspect, the anti-CD22 binding domain of the CD22 CAR of the present invention is an optimized anti-CD22 binding domain.

[0467] As used herein, an "optimized" antibody (or scFv) is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (eg, Fv, Fab, Fab', F(ab') 2 Preferably, the antibody is a human immunoglobulin (receptor antibody) in which residues from the complementary determining regions (CDRs) are replaced with residues from the CDRs to achieve the desired specificity, affinity and capacity.

[0468] Donor CDR may be subjected to few amino acid changes, which can significantly affect or change the binding properties of the CD22 CAR of the present invention. In fact, one of the present invention provides is a CD22 CAR that maintains binding (and cell lysis ability) to cells expressing CD22, but whose affinity is changed to reduce the intensity of the response (cytokine release).

[0469] Amino acid modifications are generally conservative modifications, including amino acid substitutions, additions and deletions in any of the antibody fragments and / or other parts of the CAR molecule of the CAR. Modifications can be introduced into any of the other parts of antibodies, antibody fragments or CAR molecules of the present invention by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutations or by using optimized germline sequences.

[0470] In general, an optimized CAR will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDR regions correspond to those of the original human immunoglobulin.

[0471] Conservative amino acid substitution refers to the substitution of an amino acid residue by an amino acid residue with a similar side chain. Amino acid residue families with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, aspartic acid, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CAR of the present invention can be replaced by other amino acid residues from the same side chain family, and the functional assay described herein can be used to test the changed CAR.

[0472] In a preferred embodiment, the present invention provides a CD22 CAR having a conservative sequence modification (or an amino acid sequence change) compared to the amino acid sequence of the polypeptide of SEQ ID N°15.

[0473] In a preferred embodiment, the present invention provides a CD22 CAR having 2 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID N°15.

[0474] In a preferred embodiment, the present invention provides a CD22 CAR having 3 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID N°15.

[0475] In a preferred embodiment, the present invention provides a CD22 CAR having 4 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID N°15.

[0476] In a preferred embodiment, the present invention provides a CD22 CAR having 5 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID N°15, and optionally comprising at least one sequence of SEQ ID N°20.

[0477] In a more preferred embodiment, the present invention provides a CD22 CAR having 5 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID N°15 and at least one CDR in SEQ ID N°15 is conserved.

[0478] In a more preferred embodiment, the present invention provides a CD22 CAR having 1 to 15 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID N°15 and at least one CDR in SEQ ID N°15 is conserved.

[0479] In a preferred embodiment, the CD22 CAR sequence of the present invention is modified by changing 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids compared to the wild-type CD22 CAR to reduce HAMA (human anti-mouse response) without affecting the binding ability of the CAR to its target (CD22).

[0480] In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence having at least one amino acid change compared to the wild type (the wild type is m971), wherein the at least one amino acid change does not affect or improves the binding and / or activity of the CD22 CAR in primary T cells.

[0481] Binding properties can be modified using adaptive techniques originally 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 print June 8, 2006, doi: 10.1073 / pnas.0603653103, which is incorporated herein by reference.

[0482] These optimized scFvs also carry at least one mutation corresponding to the mutations Pro-91–Thr-92 (PT) Gly-91–Ala-92 and Val-91–Phe-92.

[0483] In one embodiment, the present invention provides an anti-CD22 CAR comprising:

[0484] - Extracellular domain, comprising:

[0485] Signal peptide

[0486] An optionally optimized ligand binding domain comprising a VH domain and a VL domain 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 equivalent,

[0487] Hinge, including CD8α hinge

[0488] -CD8α transmembrane domain and

[0489] - A cytoplasmic domain containing the CD3ζ signaling domain and the co-stimulatory domain from 4-1BB.

[0490] In a preferred embodiment, the present invention provides an anti-CD22 CAR as described above, wherein the monoclonal anti-CD22 antibody is derived from an m971 antibody having a hotspot in the CDR. Preferably, one of the following Pro-91–Thr-92 (PT), Gly-91–Ala-92, or Val-91–Phe-92 mutations.

[0491] In a more preferred embodiment, the present invention provides an isolated engineered (TCR and dCK KO) immune T cell comprising an anti-CD22 CAR, comprising:

[0492] The extracellular domain contains:

[0493] A signal peptide comprising a ligand binding domain of a VH domain and a VL domain from a monoclonal anti-CD22 antibody, having at least one of the following: a Pro-91–Thr-92, a Gly-91–Ala-92 mutation; a Val-91–Phe-92 mutation,

[0494] Hinge, including CD8α hinge

[0495] -CD8α transmembrane domain, and

[0496] - A cytoplasmic domain containing the CD3ζ signaling domain and the co-stimulatory domain from 4-1BB.

[0497] In a more preferred embodiment, the present invention provides an isolated engineered (TCR and CD52 KO) immune T cell comprising an anti-CD22 CAR, comprising:

[0498] The extracellular domain contains:

[0499] A signal peptide comprising a ligand binding domain of a VH domain and a VL domain from a monoclonal anti-CD22 antibody, having at least one of the following: a Pro-91–Thr-92, a Gly-91–Ala-92 mutation; a Val-91–Phe-92 mutation,

[0500] Hinge, including CD8α hinge

[0501] -CD8α transmembrane domain and

[0502] - A cytoplasmic domain containing the CD3ζ signaling domain and the co-stimulatory domain from 4-1BB.

[0503] In a more preferred embodiment, the present invention provides an isolated engineered (TCR, CD52 and dCK KO) immune T cell comprising an anti-CD22 CAR, comprising:

[0504] The extracellular domain contains:

[0505] A signal peptide comprising a ligand binding domain of a VH domain and a VL domain from a monoclonal anti-CD22 antibody, having at least one of the following: a Pro-91–Thr-92, a Gly-91–Ala-92 mutation; a Val-91–Phe-92 mutation,

[0506] Hinge, including CD8α hinge

[0507] -CD8α transmembrane domain and

[0508] - A cytoplasmic domain containing the CD3ζ signaling domain and the co-stimulatory domain from 4-1BB.

[0509] On the one hand, CAR22 can be co-expressed on the cell surface with at least one, preferably two, more preferably three monoclonal antibody (mAb) specific epitopes, and the mAb specific epitopes can be fused with the transmembrane domain of CD8. In one embodiment, the mAb specific epitope is an epitope recognized by rituximab, and / or an epitope from QBEN 10, and the peptide co-expressed with CD22 CAR is RQR8.

[0510] In another embodiment, at least one, preferably two, and more preferably three monoclonal antibody (mAb) specific epitopes can be inserted into the linker L (binding VH to VL) of the scFv specific for CD22 and / or the hinge of the CD22 CAR.

[0511] The mAb-specific epitope can be one of the mAb-specific epitopes specifically recognized by the following monoclonal antibodies: ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab Monoclonal antibody, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and ustekinumab, preferably rituximab (R) and / or QBEND-10.

[0512] Epitope-specific mAbs can be used for in vitro cell sorting and / or in vivo cell depletion of immune cells expressing CD22.

[0513] In a specific embodiment, the extracellular binding domain of the CD22 CAR of the present invention may comprise one of the following sequences:

[0514] V 1 -L 1 -V 2 -(L) x -Epitope 1-(L) x -;

[0515] V 1 -L 1 -V 2 -(L) x -Epitope 1-(L) x -Epitope 2-(L) x -;

[0516] V 1 -L 1 -V 2 -(L) x -Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -;

[0517] (L) x -Epitope 1-(L) x -V 1 -L 1 -V 2 ;

[0518] (L) x -Epitope 1-(L) x -Epitope 2-(L)x -V 1 -L 1 -V 2 ;

[0519] Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -V 1 -L 1 -V 2 ;

[0520] (L) x -Epitope 1-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope 2-(L) x ;

[0521] (L) x -Epitope 1-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope 2-(L) x -Epitope 3-(L) x -;

[0522] (L) x -Epitope 1-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -;

[0523] (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V 1 -L 1 -V 2 -(L) x -Epitope 3-(L) x -;

[0524] (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V 1 -L 1 -V 2 -(L)x -Epitope 3-(L) x -Epitope 4-(L) x -;

[0525] V 1 -(L) x -Epitope 1-(L) x -V 2 ;

[0526] V 1 -(L) x -Epitope 1-(L) x -V 2 -(L) x -Epitope 2-(L) x ;

[0527] V 1 -(L) x -Epitope 1-(L) x -V 2 -(L) x -Epitope 2-(L) x -Epitope 3-(L) x ;

[0528] V 1 -(L) x -Epitope 1-(L) x -V 2 -(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x ;

[0529] (L) x -Epitope 1-(L) x -V 1 -(L) x -Epitope 2-(L) x -V 2 ;or,

[0530] (L) x -Epitope 1-(L) x -V 1 -(L) x -Epitope 2-(L) x -V 2 -(L) x -Epitope 3-(L) x ;

[0531] in,

[0532] V1 is VL, preferably having SEQ ID N°12, V2 is VH, preferably having SEQ ID N°13, or V1 is VH and V2 is VL;

[0533] L1 is a linker suitable for connecting the VH chain to the VL chain, preferably SEQ ID N° 10

[0534] L is a linker comprising glycine and serine residues, each occurrence of L in the extracellular binding domain may be the same as or different from other occurrences of L in the same extracellular binding domain, and

[0535] X is 0 or 1, and each occurrence of x is chosen independently of each other; and,

[0536] Epitope 1, Epitope 2, and Epitope 3 are mAb specific epitopes and may be the same or different.

[0537] In one embodiment, the extracellular binding domain comprises one of the following sequences:

[0538] V 1 -L 1 -V 2 -L-epitope 1; V 1 -L 1 -V 2 -L-epitope 1-L; V 1 -L 1 -V 2 -L-epitope 1 -L-epitope 2; V 1 -L 1 -V 2 -L-epitope 1-L-epitope 2-L; V 1 -L 1 -V 2 -L-epitope 1-L-epitope 2-L-epitope 3; V 1 -L 1 -V 2 -L-epitope 1-L-epitope 2-L-epitope 3-L; V 1 -L 1 -V 2 -Epitope 1; V 1 -L 1 -V 2 -Epitope 1-L; V 1 -L 1 -V 2 -epitope 1-L-epitope 2; V 1 -L 1 -V 2 -Epitope 1-L-Epitope 2-L; V 1 -L 1 -V 2-epitope 1-L-epitope 2-L-epitope 3; V 1 -L 1 -V 2 -Epitope 1-L-Epitope 2-L-Epitope 3-L; Epitope 1-V 1 -L 1 -V 2 ; Epitope 1-LV 1 -L 1 -V 2 ; L-epitope 1-V 1 -L 1 -V 2 ; L-epitope 1-LV 1 -L 1 -V 2 ; Epitope 1-L-Epitope 2-V 1 -L 1 -V 2 ; Epitope 1-L-Epitope 2-LV 1 -L 1 -V 2 ; L-epitope 1-L-epitope 2-V 1 -L 1 -V 2 ; L-epitope 1-L-epitope 2-LV 1 -L 1 -V 2 ; Epitope 1-L-Epitope 2-L-Epitope 3-V 1 -L 1 -V 2 ; Epitope 1-L-Epitope 2-L-Epitope 3-LV 1 -L 1 -V 2 ; L-epitope 1-L-epitope 2-L-epitope 3-V 1 -L 1 -V 2 ; L-epitope 1-L-epitope 2-L-epitope 3-LV 1 -L 1 -V 2 ; V 1 -L-Epitope 1-LV 2 ; L-epitope 1-LV 1 -L-Epitope 2-LV 2 ; V 1 -L-Epitope 1-LV 2 -L-epitope 2-L; V 1 -L-epitope 1-LV 2 -L-epitope 2 -L-epitope 3; V 1 -L-epitope 1-LV 2 -L-epitope 2-epitope 3; V1 -L-epitope 1-LV 2 -L-epitope 2-L-epitope 3-epitope 4; L-epitope 1-LV 1 -L-Epitope 2-LV 2 -L-epitope 3-L; epitope 1-LV 1 -L-Epitope 2-LV 2 -L-epitope 3-L; L-epitope 1-LV 1 -L-Epitope 2-LV 2 -L-epitope 3; L-epitope 1-LV 1 -L 1 -V 2 -L-epitope 2-L; L-epitope 1-LV 1 -L 1 -V 2 -L-epitope 2-L-epitope 3; L-epitope 1-LV 1 -L 1 -V 2 -L-epitope 2-epitope 3 or epitope 1-LV 1 -L 1 -V 2 -L-epitope 2-L-epitope 3-epitope 4; wherein

[0539] V 1 Yes V L And V 2 Yes V H ; or V 1 Yes V H And V 2 Yes V L ; and VH and VL are optimized VH and VL from m971.

[0540] L 1 is suitable for V H Chain connected to V L any joints in the chain;

[0541] L is a linker comprising glycine and serine residues, each occurrence of L in the extracellular binding domain may be the same as or different from other occurrences of L in the same extracellular binding domain, and

[0542] Epitope 1, Epitope 2, and Epitope 3 are mAb specific epitopes and may be the same or different.

[0543] L 1 is a linker comprising glycine and / or serine, preferably, L 1 It contains the amino acid sequence (Gly-Gly-Gly-Ser) nor (Gly-Gly-Gly-Gly-Ser) n wherein n is 1, 2, 3, 4 or 5, or the linker comprises an amino acid sequence (Gly 4 Ser) 4 or (Gly 4 Ser) 3 .

[0544] Similarly, L is a linker comprising glycine and / or serine, preferably, L is a linker having an amino acid sequence selected from the group consisting of SGG, GGS, SGGS, SSGGS, GGGG, SGGGG, GGGGS, SGGGGGS, SGGGGG, GSGGGGGS, GGGGGGGS, SGGGGGG, SGGGGGGGS and SGGGGSGGGGS, preferably L is SGGGG, GGGGS or SGGGGS.

[0545] In a preferred embodiment, epitope 1, epitope 2, epitope 3 and epitope 4 are independently selected from mAb specific epitopes specifically recognized by: ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab, 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, preferably rituximab (R) and / or QBEND-10, preferably epitope 1, epitope 2, epitope 3 and epitope 4 are recognized by rituximab or QBEND-10.

[0546] Epitope 1, Epitope 2, Epitope 3, and Epitope 4 are mAb-specific epitopes that are specifically recognized by:

[0547]

[0548]

[0549] Thus the mAb specific epitope may comprise a polypeptide selected from the group consisting of CPYSNPSLC, NSELLSLINDMPITNDQKKLMSNN, CQFDLSTRRLKC, CQYNLSSRALKC, CVWQRWQKSYVC, CVWQRWQKSYVC, SFVLNWYRMSPSNQTDKLAAFPEDR, SGTYLCGAISLAPKAQIKE, ELPTQGTFSNVSTNVSPAKPTTTA, ELPTQGTFSNVSTNVSPAKPTTTA, GQNDTSQTSSPS.

[0550]

[0551]

[0552] In a preferred embodiment, the mAb-specific epitope is a mAb-specific epitope having the amino acid sequence of ELPTQGTFSNVSTNVSPAKPTTTA and / or CPYSNPSLC (SEQ ID N° 19).

[0553] In a more preferred embodiment, the CD22 CAR of the present invention comprises three mAb-specific epitopes having the amino acid sequence of CPYSNPSLC(R) and one epitope having the amino acid sequence of ELPTQGTFSNVSTNVSPAKPTTTA(Q).

[0554] In an even more preferred embodiment, the CD22 CAR of the present invention comprises two mAb-specific epitopes located immediately after the VL, in the hinge NT portion, having the amino acid sequence of CPYSNPSLC(R).

[0555] In one aspect, at least one sequence that binds to rituximab (R) and / or a sequence that binds to QBEND10 (Q) may be inserted into the linker GGGGSGGGGSGGGGS and / or the hinge previously described in WO2016120216.

[0556] In a specific embodiment, the CD22 CAR of the present invention is a single-chain CAR (scCAR).

[0557] In a specific embodiment, the single-chain CD22 CAR of the present invention comprises an scFv from m971 and at least one other binding domain, preferably specific for the distal portion of CD22, alternatively specific for another B cell antigen, particularly in the case of B cell malignancies expressed, such as CD34, CD10, CD79a, CD20, IgD, CD5, CD23, CD19, STAT5, CD3, CD30, BCMA. In a specific embodiment, the CD22 CAR of the present invention is a multi-chain CAR (mcCAR). The multi-chain CD22 CAR is part of the present invention and can be produced as described in detail in WO2014039523, which is incorporated herein by reference. In a specific embodiment, the V of the immunoglobulin H结构域 and V L结构域 Alternatively, a portion of an immunoglobulin specific for CD22 can be carried by two different and separate (non-covalently bound) chains in the multi-chain CAR.

[0558] In the multi-chain form, the CD22 CAR of the present invention comprises at least two, preferably three transmembrane domains (not covalently bound to each other), wherein at least one transmembrane domain comprises a scFv specific for CD22 of the present invention.

[0559] In a specific embodiment, the V of an immunoglobulin specific for CD22 from m971 is preferred. H结构域 and V L结构域 Can be carried by one chain of a multi-chain CAR.

[0560] Figure 2 Examples of scCD22 CAR and mcCD22 CAR of the present invention are provided.

[0561] In a specific embodiment, the V of an immunoglobulin specific for CD22 H结构域 and V L结构域 The V sequence of another immunoglobulin specific for CD22 can be carried by one chain of the multi-chain CAR (preferably from m971). H结构域 and V L结构域 Can be carried by another chain of the multi-chain CAR.

[0562] Downregulation or mutation of target antigens is often observed in cancer cells, resulting in antigen loss escape variants. Therefore, in order to counteract tumor escape and make immune cells more specific to the target, the CD22-specific CAR according to the present invention can include another extracellular ligand binding domain to simultaneously bind to other elements in the target, thereby enhancing the activation and function of immune cells. In one embodiment, the extracellular ligand binding domains can be arranged in series on the same transmembrane polypeptide and can be optionally separated by a linker.

[0563] In another embodiment, the different extracellular ligand binding domains can be placed on different transmembrane polypeptides constituting the multi-chain CAR. In another embodiment, the present invention relates to a CAR group comprising different extracellular ligand binding domains, each of which is specific to CD22.

[0564] In one embodiment, the extracellular binding domain specific for CD22 and the second extracellular binding domain are on the same scCAR.

[0565] 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 associated transmembrane domains of the mc CAR.

[0566] The other second extracellular binding domain can be any extracellular binding domain that specifically binds to an antigen associated with (co-expressed, even transiently expressed) CD22 on pathological cells, 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.

[0567] The present invention also provides: related CD22 CAR nucleic acids, CD22 CAR recombinant expression vectors, engineered TCR KO cells (preferably comprising at least one other T cell with an edited gene of CD22 CAR), a population of said TCR KO cells with CD22 CAR, and pharmaceutical compositions related to CD22 CAR, proteins, expression vectors, and engineered TCR KO CD52 KO cells expressing said CD22 CAR of the present invention.

[0568] The present invention provides the following objects: CD22 CAR-related nucleic acids of the present invention, recombinant expression vectors, engineered TCR KO cells comprising at least one other edited gene (selected from genes conferring tolerance to hypoxia, genes conferring tolerance to alemtuzumab, genes conferring tolerance to protease inhibitors such as bortezomib, genes conferring tolerance to PNA (dCK)) and having CD22 CAR, and related nucleic acids, a population of engineered TCR KO cells comprising at least one other edited gene and having the CD22 CAR, and a pharmaceutical composition comprising the same object as a drug.

[0569] Polynucleotides, vectors:

[0570] The present invention relates to a polynucleotide and a vector encoding the above-mentioned CD22 CAR according to the present invention.

[0571] The polynucleotide may be contained 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 into 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.

[0572] In a specific embodiment, the different nucleic acid sequences may be contained in a polynucleotide or vector that contains a nucleic acid sequence encoding a ribosomal skipping sequence, such as a sequence encoding a 2A peptide. The 2A peptide identified in the parathyroid virus subgroup of picornaviruses causes the ribosome to "jump" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codons (see: Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008).

[0573] Therefore, the present invention provides Figure 4 Vector shown, encoding RQR8 and anti-CD22 CAR linked by peptide 2A.

[0574] A "codon" refers to three nucleotides on an mRNA (or the sense strand of a DNA molecule) that are translated by the ribosome into an amino acid residue. Thus, two polypeptides can be synthesized from a single continuous open reading frame within an mRNA when the polypeptides are separated by a 2A oligopeptide sequence in frame. This ribosome skipping mechanism is well known in the art and is known to be used by several vectors to express several proteins encoded by a single messenger RNA.

[0575] A vector that allows the CD22 CAR of the present invention to be expressed in a cell is another object of the present invention. In a preferred embodiment, the vector allows for transient expression of the CD22 CAR of the present invention. In a more preferred embodiment, the vector allows for constitutive and stable expression of the CD22 CAR of the present invention by inserting a sequence encoding the CD22 CAR into the cell genome.

[0576] The expression of the CD22 CAR of the present invention and / or the survival of cells expressing the CD22 CAR of the present invention can be controlled using genes under the control of inducible promoters 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.

[0577] In one embodiment, a CD22 CAR is provided, wherein the extracellular domain comprises at least two CD20 mimetic epitopes of SEQ ID N°19 (CPYSNPSLC) located between the m971 scfv domain and the hinge from human CD8α. The document patent WO2016120216A1 discloses a method for preparing such a structure, which is incorporated herein by reference.

[0578] In one embodiment, the present invention provides a vector comprising a sequence encoding CD22CAR selected from SEQ ID N°22.

[0579] In order to introduce a transmembrane polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also referred to as a leader sequence, prepro sequence or presequence) is provided in a polynucleotide sequence or a vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, that is, the two sequences are linked in the correct reading frame and positioned to guide the newly synthesized polypeptide into the secretory pathway of the host cell. The secretory signal sequence is usually located at the 5' end of the nucleic acid sequence encoding the polypeptide of interest, although some secretory signal sequences can be located at other positions in the nucleic acid sequence of interest (see, e.g., 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 sequences SEQ ID N°1 and SEQ ID N°2.

[0580] In a more preferred embodiment, the signal peptide of the CAR of the present invention comprises the amino acid sequence of SEQ ID N°1 from human CD8α.

[0581] Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, there may be considerable sequence variation between these polynucleotide molecules. Preferably, the nucleic acid sequences of the present invention are codon optimized for expression in mammalian cells, preferably in human cells. Codon optimization refers to the exchange of codons that are usually rare in the highly expressed genes of a particular species with codons that are usually common in the highly expressed genes of the species in the target sequence, such as codons encoding amino acids as the codons exchanged.

[0582] Methods for engineering immune cells with CD22 CAR:

[0583] The present invention includes a method for preparing immune cells for immunotherapy, comprising introducing a polynucleotide or vector encoding one of the CD22 CARs of the present invention into the immune cells ex vivo, preferably having SEQ ID N° 1 as described above.

[0584] In a preferred embodiment, the polynucleotide is contained in a vector in view of stable expression in immune cells.

[0585] According to additional embodiments, the method further comprises the step of genetically modifying the cells to make them more suitable for adoptive transfer and / or use with drugs that affect the survival of the immune cells, in particular for transplantation (also known as allogeneic transplantation or allogeneic transplantation) alone or in combination with drugs to which the immune cells become tolerant.

[0586] In the latter case, the engineered cells can first be isolated from the donor and reinfused into the same donor along with the drug to which the cells have become resistant.

[0587] For editing a gene, it means modifying a gene or inactivating a gene, such as mutating a gene, deleting a gene, inserting a sequence into a gene, modifying the methylation of the gene (including the promoter of the gene), etc. The method described in PA201670503 is incorporated herein by reference and is shown in the following examples.

[0588] MacLeod et al., Integration of a CD19 CAR into the TCR Alpha ChainLocus Streamlines Production of AllogeneicGene-Edited CAR T Cells, MolecularTherapy(2017), http: / / dx.doi.org / 10.1016 / j.ymthe.2017.02.005The method described in is incorporated herein by reference and is also a possible alternative to the method used in the present invention for preparing TCRKO CD22 CAR or cells with CD22 CAR resistant to hypoxia by overexpressing HIF-1a.

[0589] The method of the present invention is based on the cellular homology-directed repair (HDR) mechanism to "knock in" CD22 CAR in the TRAC gene (encoding the TCRα subunit of TCR), thereby producing a more effective product.

[0590] HDR with exogenous DNA sequences has been previously described in T cells using short oligonucleotides paired with CRISPR / Cas9. Other studies have shown that adeno-associated virus (AAV) vectors can be used as templates in combination with site-specific nucleases Crispr / Cas9 or MegaTAL to achieve gene insertion via HDR. In the present invention, a new method for inserting CAR into TCR genes by combining adeno-associated virus (AAV) vectors and TALEN is provided.

[0591] In UCART22, the AAV6 vector can be used after gene editing using TALEN specific for a gene such as the TRAC gene (gene encoding the α subunit or TCR) or any gene disclosed in PCT / EP2017 / 076798.

[0592] Because TALEN is specific to DNA sequences and allows the sequence to be integrated into a gene (preferably a TRAC gene), the present invention also provides engineered immune cells, comprising a sequence encoding a CAR (preferably a CD22 CAR located in the precise region of the TRAC gene as determined by the TALEN used as described above). Due to TALEN specificity, the sequence in the TRAC gene of the engineered immune cell is unique.

[0593] Therefore, the present invention provides an engineered immune cell comprising the following sequence;

[0594] (YYY) n -ZZZ-(XXX) m

[0595] Where n is = 1 to at least 10

[0596] m is from 1 to 100, preferably m is >100, representing the number of base pairs of the sequence to be integrated,

[0597] wherein ZZZ encodes a self-cleaving peptide, such as a 2A peptide, in frame with the TRAC coding sequence,

[0598] Y is A or T or G or C and flanks or contains the sequence of the TRAC gene targeted by the TALEN, the TALEN comprising at least Best (XXX) n It is A or T or G or C and a part of the exogenous sequence to be inserted into the TRAC gene, preferably a sequence encoding CAR, more preferably a sequence encoding CD22CAR.

[0599] In one embodiment, the TRAC gene is deleted and the inserted gene is expressed under the control of the TRAC promoter.

[0600] Additional or alternative sequences, such as an IRES internal ribosome entry site; may be interposed between the TALEN target and XXX.

[0601] In the present invention, the TALEN target is SEQ ID N°21 and the CAR CD22 is SEQ ID N°22.

[0602] In one embodiment, the sequence cleaved by the TALEN is AGAACCCTGACCCTG. Depending on the inserted sequence, the sequence AGAACCCTGACCCTG may be at least partially conserved in the engineered cells of the invention (see Fig. 9 ).

[0603] The present invention provides an engineered immune cell comprising a TRAC gene having SEQ ID N°22.

[0604] The present invention provides an engineered immune cell comprising a sequence encoding a CAR specific for CD19 or a sequence encoding a CAR specific for CD22 inserted into the TRAC gene (preferably anywhere in the gene locus AGAACCCTGACCCTG).

[0605] Therefore, in a specific embodiment, the engineered immune cells of the present invention comprise two different sequences encoding CAR and express the two different CARs. On the cell surface, no level of TCR is detected.

[0606] Therefore, in a specific embodiment, the engineered immune cells of the present invention comprise two different sequences encoding CAR and express the two different CARs. On the cell surface, TCR and MHC class I levels are undetectable.

[0607] The engineered immune cells of the present invention comprise a sequence encoding CD22 (preferably SEQ ID N°22) in the TRAC gene of their genome, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD19 is inserted.

[0608] The engineered immune cell of the present invention comprises a sequence encoding CD22 (preferably SEQ ID N°22) in its genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD34 is inserted.

[0609] The engineered immune cells of the present invention comprise a sequence encoding CD22 (preferably SEQ ID N°22) in the TRAC gene of their genome, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD79a is inserted.

[0610] The engineered immune cells of the present invention comprise a sequence encoding CD22 (preferably SEQ ID N°22) in the TRAC gene of their genome, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD79b is inserted.

[0611] The engineered immune cell of the present invention comprises a sequence encoding CD22 (preferably SEQ ID N°22) in its genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD10 is inserted.

[0612] The engineered immune cell of the present invention comprises a sequence encoding CD22 (preferably SEQ ID N°22) in its genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for IgD is inserted.

[0613] The engineered immune cell of the present invention comprises a sequence encoding CD22 (preferably SEQ ID N°22) in its genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD5 is inserted.

[0614] The engineered immune cells of the present invention comprise a sequence encoding CD22 (preferably SEQ ID N°22) in the TRAC gene of their genome, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD23 is inserted.

[0615] The engineered immune cell of the present invention comprises a sequence encoding CD22 (preferably SEQ ID N°22) in its genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD30 is inserted.

[0616] The engineered immune cells of the present invention comprise a sequence encoding CD22 (preferably SEQ ID N°22) in the TRAC gene of their genome, and another genomic sequence into which another exogenous gene encoding a CAR specific for BCMA is inserted.

[0617] The engineered immune cell of the present invention comprises a sequence encoding CD22 (preferably SEQ ID N°22) in its genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for FLT3 is inserted.

[0618] The engineered immune cells of the present invention comprise a sequence encoding CD22 (preferably SEQ ID N°22) in the TRAC gene of their genome, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD138 is inserted.

[0619] The engineered immune cell of the present invention comprises a sequence encoding CD22 (preferably SEQ ID N°22) in its genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD80 is inserted.

[0620] The engineered immune cell of the present invention comprises a sequence encoding CD22 (preferably SEQ ID N°22) in its genomic TRAC gene, and another genomic sequence into which another exogenous gene encoding a CAR specific for CD86 is inserted.

[0621] The other genomic sequence into which another exogenous gene encoding a second CAR is inserted may be any gene disclosed in PCT / EP2017 / 076798 or WO2017069958A2.

[0622] Other exogenous genes encoding CAR can be inserted into the TRAC gene in frame with the self-cleaving peptide and CD22 CAR sequence (preferably SEQ ID N°), or inserted into another gene encoding the protein described in PCT / EP2017 / 076798, or selected from PD1 (Uniprot Q15116), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG3 (Uniprot P18627), HAVCR2 (0Q7Z6A9), CD160 (Uniprot O95971), TIGIT (Uniprot Q495A1), CD96 (Uniprot P40200), CRTAM (Uniprot O95727), LAIR1 (UniprotQ6GTX8), SIGLEC7 (UniprotQ9Y286), SIGLEC9 (Uniprot Q9Y336), Uniprot O14763), TNFRSF10A (Uniprot O00220), CASP8 (Uniprot Q14790), CASP10 (Uniprot Q92851), CASP3 (Uniprot P42574), CASP6 (UniprotP55212), CASP7 (Uniprot P55210), FADD (Uniprot Q13158), FAS (UniprotP25445), TGFBRII (Uniprot P37173), TGFBRRI (Uniprot Q15582), SMAD2(UniprotQ15796), SMAD3(Uniprot P84022), SMAD4(Uniprot Q13485), (Uniprot B7ZSB5), SKI (UniprotP12755), SKIL (Uniprot P12757), TGIF1 (Uniprot Q15583), IL10RA (Uniprot Q13651), IL10RB (Uniprot Q08334), HMOX2 (UniprotP30519), IL6R (Uniprot P08887), IL6ST (Uniprot P40189), EIF2AK4 (Uniprot Q9P2K8), CSK (UniprotP41240), PAG1 (UniprotQ9Y3P8), SIT1 (UniprotQ9Y3P8), FOXP3 (Uniprot Q9BZS1), PRDM1 (UniprotQ60636), BATF (UniprotQ165 Q02108), GUCY1B2 (Uniprot Q8BXH3) and GUCY1B3 (Uniprot Q02153).

[0623] The preferred editing (KO) genes in UCART22 are NFRSF10B (Uniprot O14763), TNFRSF10A (Uniprot O00220), IL10RA (Uniprot Q13651), IL10RB (Uniprot Q08334), TGFBRII (Uniprot P37173), TGFRBRI (Uniprot Q15582), PD1 (Uniprot Q15116), CTLA4 (UniprotP16410), LAG3 (Uniprot P18627), HAVCR2 (Uniprot Q8TDQ0), and TIGIT (Uniprot Q495A1).

[0624] 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 described in Table A:

[0625] Table A

[0626]

[0627]

[0628] or in a safe harbor locus as disclosed in PCT / EP2017 / 076798 or Table AA

[0629] Table AA:

[0630] Zfp640 Zinc finger protein 640 LOC100038422 Uncharacterized LOC100038422 Zfp600 Zinc finger protein 600 Serpinb3a Serine (or cysteine) peptidase inhibitor, clade B (ovalbumin), member 3A Tas2r106 Taste receptor, type 2, member 106 Magea3 Melanoma antigen, family A, 3 Omt2a Oocyte maturation, α Cpxcr1 CPX chromosome region, candidate gene 1 Hsf3 Heat shock transcription factor 3 PbSn Probasin Sb Spermine binding protein wxya WAP tetra-disulfide core domain 6B Meiob Meiosis-specific with OB domain Dnm3os Dynamin 3, opposite chain Skint11 Selection and maintenance of intraepithelial T cells11

[0631] Another gene in which the second CAR may be inserted may be a gene encoding any one of the proteins in Table B selected from the following:

[0632] Table B

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640] UCART22 can be prepared using an AAV vector, preferably an AAV6 vector, and even more preferably an AAV6 / 2 vector for inserting CD22 CAR or another gene disclosed herein. AAV vectors can be used after genetic editing genes such as TRAC genes (genes encoding alpha subunits or TCRs) or any gene disclosed in PCT / EP2017 / 076798 using specific endonucleases.

[0641] Adoptive cell transfer is the transfer of cells into a patient. These cells may come from the patient themselves and then be altered before being transferred back (autologous transfer), or they may come from another individual. The cells are most commonly derived from the immune system, with the goal of transferring improved immune function and properties back to the patient along with the cells. Transferring autologous cells or cells from the patient can minimize graft-versus-host disease (GVHD) or tissue or organ rejection. Similarly, transferring TCR-deficient T cell CD22 CART cells can minimize GVHD. Transferring TCR-deficient MHC I-deficient CD22 CART minimizes GVHD and HVGD.

[0642] In one embodiment, the step of genetically modifying (engineering) the immune cell occurs before the step of introducing a polynucleotide or vector encoding one of the CD22 CARs of the present invention into the cell. According to a first aspect, immune cells can be made to reduce allogeneicity, for example, by inactivating at least one gene expressing one or more components of a T cell receptor (TCR) as described in WO 2013 / 176915, which can be combined with inactivation of genes (such as β2m genes) encoding or regulating HLA expression 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.

[0643] According to another aspect, the immune cells of the present invention can be further genetically engineered to improve the tolerance of the engineered immune cells to immunosuppressive drugs or chemotherapy, as disclosed in WO201575195, which is used as standard of care for treating CD22-positive malignant cells, and WO201575195 is incorporated herein by reference.

[0644] Tolerance to Campath (alemtuzumab)

[0645] In a preferred embodiment, the genes that can be inactivated to confer drug resistance to T cells are glucocorticoid receptor (GR) and CD52. Inactivating the genes makes the cells tolerant to these treatments and gives them a competitive advantage over the patient's own T cells that do not have a specific CD22 CAR.

[0646] The CD52 and TRAC genes in the engineered immune cells of the present invention are inactivated using TALE nuclease or CRISPR CAS9 system.

[0647] In a more preferred embodiment, the gene that can be inactivated to confer drug resistance to T cells is CD52 in TCR KO immune T cells with CD22 CAR.

[0648] In a preferred embodiment, the gene that can be inactivated to confer drug resistance to T cells is the glucocorticoid receptor (GR).

[0649] The expression of the CD3 gene can also be inhibited or reduced to confer tolerance to Teplizumab, another immunosuppressive drug. According to the present invention, the expression of HPRT can also be inhibited or reduced to confer tolerance to 6-thioguanine, a cytostatic commonly used in chemotherapy, particularly for the treatment of acute lymphoblastic leukemia.

[0650] Tolerance to purine nucleotide analogs by deletion of the human deoxycytidine kinase (dCK) gene.

[0651] In a preferred embodiment, the gene that can be inactivated to confer drug tolerance to T cells is the human deoxycytidine kinase (dCK) gene. This enzyme is necessary for the phosphorylation of deoxyribonucleoside deoxycytidine (dC), deoxyguanosine (dG) and deoxyadenosine (dA). Purine nucleotide analogs (PNA) are metabolized to monophosphate, diphosphate and triphosphate PNA by dCK. Their triphosphate forms, particularly clofarabine triphosphate, compete with ATP for DNA synthesis, serve as a pro-apoptotic agent, and are effective inhibitors of ribonucleotide reductase (RNR) involved in the generation of trinucleotides.

[0652] Inactivation of the dCK gene in the engineered immune cells of the present invention is mediated by TALE nucleases or CRISPR / CAS9 systems. To achieve this goal, several pairs of dCK TALE nucleases were designed, assembled at the polynucleotide level and verified by sequencing. Examples of TALE nuclease pairs that can be used according to the present invention are described in PCT / EP2014 / 075317.

[0653] This dCK inactivation in the engineered immune cells of the present invention confers resistance to purine nucleoside analogs (PNAs) such as clofarabine and fludarabine.

[0654] In another preferred embodiment, the inactivation of dCK in the engineered immune cells of the present invention is combined with the inactivation of the TRAC gene, so that these double knockout (KO) (TCR or TRAC KO and dCK KO) cells are both resistant to drugs such as clofarabine and have less allogeneicity.

[0655] In another preferred embodiment, CD52 inactivation is combined with TRAC gene inactivation in the engineered immune cells of the present invention, so that these double knockout (KO) (TCR or TRAC KO and CD52 KO) cells are both resistant to drugs such as CAMPATH (alemtuzumab) and have less allogeneicity.

[0656] This dual feature is particularly useful for therapeutic purposes, allowing "ready-made" allogeneic cells (UCART22) to be combined with chemotherapy for immunotherapy to treat cancer patients who need treatment. The dual KO inactivation dCK / TRAC or CD52 / TRAC can be performed simultaneously or sequentially. PCT / EP2014 / 075317 describes an example of a TALE nuclease dCK / TRAC pair that is successful in the present invention, particularly the target sequence (dCK and TRAC) in 2 loci. Document PCT / EP2014 / 075317 is incorporated herein in its entirety.

[0657] The present invention provides primary T cells expressing CD22 CAR of SEQ ID NO.15, wherein CD52 and TRAC genes are inactivated by deletion, so as to use them for the treatment of CLL, ALL, preferably their aggressive, relapsed refractory forms, optionally in lymphodepleted patients, more preferably relapsed refractory forms of B-ALL.

[0658] According to another aspect of the invention, immune cells can be further manipulated to be more active or limited in consumption by inactivating genes encoding 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 inhibited are listed in Table 9.

[0659] The present invention also provides primary T cells expressing the CD22 CAR of SEQ ID N°15, wherein the CD52, TRAC and dCK genes are deleted.

[0660] In another embodiment, the present invention also provides primary T cells expressing a CD22 CAR that confers tolerance to hypoxia.

[0661] The present invention also provides primary T cells expressing CD22 CAR of SEQ ID N°15, in which CD52, TRAC and dCK genes are deleted, and the sequence of HIF-1a is inserted to make the cells tolerant to hypoxia.

[0662] Engineered cells tolerant to hypoxia

[0663] In certain embodiments, the expression and cytolytic activity of the CD22 CAR T cells of the invention are maintained or maintained under hypoxic conditions (hypoxia) (reducing the oxygen content to 20% under normal oxygen conditions). 2 With 1-5% O 2 The expression and activity of CD22CAR T cells induced by CAR T cells are maintained, and the cells still target and destroy cancer cells when embedded in tissues.

[0664] Examples of hypoxia-inducible CARs in T cells are described in WO2013123061 or Juillerat, A. et al. An oxygen sensitive self-decisionmaking engineered CAR T-cell Sci. Rep. 7, 39833; doi: 10.1038 / srep39833 (2017), both of which are incorporated herein by reference: A synthetic promoter specific for OxiTF that drives CD22 CAR expression was constructed. OxiTF is a synthetic genetic element designed to activate CD22 CAR encoding. Once encountered a tumor (producing hypoxia), the engineered T cells can "detect" a drop in oxygen levels (corresponding to the level of O in the blood). 2Compared to the average level) and trigger the expression of CD22 CAR. The cell surface exposure of CD22 CAR enables the recognition of tumor antigens under hypoxic conditions, which ultimately triggers the activation and proliferation of T cells through the activation and costimulatory domains present in the CD22 CAR. Ultimately, cells expressing tumor antigens are lysed by the UCART22 of the present invention.

[0665] In the present invention, immune cells can also be engineered to remain effective under hypoxic conditions (low oxygen concentration is 1-5%) by overexpressing at least one of the following factors, preferably all of them: Oct3, Oct4, Sox2, Klf4 and c-Myc, or editing the HIF-1a factor.

[0666] In the present invention, engineered cells expressing oxygen-sensitive CD22 single-chain CAR and engineered cells expressing CD22 CAR tolerant to hypoxia were successfully constructed and tested.

[0667] Since CD22 is primarily expressed on CD22-expressing B-cell malignancies, which are "liquid" tumors and therefore do not produce hypoxia, as opposed to solid tumors, CD22 CAR-engineered immune cells that are tolerant to hypoxia are not expected to be more effective than CD22 CAR-engineered immune cells that are intolerant to hypoxia in patients' B-ALL. In fact, the UCART22 of the present invention that reaches nested cancer cells that are clustered or homed in tissues is able to lyse these cells.

[0668] Other genes in the UCART22 of the present invention may be edited, such as those listed in Table 5:

[0669] Table 5: List of genes encoding immune checkpoint proteins that can be inactivated in the CD22 CAR engineered T cells of the present invention according to the present invention

[0670]

[0671]

[0672] The present invention provides isolated engineered immune T cells expressing the CD22 CAR of SEQ ID N°15, wherein the dCK and / or CD52 and TRAC genes are edited, i.e. inactivated by deletion, to use them in the treatment of CLL, ALL, preferably their aggressive, relapsed, refractory forms in lymphodepleted patients.

[0673] The present invention provides isolated engineered immune T cells expressing CD22 CAR of SEQ ID N°15, wherein the b2m and TRAC genes are edited, i.e. inactivated by deletion, to use them in the treatment of CLL, ALL, preferably their aggressive, relapsed, refractory forms in lymphodepleted patients.

[0674] The present invention provides engineered immune T cells expressing the CD22 CAR of SEQ ID N°15, wherein the TRAC and IL-10R genes are edited, i.e., inactivated by deletion, to treat CLL, ALL, preferably their aggressive, relapsed refractory forms in patients with lymphoid depletion. The present invention provides engineered immune T cells (UCART22) expressing CD22 CAR, comprising defective TCRα genes (thereby impairing the expression of TCR on the cell surface), B2M defective genes (thereby impairing the expression of MHC class I molecules on the cell surface) and TGFβ receptor KO genes.

[0675] The present invention provides an engineered immune T cell (UCART22) expressing CD22 CAR, comprising a defective TCRα gene (thereby impairing the expression of TCR on the cell surface), a B2M defective gene (thereby impairing the expression of MHC class I molecules on the cell surface) and an IL-10 receptor KO gene.

[0676] The present invention provides an engineered immune T cell (UCART22) expressing CD22 CAR, comprising a defective TCRα gene (thereby impairing the expression of TCR on the cell surface), a B2M defective gene (thereby impairing the expression of MHC class I molecules on the cell surface) and an AHR KO gene.

[0677] The present invention provides an engineered immune T cell (UCART22) expressing CD22 CAR, comprising a defective TCRα gene (thereby impairing the expression of TCR on the cell surface), a B2M defective gene (thereby impairing the expression of MHC class I molecules on the cell surface) and a PD1 KO gene.

[0678] The present invention provides an engineered immune T cell (UCART22) expressing CD22 CAR, comprising a defective TCRα gene (thereby impairing the expression of TCR on the cell surface), a B2M defective gene (thereby impairing the expression of MHC class I molecules on the cell surface) and a LAG-3 KO gene.

[0679] The present invention provides an engineered immune T cell (UCART22) expressing CD22 CAR, comprising a defective TCRα gene (thereby impairing the expression of TCR on the cell surface), a B2M defective gene (thereby impairing the expression of MHC class I molecules on the cell surface) and a TIM-3 KO gene.

[0680] The present invention provides isolated engineered immune T cells expressing CD22 CAR of SEQ ID NO. 15, wherein CD52 and TRAC genes are inactivated by deletion, so as to use them to treat relapsed refractory forms of B-ALL in lymphodepleted patients.

[0681] In a preferred embodiment, the method of engineering immune cells involves introducing into said T cells a polynucleotide, particularly mRNA, encoding a specific rare-cutting endonuclease to selectively inactivate those genes as described above by DNA cleavage.

[0682] In a more preferred embodiment, the rare-cutting endonuclease is a TALE nuclease or a Cas9 endonuclease. To date, TAL nucleases have been shown to have higher specificity and cleavage efficiency than other types of rare-cutting endonucleases, making them the preferred endonucleases and preferred for large-scale generation of engineered immune cells with constant conversion rates.

[0683] Delivery Method

[0684] The above different methods involve introducing 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 a plasmid vector of the present invention. The plasmid vector encoding CD22 CAR of the present invention can also contain a selection marker, which is used to identify and / or select cells that accept the vector.

[0685] Methods allowing the introduction of a CD22 CAR according to the invention into isolated immune cells for subsequent expression are described elsewhere, for example in WO2013126720 or WO2015121454, which are incorporated herein by reference in their entirety.

[0686] Since the polynucleotide encoding the polypeptide is introduced into the cell, the polypeptide corresponding to the CD22 CAR of the present invention can be synthesized in situ in the cell. Alternatively, the polypeptide can be generated extracellularly and then introduced into the cell. The method of introducing a polynucleotide construct into a cell is known in the art, and includes a stable transformation method as a non-limiting example, in which the polynucleotide construct is integrated into the genome of the cell; a transient transformation method, in which the polynucleotide construct is not integrated into the genome of the cell; and a virus-mediated method. The polynucleotide can be introduced into the cell by, for example, a recombinant viral vector (e.g., a retrovirus, such as a lentivirus, adenovirus, adeno-associated virus), a liposome, etc. For example, a transient transformation method includes, for example, microinjection, electroporation, or particle bombardment. Considering expression in a cell, the polynucleotide can be contained in a vector, more specifically in a plasmid or a virus.

[0687] In a preferred embodiment, the polynucleotide encoding the CD22 CAR is inserted into an AAV6 vector and introduced into a given gene. Methods for preparing a CD22 CAR that allows the present invention to be introduced into isolated T cells and then expressed are described elsewhere, such as in WO2013126720, which is incorporated herein by reference.

[0688] Engineered Immune Cells (UCART)

[0689] Engineered immune cells with CD22 CAR (UCART22) are another object of the present invention.

[0690] Preferably, the immune cells are isolated immune cells, more preferably isolated immune T cells, more preferably isolated primary immune T cells.

[0691] UCART22 is provided as a medicament, and thus a therapeutically effective amount of UCART22 is provided as a medicament.

[0692] According to the present invention, "primary immune cells" refer to "cells derived from tissues such as blood samples or peripheral blood mononuclear cells (PBMCs), which can be passaged several times in culture and finally frozen before use, and the division capacity of the immune cells is limited compared to transformed or cancerous cells (Raulf-Heimsoth MT cell-primary culture from peripheral blood. Methods Mol Med. 2008; 138: 17-30. doi: 10.1007 / 978-1-59745-366-0).

[0693] The immune cells according to the present invention are preferably immune T cells or NK cells. Therefore, the engineered immune cells according to the present invention are isolated from a blood sample, are primary cells and are derived from immune T cells selected from the following and further engineered: inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes, natural killer T cells, preferably derived from cytotoxic T lymphocytes.

[0694] Engineering means that primary immune cells are modified so that they have CD22 CAR and at least one gene is edited, preferably the cells are modified so that they have CD22 CAR and neither express TCR nor die in the presence of purine nucleotide analogs.

[0695] In other words, the engineered immune cells refer to isolated immune T cells with TCR KO, contain at least one other edited gene, and express CD22 CAR.

[0696] In a specific embodiment, engineering refers to modifying primary immune cells so that they have CD22 CAR, preferably modifying the cells so that they have CD22 CAR and do not die in the presence of purine nucleotide analogs (1-5 micromoles / liter) or alemtuzumab (50 micrograms / ml). (Valton et al., Molecular Therapy vol. 23 no. 9, 1507–1518 sep. 2015)

[0697] Preferably, the T cell has a CD22 CAR of SEQ ID N°15.

[0698] More preferably, the T cell has a CD22 CAR of SEQ ID N°15 and comprises at least one sequence of SEQ ID N°22.

[0699] More preferably, the T cell has a CD22 CAR of SEQ ID N°15 and comprises at least one sequence of SEQ ID N°22 and at least a portion of the sequence.

[0700] More preferably, the T cell has a CD22 CAR of SEQ ID N°18 comprising at least one sequence of SEQ ID N°20.

[0701] 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 cells expressing CD22.

[0702] The present invention also provides primary T cells expressing the CD22 CAR of the present invention, which are used to lyse cells expressing CD22, particularly cancerous cells expressing CD22.

[0703] Preferably, cells targeted by T cells having a CD22 CAR of SEQ ID NO. 16 are effective in treating relapsed / refractory / aggressive ALL or CLL.

[0704] The present invention also relates to isolated cells or cell lines that are easily obtained by the method of engineering cells. In particular, the isolated cells include at least one CD22CAR of the present invention as described above. In another embodiment, the isolated cells include a group of CARs, each CAR including different extracellular ligand binding domains. In particular, the isolated cells include exogenous polynucleotide sequences encoding CARs. The genetically modified immune cells of the present invention are activated and can proliferate independently of the antigen binding mechanism.

[0705] Also included within the scope of the present invention are separated immune cells obtained according to any of the methods previously described, preferably separated immune T cells (T cells), more preferably engineered separated immune T cells. The immune cells refer to cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. The immune cells according to the present invention can be derived from stem cells. Stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells. The separated cells can also be dendritic cells, killer dendritic cells, mast cells, NK cells, B cells or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes. In another embodiment, the cells can be derived from CD4+ T lymphocytes and CD8+ T lymphocytes.

[0706] Before the cells of the present invention are amplified and genetically modified, a cell source can be obtained from a subject by a variety of non-limiting methods. Cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue, ascites, pleural effusion, spleen tissue and cancer. In certain embodiments of the present invention, any number of T cell lines accessible and known to those skilled in the art can be used. In another embodiment, the cells can be from healthy donors, patients diagnosed with cancer or patients diagnosed as infected. In another embodiment, the cells are part of a mixed cell population presenting different phenotypic characteristics. Also included within the scope of the present invention are cell lines obtained from engineered T cells according to the method. Modified cells that tolerate immunosuppressive therapy and are easy to obtain by previous methods are included within the scope of the present invention.

[0707] As a preferred embodiment, the present invention provides T cells or T cell populations having the CD22CAR of the present invention as described above, which do not express functional TCR and are reactive to CD22-positive cells, so as to be used for adoptive transfer thereof to a patient.

[0708] As a more preferred embodiment, the present invention provides a T cell or a T cell population having a CD22 CAR as described above, which does not express a functional TCR and is made resistant to chemotherapy, in particular to purine nucleotide analogs (PNA).

[0709] According to a preferred embodiment of the present invention, immune cells with CD22 CAR are engineered to be resistant to chemotherapeutic drugs, especially purine nucleotide analogs (PNA), making them suitable for cancer treatment in combination with adoptive immunotherapy and chemotherapy.

[0710] Purine nucleotide analogs enter the chemotherapy composition of many cancer treatments. It is used as a standard treatment for leukemia or lymphoma. The most widely used PNA is clofarabine, fludarabine and cytarabine used alone or in combination. PNA is metabolized to monophosphate, diphosphate and triphosphate PNA by an enzyme [EC 2.7.1.74] with deoxycytidine kinase (dCK) activity. Their triphosphate forms, particularly clofarabine triphosphate, compete with ATP for DNA synthesis, act as a pro-apoptotic agent, and are effective inhibitors of ribonucleotide reductase (RNR) involved in trinucleotide production.

[0711] Thus, the present invention includes methods for the ex vivo production of immune cells, preferably primary T cells that do not express a TCR, that are resistant to purine analog drugs and that can target CD22-positive malignant cells.

[0712] The method for preparing UCART22 according to the present invention may also be the method disclosed in WO 2013176915 or WO 2014191128, which are incorporated herein by reference in their entirety.

[0713] A method for preparing UCART22 comprises the following steps:

[0714] (a) providing immune cells from a donor, preferably isolated T cells or an isolated T cell population,

[0715] (b) introducing into the immune cells (preferably by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets the following genes:

[0716] - a gene expressing an enzyme having deoxycytidine kinase activity (dcK - EC 2.7.1.74), in particular the human deoxycytidine kinase gene (NCBI Gene ID: 1633), and / or

[0717] - a gene encoding one of the TCR α subunit and / or β subunit, preferably the α subunit, and / or

[0718] -Gene encoding human CD52

[0719] (c) expressing the endonuclease into the immune cell to obtain targeted inactivation of the gene;

[0720] (d) expanding the engineered immune cells obtained in step c), optionally in the presence of a purine analog drug;

[0721] (e) introducing the CD22 CAR of the present invention, preferably the CD22 CAR of SEQ ID N°15, into the immune cells.

[0722] A method for preparing UCART22 comprises the following steps:

[0723] (a) providing immune cells from a donor, preferably isolated T cells or an isolated T cell population,

[0724] (b) introducing into the immune cells (preferably by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets the following genes:

[0725] - a gene expressing an enzyme having deoxycytidine kinase activity (dcK - EC 2.7.1.74), in particular the human deoxycytidine kinase gene (NCBI Gene ID: 1633) and / or

[0726] - A gene encoding one of the TCR α subunit and / or β subunit, preferably the α subunit

[0727] -Gene encoding human beta 2 immunoglobulin (B2M)

[0728] (c) expressing the endonuclease into the immune cell to obtain targeted inactivation of the gene;

[0729] (d) expanding the engineered immune cells obtained in step c), optionally in the presence of a purine analog drug;

[0730] (e) introducing the CAR of the present invention, preferably CD22 CAR, more preferably CD22 CAR of SEQ ID N°15, alone or in combination with CD19 CAR, into the immune cells.

[0731] In a preferred embodiment, the present invention includes a method for producing immune cells ex vivo, preferably primary T cells that do not express TCR, which are resistant to purine analog drugs and can target CD22-positive malignant cells. The method comprises the following steps:

[0732] providing immune cells from a donor, preferably isolated T cells (or isolated T cell populations),

[0733] Introducing the CD22 CAR of the present invention, preferably the CD22 CAR selected from SEQ ID N°15, into the immune cells.

[0734] Expanding the engineered immune cells obtained in step b)

[0735] (d) introducing into the immune cell (by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets

[0736] a gene expressing an enzyme having 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 alpha subunit or beta subunit,

[0737] e) expressing the endonuclease into the immune cells to obtain targeted inactivation of the gene;

[0738] f) expanding the engineered immune cells obtained in step e), optionally in the presence of a purine analog drug.

[0739] The method for producing immune cells ex vivo, preferably primary T cells that do not express TCR (which are resistant to purine analog drugs and can target CD22-positive malignant cells), optionally includes another step of introducing a nucleic acid sequence encoding a rare-cutting nuclease that specifically targets one of the genes listed in Table 9 into the immune cells (by transfection or transduction), wherein the listed genes are preferably PD1, CD279, and more preferably PDCD1 (PD1, CD279) or CTLA4 (CD152).

[0740] The inventors of the present application have successfully constructed primary T cells expressing CD22 CAR (dCK-KO) that are resistant to purine nucleotide analogs, more particularly clofarabine and / or fludarabine, by introducing the inactivation (deletion) of dcK gene expression into cells using nucleases, particularly TAL nucleases.

[0741] 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 the cytotoxic activity of T cells against CD22-bearing cells.

[0742] The present application also provides TCR-KO, CD22 CAR (preferably SEQ ID N°15) primary T cells (whose deoxycytidine kinase expression has been inhibited or inactivated (dCK-KO)) for treating leukemia or lymphoma, preferably their aggressive, resistant, relapsed forms, more preferably B-ALL.

[0743] According to a preferred embodiment of the present invention, immune cells with CD22 CAR are engineered to be resistant to chemotherapeutic drugs, particularly alemtuzumab (CAMPATH), making them suitable for cancer treatment in combination with adoptive immunotherapy and chemotherapy.

[0744] Alemtuzumab is used in the treatment of many cancers. It is used as a standard therapy 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 by the trade names Campath, MabCampath, and Campath-1H. It is also used in certain conditioning regimens for bone marrow transplants, kidney transplants, and pancreatic islet cell transplants.

[0745] 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 are derived. After treatment with alemtuzumab, these lymphocytes with CD52 are targeted for destruction.

[0746] Alemtuzumab is also used as a second-line treatment for CLL and is approved by the U.S. Food and Drug Administration for patients with CLL who have failed prior treatment with an alkylating agent and fludarabine.

[0747] Therefore, the present invention includes a method for producing UCART22 ex vivo so that it does not express TCR (which is resistant to alemtuzumab).

[0748] The method for preparing UCART22 CD52 KO comprises the following steps:

[0749] (a) providing immune cells from a donor, preferably isolated T cells or an isolated T cell population,

[0750] (b) introducing into said immune cells (preferably by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets

[0751] - A gene encoding one of the TCR α subunit and / or β subunit, preferably the α subunit

[0752] - The gene encoding CD52,

[0753] (c) expressing the endonuclease into the immune cell to obtain targeted inactivation of the gene;

[0754] (d) expanding the engineered immune cells obtained in step c), optionally in the presence of alemtuzumab,

[0755] (e) introducing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID N°15) into the immune cells.

[0756] In a preferred embodiment, the present invention includes a method for producing immune cells, preferably primary T cells that do not express TCR, ex vivo, which are resistant to alemtuzumab and can target CD22-positive malignant cells. The method comprises the following steps:

[0757] Providing immune cells from a donor, preferably isolated T cells (or isolated T cell populations)

[0758] Introducing the CD22 CAR of the present invention, preferably the CD22 CAR of SEQ ID N°15, into the immune cells.

[0759] c) expanding the engineered immune cells obtained in step b)

[0760] d) introducing into the immune cells (by transfection or transduction) a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets the following genes:

[0761] A gene expressing CD52 and a gene expressing one of the TCR α subunit or β subunit,

[0762] e) expressing the endonuclease into the immune cells to obtain targeted inactivation of the gene;

[0763] f) expanding the engineered immune cells obtained in step e), optionally in the presence of a purine analog drug.

[0764] In one embodiment, the method for preparing UCART22 comprises the following steps:

[0765] (a) providing immune cells from a donor, preferably isolated T cells or an isolated T cell population,

[0766] (b) introducing the following into the immune cell (preferably by transfection or transduction):

[0767] - a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets a gene encoding one of the TCR alpha subunits and / or beta subunits, preferably the alpha subunit, and

[0768] - a nucleic acid encoding the sequence to be inserted, preferably encoding HIF-1α,

[0769] (c) expressing the endonuclease into the immune cell to obtain targeted insertion of the sequence to be inserted;

[0770] (d) expanding the engineered immune cells obtained in step c), optionally in a low O 2 Concentration (5% O 2 , preferably 1% O 2 ) exists,

[0771] (e) introducing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID N°15) into the immune cells.

[0772] In a preferred embodiment, the present invention includes a method for producing immune cells, preferably primary T cells that do not express TCR, in vitro, which are tolerant to hypoxia and can target CD22-positive malignant cells. The method comprises the following steps:

[0773] Providing immune cells from a donor, preferably isolated T cells (or isolated T cell populations)

[0774] Introducing the CD22 CAR of the present invention, preferably the CD22 CAR of SEQ ID N°15, into the immune cells.

[0775] c) expanding the engineered immune cells obtained in step b)

[0776] d) introducing into said immune cells (preferably by transfection or transduction)

[0777] - a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets a gene encoding one of the TCR alpha subunits and / or beta subunits, preferably the alpha subunit, and

[0778] - a nucleic acid encoding the sequence to be inserted, preferably encoding HIF-1α,

[0779] e) expressing the endonuclease into the immune cells to obtain targeted inactivation of the gene;

[0780] f) expanding the engineered immune cells obtained in step e), optionally in a low O 2 Concentration (5% O 2 , preferably 1% O 2 ) exists.

[0781] The method for producing immune cells ex vivo, preferably primary T cells that do not express TCR (which are resistant to purine analog drugs, alemtuzumab or hypoxia and can target CD22-positive malignant cells), optionally includes another step of introducing a nucleic acid sequence encoding a rare-cutting nuclease that specifically targets one of the genes listed in Table 9 into the immune cells (by transfection or transduction), preferably PD1, CD279, more preferably PDCD1 (PD1, CD279) and CTLA4 (CD152) among the listed genes.

[0782] The inventors of the present application have successfully constructed primary T cells expressing CD22 CAR (dCK-KO) that are resistant to purine nucleotide analogs, more particularly clofarabine and / or fludarabine, by introducing inactivation (deletion) of dcK gene expression into cells using nucleases, particularly TAL nucleases.

[0783] 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 the cytotoxic activity of T cells against CD22-bearing cells.

[0784] As described, the inventors applied the same approach to the deletion of human CD52 using specific TALENs.

[0785] The present application also provides TCR KO, CD22 CAR (preferably CD22CAR of SEQ ID N°15) primary T cells, whose deoxycytidine kinase expression has been inhibited or inactivated (dCK-KO), for treating leukemia or lymphoma, preferably their aggressive, resistant, relapsed forms, more preferably relapsed B-ALL.

[0786] Such cells are "universal" T cells (or UCART).

[0787] In one embodiment, the present application provides a CD22 CAR (preferably a CD22 CAR of SEQ ID N°15) primary T cell, the expression of which deoxycytidine kinase has been inhibited or inactivated (dCK-KO), for treating leukemia or lymphoma, preferably their aggressive, resistant, relapsed forms, more preferably B-ALL relapsed after autologous transfer.

[0788] In one embodiment, the present application provides a CD22 CAR (preferably CD22CAR of SEQ ID N°15) primary T cell, whose CD52 expression has been inhibited or inactivated (CD52-KO), for treating leukemia or lymphoma, preferably its aggressive, resistant, relapsed form, more preferably B-ALL relapsed after autologous transfer.

[0789] In one embodiment, the present application provides a CD22 CAR (preferably a CD22 CAR of SEQ ID N°15) primary T cell, which increases the expression of HIF-1α by inserting the coding sequence into the TRAC sequence without knocking out the TCR.

[0790] For the treatment of leukemia or lymphoma, preferably their aggressive, resistant, relapsed forms, more preferably B-ALL relapsed after autologous transfer.

[0791] In one embodiment, the present application provides CD22 CAR (preferably CD22 CAR of SEQ ID N°15) primary T cells, whose expression of deoxycytidine kinase and CD52 has been inhibited or inactivated (dCK and CD52 KO), for treating leukemia or lymphoma, preferably their aggressive, resistant, relapsed forms, more preferably B-ALL relapsed after autologous transfer.

[0792] In one embodiment, the present application provides a CD22 CAR (preferably a CD22 CAR of SEQ ID N°15) primary T cell, the expression of which deoxycytidine kinase and CD52 have been inhibited or inactivated (dCK and CD52 KO), and the expression of HIF-1α is increased by inserting the HIF1α coding sequence into the TRAC sequence without knocking out TCR, for treating leukemia or lymphoma, preferably their aggressive, resistant, relapsed forms, more preferably B-ALL relapsed after autologous transfer.

[0793] The method for producing immune cells ex vivo, preferably primary T cells that do not express TCR (which are resistant to purine analog drugs and / or alemtuzumab and / or hypoxia and can target CD22-positive malignant cells), optionally comprises another step of introducing a nucleic acid sequence encoding a rare-cutting endonuclease that specifically targets one of the genes listed in Table 9 into the immune cells (by transfection or transduction), preferably PD1, CD279, and more preferably PDCD1 (PD1, CD279) or CTLA4 (CD152) among the listed genes.

[0794] The inventors of the present application have successfully generated primary T cells expressing CD22 CAR (dCK-KO) that are resistant to purine nucleotide analogs, more particularly clofarabine and / or fludarabine, by using nucleases, particularly TAL nucleases, and by introducing inactivation (deletion) of dcK gene expression into cells.

[0795] The inventors of the present application have successfully generated primary T cells expressing CD22 CAR that are resistant to hypoxia by targeted insertion of the HIF-1α gene into cells using nucleases, particularly TAL nucleases.

[0796] 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 the cytotoxic activity of T cells against CD22-bearing cells.

[0797] The present application also provides TCR KO, CD22 CAR (preferably CD22 CAR of SEQ ID NO 15) primary T cells, which are resistant to hypoxia and are used to treat leukemia or lymphoma, preferably their aggressive, resistant, relapsed forms, more preferably B-ALL.

[0798] T cell activation and expansion

[0799] Whether before or after genetic modification of T cells, even if the genetically modified immune cells of the present invention are activated and proliferated independently of the antigen binding mechanism, the immune cells of the present invention, particularly T cells, can also be activated and expanded using the methods described in the following documents, for example, U.S. Patent Nos. 6,352,694, 6534055, 6905680, 6692964, 5858358, 6887466, 6905681, 7144575, 7067318, 7172869, 7232566, 7175843, 5883223, 6905874, 6797514, 6867041 and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

[0800] Typically, the T cells of the present invention are expanded by contacting with agents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of the T cells to generate activation signals 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 activation signals for the T cells.

[0801] As a non-limiting example, it is possible to contact with anti-CD3 antibodies or their antigen binding fragments or anti-CD2 antibodies fixed on the surface, or by contacting with protein kinase C activators (e.g., bryostatin) together with calcium ion carriers, stimulate T cell colonies in vitro. In order to costimulate the auxiliary molecules on the surface of T cells, a ligand bound to the auxiliary molecules is used. For example, under conditions suitable for stimulating T cell proliferation, T cell colonies are contacted with anti-CD3 antibodies and anti-CD28 antibodies. Conditions suitable for T cell culture include appropriate culture media (e.g., minimum essential culture media or RPMI culture media 1640 or X-vivo5 (Lonza)), which may contain factors required for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin 2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, -10, -2, IL-15, TGFp and TNF- or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasma products, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media can 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, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in an amount sufficient to allow T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are only included in experimental cultures and not in cell cultures to be injected into a subject. The target cells are maintained in culture under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO 2 ). T cells exposed to different stimulation times may show different characteristics.

[0802] In another specific embodiment, the cells can be expanded by co-culturing with tissues or cells. The cells can also be expanded in vivo (e.g., in the blood of a subject after the cells are administered to the subject).

[0803] Pharmaceutical composition

[0804] A pharmaceutical composition comprising engineered (TCR and dCK KO) or (TRAC and CD52 KO) immune T cells expressing CD22 CAR (preferably CD22 CAR of SEQ ID N°15) of the present invention and a pharmaceutically acceptable vehicle is another object of the present invention.

[0805] In another embodiment, the present invention provides a composition comprising UCART22 of the present invention (any of the above embodiments) and a compound of the bryostatin compound family, preferably bryostatin-1.

[0806] Bryostatins are a group of macrolides from the bryozoan Bugula neritina. The structure of bryostatin 1 was determined in the 1980s. To date, 20 different bryostatins have been isolated. In addition, identified bryostatin analogs are called "bryologs". Bryostatins are potent regulators 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 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 Society2002 124(46),13648-13649DOI:10.1021 / ja027509+)

[0807] Examples of bryostatin compounds suitable for use in combination with UCART22 of the present invention and methods for preparing these compounds are described in WO2001040214A1 or EP2737904A2, WO1997034598, which are incorporated herein by reference.

[0808] Examples of doses of bryostatin 1 that can be used in combination with UCART22 of the present invention were 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”.

[0809] Provided as a drug are engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells [UCART22] expressing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID N°15) or a pharmaceutical composition comprising the UCART22.

[0810] Another object of the present invention is to use engineered (TRAC and dCKKO or TRAC and CD52 KO) immune T cells [UCART22] expressing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID N°15) for treating cancer or reducing inflammation.

[0811] It is another object of the present invention that engineered (TRAC and dCKKO or TRAC and CD52 KO) immune T cells [UCART22] expressing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID NO 15) are used to treat ALL, CLL, refractory relapsed aggressive forms of CLL or ALL.

[0812] Provided are engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells [UCART22] expressing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID NO 15) for use in treating CD19 relapsed cancer, preferably CD19 relapsed B-ALL.

[0813] In another embodiment, an engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cell [UCART22] expressing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID N°15) that is tolerant to hypoxia or a pharmaceutical composition comprising the UCART22 is provided as a drug.

[0814] In another embodiment, the engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells [UCART22] expressing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID N°15) that are tolerant to hypoxia are used to treat cancer or reduce inflammation, which is another object of the present invention.

[0815] In another embodiment, engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells [UCART22] expressing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID N°15) that are tolerant to hypoxia are used to treat ALL, CLL, refractory relapsed invasive forms of CLL or ALL, which is another object of the present invention.

[0816] In another embodiment, engineered (TRAC and dCK KO or TRAC and CD52 KO) immune T cells [UCART22] expressing the CD22 CAR of the present invention (preferably the CD22 CAR of SEQ ID N°15) that are tolerant to hypoxia are used to treat CD19-relapsed cancer, preferably CD19-relapsed B-ALL, which is another object of the present invention.

[0817] In another embodiment, the isolated cells or cell lines derived from the isolated cells obtained by the different methods of the present invention can be used as a medicament. In another embodiment, the medicament can be used to treat cancer, in particular for the treatment of B cell lymphomas and leukemias in patients in need thereof. In another embodiment, the isolated cells or cell lines derived from the isolated cells according to the present invention can be used to prepare a medicament for the treatment of cancer in patients in need thereof.

[0818] The present invention includes autologous transfer of engineered cells. In that case, cells are isolated from a donor (human donor), engineered, and then transferred into the original donor in need thereof.

[0819] In this particular embodiment, the cells can be engineered to be resistant, for example, to drugs such as alemtuzumab (campath) and / or pna and optionally to hypoxia.

[0820] Therapeutic applications

[0821] The term "cancer" refers to a disease characterized by the uncontrolled growth of one or several types of cells.

[0822] Examples of cancers are described herein and include, but are not limited to, liquid tumors or blood cancers.

[0823] The blood cancer according to the present invention can be selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, chronic lymphocytic leukemia type B, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer acute myeloid leukemia, preferably a blood cancer expressing CD22, more preferably a relapsed or refractory blood cancer expressing CD22, and even more preferably an aggressive form of the CD22-associated blood cancer.

[0824] In a preferred embodiment, the relapsed or refractory CD22-expressing hematological cancer is relapsed and / or refractory CD22-expressing or CD22-positive B-ALL.

[0825] Therefore, a therapeutically effective amount of UCART22 according to any one of the above embodiments or a therapeutically effective amount of the pharmaceutical composition as described above is provided for use as a medicament for treating patients with relapsed and / or refractory CD22-expressing or CD22-positive B-ALL.

[0826] In another embodiment, a therapeutically effective amount of UCART22 according to any one of the above embodiments or a therapeutically effective amount of a pharmaceutical composition as described above is provided for use as a medicament for treating a patient with a CD22-positive blood cancer selected from the following: leukemia and lymphoma, hairy cell leukemia, acute lymphocytic leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia, chronic lymphocytic leukemia, B-cell chronic lymphocytic leukemia, chronic myeloid carcinoma, any of Hodgkin's lymphoma, non-Hodgkin's lymphoma and Burkitt's lymphoma, multiple myeloma.

[0827] In another embodiment, a therapeutically effective amount of UCART22 according to any one of the above embodiments or a therapeutically effective amount of a pharmaceutical composition as described above is provided for use as a medicament for treating a patient with a CD22-positive cancer selected from the following: alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck 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 tumors, 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, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer.

[0828] Other examples of CD22-mediated cancers are described herein, including, but not limited to, liver cancer, lung cancer (eg, non-small cell lung cancer), ovarian cancer, pancreatic cancer, thyroid cancer, and urothelial carcinoma.

[0829] In a specific embodiment, the invention aims to provide treatment for aggressive forms of these cancers, which are defined as growing at least 2 times faster than the typical growth rate of such cancers in the population.

[0830] In one embodiment, the invention is intended for use in the treatment of leukemia during an accelerated phase of treatment.

[0831] In a specific embodiment, the invention aims to provide a treatment for refractory / relapsed diffuse large B-cell non-Hodgkin's lymphoma - lung metastasis of breast cancer - triple cancer consisting of chronic lymphocytic leukemia and bladder and prostate cancer.

[0832] 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 cancer, acute myeloid leukemia, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma).

[0833] 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.

[0834] In one embodiment, the cancer cells are relapsed refractory CD19 negative cancer cells.

[0835] In one embodiment, the cancer cell is a relapsed refractory CD22-expressing cancer cell.

[0836] In a preferred embodiment, the cancer cell is relapsed refractory CD19-negative CD22-positive B-ALL.

[0837] B-cell ALL includes:

[0838] Early precursor B (early pre-B) ALL (also called pro-B ALL)

[0839] ·General ALL

[0840] Pre-B ALL

[0841] Mature B-cell ALL, also known as Burkitt's leukemia or non-Hodgkin's lymphoma in children.

[0842] As used herein, the term "disease associated with the expression of CD22" includes, but is not limited to, diseases associated with the expression of CD22 or disorders associated with the activity of cells expressing CD22, including tumor cells of various cancers (e.g., CD22-expressing B-ALL).

[0843] Cell destruction caused by lysis is one of the mechanisms by which the CD22 CAR T cells of the present invention act on cells expressing CD22 (reducing or eliminating tumors, promoting the infiltration of the host's immune cells into the tumor site, and enhancing / expanding anti-tumor responses).

[0844] In another aspect, the present invention relies on a method of treating a patient in need thereof, the method comprising at least one of the following steps:

[0845] ·Provide UCART22 of the present invention

[0846] administering the transformed immune cells to the patient,

[0847] In one embodiment, the UCART22 cells of the present invention can undergo robust in vivo T cell expansion and can reside in the host for an extended amount of time.

[0848] 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:

[0849] ·Treatment of patients with cancer

[0850] ·Provide UCART22 of the present invention

[0851] administering the transformed immune cells to the patient,

[0852] Conditioning includes lymphodepletion, or any suitable conditioning, which one skilled in the art, and preferably a physician, would recognize as determinative for curing the patient.

[0853] In a preferred embodiment, the method further comprises the step of bone marrow transplantation.

[0854] In one embodiment, the UCART22 cells of the present invention can undergo robust in vivo T cell expansion and can reside in the host for an extended amount of time.

[0855] The treatment may be ameliorative, curative or preventive. It may be part of an autologous immunotherapy or part of an allogeneic immunotherapy treatment. Autologous means that the cells, cell lines or cell populations used to treat the patient are derived from the patient or from a human leukocyte antigen (HLA) compatible donor. Allogeneic means that the cells or cell populations used to treat the patient are not derived from the patient but from a donor.

[0856] In one embodiment, anti-CD20 (rituximab) and / or QBEN 10 treatment is used to control the survival of the T cells of the present invention in a host.

[0857] Subjects

[0858] The compositions and methods of the present invention can be used to treat subjects that have been characterized as having pathological cells or tissues that express CD22 or are suspected of having pathological cells or tissues that express CD22. For example, subjects that benefit from treatment according to the present invention include subjects with B-ALL or CLL, refractory B-ALL, relapsed B-ALL.

[0859] In a preferred embodiment, the patient is a child with B ALL, relapsed B ALL, refractory B ALL (pediatric indication).

[0860] Treatment using engineered immune cells according to the present invention can be combined with one or more therapies against cancer selected from antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy and radiation therapy.

[0861] Therefore, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of UCART22 for treating children with B ALL, relapsed B ALL, or refractory B ALL.

[0862] The present invention also provides a pharmaceutical composition comprising UCART22 and a pharmaceutically acceptable excipient for treating children with B-ALL, relapsed B-ALL, or refractory B-ALL.

[0863] In a preferred embodiment, a pharmaceutical composition comprising UCART22 of the present invention and a bryostatin family compound (preferably bryostatin-1) and a pharmaceutically acceptable excipient is used to treat children with B-ALL, relapsed B-ALL, or refractory B-ALL.

[0864] According to a preferred embodiment of the present invention, the treatment can be given to patients receiving immunosuppressive therapy (conditioning therapy), more preferably patients receiving lymphodepletion. In fact, the present invention preferably relies on cells or cell populations that are tolerant to at least one immunosuppressant due to the inactivation of genes encoding receptors for such immunosuppressants. In this regard, immunosuppressive therapy or lymphodepletion should contribute to the selection and amplification of the T cells of the present invention and the destruction of CD22 cancerous cells in the patient.

[0865] Administration of cells or cell colonies according to the present invention can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, blood transfusion, implantation or transplantation. Compositions as described herein can be administered to patients by intravenous or intralymphatic injection or intraperitoneal, subcutaneous, intradermal, intracancer, intranodular, intramedullary, intramuscular. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

[0866] Administration of cells or cell populations may include administration of 10 4 -10 9 cells, preferably 10 per kg body weight 5 -10 6 cells, including all integer values ​​within these ranges. The cells or cell populations may be administered in one or more doses, preferably several consecutive doses (re-administration) to avoid escape (recurrent cells). In another embodiment, the effective amount of cells is administered in a single dose or multiple doses. In another embodiment, the effective amount of cells is administered in a single dose or in doses. In another embodiment, the effective amount of cells is administered in more than one dose over a period of time.

[0867] The timing of administration is within the physician's discretion and depends on the patient's clinical condition.

[0868] Cells or cell populations can be obtained from any source, such as a blood bank or donor. As individual needs vary, it is within the skill of the art to determine the optimal range of effective amounts of a given cell type for a particular disease or condition. An effective amount refers to an amount that provides a therapeutic or preventive 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 desired effect.

[0869] The current UCART22 is designed to be effective, but not active and limit cytokine storms. In the case of an overreaction in a patient, the present invention can be combined with appropriate drugs such as anti-IL-6 drugs for preventing cytokine storms.

[0870] In another embodiment, the effective amount of cells or a composition comprising those cells is administered parenterally. The administration can be intravenous. The administration can be performed directly by injection into the cancer.

[0871] In certain embodiments of the invention, cells are administered to patients with a variety of related treatment modalities (e.g., before, simultaneously, or after), including but not limited to antiviral therapy, cidofovir and interleukin 2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efalitumab therapy for psoriasis patients or other therapies for PML patients. In other embodiments, the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunoablative agents such as CAMPATH, alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase (rapamycin) which is important for growth factor-induced signal transduction (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993).

[0872] In other embodiments, the cell composition of the present invention is administered to the patient in combination with bone marrow transplantation, T cell ablation therapy (e.g., before, at the same time or after), wherein the T cell ablation therapy uses any chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell composition of the present invention is administered after B cell ablation therapy (e.g., an agent that reacts with CD20, such as Rituxan or QBEN 10). For example, in one embodiment, the subject can receive standard treatment with high-dose chemotherapy followed by peripheral blood cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In another embodiment, the expanded cells are administered before or after surgery.

[0873] In another embodiment, after transplantation, the subject receives an agent reactive with CD20, such as Rituxan or Ritiximab, preferably an agent reactive with both CD22 and CD20.

[0874] Other definitions

[0875] - Amino acid residues in a polypeptide sequence are named herein according to a one-letter code in which, for example, Q represents a GIn or glutamine residue, R represents an Arg or arginine residue, and D represents an Asp or aspartic acid residue.

[0876] -Amino acid substitution refers to the replacement of one amino acid residue by another amino acid, for example, the replacement of a glutamine residue by an arginine residue in a peptide sequence is an amino acid substitution.

[0877] - Nucleotides are named as follows: A one-letter code is used to designate the nucleoside base: a for adenine, t for thymine, c for cytosine, and g for 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.

[0878] -As used herein, "nucleic acid" or "polynucleotide" refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, shearing, endonuclease action and exonuclease action. Nucleic acid molecules can be composed of monomers of naturally occurring nucleotides (such as DNA and RNA) or analogs of naturally occurring nucleotides (such as enantiomeric forms of naturally occurring nucleotides) or a combination of both. Modified nucleotides can have changes in sugar moieties and / or pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyls, amines and azides, or sugars can be functionalized as ethers or esters. In addition, the entire sugar moiety can be replaced by spatially and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications in bases include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be single-stranded or double-stranded.

[0879] -Chimeric antigen receptor (CAR) refers to a molecule that combines a binding domain for a component present on a target cell, such as a chimeric protein produced intracellularly based on the specificity of an antibody for a desired antigen (e.g., a tumor antigen) and an activated T cell receptor, which exhibits specific anti-target cell immune activity. Typically, CAR consists of an extracellular single-chain antibody (scFvFc) fused to the intracellular signaling domain of the T cell antigen receptor complex ζ chain (scFvFc: ζ), and has the ability to redirect antigen recognition according to the specificity of a monoclonal antibody when expressed in T cells. An example of a CAR for the present invention is a CAR for a CD22 antigen, and may include an amino acid sequence as a non-limiting example: SEQ ID N°15 to SEQ ID N°18, preferably SEQ ID N°16 or 18, more preferably SEQ ID N°16.

[0880] -The term "endonuclease" refers to any wild-type or variant enzyme that is capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. An endonuclease does not cleave DNA or RNA independently of its sequence, but recognizes and cleaves DNA or RNA molecules at a specific polynucleotide sequence (further referred to as a "target sequence" or "target site"). An endonuclease can be classified as a rare-cutting endonuclease when it typically has a polynucleotide recognition site greater than 12 base pairs (bp) in length, more preferably 14-55 bp. Rare-cutting endonucleases significantly increase HR by inducing DNA double-strand breaks (DSBs) at defined loci (Perrin, Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007). The rare-cutting endonuclease can be, for example, a homing endonuclease (Paques and Duchateau 2007), a chimeric zinc finger nuclease (ZFN) (Porteus and Carroll 2005) formed by fusion of an engineered zinc finger domain with the catalytic domain of a restriction enzyme such as FokI, a Cas9 endonuclease from the CRISPR system (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) or a chemical endonuclease (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). In chemical endonucleases, a chemical or peptide cleaving enzyme binds to a nucleic acid polymer or to another DNA to recognize a specific target sequence, thereby targeting the cleavage activity to a specific sequence. Chemical endonucleases also include synthetic nucleases, such as conjugates of o-phenanthroline, 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.

[0881] -“TALE nuclease” (TALEN) means a fusion protein consisting of a nucleic acid binding domain generally derived from a transcription activator-like effector (TALE) and a 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 and Fok-1. In a specific embodiment, the TALE domain can be fused to a large range of nucleases, such as I-CreI and I-OnuI or functional variants thereof. In a more preferred embodiment, the nuclease is a monomeric TALE nuclease. Monomeric TALE nucleases are TALE nucleases that do not require dimerization for specific recognition and cleavage, such as a fusion of the engineered TAL repeat sequence described in WO2012138927 with the catalytic domain of I-TevI. Transcription activators such as effectors (TALE) are proteins from the genus Xanthomonas in bacterial species, comprising multiple repeat sequences, each repeat sequence comprising two residues (RVD) specific for each nucleotide base in the nucleic acid targeting sequence at positions 12 and 13. Binding domains with similar modular base-to-base nucleic acid binding properties (MBBBD) can also be derived from new modular proteins recently discovered by the applicant in different bacterial species. The new modular proteins have the advantage of showing more sequence variability than TAL repeat sequences. Preferably, the RVDs associated with recognizing different nucleotides are: HD that recognizes C, NG that recognizes T, NI that recognizes A, NN that recognizes G or A, NS that recognizes A, C, G or T, HG that recognizes T, IG that recognizes T, NK that recognizes G, HA that recognizes C, ND that recognizes C, HI that recognizes C, HN that recognizes G, NA that recognizes G, SN that recognizes G or A, YG that recognizes T, TL that recognizes A, VT that recognizes A or G, and SW that recognizes A. In another embodiment, the key amino acids at positions 12 and 13 can be mutated to other amino acid residues in order to adjust its specificity for the nucleotides A, T, C and G, in particular to enhance the specificity. TALE nucleases have been described and used to stimulate gene targeting and gene modification (Boch, Scholze et al. 2009; Moscow and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011). Customized TAL nucleases can be commercially available under the name TALEN. TM Commercially available (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

[0882] According to the rare-cutting endonuclease of the present invention, it can also be Cas9 endonuclease, zinc finger endonuclease, MegaTAL endonuclease. Based on the RNA-guided Cas9 nuclease from type II prokaryotic CRISPR (clustered, regularly spaced short palindromic repeats) adaptive immune system (see review (Sorek, Lawrence et al. 2013)), genome engineering tools have been developed (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 found in bacteria to resist foreign DNA from viruses or plasmids. CRISPR-mediated genome engineering is first performed by selecting a target sequence that is usually flanked by a short sequence motif (called a pre-spacer adjacent motif (PAM)). After selecting the target sequence, the specific crRNA complementary to the target sequence is engineered. The trans-activating crRNA (tracrRNA) required in the CRISPR II system is paired with the crRNA and binds to the provided Cas9 protein. Cas9 acts as a molecular anchor to promote base pairing of tracrRNA with cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the binary tracrRNA:crRNA structure acts as a guide RNA to guide the endonuclease Cas9 to the homologous target sequence. Target recognition by means of the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence to determine the homology between the target sequence and the crRNA. In addition to the complementarity of the target sequence-crRNA, DNA targeting also requires the presence of a short motif adjacent to the pre-spacer (pre-spacer adjacent motif-PAM). After pairing between the binary RNA and the target sequence, Cas9 then introduces a blunt-ended double-strand break 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).

[0883] The rare-cutting endonuclease may be a homing endonuclease, also referred to as a meganuclease. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and produce single-strand or double-strand breaks. Homing endonucleases are highly specific, recognizing DNA target sites of 12 to 45 base pairs (bp), typically 14 to 40 bp in length. The homing endonuclease according to the present invention may, for example, correspond to a LAGLIDADG endonuclease, a HNH endonuclease or a GIY-YIG endonuclease. A preferred homing endonuclease according to the present invention may be an I-Crel variant.

[0884] - "delivery vector" refers to any delivery vector that can be used in the present invention to contact (i.e., "contact") or deliver (i.e., "introduce") the reagents / chemical substances and molecules (proteins or nucleic acids) required by the present invention to cells. It includes, but is not limited to, liposome delivery vectors, viral delivery vectors, drug delivery vectors, chemical vectors, polymer vectors, lipid complexes, complexes, 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 vector. "Delivery vector" also means a delivery method for transfection.

[0885] -The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. "Vector" in the present invention includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules, which may consist of chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vectors) and / or expression of nucleic acids to which they are attached (expression vectors). A large number of suitable vectors are known to those skilled in the art and are commercially available.

[0886] Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses, particularly AAV6), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and pox viruses (e.g., vaccinia, fowlpox, and canarypox virus). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include: avian leukosis-sarcoma virus, mammalian type C, B, and D viruses, HTLV-BLV groups, lentiviruses, and bovine foamy virus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0887] -"Lentiviral vector" refers to a HIV-based lentiviral vector, which is very promising in gene delivery due to its relatively large packaging capacity, reduced immunogenicity, and its ability to efficiently and stably transduce a variety of different cell types. Lentiviral vectors are usually produced after transient transfection of three (packaging, envelope and transfer) or more plasmids into production cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with cell surface receptors. Upon entry, viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The reverse transcription product is a double-stranded linear viral DNA, which is a substrate for viral integration in the DNA of infected cells. "Integrative lentiviral vector (or LV)" refers to such vectors as non-limiting examples that can integrate the target cell genome. In contrast, "non-integrative lentiviral vector (or NILV)" refers to an effective gene delivery vector that does not integrate the target cell genome through the action of viral integrase.

[0888] - Delivery vehicles and vectors may be associated or combined with any cell permeabilization technique such as sonoporation or electroporation or derivatives of these techniques.

[0889] -Cell refers to any living eukaryotic cell, primary cell or cell line derived from these organisms used for in vitro culture.

[0890] - "Primary cells" are cells that are obtained directly from living tissue (i.e., biopsy material) and established for growth in vitro, that have undergone few population doublings compared to continuously tumorigenic or artificially immortalized cell lines and are therefore more representative of the major functional components and characteristics of the tissue from which they are derived.

[0891] As a non-limiting example, the cell line can 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.

[0892] All of these cell lines can be modified by the methods of the present invention to provide cell line models to produce, express, quantify, detect, and study genes or proteins of interest. These models can also be used for screening of bioactive molecules of interest in various fields such as chemistry, biofuels, therapeutics, and agronomy, as non-limiting examples.

[0893] - "Mutation" refers to the replacement, deletion, insertion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50 or more nucleotides / amino acids in a polynucleotide (cDNA, gene) or polypeptide sequence. A mutation may affect the coding sequence of a gene or its regulatory sequence. It may also affect the structure of the genomic sequence or the structure / stability of the encoded mRNA.

[0894] - "Variant" refers to a repeat variant, a variant, a DNA binding variant, a TALE-nuclease variant, a polypeptide variant obtained by mutating or substituting at least one residue in the amino acid sequence of the parent molecule.

[0895] - "Functional variant" refers to a catalytically active mutant of a protein or protein domain; such a mutant has the same activity, or other properties, or a higher or lower activity compared to its parent protein or protein domain.

[0896] - "Identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each sequence that are aligned for comparison. Identity can be determined by comparing the positions in each sequence that can be compared for the purpose of comparison. When a position in the compared sequence is occupied by the same base, the molecules have identity 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 the 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, which can be used as part of the GCG sequence analysis software package (University of Wisconsin, Madison, Wisconsin), and can use, for example, default settings. For example, it can be expected that a polypeptide having at least 70%, 85%, 90%, 95%, 98% or 99% identity with a specific polypeptide described herein and preferably exhibiting substantially the same function, and a polynucleotide encoding such a polypeptide. Unless otherwise indicated, similarity scores will be based on the use of BLOSUM62. When BLASTP is used, the percentage similarity is based on the BLASTP positive score, while the percentage sequence identity is based on the BLASTP identity score. BLASTP "identity" shows the number and fraction of total residues in the same high-scoring sequence pair; BLASTP "positive" indicates the number and fraction of residues that have positive alignment scores and are similar to each other. The present disclosure contemplates and encompasses amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity to the amino acid sequences disclosed herein. Polynucleotide sequences of similar polypeptides are derived using the genetic code and can be obtained by conventional methods, particularly by reverse translating their amino acid sequences using the genetic code.

[0897] - "Signal transduction domain" or "co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate co-stimulatory molecule on a T cell to provide a signal, which is a signal that mediates T cell responses in addition to the primary signal provided by, for example, the binding of a TCR / CD complex to a peptide-loaded MHC molecule, and the cell response includes, but is not limited to, proliferation activation, differentiation, etc. Co-stimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, stimulatory ligands that bind to Toll ligand receptors, agonists or antibodies and ligands that specifically bind to B7-H3. CD28 is excluded from this list. Co-stimulatory ligands also include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as but not limited to CD27, 4-IBB, OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0898] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of 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.

[0899] As used herein, a "co-stimulatory signal" 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.

[0900] As used herein, the term "extracellular ligand binding domain" is defined as an oligopeptide or polypeptide capable of binding 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 serves as a cell surface marker on a target cell associated with a particular disease state. Thus, examples of cell surface markers that may be used as ligands include those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.

[0901] 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 or refractory or relapsed ALL, or aggressive or refractory or relapsed CLL.

[0902] A mammal is any warm-blooded vertebrate of the class Mammalia, preferably a human.

[0903] "Suicide domain or switch" or "safety switch" refers to a domain, typically a cell surface domain, that is recognized by a molecule, protein, chemical, antibody in order to immunoselect expressing cells and ultimately control their function and survival.

[0904] The above written description of the invention provides the manner and process of making and using the invention to enable one skilled in the art to make and use the invention, particularly the subject matter of the appended claims which form a part of this original description.

[0905] Where numerical limitations or ranges are stated herein, the endpoints are included. Likewise, all values ​​and sub-ranges within the numerical limitations or ranges are expressly included as if expressly written out.

[0906] The above description is given to enable those 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 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. Therefore, the present invention is not intended to be limited to the embodiments shown, but rather to the widest scope consistent with the principles and features disclosed herein.

[0907] General approach

[0908] CAR screening and selection

[0909] Primary T cell culture

[0910] Ficoll gradient density medium was prepared from EFS (Etablissement, Paris, France) T cells were purified from buffy coat samples provided by duSang. The PBMC layer was recovered and X-Vivo was supplemented with 20 ng / mL human IL-2, 5% human and Dynabeads human T activator CD3 / CD28. TM T cells were activated in 5% paraformaldehyde-15 medium (Lonza) with a 1:1 bead:cell ratio (Life Technologies).

[0911] CAR mRNA transfection

[0912] Transfection of CAR mRNA encoding each CAR construct was performed on day 4 or day 11 after T cell purification and activation. Cells were immediately diluted and analyzed in X-Vivo TM -15 min in culture medium and incubate at 37°C with 5% CO 2IL-2 was added at 20 ng / mL 2 hours after electroporation.

[0913] T cell transduction

[0914] As described previously, a vector encoding CD22 CAR was introduced into T cells.

[0915] CAR detection on the surface of T cells was performed using a recombinant protein consisting of the extracellular domain of human CD22 protein (whole protein, distal part of CD22 or proximal part of CD22) fused to a mouse IgG1 Fc fragment. Binding of the protein to the CAR molecule was detected using a PE-conjugated secondary antibody (Jackson Immunoresearch) targeting the mouse Fc portion of the protein and analyzed by flow cytometry.

[0916] Inactivation of specific genes in primary T cells

[0917] Inactivation of specific genes can be performed in primary T cells before and preferably after CD22 CAR is introduced into cells using correspondingly designed endonucleases such as TAL endonucleases, optional Crispr Cas 9 endonucleases. At least one gene is inactivated, and one, two or three genes can be inactivated in one step or several consecutive steps. In a preferred embodiment, two genes are inactivated, preferably TCRα genes and genes that are deleted to confer tolerance to drugs selected from the following: purine nucleotide analogs, alemtuzumab, platinum (cisplatin or carboplatin), anti-topoisomerase I (irinotecan), anti-topoisomerase II (etoposide), methotrexate (folate analogs), preferably purine nucleotide analogs, alemtuzumab.

[0918] Typically, heterodimeric nucleases, particularly TALE-nucleases that target two long sequences (called half-targets) separated by a spacer within a target gene, are designed and produced.

[0919] Each TALE nuclease construct can be cloned in a suitable mammalian expression vector. The mRNA encoding the TALE nuclease that cuts the target genomic sequence can be synthesized from a plasmid carrying the coding sequence downstream of the promoter. Purified T cells pre-activated with anti-CD3 / CD28 coated magnetic beads are used and transfected with each of the 2 mRNAs encoding the two half TALE nucleases. Cells can be reactivated with soluble anti-CD28 to measure cell proliferation at different times, and the activation marker CD25 is detected to assess the activation state of the cells.

[0920] Degranulation assay (CD107a mobilization)

[0921] The cells were incubated with equal numbers of cells expressing various levels of the protein of interest (CD22) in 96-well plates. The co-cultures were maintained at 37°C and 5% CO. 2 The culture was maintained for 6 hours under the conditions of . CD107a staining was performed during cell stimulation by adding fluorescent anti-CD107a antibodies at the beginning of co-culture as well as anti-CD49d and anti-CD28, and 1x monensin solution was used as a control. After 6 hours of incubation, the cells were stained with fixable viability dyes and fluorescent dye-coupled anti-CD8 and analyzed by flow cytometry. Degranulation activity was determined as the percentage of CD8+ / CD107a+ cells and was performed by determining the mean fluorescence intensity signal (MFI) of CD107a staining between CD8+ cells. Degranulation was determined 24 hours after mRNA transfection.

[0922] Interferon gamma release assay

[0923] 24 h after mRNA transfection, T cells expressing CD22 CAR were incubated with cell lines expressing various levels of target protein at 37°C for 24 h. The supernatant was recovered and IFNγ was detected in the cell culture supernatant by ELISA.

[0924] Cytotoxicity assay

[0925] The cells were incubated with target cells (expressing different levels of CD22) or (negative control) cells. Before the target cells and control cells were co-cultured with CAR+T cells, they were first labeled with fluorescent intracellular dyes (CFSE or Cell Trace Violet). The co-cultures were incubated at 37 ° C for 4 hours. After the incubation period, the cells were labeled with fixable viability dyes and analyzed by flow cytometry. The viability of each cell population (target cells or negative control cells) was determined, and the percentage of specific cell lysis was calculated. Cytotoxicity tests were performed 48 h after mRNA transfection.

[0926] Tumor-resistant mouse model

[0927] Tumor cells (CD22 BALL from patients) or luciferase cells expressing target proteins are implanted into the flanks of immunodeficient mice. Subsequently, cells are implanted into the mouse brain. Continuously transplanted into more generations of mice to continue to maintain the in vivo xenograft cell line. Optionally, mice receive anticancer treatment (alemtuzumab and / or flu) before and / or simultaneously with injection of CAR+T cells. Subsequently, mice are injected with different doses of CAR+T cells to be tested or with T cells that do not express CD22 CAR iv (2 days or 7 days after injection of tumor cell lines). On the day of injection of T cells (D0), bioluminescent signals are determined on the 7th, 14th, 21st, 28th and 40th days after T cell injection to track tumor progression in different animals.

[0928] Phase I dose-escalation study to evaluate the safety, expansion, and persistence of allogeneic CD22 CART (UCART22) in patients with relapsed or refractory or MRD+CD22+ B-cell acute lymphoblastic leukemia (B-ALL).

[0929] Background and Rationale

[0930] With current multidrug chemotherapy regimens, >80% of children with acute lymphoblastic leukemia (ALL) and approximately 40% of adults with ALL achieve long-term survival. (1) Further intensification of chemotherapy has not proven to be effective. (2) Our understanding of the biology of ALL has advanced dramatically over the past few years, providing opportunities for “targeted therapies.” (3,4)

[0931] Relapsed / refractory ALL remains a challenging disease. Post-relapse therapy will result in a second CR (CR2) in 30-40% of patients with a 5-year OS of only approximately 10%. In the largest report of relapsed adult ALL patients to date, Fielding and colleagues analyzed the outcomes 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 with a median survival of 11 months. The 5-year OS of relapsed patients was only 7%. The median OS of relapsed patients was 5.5 months. Tavernier and colleagues reported the outcomes of 421 patients with first relapse of ALL 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 outcomes in 263 patients with ALL in first relapse 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%.

[0932] CD22 expression occurs in >90% of ALL patients and is an effective therapeutic target. Cellular therapies such as chimeric antigen receptor (CAR) T cell therapy are increasingly being used to treat patients with hematologic malignancies. (8-16) High complete response rates (80-90%) have been reported in patients with relapsed acute lymphoblastic leukemia (ALL) using autologous CD19 CART cells. (12) Similarly, response rates of 40-50% have been achieved in patients with relapsed chronic lymphocytic leukemia (CLL) who underwent autologous CD19 CART therapy. (9)

[0933] This study evaluated allogeneic CART cells directed against CD22 in patients with relapsed and / or refractory CD22 B-ALL.

[0934] Target

[0935] Main Objectives

[0936] Assess the safety and tolerability of allogeneic CD22 CART and determine the maximum tolerated dose (MTD)

[0937] Secondary Objectives

[0938] Determining the effect of allogeneic CD22 CART

[0939] Determine the incidence of GVHD

[0940] Exploratory goals

[0941] Determine the expansion, phenotype, trafficking, and persistence of infused CAR T cells

[0942] Inclusion criteria

[0943] 1. Relapsed or refractory CD22-positive ALL (for expansion phase: patients with MRD+ disease are allowed)

[0944] 2. Patients aged ≥ 2 years

[0945] 3. ECOG performance status ≤ 2

[0946] 4. Normal organ function including bilirubin ≤2mg / dl, ALT / AST <3x ULN and creatinine ≤2mg / dl

[0947] 5. Left ventricular ejection fraction (LVEF) ≥ 40%

[0948] Exclusion criteria

[0949] 1. The patient is pregnant or breastfeeding

[0950] 2. Patients with uncontrolled active infection

[0951] 3. Isolated extramedullary recurrence (ie, testicular, CNS)

[0952] 4. Known active CNS leukemia. Note: Patients with a history of CNS disease that has been effectively treated are eligible as long as their CNS symptoms are in remission for >4 weeks prior to enrollment

[0953] 5. Active hepatitis B or active hepatitis C

[0954] 6. HIV infection

[0955] 7. Active GVHD requiring systemic steroid therapy. Steroid replacement therapy is acceptable.

[0956] 8. Received DLI within 4 weeks after CD22 CART infusion

[0957] 9. Allo-SCT within 60 days after CD22 CART infusion

[0958] Research content

[0959] This is a Phase I study. This trial is divided into two phases. Dose escalation and dose expansion.

[0960] Patients received lymphodepleting chemotherapy followed by CD22 allogeneic CART.

[0961] Dose escalation: Four dose levels were studied in a standard 3x3 design. A total of 9-18 patients were enrolled.

[0962]

[0963] Once the R2PD level is determined, dose escalation is initiated.

[0964] Subsequently, a total of 20 patients (10 R / R ALL; 10 MRD+post-SCT) were enrolled.

[0965] Total sample size: 29-38 patients

[0966]

[0967] The second study compared allogeneic CART cells targeting CD22 with allogeneic CART cells targeting both CD22 and CD19 (2 forms) in patients with CD19+CD22+ B-ALL.

[0968] Target

[0969] Main Objectives

[0970] To evaluate the safety and tolerability of allogeneic CD19+CD22 CART and determine the maximum tolerated dose (MTD)

[0971] Secondary Objectives

[0972] Determining the effect of allogeneic CD22 CART

[0973] Determine the incidence of GVHD

[0974] Exploratory goals

[0975] Determine the expansion, phenotype, trafficking, and persistence of infused CAR T cells

[0976] Inclusion criteria

[0977] 1. Relapsed or refractory CD22-positive ALL (for expansion phase: patients with MRD+ disease are allowed)

[0978] 2. Patients aged ≥ 2 years

[0979] 3. ECOG performance status ≤ 2

[0980] 4. Normal organ function including bilirubin ≤2mg / dl, ALT / AST <3x ULN and creatinine ≤2mg / dl

[0981] 5. Left ventricular ejection fraction (LVEF) ≥ 40%

[0982] Exclusion criteria

[0983] 1. The patient is pregnant or breastfeeding

[0984] 2. Patients with uncontrolled active infection

[0985] 3. Isolated extramedullary recurrence (ie, testis, CNS)

[0986] 4. Known active CNS leukemia. Note: Patients with a history of CNS disease that has been effectively treated are eligible as long as their CNS symptoms are in remission for >4 weeks prior to enrollment

[0987] 5. Active hepatitis B or active hepatitis C

[0988] 6. HIV infection

[0989] 7. Active GVHD requiring systemic steroid therapy. Steroid replacement therapy is acceptable.

[0990] 8. Received DLI within 4 weeks after CD22 CART infusion

[0991] 9. Allo-SCT within 60 days after CD22 CART infusion

[0992] Research content

[0993] This is a Phase I study. This trial has two phases. Dose escalation and dose expansion.

[0994] Patients received lymphodepleting chemotherapy followed by CD22 allogeneic CART.

[0995] Dose escalation: Four dose levels are studied in a standard 3x3 design. A total of 9-18 patients will be enrolled.

[0996]

[0997] Once the R2PD level is determined, dose escalation is initiated.

[0998] Subsequently, a total of 20 patients (10 R / R ALL; 10 MRD+ after SCT) were enrolled.

[0999] Total sample size: 29-38 patients

[1000]

[1001]

[1002] References

[1003] 1. Inaba H, Greaves M, Mullighan CG. Acute lymphoblasticleukaemia. Lancet. 2013; 381(9881): 1943-55.

[1004] 2. Faderl S, Thomas DA, O'Brien S, Ravandi F, Garcia-Manero G, Borthakur G, et al. Augmented hyper-CVAD based on dose-intensified vincristine, dexamethasone, and asparaginase in adult acute lymphoblastic leukemia salvagetherapy. Clin Lymphoma Myeloma Leuk. 2011; 11(1): 54-9.

[1005] 3. Mullighan CG. Genome sequencing of lymphoid malignancies. Blood. 2013; 122(24): 3899-907.

[1006] 4. Mullighan CG. Genomic characterization of childhood acutelymphoblastic leukemia. Semin Hematol. 2013; 50(4): 314-24.

[1007] 5.Fielding AK,Richards SM,Chopra R,Lazarus HM,Litzow MR,Buck G,etal.Outcome of 609 adults after relapse of acute lymphoblastic leukemia(ALL);an MRC UKALL12 / ECOG 2993 study.Blood.2007;109(3):944-50.

[1008] 6.Tavernier E,Boiron JM,Huguet F,Bradstock K,Vey N,Kovacsovics T,etal.Outcome of treatment after first relapse in adults with acutelymphoblastic leukemia initially treated by the LALA-94 trial.Leukemia:official journal of the Leukemia Society of America,Leukemia Research Fund,U.K.2007;21(9):1907-14.

[1009] 7.Oriol A,Vives S,Hernandez-Rivas JM,Tormo M,Heras l,Rivas C,etal.Outcome after relapse of acute lymphoblastic leukemia in adult patientsincluded in four consecutive risk-adapted trials by the PETHEMA study Group.Haematologica.2010;95(4):589-96.

[1010] 8.Kochenderfer JN,Wilson WH,Janik JE,Dudley ME,Stetler-Stevenson M,Feldman SA,et al.Eradication of B-lineage cells and regression of lymphoma ina patient treated with autologous T cells genetically engineered to recognizeCD19.Blood.2010;116(20):4099-102.

[1011] 9.Porter DL,Levine BL,Kalos M,Bagg A,June CH.Chimeric antigenreceptor-modified T cells in chronic lymphoid leukemia.N Engl J Med.2011;365(8):725-33.

[1012] 10.Brentjens RJ,Davila ML,Riviere l,Park J,Wang X,Cowell LG,etal.CD19-targeted T cells rapidly induce molecular remissions in adults withchemotherapy-refractory acute lymphoblastic leukemia.Sci Transl Med.2013;5(177):177ra38.

[1013] 11.G rupp SA,Kalos M,Barrett D,Aplenc R,Porter DL,Rheingold SR,etal.Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.NEngl J Med.2013;368(16):1509-18.

[1014] 12.Maude SL,Teachey DT,Porter DL,Grupp SA.CD19-targeted chimericantigen receptor T-cell therapy for acute lymphoblastic leukemia.Blood.2015;125(26):4017-23.

[1015] 13.Park JH,Geyer MB,Brentjens RJ.CD19-targeted CAR T-celltherapeutics for hematologic malignancies:interpreting clinical outcomes todate.Blood.2016;127(26):3312-20.

[1016] 14.Lee DW,Kochenderfer JN,Stetler-Stevenson M,Cui YK,Delbrook C,Feldman SA,et al.T cells expressing CD19 chimeric antigen receptors for acutelymphoblastic leukaemia in children and young adults:a phase 1 dose-escalation trial.Lancet.2015;385(9967):517-28.

[1017] 15.Kochenderfer JN,Dudley ME,Kassim SH,Somerville RP,Carpenter RO,Stetler-Stevenson M,et al.Chemotherapy-refractory diffuse la rge B-celllymphoma and indolent B-cell malignancies can be effectively treated withautologous T cells expressing an anti-CD19 chimeric antigen receptor.J clinOncol.2015;33(6):540-9.

[1018] 16. Kebriaei P, Singh H, Huls MH, Figliola MJ, Bassett R, Olivares S, et al. Phase l trials using Sleeping Beauty to generate CD19-specific CAR Tcells. J Clin lnvest. 2016;126(9):3363-76.

[1019] Example

[1020] Example 1: Proliferation of TCRα-inactivated cells expressing CD22 CAR.

[1021] Heterodimer TALE-nucleases targeting 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 were designed and produced. The repeat sequences of the half-TALE-nucleases listed in Table 6 recognize each half-target.

[1022] Table 6: TAL nucleases targeting TCRα gene

[1023]

[1024]

[1025] Each TALE-nuclease construct was subcloned into a mammalian expression vector under the control of a T7 promoter using restriction digestion. mRNA encoding the TALE-nuclease that cleaves the TRAC genomic sequence was synthesized from a plasmid carrying the coding sequence downstream of the T7 promoter.

[1026] Purified T cells preactivated for 72 hours with anti-CD3 / CD28 coated magnetic beads were transfected with each of the two mRNAs encoding the two halves of the TRAC_T01 TALE nuclease. 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 was purified with anti-CD3 magnetic beads. NEG Cells were reactivated with soluble anti-CD28 (5 μg / ml) 5 days after transduction.

[1027] Cell proliferation was followed for up to 30 days after reactivation by counting cells twice weekly. Proliferation in TCRα-inactivated cells expressing the CD22 CAR was comparable to that of non-inactivated cells and was particularly increased upon reactivation with anti-CD28.

[1028] Multiple transfection of TALEN mRNA

[1029] Activated T cells were transfected with mRNA encoding TRAC TALEN (left and right) and mRNA encoding a TALEN pair specific for the B2M gene and subsequently transduced with exogenous polynucleotides encoding a CAR specific for CD22 or a CAR specific for CD19 or both.

[1030] Double KO TRAC and B2M or CD56 genes expressed undetectable levels of TCR and MHCI or TCR and CD56. After purification, the cells were transduced again.

[1031] Example 2: CD22 CAR-T

[1032] Development of engineered CAR T cells targeting CD22 for the treatment of refractory, relapsed, or aggressive ALL or CLL.

[1033] CD22 CAR: Figure 2 )

[1034] CD22 CARs were designed and prepared using different scFvs as constructs of M971 and M971 variants with reduced affinity for CD22 and high selectivity. M971 scFv was 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.2013Feb 14;121(7):1165-74.doi:10.1182 / blood-2012-06-438002.Epub 2012Dec 14)

[1035] The construct has a CAR architecture with a 4-1BB co-stimulatory domain, a CD3ζ activation domain, a CD8α transmembrane domain, and a hinge CD8α hinge (SEQ ID NO: 15) ( Figure 2 ). The construct comprises the FcγRIIIα hinge corresponding to SEQ-ID N°14.

[1036] The construct is inserted into a vector for stable expression and screening of the designed CAR.

[1037] CD22 CAR is m971-V3 CAR (SEQ ID NO.22); and SEQ ID N°24.

[1038] Sequences may also be optimized for CD22 binding and treatment of ALL and CLL, preferably their refractory relapsed and aggressive forms.

[1039] CAR expression

[1040] CD22 CAR was introduced into primary KO TCR T cells after 5 days of activation by anti-CD3CD28 coated magnetic 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 refractory or relapsed CD22+ B ALL from patients.

[1041] To test the functionality of anti-CD22 CAR, CD22-expressing B cells were used, using ALL cell lines REH, SEM, NALM6-GL, KOPN8, Daudi, Raji, and K562 (for experimental details, see Haso et al., 2013, 2013; Blood: 121(7): 1165–1174). Refractory or relapsed CD22+ B ALL was obtained from patients.

[1042] As expected, all cells expressing CD22 were positively stained and targeted by the CD22CAR from m971 of the present invention, as was the CD22 CAR (scfv2 from HA22).

[1043] Threshing test

[1044] To validate the CD22 CAR construct, a degranulation assay was performed on target cells with T cells expressing the CD22 CAR of the invention. CART degranulation was assessed by flow cytometry. The readout was the expression of CD107a on the plasma membrane of T cells after 5 hours of incubation with target cells. The results showed that CARm971-(scfv-1) degranulation was more important than CD22 CAR T cells targeting the distal portion of CD22 (SEQ ID 20) scfv-2. ( Figure 5 ).

[1045] Cytotoxicity assay

[1046] Cytotoxicity tests were performed on these same target cells using T cells expressing CD22 CAR of the present invention. Compared with cells expressing scFv against the distal part of CD22 (scFv2), CD22 CAR showed strong specific lysis of CD22 cells by UCART22 of the present invention ( Figure 6 ).

[1047] Interferon gamma test

[1048] Interferon-γ production by UCART22 (scFv-V1 targeting the proximal domain of CD22) cells in the presence of CD22-positive NALM-16 cells was increased compared with T cells that were not transduced (NT) or transduced with a CAR targeting the distal part of CD22 (scFv-V2) compared with CD22-negative SUP-T1 cells ( Figure 7 ).

[1049] Mouse survival

[1050] In the presence of UCART22 (scFv-V1 targeting the proximal domain of CD22) or CART22 (without TRAC inactivation), the survival of mice was improved compared with control cells ( Figure 8 ).

[1051] Tolerance to hypoxia and / or drugs

[1052] The engineered cells UCART22 of the present invention were not significantly affected (survival and CTL activity) in the presence of alemtuzumab (50 μg / mL) or PNA (flu) compared to non-engineered cell-sensitive cells, which died 48 hours after the addition of the drug to the cell culture or after culture under hypoxic conditions (less than 5%, preferably less than 1% O2).

[1053] Testing under hypoxic conditions (<5% or <1%) produced similar results and confirmed that UCART22 with increased HIF-1a expression can survive, express the CD22 CAR and are active under hypoxic conditions.

[1054] Similar results (survival rate, CTL activity) were obtained in mice treated with Campath (50 micrograms / mL), confirming the tolerance of UCART22 to the drug. It is shown that the UCART22 cells of the present invention may touch cancer cells nested in tissues or cancer cells forming clusters in the body, because the number of cancer cells that "recover" or "escape" UCART22 treatment is much less than that in mice treated with O2-intolerant UCART22 (about 15%). Therefore, it seems that local hypoxia caused by liquid tumors may prevent immune cells from attacking them.

[1055] Experiments were performed to determine whether UCART22 cells could be eliminated if desired. Figure 3 and Figure 4 ). The increased fluorescence intensity of tumor cells from mice treated with rituximab and UCART compared to cells alone indicated that rituximab caused a significant reduction in UCART in mice.

[1056] The first clinical data obtained showed that UCAR T cells significantly reduced relapsed and refractory ALL both in vivo and in vitro, with no or very mild (grade 1) GVHD and mild to no uncontrolled cytokine storm.

[1057] This treatment has a lower “toxicity” than autologous CD22 CAR T and can be managed with rituximab and / or QBEN 10 in patients.

[1058] The cells reside in the human body long enough to remain active (more than a month) and can be depleted using QBEN 10.

[1059] Examples of prepared CD22 CAR polypeptide sequences

[1060] The sequences in the reading frame correspond to the preferred VH and VL sequences. As described above, VH and VL can be swapped (modified in hot spots) to improve CAR efficiency

[1061] v1-m972 (FcγRIIIα-CD8αTM-41BB.IC-CD3ζ.IC) (control is not part of the present invention)

[1062]

[1063] V3-m972 (CD8α-CD8αTM-41BB.IC-CD3ζ.IC) (control is not part of the present invention)

[1064]

[1065] ·v1-m971(FcγRIIIα-CD8αTM-41BB.IC-CD3ζ.IC)

[1066]

[1067] ·v3-m971(CD8α-CD8αTM-41BB.IC-CD3ζ.IC)

[1068]

[1069]

[1070] result

[1071] The CAR construct selected for preparation in UCART22 contains the m971 anti-human CD22 scFv (described in Haso et al., 2013), the CD8α hinge (optional) and transmembrane domain, and a cytoplasmic tail consisting of a 4-1BB co-stimulatory domain and a CD3ζ signaling domain ( Figure 3 ).

[1072] Another CAR construct sequentially contains the m971 anti-human CD22 scFv sequence, two binding domains for rituximab (CD20), the CD8α hinge and transmembrane domains, and a cytoplasmic tail consisting of a 4-1BB co-stimulatory domain and a CD3ζ signaling domain ( Figure 3 ).

[1073] An alternative CAR construct comprises, in sequence, a binding domain for rituximab (CD20), a binding domain for Q-BEN10 (CD34), an m971 anti-human CD22 scFv, two binding domains for rituximab (CD20), a CD8α hinge and transmembrane domain, and a cytoplasmic tail consisting of a 4-1BB costimulatory domain and a CD3ζ signaling domain ( Figure 3 ).

[1074] In UCART22, a recombinant third-generation self-inactivating (SIN) lentiviral vector (rLV) was used to generate T cells expressing an anti-CD22 CAR and RQR8 depletion mechanism under the control of the human EF1α promoter ( Figure 4 ). Recombinant lentiviral vectors derived from HIV are genetically stable, and no evidence of pathological consequences attributed to this vector has been shown (Chang and Sadelain, 2007; Wang et al., 2009). In addition, conditions for transducing T cells with rLV are known and are compatible with preserving the functional properties of T cells.

[1075] In UCART22, AAV6 vectors ( Fig. 9 ).

[1076] Variants of the anti-CD22 CAR constructs of the present invention comprising additional CD19 scfv (before or after CD22 scfv) were also prepared, as well as multi-chain CD22 CAR, multi-chain CD22, CD19 CAR ( Figure 2) and insert it into a lentiviral vector adeno-associated virus 6 (AAV6) vector, so that the CD22 CAR construct or CD19 CAR construct to be inserted into the TRAC gene or CD25 gene or β2 microglobulin gene described below is prepared (the CAR sequence is located on the 5' and 3' sides of the sequence, so that the gene can be specifically cut using a specific TAL-protein-Fok-1 (TALEN) and then homologously recombined into the TRAC gene, CD25 gene or β2 microglobulin gene.

[1077] UCART22 that further comprises a TGFβ 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 is prepared.

[1078] Examples were prepared in which each UCART22 further contained a TGFβ 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.

[1079] To test the effect of depleting CD19+CD22+ cancer cells, an anti-CD19 construct previously described in "Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR Tcells. Waseem Qasim, et al., 2017. Science Translational Medicine 25 Jan 2017)" and also containing a suicide gene was prepared, which is different from the suicide gene carried by CD22 (as described in WO2016120216A1). In this study, the cell lysis activity of the following components was measured separately with a single cell

[1080] 1) The combination of UCART19+UCART22 (two single-chain CARs) compared with a single cell type alone (5x106 cells each),

[1081] 2) TCR-negative cells expressing bispecific anti-CD22 anti-CD19 CAR, (bispecific single-chain CAR), compared to single cells alone (all 5x106 cells),

[1082] 3) or a multi-chain CAR comprising both CD22scFv and CD19 scFv or CD19 CD22scFv.

[1083] Expression of RQR8, R2CD22 CAR, or QR3CD22 CAR to confer sensitivity to rituximab / rituximab and QBEN10

[1084] A lentiviral vector cassette driving CD22 CAR expression has been designed to co-express RQR8 (via a 2A peptide linker). RQR8 is an exhaustion ligand that can be effectively activated in uncontrollable adverse events associated with UCART22 administration. RQR8 is an artificial cell surface protein of 136 amino acids that combines antibody binding epitopes from human CD34 (recognized by QBEnd-10 antibodies) and human CD20 (Philip et al., 2014). The CD20 epitope present in the construct is recognized by rituximab, so cells expressing RQR8 can be deleted by complement-mediated cell killing (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of rituximab and / or QBEN 10.

[1085] In other experiments, constructs of anti-CD22 CARs were prepared containing 2 epitopes recognized by rituximab (R) (R2 CD22 CAR: two epitopes recognized by rituximab, inserted immediately after the scFv and immediately before the hinge, or replacing the hinge to shorten the extracellular domain compared to the entire structure with a hinge) or "QR3" QR3 CD22 CAR (Q: epitope recognized by QBEN 10, QR, N-terminus before the VH-linker-VL RR hinge TM-41BB-CD3ζ) and tested against cells expressing various levels of CD22.

[1086] TRAC knockout to limit alloreactivity in UCART22

[1087] A potential limitation of allogeneic adoptive immunotherapy is that differences in MHC recognition by the TCRαβ complex of donor cells between donor and recipient may lead to donor T cell activation / proliferation and the development of graft-versus-host disease (GvHD). TCRαβ consists of α and β subunits, with TCRα encoded by a single gene and TCRβ encoded by two homologous genes.

[1088] UCART22 cells are genetically modified to specifically disrupt the T cell receptor alpha constant (TRAC) gene. Inactivation of the TRAC gene prevents the TCRαβ complex from being expressed on the cell surface, thereby eliminating TCR-mediated recognition of tissue compatibility antigens, which can lead to GvHD. At the end of the UCART22 manufacturing process, the remaining TCRαβ+ cells will be depleted according to the release criteria, ensuring that the residual level of TCRαβ+ cells in the final product is ≤3.0%.

[1089] CD52 knockout to confer tolerance to alemtuzumab

[1090] Engineering UCART22 into CD52 - and CD52 + The mixture of cells allows the potential use of alemtuzumab in a lymphodepletion regimen prior to administration of UCART22. Alemtuzumab is a monoclonal antibody that binds to human CD52. Therefore, the CD52 of UCART22 - Some are resistant to alemtuzumab.

[1091] Technology is used to disrupt the TRAC and CD52 genes. During the production of UCART22, an electroporation system is used to disrupt the TRAC and CD52 genes. This transient expression allows Effective targeted gene knockout is performed before cells degrade it. This approach prevents potential risks associated with long-term expression of nucleases in cells infused into patients.

[1092] B2M knockout to limit alloreactivity of UCART22

[1093] Technology is used to disrupt the TRAC and B2M genes. During the production of UCART22, an electroporation system is used to disrupt the TRAC and B2M genes. is introduced into cells as messenger RNA (mRNA). This transient expression allows Perform efficient targeted gene knockout before being degraded by the cell.

[1094] Example

[1095] Sources of T cells used in nonclinical studies (pharmacology and toxicology)

[1096] Descriptions of the cell products used in the nonclinical studies are summarized in the Table.

[1097] Table C Engineered T cells used for in vitro and in vivo preclinical studies.

[1098]

[1099] DKO: double KO, KO: knockout, rLV: recombinant lentiviral vector

[1100] MHC I: major histocompatibility complex class I

[1101] B2m: beta 2 microglobulin

[1102] Summarize

[1103] Activation of UCART22 is driven by engagement between the anti-CD22 chimeric antigen receptor (CD22 CAR) and the CD22 antigen, leading to the destruction of CD22+ B-ALL cells through T cell-mediated cytotoxicity and possible production of proinflammatory cytokines.

[1104] Proof-of-concept studies conducted during the development of other UCART products (UCART19 and UCART123) demonstrated that UCART cells (TCRαβ-deficient CAR T cells) were as active as non-edited CAR T cells (TCRαβ-positive CAR T cells) in vitro and in vivo in a 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.

[1105] The in vitro pharmacology studies conducted during the nonclinical development of UCART22 are summarized in Table 2. The studies demonstrated that:

[1106] - Antitumor activity of UCART22 as tested by CD22 antigen-dependent cytotoxicity and cytokine secretion. Activity was tested against B-ALL cell lines and primary B-ALL samples.

[1107] -Effective Mediated inactivation of TRAC and CD52 genes.

[1108] -Effective Mediated inactivation of TRAC and β2-microglobulin genes.

[1109] -The CD52- T cells are resistant to alemtuzumab.

[1110] - Effective elimination of RQR8+ cells or R2 CD22 CAR+ cells using rituximab.

[1111] Table D: List of in vitro pharmacology studies.

[1112]

[1113]

[1114] CDC = complement-dependent cytotoxicity, PHA = phytohemagglutinin.

[1115] In vivo pharmacological studies (summarized in the table) demonstrated that:

[1116] - In vivo antitumor activity of UCART22 against tumor xenografts in immunodeficient mice using B-ALL cell lines. The in vivo activity of UCART22 produced under GMP conditions has been demonstrated against B-ALL cell lines.

[1117] -Using B-ALL cell lines, UCART22 / 19 has improved in vivo antitumor activity against tumor xenografts in immunodeficient mice. In vivo activity of UCART22 / 19 and 19 / 22 produced under GMP conditions against B-ALL cell lines.

[1118] - The ability of rituximab to eliminate RQR8+ cells in a normal immune mouse model.

[1119] Table E List of in vivo studies

[1120]

[1121] To demonstrate the activity of UCART22 cells, CAR-driven cytotoxicity and cytokine secretion assays were performed against tumor cell lines and primary B-ALL cells.

[1122] Evaluation of UCART22 activity by cytotoxicity assay

[1123] Cytotoxic activity against B-ALL cell lines

[1124] The potential cytotoxicity of UCART22 against several tumor cell lines from children or adult B-ALL patients was evaluated (Table F). The results showed that MHH-CALL-4, MUTZ-5, SEMK2, PALL-2, LAX2, BALL-1, NALM-6, and RS4;11 were 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.

[1125] Table F: Characteristics of cell lines.

[1126]

[1127] The specific cytotoxicity of UCART22 to CD22+ cells was assessed by detecting the viability of CD22+ target cells after co-culture with UCART22 cells. Untransduced (CAR-)TRAC and CD52 dual KO T cells were used as controls to calculate the specific cell lysis percentage of UCART22 to target CD22+B-ALL cells. The results show the cytotoxic activity of UCART22 cells to a group of CD22+B-ALL tumor cell lines. UCART22 shows specific cell lysis activity for all cell lines expressing CD22, even at the lowest levels.

[1128] Cytotoxic activity against primary B-ALL samples

[1129] The cytotoxic activity of UCART22 was confirmed against several primary B-ALL samples. The characteristics of the primary samples are listed in Table G. Only patient samples with greater than 50% blasts were used for co-incubation studies, which accounted for 14 of the 19 samples.

[1130] Table G All primary samples evaluated in preclinical studies. ND: Not Determined

[1131]

[1132]

[1133] CD22 and CD19 expression levels were also assessed in all primary B-ALL samples except Pt1. 11 CD22 surface expression was higher than the CD22 control, and 4 B-ALL patient samples showed CD22 surface expression within the range found in B-ALL cell lines (>1000 CD22 molecules / cell). With the exception of two relapse samples, those that highly expressed CD22 (>60%) also expressed CD19.

[1134] Using the same approach as for cell lines, CTL activity (greater than 10% specific cell lysis) was confirmed against eight B-ALL patient samples.

[1135] Cytokine secretion assay for B-ALL cell lines and primary B-ALL samples

[1136] The BioLegend Legend PLEX 13 cytokine assay was used to evaluate the release of interferon gamma (IFNγ) and other cytokines from UCART22 cells co-incubated with several cell lines or B-ALL patient samples. It was found that after 25 hours of co-incubation between B-ALL cells and UCART22 or control T cells (NTD DKO), IFNγ secreted into the culture medium had the highest concentration of all the cytokines tested (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)).

[1137] When UCART22 was co-incubated with B-ALL cell lines and primary B-ALL samples, high levels of interferon-γ secretion were observed.

[1138] In vitro auxiliary studies

[1139] Induced inactivation of TRAC, B2M and CD52 genes

[1140] The ability to use UCART22 in a homologous allogeneic setting depends on the ability to prevent TCRαβ and MHC CI from being expressed on the cell surface and to eliminate TCR-mediated histocompatibility antigen recognition (which may lead to GvHD and host CD8+ cell attack). To effectively eliminate TCRαβ and MHC CI from the cell surface, In addition, another to inactivate the CD52 gene, thereby allowing the use of alemtuzumab in lymphodepleting conditioning regimens. In certain settings, UCART22 was produced using To inactivate the β2 microglobulin gene (B2M) to alter the expression of major histocompatibility class I (MHC class I) molecules and prevent host T-cell rejection.

[1141] Molecular analysis of TRAC, CD52, and B2M gene disruption

[1142] In order to demonstrate the targeted modification of TRAC and CD52 genes at the molecular level, GMP-grade TRAC and CD52 mRNA or TRAC and B2M Genomic DNA was isolated from mRNA electroporated T cells, and TCRαβ-cell MHCCI- was purified. TRAC, B2M, and CD52 were amplified by polymerase chain reaction (PCR). The region near the cleavage site was analyzed by next-generation sequencing (NGS, Illumina). Before the expression of mRNA (day 6), cells were removed and subjected to the same analysis. This analysis showed that while control cells on day 6 did not show any modifications at the TRAC and CD52 loci, cells after A higher modification frequency was detected in treated T cells (Table).

[1143] Table H is performed at the TRAC, CD52 and B2M loci After cleavage, the percentage of non-homologous end joining (NHEJ).

[1144] The DNA of 3 different batches was confirmed by NGS. The percentage of T cells in all batches Control cells were sampled before mRNA electroporation.

[1145] Table H

[1146]

[1147] Further analysis of the sequences showed that The induced modifications were localized in small regions near the target site, and 93-97% of these modifications were deletions. These deletions were generally small, with an average of 97% being less than 150 bp and 81% being less than 50 bp.

[1148] Functional assays demonstrating TRAC gene disruption in UCART22

[1149] In order to prove the functional inactivation of TCRαβ, the ability of UCART22 cells to express activation markers such as CD25 and CD69 after phytohemagglutinin (PHA)-mediated TCR stimulation was analyzed. UCART22, which uses all GMP raw materials, recovers T cells before (TRAC) or after (UCART22) TCRαβ+ cell depletion. Within 24 hours, the cells were reactivated with 0.5 μg / mL PHA. The expression of activation markers (CD25 and CD69) was detected by flow cytometry. Although the control TRAC cells (65.2% TCRαβ-) upregulated CD25 and CD69, upregulation was not observed on UCART22 cells (98.9% TCRαβ-), confirming the absence of TCRαβ receptors in UCART22.

[1150] Tolerance of CD52-UCART22 cells to alemtuzumab

[1151] The ability to use the anti-CD52 monoclonal antibody alemtuzumab in lymphodepletion regimens depends on the effective clearance of CD52 glycoprotein from the cell surface of UCART22 cells to prevent their clearance by alemtuzumab. UCART22 was engineered to be a mixture of CD52+ and CD52- cells. Flow cytometric analysis of development batches of UCART22 at the end of the manufacturing process showed that an average of 72% of T cells (CD45+ / CD4+ or CD8+ cells) were CD52- (range 62.3-76.5%, N=6).

[1152] To demonstrate that CD52- cells are resistant to alemtuzumab, a complement-dependent cytotoxicity (CDC) assay was performed. Human T cells with PD-1-mediated CD52 gene inactivation were treated with 50 μg / mL of rat anti-CD52 antibody (from which the therapeutic antibody alemtuzumab is derived) or rat immunoglobulin G (IgG) as a control, in the presence or absence of rabbit complement at 37°C for 2 h, and then their viability and CD52 expression were analyzed by flow cytometry.

[1153] The results showed that cells without CD52 gene inactivation could be specifically eliminated by complement-dependent cytotoxicity in the presence of alemtuzumab.

[1154] Tolerance to host CD8+ T cells

[1155] MHC class I-deficient UCART22 cells are resistant to CD8+ T cell-mediated destruction, although interferon gamma (IFN-γ) treatment significantly induced β2-microglobulin expression and promoted CD8+ T cell-mediated killing of control UCART22 cells.

[1156] Demonstration of effective depletion of UCART22 after rituximab treatment

[1157] UCART22 is an allogeneic T cell and should therefore be cleared after the patient's immune system has recovered. In addition, UCART22 is engineered to co-express the CD22 CAR and RQR8, a short membrane protein that provides two epitopes that bind to rituximab (RTX), a therapeutic monoclonal antibody specific for human CD20. Therefore, RQR8 offers the possibility of depleting UCART22 by administering rituximab.

[1158] The ability to clear UCART22 cells by complement-dependent cytotoxicity (CDC) after treatment with RTX was studied. Frozen UCART22 (from 5 different development batches) were thawed and co-cultured with Raji CD22+ cells at a ratio of 1:0.25 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 rabbit complement (BRC). The results showed that approximately 85% of CAR+ cells were effectively cleared in vitro in the presence of both RTX and complement.

[1159] Another mechanism of action of RTX is antibody-dependent cell-mediated cytotoxicity (ADCC), through which depletion of RQR8+ cells can be achieved (Seidel et al., 2013). In this case, effector cells of the immune system (mainly natural killer cells-NK cells) can actively lyse target cells whose membrane surface antigens have been bound by specific antibodies.

[1160] Complementary experiments have been published by Martin Pule of UCL (Philip et al., 2014) demonstrating that CDC and ADCC effectively mediate the elimination of RQR8+ T cells (primary human T cells transduced with a bicistronic retroviral vector encoding RQR8 and GFP (SFG.RQR8.IRES.eGFP) and selected with Miltenyi QBEnd10 magnetic beads) in vitro. Time course and RTX dose titration demonstrated that CDC was very effective at RTX concentrations of 25 μg / mL and above, with cell killing occurring within 30 minutes. Likewise, ADCC-mediated sensitivity to RQR8+ T cells was demonstrated.

[1161] In vivo studies

[1162] Rationale for choice of animal species / model

[1163] Due to the human specificity of UCART22, studies in standard immunocompetent animal models are not applicable due to the rapid targeting and elimination of human UCART22 cells by xenogeneic immune responses.

[1164] Therefore, two approaches can be envisioned to establish in vivo models of UCART22 activity: syngeneic animal models and human tumor xenograft models in immunodeficient animals.

[1165] The syngeneic animal model approach would involve the reconstitution / redevelopment of a species-specific version of UCART22 for the model organism, followed by evaluation of the functional properties of the species-specific UCART22 replacement product when implanted into a syngeneic host. This approach is not considered relevant because the properties of individual CARs vary widely and because species-specific differences in immune function, physiology, and genetics greatly impair extrapolation of UCART22 activity in humans.

[1166] The immunodeficient mouse xenograft model was chosen because it allows the transplantation of UCART22 and human tumor CD22+ cells (B-ALL cell lines or primary samples). This animal model has been widely used to evaluate the in vivo activity of CAR T cell therapy. Although these models have the same limitations as the above-mentioned immune-normal models in terms of on-target / off-tumor cytotoxicity and off-target cytotoxicity, and completely eliminate any effects of MHC mismatching, they have been proven to be very useful for evaluating the in vivo anti-tumor efficacy of CAR. They have become the de facto standard for evaluating the in vivo activity of CAR T cell products (for example, see Carpenito et al., 2009 in many published articles; Gade et al., 2005; Gill et al., 2014; Hudecek et al., 2010; Kenderian et al., 2015; Mardiros et al., 2013; Zhou et al., 2013). They have demonstrated the ability to distinguish the relative anti-tumor activity of different CARs, highlighting their ability to capture and semi-quantitatively read out useful human T cell functions (e.g., see Milone et al., 2009). Thus, human CD22+ tumor cells have been transplanted into immunodeficient mice (NSG mice), followed by administration of UCART22, and serial assessment of cancer burden and survival time as indicators of anti-tumor activity.

[1167] Selected tumor models

[1168] Regarding tumor xenografts, the in vivo activity of UCART22 has been evaluated against Daudi cells, a CD22+ B-ALL cell line (CCL-213, ATCC) modified to express firefly luciferase (and GFP) via transduction with a lentiviral vector (amsbio LVP438-PBS) to allow tracking of cancer burden by in vivo imaging (Daudi-Luc-GFP cells).

[1169] Selected mouse strains

[1170] Mouse strains used in in vivo nonclinical studies were:

[1171] 1. Highly immunodeficient NSG mice (NOD.Cg-Prkdcscid II2rgtm1Wjl / SzJ; Jackson strain #5557), which lack mature T cells, B cells, and functional NK cells, were used to demonstrate antitumor activity.

[1172] 2. Immunologically competent mouse model (C57BL / 6x Balb / c(F1) mice) to demonstrate the effect of RQR8 depletion or R2CD22 CAR mechanism.

[1173] Analytical methods

[1174] The in vivo activity of UCART22 was evaluated using the following methods:

[1175] - Bioluminescence measurements of treated mice relative to controls (for xenograft models with cell lines expressing luciferase),

[1176] -Clinical signs

[1177] - Overall survival of mice.

[1178] Ways to Give

[1179] The route of administration of UCART22 in animals is intravenous injection (single undivided dose). In humans, UCART19 and UCART22 are also injected intravenously (undivided dose), individually once twice or three times or four times, or one after another in the following order: 19 22 19 22 19 or 22 19 22 19 or 19 / 22 19 / 22.

[1180] Demonstration of in vivo antitumor activity of UCART22

[1181] UCART22 activity was confirmed using multiple development batches of UCART22 in immunodeficient mice implanted with Daudi-Luc-GFP human tumor cells. Figure 8 An example of this study is shown. In addition, a confirmatory study is ongoing to evaluate the in vivo activity of GMP batches of UCART22.

[1182] Briefly, on day 7, NSG mice were injected intravenously with 0.5x10 6 Daudi-Luc-GFP cells and treated with UCART22 on day 0 (iv, 2 doses: 3x10 6 and 10x10 6UCART22 cells / mouse, 5 mice per group) or untreated (injected with vector). Double KO non-transduced T cells (NTD DKO) against TRAC and CD52 were injected as a control. The dose of UCART22 administered was determined based on the dose of CAR+ T cells used in the literature to demonstrate that other CD22 CAR T cells have anti-tumor activity in vivo (Haso et al., 2013). As shown in in vivo imaging, UCART22 treatment resulted in the clearance of tumor cells and prolonged survival, with all treated mice surviving at the end of the study (80 days after UCART22 treatment). These results demonstrate the anti-tumor activity of UCART22.

[1183] CD52- cells are tolerant to alemtuzumab in vivo, and CD52-CAR+ T cells show in vivo activity in the presence of alemtuzumab

[1184] Studies conducted during the development of UCART19, another UCART product containing the same TRAC / CD52 double knockout, demonstrated that CD52- T cells are resistant to alemtuzumab in vivo (Poirot et al., 2015).

[1185] In addition, the activity of UCART19 cells in the presence of alemtuzumab was demonstrated in a tumor xenograft model of NSG mice. All mice that received tumor cells but did not receive UCART19 cells showed tumor progression, resulting in being killed 13 days after injection. Of the 7 mice that received tumors and UCART19 cells, 5 had completely controlled tumors on the 13th day, while in the remaining two mice, partial responses were observed. In contrast, alemtuzumab treatment was observed to only delay tumor progression (6 / 6 mice) without infusion of UCART19 cells. In mice treated with alemtuzumab in combination with UCART19 cells, tumor cells were cleared from the bone marrow as assessed by photometry or flow cytometry of cell suspensions obtained from the bone marrow 13 days after infusion.

[1186] In conclusion, mice treated with alemtuzumab 2 days before infusion of UCART19 cells demonstrated the antitumor efficacy of UCART19 cells in vivo in the presence of alemtuzumab. In addition, spleen implant data at day 13 showed that CD52-T-cells were tolerant to alemtuzumab in vivo.

[1187] Demonstration of effective depletion of RQR8+ cells induced by rituximab in vivo

[1188] The sensitivity of RQR8+UCART22 to RTX depletion has been previously demonstrated in vitro. In addition, Martin Pule's group has demonstrated that RTX can eliminate RQR8+ cells in an immunocompetent mouse model using RTX reengineered to mouse IgG2a (functionally equivalent to human IgG1) (mRtx-IgG2a) (Philip et al., 2014). The antibody was adapted for use in the murine system by transferring the binding portion of RTX to the murine Fc, thereby binding to the murine Fc receptor with an affinity close to that of RTX binding to the human Fc receptor. The study was performed using an immunocompetent haploid adoptive transfer model, in which RQR8-transduced C57BL / 6 splenocytes were transferred to non-lethally irradiated C57BL / 6 x Balb / c hybrid (F1) recipients. This model enables the maintenance of good engraftment levels in all lymphoid tissues through allogeneic stimulation, and also preserves endogenous lymphocytes.

[1189] On day 1, 4 hours after pretreatment with 5 Gy X-ray irradiation, 1.5 × 10 6 RQR8+ donor splenocytes (transduced with retrovirus encoding the RQR8-2A-GD2CAR construct and purified using MiltenyiCD34 magnetic beads) were injected intravenously into F1 mice (C57BL / 6x Balb / c). On day 7, engraftment of donor cells in peripheral blood was confirmed by flow cytometry. Mice were treated 3 times with allosteric RTX (ritux-mIgG2a 150μg, intravenous injection through the tail vein on days 7, 10 and 12) or PBS. There were 5 mice in each cohort. 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 were observed in the spleen, bone marrow and blood, respectively, within 6 hours after treatment of mice with rituximab-mIgG2a.

[1190] The same study was also performed using the anti-CD19 CAR used in the UCART19 product, which is also an allogeneic engineered CAR T-cell product containing the same RQR8 construct as UCART22.

[1191] Re-administration

[1192] This study evaluated the antitumor activity of UCART22 given single or multiple injections in a CD22-expressing B-ALL tumor cell line (Daudi, which expresses high levels of CD22). Cells were transduced with a lentiviral vector to express GFP and firefly luciferase.

[1193] On day 7, tumor cells (0.5 × 10 6 Daudi-Luc-GFP) was injected intravenously into NSG mice (NOD.Cg-Prkdcscid II2rgtm1Wjl / SzJ, strain 005557, Jackson Laboratory). On day 0, mice were randomly divided into 11 groups with 6 mice in each group according to the bioluminescent signal and body weight measured on day -1.

[1194] 1, 2 or 3 UCART22 treatments were given (at 1 or 3 million CAR+ cells / mouse). Mice were injected intravenously with UCART22 on days 0+ / -10+ / -20.

[1195] The antitumor activity of UCART22 was evaluated by tracking tumor burden by bioluminescence imaging on days 1, 7, 14, 21, 28, and 35, and observing clinical signs and survival of mice.

[1196] Daudi model, with 1x 0 6 UCART22g cells / mouse treated

[1197] With 1x 10 6 Compared with mice treated once with 1x 10 6 Mice treated with UCART22 2 or 3 times showed better control of tumor progression and longer survival (60-day survival rate was 80% for 3 doses and 0% for 1 dose).

[1198] In one experiment, UCART19 was given intravenously to mice on day 30. In those mice, which remained alive, levels of cancer cells were below detectable levels on day 90.

[1199] Summarize

[1200] The activity of UCART22 has been demonstrated in vitro and in vivo against B-ALL cell lines and in vitro against primary B-ALL samples.

[1201] UCART22 is an allogeneic T cell and should therefore be cleared after the patient's immune system recovers. In addition, UCART22 is engineered to co-express CD22 CAR and RQR8, a short membrane protein that provides two epitopes that bind to rituximab (RTX). Therefore, RQR8 provides a method for depleting RQR8+UCART22 by administering rituximab in the event of uncontrolled UCART22-related toxicity (e.g., cytokine release syndrome (CRS) or GvHD) or before allo-HSCT. Using an immunocompetent mouse model and a variant version of rituximab, the effectiveness of in vitro clearance of RQR8+ cells was confirmed by CDC and ADCC assays. In addition, the efficiency of rituximab-induced RQR8+ cell clearance was confirmed in the blood, spleen, bone marrow, and lymph nodes using an immunocompetent mouse model and a variant version of rituximab.

[1202] Similar results were obtained using R2 or QR3 anti-CD22 CARs expressed in TCR-deficient MHC CI-deficient T cells.

[1203] Tolerance of CD52- cells to alemtuzumab was demonstrated in vitro and in vivo, allowing for the potential use of alemtuzumab in lymphodepletion regimens.

[1204] 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.

[1205] Other studies conducted to assess various risks are summarized in Tables I and J and described in detail in the following sections.

[1206] Table 1 List of in vitro toxicology studies performed.

[1207]

[1208]

[1209] Table J List of in vivo toxicology studies.

[1210]

[1211] During the development of the aforementioned UCART products, the potential risk of GvHD was evaluated in immunodeficient mice. This animal model allows for efficient engraftment of human T cells and has previously been shown to produce persistent xenogeneic GvHD after infusion of human PBMCs or T cells (Ali et al., 2012; Schroeder and DiPersio, 2011).

[1212] Off-target activity of CD22 CAR

[1213] GLP-compliant tissue hybridization reactivity studies to evaluate the CD22 specificity of the CD22 CAR showed no significant unexpected off-target binding between the scFv component of the CD22 CAR and human tissues, and Retrogenix screening is ongoing. In addition, published clinical data on patients treated with m971-derived CAR T cells showed no off-target toxicity (Fry et al., 2017).

[1214] Since CD22 is expressed on normal B cells, the risk of B-cell aplasia due to on-target / off-target activity of UCART22 can be expected as long as UCART22 persists.

[1215] GVHD in UCART22 CAR

[1216] The basic safety feature of the UCART22 product is whether the possibility of UCART22-mediated GvH reactions has been eliminated due to the destruction of the TRAC gene and the purification of TCRαβ-cells. This risk was evaluated during the development of the aforementioned UCART products (UCART19 and UCART123) in in vivo preclinical studies. GvHD signs were observed in all NSG mice infused with unmodified T cells, which were found to be more severe at the highest dose, but no treatment-related changes were observed in mice infused with UCART. So far, in the clinic, 4 of the 14 patients treated with UCART19 have developed mild to moderate skin GvHD (3 grade 1 and 1 grade 2) (Qasim et al., 2017), and R. Benjamin et W. Qasim presented data at the 2017 ASH meeting, and these GvHD can be controlled without systemic treatment.

[1217] CRS

[1218] Other potential safety risks related to the administration of immunotherapy products and underlying diseases have been identified: infusion-related reactions, cytokine release syndrome (CRS), tumor lysis syndrome, infection, and neurotoxicity. CRS is a common adverse event in immunotherapy, especially in CAR T-cell trials. In clinical trials of autologous anti-CD19 CAR T cells, the intensity of cytokine release observed is the result of a combination of several parameters, such as the dose of injected CAR T cells, the CAR (4-1BB / CD28) signaling domain, the level of CAR T-cell proliferation, and the patient's tumor burden on the day of administration. These risks were assessed in vivo in a model similar to a recently published model (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).

[1219] Recommended starting dose for FIH studies

[1220] Standard approaches to clinical dose determination are not applicable for CAR T-cell-based immunotherapies due to the limited relevance of animal models, the biodynamic nature of the product, and the immune adaptive mechanisms at play.

[1221] Therefore, the selection of the starting clinical dose was mainly based on the following aspects: (i) the experience gained from the previous administration of CAR T cells in humans (the literature on autologous CART cells); (ii) loving cases and patients treated in ongoing clinical studies with UCART19 at University College London (UCL) and King's College London (KCL) (reference (Qasim et al., 2017) and R. Benjamin and W. Qasim's report at the 2017 ASH meeting) and (iii) doses currently studied or approved in other B-ALL autologous CAR studies. The method is described in the clinical section.

[1222] Preliminary results from clinical studies have shown that in patients treated with UCART22, tumor masses shrank significantly (>80%) in both trials. In addition, the data suggest that even short but effective treatment (starting with two doses) can interrupt and get rid of malignant B cells (73% remission at 150 days), especially after re-dosing and / or use of UCART22 and 19.

Claims

1. An engineered human T cell that is T cell receptor knockout and expresses a CD22-specific chimeric antigen receptor and a safety switch on its surface, The CD22-specific chimeric antigen receptor consists of: i) The extracellular domain, which consists of: - the hinge domain from CD8α of SEQ ID No. 4, - an antigen-binding domain specific for CD22 consisting of a heavy chain variable fragment of SEQ ID No. 12, a linker of SEQ ID No. 10 and a light chain variable fragment of SEQ ID No. 13, ii) the transmembrane domain from CD8α of SEQ ID No. 6, and iii) an intracellular signaling domain consisting of a CD3ζ signaling domain of SEQ ID No. 9 and a co-stimulatory domain from 4-1BB of SEQ ID No. 8, The safety switch comprises: - RQR8 transiently linked to the CD22-specific chimeric antigen receptor by peptide 2A, wherein the RQR8 consists of and is arranged from N-terminus to C-terminus: a rituximab mAb specific epitope, a QBEN-10 mAb specific epitope, a rituximab mAb specific epitope, a hinge domain from CD8α, and a transmembrane domain from CD8α; or - at least two rituximab mAb specific epitopes, or 3 rituximab mAb specific epitopes or 3 rituximab mAb specific epitopes and a QBEN-10 mAb specific epitope linked to said CD22-specific chimeric antigen receptor.

2. An engineered human T cell according to claim 1, wherein a polynucleotide sequence consisting of the polynucleotide sequence of SEQ ID No. 11 is inserted into the genome and further comprises an inactivated TRAC gene with an insertion, deletion or mutation within SEQ ID No. 18, and having an undetectable level of T cell receptors on the cell surface as measured by flow cytometry, and an undetectable level of off-site events as measured by guide sequence technology.

3. The engineered human T cell according to claim 2, wherein The polynucleotide sequence is inserted into the TRAC gene of SEQ ID No.

18.

4. The engineered human T cell according to claim 1, comprising another inactivated gene selected from the group consisting of dCK gene, B2M gene, and CD52 gene.

5. The engineered human T cell according to claim 4, wherein the other inactivated gene is the CD52 gene.

6. The engineered human T cell of claim 4, wherein at least one additional gene is inactivated, the gene being selected from genes encoding: aryl hydrocarbon receptor, transforming growth factor beta receptor, interleukin 10 receptor, programmed cell death protein 1, and combinations thereof.

7. The engineered human T cells of claim 1, comprising an additional scFv specific for a tumor-associated surface antigen selected from the group consisting of CD19, CD20, CD30, a major histocompatibility complex molecule, an immunoglobulin, CD3, CD5, CD34, CD79, CD138, B7-1, BCMA, FLT-3, or PAX5.

8. The engineered human T cell according to claim 7, wherein The additional scFv is specific for CD79b.

9. The engineered human T cell according to claim 7, wherein The additional scFv is specific for CD19.

10. The engineered human T cell of claim 1, wherein the CD22-specific chimeric antigen receptor further comprises an antigen binding domain specific for CD19, or wherein the engineered human T cell further comprises a CD19-specific chimeric antigen receptor.

11. The engineered human T cell according to claim 10, comprising a CD19-specific chimeric antigen receptor consisting of a polynucleotide sequence of SEQ ID No. 25 or SEQ ID No.

26.

12. A population of cells comprising the engineered human T cells according to any one of claims 1 to 11.

13. The cell population according to claim 12, comprising universal chimeric antigen receptor T cells expressing a CD19-specific chimeric antigen receptor on the cell surface, wherein the CD19-specific chimeric antigen receptor comprises the sequence of SEQ ID No. 25 or SEQ ID No.

26.

14. A pharmaceutical composition comprising the engineered human T cells according to any one of claims 1 to 11 or the population of cells according to claim 12 or 13 and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, further comprising bryostatin.

16. Use of the pharmaceutical composition according to claim 14 or 15 in the preparation of a medicament for preventing or treating a patient suffering from a CD22+ mediated cancer, wherein the CD22+ mediated cancer is B-cell acute lymphoblastic leukemia (B-ALL). 17 . The use according to claim 16 , wherein the medicament is used for treating relapsed or refractory CD22-positive B-cell acute lymphoblastic leukemia (B-ALL).

18. The use according to claim 16, wherein the medicament is used for treating relapsed or refractory CD22-expressing B-cell acute lymphoblastic leukemia (B-ALL) as a pediatric indication.

19. The use according to claim 16, wherein treating a patient comprises administering to the patient at least one monoclonal antibody in a dose that allows contact of the engineered human T cells with the at least one monoclonal antibody, the at least one monoclonal antibody being selected from QBEN 10 and rituximab.

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