Method for generating t-cells compatible for allogenic transplantation

Abstract

The present invention pertains to engineered T-cells, method for their preparation and their use as medicament, particularly for immunotherapy. The engineered T-cells of the invention are characterized in that the expression of beta 2-microglobulin (B2M) and/or class II major histocompatibility complex transactivator (CIITA) is inhibited, e.g., by using rare-cutting endonucleases able to selectively inactivating by DNA cleavage the gene encoding B2M and/or CIITA, or by using nucleic acid molecules which inhibit the expression of B2M and/or CIITA. In order to further render the T-cell non-alloreactive, at least one gene encoding a component of the T-cell receptor is inactivated, e.g., by using a rare-cutting endonucleases able to selectively inactivating by DNA cleavage the gene encoding said TCR component. In addition, expression of immunosuppressive polypeptide can be performed on those modified T-cells in order to prolong the survival of these modified T cells in host organism. Such modified T-cell is particularly suitable for allogeneic transplantations, especially because it reduces both the risk of rejection by the host's immune system and the risk of developing graft versus host disease. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer, infections and auto-immune diseases.

1 . A method for preparing an engineered T-cell comprising the steps of:
 a) providing a T-cell; and   b) inhibiting the expression of beta 2-microglobulin (B2M) and/or class II major histocompatibility complex transactivator (CIITA) in said T-cell by using a rare-cutting endonuclease able to selectively inactivate by DNA cleavage gene encoding said B2M and/or CIITA.   
     
     
         2 . The method according to  claim 1 , wherein step b) is performed by using a rare-cutting endonuclease able to selectively inactivate by DNA cleavage the gene encoding B2M. 
     
     
         3 . The method according to  claim 1 , wherein step b) is performed by using a rare-cutting endonuclease able to selectively inactivate by DNA cleavage the gene encoding CIITA. 
     
     
         4 . The method according to any one of  claim 1  to  claim 3 , wherein step b) is performed by using a TAL-nuclease, meganuclease, zing-finger nuclease (ZFN), or RNA guided endonuclease. 
     
     
         5 . The method according to  claim 4 , wherein step b) is performed using a TAL-nuclease. 
     
     
         6 . The method according to  claim 4 , wherein step b) is performed by using a RNA-guided endonucleases. 
     
     
         7 . The method according to  claim 6 , wherein the RNA-guided endonuclease is Cas9. 
     
     
         8 . The method according to any one of  claims 1  to  7 , further comprising the step of:
 c) inactivating at least one gene encoding a component of the T-cell receptor (TCR). 
 
     
     
         9 . The method according to  claim 8 , wherein step c) is performed by using a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably double-strand break, at least one gene encoding a component of the T-cell receptor (TCR). 
     
     
         10 . The method according to  claim 9 , wherein the rare-cutting endonuclease is a TAL-nuclease, meganuclease, zing-finger nuclease (ZFN), or RNA guided endonuclease like Cas9/CRISPR. 
     
     
         11 . The method according to any one of  claims 8  to  10 , wherein the component of the TCR is TCR alpha. 
     
     
         12 . The method according to any one of  claims 1  to  11 , further comprising the step of:
 d) introducing into said T-cell an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell. 
 
     
     
         13 . The method according to  claim 12 , wherein said Chimeric Antigen Receptor is directed against the B-lymphocyte antigen CD19. 
     
     
         14 . The method according to  claim 12 , wherein said Chimeric Antigen Receptor is directed against an antigen selected from a cluster of differentiation molecule, such as CD16, CD64, CD78, CD96,CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial nnucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), multiple myeloma or lymphoblastic leukaemia antigen, such as one selected from TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), and FCRL5 (UNIPROT Q68SN8), a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen as well as any derivate or variant of these surface antigens. 
     
     
         15 . The method according to any one of  claims 1  to  14 , further comprising the step of :
 d′) expressing at least one non-endogenous immune-suppressive polypeptide. 
 
     
     
         16 . The method according to  claim 15 , wherein said non-endogenous immune-suppressive polypeptide is a viral MHC homolog. 
     
     
         17 . The method according to  claim 16 , wherein said viral MHC homolog is UL18. 
     
     
         18 . The method according to any one of  claims 15  to  claim 17 , wherein the non-endogenous immune-suppressive polypeptide comprises an amino acid sequence sharing at least 80%, preferably at least 90% and more preferably at least 95% of identity with SEQ ID NO: 89. 
     
     
         19 . The method according to  claim 15 , wherein said non-endogenous immune-suppressive polypeptide is a NKG2D ligand. 
     
     
         20 . The method according to  claim 15  or  19 , wherein the non-endogenous immune-suppressive polypeptide comprises an amino acid sequence sharing at least 80%, preferably at least 90% and more preferably at least 95% of identity with any one of SEQ ID NO: 90-97. 
     
     
         21 . The method according to any one of  claims 1  to  20 , further comprising the step of:
 e) expanding the resulting engineered T-cell. 
 
     
     
         22 . The method according to any one of  claims 1  to  21 , wherein said T-cell in step a) is derived from an inflammatory T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte or a helper T-lymphocyte. 
     
     
         23 . The method according to  claim 22 , wherein said T-cell is derived from a CD4+ T-lymphocyte or a CD8+ T-lymphocytes. 
     
     
         24 . An engineered, preferably isolated, T-cell, wherein said T-cell expresses a rare-cutting endonuclease able to selectively inactivate by DNA cleavage the gene encoding B2M. 
     
     
         25 . An engineered, preferably isolated, T-cell, wherein said T-cell expresses a rare-cutting endonuclease able to selectively inactivate by DNA cleavage the gene encoding CIITA. 
     
     
         26 . The engineered T-cell according to  claim 24  or  25 , wherein said T-cell comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding said rare-cutting endonuclease. 
     
     
         27 . The engineered T-cell according to  claim 26 , wherein said rare-cutting endonuclease is a TAL-nuclease, meganuclease, zing-finger nuclease (ZFN), or RNA guided endonuclease. 
     
     
         28 . The engineered T-cell according to  claim 27 , wherein said rare-cutting endonuclease is a TAL-nuclease. 
     
     
         29 . The engineered T-cell according to  claim 27 , wherein said rare-cutting endonuclease is a RNA-guided endonucleases. 
     
     
         30 . The engineered T-cell according to  claim 29 , wherein the RNA-guided endonuclease is Cas9. 
     
     
         31 . The engineered T-cell according to any one of  claims 26  to  30 , wherein said nucleic acid is a vector allowing said rare-cutting endonucleases to be expressed by said T-cell. 
     
     
         32 . The engineered T-cell according to any one of  claims 26  to  30 , wherein said nucleic acid is a transfected mRNA. 
     
     
         33 . The engineered T-cell according to  claim 24 , wherein said T-cell comprises an exogenous nucleic acid molecule that inhibits the expression of B2M. 
     
     
         34 . The engineered T-cell according to  claim 34 , wherein the nucleic acid molecule comprises at least 10 consecutive nucleotides of the complement of SEQ ID NO: 3. 
     
     
         35 . The engineered T-cell according to  claim 25 , wherein said T-cell comprises an exogenous nucleic acid molecule that inhibits the expression of CIITA. 
     
     
         36 . The engineered T-cell according to  claim 35 , wherein the nucleic acid molecule comprises at least 10 consecutive nucleotides of the complement of SEQ ID NO: 5. 
     
     
         37 . The engineered T-cell according to any one of  claims 33  to  36 , wherein the nucleic acid molecule is an antisense oligonucleotide, ribozyme or interfering RNA (RNAi) molecule. 
     
     
         38 . The engineered T-cell according to any one of  claims 24  to  37 , further characterized in that at least one gene encoding a component of the TCR receptor is inactivated. 
     
     
         39 . The engineered T-cell according to  claim 38  wherein said T-cell expresses a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably double-strand break, said at least one gene encoding a component of the T-Cell receptor (TCR). 
     
     
         40 . The engineered T-cell according to  claim 39 , wherein said T-cell comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding said rare-cutting endonuclease. 
     
     
         41 . The engineered T-cell according to  claim 40 , wherein said said rare-cutting endonuclease is a TAL-nuclease, nneganuclease, zing-finger nuclease (ZFN), or RNA guided endonuclease. 
     
     
         42 . The engineered T-cell according to any one of  claims 24  to  41 , wherein said T-cell expresses a Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell. 
     
     
         43 . The engineered T-cell according to  claim 42 , wherein said T-cell comprises an exogenous nucleic acid molecule comprising a nucleotide sequence encoding said CAR. 
     
     
         44 . The engineered T-cell according to  claim 42  or  43 , wherein said CAR is directed against the B-lymphocyte antigen CD19. 
     
     
         45 . The engineered T-cell according to  claim 42  or  43 , wherein said CAR is directed against an antigen selected from a cluster of differentiation molecule, such as CD16, CD64, CD78, CD96,CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), multiple myeloma or lymphoblastic leukaemia antigen, such as one selected from TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), and FCRL5 (UNIPROT Q68SN8), a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen as well as any derivate or variant of these surface antigens. 
     
     
         46 . The engineered T-cell according to any one of  claims 24  to  45 , wherein said T-cell expresses at least one non-endogenous immune-suppressive polypeptide. 
     
     
         47 . The engineered T-cell according to  claim 46 , wherein said non-endogenous immune-suppressive polypeptide is a viral MHC homolog. 
     
     
         48 . The engineered T-cell according to  claim 47 , wherein said a viral MHC homolog is UL18. 
     
     
         49 . The engineered T-cell according to any one of  claims 46  to  48 , wherein said T-cell comprises an exogenous nucleic acid molecule comprising a nucleotide sequence coding fora polypeptide sharing at least 80%, preferably at least 90% and more preferably at least 95% of identity with SEQ ID NO: 89. 
     
     
         50 . The engineered T-cell according to  claim 46 , wherein said non-endogenous immune-suppressive polypeptide is a NKG2D ligand. 
     
     
         51 . The engineered T-cell according to  claim 46  or  50 , wherein said T-cell comprises an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a polypeptide sharing at least 80%, preferably at least 90% and more preferably at least 95% of identity with any one of SEQ ID NO: 90-97. 
     
     
         52 . The engineered T-cell according to any one of  claims 24  to  51 , wherein said T-cell is derived from an inflammatory T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte or a helper T-lymphocyte. 
     
     
         53 . The engineered T-cell according to  claim 52 , wherein said T-cell is derived from a CD4+ T-lymphocyte or a CD8+ T-lymphocytes. 
     
     
         54 . The engineered T-cell according to any one of  claims 24  to  53  for use as a medicament. 
     
     
         55 . The engineered T-cell according to any one of  claims 24  to  53  for use in the treatment of a cancer or viral infection. 
     
     
         56 . The engineered T-cell according to any one of  claims 24  to  53  for use in the treatment of lymphoma. 
     
     
         57 . The engineered T-cell according to any one of  claims 48  to  56 , wherein said T-cell originates from a patient to be treated. 
     
     
         58 . The engineered T-cell according to any one of  claims 48  to  56 , wherein said T-cell originates from a donor. 
     
     
         59 . A composition comprising at least one engineered T-cell according to any one of  claims 24  to  53 .