Method for generating T-cells compatible for allogenic transplantation
Inventors
Poirot, Laurent • Sourdive, David • Duchateau, Philippe • Cabaniols, Jean-Pierre
Assignees
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Publication Number
US-11820996-B2
Publication Date
2023-11-21
Expiration Date
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.
Core Innovation
The invention relates to a therapeutic composition comprising engineered human T-cells expressing a Chimeric Antigen Receptor (CAR) that have lost surface expression of HLA-A, HLA-B, and HLA-C. At least one gene encoding beta 2-microglobulin (B2M) is inactivated in the T-cells, resulting in lack of surface expression of the HLA class I proteins.
The inactivation of the gene encoding B2M is achieved through expression in the T cells of a rare-cutting endonuclease able to selectively inactivate the gene encoding B2M. In particular, the rare-cutting endonuclease is a TALE-nuclease encoded by a polynucleotide having at least 90% identity with SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:87, or SEQ ID NO:88.
The engineered human T-cells also include additional immune-related features, including TCR loss and optional non-endogenous immunosuppressive polypeptides such as a viral MHC homolog and/or a NKG2D ligand. The approach is intended to address issues associated with T-cell allorecognition/proliferation when β2-microglobulin is absent, and to modulate NK-mediated lysis when decoy molecules are expressed in engineered T cells.
Claims Coverage
The claim coverage centers on one independent claim directed to engineered human CAR T-cells lacking surface expression of HLA-A, HLA-B, and HLA-C, together with B2M inactivation by a rare-cutting TALE-nuclease defined by sequence identity to specified SEQ ID NOs. The merged inventive features also include related dependent limitations involving TCR loss, optional immune-related polypeptides, and CAR target specificity.
Engineered CAR T-cells lacking HLA-A, HLA-B, and HLA-C surface expression
A therapeutic composition comprising engineered human T-cells expressing a Chimeric Antigen Receptor (CAR) that have lost surface expression of HLA-A, HLA-B, and HLA-C.
B2M gene inactivation using a rare-cutting endonuclease selectively inactivating B2M
At least one gene encoding beta 2-microglobulin (B2M) is inactivated through expression in the T cells of a rare-cutting endonuclease able to selectively inactivate the gene encoding B2M.
TALE-nuclease defined by at least 90% identity to specified SEQ IDs
The rare-cutting endonuclease inactivating the gene encoding B2M is a TALE-nuclease encoded by a polynucleotide having at least 90% identity with SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:87, or SEQ ID NO:88.
TCR loss and optional immune-related polypeptides
The engineered human T-cell phenotypes include TCR loss and optional non-endogenous immunosuppressive polypeptides such as a viral MHC homolog and/or a NKG2D ligand.
CAR targeting CD19
The therapeutic composition includes a chimeric antigen receptor (CAR) that targets the B-lymphocyte antigen CD19.
Overall, the claim coverage is directed to engineered human CAR T-cells whose HLA-A, HLA-B, and HLA-C surface expression is lost through B2M inactivation by a rare-cutting TALE-nuclease defined by sequence identity to specified SEQ ID NOs, with dependent limitations including TCR loss, optional viral MHC homolog and/or NKG2D ligand, and CD19 targeting.
Stated Advantages
Reduced T-cell allorecognition/proliferation when β2-microglobulin is absent.
Reduced NK-mediated lysis when decoy molecules are expressed in engineered T cells.
Documented Applications
Use of engineered CAR T-cells lacking B2M surface expression in co-culture settings to assess T-cell allorecognition/proliferation outcomes.
Use of engineered T cells expressing decoy molecules to reduce NK-mediated lysis, as assessed in NK cell-related functional assays.
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