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-12252699-B2
Publication Date
2025-03-18
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.
Core Innovation
The invention provides engineered human T-cells expressing a Chimeric Antigen Receptor (CAR) in which surface expression of HLA-A, HLA-B, and HLA-C is lost. The loss of HLA surface expression is induced by selectively inactivating the gene encoding beta 2-microglobulin (B2M) using a rare-cutting endonuclease that cleaves the B2M gene in the human T-cells.
In particular, the rare-cutting endonuclease is a TALE-nuclease encoded by a polynucleotide having at least 90% identity with specified SEQ ID sequences. After cleaving the B2M gene, the engineered T-cells exhibit loss of surface expression of B2M, HLA-A, HLA-B, and HLA-C, and a CAR is introduced into the engineered T-cells, or the CAR is already present, depending on the method.
The disclosed approach is further supported by engineering strategies that target T-cell alloreactivity by inhibiting B2M and/or CIITA expression, and inactivating TCR genes to produce non-alloreactive allogeneic T-cells. Optional stealth modifications are described by expressing non-endogenous immunosuppressive polypeptides, including a viral MHC homolog UL18 or NKG2D ligand variants, to enhance persistence and reduce host rejection/GvHD risk.
Claims Coverage
The independent claims cover methods for preparing engineered human T-cells with CAR expression and lost surface expression of HLA-A, HLA-B, and HLA-C, using selective B2M gene inactivation by a TALE-nuclease defined by polynucleotide identity thresholds and then introducing or maintaining CAR expression. Across the independent claims, the inventive features focus on CAR-expressing human T-cells, rare-cutting endonuclease-mediated B2M inactivation to remove HLA surface expression, and a specific ordering option in which the CAR is introduced after HLA loss.
CAR-expressing T-cells with lost HLA-A/HLA-B/HLA-C surface expression
A method for preparing engineered human T-cells expressing a Chimeric Antigen Receptor (CAR) that have lost surface expression of HLA-A, HLA-B, and HLA-C.
Selective TALE-nuclease inactivation of B2M to induce loss of HLA surface expression
Introducing into said human T-cells a rare-cutting endonuclease able to selectively inactivate the gene encoding B2M, wherein said 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; and cleaving the B2M gene with said rare-cutting endonuclease in said human T-cells to induce loss of surface expression of B2M, HLA-A, HLA-B, and HLA-C.
CAR introduction after B2M/HLA inactivation
Providing a population of primary human T-cells; inactivating B2M using a TALE-nuclease to induce loss of surface expression of B2M, HLA-A, HLA-B, and HLA-C; and introducing a CAR into the human T-cells that have lost surface expression of B2M, HLA-A, HLA-B, and HLA-C.
Overall, the independent claims require engineered human T-cells with CAR expression and lost surface expression of HLA-A/HLA-B/HLA-C, achieved by TALE-nuclease-mediated cleavage and inactivation of the B2M gene using TALE-nuclease polynucleotides meeting specified identity relationships to named SEQ ID sequences, with one independent claim emphasizing the timing of introducing the CAR after B2M/HLA inactivation.
Stated Advantages
Enhance persistence.
Reduce host rejection/GvHD risk.
Produce non-alloreactive allogeneic T-cells.
Documented Applications
Allogeneic transplantation use cases are addressed through preparation of non-alloreactive engineered T-cells intended to reduce host rejection/GvHD risk.
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