Use of pre T alpha or functional variant thereof for expanding TCR alpha deficient T cells
Inventors
Galetto, Roman • Gouble, Agnes • Grosse, Stephanie • Mannioui, Cecile • Poirot, Laurent • Scharenberg, Andrew • Smith, Julianne
Assignees
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Publication Number
US-12577581-B2
Publication Date
2026-03-17
Expiration Date
Abstract
A method of expanding TCRalpha deficient T-cells by expressing pTalpha or functional variants thereof into said cells, thereby restoring a functional CD3 complex. This method is particularly useful to enhance the efficiency of immunotherapy using primary T-cells from donors. This method involves the use of pTalpha or functional variants thereof and polynucleotides encoding such polypeptides to expand TCRalpha deficient T-cells. Such engineered cells can be obtained by using specific rare-cutting endonuclease, preferably TALE-nucleases. The use of Chimeric Antigen Receptor (CAR), especially multi-chain CAR, in such engineered cells to target malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.
Core Innovation
The invention relates to adoptive immunotherapy using genetically engineered T cells rendered non-alloreactive and immunosuppressive-resistant by ex vivo targeted mutagenesis. Mutations are introduced to inactivate immunosuppressive targets, including CD52 and glucocorticoid receptor (GR), and the engineered cells are designed to avoid adverse immune interactions associated with those targets.
The invention further includes introduction of preT3 (pT3/preTCR3) or functional variants to restore a functional CD3 complex and enable expansion of TCR3-deficient cells. This supports retaining or re-establishing functional T-cell signaling associated with the CD3 complex while the targeted mutagenesis maintains immunosuppressive resistance.
In addition, the invention describes the use of multi-chain Fc5RI-derived CARs to redirect specificity to tumor or infected cells independently of MHC. The CAR architecture enables cytotoxic function following genome engineering, and the approach is supported by analysis of nuclease activity, functional outcomes, and off-target evaluation.
The disclosed engineering framework uses TALE-nuclease-mediated inactivation by targeted mutagenesis, with support for selecting TALE-nuclease targets and demonstrating nuclease mutagenesis activity and downstream functional effects. Off-target safety is addressed through deep sequencing evaluation, and multiple constructs, including pT3 variants and Fc5RI-based CAR formats, are reported for restoring function and enabling redirection of cell specificity.
Claims Coverage
The independent claim set in the provided material includes 1 independent claim. The main inventive features focus on ex vivo targeted mutagenesis to generate CD52-negative primary immunotherapy cells by introducing mutations in both alleles, with the engineered cell being a specified immune/stem cell type.
Ex vivo CD52-negative engineering by biallelic mutagenesis
A method of engineering a primary cell for immunotherapy by introducing mutations in the cell ex vivo by targeted mutagenesis to generate mutations in both alleles of the CD52 gene to render the cell CD52-negative.
Engineered cell type for immunotherapy
The method wherein the genetically modified cell is a stem cell, an iPS cell, a dendritic cell, an NK-cell, a B-cell, or a T-cell.
In the provided independent claim, the core coverage is directed to ex vivo, targeted mutagenesis that introduces mutations in both alleles of the CD52 gene to produce a CD52-negative immunotherapy cell, where the modified cell is selected from stem and multiple immune cell types.
Stated Advantages
Renders the genetically modified cell CD52-negative.
Documented Applications
Adoptive immunotherapy using engineered primary cells made CD52-negative and, in the disclosed description, designed to be non-alloreactive and immunosuppressive-resistant.
Redirecting specificity to tumor or infected cells using multi-chain Fc5RI-derived CARs independent of MHC.
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