Targeted gene integration of NK inhibitors genes for improved immune cells therapy
Inventors
BUSSER, Brian • Duchateau, Philippe • Juillerat, Alexandre • Poirot, Laurent • Valton, Julien
Assignees
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Publication Number
US-12209125-B2
Publication Date
2025-01-28
Expiration Date
Abstract
The invention pertains to the field of adaptive cell immunotherapy. It provides with the genetic insertion of exogenous coding sequence(s) that help the immune cells to direct their immune response against infected or malignant cells. These exogenous coding sequences are more particularly inserted under the transcriptional control of endogenous gene promoters that are sensitive to immune cells activation. Such method allows the production of safer immune primary cells of higher therapeutic potential.
Core Innovation
The invention provides a method for preparing at least 10^6 engineered primary T cells for cell immunotherapy. The method includes introducing into a proportion of primary T cells an exogenous polynucleotide sequence encoding an HLA-E heavy chain for targeted gene integration into the endogenous b22m locus, using at least one sequence-specific reagent that specifically targets the endogenous b22m locus.
The exogenous polynucleotide sequence encoding HLA-E heavy chain comprises SEQ ID NO:68 or SEQ ID NO:70. The integrated sequence is transcribed under control of an endogenous b22m promoter, generating engineered primary T cells with the exogenous polynucleotide sequence inserted into the endogenous b22m locus.
The disclosure further associates this concept with TALEN constructs and B2M-targeting matrices, including variants targeting B2M exons and an HLAE/HLAG fusion design element. It also describes CAR+/HLAE+ T-cell populations and related modifications in which B2M and TCR targeting are combined with insertion of an anti-CD22 CAR at the TRAC locus and an NK inhibitor fusion at the B2M locus.
Claims Coverage
The claim coverage centers on independent claims for targeted integration of an exogenous HLA-E heavy-chain polynucleotide into the endogenous b22m locus using a sequence-specific reagent targeting b22m, to generate at least 10^6 engineered primary T cells for cell immunotherapy. The inventive features include sequence-limited HLA-E heavy chain insertions, endogenous b22m promoter control, specified integration mechanisms, and CAR inclusion.
Targeted integration of an HLA-E heavy chain into the endogenous b22m locus
Introducing into a proportion of primary T cells an exogenous polynucleotide sequence encoding an HLA-E heavy chain for targeted gene integration into the endogenous b22m locus, wherein the exogenous polynucleotide sequence encoding HLA-E heavy chain comprises SEQ ID NO:68 or SEQ ID NO:70, and is inserted by targeted gene integration into the endogenous b22m locus.
Preparation of at least 10^6 engineered primary T cells with b22m-locus HLA-E insertion
Generating at least 10^6 engineered primary T cells with the exogenous polynucleotide sequence encoding an HLA-E heavy chain inserted into the endogenous b22m locus for cell immunotherapy.
Endogenous b22m promoter control of the integrated HLA-E heavy chain
The exogenous polynucleotide sequence encoding an HLA-E heavy chain is integrated at a locus so it is transcribed under control of an endogenous b22m promoter.
Targeted gene integration by homologous recombination or NHEJ
Integrating a targeted gene into primary T cells using homologous recombination or NHEJ.
Engineered primary T-cells further comprising a CAR
The engineered primary T-cells are endowed with a chimeric antigen receptor (CAR).
Overall, the claim coverage focuses on producing at least 10^6 engineered primary T cells by integrating an exogenous HLA-E heavy chain, limited to SEQ ID NO:68 or SEQ ID NO:70, into the endogenous b22m locus using a sequence-specific reagent, with additional refinements for promoter-linked transcriptional control, integration mechanism, and CAR inclusion.
Stated Advantages
Enhanced CAR T-cell persistence by knocking out B2M and inserting an HLA-E heavy chain into the endogenous b22m locus.
Assaying resistance to NK/alloreactive T-cell attack using the engineered T cells.
Enhance persistence of the engineered CAR+/HLAE+ T-cells.
Reduce graft-versus-host attack.
Reduce NK-cell mediated depletion.
Documented Applications
Use for assaying resistance to NK/alloreactive T-cell attack.
Cell immunotherapy using engineered primary T cells prepared with exogenous HLA-E heavy-chain integration at the endogenous b22m locus.
Simultaneous B2M and TCR targeting with insertion of an anti-CD22 CAR at the TRAC locus and an NK inhibitor fusion at the B2M locus to generate CAR+/HLAE+ T-cells.
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