Targeted gene integration of CRS inhibitor genes for improved immune cells therapy
Inventors
BUSSER, Brian • Duchateau, Philippe • Juillerat, Alexandre • Poirot, Laurent • Valton, Julien • SACHDEVA, Mohit
Assignees
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Publication Number
US-12221478-B2
Publication Date
2025-02-11
Expiration Date
Abstract
The invention pertains to the field of adaptive cell immunotherapy. It provides with the genetic insertion of exogenous coding sequence(s) into genetically engineered immune cells to prevent cytokine release syndrome to arise during the course of cell therapy. These exogenous coding sequences are more particularly soluble human polypeptides placed 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 relates to a method for preparing engineered immune cells for cell immunotherapy by introducing genome edits into T-cells using sequence-specific reagents that specifically target a PD-1 endogenous locus. An exogenous polynucleotide encoding a soluble form of GP130 is integrated into a human endogenous PD-1 gene locus, thereby eliminating expression of the PD-1 protein and placing soluble GP130 expression under control of the endogenous PD-1 promoter.
The method further integrates a nucleic acid encoding a CAR directed against a tumor antigen into the genome of the T-cells under the control of a constitutive promoter. Tumor cell engagement by the CAR induces expression and secretion of the soluble form of GP130 from the PD-1 promoter, while PD-1 protein expression is prevented by the integration of the exogenous polynucleotide.
Claims Coverage
The document provides one independent claim and centers on three inventive features: PD-1 locus integration of soluble GP130 under endogenous PD-1 promoter control, elimination of PD-1 protein expression, and constitutive integration of a tumor-antigen-directed CAR that induces soluble GP130 expression and secretion upon tumor cell engagement.
Pd-1 locus integration of soluble GP130 to eliminate PD-1 protein
Introducing into a proportion of T-cells at least one nucleic acid comprising an exogenous polynucleotide sequence expressing a soluble form of GP130 consisting of the amino acid sequence of SEQ ID NO:61, integrated into a human endogenous PD-1 gene locus and eliminating expression of the PD-1 protein, wherein expression of the soluble form of GP130 is under control of the endogenous PD-1 promoter.
Car integrated under constitutive promoter with tumor engagement–induced soluble GP130 secretion
Introducing at least one nucleic acid encoding a CAR directed against a tumor antigen integrated into the genome of the T-cells under the control of a constitutive promoter, wherein tumor cell engagement by the CAR induces expression and secretion of the soluble form of GP130 from the PD-1 promoter, while expression of PD-1 protein is prevented by the integration of the exogenous polynucleotide.
Sequence-specific PD-1 locus cleavage using targeted endonucleases
Introducing into a proportion of said T-cells by cleavage by at least one sequence-specific reagent selected from an RNA-guided endonuclease, a TAL-endonuclease, a zinc finger nuclease, a homing endonuclease, or any combination thereof that specifically targets the PD-1 endogenous locus.
Overall, the claim coverage centers on a coupled genetic design in which exogenous soluble GP130 is integrated into the human endogenous PD-1 locus to eliminate PD-1 protein expression while retaining regulation by the endogenous PD-1 promoter, and a tumor-antigen-directed CAR is integrated under a constitutive promoter so that CAR engagement drives soluble GP130 expression and secretion from the PD-1 promoter.
Stated Advantages
Tumor cell engagement by the CAR induces expression and secretion of the soluble form of GP130 from the PD-1 promoter while preventing expression of the PD-1 protein.
Documented Applications
Cell immunotherapy using engineered immune cells comprising T-cells.
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