US 12,221,478 B2
Targeted gene integration of CRS inhibitor genes for improved immune cells therapy
Brian Busser, New York, NY (US); Philippe Duchateau, Draveil (FR); Alexandre Juillerat, New York, NY (US); Laurent Poirot, Paris (FR); Julien Valton, New York, NY (US); and Mohit Sachdeva, New York, NY (US)
Assigned to CELLECTIS, Paris (FR)
Appl. No. 16/755,093
Filed by CELLECTIS, Paris (FR)
PCT Filed Apr. 16, 2018, PCT No. PCT/EP2018/059692
§ 371(c)(1), (2) Date Apr. 9, 2020,
PCT Pub. No. WO2019/076489, PCT Pub. Date Apr. 25, 2019.
Claims priority of application No. PCT/EP2017/076798 (WO), filed on Oct. 19, 2017; application No. PCT/EP2018/053343 (WO), filed on Feb. 9, 2018; and application No. PCT/EP2018/055957 (WO), filed on Mar. 9, 2018.
Prior Publication US 2020/0407694 A1, Dec. 31, 2020
Int. Cl. C07K 16/28 (2006.01); A61K 39/00 (2006.01); C07K 16/30 (2006.01); C12N 5/0783 (2010.01); C12N 5/10 (2006.01)
CPC C07K 16/2803 (2013.01) [A61K 39/4611 (2023.05); A61K 39/4631 (2023.05); A61K 39/4636 (2023.05); A61K 39/464413 (2023.05); A61K 39/464419 (2023.05); C07K 16/30 (2013.01); C12N 5/0636 (2013.01); C12N 5/0638 (2013.01); C12N 5/0646 (2013.01); C12N 5/10 (2013.01); C07K 2317/622 (2013.01); C07K 2319/03 (2013.01); C07K 2319/33 (2013.01)] 7 Claims
 
1. A method for preparing engineered immune cells for cell immunotherapy, said method comprising:
providing a population of cells comprising T-cells;
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 PD-1 endogenous locus,
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 such that the exogenous polynucleotide sequence is integrated into a human endogenous PD-1 gene locus and eliminates expression of the PD-1 protein, such that expression of the soluble form of GP130 is under control of the endogenous PD-1 promoter and
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 the 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.