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US 12,698,317 B2Granted
Manufacturing process for making t cells expressing chimeric antigen receptors
AssigneeCrispr Therapeutics AG
InventorsHui Yu, Demetrios Kalaitzidis, Siyuan Tan
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Abstract
Aspects of the present disclosure relate to methods for manufacturing genetically engineered T cells expressing a chimeric antigen receptor (CAR) that provide several improvements over conventional manufacturing methods, thereby enabling production of a robust supply of clinically useful CAR T-cell therapies.
Claims40
A method for manufacturing genetically engineered T cells, the method comprising:
- (i) providing a first population of T cells obtained from human blood cells;
- (ii) incubating the first population of T cells in the presence of a T cell activating agent in a cell culture vessel to produce a second population of T cells, wherein the second population of T cells comprises activated T cells;
- (iii) introducing into the second population of T cells a first ribonucleoprotein (RNP) complex comprising a first Cas9 enzyme and a first guide RNA (gRNA) targeting a T cell receptor alpha chain constant region (TRAC) gene, and a second RNP complex comprising a second Cas9 enzyme and a second gRNA targeting a beta-2 microglobulin (β2M) gene to produce a third population of T cells, wherein the third population of T cells comprises T cells having the TRAC gene disrupted and the β2M gene disrupted, wherein the first RNP complex and the second RNP complex are introduced into the second population of T cells in one electroporation event;
- (iv) incubating the third population of T cells with an adeno-associated viral (AAV) vector to produce a fourth population of T cells, wherein the AAV vector comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and wherein the nucleic acid sequence is flanked by homologous sequences to the TRAC gene locus, and wherein the fourth population of T cells comprises activated T cells expressing the CAR and having the TRAC gene disrupted and the β2M gene disrupted;
- (v) expanding the fourth population of T cells thereby producing an expanded T cell population;
- (vi) removing TCRαβ+ T cells from the expanded T cell population to produce a population of genetically engineered T cells, wherein the population of genetically engineered T cells comprises T cells expressing the CAR and having the TRAC gene and the β2M gene disrupted;
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- (vii) harvesting the population of genetically engineered T cells.
5 more elements
Dependencies are inferred from the claim text.