JPWO2020095249A5 - - Google Patents

Claims (25)

below:
(i) a nucleic acid encoding a chimeric antigen receptor (CAR) , wherein said CAR comprises an ectodomain that specifically binds to LIV1, wherein the ectodomain of said CAR is an anti-LIV1 single chain variable fragment (scFv ); and
(ii) a disrupted T-cell receptor alpha chain constant region (TRAC) gene, a disrupted beta-2-microglobulin (β2M) gene, or a combination thereof;
The genetically engineered population of T cells comprising: said anti-LIV1 scFv comprises the same heavy chain variable domain (VH) complementarity determining regions (CDRs) and the same light chain variable domain (VL) CDRs as a reference antibody, wherein said reference antibody is (i) set forth in SEQ ID NO:55 and the VL according to SEQ ID NO:56, or (ii) the VH according to SEQ ID NO: 90 and the VL according to SEQ ID NO: 88 . 3. The population of genetically engineered T cells of claim 2 , wherein said anti-LIVl scFv comprises the same VH and VL chains as said reference antibody. 3. The population of genetically engineered T cells of claim 2, wherein said anti-LIVl scFv comprises the amino acid sequence of any one of SEQ ID NOs: 54, 70, 83 and 86. 3. The genetically engineered T cell population of claim 2, wherein said anti-LIVl scFv comprises the amino acid sequence of SEQ ID NO:83. 6. The population of genetically engineered T cells according to any one of claims 1-5 , wherein said CAR further comprises a CD28 co-stimulatory domain or a 41BB co-stimulatory domain. 7. The population of genetically engineered T cells of claim 6 , wherein said CAR further comprises a CD3zeta cytoplasmic signaling domain. 8. The population of genetically engineered T cells of any one of claims 1-7, wherein said CAR comprises the amino acid sequence of any one of SEQ ID NOS: 50, 52, 105, 109, 68 and 66. . 9. The population of genetically engineered T cells of claim 8, wherein said CAR comprises the amino acid sequence of SEQ ID NO:105. 49, 51, 104 or 108 or a nucleotide sequence comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 49, 51, 104 or 108, wherein said CAR is encoded by a nucleotide sequence comprising: 8. A population of genetically engineered T cells according to any one of 1-7 . 11. The population of genetically engineered T cells of any one of claims 1-10 , wherein said nucleic acid encoding said CAR is inserted into said disrupted TRAC gene. Claims 1- , wherein the disrupted TRAC gene comprises the nucleotide sequence of any one of SEQ ID NOs: 63, 64, 107 and 111 and/or the nucleotide sequence of any one of SEQ ID NOs: 49, 51, 104 and 108. 12. The genetically engineered T cell population according to any one of 11. 13. The genetically engineered gene of any one of claims 1-12 , wherein the disrupted β2M gene comprises at least one nucleotide sequence selected from any one of SEQ ID NOS: 9-14. A population of T cells. A population of genetically engineered T cells according to any one of claims 1 to 13 , which are human T cells. A population of cells comprising the genetically engineered T cells of any one of claims 1-14 ,
(i) at least 15% or at least 50% of the genetically engineered T cells of said population express said CAR ;
(ii) at least 50% of the genetically engineered T cells of said population do not express detectable levels of T cell receptor (TCR) protein; and/or
(iii) said population, wherein at least 50% of the genetically engineered T cells of said population do not express detectable levels of β2M protein. A method of generating a population of genetically engineered T cells according to any one of claims 1-15 , comprising:
(a) to T cells,
(i) an RNA-guided nuclease;
(ii) a gRNA targeting the TRAC gene, a gRNA targeting the β2M gene, or a combination thereof ;
(iii) a vector comprising a donor template comprising a nucleic acid encoding a CAR comprising an ectodomain that specifically binds to LIV1;
and (b) generating genetically engineered T cells that have a disrupted TRAC gene , a disrupted β2M gene, or a combination thereof and express said CAR , Method. said gRNA targeting said TRAC gene comprises the nucleotide sequence of SEQ ID NO: 18 or 19, or targets the nucleotide sequence of SEQ ID NO: 40 ; or
17. The method of claim 16 , wherein the gRNA targeting the β2M gene comprises the nucleotide sequence of SEQ ID NO:20 or 21 or targets the nucleotide sequence of SEQ ID NO:41. 18. The method of any one of claims 16 or 17, wherein the donor template comprises the nucleotide sequence of any one of SEQ ID NOs: 63, 64, 107 and 1-11. The method of any one of claims 16-18 , wherein said RNA-guided nuclease is Cas9 nuclease. 20. The method of claim 19, wherein said Cas9 nuclease is S. pyogenes Cas9 nuclease. A population of genetically engineered T cells for use in the treatment of cancer in a subject, wherein said genetically engineered T cells are the genes of any one of claims 1-15. A population of strategically engineered T cells . 22. A population of genetically engineered T cells for use according to claim 21, wherein said subject is a human cancer patient. Said cancer is pancreatic cancer, gastric cancer, ovarian cancer, uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, nasopharyngeal cancer, non-small cell lung (NSCLC), glioblastoma, neuronal, soft tissue sarcoma, leukemia, lymphoma , melanoma, colon cancer, colon adenocarcinoma, cerebral glioblastoma, hepatocellular carcinoma, liver cholangiocarcinoma, osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, advanced pancreatic cancer, lung adenocarcinoma, lung squamous cell carcinoma, small lung 23. A population of genetically engineered T cells for use according to claim 21 or 22 , selected from the group consisting of cell carcinoma, renal carcinoma and intrahepatic cholangiocarcinoma. A population of genetically engineered T cells for use according to any one of claims 21 to 23 , wherein said cancer comprises cancer cells expressing LIV1. A population of genetically engineered T cells for use according to any one of claims 21 to 24, wherein said genetically engineered T cells are allogeneic to said subject.