Compositions and methods for the treatment of cancer using a tet2 engineered t cell therapy

Abstract

Compositions comprising and methods for the treatment of cancer using a neoTCR based cell therapy with a knockout of the expression of the TET2 gene.

What is claimed is: 
     
         1 . A composition comprising an effective amount of a cell comprising:
 a. an exogenous patient-derived T cell receptor (TCR) recognizing a neoantigen; and   b. a gene disruption of a TET2 locus.   
     
     
         2 . The composition of  claim 1 , wherein the gene disruption comprises a missense mutation, a nonsense mutation, a non-frameshift deletion, a frameshift deletion, a non-frameshift insertion, a frameshift insertion, or any combination thereof. 
     
     
         3 . The composition of  claim 1 , wherein the gene disruption of the TET2 locus results in a non-functional TET2 protein or in a knockout of the TET2 gene expression. 
     
     
         4 . The composition of  claim 1 , wherein the cell further comprises a gRNA and a Cas9 nuclease. 
     
     
         5 . The composition of  claim 4 , wherein the gRNA comprises a nucleotide sequence set forth in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5. 
     
     
         6 . The composition of  claim 1 , wherein the gene disruption of the TET2 locus enhances cell persistence or memory cell differentiation. 
     
     
         7 . The composition of  claim 1 , wherein the cell is a primary cell. 
     
     
         8 . The composition of  claim 1 , wherein the cell is a patient-derived cell. 
     
     
         9 . The composition of  claim 1 , wherein the cell is a T cell. 
     
     
         10 . The composition of  claim 9 , wherein the T cell is
 a. CD45RA+, CD62L+, CD28+, CD95−, CCR7+, and CD27+;   b. CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+; or   c. CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+.   
     
     
         11 . The composition of  claim 1 , wherein the exogenous TCR comprises a signal sequence, a first and second 2A sequence, and a TCR polypeptide sequence. 
     
     
         12 . The composition of  claim 1 , wherein the neoantigen is a patient specific neoantigen. 
     
     
         13 . The composition of  claim 1 , wherein the exogenous patient-derived TCR is integrated in an endogenous TRAC or TRBC locus. 
     
     
         14 . The composition of  claim 1 , further comprising a pharmaceutically acceptable excipient. 
     
     
         15 . The composition of  claim 1 , further comprising a cryopreservation agent, serum albumin, and a crystalloid solution. 
     
     
         16 . The composition of  claim 1 , further comprising Plasma-Lyte A, HSA, and CryoStor CS10. 
     
     
         17 . A method of modifying a cell, the method comprising:
 a. introducing into the cell a non-viral homologous recombination (HR) template nucleic acid sequence comprising
 i. first and second homology arms homologous to first and second target nucleic acid sequences, and 
 ii. a patient derived T cell receptor (TCR) gene sequences positioned between the first and second homology arms; 
   b. recombining the HR template nucleic acid into an endogenous locus of the cell; and   c. disrupting a TET2 locus of the cell.   
     
     
         18 . The method of  claim 17 , wherein the HR template comprises a first P2A-coding sequence positioned upstream of the TCR gene sequence and a second P2A-coding sequence positioned downstream of the TCR gene sequence, wherein the first and second P2A-coding sequences code for the same amino acid sequence that are codon-diverged relative to each other. 
     
     
         19 . The method of  claim 18 , wherein the HR template comprises a sequence coding for the amino acid sequence Gly Ser Gly positioned immediately upstream of the P2A-coding sequences, a sequence coding for a Furin cleavage site positioned upstream of the second P2A-coding sequence, and a signal sequence positioned between the first P2A-coding sequence and the TCR gene sequence. 
     
     
         20 . The method of  claim 17 , wherein the first and second homology arms are each from about 300 bases to about 2,000 bases in length. 
     
     
         21 . The method of  claim 18 , wherein the HR template comprises a second TCR sequence positioned between the second P2A-coding sequence and the second homology arm. 
     
     
         22 . The method of  claim 21 , wherein the HR template comprises:
 a. a first signal sequence positioned between the first P2A-coding sequence and the first TCR gene sequence; and   b. a second signal sequence positioned between the second P2A-coding sequence and the second TCR gene sequence;
 wherein the first and the second signal sequences encode for the same amino acid sequence and are codon diverged relative to each other. 
   
     
     
         23 . The method of  claim 17 , wherein the HR template is a circular DNA or a linear DNA. 
     
     
         24 . The method of  claim 17 , wherein the introducing occurs via electroporation. 
     
     
         25 . The method of  claim 17 , wherein the recombining comprises:
 a. cleavage of the endogenous locus by a Cas9/gRNA ribonucleoprotein; and   b. recombination of the HR template nucleic acid sequence into the endogenous locus by homology directed repair.   
     
     
         26 . The method of  claim 17 , wherein the disrupting comprises cleavage of the TET2 locus by a Cas9/gRNA ribonucleoprotein. 
     
     
         27 . The method of  claim 26 , wherein the gRNA comprises a sequence set forth in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5. 
     
     
         28 . A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of a cell comprising:
 a. an exogenous patient-derived T cell receptor (TCR) recognizing a neoantigen; and   b. a gene disruption of a TET2 locus.   
     
     
         29 . The method of  claim 28 , wherein prior to administering the therapeutically effective amount of cells, a non-myeloablative lymphodepletion regimen is administered to the subject. 
     
     
         30 . The method of  claim 28 , wherein the cancer is selected from the group consisting of follicular lymphoma, leukemia, multiple myeloma, melanoma, thoracic cancer, lung cancer, ovarian cancer, breast cancer, pancreatic cancer, head and neck cancer, prostate cancer, gynecological cancer, central nervous system cancer, cutaneous cancer, HPV+ cancer, esophageal cancer, thyroid cancer, gastric cancer, hepatocellular cancer, cholangiocarcinomas, renal cell cancers, testicular cancer, sarcomas, and colorectal cancer.