Compositions and methods for the treatment of cancer using a tet2 engineered t cell therapy
a technology of tet2 and t cell therapy, which is applied in the direction of drug compositions, peptides, enzymology, etc., can solve the problems of poor outcome, non-functional proteins, uncontrolled growth and division of these cells, etc., and achieve the effect of enhancing memory cell differentiation and cell persisten
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example 1
tegration of the NeoTCR
[0298]Neoepitope-specific TCRs identified by the imPACT Isolation Technology described in PCT / US2020 / 17887 (which is herein incorporated by reference in its entirety) were used to generate homologous recombination (HR) DNA templates. These HR templates were transfected into primary human T cells in tandem with site-specific nucleases (see FIGS. 2A-2C, and FIG. 3). The single-step non-viral precision genome engineering resulted in the seamless replacement of the endogenous TCR with the patient's neoepitope-specific TCR, expressed by the endogenous promoter. The TCR expressed on the surface is entirely native in sequence.
[0299]The precision of neoTCR-T cell genome engineering was evaluated by Targeted Locus Amplification (TLA) for off-target integration hot spots or translocations, and by next generation sequencing based off-target cleavage assays and found to lack evidence of unintended outcomes.
[0300]As shown in FIGS. 2A-2C, constructs containing genes of inte...
example 2
n of TET2 Knockout NeoTCR-P1 T Cells
[0310]Materials and Methods. T cells were transfected with gRNAs targeting the first exon of TET2 as Cas9 RNPs along with the TRA and TRB gRNA-Cas9 RNPs as described in Example 1 and in PCT / US2020 / 17887 (which is herein incorporated by reference in its entirety), resulting in disruption of the endogenous TET2 coding sequence and reduced TET2 protein expression (i.e., a TET2 Product).
[0311]T cell Isolation and Editing. TET2 Products were made by first isolating CD4 and CD8 T cells from leukopaks of donors and then transfecting the cells with the TCRα and TCRβ gRNAs (using the NeoTCR integration methods disclosed in Example 1) along with a TET2 gRNA to knockout the TET2 gene. While any gRNA that specifically binds to TET2, disrupts the TET2 gene, and knocks out the TET2 gene expression can be used, the exemplary gRNAs provided in Table 2 were used herein. sgRNA 1 and 5 are negative strand sgRNAs and sgRNAs 2-4 are positive strand sgRNAs.
TABLE 2Exemp...
example 3
nd Materials Used for the Generation of TET2 Products
[0331]Materials and Methods. T cells were transfected with gRNAs targeting the first exon of TET2 as Cas9 RNPs along with the TRA and TRB gRNA-Cas9 RNPs as described in Example 1 and in PCT / US2020 / 17887 (which is herein incorporated by reference in its entirety), resulting in disruption of the endogenous TET2 coding sequence and reduced TET2 protein expression.
[0332]T cell Isolation and Editing. CD4 and CD8 T cells were isolated from healthy donor PBMCs. By means of example, isolation of such cells can be achieved using the Miltenyi Prodigy or Miltenyi MACS separation columns according to the manufacturers' instructions. Positively-selected CD4 and CD8 T cells (for example, using Miltenyi antibodies and isolation column) were used fresh or cryopreserved in 1% human serum albumin, 49% plasmalyte (Baxter), and 50% CS10 (Sigma). Cryopreserved cells were thawed, washed in TexMACS (Miltenyi)+10% human AB serum (Valley Biomedical), and ...
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