Abstract
BackgroundAdoptive transfer of engineered immune cells is a promising strategy for cancer treatment. However, low transduction efficiency particularly when large payload lentiviral vectors are used on primary T cells is a limitation for the development of cell therapy platforms that include multiple constructs bearing long DNA sequences. RB-340-1 is a new CAR T cell that combines two strategies in one product through a CRISPR interference (CRISPRi) circuit. Because multiple regulatory components are included in the circuit, RB-340-1 production needs delivery of two lentiviral vectors into human primary T cells, both containing long DNA sequences. To improve lentiviral transduction efficiency, we looked for inhibitors of receptors involved in antiviral response. BX795 is a pharmacological inhibitor of the TBK1/IKKɛ complex, which has been reported to augment lentiviral transduction of human NK cells and some cell lines, but it has not been tested with human primary T cells. The purpose of this study was to test if BX795 treatment promotes large payload RB-340-1 lentiviral transduction of human primary T cells.MethodsTo make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels named RB-340-1F. We incorporated BX795 treatment into the human primary T cell transduction procedure that was optimized for RB-340-1F. We tested BX795 with T cells collected from multiple donors, and detected the effect of BX795 on T cell transduction, phenotype, cell growth and cell function.ResultsWe found that BX795 promotes RB-340-1F lentiviral transduction of human primary T cells, without dramatic change in cell growth and T cell functions. Meanwhile, BX795 treatment increased CD8+ T cell ratios in transduced T cells.ConclusionsThese results indicate that BX795 treatment is effective, and might be a safe approach to promote RB-340-1F lentiviral transduction of human primary T cells. This approach might also be helpful for other T cell therapy products that need delivery of complicated platform via large payload lentiviral vectors.
Highlights
Adoptive transfer of engineered immune cells is a promising strategy for cancer treatment
RB-340-1 is engineered to express an anti-Human epidermal growth factor receptor 2 (HER2) Chimeric antigen receptor (CAR) single chain variable fragment (4D5 clone) [16], with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a Programmed cell death protein 1 (PD-1) promoter regiontargeting short guide RNA (PD1sg)
Establishment of human primary T cell transduction procedure for RB‐340‐1F RB-340-1 is a combinatorial CAR/CRISPR interference (CRISPRi) circuit delivered into T cells by two lentiviral constructs, LdCK and CAR-TEV (Fig. 1a)
Summary
Adoptive transfer of engineered immune cells is a promising strategy for cancer treatment. Li et al J Transl Med (2020) 18:363 tumors [1, 2], including antigen escape [3, 4], immunosuppression in the tumor microenvironment [1, 5, 6] and lack of strictly tumor-specific targets [7] To overcome these hurdles, incorporation of multiple strategies which can enhance antitumor efficacy and improve safety are required, and increasingly more combination strategies and synthetic biological approaches emerged. Upon antigen- encounter, the LAT-dCas9-Krab complex is cleaved by the TEV allowing nuclear translocation of dCas9-Krab to the transcription start site of the PD-1 gene This conditional, non-gene editing and reversible suppression promotes resilience to checkpoint inhibition, and in vivo persistence and effectiveness against HER2-expressing oropharyngeal cancer xenografts. To make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels, which is named as RB-340-1F
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