Contextual reprogramming of CAR-T cells for treatment of HER2+ cancers

Abstract

Background Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. Methods To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. Results Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product. Conclusions As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

The KRAB Domain of ZNF10 Guides the Identification of Specific Amino Acids That Transform the Ancestral KRAB-A-Related Domain Present in Human PRDM9 into a Canonical Modern KRAB-A Domain

“…Here and throughout the manuscript amino acids with nonpolar side-chains are colored black, those with polar side-chains are in green, ones with basic side-chains are blue and those with acidic side-chains are red. [30][31][32] along with the HMM scores (E-values; the lower, the better) against the different KRAB-A models. Selection based on best hits of a group member vs the different KRAB-A models, inclusion of phylogenetically oldest species and added outliers (names in red).…”

“…Of major interest is the role of KZNF proteins in the restriction of retroviral genomic sequences, in particular during development, and for exaptation, i.e., their repurposing for novel regulatory principles [16,25,26]. In conjunction with CRISPR/Cas the ZNF10-KRAB is used as a tool for modulating and redirecting transcriptional gene regulation in basic research (CRISPRi; [27,28]), and applied in oncology (e.g., [29,30]), in human genetics (e.g., [31]) and virology (e.g., [32]).…”

Standardized in-vitro evaluation of CAR-T cells using acellular artificial target particles

“…We next tried to apply aaTPs in the characterization of a more complex CAR-T cell product. RB-340 is a ‘next-generation’ CAR-T cell in which activation of the CAR receptor by its respective target antigen (HER2) not only stimulates T-cell activation but also inhibits upregulation of PD-1 using CAR-dependent activation of dCAS9-KRAB element which specifically targets PD-1 ( 19 ). Upon co-incubation with HER2-Mid aaTPs, both RB-340 and conventional CAR-T cells showed very similar trend in the effect on viability with high antigen doses ( Figure 6A ) as well as induction of all the previously described markers of activation, namely CD69 ( Figure 6B ) and downstream cytokine secretion (data not shown).…”

Tumor microenvironment and immunotherapy of oral cancer

“…Recently, Yang et al constructed an all-in-one CAR-T cells by coupling anti-human epidermal growth factor receptor 2 (anti-HER2) CAR signaling and clustered regularly interspaced short palindromic repeats interference (CRISPRi)-mediated PD-1 gene suppression. These symphysis CAR-T cells reversed PD-1/PD-L1 immune checkpoint inhibition and promoted the persistence and effectiveness against HER2-expressing human head and neck squamous cell carcinoma [ 154 ].…”