Contextual Reprogramming of CAR-T Cells for Treatment of HER2+ Cancers

Zhifen Yang,Veena Vinod,Alessandra Cesano,Francesco M Marincola,Bing Wang,Damla Inel,Pohan Chen,Ahu Turkoz,Hana Choi,Vitaly Balan,Lei S Qi,Alper Kearney,Rona Harari-Steinfeld,Maggie Bobbin,Kyung-Ho Roh,Dharmesh Patel,Ofir Stefanson,Lingyu Li

doi:10.2139/ssrn.3656606

Abstract

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. 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. 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.

Highlights

Success of adoptive cell therapy (ACT) depends upon T cell persistence and engraftment [1, 2]
RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of chimeric antigen receptor (CAR)-T cells in vitro, prolonged in vivo persistence and more efficient suppression of human epidermal growth factor receptor 2 (HER2)+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product
The first (HER2-TEV) encodes an anti-HER2 (4D5 clone) [17] Single chain variable fragment (scFv) combined to the CD28 and CD3ζ costimulatory domains, the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the transcription start site (TSS) of the endogenous PD-1 gene (PD-1sg)

Summary

Introduction

Success of adoptive cell therapy (ACT) depends upon T cell persistence and engraftment [1, 2]. Lynn et al [10] proposed that overexpression of c-Jun suppresses PD-1 and other checkpoints resulting in enhanced T cell persistence and improved anti-tumor efficacy. These constitutive approaches lead to permanent alterations of the DNA structure and increased risk for neoplastic transformation preventable by a conditional system, where gene expression is modulated in a context-dependent manner without permanent alterations of the DNA structure. We previously described a non-editing gene expression regulation strategy based on the nuclease de-activated CRISPR-associated (dCas9) protein, which offers a platform for RNA-guided DNA targeting [11,12,13]. The approach is limited by cumula‐ tive costs and toxicities

Methods

Results

Conclusion