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

• Published in: Journal of translational medicine 2021-11, Vol.19 (1), p.459-18, Article 459
• Main Authors: Yang, Zhifen, Li, Lingyu, Turkoz, Ahu, Chen, Pohan, Harari-Steinfeld, Rona, Bobbin, Maggie, Stefanson, Ofir, Choi, Hana, Pietrobon, Violena, Alphson, Bennett, Goswami, Angshumala, Balan, Vitaly, Kearney, Alper, Patel, Dharmesh, Yang, Jin, Inel, Damla, Vinod, Veena, Cesano, Alessandra, Wang, Bing, Roh, Kyung-Ho, Qi, Lei S, Marincola, Francesco M
• Format: Article
• Language: English
• Subjects: Adoptive transfer; Antigen receptors, T cell; Antigens; Apoptosis; Cancer; Care and treatment; CD28 antigen; CD28 Antigens - genetics; Cell activation; Cell death; Cell Line, Tumor; Chimeric antigen receptors; CRISPR; Cytokines; Cytotoxicity; Dosage and administration; Efficiency; Epidermal growth factor; ErbB-2 protein; Experiments; Gene expression; Genetic aspects; Genome editing; Growth factors; Humans; Immunotherapy; Immunotherapy, Adoptive; Kruppel protein; Ligands; Lymphocytes; Lymphocytes T; Methods; Neoplasms; Nuclear reprogramming; Nuclease; Oropharyngeal cancer; PD-1 protein; Proteins; Receptors; Receptors, Antigen, T-Cell - genetics; RNA, Guide, CRISPR-Cas Systems; Single-Chain Antibodies; T cells; T-Lymphocytes; Transcription; Tumor necrosis factor-TNF; Xenografts
• ISSN: 1479-5876; 1479-5876
• DOI: 10.1186/s12967-021-03132-6

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. 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. 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.