Base-edited CAR T Cells for combinational therapy against T cell malignancies

Authors:
Christos Georgiadis, Jane Rasaiyaah,  Soragia Athina Gkazi, Roland Preece, Aniekan Etuk, Abraham Christi,  Waseem Qasim
In:
Source: BioResearch Open Access
Publication Date: (2020)
Issue: 1: 1
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Regenerative medicine
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofector™ X-Unit
4D-Nucleofector™ LV-Unit
Experiment

Activated T cells were electroporated with TRBC sgRNA (Synthego), CD7 sgRNA (Synthego) and codon optimised BE3 mRNA (TriLink Biotechnologies) in a Lonza 4D or LV nucleofector (Lonza, Slough, UK) and cultured at 30°C 5% CO2 overnight before returning to 37°C. Cells were transduced with 3CAR or 7CAR lentiviral vector preparations at an MOI of 5 the following day. Cells were cultured in G-Rex 10 or G—Rex 100 chambers as per manufacturer’s instructions (Wilson Wolf, MN, USA).

Abstract

Targeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Genome-editing can overcome such hurdles through targeted disruption of problematic shared antigens. Base editing offers the possibility of seamless disruption of gene expression through the creation of stop codons or elimination of splice donor or acceptor sites. We describe the generation of fratricide resistant, T cells by orderly removal of shared antigens such as TCR/CD3 and CD7 ahead of lentiviral mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base edited cells confirmed virtual elimination of chromosomal translocation events detected in conventional Cas9 treated cells. Interestingly, co-culture of 3CAR and 7CAR cells resulted in ‘self-enrichment’ yielding populations that were 99.6% TCR-/CD3/-CD7-. 3CAR or 7CAR cells were able to exert specific cytotoxicity against their relevant target antigen in leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited the highest level of cytotoxicity against T-ALL targets expressing both target in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no evidence of promiscuous base conversion activity affecting CAR antigen specific binding regions which may otherwise redirect T cell specificity. Combinational infusion of fratricide resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allogeneic haematopoietic stem cell transplantation for T cell malignancies.