Preclinical studies of the off-target reactivity of AFP158-specific TCR engineered T cells

Abstract

Abstract Autologous T cells engineered with T receptor genes (TCR-T) are being studied to treat cancers. We have recently identified a panel of mouse TCRs specific for the HLA-A0201/alpha fetoprotein epitope (AFP158) complex and have shown that the TCR-engineered human T cells can eradicate hepatocellular carcinoma xenografts in NSG mice. In this current study, we evaluate their off-target toxicity. We found that 3 AFP158-specific TCR-Ts could be cross-activated by ENPP1436 peptide and that the TCR3-Ts could also be activated by another off-target peptide, RCL1215. However, compared to AFP158, it requires 250 times more ENPP1436 and 10,000 times more RCL1215 peptides to achieve the same level of activation. The EC50 of ENPP1436 peptide for activating TCR-Ts is approximately 17–33 times higher than AFP158. Importantly, the ENPP1+ tumor cells did not activate TCR1-Ts and TCR2-Ts, and only weakly activated TCR3-Ts. The IFNγ produced by TCR3-Ts after ENPP1+ cell stimulation was 22 times lower than that after HepG2 cells. And, all TCR-Ts did not kill ENPP1+ tumor cells. Furthermore, ectopic over-expression of ENPP1 protein in HLA-A0201+ tumor cells did not activate TCR-Ts. In silico analysis showed that the ENPP1436 peptide affinity for HLA-A0201 was ranked 40 times lower than AFP158 and the chance of ENPP1436 peptide being processed and presented by HLA-A0201 was 100 times less likely than AFP158. In contrast, the two off-targets (Titin and MAGE-A3) that did cause severe toxicity in previous trials have the same or higher MHC-binding affinity and the same or higher chance of being processed and presented. In conclusion, our data showed that these TCR-Ts, especially TCR1-Ts and TCR2-Ts, will unlikely cause significant off-target toxicity.