Preclinical Studies of the Off-Target Reactivity of AFP158-Specific TCR Engineered T Cells

Autologous T cells engineered with T receptor genes (TCR) are being studied to treat cancers. We have recently identified a panel of mouse TCRs specific for the HLA-A0201/alpha fetoprotein epitope (AFP(158)) complex and have shown that human T cells engineered with these TCR genes (TCR-Ts) can eradicate hepatocellular carcinoma (HCC) xenografts in NSG mice. However, due to TCR's promiscuity, their off-target cross-reactivity must be studied prior to conducting clinical trials. In this study, we conducted in vitro X-scan assay and in silico analysis to determine the off-target cross-reactivity of 3 AFP(158)-specific TCR-Ts. We found that the 3 AFP(158)-specific TCR-Ts could be cross-activated by ENPP1(436) peptide and that the TCR3-Ts could also be activated by another off-target peptide, RCL1(215). However, compared to AFP(158), it requires 250 times more ENPP1(436) and 10,000 times more RCL1(215) peptides to achieve the same level of activation. The EC50 of ENPP1(436) peptide for activating TCR-Ts is approximately 17-33 times higher than AFP(158). Importantly, the ENPP1+ tumor cells did not activate TCR1-Ts and TCR2-Ts, and only weakly activated TCR3-Ts. The IFN gamma produced by TCR3-Ts after ENPP1+ cell stimulation was >22x 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-A2+ tumor cells did not activate TCR-Ts. In silico analysis showed that the ENPP1(436) peptide affinity for HLA-A0201 was ranked 40 times lower than AFP(158) and the chance of ENPP1(436) peptide being processed and presented by HLA-A0201 was 100 times less likely than AFP(158). 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 shows that TCR-Ts can be activated by off-target ENPP1(436) peptide. But, compared to target AFP(158), it requires at least 250 times more ENPP1(436) to achieve the same level of activation. Importantly, ENPP1(436) peptide in human cells is not processed and presented to a sufficient level to activate the AFP(158)-specific TCR-Ts. Thus, these TCR-Ts, especially the TCR1-Ts and TCR2-Ts, will unlikely cause significant off-target toxicity.