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 crossreactivity 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 EC 50 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 IFNγ 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.

Liver cancers, majority of which are primary hepatocellular carcinoma (HCC), continue to be on the rise in the world. Furthermore, due to the lack of effective treatments, liver cancer ranks the 4 th most common cause of male cancer deaths. Novel therapies are urgently needed. Over last few years, immunotherapies, especially the checkpoint blockades and adoptive cell therapies of engineered T cells, have demonstrated a great potential for treating malignant tumors including HCC. In this review, we summarize the current ongoing research of antigen-specific immunotherapies including cancer vaccines and adoptive cell therapies for HCC. We briefly discuss the HCC cancer vaccine and then focus on the antigen-specific T cells genetically engineered with the T cell receptor genes (TCRTs) and the chimeric antigen receptor genes (CARTs). We first review the current options of TCRTs and CARTs immunotherapies for HCC, and then analyze the factors and parameters that may help to improve the design of TCRTs and CARTs to enhance their antitumor efficacy and safety. Our goals are to render readers a panoramic view of the current stand of HCC immunotherapies and provide some strategies to design better TCRTs and CARTs to achieve more effective and durable antitumor effects.

Background and Aims: Chimeric antigen receptor engineered T cells (CARTs) for HCC and other solid tumors are not as effective as they are for blood cancers. CARTs may lose function inside tumors due to persistent antigen engagement. The aims of this study are to develop low-affinity monoclonal antibodies (mAbs) and low-avidity CARTs for HCC and to test the hypothesis that low-avidity CARTs can resist exhaustion and maintain functions in solid tumors, generating durable antitumor effects. Methods and Results: New human glypican-3 (hGPC3) mAbs were developed from immunized mice. We obtained three hGPC3-specific mAbs that stained HCC tumors, but not the adjacent normal liver tissues. One of them, 8F8, bound an epitope close to that of GC33, the frequently used high-affinity mAb, but with approximately 17-fold lower affinity. We then compared the 8F8 CARTs to GC33 CARTs for their in vitro function and in vivo antitumor effects.In vitro, low-avidity 8F8 CARTs killed both hGPC3 high and hGPC3 low HCC tumor cells to the same extent as high-avidity GC33 CARTs. 8F8 CARTs expanded and persisted to a greater extent than GC33 CARTs, resulting in durable responses against HCC xenografts. Importantly, compared with GC33 CARTs, there were 5-fold more of 8F8-BBz CARTs in the tumor mass for a longer period of time. Remarkably, the tumor-infiltrating 8F8 CARTs were less exhausted and apoptotic, and more functional than GC33 CARTs.