Adoptive immunotherapy using lymphocytes genetically-modified to express a chimeric antigen receptor (CART) holds considerable promise for the treatment of cancer. However, CAR-based therapies may involve on-target toxicity against normal tissues expressing low amounts of the targeted tumor-associated antigen (TAA). To specify T cells for robust effector function that is selective for tumor but not normal tissue, we developed a trans-signaling CAR strategy whereby T cell activation signal 1 (CD3ζ) is physically dissociated from costimulatory signal 2 (CD28) in two CARs of differing antigen specificity; mesothelin and a-folate receptor (FRa). Human T cells were genetically modified to co-express signal 1 (Anti-Meso scFv-CD3ζ) and signal 2 (Anti-FRa scFv-CD28) CARs in trans. Trans-signaling CART cells showed weak cytokine secretion against target cells expressing only one TAA in vitro, similar to first generation CART cells bearing CD3ζ only, but demonstrated enhanced cytokine secretion upon encountering natural or engineered tumor cells co-expressing both antigens, equivalent to that of second generation CART cells with dual signaling in cis. CART cells with dual specificity also showed potent anti-cancer activity and persistence in vivo which was superior to first generation CART cells and equivalent to second generation CARs. Importantly, second generation CART cells exhibited potent activity against cells expressing mesothelin alone, recapitulating normal tissue, whereas trans-signaling CART cells did not. Thus, a dual specificity, trans-signaling CAR approach can potentiate the therapeutic efficacy of CART cells against cancer while minimizing parallel reactivity against normal tissues bearing single antigen.

DNA endonuclease eight-like glycosylase 3 (NEIL3) is one of the DNA glycosylases that removes oxidized DNA base lesions from single-stranded DNA (ssDNA) and non-B DNA structures. Approximately seven percent of human tumors have an altered NEIL3 gene. However, the role of NEIL3 in replication-associated repair and its impact on modulating treatment response is not known. Here, we report that NEIL3 is localized at the DNA double-strand break (DSB) sites during oxidative DNA damage and replication stress. Loss of NEIL3 significantly increased spontaneous replication-associated DSBs and recruitment of replication protein A (RPA). In contrast, we observed a marked decrease in Rad51 on nascent DNA strands at the replication fork, suggesting that HR-dependent repair is compromised in NEIL3-deficient cells. Interestingly, NEIL3-deficient cells were sensitive to ataxia–telangiectasia and Rad3 related protein (ATR) inhibitor alone or in combination with PARP1 inhibitor. This study elucidates the mechanism by which NEIL3 is critical to overcome oxidative and replication-associated genotoxic stress. Our findings may have important clinical implications to utilize ATR and PARP1 inhibitors to enhance cytotoxicity in tumors that carry altered levels of NEIL3.

DNA polymerase beta (Pol β) is a key enzyme in the base excision repair (BER) pathway. Pol β is mutated in approximately 40% of human tumors in small-scale studies. The 5´-deoxyribose-5-phosphate (dRP) lyase domain of Pol β is responsible for DNA end tailoring to remove the 5’ phosphate group. We previously reported that the dRP lyase activity of Pol β is critical to maintain DNA replication fork stability and prevent cellular transformation. In this study, we tested the hypothesis that the human gastric cancer associated variant of Pol β (L22P) has the ability to promote spontaneous chromosomal instability and carcinogenesis in mice. We constructed a Pol β L22P conditional knock-in mouse model and found that L22P enhances hyperproliferation and DNA double strand breaks (DSBs) in stomach cells. Moreover, mouse embryonic fibroblasts (MEFs) derived from L22P mice frequently induce abnormal numbers of chromosomes and centrosome amplification, leading to chromosome segregation errors. Importantly, L22P mice exhibit chronic inflammation accompanied by stomach tumors. These data demonstrate that the human cancer-associated variant of Pol β can contribute to chromosomal instability and cancer development.