利用Oncolinkage平台鉴定的新型高选择性实体瘤靶点的CAR的发现与表征

CAR T cell therapy has demonstrated remarkable efficacy in treating hematologic malignancies. However, these therapies have been largely unsuccessful in solid tumors, which account for approximately 90% of cancer diagnoses. A critical obstacle to developing CAR T cell therapies for solid tumors is the lack of surface-expressed targets that are selectively present on tumors and not healthy tissues. Identification of tumor-restricted targets is essential to avoid on-target, off-tumor toxicity, which has proven therapeutically limiting for several CAR T therapies, including anti-PSMA and MSLN CARs. To address this challenge, we developed Oncolinkage, a computational platform to discover novel, selectively expressed surface targets whose upregulation is linked to an inciting oncogenic event through the non-specific nature of genomic mutation. As these events drive tumor formation, oncolinked targets are under positive selection, resulting in uniform expression and resistance to loss, making them ideal CAR targets. We first selected a set of ten surface targets that exhibited oncolinked properties--restricted expression in normal tissues, but dysregulated expression in tumors-and identified antibodies that bound cells for 9/10 targets examined. Binding was confirmed by CRISPR/Cas9 knockout of the gene of interest and assessed by flow cytometry. Next, 4-1BB/CD3z CARs were generated for each target using the VH/VL regions of the identified antibodies for scFv generation. After lentiviral transduction of CAR constructs into Jurkat E6.1 cells, active CARs were identified for multiple targets by assessing antigen-dependent CD69 upregulation in overnight co-culture experiments with target-expressing cell lines. The most advanced CAR binds a surface protein whose cancer-specific amplification is observed in subsets of multiple solid tumors, including lung and prostate cancers. This CAR was advanced into primary donor T cells for functional validation. Cytotoxicity was assessed against cell lines exhibiting varying antigen densities of the target protein to demonstrate anti-tumor efficacy at expression levels comparable to primary human tumors. Together, this study highlights the potential of the Oncolinkage platform to efficiently identify and validate multiple novel CAR targets and demonstrates pre-clinical efficacy of solid tumor-directed CARs based on this platform.