Generation of CART cells targeting oncogenic TROP2 for the elimination of epithelial malignancies

Abstract

Background & Aim TROP2 (also referred to as Tacstd2) is a membrane-associated glycoprotein highly expressed in many epithelial cancers, including oral, head-and-neck, thyroid, lung, esophageal, gastric, colorectal, pancreatic, breast, renal, uterine, cervical, ovarian, prostate, and glioma, and expression of TROP2 negatively correlates with prognosis and survival in many of these malignancies. TROP2 signaling supports tumor growth and progression through the induction of pathways controlling cell cycle progression, invasion and metastasis, proliferation, angiogenesis, cell growth and survival. In addition, tumor release of TROP2-containing exosomes can promote the migration of TROP2-negative tumor cells. Here, we generated several TROP-2-targeting CAR molecules and evaluated their activity as CART cells against various epithelial tumor cell lines. Methods, Results & Conclusion Single-chain variable fragments targeting TROP2 were produced through domain sequencing of hybridomas and through other publically available sources and cloned into lentiviral transfer plasmids containing second-generation CAR backbones. Transduction of human T cells routinely leads to the expression of >50% CAR+ cells, including the generation of TROP2-CART cells from T cells of a mTNBC patient. We evaluated interferon-gamma production as well as cytotoxicity of TROP2-CART cells cultured with TROP2+ breast, prostate and pancreatic tumor cells to choose a lead TROP2-CAR. In addition, we generated an isogenic TROP2- prostate cancer through Cas9-based gene editing and observed loss of cytotoxicity, demonstrating specificity of the TROP2-CART. However, TROP2-CART can kill TROP2- cells in the presence of TROP2+ cells, a feature also observed by CART19 when cultured with CD19+ and CD19- cells, consistent prior reports that activated CART cells induce tumor apoptosis through death receptors. Prior reports demonstrated that blockade of death receptor ligands abolished the non-specific cytotoxicity of CART19 cells, but this approach provided only partial blockade of TROP2-CART cytotoxicity. Instead, we describe and demonstrate a new mechanism by which transfer of TROP2+ tumor exosomes to TROP2- tumor cells increases the proportion of tumor targetable by TROP2-CART and may allow TROP2-CART to overcome the challenge of antigen heterogeneity in solid tumors.