Improved Antitumor Activity of Interleukin-12-Secreting Chimeric Antigen Receptor T Cells Targeting CD176 across Different Carcinomas.

INTRODUCTION: Effective therapeutic options for advanced solid tumors remain severely limited, causing high fatality rates especially after metastasis. The carbohydrate structure CD176 has been identified as a promising target for precise immunotherapy in multiple carcinomas, as it is present in about 90% of carcinomas but unavailable for binding on healthy tissue. Here, we report the development of CD176-specific 4th-generation chimeric antigen receptor T cells (CAR-Ts), also known as T cells redirected for antigen-unrestricted cytokine-initiated killing (TRUCKs). To address the immunosuppressive tumor microenvironment (TME) and the heterogeneous antigen expression of solid tumors, which limit the efficacy of CAR-Ts, they were endowed with NFAT-inducible interleukin-12 (iIL12) release to improve pro-inflammatory autocrine and paracrine effects. METHODS: The CD176-iIL12-TRUCK construct was tested for target specificity in a reporter cell assay using a JE6-1-derived reporter cell line. Afterward, CD176-iIL12-TRUCKs were manufactured using primary CD8(+) T cells. The influence of iIL12 on functionality of CD176-iIL12-TRUCKs, including T-cell activation levels, cytotoxic capacity, and recruitment of bystander immune cells, was evaluated following cocultures with CD176(+) cell lines from different carcinomas. RESULTS: Upon recognition of CD176(+) cancer cell lines, CD176-iIL12-TRUCKs specifically released pro-inflammatory mediators (interferon-γ, tumor necrosis factor-α) and showed an increased activation marker expression (CD25, CD69). Using both a 7-AAD-based viability assay and an impedance-based cytotoxicity assay, elimination of CD176(+) cell lines from different tumor entities by CD176-iIL12-TRUCKs was shown. Additionally, iIL12 released by CD176-iIL12-TRUCKs led to recruitment of monocyte and NK cell lines in a chemotaxis chamber assay. DISCUSSION/CONCLUSION: Overall, the IL-12 release substantially improved effector functionality against CD176(+) cells but not CD176(-) cells, indicating efficacy while maintaining specificity. Thus, CD176-iIL12-TRUCKs, with their potent antitumor efficacy and TME modulation potential, are a promising treatment option for patients with a variety of advanced solid tumors.