Supplementary Material for: Improved antitumor activity of IL-12-secreting CAR-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 TRUCKs (T cells redirected for antigen-unrestricted cytokine-initiated killing). 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. Afterwards, 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 co-cultures 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 anti-tumor efficacy and TME modulation potential, are a promising treatment option for patients with a variety of advanced solid tumors.