TROP2-circular RNA vaccine and IL7 synergistically inhibit TROP2+ tumor growth in mouse models

Human trophoblast cell surface antigen 2 (hTROP2), a transmembrane glycoprotein encoded by the Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) gene, is a promising tumor-associated antigen (TAA) in several types of cancers for targeted immunotherapy. We developed hTROP2 protein and RNA vaccines that effectively suppressed the growth of TROP2-positive tumors in mouse models. However, the efficacy of TAA-targeted vaccines in humans may be limited by self-tolerance and poor tumor infiltration of immune effector cells. Given the critical role of interleukin-7 (IL7) in promoting T-cell proliferation, survival, and repertoire diversity, we hypothesized that IL7 could enhance hTROP2 vaccine efficacy by expanding and diversifying the T-cell pool, thereby improving tumor infiltration and overcoming self-tolerance. To test this, we co-administered an hTROP2-expressing circular RNA-lipid nanoparticle (hTROP2-circRNA-LNP) vaccine with an IL7-expressing circRNA-LNP (IL7-circRNA-LNP) in mouse models. Cellular immune responses were assessed using IFNgamma ELISpot assays on splenocytes from vaccinated mice. Co-administration with IL7-circRNA-LNP enhanced the cellular immune response by 2.3- and 2.7-fold for the two predominantly active stimulating peptides compared with the response induced by hTROP2-circRNA-LNP alone. Comparable results were observed when IL7-circRNA-LNP was administered either before or after the hTROP2-circRNA-LNP vaccine. In mice bearing tumors derived from TROP2-humanized cancer cell lines (MC38-hTROP2 and 4T1-hTROP2), co-administration of the hTROP2-circRNA-LNP vaccine with IL7-circRNA-LNP significantly enhanced anti-tumor efficacy compared with hTROP2-circRNA-LNP alone. Tumor growth inhibition increased from 57.4% to 87.0% in the MC38-hTROP2 model and from negligible effect to 63.4% in the 4T1-hTROP2 model. In control mice that received IL7-circRNA-LNP alone, IL7 monotreatment achieved 20% tumor growth inhibition (without statistical significance) in the MC38-hTROP2 model but had no effect in the 4T1-hTROP2 model. Analysis of tumor-infiltrating lymphocytes revealed that co-administration of the hTROP2-circRNA-LNP vaccine with IL7-circRNA-LNP significantly increased the proportions of cytotoxic CD8alpha+ and helper CD4+ T-cells, as well as their activated subsets (CD8alpha+IFNgamma+ and CD4+IFNgamma+ cells). Neither the hTROP2- circRNA-LNP vaccine nor IL7-circRNA-LNP alone significantly altered these T-cell populations. Collectively, our findings demonstrate that IL7 enhances the anti-tumor activity of the hTROP2-circRNA-LNP vaccine by the expansion of cytotoxic and helper T cells. These results suggest that co-administration of IL7-circRNA-LNP with hTROP2-circRNA-LNP represents a promising strategy for human cancer immunotherapy.