Abstract 2252: ASO targeting of intratumoral human FOXP3+ Tregs in humanized SGM3-IL15 mice

Abstract Introduction: Humanized mice are an indispensable tool for pre-clinical cancer immunotherapy research, but there are limited data regarding human antitumor T cell immunity in such models, especially regarding the development of intratumoral T cell exhaustion. Methods: We wanted to develop a Treg-dependent tumor model in humanized mice to study the effect of AUMsilence FOXP3 antisense oligonucleotides (ASO) targeting of intratumoral human CD4+ FOXP3+ regulatory T cells (Tregs) in vivo. The ideal model would support development and function of human Tregs, CD4+ and CD8+ T cells, allow tumor growth without apparent allogeneic reactivity, demonstrate the development of a suppressive tumor microenvironment, including with exhaustion of tumor-infiltrating T cells, and prove to be a Treg dependent tumor model. To do so, we evaluated two novel JAX strains of humanized CD34+ engrafted mice, NSG-SGM3 and NSG-SGM3-IL15. Results: Both strains of mice had high (>80%) reconstitution by human CD45+ cells, no signs of GVHD, and increased proportions of FOXP3+ Tregs in their spleens, blood and livers (23-35% of CD4+ cells). Suppressive function of isolated liver and splenic Tregs were comparable with healthy donor blood Tregs. Both strains of mice supported similar growth of A549 lung tumor cells without rejection, and tumors were infiltrated by human CD45+ cells (8-20%), including 15-35% of FOXP3+ Tregs. Expression of T cell exhaustion markers was observed in tumors, but not in spleens, livers or blood. FOXP3 ASO treatment resulted in a decrease in tumor growth, while scramble treated mice developed multiple lung metastases and larger tumors. FOXP3 expression was also decreased in tumors (2.7 times, p =0.02) but not in the spleens and livers of ASO treated mice. Of note, targeting of intratumoral Tregs led to decreased expression of most T cell exhaustion markers in tumors, confirming our previous report that intratumoral Treg may promote T cell exhaustion. Conclusions: NSG-SGM3 and NSG-SGM3-IL15 mice provide a platform for preclinical drug testing involving an immunosuppressive tumor microenvironment and accumulation of exhausted T cells and fully functional Tregs. AUMsilence FOXP3 ASO therapy has powerful anti-tumoral effects in vivo, decreases metastases and reverses features of T cell exhaustion. Citation Format: Wayne W. Hancock, Tatiana Akimova, Liqing Wang, Zhanna Bartosh, Lanette M. Christensen, Pali Kaur, Veenu Aishwarya. ASO targeting of intratumoral human FOXP3+ Tregs in humanized SGM3-IL15 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2252.