Abstract 3477: Engineered nanoparticles promote direct <i>in vivo</i> expansion and activation of tumor infiltrating lymphocytes for cancer growth inhibition

Abstract INTRODUCTION: TILs (Tumor infiltrating lymphocytes) therapy has been shown to be highly effective in melanoma and holds great potential for improving cancer immunotherapy in other cancers. The key limitation for TIL therapy is the complex process to activate and expand TILs. Thus, there is a strong need for refining strategies to enable TIL immunotherapy. To address this challenge, we hypothesized that we can directly activate and expand TILs in vivo, thereby eliminating the complex production process. We took advantage of a multifunctional nanoparticle (NP) platform to engineer NPs with multiple co-stimulatory molecules. We aimed to show that our in vivo approach can effectively stimulate TILs. METHODS: Co-stimulatory molecules functionalized NPs (TIL-NPs) were synthesized through reacting Azide-functionalized NP cores with DBCO-functionalized molecules. TIL-NPs were injected intratumorally to promote the expansion and activation of TILs in vivo. The MC38 colorectal tumor model was used for evaluating the effects of TIL-NPs on TILs activation. For the in vitro experiment, TILs from tumors of MC38-bearing mice were incubated with MC38 cells following various stimulation strategies. For the in vivo experiment, a bilateral tumor model was established, with only the right flank receiving TIL-NP injection. RESULTS: We successfully engineered NPs containing anti-CD3, anti-41BB and IL-2. These NPs were 126.2 nm in size. Per miligram NP conjugated with approximately 50μg immune activating molecules. TILs from MC38 tumors were incubated with TIL-NPs. Free antibodies were used as control. We found TILs stimulated by TIL-NPs demonstrated greater cytotoxicity against MC38 cells compared to the groups treated with free antibodies or unstimulated TILs. IFN-γ levels were elevated in all treatment groups, with the most significant increase observed in the TIL-NPs group. To determine if TIL-NPs increased T-cell activation in vivo, we evaluate the antitumor effects of TIL-NPs in a bilateral tumor model. One tumor received TIL-NPs while the other tumor was used to assess systemic treatment responses. Intratumorally injected TIL-NPs were highly effective in eradicating the injected tumor when compared to that of control and free antibodies groups. All of the tumors on the right flank/injected completely regressed, achieving a 100% complete response (CR) rate. Importantly, the TIL-NPs also provided a robust systemic response, providing 60% CRs in the contralateral flank tumors. Overall, TIL-NPs significantly prolonged survival of mice. Functionalizing these molecules on NPs also induced clustering of their respective receptors and improved signaling. CONCLUSION: TIL-NPs engineered with multiple co-stimulatory molecules can directly stimulate TILs in vivo, providing a systemic immunotherapy response against cancer. Citation Format: Xin Wen, Lifu Ruan, Longzhen Zhang, Tian Zhang, Andrew Wang. Engineered nanoparticles promote direct in vivo expansion and activation of tumor infiltrating lymphocytes for cancer growth inhibition [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 3477.