Direct in vivo activation and functional reprogramming of human TILs using a multifunctional nanoengager in humanized renal carcinoma

INTRODUCTION: Tumor-infiltrating lymphocytes (TILs) therapy has demonstrated remarkable clinical success in melanoma. However, its application in other cancers has been limited by high manufacturing cost and toxic conditioning regimen. To overcome these limitations, we aimed to develop a novel strategy to directly activate and expand TILs in vivo using a multifunctional nanoengager (TIL-NPs). In this study, we report the validation of TIL-NPs using mouse models of cancer, including a humanized renal carcinoma model that closely recapitulates human immune-tumor interactions. METHODS: TIL-NPs were generated by conjugating azide-functionalized PEG-PLGA nanoparticles with DBCO-modified anti-CD3, anti-4-1BB, and IL-2. Their ability to activate and expand TILs was assessed using both MC38 and 786-O models. For the MC38 study, mice bearing bilateral tumors received intratumoral TIL-NPs in the right flank tumor only, and TIL activation was further evaluated using isolated MC38 TILs co-cultured with MC38 cells in vitro. To assess human relevance, TIL-NP-mediated expansion of human TILs was tested using renal cell carcinoma (RCC) patient samples, and a humanized RCC model was established by inoculating 786-O cells into NSG mice engrafted with human PBMCs. RESULTS: We engineered multifunctional nanoparticles (TIL-NPs) displaying anti-CD3, anti-4-1BB, and IL-2, measuring ~126 nm with ~50 μg of immune agonists conjugated per mg NP. In vitro, MC38 tumor-derived TILs stimulated with TIL-NPs showed markedly enhanced cytotoxicity against MC38 cells and produced the highest IFN-gamma levels compared with free antibodies or unstimulated TILs. In vivo, intratumoral delivery of TIL-NPs in a bilateral MC38 model induced complete regression of all injected tumors and generated a strong systemic response, with substantial regression of contralateral tumors and improved survival. Using RCC patient-derived TILs, TIL-NPs also enhanced cytotoxicity against 786-O and ACHN cells relative to free antibodies. In a humanized RCC model, TIL-NPs produced near-complete regression of treated tumors and markedly suppressed contralateral tumor growth. Immunohistochemistry confirmed dense CD8⁺ T-cell infiltration in TIL-NP-treated tumors, indicating enhanced activation and recruitment of cytotoxic T cells. Overall, TIL-NPs strengthened T-cell signaling through receptor clustering, prolonged survival, and elicited potent local and systemic antitumor responses. CONCLUSION: TIL-NPs potently activate and reprogram TILs in vivo, enhancing cytotoxicity and systemic antitumor immunity, highlighting their translational potential for adoptive T-cell therapy in solid tumors.