In Situ Genetic Engineering of Tumors for Long-Lasting and Systemic Immunotherapy

Purpose: Cancer immunotherapy has shown clinical success but remains limited by low response rates, high cost, and the need for ex vivo cell manipulation or prior knowledge of patient-specific tumor antigens. A broadly applicable, antigen-agnostic strategy that genetically reprograms tumor cells in situ—without ex vivo cellular manufacturing—could reduce cost and broaden accessibility. ---

Hypothesis: If biodegradable poly(beta-amino ester) (PBAE) nanoparticles deliver plasmid DNA encoding the costimulatory molecule 4-1BBL (signal 2) and the cytokine IL-12 (signal 3) directly into tumor cells in situ, then tumor cells will coexpress these signals alongside intrinsic tumor antigen/MHC (signal 1), reprogramming them into “tumor-associated antigen-presenting cells” (tAPCs). This should activate cytotoxic T cells, reduce tumor growth, and generate long-lasting, systemic antitumor immunity without requiring a priori knowledge of tumor antigens or ex vivo cell manipulation. ---

Aims: - Synthesize and screen a library of PBAE polymers for safe and effective transfection of B16-F10 melanoma and MC38 colorectal carcinoma cells in vitro and in vivo. - Evaluate in vitro coexpression of 4-1BBL and IL-12 and activation of primary T cells and NK cells. - Test intratumoral tAPC-reprogramming nanoparticles, alone and with anti-PD-1 checkpoint blockade, in B16-F10 and MC38 mouse tumor models. - Analyze local and systemic immune responses, including tumor-infiltrating lymphocytes and splenic T cell specificity. - Assess whether treated long-term survivors resist tumor rechallenge at a distant site. ---

Delivery system: Platform: Synthetic, biodegradable, nonviral PBAE nanoparticles. Lead polymer: PBAE 5-3-49, selected from an array of PBAEs. Formulation: PBAE mixed with plasmid DNA at a 30:1 (w/w) polymer:DNA ratio; final DNA dose 5 µg per intratumoral injection. Payloads: - Plasmid DNA encoding 4-1BBL (costimulatory signal 2) - Plasmid DNA encoding IL-12 (cytokine signal 3) - Control plasmids: firefly luciferase (fLuc), GFP Physicochemical properties: - TEM: ~50–100 nm when dry - Number-average hydrodynamic diameter: 143 ± 6 nm - Intensity-weighted Z-average: 231 ± 3 nm - Zeta potential: +23.3 ± 0.9 mV Administration: Intratumoral (i.t.) injection; anti-PD-1 antibody administered intraperitoneally (i.p.). ---

Approach: In vitro: - B16-F10 and MC38 cells transfected with GFP/fLuc, 4-1BBL, IL-12, or both. - Co-culture with primary splenic CD8+ T cells or NK cells; IFN-γ measured by ELISA. - MTS assay for toxicity; flow cytometry for transfection efficiency and surface 4-1BBL. In vivo: - C57BL/6J mice bearing subcutaneous B16-F10 melanoma or MC38 colorectal carcinoma. - Treatment days 7, 9, and 11 after tumor inoculation: i.t. nanoparticles encoding control, 4-1BBL, IL-12, or 4-1BBL/IL-12. - Anti-PD-1 (200 µg on day 7, 100 µg on day 9, i.p.) or PBS control. - Efficacy groups: n = 7 mice; immune analysis groups: n = 4 mice. - Long-term survivors (no disease at day 50) rechallenged with tumor cells on the opposite flank at day 66. - Tumor size measured by caliper; survival recorded. ---