502 Delivery of hexameric TIGIT-Fc-LIGHT as lipid-encapsulated mRNA achieves therapeutic exposures that potentiate immune cell activation and anti-tumor activity in the setting of CPI acquired resistance

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Background Previously, we characterized the bispecific Fc fusion protein (TIGIT-Fc-LIGHT) that blocks the PVR checkpoint axis and agonizes TNFRs through LIGHT. In a preclinical tumor model of acquired resistance to PD-1 blockade (CT26/AR), exposure to TIGIT-Fc-LIGHT promoted antitumor activity, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade. LIGHT can promote vascular remodeling through LTbR/HVEM and linking LIGHT to a vascular targeting peptide (VTP) is one strategy to enrich HEV/TLS formation in the TME. TNFRs (e.g. HVEM/LTbR) are kinetically regulated and localizing co-stimulation to a tissue may not be sufficient for maximum efficacy in the absence of durable exposure to the TNF-ligand that aligns with the expression pattern of the target receptor. We hypothesized that tumor checkpoint receptor engagement via TIGIT could also localize TIGIT-Fc-LIGHT to the TME and when TIGIT-Fc-LIGHT is delivered as an mRNA, could prolong therapeutic exposures that match the expression of the cognate TNF-receptors; to promote combined HEV/TLS formation, checkpoint blockade, and anti-tumor immune responses. Methods Mouse TIGIT-Fc-LIGHT fusion protein and mRNA/LNP were synthesized and delivered into naïve or CT26/AR bearing Balb/c mice. Serum/tissue expression of TIGIT-Fc-LIGHT and cytokines were assessed using MSD. TIL were immune phenotyped using flow cytometry. Tumors were measured 3x weekly. For HEV/TLS analysis, RIP-Tag mice bearing established pancreatic neuroendocrine tumors were given mTIGIT-Fc-LIGHT IV. After 2 weeks of treatment, tumors were isolated and assessed by IF for MECA79, CD3, CD31, CD68 and F4/80. Results In vivo delivery of mRNA/LNP encoding TIGIT-Fc-LIGHT led to rapid production of functional hexameric fusion proteins in vivo, increasing overall exposure (AUC) relative to the recombinant protein by ~28-fold over 96 hours in serum and 135-fold higher within tumors. This resulted in significant tumor growth delay, which improved in combination with anti-PDL1 in CT26/AR. This anti-tumor activity was associated with increased serum innate and adaptive immune cytokines and tumor infiltration of antigen-specific CD8+ T cells. In the RIP-Tag pancreatic neuroendocrine model, TIGIT-Fc-LIGHT increased intratumoral MECA79 staining (HEV), TLS formation, activation of intratumoral myeloid cells, and infiltration of cytotoxic T cells. Conclusions Our findings underscore the potential of biologics as lipid-encapsulated mRNAs. The delivery of TIGIT-Fc-LIGHT mRNA resulted in high expression in the serum and tumor following a single administration. TIGIT-Fc-LIGHT protein exposure was associated with tumor vasculature remodeling, promoting immune cell infiltration and anti-tumor efficacy. The versatility of mRNA therapeutics and the ability to fine-tune expression/exposure could mark a significant advancement in immunotherapy. Ethics Approval All murine animal studies have been conducted by and with the approval of an Institutional Animal Care and Use Committee (IACUC) and reviewed and approved by a licensed veterinarian.

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