P18.12.a CTX-Cnf1 Boosts Glioblastoma Survival by Reprogramming the Immune Microenvironment

Abstract BACKGROUND Glioblastoma (GB) is an aggressive brain tumor with limited treatment options and poor prognosis. Immune evasion is a hallmark of GB, contributing to resistance against conventional and emerging therapies. This study investigates the therapeutic potential of CTX-CNF1, a novel recombinant protein combining cytotoxic necrotizing factor 1 (CNF1)—which impairs glioma cell proliferation, preserves neuronal function, and acts as an immune adjuvant—with chlorotoxin (CTX), a peptide capable of crossing the blood-brain barrier and selectively targeting glioma cells. MATERIAL AND METHODS Two syngeneic GB mouse models (GL261 and CT-2A) were used to evaluate the in vivo effects of systemic CTX-CNF1 administration on survival, tumor burden, motor performance, and immune response. Immune modulation was assessed using flow cytometry, immunohistochemistry, ELISA, and PCR-based transcriptomic analyses. CTX-CNF1 was also tested in combination with anti-PD-1 immune checkpoint blockade. RESULTS CTX-CNF1 treatment significantly prolonged survival, reduced tumor volume, and improved motor deficits in tumor-bearing mice. Notably, over 50% of treated GL261 mice achieved complete tumor eradication and resisted glioma rechallenge, indicating durable immune memory. Mechanistically, CTX-CNF1 enhanced intratumoral infiltration and activation of cytotoxic CD8+ T cells and promoted pro-inflammatory reprogramming of tumor-associated macrophages. No signs of systemic toxicity were observed. Co-administration with anti-PD-1 further extended survival. CONCLUSION CTX-CNF1 effectively remodels the GB immune landscape, converting the tumor into a more immunogenic state and enhancing anti-tumor responses. These findings support its potential as a novel immunotherapeutic strategy for GB, warranting further clinical translation.