Abstract 1795: Targeting the blood-brain tumor barrier with engineered tumor necrosis factor synergizes with temozolomide antitumor activity against glioblastoma

Abstract Background: The blood-brain tumor barrier (BBTB) is an obstacle in delivering anticancer drugs to brain tumors. Therefore, novel strategies aimed at targeting and breaching this structure are of high experimental and clinical interest. We developed a derivative of tumor necrosis factor-α (TNF) that can target and alter the BBTB. This drug, called NGR-TNF, consists of a TNF molecule fused to the Cys-Asn-Gly-Arg-Cys-Gly peptide (called NGR), a ligand of aminopeptidase N (CD13)-positive tumor blood vessels. Administration of low-dose NGR-TNF to patients with primary central nervous system lymphomas promotes BBTB alteration, increases the efficacy of R-CHOP (a chemo-immunotherapy regimen), and improves patient survival. Unfortunately, the inherent instability and heterogeneity of NGR-TNF have limited the further development of this drug. We have shown that fusing a serine to its N-terminus generates a new drug (called S-NGR-TNF) with increased stability, reduced heterogeneity and improved antitumor activity in preclinical lymphoma and sarcoma models, alone or in combination with chemotherapy. Methods: We evaluated the antitumor activity of murine S-NGR-TNF (5 ng/kg) alone or with temozolomide (TMZ, 20 mg/kg, administered 48 h and 96 h post S-NGR-TNF) in 2 glioblastoma (GBM) mouse models. These models involved intracranial implantation of GL261 cells engineered to express Luc2 firefly luciferase (GL261-Luc2) or Luc2 firefly luciferase along with the truncated human nerve growth factor-receptor (GL261/SR-Luc2 cells). Tumor growth was monitored using bioluminescent imaging, with pharmacological treatments starting 7 d after tumor implantation and repeated every 11-13 d for 2-4 cycles. Major immune cell populations infiltrating the tumors were assessed by FACS 48 h after S-NGR-TNF administration. Results: In the GL261-Luc2 model, S-NGR-TNF-treated mice showed a significant antitumor response compared to controls, with complete tumor regression in 4 out of 10 treated mice, while none in the control group showed a similar response. No treatment-related toxicity was observed. Cumulative results from 3 independent experiments using the GL261/SR-Luc2 model also showed significant antitumor effects. The S-NGR-TNF/TMZ combination group showed stronger antitumor effects than the groups treated with TMZ, S-NGR-TNF, or vehicle (19/30, 12/28, 12/29, and 6/28 cured mice, respectively). Analysis of GL261-Luc2 tumors after S-NGR-TNF treatment showed reduced tumor-infiltrating B lymphocytes, CD8+ T cells, Tregs and M2 macrophages. Conclusion: S-NGR-TNF synergizes with TMZ in GBM models, potentially by decreasing tumor-infiltrating immunosuppressive cells. Further preclinical studies combining S-NGR-TNF with TMZ and radiotherapy, the current standard of care for GBM, are warranted to evaluate its clinical application potential. Citation Format: Giulia Taiè, Anna Maria Gasparri, Federica Pozzi, Barbara Colombo, Federico Rossari, Nadia Coltella, Elisa Sangiovanni, Giulia Anderluzzi, Matteo Monieri, Tamara Canu, Elisa Bruno, Antonello Spinelli, Anna Palmisano, Antonio Esposito, Andres J. Ferreri, Imane Nafia, Corti Angelo, Flavio Curnis. Targeting the blood-brain tumor barrier with engineered tumor necrosis factor synergizes with temozolomide antitumor activity against glioblastoma [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 1795.