Amplification Of The Cxcr3/Cxcl9 Axis Via Intratumoral Electroporation Of Cxcl9 Synergizes With Il-12 Gene Therapy (Tavo) To Elicit Robust Anti-Tumor Immunity

Clinical and preclinical studies have demonstrated that plasmid IL-12 (tavokinogene telseplasmid; TAVO) delivered intratumorally via electroporation drives IFN-γ expression and recruits T cells to the tumor microenvironment, ultimately yielding durable systemic T cell responses. Interrogation of longitudinal biomarker data from our IL-12/anti-PD-1 clinical trial has identified that clinical responses are closely tied to intratumoral CXCR3 levels. While all patients had a similar frequency of activated CD8+ T cells in the periphery, responding patients had a significant increase of intratumoral CXCR3 transcripts post-treatment (p=0.03) compared to nonresponding patients (p=0.4), underscoring the clinical relevance of tumor-infiltrating CXCR3+ immune cells. Since the IFN-γ/CXCL9/CXCR3 axis is known to increase sensitivity to anti-PD-1 therapies, we hypothesized that combining intratumoral TAVO with a DNA-encoded locally secreted CXCL9 (cognate ligand for CXCR3) would further augment/restore this axis and ‘license9 a robust anti-PD-1 response beyond the treated lesion. An appropriate CXCL9 gradient can productively modulate frequencies of tumor infiltrating tumor-reactive CXCR3+ T cells. We have previously demonstrated that intratumoral electroporation of plasmid IL-12 and CXCL9 elicits a robust antitumor immune response evidenced by increased systemic antigen-specific CD8+ T cells and improved regression of both treated and contralateral CT26 tumors. In the current study, we demonstrate that an enhanced CXCL9 gradient via intratumoral electroporation leads to efficient trafficking of CXCR3+ CD8+ T cells into CT26 tumors. We further explored how DNA-encodable IL-12/CXCL9 can work together to improve checkpoint inhibitor response. We demonstrated that intratumoral TAVO rapidly drives a population of CXCR3+ CD8+ T cells in the lymph node and importantly, depletion of these CXCR3+ immune cells abrogated an IL-12-mediated anti-tumor response. Furthermore, upon electroporation of IL-12 and CXCL9, transcriptomic analysis of the tumor microenvironment revealed an enrichment of genes associated with immune-related pathways (IFN-γ signaling, interleukin signaling, GPCR signaling), antigen presentation machinery, and TCR signaling, indicating that this combination therapy augments anti-tumor immunity. Lastly, leveraging the partially responsive anti-PD-1 CT26 tumor model, we demonstrated that intratumoral electroporation of plasmid IL-12 with CXCL9 significantly improved anti-PD1 response, providing a strong rationale for filing an Investigational New Drug application based on this intratumoral DNA-encodable combination approach.Intratumoral CXCL9 with IL12 Citation Format: Anandaroop Mukhopadhyay, Bianca Nguyen, Jack Y. Lee, Mia Han, Jon Salazar, Reneta Hermiz, Lauren Svenson, Erica Browning, Daniel J. O9Connor, David A. Canton, Daniel Fisher, Joseph Skitzki, Christopher G. Twitty. Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of CXCL9 synergizes with IL-12 gene therapy (TAVO) to elicit robust anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-396.