Intratumoral Electroporation Of Plasmid-Encoded Il-12 And Membrane-Bound Anti-Cd3 Increases Tumor Immunogenicity And Augments The Function Of T Cell Subsets

Intratumoral (IT) delivery of plasmid IL-12 (tavokinogene telseplasmid; tavo) via electroporation (EP), collectively referred as IT-tavo-EP, generates immunologically-relevant levels of localized IL-12, triggering regression of both treated and distant tumors with minimal toxicity in preclinical and clinical studies. Our previous clinical trial data from melanoma patients treated with IT-tavo-EP identified a treatment-related increase of infiltrating T cells and transcripts related to immune activation, as well as a significant increase in the IFN-γ score of patients with a clinical benefit, suggesting that CD3+ tumor-infiltrating lymphocytes (TIL) may be critical in maximizing the anti-tumor effects of IT-tavo-EP. Furthermore, in-house biomarker data have identified an abundance of non-tumor reactive TIL that, if mobilized could additionally contribute to a clinical response. Accordingly, a plasmid-encoded membrane-bound polyclonal T cell-stimulating anti-CD3 (αCD3) hybrid antibody (scFv) was developed and used in combination with tavo (IT-tavo-αCD3-EP) to broaden the scope and depth of the T cell response. We previously demonstrated that membrane expression of αCD3 on neoplastic and stromal cells could activate CD3+ TIL, driving enhanced proliferation and cytotoxicity in a B16-OVA murine model. Here, using immune profiling of the tumor microenvironment (TME), we have demonstrated that this membrane-bound αCD3 therapeutic can significantly upregulate frequencies of CXCR3+CD8+ T cells and short-lived effector T cells, while reducing PD-1 expression on CD8+ T cells in vivo. Critically, naive T cells, Treg cells, and exhausted T cells (subsets not typically associated with strong anti-tumor responses) displayed enhanced effector function (IFN-γ and granzyme B release) with engagement of membrane-bound αCD3 and IL-12. Furthermore, we found that this therapeutic approach could equally enhance proliferation of T cells regardless of the affinity for their cognate peptide:MHC, suggesting a TCR independent mechanism. Collectively, these observations demonstrate that IT-tavo-αCD3-EP can mobilize broad subsets of T cells beyond dominant anti-tumor effectors demonstrated. Thus, while enhanced cytolytic function is associated with this therapy, inclusion of additional atypical anti-tumor T cell subsets may also promote reshaping of the TME by production of effector cytokines upon engagement of surface-bound αCD3. Moreover, functional restoration of TIL isolated from a melanoma patient with active clinical progression on anti-PD-1 therapy, was possible with engagement of membrane-bound αCD3 in the presence of IL-12. Collectively, these data continue to support the utility of IT-tavo-αCD3-EP as a promising therapeutic approach for patients with melanoma and other accessible solid tumors. Citation Format: Mia Han, Anandaroop Mukhopadhyay, Bianca Nguyen, Jack Y. Lee, Erica Browning, Jon Salazar, Reneta Hermiz, Lauren Svenson, Chris Baker, Daniel O9Connor, Kellie Malloy, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of plasmid-encoded IL-12 and membrane-bound anti-CD3 increases tumor immunogenicity and augments the function of T cell subsets [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-390.