Abstract Immunotherapy is increasingly used in combination with current standard-of-care treatments such as chemotherapy that likely provides danger signals via immunogenic tumor cell death and activation of STING or TLR pathways. However, both the toxicity and immunosuppressive effects of chemotherapy risks hindering clinical benefit. Agonistic CD40 antibody is a potent activator of immune responses, acting upstream of immune checkpoint blockade (ICB) by driving T-cell priming. Here, we show that anti-CD40 is sufficient to drive potent T-cell responses against murine KPC (KrasG12D;Trp53R172H;Pdx-Cre) pancreatic tumors if combined with ICB. CD40/ICB was more effective than CD40/ICB/chemotherapy (median overall survival 76 days vs. 44 days in CD40/ICB vs. CD40/ICB/chemotherapy-treated mice, p<0.01). Therapeutic efficacy of CD40/ICB was fully independent of the STING, MyD88, and IFNAR pathways, requiring instead host expression of CD40, cross-presenting dendritic cells, and an intact T-cell compartment. Therapy-induced CD4 and CD8 T cell responses displayed enhanced polyfunctional cytokine secretion (48.9% vs. 29.5% of CD8+ T cells in vehicle control treated mice), cleared established primary tumors (43% vs. 0% in vehicle control or single-treatment cohorts p<0.0001), and formed protective memory responses in 57% of CD40/ICB cured mice. Immunosuppressive regulatory T cells were noticeably absent from the tumor site after CD40 treatment (7% of CD4+ T cells vs. 33% in mice treated with ICB alone, p<0.01), concomitant with improved T-cell activation and proliferation, whereas ICB was needed to potentiate the full activation and reinvigoration of the T-cell response both locally and systemically. The efficacy of CD40/ICB in in this ICB-resistant KPC model opens the possibility of optimized immunotherapy regimens free of the toxicity and immunosuppressive effects of cytotoxic therapy. This abstract is also being presented as Poster B08. Citation Format: Katelyn T. Byrne, Alexander H. Morrison, Robert H. Vonderheide. STING and TLR-independent activation of T-cell responses against pancreatic cancer using agonistic CD40 antibody [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR04.