Abstract Immunotherapy has come of age and is truly a breakthrough in the treatment of cancer. Immune checkpoint inhibition is now an established and effective modality for treatment of multiple tumor types. Combination immunotherapy creates even more potential for efficacy by targeting synergistic immune pathways. However, systemically administered immunomodulators can activate T-cells non-specifically resulting in off-target toxicity, which is dose-limiting and potentially lethal in combination. Intratumoral administration of immunotherapeutic agents has several advantages including 1) higher concentrations of agents in close proximity to target antigens and tumor-infiltrating lymphocytes 2) lower doses overall with less systemic exposure and toxicity 3) potentially a novel mechanism of action such as depletion of intratumoral Tregs 4) potential for an abscopal effect, acting as an in-situ cancer vaccine. Here we report a novel combination of immunomodulators, anti-CTLA4, CD137 and OX40 administered by intratumoral route in a mouse lymphoma model (A20) as well as a colon cancer model (MC38). CTLA4 is an immune checkpoint, and CD137 and OX40 are members of the TNF receptor superfamily - all agents in clinical practice or clinical trials. For both the A20 and MC38 cell lines, a dual tumor model was used in which tumor cells were inoculated bilaterally on the flanks. BALBC mice were used for the A20 model and C57BL/6 mice for MC38. Treatment was started approximately day 8 when there were visible tumors between 5-7mm in diameter. Intratumoral injection was to the left tumor only. Injection of the triple combination at doses about 1/10 that of usual systemic doses (30 ug each) resulted in significant tumor growth inhibition/regression and a significant survival advantage for both the A20 and MC38 models. For the A20 model, at doses of approximately 1/40 systemic doses (10ug each) the triple combination resulted in tumor clearance in 70-100% of mice, and significantly prolonged survival. There was no toxicity observed. The majority of the mice remained tumor free, and did not grow tumor when rechallenged with A20 cells. The response was shown to be CD8 T-cell dependent, and in vivo CD8 depletion, but not CD4 depletion led to complete abrogation of the treatment effect. A memory t-cell response was confirmed in vitro, with CD8CD44(hi) T-Cells demonstrating increased IFN-g production when incubated with A20 cells, but not control cells (4T1 breast ca). Intratumoral administration at low doses was more effective than 2 systemic routes - intraperitoneal (IP) and subcutaneous (SC), supporting a local in situ vaccine effect. Interestingly, in single tumor model, SC injection in proximity to the tumor draining lymph node (DLN) was significantly more effective than SC injection close to a non-tumor DLN, suggesting that the tumor DLN may be equally involved as the tumor microenvironment in establishing an in situ vaccine effect. Finally, in a single tumor model, a cell based vaccine of irradiated A20 cells co-injected subcutaneously with the low dose triple combination at a site distant from the tumor and tumor DLN had anti-tumor effects greater than the triplet alone SC at the same location, but less than IT or when injected SC in proximity to the tumor DLN. Thus we demonstrate that this novel combination of immunomodulators delivered intratumorally induces a dramatic anti-tumor effect. At low doses, the triple combination is more effective when delivered IT than by systemic routes and the anti-tumor immune response is completely dependent on CD8 T-cells, supporting an in situ vaccine effect. Importantly, it appears both the tumor microenvironment and DLN are important to the anti-tumor immune response. Injecting in the region of the tumor DLN may be a viable and effective option clinically when an injectable tumor site is not easily accessible. The A20 cellular vaccine delivered together with the low dose triple combo at a site distant from the tumor was more effective than triple combo alone, but less effective than intratumoral, suggesting that elements of the tumor microenvironment and/or tumor DLN are important for full effect. Overall these results demonstrate potent anti-tumor effects of the triple combination anti-CTLA4, -CD137 and -OX40, all agents in clinical practice or clinical trials, and support the intratumoral route as safe and highly efficacious route of administration. Disclosures Hebb: Alligator Biosciences: Research Funding. Ellmark:Alligator Biosciences: Employment. Norlen:Alligator Biosciences: Employment. Felsher:Alligator Biosciences: Research Funding.