This proposal seeks to clinically evaluate a novel tumor cell vaccination strategy, with human melanoma as the preferred tumor target. The ?costimulator-expressing? tumor cell vaccine advocated here breaks new ground in number of ways, by virtue of its: 1) reliance on protein transfer, as opposed to gene transfer, for neo-expressing costimulators on the tumor vaccine cells, which enables unprecedented quantitative control of surface costimula levels and simplifies the simultaneous delivery of more than one costimulator; 2) combinatorial use of costimulators directed towards the activation of multiple anti-tumor immune effectors, including T, NK, and dendritic cells; and 3) shift to intratumoral administration of the multi-component costimulator therapeutic, which constitutes a move to in vivo, from the more typical (and inherently more cumbersome) ex vivo, costimulator engineering of tumor cells. The preliminary data document the feasibility of applying an innovative Fc fusion protein transfer method for this in situ cancer vaccination in a murine tumor model. The major observation is that the injection of a ?tetra-costimulator? combination (with costimulators chosen to activate T, NK, and dendritic cells), in the form of palmitated protein A: costimulator-Fc conjugates, directly into a tumor bed results in both local tumor regression (with a prolific local inflammatory response) and signs of systemic anti-tumor immunity. The four specific aims of this proposal now seek to develop this vaccination strategy in greater depth and move into clinical trials. The first two preclinical aims call for casting a wider net amidst the pool of available costimulators and identifying ones that are optimal for melanoma (Specific Aim 1), along with resolving dosing and other issues that are peculiar to the intratumoral costimulator inoculation method (Specific Aim 2). In turn the baseline information gained will enable a Phase I clinical trial, which encompasses the evaluation of toxicity under Specific Aim 3) and immunological responses (under Specific Aim 4) in patients with metastatic whose tumors are directly injected with the preferred "multi-costimulator paints."