a Hit compounds from the primary screen were tested on T-cell/spheroid co-cultures where both cell types were primed with the same antigen (CEF Peptide Pool), cell types were primed with different antigens (CEF Scramble) or spheroid monocultures with no T-cells (No T-cells). Anti-inflammatory compounds (NF-κB and IL-4 inhibitors) were shown to enhance T-cell spheroid-killing properties, where antigen-dependent interactions significantly increased compound efficacies. Other compounds, such as CDK4/6 or BRD inhibitors, demonstrated enhanced T-cell anti-tumor function in a mainly antigen-independent manner (Cytostatic). Several compounds targeting novel proteins not reportedly associated with enhanced T-cell cytotoxicity demonstrated potent antigen-dependent phenotypes. Spheroid sizes are quantitated in bar graphs on right, relative to DMSO controls. Scale bars indicate 100 µm. An independent sample t-test was used to calculate probability where *p < 0.05, **p < 0.01; n = 3 biologically independent samples and error bars represent standard deviation. b Viability readout of spheroids from a using HCT116-Luc reporter spheroids in the presence of T-cells and hit compounds with matching viral antigens (CEF Peptide Pool). NF-κB inhibitor demonstrated toxicity but limited potency, while the CDK4/6 inhibitor showed a predictably cytostatic response. Sonic Hedgehog inhibitor (SHHi, Cyclopamine), as well as a compound targeting an unidentified protein (GNF001), demonstrated high potencies and sigmoidal dose–response curves.