T-cell receptor (TCR)-based adoptive therapy is establishing itself as an effective approach in the field of cancer immunotherapy. Many studies have demonstrated that genetically engineered T cells with affinity-enhanced TCRs have higher antitumor activity and have demonstrated important promise in the clinic. Finding an optimal balance between TCR affinity and specificity, to optimize function but avoid autoreactivity of the engineered T cells, is key to ensure the safety of cancer patients. Therefore, the Michielin/Zoete lab previously developed a panel of affinity-enhanced TCRs targeting the HLA-A2 restricted NY-ESO-1157-165 peptide by computational design. In order to better understand the effect of TCR-peptide-major histocompatibility complex (pMHC) binding affinity on CD8+ T cell responsiveness against tumor cells in vitro, we assessed the killing capacity of NY-ESO-1-TCR-engineered T cells along with the expression of activation, inhibition and proliferation markers and cytokine production in human and murine models. Our findings can provide insight for improving immunotherapy of genetically engineered T cells with carefully selected tumor-specific affinity-optimized NY-ESO-1 TCRs.