ARTICLE ABSTRACTDespite progress in adoptive T-cell therapies, the identification of targets remains a challenge. Although chimeric antigen receptors recognize cell-surface antigens, T-cell receptors (TCR) have the advantage that they can target the array of intracellular proteins by binding to peptides associated with major histocompatibility complex (MHC) products (pepMHC). Although hundreds of cancer-associated peptides have been reported, it remains difficult to identify effective TCRs against each pepMHC complex. Conventional approaches require isolation of antigen-specific CD8+ T cells, followed by TCRαβ gene isolation and validation. To bypass this process, we used directed evolution to engineer TCRs with desired peptide specificity. Here, we compared the activity and cross-reactivity of two affinity-matured TCRs (T1 and RD1) with distinct origins. T1-TCR was isolated from a melanoma-reactive T-cell line specific for MART-1/HLA-A2, whereas RD1-TCR was derived de novo against MART-1/HLA-A2 by in vitro engineering. Despite their distinct origins, both TCRs exhibited similar peptide fine specificities, focused on the center of the MART-1 peptide. In CD4+ T cells, both TCRs mediated activity against MART-1 presented by HLA-A2. However, in CD8+ T cells, T1, but not RD1, demonstrated cross-reactivity with endogenous peptide/HLA-A2 complexes. Based on the fine specificity of these and other MART-1 binding TCRs, we conducted bioinformatics scans to identify structurally similar self-peptides in the human proteome. We showed that the T1-TCR cross-reacted with many of these self-peptides, whereas the RD1-TCR was rarely cross-reactive. Thus, TCRs such as RD1, generated de novo against cancer antigens, can serve as an alternative to TCRs generated from T-cell clones.