Immunopeptidomic discovery of fetal WNT-associated antigen NKD1 enables HLA-A2+ restricted TCR-T therapy for MSS mCRC

T cell-engaging therapies have achieved limited success in microsatellite stable (MSS) metastatic colorectal cancer (mCRC), in part because of a paucity of truly cancer-specific targets. Systematic interrogation of the immunopeptidome, the repertoire of peptides presented by human leukocyte antigen (HLA) class I molecules, can expand the pool of druggable antigens beyond conventional surface receptors. Nonetheless, both neoantigenic driver mutations and recently described oncofetal peptides are rare or absent in most CRCs. Our recent work shows that mCRC cells adopt a highly stereotyped fetal-like phenotype, characterized by activation of a developmental WNT signaling program that is further enriched in metastasis-initiating cells and conserved across diverse patients. We hypothesized that this transcriptional reprogramming generates a cancer-specific, developmentally fixed HLA-I ligandome that can be exploited for T cell therapy. Using an integrated platform for systematic collection and multimodal profiling of matched normal colon, primary tumor, and metastases from patients undergoing CRC surgery, we established ex vivo patient-derived organoids (PDOs) that faithfully capture patient-specific CRC cell states and provide an effectively inexhaustible source of tumor cells for immunopeptidomic discovery and functional validation. In HLA-A2 + PDOs, representing the most common HLA allele, immunopeptidomic analyses identified recurrent presentation of peptides derived from fetal WNT program genes, in particular NKD1, that are prevalent across mCRC PDOs from HLA-A2 + patients and undetectable in healthy tissues. These peptides were immunogenic in vitro, eliciting robust reactivity from healthy donor T cells and supporting their suitability as therapeutic targets. Moreover, naive CD8 + T cells engineered with NKD1-specific T cell receptors (TCRs) showed strong cytokine activation and potent, antigen-dependent cytotoxicity against mCRC PDOs without detectable off-target activity, consistent with a favorable therapeutic index. Together, these data nominate NKD1 as a conserved antigen in mCRC and support the clinical advancement of an NKD1-directed TCR-T cell product as a first-in-class precision therapy for patients with MSS mCRC.