Targeting the evolving immunopeptidome of metastatic colorectal cancer There has been limited success of chimeric antigen receptor (CAR) T cell therapies in solid tumors, in part, due to the inability to identify cancer-specific targets. Exploring the immunopeptidome, peptides presented by human leukocyte antigen (HLA) class I molecules, can expand the repertoire of targetable antigens. Our recent findings show that metastatic colorectal cancer (mCRC) cells adopt a highly stereotyped fetal-like phenotype, characterized by activating a developmental WNT-signaling signature as a mechanism of cell-fate reprogramming. This transcriptional signature is accentuated in metastasis initiating cells relative to the primary tumor and is highly conserved across diverse patients. Therefore, we hypothesize that these changes will translate into a cancer specific immunopeptidome and reveal a conserved and targetable sequence. Using our established integrated platform for the collection and multimodal analysis of resected normal colon, primary and metastatic lesions from patients undergoing CRC surgery, we have harnessed ex vivo patient-derived organoids (PDOs) that recapitulate patient- specific CRC cell states. Preliminary analysis using PDOs from HLA-A*02:01+ patients, the most common HLA allele, identified peptide targets derived from these fetal WNT-pathway gene states, specifically NKD1, that are both specific and prevalent in CRC and are lacking in healthy tissues. Furthermore, we showed that these peptides retain their immunogenicity based on healthy donor T cell reactivity. In Aim 1 (K99), we will systematically elucidate the evolving immunopeptidome from normal colon, primary tumor and metastatic lesion PDOs through mass-spectrometry and identify conserved intracellular targets for therapeutic interventions. In Aim 2 (K99), we will validate the biological role of NKD1 in CRC development, invasion, and metastasis by modulating its expression in metastatic lesion PDOs in an orthotopic xenograft mouse model. In Aim 3 (R00), we will identify NKD1-reactive T cell receptor (TCR) clones from HLA-A*02:01+ CRC patient’s peripheral blood to develop engineered TCR- T cell therapies. Drawing on my expertise in human immunology and medical oncology, along with the mentorship of prominent CRC biologist Dr. Karuna Ganesh and immunopeptidome-based therapy expert Dr. David Scheinberg at the outstanding Memorial Sloan Kettering Cancer Center, I am well- equipped to address these research questions. This foundation will enable me to master the necessary techniques, acquire knowledge, and develop the leadership skills required to emerge as a leading independent researcher and physician-scientist in the field of immuno-oncology.

Metastatic colorectal cancer (mCRC) cells exhibit an essential and unique fetal WNT-signaling signature for dissemination and renewal, consistently observed across multiple patients despite varying genomic mutations. Immunopeptidomic analysis of patient-derived organoids (PDOs), which provide a an effectively inexhaustive reservoir of clinically relevant cancer cells, reveals the presentation of peptides associated with fetal WNT signaling on the cell surface, offering insights into novel cancer targets. Through detailed and comprehensive studies of PDOs, we aim to uncover the evolving immunopeptidomes in advanced disease and the role of NDK1 in the establishment of metastases, paving the way for identifying T cell receptors for novel cellular therapeutic strategies.