Discovery and Targeting of a Cryptic Human Proteome

First-in-class therapeutics require first-in-class biology. Yet despite decades of genomic and proteomic cataloging, vast regions of the human transcriptome remain dark and their encoded proteins invisible. Here we present RyboCypher™, an integrated RNA-sequencing and AI-assisted proteogenomics platform that systematically maps the RyboCypher-derived “dark” transcriptome to unannotated peptides, predicting and empirically identifying cryptic proteins across the uncharted genome. Applied to cancer cell lines, patient tumors, and matched healthy tissues, RyboCypher resolved ∼8.3 million dark RNA isoforms and ∼16 million candidate ORFs. Interrogating these against ∼0.5 billion MS/MS spectra from cellular proteomics, membrane proteomics, and immunopeptidomics datasets (comprising a total of >8,000 raw MS data files (∼7TB of MS data), derived from 2,229 patient samples), we empirically identified ∼80,000 cryptic peptides (∼10,000 cancer-associated or cancer-upregulated) at <1% FDR. Altogether, these datasets establish the CypherAtlas™, a comprehensive proteogenomic atlas of an unreported proteome comprising thousands of novel proteins, including membrane proteins with targetable extracellular domains, and intracellular proteins accessible through antigen presentation. By linking dark-RNA transcripts, predicted proteins, and patient-level metadata across RyboDyn’s proprietary experimental data, CypherAtlas further provides the training substrate for multi-modal models such as DarkCypher™, which is being developed to prioritize cryptic targets and to forecast their expression in new patient samples. As proof of therapeutic potential, we disclose evidence for a cancer-associated, cryptic protein expressed from the YBX1 locus, (cryptic YBX1; cYBX1) and demonstrate selective in vitro tumor cell killing through a cryptic peptide-MHC (pMHC) complex derived from this protein with a TCR-mimic (TCRm) antibody when formatted as antibody drug conjugates (ADCs). Together, RyboCypher and CypherAtlas establish the dark proteome as a vast and previously inaccessible reservoir of novel targetable biology, laying the foundation for the next generation of first-in-class therapeutics.