ABSTRACT Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrent mutations in driver genes that are restricted by prevalent HLA alleles. Here, we report on a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant (Mut) PIK3CA , among the most common genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this method, we developed a library of TCRs that recognize an endogenously processed neoepitope containing a common PIK3CA hotspot mutation that is restricted by HLA-A*03:01. Mechanistically, immunogenicity to this public NeoAg arises primarily from enhanced stability of the neopeptide/HLA complex caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a relatively “featureless” surface dominated by the peptide’s backbone. To overcome the challenge of binding such an epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface aided by an unusually long β-chain complementarity-determining region 3 (CDR3β) loop. In a pan-cancer cohort of patients with diverse malignancies that express the PIK3CA public NeoAg, we observed spontaneous immunogenicity, NeoAg clonal conservation, and in a limited number of cases, evidence of targeted immune escape. Together, these results establish the immunogenic potential of Mut PIK3CA , creating a framework for off-the-shelf immunotherapies targeting this public NeoAg.

Immunogenicity of a public neoantigen derived from mutatedPIK3CA