BACKGROUND: Neoantigen-specific peripheral blood lymphocytes (NeoPBL) are the circulating counterparts of neoantigen-specific tumor-infiltrating lymphocytes (TILs). A recent study showed NeoPBL exist in a less differentiated state in vivo than TIL, which, along with their non-invasive potential, makes them attractive for cell therapy. However, NeoPBL circulate at ultra-low frequencies, usually less than 0.005%, posing a major translational barrier for their clinical use. METHODS: Here, we used NeoSelect, a peptide-based cyclic enrichment and expansion strategy that leverages magnetic selection of antigen-presenting cell-NeoPBL conjugates ex vivo to increase NeoPBL frequency by several thousand-fold over 28 days while also achieving large-scale expansion. We then evaluated expanded cells using functional, transcriptomic, and epigenetic profiling. RESULTS: Most NeoPBL clonotypes amplified by NeoSelect are detectable as exhausted TIL in vivo. In addition, neoantigen-specific TIL that failed to expand during ex vivo culture could be recovered as NeoPBL. Transcriptional and epigenetic analyses reveal that ex vivo NeoPBL have distinct transcriptional and epigenetic programs compared with TIL and retain expression of and chromatin accessibility at genes associated with stemness (TCF7, KLF2) and costimulation (CD28, CD40LG). CONCLUSIONS: NeoPBL encompass physiologically relevant antitumor clonotypes, can be rapidly expanded to clinically useful numbers and frequencies ex vivo, and can be used to expand the targetable neoantigen repertoire. By combining broad potential applicability and minimal invasiveness, these results position NeoPBL as a clinically streamlined and biologically distinct autologous cell platform that may be useful for adoptive cell transfer.