Neoantigen-specific TCR sequences are identified from patients with melanoma immunized with personal neoantigen vaccines. (A) TCR repertoire was assessed in PBMCs collected from melanoma patients (patients 1 and 3) ∼16 weeks after initiation of personalized neoantigen vaccination.²⁷  (B) Neoantigen-reactive CD4⁺ and CD8⁺ T cells from patients 1 and 3, respectively, were isolated on the basis of IFNγ secretion after exposure of PBMCs to pools of 15 to 16 mer-length (CD4) and 9 to 10 mer-length (CD8) peptides corresponding to the immunizing peptides (*neoantigens previously demonstrated to be immunogenic based on bulk PBMC analysis). (C) Targeted single-cell TCR sequencing reveals multiple paired TCR sequences enriched in the neoantigen-reactive repertoires of patients 1 and 3. Red for downstream cloning and expression; open triangle for antigen-matched TCRs. (D) One of 6 reconstructed CD4⁺ TCRs from patient 1 was matched with mut-RUSC2-2 (mapping of this peptide within the original IMP indicated) per detection of reactivity by IL-2 ELISA. Functional avidity of the mut-RUSC2-2-specific P1.6 TCR against mutant and corresponding wild-type (WT) peptide was determined using autologous APCs (P = .0003, by 2-sample t-test with Welch’s correction from 4 technical replicates). (E) Three reconstructed CD8⁺ TCRs specific for mut-VPS16 were identified from patient 3 per screening by IL-2 ELISA, and mapping of the cognate peptide within the IMP is shown. The peptides to test against the TCRs were selected on the basis of reactivities against the peptides in bulk in vitro cultures (supplemental Figure 3). Functional avidity for mut- and WT-VPS16 by the P3.3, P3.6, and P3.7 TCRs was determined using B721.221 HLA-B*27:05 cell line pulsed with VPS16 peptides by IL-2 ELISA. Significant difference in IL-2 response was measured at 1 μg/mL mut-VPS16 peptide and corresponding WT peptide. (F) HLA-B*27:05 restriction of mut-VPS16 was verified by coculture of TCR-expressing reporter cells with peptide-pulsed K562 cells expressing HLA-A*02:01 or B721.221 cells expressing HLA-A*03:01 or HLA-B*27:05.