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Background Patients with melanoma refractory to PD-1 inhibitors have limited therapeutic options. Personalized neoantigen vaccines can induce tumor-specific T-cell responses, but their efficacy in the PD-1-refractory setting remains uncertain. This phase I study evaluated the immunologic impact of the neoantigen peptide vaccine PNV-021 in PD-1-refractory melanoma.Methods Eight patients received up to 20 neoantigen peptides, predicted to bind HLA class I/II, every four weeks alongside Poly-ICLC and nivolumab. Peripheral blood mononuclear cells (PBMCs) were collected every 4 weeks and tumor biopsies were collected before and 12 weeks post-vaccination. Tumor biopsies underwent single-cell RNA and TCR sequencing. In four patients with either progressive disease (Pt5), stable disease (Pt13), or partial responses (Pt6, Pt16), vaccine-specific T cell responses were assessed by ELISpot using PBMCs. The 25–55 most expanded or de novo tumor-associated TCRs at week 12 were reconstituted in Jurkat reporter cells and tested for reactivity against patient-derived tumor cell lines and autologous B cells pulsed with vaccine peptides or TAAs. Reactivity was defined by reporter activation.Results Vaccination induced expansion and persistence of neoantigen-reactive T cells in peripheral blood ( figure 1a). Week 12 biopsies from all four patients showed increased immune-cell infiltration compared to enrollment biopsies (figure 1b). In Pt5 (PD), 47% (26/55) of reconstituted tumor expanded lymphocyte (TEL) TCRs were reactive to the tumor cell line; eight targeted vaccine neoantigens (HSP90AB1 A498V, CDC37 A232V), and three were cross-reactive to the MelanA TAA. In Pt13 (SD), 64% (16/25) of TELs were tumor-reactive; none were specific vaccine peptide, while three targeted TAAs (tyrosinase, MelanA, PRAME). For Pt16 (PR), 32% (8/25) of TELs were tumor-reactive; the most abundant TCR recognized a vaccine-targeted neoantigen (ERN1.00 P97S), and one recognized tyrosinase. For Pt6 (PR), 70% (33/47) of TELs were tumor-reactive; one recognized a non-vaccine neoantigen (LDLR G287S), and ten targeted TAAs including gp100, tyrosinase, or tyrosinase-related protein 2 (TYRP2). Across patients, tumor-reactive clones—regardless of antigen specificity—expressed activation/exhaustion-associated genes (CXCL13, ENTPD1) (figure 2).Conclusions PNV-021 neoantigen vaccination induced strong immune activation in PD-1 refractory melanoma. All patients showed increased immune infiltration within the tumor, with TILs recognizing both vaccine-targeted neoantigens and non-targeted neoantigens/TAAs. These results suggest that PNV-021 alongside polyICLC and nivolumab induces/expands tumor-specific responses beyond targeted epitopes. Analyses of response and resistance mechanisms are ongoing.Abstract 602 Figure 1Immune responses in patients assessed by ex vivo IFN-γ ELISpot of PBMCs at multiple time points and multiplex immunofluorescence staining of tumor biopsies before and after vaccine treatment. a) Neoantigen-reactive T cells expanded in the blood of all patients post-vaccination (mean ± SEM, 2-3 wells/timepoint). b) Week 12 biopsies from all patients showed increased CD163+ macrophage and CD3+ T cell infiltration compared to enrollment biopsiesAbstract 602 Figure 2Uniform Manifold Approximation and Projection (UMAP) of the phenotypic distribution of tumor-reactive T cells. Tumor-reactive TCRs expressed genes associated with T cell activation and exhaustion, including the previously characterized tumor-reactive T cell signature markers CXCL13 and ENTPD1