Background: Nivolumab,an IgG4 fully human monoclonal antibody against checkpoint protein PD-1,is active in metastatic melanoma,renal cell and non-small cell lung cancer.It was administered with a multi-peptide vaccine to patients(pts)with unresectable melanoma who failed at least one regimen for metastatic disease and were ipilimumab na(i)ve,or failed ipilimumab,to assess the toxicity and tolerability of the combination and perform correlative immune assays.Methods: Three cohorts of 10 HLA A0201 positive ipilimumab-na(i)ve pts received nivolumab at 1,3 or 10 mg/kg,then three additional cohorts of pts who had failed prior ipilimumab received nivolumab at 3 mg/kg: two cohorts of 10 pts each who were A0201 positive and had either grade 2 or less ipilimumab toxicity,or grade 3 dose limiting ipilimumab toxicity; finally 40 pts were treated with antibody who had grade 2 or less ipilimumab toxicity and were not HLA restricted.Pre-treatment archived tumor tissue as well as pre-and post-treatment peripheral blood cells were collected.Results: Median age for all pts was 59;76%were M1c.Response rates by RECIST were 28%in 34 pts na(i)ve to,and 32%for 46 pts who failed prior ipilimumab.Nivolumab did not induce the same irAEs in pts with prior ipilimumab induced toxicity.No cohort had more than one dose limiting toxicity.2 pts had grade 3 pneumonitis.Three of ten pts who failed nivolumb had stable disease or a partial response to subsequent ipilimumab.Biomarker studies showed that elevated NY-ESO 1 and MART-1 specific CD8 T cells pre-treatment were associated with non-response(p,0.005 and,0.001),and that CTLA-4 positive CD4 T cells and T regulatory cells were elevated after treatment in non-responders(p,0.01).Immunohistochemical analysis of pre-treatment tumors indicated that PD-L1 staining was associated with response,but responses were also observed in pts whose tumors did not stain.Conclusions: Objective responses to nivolumab were observed after failing ipilimumab,and to ipilimumab after failing nivolumab.Elevation of CTLA-4 after nivolumab in non-responders suggest that sequential therapy with the combination should be tested.Tumor PD-L1 was associated with but not predictive of response.