Background: Despite improvements in treatment with targeted agents and immunotherapeutics metastatic melanoma still has a guarded prognosis. Many melanoma cells upregulate the disialoganglioside GD2. Early GD2 chimeric antigen receptor (CAR) T-cell studies in neuroblastoma demonstrated no toxicity but limited ability to expand in vivo. Strategies to expand these GD2.CAR T cells might improve efficacy while retaining safety. This pilot study aimed to evaluate the safety and efficacy of first generation 14g2a.zeta chimeric antigen receptor (GD2.CAR) transduced, activated T cells enriched for vaccine specific cytotoxic T-Lymphocytes (tvs-CTL). Methods: Patients with metastatic melanoma in which standard therapy had failed were eligible if they had recovered from effects of prior therapy, did not have rapidly progressive disease, were free of melanoma involving the CNS and did not have a contraindication to receiving Hepatitis B, Polio or DTAP vaccine. Patients received each of these vaccines prior to cell harvest, 4 days before and 28 days after autologous T cell infusion. Patient 1 was treated with 2 x 10⁸ cell dose and patients 2 and 3 were treated with 4x10⁸ cell dose. We used QPCR to measure transgene copy number in patients before and after infusion. Interferon-gamma enzyme linked immunospot (ELISPOT) assay was used to measure the frequencies of tetanus, pertussis, diphtheria, poliovirus and tumor antigens-specific T cells in peripheral blood. Results: GD2.CAR-tvs-CTL were manufactured and infused in 3 patients. Overall the infusions were safe. Seven low grade adverse events possibly related to study participation were reported. The first 2 patients did not demonstrate robust in vivo expansion of GD2.CAR-tvs-CTLs by QPCR and had rapid disease progression. In patient 3 a significant expansion of GD2.CAR-tvs-CTLs, i.e. 18,250 copies/ug genomic DNA was observed on day 7 and cells persisted at 159 copies/ug DNA for up to 12 months (latest measured time point). High pertussis-specific responses were also observed by INF-gamma ELISPOT in this patient starting from day 14 after the vaccination through month 12. Conclusions: We have demonstrated that GD2.CAR T cells expanded and persisted in melanoma patient for up to 12 months. The use of vaccination before blood procurement for T cell manufacture and boosting virus-specific T cell after CAR T cell infusion is a safe strategy and may have helped induced higher transgene levels in one of three patients. Clinical trial information: NCT02482532.