Clinical-grade myeloma Ag pre-loaded DC vaccines retain potency after cryopreservation S Szmania 1 , Q Yi 1 , M Cottler-Fox 2 , NA Rosen 1 , J Freeman 1 , BJ Kordsmeier 1 , A Moreno 1 , J Shi 1 , B Barlogie 1 , G Tricot 1 and F van Rhee 1 1 Myeloma Institute for Research and Therapy and 2 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA Background The use of myeloma Ag-loaded mature DC vaccines, cryopreserved in single-use aliquots, is an attractive immunotherapeutic strategy. In this study we investigated the retention of phenotype, viability and potency of DC vaccines after freezing and thawing. Methods Plastic-adherent monocytes, derived from a steady-state leukapheresis, were cultured in serum-free media containing GM-CSF and IL-4. DC were loaded on day 6 with myeloma lysate (ML) or idiotype (Id) Ag and keyhole limpet hemocyanin (KLH), induced to mature on day 7 with CD40-ligand and cryopreserved on day 9. Seventeen clinical-scale cultures were evaluated for DC yield, recovery and immunophenotype after potency was validated with allogeneic mixed lymphocyte culture and Ag presentation assays. Results We produced 88 individual vaccines from 17 clinical-scale cultures. Median DC yield at harvest was 131  /10 6 (range 37 /375  /10 6 ) and median recovery of viable DC after thawing was 69% (range 11 /100%). We confirmed viability (7AAD  ), phenotype (CD14  , CD83  /CD40  , CD83  /CD80  , CD83  /CD86  , CD83  / CD54  , HLA-DR  ) and the ability of the DC to present Ag and stimulate allogeneic T cells post-thawing. Discussion We have validated a serum-free culture system for the production of DC. Cryopreservation did not interfere with DC activity, allowed time for rigorous quality control (QC) and flexible scheduling of intranodal vaccination, and reduced the time to prepare multiple vaccines. Keywords cellular immunotherapy, clinical trial, DC, multiple myeloma, tumor lysate. Introduction DC are potent APC that play a central role in the induction of anti-tumor responses, with the unique ability to stimulate naive T cells [1,2]. Several investigators have demonstrated that vaccines comprising idiotype (Id)- loaded DC can induce immunologic responses in multiple myeloma (MM) patients [3  /11]. In myeloma, DC are rendered dysfunctional in vivo by immunosuppressive cytokines such as IL-6, transforming growth factor-b1, vascular endothelial growth factor-b and IL-10, secreted by myeloma cells or the microenvironment as a means of tumor escape [12  /17]. Ratta et al. have demonstrated that circulating myeloid DC from MM patients have a reduced capacity to present Id to naive T cells [13]. We have recently demonstrated that serum b2-microglobulin present at high levels in MM negatively affects Ag processing and presentation by DC [18]. Further, the number of circulating myeloid DC is reduced in MM [19]. These considerations make DC derived directly from the peripheral blood less suitable for myeloma vaccine preparation. In contrast, monocytes are abundant in the peripheral blood, are easily enriched by adherence, and when cultured in vitro with GM-CSF and IL-4 differentiate into DC and Correspondence to: Frits van Rhee, MD, PhD, MRCP(UK), FRCPath, Associate Professor of Medicine, Director of Immunotherapy and Allogeneic Transplantation, Myeloma Institute for Research and Therapy, Section for Gene and Immunotherapy, Arkansas Cancer Research Center #776, University of Arkansas For Medical Sciences, 4301 W. Markham Street, Little Rock, AK 72205, USA. Cytotherapy (2005) Vol. 7, No. 4, 374 /384 – 2005 ISCT DOI: 10.1080/14653240510027235