Development of a large scale manufacturing process of dendritic cell vaccine for colorectal cancer using tumor derived peptides

Proc Amer Assoc Cancer Res, Volume 45, 2004 1252 Patients with advanced colorectal cancer have limited options for treatment. Immunotherapy, and especially dendritic cell (DC)-based vaccines, appears to be one of the most promising and safe therapies. In a pre-clinical study to develop a DC vaccine for colorectal cancer patients we previously evaluated a series of wild type and analog MHC Class I peptides from the tumor antigens including CEA, MAGE, HER-2/neu, and p53. Matured DC (mDC) generated from HLA-A2001+ healthy donor apheresis product were pulsed with individual peptides, cryopreserved, and used to stimulate autologous CD8+ T cells. After three in vitro stimulations, the wells were screened for CTL activity. Peptides shown to be immunogenic in CTL stimulation were selected for further development of the vaccine. In order to scale up the process for manufacturing the DC vaccine for a clinical trial, we further evaluated a large scale manufacturing process to produce adequate number of DC loaded with the immunogenic peptides. Monocytes in bulk mononuclear cells (MNC) from healthy donors were cultured in serum-free VacCell™ media containing GM-CSF (500 U/mL) and IL-13 (50 ng/mL). On day 7 non-matured DC were enriched by elutriation. This process generated 5.5-9.3 x 108 viable DC from 1 x 1010 MNC with the DC purity of 91-98% (defined by CD45+/CD11c+) and viability of 94-98% (n=5). The enriched DC population was matured with a clinical grade bacterial membrane fraction (FMKp, 1 μg/mL), in association with IFN-γ (500 U/mL). The mDC cell product has shown the up-regulation of CD83 and co-stimulatory molecules (CD86 and CD80) and the down regulation of CD14. This cell product has been shown to contain potent antigen presenting cells that produce IL-12 in vitro . The mDC were pulsed with relevant peptides, washed, and were cryopreserved in a dose of 35 x 106 viable DC/vial. Studies were done to demonstrate that individual peptides were presented in the mixture using the CTL lines. In our manufacturing process we generated 4-6.9 x108 viable DC (viability of 88±0.8%, n=5) that are equivalent to 10-21 doses as the final formulation product. Upon thawing and reconstitution, we still obtained 29±3 x 106 viable DC per dose. These data demonstrate that our manufacturing process adequately generates enough DC product loaded with peptides for a clinical trial (4 treatment doses plus 2 release testing doses) and for other quality control testing. Based on these data, a Phase I / II clinical trial is underway to evaluate a peptide pulsed DC vaccine for colorectal cancer patients.