Pharmaceutical development of the plasmid DNA vaccine pDERMATT

Abstract

The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) is a plasmid that encodes for MART-1, a melanoma associated antigen that is expressed in a large fraction of melanomas. In animal models administration of this plasmid by intradermal tattooing induces a significant immune response against MART-1, indicating that the self tolerance mechanism indeed can be broken. This thesis describes the development of the pharmaceutical production of pDERMATT and its dosage form for a phase I clinical study. A production process using E.coli fermentation, which complies to Good Manufacturing Practice (GMP) guidelines and avoids the use of RNase and organic solvents, was developed. Using this process, pDERMATT is successfully purified from its main contaminants (endotoxins, bacterial DNA, RNA and proteins) to levels below set specifications. Since endotoxins or lipopolysaccharides (LPS) are known for their immunostimulatory properties, the influence of LPS on the efficacy and toxicity of pDERMATT was assessed in vivo. It was found that upon tattooing, LPS serves as a non-toxic contaminant, with the potential to act as an adjuvant at levels above pharmaceutical acceptable limits (> 100 IU/mg pDNA). For the content and purity analysis of pDERMATT an anion-exchange high pressure liquid chromatography (AEX-HPLC) method with ultraviolet (UV) detection was developed. The method proved to be precise, accurate, linear (5-150 g/mL) and stability indicating as supercoiled (SC) pDNA is well separated from the degraded topoisoforms (open circular (OC) and linear pDNA). To improve shelf-life stability, pDERMATT was lyophilised using various disaccharides as lyoprotectants. Upon accelerated stability studies in terms of elevated temperatures and increased residual moisture content sucrose performed best as stabiliser with a resulting shelf-life of >2 years at +2-8?C. Reconstitution of the dried product results in an isotonic product with a DNA concentration of 5 mg/mL. Disaccharides significantly reduce transfection efficiency of pDNA (up to 3%) which, however, is considered clinically irrelevant. With the addition of t-butanol (TBA) to the formulation solution, lyophilisation time could be reduced with more than 40%. A concentration of 30% v/v TBA was found most favourable in terms of residual TBA content, which appeared to mainly determine stability. It was demonstrated that tattoo administration only causes minor damage to the plasmid (?3% SC pDNA reduction to OC pDNA) which most likely will not affect clinical antigen expression and T-cell responses. Antigen expression and T-cell responses upon tattoo administration of SC and OC pDNA are equal in a murine model, but SC pDNA antigen expression is significantly higher than OC and linear pDNA in an ex vivo human skin model. pDNA topology does not influence antigen expression when formulated as PEGylated polyplexes. For intradermal tattoo administration the ex vivo skin model might be more suitable than the standard murine model for distinguishing subtle alterations in antigen expression of clinical pDNA formulations.