Antibody Directed Enzyme Prodrug Therapy: Discovery of novel genes, isolation of novel gene variants and production of long acting drugs for efficient cancer treatment

Back Ground Antibody‐Directed Enzyme Prodrug Therapy (ADEPT) is a novel strategy to improve the selectivity of cancer treatment. The ADEPT uses the bacterial enzyme, glucarpidase to produce the antibody‐enzyme complex. Repeated cycles of ADEPT and the use of wild type glucarpidase in detoxification are essential but are hampered by the human antibody response to the enzyme. Objectives 1. Production of novel glucarpidases and glucarpidases variants by different strategies 2. Production of long acting glucarpidase(s) 3. Synthesis of Tubulysin derivatives, as a Prodrug, on a gram scale for conjugation experiments Methods Novel glucarpidase producers were isolated from soil using minimal plates with folate as the only carbon source. Genomic library and PCR were used to clone two novel glucarpidase genes. The two genes were overexpressed in E. coli using pET28a vector. DNA shuffling was used to produce novel glucarpidase variants. Long acting novel glucarpidase was prepared using different PEGylation techniques. Novel prodrug was prepared by novel multicomponent approach which involves the condensation of dieptide acid, β‐amino aldehyde and isocyanide derived from Cys(Trt)/OMe. Results We successfully produced, by DNA shuffling an ultra‐active glucarpidase that degrades MTX with a high efficiency. We also isolated three novel glucarpidase producing bacteria from Qatari soil. Two novel glucarpidases were successful cloned, overexpressed and purified. We managed to introduce a new mutation into one of the newly isolated glucarpidase gene which led to a 500% increase in the glucarpidase activity. The talk will cover many the production of long acting glucarpidase and many other issues.