Studies on Ligand Directed Enzyme Prodrug Therapy and Production of Long Acting Protein Therapeutics for Targeted Cancer Treatment

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Targeting cancer therapy is one of the powerful tools that has been implemented for cancer treatment. One of the targeted cancer strategies is known as Antibody Directed Enzyme Prodrug Therapy (ADEPT). Three main components in this strategy, enzyme, cancer-specific antibody and a prodrug. The enzyme that has been used so far and reached clinical trials is carboxypeptidase G2 (CPG2) and the antibody chosen depends on the type of cancer to be treated. To overcome the need to conjugate a cancer-specific antibody to CPG2, our work showed that a small known peptide motif Aspargine-Glycine-Arginine (NGR) in place of an antibody fused to CPG2 can direct the enzyme to aminopeptidase N (APN ) that is highly expressed tumor. The CPG2 then converts the prodrug into the drug near the cancerous tissue. We called this strategy Ligand-targeted enzyme prodrug therapy (LDEPT). We have successfully demonstrated the ability of our new conjugates to bind specifically to cancer cells and CPG2 to produce the cytotoxic drug to kill the tumor cells. The ADEPT or LDEPT have disadvantages such as immunogenicity over CPG2 that undermine the efficacy of the therapy. We have therefore started the tasks to produce long acting CPG2 new variants. First and for the first time, we produced two CPG2 conjugates using life extenders PEGylation and gene fusion of HSA Our produced CPG2 variants (PEG-CPG2 and HSA-CPG2) showed a longer half-life under physiological conditions in addition to lower ex-vivo immunotoxicity . Second, we produced a long acting peptide-CPG2 that can specifically bind to tumor and has a long half-life, these are single and double fusion of PEG-CPG2-CNGRC. The resulting conjugates; single (CNGRC-CPG2) and double (CNGRC-CPG2-CNGRC) have been found to specifically target and bind APN, which is known to be highly expressed on the surface of tumor cells. In addition, the conjugates generated showed higher stability and significantly reduced ex vivo immunotoxicity. CPG2 in both conjugates has the ability to convert prodrug into a cytotoxic drug to kill the cancer cells. The work presented in this thesis paves the way for further in vivo and clinical studies to improve two of the important cancer targeting strategies, ADEPT and LDEPT.