Characterization of a fusion protein of RGD4C and the ß-lactamase variant for antibody-directed enzyme prodrug therapy

Abstract

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue, while minimizing systemic drug exposure. ADEPT has been reported to be an attractive approach for improving the efficacy of cancer therapy. A previously reported β-lactamase was found to contain four cluster of differentiation (CD)4+ T cell epitopes; however, single amino acid changes in the enzyme resulted in significantly reduced proliferative responses, while retaining stability and activity of the enzyme. The β-lactamase variant with reduced immunogenicity is an attractive alternative for constructing the ADEPT fusion protein. In this study, we fused the peptide, RGD4C, known to target integrin αvβ3, to the β-lactamase variant for use in ADEPT. Biological function studies revealed that RGD4C-β-lactamase had a high hydrolytic effect on nitrocefin and cephalosporin–melphalan, and high plasma stability was observed. In addition, fusion of the RGD4C moiety to β-lactamase had little effect on immunogenicity compared with β-lactamase in the proliferation of peripheral blood mononuclear cells. The ability of this fusion protein to both target the central region of αvβ3 and induce toxicity in the non-small-cell lung cancer cell NCI-H460 makes it a promising therapeutic approach in the treatment of cancer.

Somatostatin Receptor–Mediated Specific Delivery of Paclitaxel Prodrugs for Efficient Cancer Therapy

“…10 Receptor-targeted polymeric prodrugs have been shown to improve therapeutic responses both in vitro and in vivo. 11 Various ligands have been investigated, including folate, 12 transferrin, 13 antibodies, 14 peptides, 15 and aptamers. 16 Somatostatin (SST) is a polypeptide that is released in the gastrointestinal tract by delta cells and the hypothalamus.…”

“…Receptor‐targeted polymeric prodrugs have been shown to improve therapeutic responses both in vitro and in vivo . Various ligands have been investigated, including folate, transferrin, antibodies, peptides, and aptamers …”

A Fusion Protein of RGD4C and β-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy

“…Fusion gene was cloned into E. coli BL21 (DE3); the protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) resin, and was further confirmed by western blotting [16,17]. …”

Prodrug Strategy in Drug Development

“…In the first phase the exogenous enzyme is coupled to antibody and administered by infusion, leaving enough time for its localization and accumulation on the tumor cell. In the second phase the inactive prodrug, a selective substrate of the enzyme is administered, being activated by this to cytostatic drug [50][51][52][53].…”