Improved yield and stability of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation, by protein engineering.

Abstract

The L49 single-chain Fv fused to beta-lactamase (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen. However, large-scale production of L49-sFv-bL from refolded E. coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein. Sequence analysis of the L49-sFv framework regions revealed three residues in the framework regions at positions L2, H82B, and H91, which are not conserved for their position, occurring in <1% of sequences in Fv sequence databases. One further unusual residue, found in <3% of variable sequences, was observed at position H39. Each unusual residue was mutated to a conserved residue for its position and tested for refolding yield from inclusion bodies following expression in E. coli. The three V(H) single mutants showed improvement in the yield of active protein and were combined to form double and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein. In an attempt to further improve yield, the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resulting in a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein. The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations. Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts, respectively. These results show that a rational protein-engineering approach improved the yield, stability, and refolding characteristics of L49-sFv-bL while maintaining binding affinity and therapeutic efficacy.

• Authors: Charlotte F McDonagh, Kevin S Beam, Gabrielle J S Wu, Judy H Chen, Dana F Chace, Peter D Senter, Joseph A Francisco
• Journal: Bioconjugate chemistry (Bioconjug Chem) 2003 Sep-Oct Vol. 14 Issue 5 Pg. 860-9 ISSN: 1043-1802 [Print] United States
• PMID: 13129388 (Publication Type: Journal Article)

Chemical References

• Antibodies, Monoclonal
• Antibodies, Neoplasm
• Antigens, Neoplasm
• Antineoplastic Agents
• Immunoglobulin G
• Melanoma-Specific Antigens
• Neoplasm Proteins
• Prodrugs
• Recombinant Fusion Proteins
• L49-sFv-beta-lactamase
• beta-Lactamases

Topics

• Animals
• Antibodies, Monoclonal (genetics, therapeutic use)
• Antibodies, Neoplasm (administration & dosage, biosynthesis, genetics, metabolism)
• Antigens, Neoplasm
• Antineoplastic Agents (administration & dosage, chemistry, metabolism)
• Binding Sites, Antibody (genetics)
• Dose-Response Relationship, Drug
• Drug Stability
• Female
• Humans
• Immunoglobulin G (administration & dosage, biosynthesis, genetics, metabolism)
• Melanoma-Specific Antigens
• Mice
• Mice, Inbred BALB C
• Mutagenesis, Site-Directed
• Neoplasm Proteins (genetics, immunology)
• Prodrugs (administration & dosage, chemistry, metabolism)
• Protein Engineering (methods)
• Recombinant Fusion Proteins (administration & dosage, biosynthesis, genetics, metabolism)
• Tumor Cells, Cultured
• Xenograft Model Antitumor Assays (methods)
• beta-Lactamases (administration & dosage, biosynthesis, genetics, metabolism)