Improved Yield and Stability of L49-sFv−β-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.

Do Proteins Always Benefit from a Stability Increase? Relevant and Residual Stabilisation in a Three-state Protein by Charge Optimisation

“…[15][16][17] The issue even shows a promising ramification to the emerging field of molecular remediation of folding diseases. 18 Among the methods proposed and tested in order to rationally stabilise proteins, some exploit entropic differences between folded and unfolded polypeptides to achieve an overall stabilisation through the engineering of disulfide bonds [19][20][21] or the removal of glycine residues or introduction of proline residues into the sequence.…”

Novel β-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy

“…In this type of targeted therapy, the β-lactamase enzyme protein is chemically (Meyer et al, 1993; Svensson et al, 1995) or biologically (Rodrigues et al, 1995; McDonagh et al, 2003; Cortez-Retamozo et al, 2004; Alderson et al, 2006) conjugated to a cancer-specific ligand (usually an antibody) for delivering the enzyme to the tumor site. The presence of β-lactamase enzyme predominantly at the tumor site amplifies availability of the active drug concentrations locally following a cephalosporin prodrug infusion.…”

Expression of antibody variable region-human alkaline phosphatase fusion proteins in mammalian cells

“…Among them, enzyme-labeled antibody is an essential component in immunoassays such as ELISA and Western blotting (Porstmann and Kiessig, 1992). To date, they have been produced either by chemical-conjugation, or more recently by expression systems for recombinant antibody-enzyme fusion proteins, mainly by microbial expression systems (Ducancel et al, 1993;Weiss and Orfanoudakis, 1994;Suzuki et al, 1997;McDonagh et al, 2003;Medzihradszky et al, 2004;Alderson et al, 2006;Mayer et al, 2006;Venisnik et al, 2006;Coelho et al, 2007;Mousli et al, 2008). However, chemical conjugation needs multiple steps of reaction/purification that might affect the antibody activity (Ishikawa et al, 1983).…”