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. 2014 Apr 10:7:535-41.
doi: 10.2147/OTT.S59346. eCollection 2014.

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

Affiliations

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

Xiaoliang Zhou et al. Onco Targets Ther. .

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.

Keywords: ADEPT; immunogenicity; integrin αvβ3.

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Figures

Figure 1
Figure 1
Purification and characterization of RGD4C-βL. Notes: (A) Purification of RGD4C-βL by ion-exchange column chromatography. Lane 1: load sample; lanes 2–5: “flow through;” lanes 6–8: elution. (B) SDS-PAGE analysis of the final product. RGD4C-βL appeared as a single monomer band under reducing conditions (lane 1), and as a monomer and dimer under nonreducing SDS-PAGE (lane 2). Abbreviations: RGD4C-βL, RGD4C-β-lactamase fusion protein; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; WM, weight marker.
Figure 2
Figure 2
Stability of RGD4C-βL in human plasma at 37°C. Notes: The activity was monitored for 14 days. RGD4C-βL showed more than 60% activity after 14 days. Abbreviation: RGD4C-βL, RGD4C-β-lactamase fusion protein.
Figure 3
Figure 3
(A) Human PBMC proliferation assay. In eight tests, the RGD4C-βL groups showed no significant differences compared with the vehicle control groups (P>0.05), and the LPS groups were significantly higher than the control groups (P<0.05), with the exception of two donors (No 3 and No 7). (B) Mouse splenocyte proliferation assay. In five tests, the SI of the RGD4C-βL groups was lower than 2, and of the LPS groups was significantly higher (>2). A positive response was observed if the proliferative response reached an SI (average experimental cpm/average control cpm) of ≥1.99. Abbreviations: LPS, lipopolysaccharide; PMBC, human peripheral blood mononuclear cells; RGD4C-βL, RGD4C-β-lactamase fusion protein; SI, stimulation index.
Figure 4
Figure 4
Fluorescence imaging of NCI-H460 cells binding to FITC-RGD4C-βL. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; RGD4C-βL, RGD4C-β-lactamase fusion protein.
Figure 5
Figure 5
In vitro activation of cephalosporin–melphalan by RGD4C-βL. Notes: The final concentration of RGD4C-βL was 2 nM (diamonds 0 and 2 nM), and there was no significant decrease in cell viability. The concentrations of cephalosporin–melphalan and melphalan were 0, 0.4, 1.6, 6.4, 25.6, 102.4, and 200 µM; cephalosporin–melphalan had no significant cytotoxicity when used in combination with RGD4C-βL, and its cytotoxic effect on tumor cells was equal to equimolar amounts of melphalan. This demonstrated activation of cephalosporin–melphalan by RGD4C-βL. Abbreviations: C-Mel, cephalosporin–melphalan; RGD4C-βL, RGD4C-β-lactamase fusion protein.

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