Comparison of antibody-scTRAIL Fc fusion proteins with varying valency for EGFR and TRAIL receptors

Abstract

Fusion proteins combining TNF-related apoptosis inducing ligand (TRAIL) and antibody building blocks have emerged as a strategy for the targeted treatment of cancer cells. Using a single-chain derivative of homotrimeric TRAIL (scTRAIL), several targeted and non-targeted scTRAIL fusion proteins of varying geometries and valencies for TRAIL receptors and target antigens, all comprising an Fc region, were generated. These fusion proteins comprised either 1 or 2 scTRAIL units, i.e. are tri- or hexavalent for TRAIL receptors and in the targeted versions, 1 or 2 binding sites for EGFR. These fusion proteins were analyzed for cell binding and cell death induction using the EGFR-expressing colorectal cancer cell lines Colo205 and HCT116. In line with previous findings, all fusion proteins that were hexavalent for TRAIL receptors exhibited a strongly increased cell killing activity compared to the trivalent ones. Interestingly, the fusion proteins comprising one scTRAIL unit, did not benefit from targeting to EGFR. In contrast, the hexavalent scTRAIL fusion proteins further benefited from EGFR targeting, resulting in an approximately 6- to 30-fold increase in cell killing. In summary, this study shed further light on the influence of geometry and valency of TRAIL fusion proteins and confirmed IgG-scTRAIL fusion proteins as highly potent cell death inducers.

Keywords: Antibody fusion protein; Cancer; ScTRAIL; TNF-related apoptosis-inducing factor; TRAIL; Targeting.

Fig. 1

Overview of scTRAIL fusion proteins with different numbers of scTRAIL units (red) and antigen-binding site for EGFR (light and dark green). Constant antibody regions are shown in grey, the VL of a dummy antibody is shown in cyan. The first number refers to the number of antigen binding sites, the second number to the number scTRAIL units. The following molecules have been generated: (1) Fc-scTRAIL (0 + 2); (2) DIgG-Fc-scTRAIL (0 + 1); (3) IgG-scTRAIL (2 + 2); (4) scFv-Fc-scTRAIL (2 + 2); (5) IgG-scTRAIL (2 + 1); (6) scFv-Fc-scTRAIL (2 + 1); (7) scTRAIL-Fc (0 + 2); (8) DFab-scTRAIL-Fc (0 + 1) 9); scTRAIL-Fc-scFv (2 + 2); 10) Fab-scTRAIL-Fc (1 + 1); 11) scFv-scTRAIL-Fc (1 + 1).

Fig. 2

Binding of antibody-scTRAIL fusion proteins to immobilized DR5 and EGFR analyzed by ELISA. (A) Hexavalent TRAIL molecules on DR5. (B) Trivalent TRAIL molecules on DR5. (C) Bivalent targeted molecules to EGFR. (D) Monovalent targeted molecules to EGFR. scTRAIL-antibody fusion proteins were titrated at a 1:4 dilution between concentrations starting at 100 nM, coated with DR5- or EGFR-moFc (0.3 µg/well), detected with HRP-conjugated anti-human Fc antibody. n = 3, mean ± SD.

Fig. 3

Binding of antibody-scTRAIL fusion proteins to Colo205 and HCT116 cells analyzed by flow cytometry. (A) Hexavalent TRAIL molecules on Colo205. (B) Trivalent TRAIL molecules on Colo205. (C) Hexavalent TRAIL molecules on HCT116. (D) Trivalent TRAIL molecules on HCT116. scTRAIL-antibody fusion proteins were titrated at a 1:4 dilution between concentrations starting at 100 nM, 100,000 cells/well, detection with PE-conjugated anti-human Fc antibody. n = 3, mean ± SD.

Fig. 4

Cell death induction assay of hexavalent and trivalent TRAIL molecules on Colo205 and HCT116 cells. (A) Hexavalent TRAIL molecules on Colo205. (B) Trivalent TRAIL molecules on Colo205. (C) Hexavalent TRAIL molecules on HCT116. (D) Trivalent TRAIL molecules on HCT116. scTRAIL-antibody fusion proteins were titrated at a 1:3 dilution between concentrations starting at 1 nM (2 + 2 formats) or 10 nM (0 + 2 formats), 50,000 cells/well, detection by crystal violet staining. n = 3, mean ± SD.

Fig. 5

Caspase-3/7-assay of hexavalent and trivalent TRAIL molecules on Colo205. (A) Hexavalent TRAIL molecules on Colo205. (B) Trivalent TRAIL molecules on Colo205. scTRAIL-antibody fusion proteins were added at 1 nM final concentration to 15,000 cells/well, luminescence signal is proportional to the amount of caspase activity. n = 1, mean of duplicates.

Fig. 6

Correlation between cell binding and killing. Cell binding and cell killing activity of the different scTRAIL fusion proteins are plotted, demonstrating the beneficial effects of targeting hexavalent scTRAIL fusion proteins on cell killing activity.