Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

Figures & data

Figure 1 SNAP-tag technology and the mechanism of action of the scFv-SNAP-AURIF ADCs.

Notes: SNAP-tag technology allows site-specific covalent and irreversible coupling of BG-modified cytotoxic payloads such as AURIF to scFv-SNAP fusion proteins. (A) The SNAP-tag undergoes a self-labeling reaction to form a covalent bond with BG derivatives. (B) (A and B) The scFv-SNAP-AURIF ADC binds via its scFv specifically to the extracellular receptor of the target tumor cell and (C and D) is internalized receptor mediated into the lysosomal compartment. (E and F) Due to acidification and enzymatic reactions within the lysosomes, the fusion protein is degraded and the toxin (AURIF) is set free into the cytosol. (G) Auristatin-based toxins have an effect on the microtubule structure and cause cell death by apoptosis.
Abbreviations: ADC, antibody–drug conjugate; BG, benzylguanine; AURIF, auristatin F; EGFR, epidermal growth factor receptor; scFv, single-chain fragment variable; V_H, heavy chain variable domain; V_L, light chain variable domain.

Figure 2 Generation and binding activity of scFv-SNAP-AURIF.

Notes: Purified EGFR-specific scFv-SNAP fusion proteins were coupled to AURIF and the conjugation was verified by postincubation with BG-VG before separation by SDS-PAGE under denaturing conditions. (A) Coomassie Brilliant Blue staining of BG-AURIF or BG-VG coupled to the scFv-SNAP fusion proteins and (B) corresponding in-gel fluorescence visualization of the BG-VG conjugation. The black arrow indicates free uncoupled VG. M: prestained protein marker (broad range); 1: (−) 425(scFv)-SNAP incubated with BG-VG; 2: (+) 425(scFv)-SNAP incubated with a twofold molar excess of AURIF for 2 h and postincubation with BG-VG; 3: (−) 1711(scFv)-SNAP incubated with BG-VG; 4: (+) 1711(scFv)-SNAP incubated with AURIF, following postincubation with BG-VG; 5: (−) mock-SNAP incubated with BG-VG; and 6: (+) mock-SNAP incubated with AURIF, following postincubation with BG-VG. (C) Binding of the scFv-SNAP fusion proteins and corresponding scFv-SNAP-AURIF conjugations to EGFR+ cell lines representing different tumors. Bound scFv-SNAP (with and without AURIF) fusion proteins were detected using an anti-His6 PEantibody (background control). Specific binding of fusion proteins to the target cell lines A431, MDA-MB-468, RD, and A549 was detected. Mock-SNAP (with and without AURIF) and A2058 cells served as controls.
Abbreviations: ADC, antibody–drug conjugate; AURIF, auristatin F; BG, benzylguanine; EGFR, epidermal growth factor receptor; scFv, single-chain fragment variable; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; VG, VistaGreen.

Figure 3 Cytotoxic activity of scFv-SNAP-AURIF to various solid cancer cells.

Notes: The cytotoxic activity of the novel ADCs toward EGFR⁺ A431, MDA-MB-468, and RD cells was assessed using an XTT-based viability assay after incubation for 72 h. Cells were treated with decreasing concentrations of ADC and the EC₅₀ values relative to PBS-treated cells were calculated using GraphPad Prism v5. Graphs demonstrate one representative XTT assay for both ADCs in each cell line. Data are mean ± SD of each measurement, and the measurements were performed in duplicate at least three times.
Abbreviations: ADC, antibody–drug conjugate; AURIF, auristatin F; EC₅₀, concentration required to achieve a 50% reduction of cell viability; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; scFv, single-chain fragment variable.

Table 1 EC₅₀ values for AURIF and EGFR-specific ADCs

ADC	A431, EC50 (95% CI)	MDA-MB-468, EC50 (95% CI)	RD, EC50 (95% CI)	A2058, EC50 (95% CI)
425(scFv)-SNAP-AURIF	8 nM (2.6–22.0)	4 nM (3.5–4.6)	8 nM (6.7–8.8)	ND
1711(scFv)-SNAP-AURIF	12 nM (2.3–66.8)	4 nM (3.5–4.4)	4 nM (2.9–5.8)	ND
AURIF	19 nM (13.0–28.0)	10 nM (3.8–28.0)	26 nM (13.4–48.9)	7.8 nM (5.3–11.5)

Notes: The EC₅₀ values (95% CI) for scFv-SNAP-AURIF are derived from the XTT-based cell viability assays and indicate the ADC concentration required to achieve a 50% reduction in cell viability relative to the PBS-treated control cells (ND).

Abbreviations: ADC, antibody–drug conjugate; AURIF, auristatin F; CI, confidence interval; EC₅₀, concentration required to achieve a 50% reduction of cell viability; EGFR, epidermal growth factor receptor; ND, not defined; PBS, phosphate-buffered saline; scFv, single-chain fragment variable.

Figure 4 The effect of the EGFR-specific ADCs on cell cycle behavior.

Notes: (A, C, and E) The induction of apoptosis by 10 nM EGFR-specific ADCs or free AURIF measured by annexin V-EGFP/PI staining after incubation for 72 h. The bars represent the sum of early and late apoptotic/necrotic cells for each construct and each cell line. The data are shown as mean ± SEM of at least three independent experiments performed in duplicate. The statistical significance compared to mock-SNAP-AURIF was determined by one-way ANOVA followed by a post hoc Bonferroni test (***P≤0.001). (B, D, and F) The induction of cell cycle arrest by 10 nM scFv-SNAP-AURIF after incubation of 48 h. Representative flow cytometry data are presented for each cell line and construct after PI staining. (B) MDA-MB-468; (D) RD; and (F) A2058. The G0/G1 phase (1), S phase (2), and G2/M phase (3) are separated by dashed lines.
Abbreviations: ADC, antibody–drug conjugate; ANOVA, analysis of variance; AURIF, auristatin F; EGFP, enhanced green fluorescent protein; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; PI, propidium iodide; scFv, single-chain fragment variable; SEM, standard error of the mean.

Figure 5 Cytotoxicity and microtubule dynamics assays in A549 cells.

Notes: (A) Cytotoxicity of 1711(scFv)-SNAP-AURIF toward A549 cells after incubation for 72 h assessed using an XTT-based viability assay after incubation for 72 h. Cells were treated with 50, 150, or 250 nM of the ADC at least three times, and measurements were taken in duplicate. (B) Induction of microtubule disassembly in A549 cells stably expressing SNAP-TubB3. The cells were treated for up to 48 h with 50, 150, or 250 nM of 1711(scFv)-SNAP-AURIF and free AURIF. The microtubule structures were visualized by conjugating SNAP-Cell TMR-Star to SNAP-TubB3 (green), and the nuclei were counterstained with DAPI (blue). Scale bar =50 µm and applies to all images.
Abbreviations: ADC, antibody–drug conjugate; AURIF, auristatin F; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; scFv, single-chain fragment variable; TubB3, tubulin B3.

Figure 6 Specific ex vivo binding studies.

Notes: Tumor tissue biopsies from breast cancer patients were used to test the ex vivo binding of two different ADCs. Representative images show FFPE tumor sections stained with new fuchsin and counterstained with hematoxylin and eosin (objective =40×, scale bars =50 µm). The specific binding of scFv-SNAP-AURIF is indicated by red-stained EGFR⁺ clusters, and areas of interest are marked with arrows. A mock-SNAP-AURIF construct was used as a control. Further controls were prepared by staining with the detection antibody only (anti-SNAP antibody M2D11 and GAM^AP).
Abbreviations: ADC, antibody–drug conjugate; AURIF, auristatin F; EGFR, epidermal growth factor receptor; FFPE, formalin-fixed paraffin-embedded; scFv, single-chain fragment variable.

Figure S1 Cytotoxic effects of free AURIF and mock-SNAP-AURIF.

Notes: The cytotoxicity of free AURIF and mock-SNAP-AURIF assessed using an XTT-based viability assay after incubation for 72 h. Representative data from one assay are shown. Cells were incubated with decreasing concentrations of free AURIF and mock-SNAP-AURIF. The EC₅₀ values relative to cells treated with PBS (negative control) and Zeocin (positive control) were calculated using GraphPad Prism v5. The data are shown as mean ± SD of each measurement.

Abbreviations: AURIF, auristatin F; EC₅₀, concentration required to achieve a 50% reduction of cell viability; PBS, phosphate-buffered saline.