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. 2021 Apr;15(4):1054-1068.
doi: 10.1002/1878-0261.12919. Epub 2021 Feb 20.

A novel diphtheria toxin-based bivalent human EGF fusion toxin for treatment of head and neck squamous cell carcinoma

Zeng Qi  1   2 , Yue Qiu  3 , Zhaohui Wang  1   2 , Huiping Zhang  1   2 , Ling Lu  3 , Yanqiu Liu  3 , David Mathes  1 , Elizabeth A Pomfret  2 , Dexiang Gao  4 , Shi-Long Lu  3 , Zhirui Wang  1   2
Affiliations

A novel diphtheria toxin-based bivalent human EGF fusion toxin for treatment of head and neck squamous cell carcinoma

Zeng Qi et al. Mol Oncol. 2021 Apr.

Abstract

Epidermal growth factor receptor (EGFR) is often overexpressed in head and neck squamous cell carcinoma (HNSCC) and represents a top candidate for targeted HNSCC therapy. However, the clinical effectiveness of current Food and Drug Administration (FDA)-approved drugs targeting EGFR is moderate, and the overall survival rate for HNSCC patients remains low. Therefore, more effective treatments are urgently needed. In this study, we generated a novel diphtheria toxin-based bivalent human epidermal growth factor fusion toxin (bi-EGF-IT) to treat EGFR-expressing HNSCC. Bi-EGF-IT was tested for in vitro binding affinity, cytotoxicity, and specificity using 14 human EGFR-expressing HNSCC cell lines and three human EGFR-negative cancer cell lines. Bi-EGF-IT had increased binding affinity for EGFR-expressing HNSCC compared with the monovalent version (mono-EGF-IT), and both versions specifically depleted EGFR-positive HNSCC, but not EGFR-negative cell lines, in vitro. Bi-EGF-IT exhibited a comparable potency to that of the FDA-approved EGFR inhibitor, erlotinib, for inhibiting HNSCC tumor growth in vivo using both subcutaneous and orthotopic HNSCC xenograft mouse models. When tested in an experimental metastasis model, survival was significantly longer in the bi-EGF-IT treatment group than the erlotinib treatment group, with a significantly reduced number of metastases compared with mono-EGF-IT. In addition, in vivo off-target toxicities were significantly reduced in the bi-EGF-IT treatment group compared with the mono-EGF-IT group. These results demonstrate that bi-EGF-IT is more effective and markedly less toxic at inhibiting primary HNSCC tumor growth and metastasis than mono-EGF-IT and erlotinib. Thus, the novel bi-EGF-IT is a promising drug candidate for further development.

Keywords: EGF; EGFR; HNSCC; diphtheria toxin; fusion toxin; head and neck cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Schematic diagrams, SDS/PAGE, and western blot analysis of the mono‐EGF‐IT and bi‐EGF‐IT. (A) Schematic diagrams of the mono‐EGF‐IT and bi‐EGF‐IT. N, N‐terminal; C, C‐terminal. (B) SDS/PAGE (4–12% NuPAGE) of the mono‐EGF‐IT and bi‐EGF‐IT. (C) Western blot analysis using a mouse anti‐His mAb. (D) Western blot analysis using a mouse anti‐DT mAb. Lane 1: protein marker; lane 2: mono‐EGF‐IT (50.1 kDa); lane 3: bi‐EGF‐IT (57.2 kDa). The weak high molecular‐weight bands in lane 3 are dimers of bi‐EGF‐IT due to the formation of disulfide bonds.
Fig. 2
Fig. 2
In vitro binding affinity and efficacy analysis of the mono‐EGF‐IT and bi‐EGF‐IT to the human EGFR+ HNSCC cell line. (A) Binding affinity analysis of the mono‐EGF‐IT and bi‐EGF‐IT to the human EGFR+ HNSCC cell line, Cal27, using flow cytometry. Anti‐human EGFR mAb was used as a positive control, and biotinylated anti‐murine PD‐1 immunotoxin served as a negative background control for protein biotinylation. The data are representative of three individual experiments. (B) K D determination of the human EGF fusion toxins for Cal27 cells using flow cytometry and nonlinear least‐squares fitting. The MFI was plotted over a wide range of biotinylated mono‐EGF‐IT or bi‐EGF‐IT concentrations. Nonlinear regression was based on the equation Y = B max × X/(K D + X), where Y = MFI at the given biotinylated fusion toxin concentration after subtracting the background, X = biotinylated fusion toxin concentration, and B max = the maximum specific binding in the same units as Y. (C) Analysis of the blocking of anti‐human EGFR mAb binding to the human EGFR+ HNSCC cell line, Cal27, by mono‐EGF‐IT and bi‐EGF‐IT using flow cytometry. (D) The percentage inhibition of anti‐human EGFR mAb binding to Cal27 cells is plotted versus the concentration of binding competitor (mono‐EGF‐IT or bi‐EGF‐IT). The data are representative of three individual experiments. (E) In vitro efficacy of the mono‐EGF‐IT and bi‐EGF‐IT in the human EGFR+ HNSCC cell line, Cal27 determined by the CellTiter‐Glo® Luminescent Cell Viability Assay (red line: mono‐EGF‐IT group; green line: bi‐EGF‐IT group; blue line: anti‐murine PD‐1 immunotoxin group as the negative control). Y‐axis: percent inhibition of cell viability determined by the number of viable cells based on the quantification of ATP. X‐axis: fusion toxin concentration. Cycloheximide (1.25 mg·mL−1) was used as a positive control. The negative control wells contained cells without fusion toxin. Data are from three individual experiments. Error bars indicate SD.
Fig. 3
Fig. 3
Off‐target analysis of the human EGF fusion toxins using three human EGFR– tumor cell lines (JeKo‐1, Jurkat, and EL4). The human EGFR+ HNSCC cell line, UMSCC10B, was included as a positive control. (A) Binding affinity analysis of the human EGF fusion toxins to three human EGFR– tumor cell lines using flow cytometry. The data are representative of three individual experiments. (B‐E) In vitro efficacy of human EGF fusion toxins in three human EGFR– and one EGFR+ tumor cell lines using the CellTiter‐Glo® Luminescent Cell Viability Assay. (B) UMSCC‐10B (EGFR+). (C) Jeko‐1 (EGFR−). (D) Jurkat (EGFR−). (E) EL4 (EGFR−). Data are from three individual experiments. Error bars indicate SD.
Fig. 4
Fig. 4
In vivo efficacy of human EGF fusion toxins against subcutaneous xenografts in NSG mice. (A) Cal27 cells were subcutaneously injected into the right flank and treated with DT390 (n = 10), mono‐EGF‐IT (n = 10), bi‐EGF‐IT (n = 10), or erlotinib (n = 10) once daily for 10 consecutive days beginning on day 4 after the tumor cell injection. Kaplan–Meier survival curves were recorded for the DT390 (blue line), mono‐EGF‐IT (red line), bi‐EGF‐IT (green line), and erlotinib (purple line) groups. (B‐D) Cal27 cells were subcutaneously injected into the flanks of a second cohort of NSG mice that were then treated with DT390 (n = 8), mono‐EGF‐IT (n = 8), bi‐EGF‐IT (n = 8), or erlotinib (n = 8). Mice were euthanized on day 14 after tumor cell injection when the first mouse in the DT390 group reached the end point. (B) Tumor volumes were measured periodically, and the growth kinetics of the four groups were plotted. (C) Image of harvested tumors on day 14 and (D) the mean tumor weight for each group. Scale bar: 1 cm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant. The P‐values for the survival curves in panel A were calculated using the Mantel–Cox log‐rank test and that for the comparisons in panels B and D were calculated using the two‐tailed Student t‐test (graphpad prism 9.0.0).
Fig. 5
Fig. 5
In vivo efficacy of EGF fusion toxins against tongue SCCs in NSG mice. (A) Cal27 cells were injected into the tongues of NSG mice and treated with DT390 (n = 10), mono‐EGF‐IT (n = 10), bi‐EGF‐IT (n = 9), or erlotinib (n = 8) once daily for 10 consecutive days starting on day 4 after the tumor cell injection. Kaplan–Meier survival curves were recorded for the DT390 (blue line), mono‐EGF‐IT (red line), bi‐EGF‐IT (green line), and erlotinib (purple line) groups. The timeline and detailed schedules for tumor cell injection and treatments are shown under the survival curve. The vertical arrows indicate the days on which the tumor cells or the treatments were administered. (B‐C) Cal27 cells were injected into the tongues of a second cohort of NSG mice that were then treated with DT390 (n = 6), mono‐EGF‐IT (n = 6), bi‐EGF‐IT (n = 6), or erlotinib (n = 6). The mice were euthanized on day 8 after tumor cell injection when the first mouse in the DT390 group reached the end point. (B) Image of tongue SCCs (circled by the black dotted lines) and (C) tumor volume comparison of the treatment groups. Scale bar: 1 cm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant. The P‐values for the survival curves in panel A were calculated using the Mantel‐Cox log‐rank test and that for the comparisons in panel C were calculated using the two‐tailed Student t‐test (graphpad prism 9.0.0).
Fig. 6
Fig. 6
In vivo efficacy of EGF fusion toxins against an experimental lung metastasis mouse model. (A) Cal27 cells were intravenously injected into NSG mice that were then treated with DT390 (n = 18), mono‐EGF‐IT (n = 13), bi‐EGF‐IT (n = 13), or erlotinib (n = 15) once daily for 10 consecutive days starting on day 4 after the tumor cell injection. Kaplan–Meier survival curves were recorded for the DT390 (blue line), mono‐EGF‐IT (red line), bi‐EGF‐IT (green line), and erlotinib (purple line) groups. The timeline and detailed schedules for tumor cell injection and treatments are shown under the survival curve. The vertical arrows indicate the days on which the tumor cells or the treatments were administered. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The P‐values for the survival curves in panel A were calculated using the Mantel–Cox log‐rank test (graphpad prism 9.0.0). (B‐C) Cal27 cells were intravenously injected into a second cohort of NSG mice that were then treated with DT390 (n = 12), mono‐EGF‐IT (n = 12), bi‐EGF‐IT (n = 6), or erlotinib (n = 6). The mice were euthanized on day 10 after tumor cell injection when the first mouse in the DT390 group reached the end point. (B) H&E pictures of lung metastases (black arrowheads, upper panels), with higher magnification pictures of the black rectangles (lower panels). (C) The numbers of lung metastasis present in the DT390, mono‐EGF‐IT, bi‐EGF‐IT, and erlotinib treatment groups. Scale bar: 500 µm. Error bars indicate SD.
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