An EGFR-targeting antibody-drug conjugate LR004-VC-MMAE: potential in esophageal squamous cell carcinoma and other malignancies

Xin-Yue Hu, Rong Wang, Jie Jin, Xiu-Jun Liu, A-Long Cui, Lian-Qi Sun, Yan-Ping Li, Yi Li, Yu-Cheng Wang, Yong-Su Zhen, Qing-Fang Miao, Zhuo-Rong Li, Xin-Yue Hu, Rong Wang, Jie Jin, Xiu-Jun Liu, A-Long Cui, Lian-Qi Sun, Yan-Ping Li, Yi Li, Yu-Cheng Wang, Yong-Su Zhen, Qing-Fang Miao, Zhuo-Rong Li

Abstract

Epidermal growth factor receptor (EGFR) is a rational target for cancer therapy, because its overexpression plays an important oncogenic role in a variety of solid tumors; however, EGFR-targeted antibody-drug conjugate (ADC) therapy for esophageal squamous cell carcinoma (ESCC) is exceedingly rare. LR004 is a novel anti-EGFR antibody with the advantages of improved safety and fewer hypersensitivity reactions. It may be of great value as a carrier in ADCs with high binding affinity and internalization ability. Here, we prepared an EGFR-targeting ADC, LR004-VC-MMAE, and evaluated its antitumor activities against ESCC and EGFR-positive cells. LR004 was covalently conjugated with monomethyl auristatin E (MMAE) via a VC linker by antibody interchain disulfide bond reduction. VC-MMAE was conjugated with LR004 with approximately 4.0 MMAE molecules per ADC. LR004-VC-MMAE showed a potent antitumor effect against ESCC and other EGFR-positive cells with IC50 values of nM concentrations in vitro. The in vivo antitumor effects of LR004-VC-MMAE were investigated in ESCC KYSE520 and A431 xenograft nude mice models. Significant activity was seen at 5 mg·kg-1 , and complete tumor regression was observed at 15 mg·kg-1 in the KYSE520 xenograft nude mice after four injections, while the naked antibody LR004 had little effect on inhibiting tumor growth. Similar promising results were obtained in the A431 models. In addition, the tumors also remained responsive to LR004-VC-MMAE for large tumor experiments (tumor volume 400-500 mm3 ). The study results demonstrated that LR004-VC-MMAE could be a potential therapeutic agent for ESCC and other EGFR-expressing malignancies. We also evaluated PK profile of LR004-VC-MMAE ADC in the mice model, which would provide qualitative guiding significance for the further research.

Keywords: EGFR; ESCC; LR004; LR004-VC-MMAE; antitumor activity; preparation.

© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Figures

Figure 1
Figure 1
Characterization of LR004 and LR004‐VC‐MMAE. (A) The structure of LR004‐VC‐MMAE. (B) SDS/PAGE analysis of LR004 and LR004‐VC‐MMAE under the reducing and nonreducing conditions (gradient 4–12%). (C) SEC‐HPLC analysis of LR004 and LR004‐VC‐MMAE. LR004, RT (retention time) = 3.998 s; LR004‐VC‐MMAE, RT (retention time) = 4.031 s. (D) HIC analysis of LR004 and LR004‐VC‐MMAE. The HIC‐HPLC spectrum of LR004‐VC‐MMAE displays five major peaks, corresponding to zero, two, four, six, and eight drugs per antibody. The average DAR of LR004‐VC‐MMAE is approximately 4.0 after integration of the observed peaks.
Figure 2
Figure 2
Binding ability of LR004 and LR004‐VC‐MMAE in vitro. (A) The binding activity of LR004 and LR004‐VC‐MMAE to the recombinant human EGFR antigen by ELISA. (B) The sensorgram of Biacore analysis. The CM5 sensor chip was pre‐immobilized with LR004 and LR004‐VC‐MMAE at a concentration of 1 μg·mL −1. The antigen EGFR was injected at a flow of 30 μL·min−1 at concentrations ranging from 15.625 to 1000 ng·mL −1 in HEPES buffer. The dissociation constant, KD, was calculated as the ratio of these two constants (koff/kon). (C) The expression level of EGFR on various cells surface under the saturation state by FACS analysis (the concentration of LR004 was 10 μg·mL −1). The horizontal axis represents the values of relative fluorescence intensity. (D) The binding curves of different concentrations of LR004 and LR004‐VC‐MMAE to the EGFR high‐expression cells by FACS analysis. The vertical axis represents the values of mean fluorescence intensity.
Figure 3
Figure 3
Cytotoxicity in vitro of LR004‐VC‐MMAE. (A) The cell viability analysis of LR004 (blue line), LR004‐VC‐MMAE (red line), and rituximab‐VC‐MMAE (purple line) to KYSE520 and KYSE150 cells. The cell viability analysis of LR004‐VC‐MMAE to A431 and Karpas 299 cells. Karpas 299 cells were used as the negative control of cell line which expressed CD30 antigen on the cell surface. The cell viability was assessed using the CCK‐8 assay for 48 h. (B) The induction of apoptosis analysis in the KYSE520 and KYSE150 cells was detected by flow cytometry. The cells were treated with various concentrations of LR004‐VC‐MMAE for 24 h. (C) The cell cycle arrest analysis in the KYSE520 and KYSE150 cells was detected by flow cytometry. The cells were treated with various concentrations of LR004‐VC‐MMAE for 24 h.
Figure 4
Figure 4
Confocal analysis for intracellular localization and fluorescence imaging in KYSE520 model. (A) The internalization and lysosomal localization of LR004 and LR004‐VC‐MMAE in the KYSE520 cells by laser scanning confocal microscope. The KYSE520 cells were treated with 5 μg·mL −1 LR004 and LR004‐VC‐MMAE at 4 °C for 30 min or at 37 °C for 2 and 10 h. The lysosomes were labeled with a LAMP‐1 antibody followed by an Alexa Fluor 555‐labeled goat anti‐rabbit IgG (H+L) antibody. The cell nuclei were stained with DAPI. (B) In vivo fluorescence imaging of LR004 and LR004‐VC‐MMAE in KYSE520 nude mice xenograft model. Mice in the three DyLight 680‐labeled groups (LR004, LR004‐VC‐MMAE, and rituximab groups) were injected via the tail veins with the dose of 20 mg·kg−1 each. Representative in vivo fluorescence imaging at the indicated time points. Color scale represents photons/s/cm2/steradian.
Figure 5
Figure 5
Therapeutic efficacy of LR004‐VC‐MMAE against KYSE520 tumor xenograft model in nude mice. (A) Tumor growing curves of ESCC KYSE520 tumor xenograft model (n = 6). The mice were treated with various doses of LR004‐VC‐MMAE (5, 10, 15 mg·kg−1), LR004 (15 mg·kg−1), and rituximab‐VC‐MMAE (15 mg·kg−1) every 4 days for a total of four injections. (B) Body weight change of the KYSE520 tumor xenograft model. (C) Histopathological examination (H&E staining, ×200) of various organs and tumors (skin, heart, liver, spleen, lung, kidney, stomach, intestine, and bone) of the KYSE520 xenograft tumor nude model treated with the medicated groups.
Figure 6
Figure 6
Therapeutic efficacy of LR004‐VC‐MMAE against A431 tumor xenograft model in nude mice. (A) Tumor growing curves of the A431 tumor xenograft model (n = 7). The mice were treated with various doses of LR004‐VC‐MMAE (1, 5, 10, 15 mg·kg−1), LR004 (15 mg·kg−1) and MMAE (0.3 mg·kg−1) every 4 days for a total of six injections. ***P < 0.0001, compared with the control and MMAE (0.3 mg·kg−1) groups on day 40. **P < 0.01, compared with the LR004 (15 mg·kg−1) on day 60. (B) Tumor growing curves for the large tumor group of the A431 tumor xenograft model. ***P < 0.0001, compared with the control. (C) Histopathological examination (H&E staining, ×200) of various organs and tumors (heart, liver, spleen, lung, kidney, stomach, intestine, and bone) of the A431 xenograft tumor nude model treated with LR004 and LR004‐VC‐MMAE at a dosage of 15 mg·kg−1, respectively.
Figure 7
Figure 7
Concentration–time curves of single dose administrated with LR004‐VC‐MMAE in nude mice model. The BALB/c nude mice were injected subcutaneously with 15 mg·kg−1 of LR004‐VC‐MMAE and then sacrificed at 0, 0.5, 1, 2, 6, 24, 48, 72, 120, and 216 h after serum collection. (A) The mean total antibody concentrations (by ELISA) in serum of LR004‐VC‐MMAE ADC in nude mice. (B) The mean free MMAE (blue curve) and conjugated MMAE (red curve) concentrations (by LC‐MS/MS) in serum following administration of LR004‐VC‐MMAE in nude mice. Data points represent mean ± SD, n = 4 in both (A) and (B).

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