Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype and occurs in approximately 15％–20％ of diagnosed breast cancers. TNBC is characterized by its highly metastatic and recurrent features, as well as a lack of specific targets and targeted therapeutics. Epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors, especially in TNBC. LR004-VC-MMAE is a new EGFR-targeting antibody–drug conjugate produced by our laboratory. This study aimed to evaluate its antitumor activities against EGFR-positive TNBC and further studied its possible mechanism of antitumor action. Methods: LR004-VC-MMAE was prepared by coupling a cytotoxic payload (MMAE) to an anti-EGFR antibody (LR004) via a linker, and the drug-to-antibody ratio (DAR) was analyzed by HIC-HPLC. The gene expression of EGFR in a series of breast cancer cell lines was assessed using a publicly available microarray dataset (GSE41313) and Western blotting. MDA-MB-468 and MDA-MB-231 cells were treated with LR004-VC-MMAE (0, 0.0066, 0.066, 0.66, 6.6 nmol/L), and the inhibitory effects of LR004-VC-MMAE on cell proliferation were examined by CCK-8 and colony formation. The migration and invasion capacity of MDA-MB-468 and MDA-MB-231 cells were tested at different LR004-VC-MMAE concentrations (2.5 and 5 nmol/L) with wound healing and Transwell invasion assays. Flow cytometric analysis and tumorsphere-forming assays were used to detect the killing effects of LR004-VC-MMAE on cancer stem cells (MDA-MB-468 and MDA-MB-231 cells). The mouse xenograft models were also used to evaluate the antitumor efficacy of LR004-VC-MMAE in vivo. Briefly, BALB/c nude mice were subcutaneously inoculated with MDA-MB-468 or MDAMB-231 cells. Then they were randomly divided into 4 groups (n=6 per group) and treated with PBS, naked LR004 (10 mg/kg), LR004-VC-MMAE (10 mg/kg), or doxorubicin, respectively. Tumor sizes and the body weights of mice were measured every 4 d. The effects of LR004-VC-MMAE on apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blotting was used to detect the effects of LR004-VC-MMAE on EGFR, ERK, MEK phosphorylation and tumor stemness marker gene expression. Results: LR004-VC-MMAE with a DAR of 4.02 were obtained. The expression of EGFR was found to be significantly higher in TNBC cells compared with non-TNBC cells (P<0.01). LR004-VC-MMAE inhibited the proliferation of EGFR-positive TNBC cells, and the IC50 values of MDA-MB-468 and MDA-MB-231 cells treated with LR004-VC-MMAE for 72 h were (0.13±0.02) nmol/L and (0.66±0.06) nmol/L, respectively, which were significantly lower than that of cells treated with MMAE [(3.20±0.60) nmol/L, P<0.01, and (6.60±0.50) nmol/L, P<0.001]. LR004-VC-MMAE effectively inhibited migration and invasion of MDA-MB-468 and MDA-MB-231 cells. Moreover, LR004-VC-MMAE also killed tumor stem cells in EGFR-positive TNBC cells and impaired their tumorsphere-forming ability. In TNBC xenograft models, LR004-VC-MMAE at 10 mg/kg significantly suppressed tumor growth and achieved complete tumor regression on day 36. Surprisingly, tumor recurrence was not observed until the end of the experiment on day 52. In a mechanistic study, we found that LR004-VC-MMAE significantly induced cell apoptosis and cell cycle arrest at G2/M phase in MDAMB-468 [(34±5)％ vs. (12±2)％, P<0.001] and MDA-MB-231 [(27±4)％ vs. (18±3)％, P<0.01] cells. LR004-VC-MMAE also inhibited the activation of EGFR signaling and the expression of cancer stemness marker genes such as Oct4, Sox2, KLF4 and EpCAM.Conclusions: LR004-VC-MMAE showed effective antitumor activity by inhibiting the activation of EGFR signaling andthe expression of cancer stemness marker genes. It might be a promising therapeutic candidate and provides a potential therapeutic avenue for the treatment of EGFR-positive TNBC.

Dan?Dan Zhou;Wei?Qi Bai;Xiao?Tian Zhai;Li?Ping Sun;Yong?Su Zhen;Zhuo?Rong Li;Qing?Fang Miao

NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College,No.1 Tiantan Xili, Beijing 100050, China;Department of Organic Chemistry, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, No.1 Tiantan Xili,Beijing 100050, China

军事医学研究（英文）

[1]Dan?Dan Zhou;Wei?Qi Bai;Xiao?Tian Zhai;Li?Ping Sun;Yong?Su Zhen;Zhuo?Rong Li;Qing?Fang Miao-.Excellent effects and possible mechanisms of action of a new antibody–drug conjugate against EGFR?positive triple?negative breast cancer)[J].军事医学研究（英文）,2022(04):419-431

LR004,GSE41313,tumorsphere,MDAMB,andthe

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