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.
Mil Med Res. 2021 Dec 9;8(1):63. doi: 10.1186/s40779-021-00358-9.
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.
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 in 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 MDA-MB-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 days. 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.
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 IC 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 G/M phase in MDA-MB-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.
LR004-VC-MMAE showed effective antitumor activity by inhibiting the activation of EGFR signaling and the 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.
三阴性乳腺癌（TNBC）是最具侵袭性的亚型，约占诊断出的乳腺癌的 15-20%。TNBC 的特点是其高度转移性和复发性，以及缺乏特异性靶点和靶向治疗药物。表皮生长因子受体（EGFR）在多种肿瘤中高度表达，尤其是在 TNBC 中。LR004-VC-MMAE 是我们实验室生产的一种新型 EGFR 靶向抗体药物偶联物。本研究旨在评估其对 EGFR 阳性 TNBC 的抗肿瘤活性，并进一步研究其可能的抗肿瘤作用机制。
LR004-VC-MMAE 通过连接子将细胞毒药物（MMAE）偶联到抗 EGFR 抗体（LR004）上制备，并用 HIC-HPLC 分析药物抗体比（DAR）。使用公开的微阵列数据集（GSE41313）和 Western blot 评估一系列乳腺癌细胞系中 EGFR 的基因表达。用 LR004-VC-MMAE（0、0.0066、0.066、0.66、6.6 nmol/L）处理 MDA-MB-468 和 MDA-MB-231 细胞，用 CCK-8 和集落形成实验检测 LR004-VC-MMAE 对细胞增殖的抑制作用。用划痕愈合和 Transwell 侵袭实验检测不同浓度 LR004-VC-MMAE（2.5 和 5 nmol/L）对 MDA-MB-468 和 MDA-MB-231 细胞迁移和侵袭能力的影响。用流式细胞术分析和肿瘤球形成实验检测 LR004-VC-MMAE 对 MDA-MB-468 和 MDA-MB-231 细胞中癌症干细胞的杀伤作用。还使用小鼠异种移植模型评估 LR004-VC-MMAE 在体内的抗肿瘤疗效。简而言之，将 MDA-MB-468 或 MDA-MB-231 细胞皮下接种于 BALB/c 裸鼠。然后将它们随机分为 4 组（每组 n=6），分别用 PBS、裸 LR004（10 mg/kg）、LR004-VC-MMAE（10 mg/kg）或阿霉素处理。每隔 4 天测量一次肿瘤大小和小鼠体重。用流式细胞术分析 LR004-VC-MMAE 对细胞凋亡和细胞周期分布的影响。用 Western blot 检测 LR004-VC-MMAE 对 EGFR、ERK、MEK 磷酸化和肿瘤干细胞标志物基因表达的影响。
获得了 DAR 为 4.02 的 LR004-VC-MMAE。与非 TNBC 细胞相比，TNBC 细胞中 EGFR 的表达明显更高（P<0.01）。LR004-VC-MMAE 抑制 EGFR 阳性 TNBC 细胞的增殖，MDA-MB-468 和 MDA-MB-231 细胞用 LR004-VC-MMAE 处理 72 h 的 IC 值分别为（0.13±0.02）nmol/L 和（0.66±0.06）nmol/L，明显低于用 MMAE 处理的细胞[（3.20±0.60）nmol/L，P<0.01，和（6.60±0.50）nmol/L，P<0.001]。LR004-VC-MMAE 有效抑制 MDA-MB-468 和 MDA-MB-231 细胞的迁移和侵袭。此外，LR004-VC-MMAE 还杀伤 EGFR 阳性 TNBC 细胞中的肿瘤干细胞，并损害其肿瘤球形成能力。在 TNBC 异种移植模型中，LR004-VC-MMAE 以 10 mg/kg 显著抑制肿瘤生长，并在第 36 天达到完全肿瘤消退。令人惊讶的是，直到第 52 天实验结束才观察到肿瘤复发。在机制研究中，我们发现 LR004-VC-MMAE 显著诱导 MDA-MB-468[（34±5）% vs. （12±2）%，P<0.001]和 MDA-MB-231[（27±4）% vs. （18±3）%，P<0.01]细胞中的细胞凋亡和 G/M 期细胞周期阻滞。LR004-VC-MMAE 还抑制了 EGFR 信号的激活和癌症干细胞标志物基因如 Oct4、Sox2、KLF4 和 EpCAM 的表达。
LR004-VC-MMAE 通过抑制 EGFR 信号的激活和癌症干细胞标志物基因的表达显示出有效的抗肿瘤活性。它可能是一种有前途的治疗候选药物，为治疗 EGFR 阳性 TNBC 提供了一种潜在的治疗途径。
