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

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.

Hu XY ，Wang R ，Jin J ，Liu XJ ，Cui AL ，Sun LQ ，Li YP ，Li Y ，Wang YC ，Zhen YS ，Miao QF ，Li ZR ... -  《-》

Excellent effects and possible mechanisms of action of a new antibody-drug conjugate against EGFR-positive triple-negative breast cancer.

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 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 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.

Zhou DD ，Bai WQ ，Zhai XT ，Sun LP ，Zhen YS ，Li ZR ，Miao QF ... -  《-》

Monomethyl auristatin E-conjugated anti-EGFR antibody inhibits the growth of human EGFR-positive non-small cell lung cancer.

Li Z ，Wang M ，Yu D ，Luo W ，Fang J ，Huang C ，Yao X ... -  《-》

HER2-targeted antibody drug conjugates for ovarian cancer therapy.

HER2 targeted delivery of ovarian cancer therapy has been beneficial for some patients, although, its efficacy is yet to be confirmed in large populations. We generated a novel anti-HER2 humanized antibody (Hertuzumab) and conjugated it to a microtubule-disrupting drug monomethyl auristatin E conjugate (MMAE) with a lysosomal protease-cleavable valine-citrulline linker. The average drug to antibody ratio (DAR) of Hertuzumab-vc-MMAE was varied by conjugating Hertuzumab antibodies with increasing linker-drugs (LDs) from D0-D8. The resulting conjugates were tested for kinetic affinity for soluble HER2-ECD, cytotoxicity, and in vivo pharmacokinetics. The kinetic binding constant values (KD) were obtained by the bio-layer interference (BLI) method. The half time (t1/2) and clearance (Cl) results of the pharmacokinetic profile in rats were DAR-dependent. Hertuzumab-vc-MMAE with DAR4 was selected for further evaluation. Both Hertuzumab and Hertuzumab conjugates could bind to HER2 antigen, and exhibited significant cytotoxicity on HER2 positive tumor cells after internalization by receptor-mediated endocytosis. Hence, Hertuzumab-vc-MMAE conjugates were significantly selective both in vitro and in vivo as compared to other ovarian cancer clinical therapies that are currently used. Cell signal transduction and cell cycle were also affected, as shown by down regulation of PI3K/AKT pathway and arrested mitosis in the G2/M phase. The pharmacokinetics and pharmacodynamics (PK-PD) of the conjugates in nude mouse xenograft model demonstrated a correlation between efficacy and drug concentration. These results show that Hertuzumab-vc-MMAE is a potential therapeutic agent for HER2 positive ovarian cancer.

Jiang J ，Dong L ，Wang L ，Wang L ，Zhang J ，Chen F ，Zhang X ，Huang M ，Li S ，Ma W ，Xu Q ，Huang C ，Fang J ，Wang C ... -  《-》

A glypican-1-targeted antibody-drug conjugate exhibits potent tumor growth inhibition in glypican-1-positive pancreatic cancer and esophageal squamous cell carcinoma.

An antibody-drug conjugate (ADC) is a promising therapeutic modality because selective and effective delivery of an anti-cancer drug is achieved by drug-conjugated antibody-targeting cancer antigen. Glypican 1 (GPC1) is highly expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC) and esophageal squamous cell carcinoma (ESCC). Herein, we describe the usefulness of GPC1-targeting ADC. Humanized anti-GPC1 antibody (clone T2) was developed and conjugated with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PABC) linkers (humanized GPC1-ADC[MMAE]). Humanized GPC1-ADC(MMAE) inhibited the growth of GPC1-positive PDAC and ESCC cell lines via inducing cycle arrest in the G2/M phase and apoptosis in vitro. The binding activity of humanized GPC1-ADC(MMAE) with GPC1 was comparable with that of the unconjugated anti-GPC1 antibody. The humanized GPC1-ADC(MMAE) was effective in GPC1-positive BxPC-3 subcutaneously xenografted mice but not in GPC1-negative BxPC-3-GPC1-KO xenografted mice. To assess the bystander killing activity of the humanized GPC1-ADC(MMAE), a mixture of GPC1-positive BxPC-3 and GPC1-negative BxPC-3-GPC1-KO-Luc cells were subcutaneously inoculated, and a heterogenous GPC1-expressing tumor model was developed. The humanized GPC1-ADC(MMAE) inhibited the tumor growth and decreased the luciferase signal, measured with an in vivo imaging system (IVIS), which suggests that the suppression of the BxPC-3-GPC1-KO-Luc population. The humanized GPC1-ADC(MMAE) also inhibited the established liver metastases of BxPC-3 cells and significantly improved the overall survival of the mice. It exhibited a potent antitumor effect on the GPC1-positive PDAC and ESCC patient-derived xenograft (PDX) models. Our preclinical data demonstrate that GPC1 is a promising therapeutic target for ADC.

Munekage E ，Serada S ，Tsujii S ，Yokota K ，Kiuchi K ，Tominaga K ，Fujimoto M ，Kanda M ，Uemura S ，Namikawa T ，Nomura T ，Murakami I ，Hanazaki K ，Naka T ... -  《NEOPLASIA》