(S)-Afatinib (BIBW2992) is an irreversible EGFR family inhibitor with IC50s of 0.5/0.4/10/14/1 nM for EGFRwt, EGFR (L858R), EGFR (L858R/T790M), HER2, and HER4, respectively.

HER2:14 nM (cell free), EGFR (WT):0.5 nM (cell free), EGFR (L858R):0.4 nM (cell free), EGFR (L858R/T790M):10 nM (cell free), ERB4:1 nM (cell free)

(S)-Afatinib对EGFR与HER2的野生型及其突变形式展示出强大活性（IC50分别为：EGFRwt 0.5 nM、EGFR L858R 0.4 nM、EGFR L858R/T790M 10 nM、HER2 14 nM）。在人类乳腺癌细胞系中，使用100 nM (S)-Afatinib足以阻止hereregulin刺激的HER3磷酸化作用[1]。食管鳞状细胞癌（ESCC）细胞系对于afatinib敏感，IC50浓度处于较低μM范围内（72小时培养下：HKESC-1 = 0.002 μM、HKESC-2 = 0.002 μM、KYSE510 = 1.090 μM、SLMT-1 = 1.161 μM与EC-1 = 0.109 μM）。ErbB家族下游效应物如pAKT、pS6与pMAPK的磷酸化在HKESC-2与EC-1中被显著抑制。两个细胞系在暴露于afatinib后24小时观察到凋亡现象[2]。

每日口服BIBW2992，剂量为20 mg/kg，连续25天治疗，显著促进肿瘤退化并下调EGFR和AKT的磷酸化水平。通过NCIH1975细胞系（表达EGFR L858R/T790M）引起的异种移植瘤形成，经BIBW2992处理后被有效控制，20 mg/kg剂量的T/C值为12% [1]。Afatinib能有效抑制小鼠体内HKESC-2肿瘤生长，且无明显毒副作用；单独使用Afatinib在体内模型中对食管鳞状细胞癌（ESCC）表现出优秀的生长抑制效果 [2]。

The wild type tyrosine kinase domain of the human EGFR, as well as the EGFR L858R/T790M double mutant, were fused to Glutathione-S-transferase (GST) and extracted as described in Supplementary methods. The L858R mutant was purchased from Upstate. Enzyme activity was then assayed in the presence or absence of serial inhibitor dilutions performed in 50% Me2SO. A random polymer pEY (4:1) from Sigma was used as substrate. Biotinylated pEY was added as a tracer substrate. The kinase domain of HER2 was cloned using baculovirus system and extracted similarly to that of EGFR kinase domain. Detailed procedures for EGFR, HER2, SRC, BIRK and VEGFR2 kinase activity assays are included in Supplementary information [1].

Cells (1×10^4) were transferred into each well of a 96-well plate and cultured over night in serum-free media for EGFR phosphorylation assay. After addition of test compounds on the next day, the plates were then incubated at 37°C for 1 hour. EGF-stimulation was done at 100 ng/ml for 10 min at room temperature. Cells were washed with ice cold PBS before extraction with 120 μl per well HEPEX buffer and shaken for 1 h at room temperature. In all 2×10^4 cells per well was used for HER2 phosphorylation assay. Streptavidin precoated plates were coated with anti-EGFR-biotin at 1:100 dilution with blocking buffer and c-erb2/HER2 oncoprotein Ab-5(Clone N24)-Biotin. Extracts from above steps were then transferred to the antibody-coated wells and incubated for 1 h at room temperature. Assessment of color development is described in Supplementary information. Extinction was measured at 450 nm. The data generated were analysed by the program PRISM. Normalized values were used to calculate the IC50 by a nonlinear regression curve fit (variable slope) [1].

Six weeks old female athymic nude mice (nu/nu) weighing about 16-20 gram were housed by Laboratory Animal Services Centre of The Chinese University of Hong Kong. The experiment was conducted by researchers under license from the Hong Kong Government Department of Health and according to approval given by Animal Experimentation Ethics Committee of the Chinese University of Hong Kong. ESCC xenografts were established by inoculating HKESC-2 (0.6 × 10^5 cells re-suspended in 50 μl of HBSS-buffer) subcutaneously into both flanks of the nude mice. When tumor size reached to 4-6 mm diameter, they were randomized in either treatment (15 mg/kg) or vehicle control group. Afatinib for treatment was prepared by dissolving in 0.5% methylcellulose before administration. Either drug or vehicle was administered to mouse by oral gavage in a schedule of 5 days on plus 2 days off for two weeks. Drug efficacy was evaluated by monitoring the change in tumor size with caliper. Tumor volume was calculated with the formula Tumor Volume = (width2 × length)/2 [2].