Afatinib(BIBW2992)

Afatinib(BIBW2992) is an oral, selective inhibitor of the receptor tyrosine kinases (RTK) of the ErbB family with IC₅₀ values of 0.0.5nM for EGFR kinase, 14nM for HER2 kinase, and 1nM for HER4 kinase. The substance irreversibly binds to cysteine 797 of the EGF receptor and the corresponding cysteines 805 and 803 in HER2 and HER4, respectively^[1][2].

In vitro, T cells were exposed to 1µM Afatinib for 3 days and then analyzed by FCM for CD62L expression and ROS content. Compared with the negative control, Afatinib significantly increased the expression levels of CD62L and decreased the concentration of ROS. CAR-T cells were exposed to 1µM Afatinib for 3 days and then subjected to analysis or in vivo experiments. Afatinib could modulate CAR-T metabolism and differentiation, which further inhibit exhaustion and enhance persistence, and significantly improve the antitumor performance^[3]. Afatinib treatment (400nM) for 24h completely blocked EGF-induced phosphorylation of EGFR, MAPK (ERK1/2) and Akt in human pancreatic cancer BxPC-3 cells. Afatinib also increased proportion of cells in subG1 phase of the cell cycle and reduced the percentage of cells in G0/G1 phase^[4]. FaDu human squamous-cell carcinoma model was incubated with Afatinib (3, 30 and 300nM) for up to 9 days. Afatinib revealed a significant and dose-dependent inhibitory effect on tumor cell proliferation and increase of the G0/G1 fraction^[5]

In vivo, Afatinib (20 mg/kg/day) was orally administrated as a single agent or in combination with rapamycin (2mg/kg, i.p.) in H1781 non-small cell lung cancer (NSCLC) xenograft mice. The Afatinib/rapamycin combination treatment of mice carrying large (~200mm³) H1781 tumors resulted in significant tumor regressions in all treated animals whereas single agents delayed or stopped tumor growth^[6]. Egfr exon 19 mutation induced lung cancer models were treated with oral Afatinib (5 mg/kg/day),gefitinib (5 mg/kg/day), or vehicle alone from 11 to 15 weeks of age. Afatinib-treated mice showed significantly less tumor burden. Afatinib is more effective than gefitinib for treatment of lung cancer induced by the Egfr mutation in exon 19 because it suppresses not only pEGFR but also pHER2 and induces apoptosis over a longer period than gefitinib^[7].

References:
[1] Wecker H, Waller C F. Afatinib. Recent Results Cancer Res. 2018:211:199-215.
[2] Regales L, Gong Y, Shen R, de Stanchina E, Vivanco I, Goel A et al (2009) Dual targeting ofEGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer. J Clin Invest
[3] Dai Y Y, Liu Y, An L N, et al. Afatinib boosts CAR-T cell antitumor therapeutic efficacy via metabolism and fate reprogramming. J Immunother Cancer. 2024 Nov 17;12(11):e009949.
[4] Ioannou N, Dalgleish A G, Seddon A M, et al. Anti-tumour activity of afatinib, an irreversible ErbB family blocker, in human pancreatic tumour cells. Br J Cancer. 2011 Nov 8;105(10):1554-62.
[5] Schütze C, Dörfler A, Eicheler W,et al. Combination of EGFR/HER2 tyrosine kinase inhibition by BIBW 2992 and BIBW 2669 with irradiation in FaDu human squamous cell carcinoma. Strahlenther Onkol. 2007 May;183(5):256-64.
[6] Perera S A, Li D, Shimamura T,et al. HER2YVMA drives rapid development of adenosquamous lung tumors in mice that are sensitive to BIBW2992 and rapamycin combination therapy. Proc Natl Acad Sci U S A. 2009 Jan 13;106(2):474-9.
[7] Ninomiya T, Takigawa N, Ichihara E, et al. Afatinib prolongs survival compared with gefitinib in an epidermal growth factor receptor-driven lung cancer model. Mol Cancer Ther. 2013. 12(5):589–597.

Afatinib(BIBW2992)是一种口服、选择性ErbB家族受体酪氨酸激酶（RTK）抑制剂，其对EGFR激酶的IC₅₀值为0.05nM，对HER2激酶为14nM，对HER4激酶为1nM。该物质不可逆地结合于EGF受体的半胱氨酸797以及HER2和HER4中对应的半胱氨酸805和803^[1][2]。

体外实验中，T细胞以1µM Afatinib处理3天后用流式细胞术检测CD62L表达和ROS含量。与阴性对照相比，Afatinib显著上调CD62L表达水平并降低ROS浓度。CAR-T细胞以1µM Afatinib处理3天后进行分析或体内实验；Afatinib可调控CAR-T代谢与分化，进一步抑制耗竭并增强持久性，在体内显著改善抗肿瘤表现^[3]。400nM Afatinib处理24h即可完全阻断人胰腺癌BxPC-3细胞中EGF诱导的EGFR、MAPK(ERK1/2)和Akt磷酸化；Afatinib还提高细胞周期subG1期比例，降低G0/G1期比例^[4]。FaDu人鳞状细胞癌模型以3、30、300nM Afatinib孵育至9天，Afatinib呈剂量依赖性显著抑制肿瘤细胞增殖并增加G0/G1期比例^[5]。

体内实验，Afatinib(20mg/kg/day)口服单药或与rapamycin(2mg/kg，i.p.)联合用于H1781非小细胞肺癌(NSCLC)异种移植小鼠。对携带约200mm³大型H1781肿瘤的鼠进行Afatinib/rapamycin联合治疗，所有受治动物均出现显著肿瘤消退，而单药仅延缓或阻止肿瘤生长^[6]。Egfr exon 19突变诱导的肺癌模型在11至15周龄时分别口服阿法替尼（5mg/kg/day）、吉非替尼（5mg/kg/day）或空白对照；Afatinib治疗组肿瘤负荷显著减少。Afatinib对由Egfr exon19突变诱导的肺癌疗效优于gefitinib，原因在于其不仅抑制pEGFR，还抑制pHER2，且诱导凋亡的持续时间更长^[7]。