非天然氨基酸定点偶联抗人类表皮生长因子受体2-抗体偶联药物的药理学活性

doi:10.19723/j.issn.1671-167X.2019.05.002

摘要/Abstract

摘要：

目的:考察用非天然氨基酸定点偶联技术获得的均一性良好的抗人类表皮生长因子受体2(human epidermal growth factor receptor 2, HER2)-抗体偶联药物(antibody drug conjugate, ADC), 对9种不同HER2表达量的肿瘤细胞的增殖抑制活性,以及对5种异体移植的肿瘤小鼠模型的肿瘤生长抑制效果。方法:用QIFI试剂盒通过流式细胞仪检测HER2在BT-474、Calu-3、MCF-7、MDA-MB-231、MDA-MB-468、SK-BR-3、SK-OV-3、HCC1954、NCI-N87共9种肿瘤细胞中的表达。对9种肿瘤细胞进行培养,铺板过夜后分别加入梯度稀释的抗HER2-ADC、曲妥珠单抗-美坦新偶联物、AS269、pAF-AS269、紫杉醇5种药物,然后培养72 h或96 h,检测这5种药物对肿瘤细胞的增殖抑制活性。选择HER2阳性肿瘤细胞HCC1954、BT-474、SK-OV-3、NCI-N87和HER2阴性肿瘤细胞MDA-MB-468,分别接种到5~6周龄的BALB/c裸小鼠身上,待肿瘤长到一定体积后,分别注射抗HER2-ADC、曲妥珠单抗-美坦新偶联物、曲妥珠单抗、紫杉醇4种药物和空白对照磷酸盐缓冲液,考察药物的抑瘤效果。结果:流式细胞仪检测结果显示,SK-OV-3、NCI-N87、SK-BR-3、Calu-3、HCC1954、BT-474这6株肿瘤细胞的HER2表达量较高,每个细胞表面HER2受体数在43~80万个,比另外3株MDA-MB-231、MCF-7、MDA-MB-468肿瘤细胞的HER2表达水平高50倍以上。药物对9种肿瘤细胞的增殖抑制活性结果显示,抗HER2-ADC对HER2高表达的细胞有很强的抑制细胞生长活性,对SK-OV-3、NCI-N87、SK-BR-3、Calu-3、HCC1954、BT-474的半数抑制浓度分别为46、17、17、161、125、50 pmol/L。在动物体内药效试验中,抗HER2-ADC在所有HER2阳性表达的肿瘤模型中都表现了较强的呈剂量依赖的抗肿瘤活性,在NCI-N87异种移植肿瘤模型中,与曲妥珠单抗及曲妥珠单抗-美坦新偶联物相比,相同剂量的抗HER2-ADC表现出更好的抗肿瘤活性,其相对肿瘤增殖率约为二者的1/30至1/20,在HCC1954模型中表现出肿瘤的完全消退和治愈效果;抗HER2-ADC对HER2低表达MDA-MB-468肿瘤细胞移植瘤模型没有效果。与曲妥珠单抗-美坦新偶联物相比,抗HER2-ADC表现出相同或更好的抗肿瘤活性。结论:用非天然氨基酸定点偶联技术获得的抗HER2-ADC在细胞体外和动物体内实验中对HER2高表达的肿瘤具有明显的抑制效果。

关键词: 抗体偶联药物, 人类表皮生长因子受体2, 非天然氨基酸

Abstract:

Objective: To investigate inhibitory activities of a homogenous anti-human epidermal growth factor receptor 2 (HER2)-antibody drug conjugate (ADC) on the proliferation of nine tumor cell lines with different levels of HER2 expressions, and its activities on the tumor growth of five xenograft mouse models. Methods: The HER2 expression levels of BT-474, Calu-3, MCF-7, MDA-MB-231, MDA-MB-468, SK-BR-3, SK-OV-3, HCC1954, NCI-N87 tumor cell lines were measured using QIFI KIT. For the in vitro anti-proliferation assay, serial diluted anti-HER2-ADC, ado-trastuzumab emtansine, AS269, pAF-AS269 and paclitaxel were added to the seeded cells, and after 72 or 96 hours of incubation, the cell proliferation was analyzed. For the in vivo activity, 5-6 weeks old mice were inoculated with four HER2 positive tumor cell lines HCC1954, BT-474, SK-OV-3, NCI-N87 or one HER2 negative tumor cell line MDA-MB-468. Different amounts of anti-HER2-ADC, ado-trastuzumab emtansine, trastuzumab, paclitaxel and phosphate buffered saline control were injected after the tumor volume reached a certain size, then the tumor growth inhibition was analyzed. Results: The expression levels of the six high HER2-expression cell lines SK-OV-3, NCI-N87, SK-BR-3, Calu-3, HCC1954, BT-474 were between 430 000 to 800 000 receptors per cell, which were 50 times higher than those of the other three low HER2 expression tumor cell lines MDA-MB-231, MCF-7, MDA-MB-468. Anti-HER2-ADC had inhibition effects on cell lines with high level of HER2 expression in the in vitro anti-proliferation assay. The half maximal inhibitory concentrations of anti-HER2-ADC on SK-OV-3, NCI-N87, SK-BR-3, Calu-3, HCC1954, BT-474 tumor cell lines were 46 pmol/L, 17 pmol/L, 17 pmol/L, 161 pmol/L, 125 pmol/L, 50 pmol/L, respectively. Anti-HER2-ADC had a dose dependent antitumor activity in vivo in all the HER2 positive xenograft mouse models. In NCI-N87 xenograft tumor model, the same dose of anti-HER2-ADC showed better anti-tumor activity compared with trastuzumab and ado-trastuzumab emtansine, and its relative tumor proliferation rates were about 1/30 to 1/20 of the two. In HCC1954 xenograft tumor model, the complete regression of the tumor was observed. As expected, anti-HER2-ADC had no tumor inhibitory effects on MDA-MB-468 xenograft models with low HER2 expression. The antitumor activities of anti-HER2-ADC in HER2 positive xenograft tumor models were the same as or better than the activities of ado-trastuzumab emtansine. Conclusion: The homogenous site-specific anti-HER2-ADC obtained using unnatural amino acid technology can inhibit the growth of high HER2-expression tumor cells with high potency both in vivo and in vitro.

Key words: Antibody drug conjugate, Human epidermal growth factor receptor 2, Unnatural amino acid

中图分类号:

R966

梁学军,宫丽颖,周菲,周德敏,祝静静. 非天然氨基酸定点偶联抗人类表皮生长因子受体2-抗体偶联药物的药理学活性[J]. 北京大学学报（医学版）, 2019, 51(5): 797-804.

Xue-jun LIANG,Li-ying GONG,Fei ZHOU,De-min ZHOU,Jing-jing ZHU. Pharmacological effects of site specific conjugated anti-human epidermal growth factor receptor 2-antibody drug conjugate using unnatural amino acid technology[J]. Journal of Peking University(Health Sciences), 2019, 51(5): 797-804.

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Cell lines	Cancer type	HER2 level	IC₅₀ /(nmol/L)
Cell lines	Cancer type	HER2 level	Anti-HER2-ADC	Ado-trastuzumab emtansine	AS269	pAF-AS269	Paclitaxel
MDA-MB-468	Breast cancer	127	>30	10.160	>30	>30	6.857
MCF-7		7637	>30	>30	>30	>30	3.017
MDA-MB-231		7737	>30	19.960	>30	>30	6.286
BT-474		428877	0.050	0.180	>30	>30	10.280
HCC1954		448095	0.125	0.294	>30	>30	11.500
Calu-3	Lung cancer	537491	0.161	4.557	>30	>30	12.950
SK-BR-3	Breast cancer	719797	0.017	0.044	>30	>30	6.468
NCI-N87	Gastric cancer	748406	0.017	0.032	>30	>30	7.189
SK-OV-3	Ovarian cancer	799943	0.046	0.065	>30	>30	8.475

Drugs	Dose/(mg/kg)	T/C in the xenograft models/%
Drugs	Dose/(mg/kg)	BT-474	HCC1954	SK-OV-3	NCI-N87	MDA-MB-468
Control	0	100.0	100.0	100.0	100.0	100.0
	0.55	38.1±0.8^*	25.8±1.0^*	79.2±1.4	65.5±1.2	101.3±1.4
Anti-HER2-ADC	1.67	5.0±0.2^*	1.0±0.0^*	26.0±1.1^*	56.6±1.3	106.1±2.2
	5.00	1.0±0.1^*	0.2±0.0^*	10.9±1.3^*	2.8±0.3^*	110.5±1.4
	0.55		50.8±1.8	96.0±3.3	90.2±1.8	122.4±2.0
Ado-trastuzumab emtansine	1.67		7.9±0.5^*	66.1±1.3	71.8±2.1	111.2±2.4
	5.00		7.1±0.6^*	24.0±0.8^*	54.9±1.7	127.0±1.9
Trastuzumab	5.00	62.1±1.9	42.9±1.6	72.7±1.2	79.1±1.9	149.1±1.8
Paclitaxel	15.00	53.3±0.8	26.1±0.6^*	71.7±2.5	59.5±1.4	32.9±1.0^*