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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (5): 797-804. doi: 10.19723/j.issn.1671-167X.2019.05.002

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Pharmacological effects of site specific conjugated anti-human epidermal growth factor receptor 2-antibody drug conjugate using unnatural amino acid technology

Xue-jun LIANG1,Li-ying GONG1,Fei ZHOU1,De-min ZHOU2,Jing-jing ZHU1,△()   

  1. 1. Zhejiang NovoCodex Biopharmaceuticals Company Limited, Shaoxing, Zhejiang 312000, China
    2. Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100871, China
  • Received:2018-08-21 Online:2019-10-18 Published:2019-10-23
  • Contact: Jing-jing ZHU E-mail:zhujingjing@zmc-china.com
  • Supported by:
    Supported by the Special Fund from Science Technology Department of Zhejiang Province

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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

CLC Number: 

  • R966

Figure 1

Site specific conjugate anti-HER2 mAb and cytotoxic AS269 HER2, human epidermal growth factor receptor 2; mAb, monoclonal antibody."

Figure 2

Conjugation rate profile of anti-HER2-ADC by high performance hydrophobic interaction chromatography mAb, monoclonal antibody."

Table 1

The inhibitory activities in cell proliferation assay"

Cell lines Cancer type HER2 level IC50 /(nmol/L)
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

Figure 3

Internalization of anti-HER2-ADC in HCC1954 cell line after DRAQ5 staining and Pacific OrangeTM fluorescence labeling(×200)"

Figure 4

The tumor volume after administering drugs to xenograft model NCI-N87"

Table 2

The relative tumor volume proliferation rate of test articles in the xenograft models"

Drugs Dose/(mg/kg) T/C in the xenograft models/%
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*

Figure 5

The relative tumor volume proliferation rate after administering anti-HER2-ADC to xenograft models"

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