HER2-Specific Pseudomonas Exotoxin A PE25 Based Fusions: Influence of Targeting Domain on Target Binding, Toxicity,
Haozhong Ding1, Mohamed Altai2, Wen Yin1
1Department of Protein Science, KTH Royal Institute of Technology, Roslagstullsbacken 21, 114 17 Stockholm, Sweden.
Abstract:
The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT6, can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing. In this study we expanded the investigation for an optimal targeting domain and constructed two fusion toxins where a HER2-binding affibody molecule, ZHER2:2891, or the dual-HER2-binding hybrid ZHER2:2891-ADAPT6 were used for cancer cell targeting. We found that both targeting domains conferred strong binding to HER2; both to the purified extracellular domain and to the HER2 overexpressing cell line SKOV3. This resulted in fusion toxins with high cytotoxic potency toward cell lines with high expression levels of HER2, with EC50 values between 10 and 100 pM. For extension of the plasma half-life, an albumin binding domain was also included. Intravenous injection of the fusion toxins into mice showed a profound influence of the targeting domain on biodistribution. Compared to previous results, with ADAPT6 as targeting domain, ZHER2:2891 gave rise to further extension of the plasma half-life and also shifted the clearance route of the fusion toxin from the liver to the kidneys. Collectively, the results show that the targeting domain has a major impact on uptake of PE25-based fusion toxins in different organs. The results also show that PE25-based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxicity beyond a certain point in affinity. In conclusion, ZHER2:2891 has the most favorable characteristics as targeting domain for PE25.
Insights
The study compared HER2-targeting domains for fusion toxins, finding ZHER2:2891 optimal for extending plasma half-life and improving biodistribution in HER2-targeted cancer therapy.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Human epidermal growth factor receptor 2 (HER2) is a key target in cancer therapy.
- Current HER2-targeted therapies show success but have limitations, necessitating novel approaches.
- Previous work demonstrated the efficacy of ADAPT6-PE25 fusion toxins for HER2-positive cancer cells.
Purpose of the Study:
- To evaluate novel HER2-targeting domains for PE25-based fusion toxins.
- To compare the binding affinity, cytotoxic potency, and pharmacokinetic properties of different targeting domains.
- To identify an optimal targeting domain for enhanced HER2-targeted cancer therapy.
Main Methods:
- Construction of fusion toxins using ZHER2:2891 and ZHER2:2891-ADAPT6 targeting domains.
- Assessment of HER2 binding to purified extracellular domain and HER2-overexpressing cell lines (SKOV3).
- Evaluation of in vitro cytotoxicity (EC50 values) and in vivo biodistribution in mice.
Main Results:
- Both ZHER2:2891 and ZHER2:2891-ADAPT6 showed strong HER2 binding.
- Fusion toxins exhibited high cytotoxic potency (EC50: 10-100 pM) against HER2-high cells.
- ZHER2:2891 significantly extended plasma half-life and shifted clearance from liver to kidneys compared to ADAPT6.
Conclusions:
- The targeting domain profoundly influences the biodistribution and pharmacokinetics of PE25-based fusion toxins.
- Optimal HER2 affinity does not always correlate with increased cytotoxicity beyond a certain threshold.
- ZHER2:2891 demonstrates the most favorable characteristics as a targeting domain for PE25-based HER2-targeted cancer therapy.
