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Synthesized Anti-HER2 Trastuzumab-MCC-DM1 Conjugate: An Evaluation of Efficacy and Cytotoxicity
Soodabeh Shafiee1, Roya Mirzaei1, Malihe Salehi1
1Department of Recombinant Proteins, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran.
Background:
Trastuzumab is a humanized monoclonal antibody that targets site-specifically human epidermal growth factor-2 receptor (HER2) cell surface antigen overexpressed in approximately 20% of human breast carcinomas. Despite its positive therapeutic outcomes, a large proportion of individuals are unresponsive to the treatment with the trastuzumab or develop resistance to it.
Objective:
To evaluate a chemically synthesized trastuzumab-based antibody-drug conjugate (ADC) to improve the trastuzumab therapeutic index.
Methods:
The current study explored the physiochemical characteristics of the trastuzumab conjugated to a cytotoxic chemotherapy agent DM1 via Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, created in our earlier study, using SDS-PAGE, UV/VIS, and RP-HPLC analyses. The antitumor effects of the ADCs were analyzed using MDA-MB-231 (HER2-negative) and SK-BR-3 (HER2-positive) cell lines utilizing in vitro cytotoxicity, viability, and binding assays. Three different formats of a HER2-targeting agent: trastuzumab, synthesized trastuzumab-MCC-DM1, and commercially available drug T-DM1 (Kadcyla®) were compared.
Results:
UV-VIS spectroscopic analysis showed that the trastuzumab-MCC-DM1 conjugates, on average, entailed 2.9 DM1 payloads per trastuzumab. A free drug level of 2.5% was determined by RP-HPLC. The conjugate appeared as two bands on a reducing SDS-PAGE gel. MTT viability assay showed that conjugating trastuzumab with DM1 significantly improved the antiproliferative effects of this antibody in vitro. Importantly, the evaluations using LDH release and cell apoptosis assays confirmed that trastuzumab maintains its ability to induce cell death response while conjugating with the DM1. The binding efficiency of trastuzumab-MCC-DM1 was comparable to that of the naked trastuzumab.
Conclusion:
Trastuzumab-MCC-DM1 was found effective against HER2+ tumors. The potency of this synthesized conjugate brings it closer to the commercially available T-DM1.
Insights
A new trastuzumab-based antibody-drug conjugate (ADC) effectively targets HER2+ breast cancer cells. This synthesized conjugate shows potency comparable to T-DM1, offering a promising alternative for patients unresponsive to trastuzumab.
Area of Science:
- Oncology
- Biotechnology
- Pharmacology
Background:
- Trastuzumab is a monoclonal antibody targeting HER2, crucial in treating HER2-positive breast cancer.
- Resistance and unresponsiveness to trastuzumab limit its therapeutic efficacy in many patients.
Purpose of the Study:
- To evaluate a chemically synthesized trastuzumab-based antibody-drug conjugate (ADC) for improved therapeutic index.
- To assess the physiochemical properties and in vitro antitumor effects of the novel ADC.
Main Methods:
- Synthesized trastuzumab conjugated to DM1 via SMCC linker (trastuzumab-MCC-DM1).
- Analyzed conjugate characteristics using SDS-PAGE, UV/VIS, and RP-HPLC.
- Evaluated in vitro cytotoxicity, viability, and binding assays on HER2-negative (MDA-MB-231) and HER2-positive (SK-BR-3) cell lines.
Main Results:
- Trastuzumab-MCC-DM1 conjugates carried an average of 2.9 DM1 payloads per trastuzumab.
- Conjugation significantly enhanced antiproliferative effects in vitro while maintaining HER2 binding.
- The synthesized ADC demonstrated efficacy against HER2+ cells, comparable to T-DM1.
Conclusions:
- The synthesized trastuzumab-MCC-DM1 conjugate is effective against HER2-positive tumors.
- This novel ADC exhibits potency nearing that of the commercial drug T-DM1, suggesting therapeutic potential.
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