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A Novel Affibody-Auristatin E Conjugate With a Potent and Selective Activity Against HER2+ Cell Lines
Alicja M Sochaj-Gregorczyk1, Anna M Serwotka-Suszczak, Jacek Otlewski
1Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
Abstract:
Targeted therapy is a new type of cancer treatment that most often uses biologically active drugs attached to a monoclonal antibody. This so called antibody-drug conjugate strategy allows the use of highly toxic substances that target tumor cells specifically, leaving healthy tissues largely unaffected. Over the last few years, antibody-drug conjugates have become a powerful tool in cancer treatment. We developed and characterized a novel cytotoxic conjugate against HER2 tumors in which the antibody has been substituted with a much smaller molecule: the affibody. The conjugate is composed of the ZHER2:2891 affibody that recognizes HER2 and a highly potent cytotoxic drug auristatin E. The ZHER2:2891 molecule does not contain cysteine(s) in its amino acid sequence. We generated 3 variants of ZHER2:2891, each containing a single cysteine to allow conjugation through the maleimide group that is present in the cytotoxic component. In 2 variants, we introduced single S46C and D53C substitutions. In the third variant, a short Drug Conjugation Sequence (DCS) containing a single cysteine was introduced at the C-terminus of ZHER2:2891, resulting in ZHER2:2891-DCS. The latter variant exhibited a significantly higher conjugation yield, and therefore its cytotoxicity has been studied more thoroughly. The ZHER2:2891-DCS-MMAE conjugate killed the HER2-overexpressing SK-BR-3 and MDA-MB-453 cells efficiently (IC50 values of 5.2 and 24.8 nM, respectively). The T-47-D and MDA-MB-231 cells that express normal levels of HER2 were significantly less sensitive to the conjugate (IC50 values of 135.6 and 161.5 nM, respectively). Overall, we have demonstrated for the first time that proteins other than antibodies/antibody fragments can be successfully combined with a linker-drug module, resulting in conjugates that eliminate cancer cells selectively.
Insights
Researchers created a novel targeted cancer therapy using affibody-drug conjugates. This new approach selectively eliminates HER2-positive cancer cells, showing promise for more effective treatments.
Area of Science:
- Oncology
- Biotechnology
- Drug Development
Background:
- Antibody-drug conjugates (ADCs) are effective cancer therapeutics, utilizing monoclonal antibodies to deliver cytotoxic drugs specifically to tumor cells.
- The development of targeted cancer therapies is crucial for improving treatment efficacy and minimizing off-target toxicity.
- HER2-positive tumors represent a significant area of focus for targeted treatment strategies.
Purpose of the Study:
- To develop and characterize a novel cytotoxic conjugate using a small affibody molecule instead of a full antibody for HER2-targeted cancer therapy.
- To evaluate the conjugation efficiency and cytotoxicity of the novel affibody-drug conjugate against HER2-expressing cancer cells.
- To demonstrate the potential of non-antibody proteins in targeted drug delivery systems.
Main Methods:
- Engineered three variants of the ZHER2:2891 affibody, each with a single cysteine for conjugation.
- Introduced cysteine substitutions (S46C, D53C) or a C-terminal Drug Conjugation Sequence (DCS) for maleimide-based conjugation with auristatin E.
- Assessed conjugation yield and in vitro cytotoxicity (IC50 values) of the ZHER2:2891-DCS-MMAE conjugate against HER2-overexpressing and HER2-normal cell lines.
Main Results:
- The ZHER2:2891-DCS variant showed significantly higher conjugation yield compared to other variants.
- The ZHER2:2891-DCS-MMAE conjugate demonstrated potent and selective cytotoxicity against HER2-overexpressing cell lines (SK-BR-3, MDA-MB-453) with low nanomolar IC50 values.
- Cell lines expressing normal levels of HER2 (T-47-D, MDA-MB-231) were significantly less sensitive to the conjugate.
Conclusions:
- Proteins other than antibodies can be successfully utilized in linker-drug conjugates for targeted cancer therapy.
- Affibody-drug conjugates represent a promising alternative to traditional antibody-drug conjugates for selective cancer cell elimination.
- This study validates a novel platform for developing targeted therapeutics against HER2-positive cancers.
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