Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Therapeutics

Background:

• Immunotoxins, combining targeting proteins with toxins, are promising cancer therapeutics.
• Current methods like chemical conjugation or genetic fusion have limitations, including reduced activity, stability issues, low yields, and solubility problems.
• A need exists for efficient and modular immunotoxin engineering strategies.

Purpose of the Study:

• To develop a straightforward, noncovalent conjugation process for engineering modular immunotoxins.
• To utilize coassociating peptides (K3 and E3) from p53 for modular immunotoxin assembly.
• To evaluate the efficacy and specificity of the engineered immunotoxins against HER2-overexpressing cancer cells.

Main Methods:

• Genetically fused nanobodies targeting HER2 (nano-HER2) and a toxin fragment (TOX) to coassembling peptides (E3 and K3).
• Produced constructs separately in *Escherichia coli*, achieving soluble forms and high yields.
• Assessed binding specificity, affinity, cytotoxicity, and potency on HER2-overexpressing cells and breast cancer cell lines.

Main Results:

• Engineered immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) demonstrated specific binding to HER2-overexpressing cells with low nanomolar to picomolar binding constants.
• Toxin modules and assembled immunotoxins showed comparable cytotoxicity to the toxin alone.
• Immunotoxins exhibited high potency and specificity against HER2-overexpressing breast cancer cells, with IC50 values in the picomolar range.

Conclusions:

• The described noncovalent conjugation method using coassembling peptides is a straightforward and modular approach for immunotoxin engineering.
• This method facilitates the development of targeted cancer therapeutics with high efficacy and specificity.
• The strategy holds potential for targeting various cancer types by modifying the targeting moiety.