Listeriolysin O as cytotoxic component of an immunotoxin

Area of Science:

• Oncology
• Immunology
• Biochemistry

Background:

• Monoclonal antibodies (mAbs) are promising anticancer therapeutics.
• Conjugating tumor-specific mAbs with cytotoxic molecules enhances efficacy.
• Bifunctional immunotoxins offer significant potential in cancer therapy.

Purpose of the Study:

• To evaluate the suitability of bacterial Listeriolysin O (LLO) as a cytotoxic component for immunotoxins.
• To investigate the characteristics of LLO, including its pH optimum and regulated cytolytic activity.
• To develop and test a novel immunotoxin targeting tumor-specific antigens.

Main Methods:

• Assessed LLO's cytolytic activity via hemolysis, comparing oxidized and reduced forms.
• Determined cell-type-specific toxicity of LLO in cell culture.
• Coupled LLO to the dsFv fragment of the B3 antibody (recognizing Lewis Y antigen) using fusion peptides.
• Evaluated the efficacy of the resulting B3-LLO immunotoxin against antigen-positive and antigen-negative cell lines.

Main Results:

• Oxidized LLO exhibited significantly reduced cytolytic activity compared to reduced LLO.
• Both forms of LLO showed cell-type-unspecific toxicity, with reduced LLO being more potent.
• The novel B3-LLO immunotoxin specifically eliminated antigen-positive MCF7 cells (EC(50) = 2.3 nM).
• Antigen-negative cell lines were 80- to 250-fold less sensitive to B3-LLO.

Conclusions:

• Listeriolysin O is a viable and tunable toxophore for immunotoxin development.
• The B3-LLO immunotoxin demonstrates specific and potent targeting of Lewis Y-expressing tumor cells.
• This approach holds promise for developing targeted cancer therapies with improved efficacy and reduced off-target effects.