A recombinant immunotoxin engineered for increased stability by adding a disulfide bond has decreased immunogenicity

Area of Science:

• Biotechnology
• Immunology
• Oncology

Background:

• Recombinant immunotoxins (RITs) are promising anti-cancer agents.
• Immunogenicity of non-human proteins in RITs poses a significant challenge.
• HA22-LR, an anti-CD22 Fv fused to Pseudomonas exotoxin A, is a RIT candidate.

Purpose of the Study:

• To test the hypothesis that increased RIT stability reduces immunogenicity.
• To engineer a more stable and less immunogenic RIT variant.

Main Methods:

• Computer modeling identified sites for disulfide bond introduction in HA22-LR's domain III.
• Mutagenesis introduced cysteines to form a disulfide bond, creating HA22-LR-DB.
• Assessed thermal stability, trypsin resistance, cytotoxicity, anti-tumor activity, and immunogenicity in mice.

Main Results:

• HA22-LR-DB exhibited significantly increased thermal stability and trypsin resistance.
• The engineered RIT, HA22-LR-DB, maintained potent cytotoxic and anti-tumor activity.
• HA22-LR-DB demonstrated substantially lower immunogenicity in murine models.

Conclusions:

• Protein engineering can enhance RIT stability.
• Increased stability correlates with reduced immunogenicity in RITs.
• This strategy offers a pathway to safer and more effective RIT-based cancer therapies.