Protein deimmunization via structure-based design enables efficient epitope deletion at high mutational loads.

Area of Science:

• Biochemistry
• Immunology
• Protein Engineering

Background:

• Anti-drug immune responses pose a significant risk to biotherapeutic efficacy and patient safety.
• Undesired immunogenicity can alter drug pharmacokinetics and compromise therapeutic outcomes.
• Efficient protein deimmunization tools are essential to harness the full potential of biotherapeutics.

Purpose of the Study:

• To develop and validate a structure-based protein design algorithm, EpiSweep, for reducing immunogenicity.
• To engineer biotherapeutic candidates with reduced T cell epitope content while preserving protein structure and function.
• To compare the efficacy of structure-based versus sequence-based deimmunization strategies.

Main Methods:

• Development of EpiSweep, a structure-based algorithm for protein deimmunization.
• Application of EpiSweep to engineer seven beta-lactamase variants with reduced predicted epitope content.
• Experimental validation of engineered variants for stability, activity, and interaction with human class II MHC proteins.
• Comparative analysis against beta-lactamase variants designed using a sequence-based algorithm.

Main Results:

• Seven engineered beta-lactamase variants showed 27-47% reduction in predicted epitope content with 8 mutations each.
• All engineered variants maintained good stability and enzymatic activity.
• Variants exhibited significantly reduced interaction with human class II MHC proteins.
• Structure-based designs demonstrated superior thermostability and fewer high-affinity epitopes compared to sequence-based designs.

Conclusions:

• EpiSweep is the first validated structure-based deimmunization algorithm for mapping optimal biotherapeutic design space.
• Structure-based design enables simultaneous targeting of multiple epitopes with high mutational loads by optimizing mutations for reduced immunogenicity and favorable intramolecular interactions.
• This approach enhances biotherapeutic safety and efficacy by engineering immunotolerance.