Engineering an anti-CD52 antibody for enhanced deamidation stability.

Area of Science:

• Biochemistry
• Protein Engineering
• Immunology

Background:

• Deamidation of therapeutic antibodies, particularly at asparagine residues, is a critical developability issue.
• Investigating deamidation in antibody complementarity-determining regions (CDRs) is essential for maintaining therapeutic efficacy.
• Understanding the structural basis of deamidation impacts is key to designing more stable antibody therapeutics.

Purpose of the Study:

• To evaluate the impact of Asn-Gly deamidation on the structure and function of a human anti-CD52 IgG1 antibody.
• To explore protein engineering strategies for mitigating deamidation risks in therapeutic antibodies.
• To utilize structure-based design for enhancing antibody developability.

Main Methods:

• Co-crystallography of the antigen-binding fragment (Fab) with a CD52 peptide mimetic.
• Site-directed mutagenesis at Asn33 and Gly34 residues within the CDR1 of the antibody light chain.
• Forced deamidation studies using elevated pH and temperature.
• Liquid chromatography-mass spectrometry (LC-MS) peptide mapping for deamidation assessment.
• Biacore analysis for measuring antigen binding affinity.
• Complement-dependent cytotoxicity (CDC) assays to evaluate effector function.

Main Results:

• Mimicking deamidation at Asn33 by introducing an Asp residue caused a ~400-fold decrease in antigen binding affinity.
• Mutagenesis at Asn33 significantly reduced antibody binding affinity, highlighting its critical role in antigen interaction.
• The crystal structure revealed Asn33's direct interaction with the CD52 antigen's phosphate group.
• Mutants at Gly34 (G34R, G34K, G34Q) demonstrated increased resistance to deamidation.
• These Gly34 mutants retained high binding affinity to the CD52 antigen and exhibited functional antibody effector activity in CDC assays.
• Gly34 was found to be away from the binding interface, allowing for successful mutagenesis without compromising affinity.

Conclusions:

• Asn33 is crucial for maintaining the binding affinity of the anti-CD52 antibody.
• Gly34 is amenable to mutagenesis for enhancing deamidation resistance without impacting antigen binding or effector function.
• Structure-based protein engineering of residues like Gly34 is an effective strategy for mitigating deamidation and improving therapeutic antibody developability.