Laila Shehata, Daniel P Maurer, Anna Z Wec, Asparouh Lilov, Elizabeth Champney, Tingwan Sun, Kimberly Archambault, Irina Burnina, Heather Lynaugh, Xiaoyong Zhi, Yingda Xu, Laura M Walker

Cell Reports, 28(3), 3300–3308.e4. DOI: 10.1016/j.celrep.2019.08.056

September 24, 2019

Somatic hypermutation (SHM) is a key process in antibody evolution that enables the immune system to refine antigen recognition by increasing specificity while optimizing binding affinity and functional activity. However, the broader biophysical consequences of this in vivo selection process remain less understood, particularly regarding stability, aggregation propensity, expression titer, and hydrophobicity, as well as how these traits differ among antibodies derived from distinct B cell subpopulations. This study, conducted by Adimab scientists, examines how somatic hypermutation (SHM) in human B cell-derived monoclonal antibodies shapes specificity, polyreactivity, and stability, revealing trade-offs that have important implications for therapeutic antibody development.

Key hypotheses and objectives

• Antibodies derived from human memory B cells and long-lived plasma cells (LLPCs) are expected to exhibit reduced polyreactivity and hydrophobicity compared to naïve B cell-derived antibodies due to higher levels of SHM.
• SHM, while improving antigen specificity, can negatively impact conformational stability, leading to a reduction in thermal stability.
• SHM may also be associated with decreased B cell receptor (BCR) expression levels.
• The developability profiles of most B cell-derived antibodies examined in this study are broadly comparable to those of clinically approved monoclonal antibodies (mAbs), despite the trade-offs observed between affinity, specificity, and stability.

Approach and techniques

1. Antibody isolation and cloning: 400 human monoclonal antibodies were cloned and expressed from naïve, memory, and LLPC subsets using a high-throughput single B cell isolation and characterization platform.
2. Biophysical characterization:
   • Polyreactivity: Assessed using a polyspecificity reagent (PSR) assay, which provides a strong correlation between the degree of polyreactivity and human serum half-life, to evaluate off-target binding.
   • Hydrophobicity: Measured via hydrophobic interaction chromatography (HIC) to determine aggregation potential.
   • Thermal stability: Evaluated using differential scanning fluorimetry (DSF) to assess resistance to unfolding and therefore conformational stability.
3. Correlation analysis: SHM load was mapped against polyreactivity, hydrophobicity, and stability to determine the impact of affinity maturation on developability.
4. Evaluation of SHM load and thermal stability: Antibodies derived from B cells with varying BCR densities were assessed by flow cytometry and analysis of IgG₁ expression levels.

Major findings and impact

• Reduced polyreactivity and hydrophobicity: Antibodies from memory B cells and LLPCs displayed lower non-specific binding and lower hydrophobicity compared to germline-encoded mAbs; these enhancements may reduce their aggregation risk.
• Decreased conformational stability: Although SHM was inversely correlated with thermal stability, indicating a trade-off between specificity and structural integrity, this generally did not decrease below the in vivo BCR stability threshold of 60˚C. 
• Preferential selection of clones with higher BCR stability: LLPC-derived mAbs showed significantly higher thermostability compared with IgG+ memory B cell-derived mAbs, despite containing overall higher levels of SHM.
• Lower BCR expression associated with reduced Fab stability: B cells with low BCR expression levels encoded antibodies with higher levels of SHM correspondingly lower thermal stability than those from high-expressing B cells.
• Comparable developability to clinical mAbs: Despite stability concerns, the vast majority of human B cell-derived mAbs retained acceptable "drug-like" profiles, reinforcing their therapeutic potential.

Implications for therapeutic antibody engineering

This study highlights the importance of balancing affinity, specificity, expression, and stability in antibody design. While SHM-driven improvements in antigen binding are essential for therapeutic efficacy, compensatory engineering strategies may be required to restore stability. Approaches such as germline reversion (removing non-essential mutations) and stabilizing framework modifications could mitigate these trade-offs, optimizing antibodies for clinical application.

For more details, read the full article in Cell Reports.