Journal
MABS
Volume 14, Issue 1, Pages -Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/19420862.2022.2146629
Keywords
mAb; antibody; self-interaction; affinity; directed evolution; complementarity-determining regions; CDR; developability; viscosity; aggregation; antibody engineering; protein design; AC-SINS; CS-SINS; polyspecificity; polyreactivity; non-specific binding; off-target binding
Categories
Funding
- National Science Foundation
- National Institutes of Health
- University of Michigan [1T32GM140223-01]
- Pfizer
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This study reports a high-throughput protein engineering method for rapidly identifying antibody candidates with low self-association and high affinity. By conjugating quantum dots to strongly self-associating antibodies, the researchers were able to detect other high self-association antibodies. Using this method, rare variants with co-optimized levels of low self-association and high affinity were identified, and several of these variants also displayed improved folding stability and reduced nonspecific binding.
Self-association governs the viscosity and solubility of therapeutic antibodies in high-concentration formulations used for subcutaneous delivery, yet it is difficult to reliably identify candidates with low self-association during antibody discovery and early-stage optimization. Here, we report a high-throughput protein engineering method for rapidly identifying antibody candidates with both low self-association and high affinity. We find that conjugating quantum dots to IgGs that strongly self-associate (pH 7.4, PBS), such as lenzilumab and bococizumab, results in immunoconjugates that are highly sensitive for detecting other high self-association antibodies. Moreover, these conjugates can be used to rapidly enrich yeast-displayed bococizumab sub-libraries for variants with low levels of immunoconjugate binding. Deep sequencing and machine learning analysis of the enriched bococizumab libraries, along with similar library analysis for antibody affinity, enabled identification of extremely rare variants with co-optimized levels of low self-association and high affinity. This analysis revealed that co-optimizing bococizumab is difficult because most high-affinity variants possess positively charged variable domains and most low self-association variants possess negatively charged variable domains. Moreover, negatively charged mutations in the heavy chain CDR2 of bococizumab, adjacent to its paratope, were effective at reducing self-association without reducing affinity. Interestingly, most of the bococizumab variants with reduced self-association also displayed improved folding stability and reduced nonspecific binding, revealing that this approach may be particularly useful for identifying antibody candidates with attractive combinations of drug-like properties.
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