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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. Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CS-SINS: charge-stabilized self-interaction nanoparticle spectroscopy; FACS: fluorescence-activated cell sorting; Fab: fragment antigen binding; Fv: fragment variable; IgG: immunoglobulin; QD: quantum dot; PBS: phosphate-buffered saline; V H : variable heavy; V L : variable light.

Sarcoidosis is a granulomatous disorder that frequently affects the lungs. Cough is commonly reported by patients and can significantly reduce health-related quality of life. The mechanism of cough is unknown but airway inflammation, mechanical distortion from pulmonary fibrosis and disruption of the vagus nerve are possible. Recent evidence suggests cough reflex hypersensitivity may also be an important mechanism and predictor of the frequency of cough. The investigation of cough should evaluate common causes such as asthma, gastro-oesophageal reflux and rhinitis. In patients with suspected cough due to sarcoidosis, a trial of corticosteroids should be considered. The severity of cough should be evaluated with validated outcome measures such as visual analogue scales, cough severity diary, health-related quality of life questionnaires such as the Leicester Cough Questionnaire and objective cough monitors. Future studies are needed to identify targets for therapeutic development.

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