(D-141) Engineering Antibody–Invertase Fusion Proteins for Enhanced Detection of Diseases Targeted Antibodies Using Commercial Glucometers

Introduction:: To effectively monitor and mitigate the spread of SARS-CoV-2 and future outbreaks, it is crucial to design rapid, cost-effective, and widely accessible diagnostic tools. An assay using an engineered antibody-enzyme fusion protein has been developed to link recognition of SARS-CoV-2-specific antibodies to catalytic conversion of sucrose to glucose, which enables the quantification of antibodies against disease antigens using commercial glucometers. Engineering the detection antibody, along with substitution of the capture antigen, has expanded the diagnostic's applications and efficacy.


Materials and Methods:: To study protection against SARS-CoV-2 infection, it is necessary to assess both immunoglobulin (Ig) G and IgA levels, as both are known to carry out anti-viral activities. To measure the levels of both Igs, we affinity matured HP6017, an antibody that specifically binds to the Fc domain of human IgG using the yeast surface display platform. We generated an error-prone library of HP6017 variants that mutagenized complementarity-determining regions of both the heavy and light chain variable domains. We then selected for mutants with improved affinity and association rates for human IgG (hIgG) Fc domains. Additionally, we selected for mutants with affinity for hIgA Fc domains. Later rounds of sorting with higher stringency were performed using lower concentrations of IgG and IgA, as well as with shorter incubation times to further select for stronger binders. We produced full-length antibodies and fusion proteins that linked these antibodies to the enzyme invertase (Ab+Invertase), which catalyzes the conversion of sucrose to glucose. The lead candidates were compared with respect to binding affinity by biolayer interferometry (BLI), and in the context of assay performance for detecting antigen-specific hIgA and hIgG.


Results, Conclusions, and Discussions:: Results:

After five rounds of sorting, we successfully enriched mutants with both enhanced anti-hIgG binding and that introduced anti-hIgA reactivity. We found that as their affinities increased, their cross-reactivity decreased, which was unsurprising as the Fc domains of hIgG and hIgA share very few solvent-accessible residues. Our hIgG binders had significantly higher binding and association rates compared to parental HP6017, in both full-length and invertase-fused antibody formats. The invertase-fused antibodies also preserved the catalytic activity of the component invertase enzyme and demonstrated enhanced catalytic performance in the anti-SARS-CoV-2 antibody detection assay. We also selected variants of HP6017 with detectable hIgA binding, and we further affinity matured the best candidates from the initial library.

Conclusions:

We have developed Ab+Invertase fusion proteins that allow for the quantitative assessment of serum levels of disease-protective antibodies in patients. This detection approach enables facile measurement of these levels using a commercially available glucometer. Through affinity maturation, we selected anti-hIgG and anti-hIgA-binding variants of the parent antibody that detect these Ig isotypes with greater sensitivity. We have also engineered these variants to have faster association rates compared to the original HP6017, allowing for shorter incubation times and leading to a more expedient diagnostic assay.

Discussions:

We have designed, produced, and validated the function of an Ab+Invertase fusion protein for detection of disease-specific antibodies, demonstrating its efficacy as a diagnostic tool. Importantly, our assay can applied to detect for protection against new Variants of Concern by simple substitution of the disease antigen associated with those variants as the capture moiety. This workflow enables rapid procurement of vital information in light of the continued emergence of new variants, especially in individuals whose immunity may wane more quickly. We plan to test longitudinal serum samples from patients with known vaccination and/or infection times using assays with relevant disease antigens to validate this approach. Overall, the development of this SARS-CoV-2 antibody assay represents a significant advancement in the field of disease monitoring, and has the potential to revolutionize our approach to combating pandemics.


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