(174u) Engineering Antibody–Invertase Fusion Proteins for Enhanced Detection of Diseases Targeted Antibodies Using Commercial Glucometers

Introduction and significance:

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. We previously developed an assay that uses an engineered antibody-enzyme fusion protein to recognize SARS-CoV-2-specific patient antibodies and catalyze the conversion of sucrose to glucose, which enables the quantification of antibodies against disease antigens using commercial glucometers. To enhance the diagnostic’s applications and efficacy, we have engineered the detection antibody to improve sensitivity and to expand recognition to other antibody classes.

Our ultimate goal is to develop a novel antibody detection platform for dynamic population-scale monitoring of diseases spread that can be commercialized and translated to the clinic. To achieve this, we are working with our collaborators to integrate our antibody detection assay with a single-channel microfluidic device that will allow automatic processing of the blood samples. This versatile platform can be applied to detect antibodies associated with other diseases, such as autoimmune and infectious diseases, through straightforward substitution of the capture antigen. Overall, this antibody detection platform could allow for rapid and facile assessment of immune protection, which can transform our understanding and targeting of disease.

Materials and Methods:

To accurately predict protection against SARS-CoV-2 infection, it is necessary to assess both immunoglobulin G (IgG) and IgA levels with high sensitivity, as both are known to confer protection. To achieve sensitive detection of IgG and introduce binding to IgA, we created a yeast surface-displayed error-prone library based on the anti-human IgG Fc antibody HP6017 that mutagenized complementarity-determining regions of both the heavy and light chains. We then conducted selections against human IgG Fc to evolve enhanced affinity binders and, in parallel, conducted selections against human IgA Fc to discover binders against this antibody class. We reformulated promising clones as full-length antibodies and antibody-invertase (Ab+Invertase) fusion proteins and determined their binding affinity by biolayer interferometry. We then assessed their in-assay performance for detecting human IgG and IgA compared to the parent clone HP6017.

Results:

After five rounds of sorting, we achieved excellent enrichment of our libraries against both human IgG and human IgA. Our human IgG binders exhibited significantly tighter binding compared to parental HP6017, as both full-length antibodies and Ab+Invertase fusion proteins. 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. Our IgA binders showed reactivity against human IgA, whereas the parental HP6017 did not, and these clones are also being affinity matured via DNA shuffling.

In conclusion, we have designed, produced, and validated Ab+Invertase fusion proteins that allow for the quantitative assessment of disease-protective antibodies in patient serum using a commercially available glucometer. Through affinity maturation, we selected anti-IgG and anti-IgA variants that detect their respective targets with greater sensitivity. In particular, these molecules show faster association rates compared to the original HP6017, allowing for shorter incubation times to expedite the diagnostic assay. The development of this novel antibody detection platform represents a significant advance in the field of disease monitoring, and has the potential to revolutionize our ability to combat diseases and future pandemics.