Studying protein interactions in high throughput (HTP) is critical for advancing many aspects of drug discovery, biomarker identification, and diagnostic development. However, existing methods for producing functional protein libraries are costly, time-consuming, and lack real-time kinetic screening capabilities. To address these limitations, we developed an automated platform for HTP production and screening of a library of proteins on biosensor surfaces, facilitating large-scale measurement of interaction kinetics. This technology, Sensor-Integrated Proteome On Chip (SPOC®), involves cell-free protein expression in nanowells directly from customizable libraries of DNA, facilitating simultaneous capture-purification of up to 2400 unique full-length folded proteins or proteoforms onto a single gold biosensor chip. SPOC protein biosensors can then be screened by real-time label-free analysis, including surface plasmon resonance (SPR) to generate kinetic data. Fluorescent and SPR assays were used to demonstrate zero crosstalk between protein spots. The functionality of SPOC proteins was validated using a variety of assays. Monoclonal antibodies were found to selectively bind their SPOC protein targets and anti-RBD antibody was used to demonstrate discriminated binding kinetics to numerous SARS-CoV-2 RBD variants on-chip. With advantages of HTP, flexibility, low-cost, and real-time kinetic profiling, the SPOC platform addresses challenges of interrogating protein interactions at scale for research and clinical applications.

Accurate detection of biomolecular interactions is essential in many areas, from the detection of the presence of biomarkers in the clinic to the development of therapeutic drugs and biologics in biopharma to the understanding of various biological processes in basic research. Traditional endpoint approaches can suffer from false-negative results for biomolecular interactions with fast kinetics. By contrast, real-time detection techniques like surface plasmon resonance (SPR) monitor interactions as they form and disassemble, reducing the risk of false-negative results. By leveraging cell-free expressed proteins captured on either glass or SPR biosensors and using two different commercial antibodies with variable off-rates that both target HaloTag antigens as a model, we compare and contrast results from a fluorescence endpoint assay versus real-time sensor-integrated proteome on chip (SPOC^®) SPR-based detection. In this study, we illustrate the limitations of the representative immunofluorescent endpoint assay when investigating transient interactions characterized by fast dissociation rates. We highlight the importance of choosing reagents well suited to the selected assay, as well as the importance of considering binding kinetics and protein ligand conformational states when interpreting results from binding assays, especially for applications as critical as the off-target screening of therapeutics.

Epitope mapping is central to rational antibody drug design, affinity optimization and the anticipation of therapeutic resistance mechanisms. Here, we demonstrate the use of Sensor Integrated Proteome on Chip (SPOC) technology for single amino acid resolution epitope mapping. By generating high throughput (HTP) binding kinetics data, we identify important residues within the target epitope whose mutations alter drug-target interactions. The SPOC platform integrates simultaneous HTP cell-free production of folded proteins in nanowells from immobilized plasmid DNAs or linear expression cassettes and capture onto biosensor chips for subsequent label-free binding kinetic analysis using surface plasmon resonance (SPR). The model system comprised the extracellular domain (ECD) of CD20, a membrane-spanning 4-domain family protein, screened against its FDA-approved therapeutic monoclonal antibodies (thAbs) - rituximab and ocrelizumab. Using our proprietary POC protein nanofactory system, a partial deep mutationally scanned (DMS) CD20 ECD mutant library of 79 variants was produced on SPOC biosensor chips via rational single amino acid substitutions of the epitope and surrounding residues with alanine, aspartic acid, lysine, and serine, collectively representing four broad classes of amino acid side chain chemistries: nonpolar, acidic, basic, and polar neutral. The SPOC protein biosensor chip was then screened with both thAbs using SPOC SPR to generate kinetic affinity data, evaluate mutations that led to affinity loss or gain, and ultimately identify critical epitope residues that interface with the antibodies. Most mutations within the rituximab and ocrelizumab epitopes - EPANPSEK and YNCEPANPSEKNSPST, respectively - resulted in complete loss of binding or >25% increase in apparent K _D . Notably, N171, P172, and S173 mutations, irrespective of side chain substitution, resulted in complete loss of rituximab binding while at least three diverse side chain substitutions at E168, P169, N171, P172, S173, E174, K175, and T180, led to complete loss of binding for ocrelizumab. These outcomes identify the listed residues as the most critical contact points for their respective antibodies. Interestingly, we also found that functional side-chain substitutions at some residues flanking the epitope increased affinity. This indicates that these non-epitope residues contribute to antibody contact, and that polarity at these sites is a tractable lever for affinity modulation by targeting the corresponding contact residues on the antibody CDRs. The proposed SPOC approach of screening drug candidates against on-chip library of mutationally-scanned therapeutic targets is relevant in the early phase of drug development to resolve epitopes at the residue-level to support more informed down-selection of candidates. It facilitates cost-effective improvement of thAbs, enhancing therapeutic efficacy across a wide array of therapeutic targets, including rare variants that might otherwise lead to therapeutic resistance.