Discovery of antibodies targeting multipass transmembrane proteins using a suspension cell-based evolutionary approach.

Due to their critical functions in cell sensing and signal processing, membrane proteins are highly preferred as pharmacological targets, and antibody drugs constitute the fastest growing category of therapeutic agents on the pharmaceutical market. However, major limitations exist in developing antibodies that recognize complex, multipass transmembrane proteins, such as G-protein-coupled receptors (GPCRs). These challenges, largely due to difficulties with recombinant expression of multipass transmembrane proteins, can be overcome using whole-cell screening techniques, which enable presentation of the functional antigen in its native conformation. Here, we developed suspension cell-based whole-cell panning methodologies to screen for specific binders against GPCRs within a naive yeast-displayed antibody library. We implemented our strategy to discover high-affinity antibodies against four distinct GPCR target proteins, demonstrating the potential for our cell-based screening workflow to advance the discovery of antibody therapeutics targeting membrane proteins.

A Hybrid Adherent/Suspension Cell-Based Selection Strategy for Discovery of Antibodies Targeting Membrane Proteins.

Membrane proteins are favored drug targets and antibody therapeutics represent the fastest-growing category of pharmaceuticals. However, there remains a need for rapid and effective approaches for the discovery of antibodies that recognize membrane proteins to develop a robust clinical pipeline for targeted therapeutics. The challenges associated with recombinant expression of membrane proteins make whole cell screening techniques desirable, as these strategies allow presentation of the target membrane proteins in their native conformations. Here, we describe a workflow that employs both adherent cell-based and suspension cell-based whole cell panning methodologies to enrich for specific binders within a yeast-displayed antibody library. The first round of selection consists of an adherent cell-based approach, wherein a diverse library is panned over target-expressing mammalian cell monolayers in order to debulk the naïve library. Subsequent rounds involve the use of suspension cell-based approaches, facilitated with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS), to achieve further library enrichment. Finally, we describe a high-throughput approach to screen target binding and specificity of individual clones isolated from selection campaigns.

Suspendable Hydrogel Nanovials for Massively Parallel Single-Cell Functional Analysis and Sorting.

Techniques to analyze and sort single cells based on functional outputs, such as secreted products, have the potential to transform our understanding of cellular biology as well as accelerate the development of next-generation cell and antibody therapies. However, secreted molecules rapidly diffuse away from cells, and analysis of these products requires specialized equipment and expertise to compartmentalize individual cells and capture their secretions. Herein, we describe methods to fabricate hydrogel-based chemically functionalized microcontainers, which we call nanovials, and demonstrate their use for sorting single viable cells based on their secreted products at high-throughput using only commonly accessible laboratory infrastructure. These nanovials act as solid supports that facilitate attachment of a variety of adherent and suspension cell types, partition uniform aqueous compartments, and capture secreted proteins. Solutions can be exchanged around nanovials to perform fluorescence immunoassays on secreted proteins. Using this platform and commercial flow sorters, we demonstrate high-throughput screening of stably and transiently transfected producer cells based on relative IgG production. Chinese hamster ovary cells sorted based on IgG production regrew and maintained a high secretion phenotype over at least a week, yielding >40% increase in bulk IgG production rates. We also sorted hybridomas and B lymphocytes based on antigen-specific antibody production. Hybridoma cells secreting an antihen egg lysozyme antibody were recovered from background cells, enriching a population of ∼4% prevalence to >90% following sorting. Leveraging the high-speed sorting capabilities of standard sorters, we sorted >1 million events in <1 h. IgG secreting mouse B cells were also sorted and enriched based on antigen-specific binding. Successful sorting of antibody-secreting B cells combined with the ability to perform single-cell RT-PCR to recover sequence information suggests the potential to perform antibody discovery workflows. The reported nanovials can be easily stored and distributed among researchers, democratizing access to high-throughput functional cell screening.

Emerging technologies in protein interface engineering for biomedical applications.

Protein interactions communicate critical information from the environment into cells to orchestrate functional responses relevant to health and disease. Whereas the natural repertoire of protein interfaces is finite, biomolecular engineering tools provide access to an unlimited scope of potential interactions that can be custom-designed for affinity, specificity, mechanism, or other properties of interest. This review highlights recent developments in protein interface engineering that offer insight into human physiology to inform the design of new pharmaceuticals, with a particular focus on immunotherapeutics. We cover three innovative and translationally promising approaches: (1) reprogramming receptor oligomerization to manipulate signaling pathways; (2) computational protein interface design strategies; and (3) engineering bioorthogonal protein interaction networks.

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model.

OBJECTIVE: The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis. APPROACH AND RESULTS: A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer. CONCLUSIONS: A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.