Recent Advances in Microfluidic Technology for Bioanalysis and Diagnostics.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells. This protocol describes a microfluidic platform for dynamic high-throughput analysis of the phenotypes of single cells. Cell-surface markers and secreted proteins are quantified and characterized by fluorescence detection using tailored immunoassays.

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Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap

Drug development is a long process whose main content includes drug synthesis, drug delivery, and drug evaluation. Compared with conventional drug development procedures, microfluidics has emerged as a revolutionary technology in that it offers a miniaturized and highly controllable environment for bio(chemical) reactions to take place. It is also compatible with analytical strategies to implement integrated and high-throughput screening and evaluations. In this review, we provide a comprehensive summary of the entire microfluidics-based drug development system, from drug synthesis to drug evaluation. The challenges in the current status and the prospects for future development are also discussed. We believe that this review will promote communications throughout diversified scientific and engineering communities that will continue contributing to this burgeoning field.

Microfluidics for Drug Development: From Synthesis to Evaluation.

Currently, high-throughput approaches are lacking in the isolation of antibodies with functional readouts beyond simple binding. This situation has impeded the next generation of cancer immunotherapeutics, such as bispecific T cell engager (BiTE) antibodies or agonist antibodies against costimulatory receptors, from reaching their full potential. Here, we developed a highly efficient droplet-based microfluidic platform combining a lentivirus transduction system that enables functional screening of millions of antibodies to identify potential hits with desired functionalities. To showcase the capacity of this system, functional antibodies for CD40 agonism with low frequency (<0.02%) were identified with two rounds of screening. Furthermore, the versatility of the system was demonstrated by combining an anti-Her2 × anti-CD3 BiTE antibody library with functional screening, which enabled efficient identification of active anti-Her2 × anti-CD3 BiTE antibodies. The platform could revolutionize next-generation cancer immunotherapy drug development and advance medical research.

High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics.

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases.

/pdf/a-sars-cov-2-neutralizing-antibody-with-extensive-spike-4d8e7f1vt5.pdf

A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes.

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

/pdf/high-throughput-single-cell-activity-based-screening-and-e8xoc2dwow.pdf

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

/pdf/author-correction-high-throughput-single-cell-activity-based-4znh8xv0je.pdf

Scott McNamara

Papers

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

Author Correction: High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.