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

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.

Author Correction: 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.

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

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.

DNA sequence analysis with droplet-based microfluidics.

Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence.

Experimental validation of plugging during drop formation in a T-junction.

At low capillary number, drop formation in a T-junction is dominated by interfacial effects: as the dispersed fluid flows into the drop maker nozzle, it blocks the path of the continuous fluid; this leads to a pressure rise in the continuous fluid that, in turn, squeezes on the dispersed fluid, inducing pinch-off of a drop. While the resulting drop volume predicted by this "squeezing" mechanism has been validated for a range of systems, as of yet, the pressure rise responsible for the actual pinch-off has not been observed experimentally. This is due to the challenge of measuring the pressures in a T-junction with the requisite speed, accuracy, and localization. Here, we present an empirical study of the pressures in a T-junction during drop formation. Using Laplace sensors, pressure probes we have developed, we confirm the central ideas of the squeezing mechanism; however, we also uncover other findings, including that the pressure of the dispersed fluid is not constant but rather oscillates in anti-phase with that of the continuous fluid. In addition, even at the highest capillary number for which monodisperse drops can be formed, pressure oscillations persist, indicating that drop formation in confined geometries does not transition to an entirely shear-driven mechanism, but to a mechanism combining squeezing and shearing.

Analysis of gene expression at the single-cell level using microdroplet-based microfluidic technology.

In the present work, we have measured the messenger RNA expression of specific genes both from total RNA and cells encapsulated in droplets. The microfluidic chip introduced includes the following functionalities: RNA∕cell encapsulation, lysis, reverse transcription and real-time polymerase chain reaction. We have shown that simplex and duplex gene expression measurements can be carried out over a population of 100 purified RNA samples encapsulated simultaneously in 2 nl droplets in less than 2 h. An analysis of 100 samples containing one to three cells has shown excellent consistency with standard techniques regarding average values. The cell-to-cell distributions of the E-cadherin expression suggest fluctuations on the order of 80% in the number of transcripts, which is highly consistent with the general findings from the literature. A mathematical model has also been introduced to strengthen the interpretation of our results. The present work paves the way for the systematic acquisition of such information in biological and biomedical studies.

Controlling droplet incubation using close-packed plug flow.

Controlling droplet incubation is critical for droplet-based microfluidic applications; however, current techniques are either of limited precision or place strict limits on the incubation times that can be achieved. Here, we present a simple technique to control incubation time by exploiting close-packed plug flow. In contrast to other techniques, this technique is applicable to very short and very long incubation times.

On-chip background noise reduction for cell-based assays in droplets.

Droplet-based microfluidics provides an excellent platform for high-throughput biological assays. Each droplet serves as a reaction vessel with a volume as small as a few picolitres. This is an important technology for a high variety of applications. However this technology is restricted to homogeneous assays as it is very difficult to wash reagents from the reaction vessel. To help overcome this limitation, we introduce a method to effectively dilute the content of a droplet while retaining the high throughput. We use electrocoalescence to merge the parent drop with a much larger drop containing only solvent, thereby increasing the volume of the drop by as much as a factor of 14. Three T-junctions then break the larger drop into eight smaller droplets. This dilution and break-up process can be repeated, thus leading to many drops comparable in size to the original one but with much lower concentration of reagents. The system is fully integrated in a PDMS device. To demonstrate its power, we perform a labelling reaction at the surface of the cells by coencapsulating yeast cells expressing S6 peptide tags with the enzyme SFP synthase and the fluorescent substrate CoA 488. After reaction, the droplets are diluted twice using the system and the intensity of their fluorescence is measured. This noise reduction method enables us to more easily distinguish the fluorescence at the surface of a single cell from the fluorescent background inside the droplet.

High-throughput injection with microfluidics using picoinjectors.

Adding reagents to drops is one of the most important functions in droplet-based microfluidic systems; however, a robust technique to accomplish this does not exist. Here, we introduce the picoinjector, a robust device to add controlled volumes of reagent using electro-microfluidics at kilohertz rates. It can also perform multiple injections for serial and combinatorial additions.

Monodisperse colloids synthesized with nanofluidic technology.

Limitations in the methods employed to generate micrometric colloidal droplets hinder the emergence of key applications in the fields of material science and drug delivery. Through the use of dedicated nanofluidic devices and by taking advantage of an original physical effect called capillary focusing, we could circumvent some of these limitations. The nanofluidic (i.e., submicrometric) devices introduced herein are made of soft materials, and their fabrication relies upon rapid technologies. The objects that we have generated are simple droplets, multiple droplets, particles, and Janus particles whose sizes lie between 900 nm and 3 microm (i.e., within the colloidal range). Colloidal droplets have been assembled on-chip into clusters and crystals, yielding discrete diffraction patterns. We illustrate potential applications in the field of drug delivery by demonstrating the ability of multiple droplets to be phagocytosed by murine macrophage-type cells.

Microfluidic droplet-based liquid-liquid extraction.

We study microfluidic systems in which mass exchanges take place between moving water droplets, formed on-chip, and an external phase (octanol). Here, no chemical reaction takes place, and the mass exchanges are driven by a contrast in chemical potential between the dispersed and continuous phases. We analyze the case where the microfluidic droplets, occupying the entire width of the channel, extract a solute-fluorescein-from the external phase (extraction) and the opposite case, where droplets reject a solute-rhodamine-into the external phase (purification). Four flow configurations are investigated, based on straight or zigzag microchannels. Additionally to the experimental work, we performed two-dimensional numerical simulations. In the experiments, we analyze the influence of different parameters on the process (channel dimensions, fluid viscosities, flow rates, drop size, droplet spacing, ...). Several regimes are singled out. In agreement with the mass transfer theory of Young et al. (Young, W.; Pumir, A.; Pomeau, Y. Phys. Fluids A 1989, 1, 462), we find that, after a short transient, the amount of matter transferred across the droplet interface grows as the square root of time and the time it takes for the transfer process to be completed decreases as Pe-2/3, where Pe is the Peclet number based on droplet velocity and radius. The numerical simulation is found in excellent consistency with the experiment. In practice, the transfer time ranges between a fraction and a few seconds, which is much faster than conventional systems.

Interactions between sulfobetaine-based polyzwitterions and polyelectrolytes.

We investigate the interactions between sulfobetaine-based polyzwitterions and polyelectrolytes, either positive or negative ones, i.e., poly(DADMAC)s and poly(AA)s. Three different sulfobetaine motifs denoted SPE, SPP, and SHPP have been considered, presenting slight chemical changes either in the function carrying the zwitterionic group or in the zwitterionic motif itself. All three poly(sulfobetaine)s normally present critical temperatures (T(c)) above which they become fully soluble. The association with polyelectrolytes directly affects the critical temperature in a highly nonmonotonic fashion as the mixture composition is varied. Thanks to layer-by-layer deposition in a reflectometric cell, we demonstrate that a selective attraction exists between polyzwitterions and polyelectrolytes, from which an association follows at a nanoscopic scale as demonstrated by small-angle X-ray scattering and atomic force microscopy. The association of polyzwitterions with polyelectrolytes, however, is site-specific since it exists only between positive polyelectrolytes (i.e., polycations) and polyzwitterions based on SPE or SPP motifs. The range in which the association affects the critical temperature, T(c), is found to largely depend on the molecular weights of both zwitterionic and cationic species. As a result, the complexation and the creation of a hybrid object, referred to as a complex, also depend on the same parameters. By varying the latter from a few thousands to several millions, we define rules for the existence of this complex. In particular, a minimum polyzwitterion molecular weight is needed to observe alterations of the critical temperatures and closure of the complexation cone. Finally, within a Flory-like approach, we consider the polyzwitterion/polyelectrolyte complex as an effective statistical copolymer, whose composition comprises a fraction phi(A) of excess zwitterionic motifs as the majority species and a fraction 1 - phi(A) of complex motifs. We thereby reduce a polymer/polymer/solvent ternary system to a copolymer/solvent binary one, an assumption valid within the limit of small additions of cationic species. The approach predicts the reciprocal critical temperature 1/T(c) to be quadratic in phi(A), which agrees very well with all experimental results, even for a large mismatch between the molecular weights of both species, and regardless of the zwitterionic motif, SPE or SPP.

Reconciling low- and high-salt solution behavior of sulfobetaine polyzwitterions.

We investigate the water-solubility upon salt additions, of homogeneous families of sulfobetaine-based polyzwitterions. These polymers bear both positive ammonium, and negative sulfonate charges on each monomer and as a result present an upper critical solution temperature (UCST) in the 0-100 degrees C temperature range. Two chemistries are investigated, with either a carboxylate-carrying function (SPE) or an amido-carrying one (SPP). In agreement with the literature published on pSPEs, we find that an addition of simple salts improves the water-solubility of pSPEs, as well as that of pSPPs, yet only once a threshold concentration of added salt has been reached in the solution. We verify using scaling arguments that the onset of solubility promotion, corresponds exactly to the complete screening of the attraction between positive and negative charges inside a polyzwitterionic coil. On the contrary, for salt concentrations smaller than the threshold concentration, we observe that an addition of salt can be adverse to the solubility of polyzwitterions, depending on the degree of polymerization, the type of salt, and the type of zwitterionic motive. Thanks to zeta-potential measurements and systematic variations of these three parameters, we demonstrate, in agreement with theoretical prediction, that this molecular weight-dependent enhanced solubility at small salt concentrations is due to charge asymmetry resulting from partial hydrolysis, combined with specific interactions between salts and zwitterion constituents, evidencing the complexity of the solution behavior of these macromolecules. We thereby reconcile the different behaviors in the domains of low- and high-salinity.