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

Abstract

Currently high throughput approaches are lagged for isolation of antibodies whose function goes beyond simple binding, which have prevented the next generation cancer immunotherapeutics, such as bispecific T cell engager antibodies or agonist antibody of costimulatory receptor, from reaching their full potential. Here we developed a highly efficient droplet-based microfluidics platform combining with lentivirus transduction system that enables functional screening of millions of antibodies. To showcase the capacity of the system, functional antibodies for CD40 agonism with low frequency (<0.02%) were identified with 2 rounds of screening. To demonstrate its versatility, an anti-Her2/anti-CD3 bispecific antibody library was established using bispecific T cell Engager (BiTE) platform and functional screening enabled efficient identification of potent anti-Her2/anti-CD3 BiTE antibodies. The platform could revolutionize the next generation cancer immunotherapy drug development and research world.

Versatile and rapid microfluidics-assisted antibody discovery

“…Future development warrants optimization of several device properties, workflow steps, and methodologies, as discussed above. Application of homogeneous monoclonal secretion libraries 28 could enable repeated interrogation of the full B cell diversity in robust in-format screens. Stand-alone devices 16 , 33 may accelerate the full establishment of microfluidics as a standard methodology for the discovery of differentiated therapeutic antibody candidates with unique properties, identified in broad functional or target-agnostic phenotypic screens 7 against diverse antigens, including those that are difficult to target.…”

“…Pioneering early works mainly using autocrine, i.e., self-labeling, one cell systems [41][42][43][44] or mammalian display, with focus on antibody developability such as affinity maturation 45 and beyond, 5 are all compatible with flow cytometry for high throughput variant screening. The advent of microfluidics broadened opportunities for functional screening of secreted antibodies for internalization and intracellular signaling, 20 virus neutralization, 29 optimal agonistic properties, 28 and further biotherapeutics 23 when combined with suitable, mostly fluorescence-based cellular reporter assays. In evaluating function-first screening capacities, the anti-CD3 agonist antibody OKT3-secreting hybridoma cell line was spiked into irrelevant antibodysecreting hybridoma and could be highly enriched from 0.55% to 88% in a single run sorting for the GFP reporter signal (Figure 4), indicating successful screening toward the agonistic function.…”

“…25 Compatible with the high throughput of microfluidic setups, mostly fluorescence-based methods are applied for real-time adjustable mining of large diversities. Methodologies range from selection for binding on the recombinant target or cells 26 to internalization or functional screening using reporter gene target cells, 23,27,28 as exemplified by the recent work from Gérard et al 20 While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, 22,29,30 microfluidics can also yield tools 16 as well as therapeutic antibodies targeting cancer or immunological diseases. 31,32 Several companies offer custom and customizable microfluidic chips for commercial and academic antibody screening campaigns.…”

A high throughput bispecific antibody discovery pipeline

“…Other droplet microfluidic systems [65][66][67][68][69] , microwell assays 70 , optofluidics 71 and microcapillary arrays 72 have been applied previously for antibody discovery but they either operate with bindingbased assays with a moderate throughput or otherwise focused on other areas of discovery not directly applicable to BsAb. A recent study employed single-cell-based functional screening but required multiple rounds of enrichment 73 , whereas innovations in the single cell BsAb discovery platform reported here, including single variant library construction via modular, site-directed landing-pad system, multiplexed orthogonal assay chemistry, and multi-point detection and droplet-indexing strategy enable us to minimize false positives and interrogate large libraries at a much greater depth and lower abundance (0.001%). We have demonstrated that our platform can discover extremely rare clones with unique properties that would otherwise be untractable using conventional low-throughput, biased, and trial-and-error methods.…”

High-throughput single-cell TCR - pMHC dissociation rate measurements performed by an autonomous microfluidic cellular processing unit

“…While microfluidic platforms have been applied to single-cell applications, including peptidemajor histocompatibility complexes (pMHC) and T cell receptors (TCR) interactions [7,8], no platforms have so far enabled a closed-loop selection system with spatial and temporal resolution at the single-cell level. Droplet microfluidic technologies enabled high-throughput screening and sorting of immune cells [9,10] but rely on a single time point measurements for analysis and decision making, which limits the applicability to static or slow biological processes. On the other hand, microfluidic platforms capable of analysing single cells in response to stimuli have been described but have achieved limited throughput analyzing tens of cells per experiment and do not enable specific cell recovery [11].…”