![]() The divergence of reporter cell activation in the plate well and droplets demonstrates that antibodies secreted by cells in the droplet cannot diffuse between different droplets thus, the system is suitable for screening a library. In contrast, when the anti-Her2 × anti-CD3 lentivirus-infected K562-Her2 cells were cocultured with reporter cells in the plate well, 72.7% of the reporter cells were activated after 16 hours of incubation ( Fig. After 16 hours of incubation, the droplets were reinjected into the sort chip to analyze the activation of reporter cells in the droplet, and approximately 9.5% of the reporter cells were activated. The results were in good agreement with the theoretical double Poisson distribution, with 4524 droplets analyzed (fig. The number of cells per droplet was analyzed on the basis of the fluorescent signal from the antibody-secreting cell labeled with CellTrace Violet dye and the reporter cell labeled with CellTrace Yellow dye. S1, A and B) with 0.5 antibody-secreting cells and 1 reporter cell per droplet on average. ![]() Each infected K562-Her2 cell was encapsulated with a Jurkat/pIL2–enhanced green fluorescent protein (eGFP) reporter cell (fig. K562-Her2 played a dual role in expressing antibodies and providing Her2-mediated cross-linking of the secreted antibodies. Her2-overexpressing K562-Her2 cells were infected with the anti-Her2 × anti-CD3 BiTE positive control lentivirus at a low MOI, resulting in less than 10% of cells being infected. The anti-Her2 × anti-CD3 BiTE positive control was engineered to recognize Her2 on tumor cells and CD3 on T cells using the variable domain sequences of trastuzumab and blinatumomab, respectively ( 28). Next, we used an anti-Her2 × anti-CD3 BiTE antibody as an example for the development and optimization of the droplet-based assay. No merging of droplets was observed, indicating that the droplets were stable (Fig. However, improvement is urgently needed to screen for functional antibodies.ĭroplets generated by the microfluidic chip were incubated for 24 hours at 37☌. Millions of plasma cells can be screened for antibodies bound to vaccines or cancer targets ( 21). With the sophistication of the microfluidic droplet system, simultaneous analyses of millions of individual antibody-secreting plasma cells for their antibody secretion rate and affinity can be achieved ( 20). ( 18, 19) described application of the microfluidic droplet system for screening hundreds of thousands of hybridoma cells for antibodies that inhibit enzyme angiotensin-converting enzyme 1 (ACE-1) or bind to target cells ( 18, 19). Last, droplets containing desirable cells are sorted by fluorescence-activated droplet sorting (FADS). Antibodies generated by the cells are contained in the droplet, enabling the maintenance of phenotype and genotype linkage within the droplet ( 15– 17). The microfluidic droplet system can encapsulate single cells in water-in-oil droplets at a rate of thousands of droplets per second ( 14). The platform could revolutionize next-generation cancer immunotherapy drug development and advance medical research.ĭroplet microfluidic technology allows analysis and screening of cells at the single-cell level with unprecedented throughput, something that cannot be obtained using bulk population-based assays. 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. To showcase the capacity of this system, functional antibodies for CD40 agonism with low frequency (<0.02%) were identified with two rounds of screening. 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. 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. Yuan Wang, Ruina Jin, , Bingqing Shen, Na Li, , He Zhou, Wei Wang, Yingjie Zhao, Mengshi Huang, Pan Fang, , Shanshan Wang, Pascaline Mary, Ruikun Wang, Peixiang Ma, Ruonan Li, Yujie Tian, Youjia Cao, Fubin Li, Liang Schweizer, and Hongkai Zhang +16 authors +14 authors +9 authors fewer Authors Info & AffiliationsĬurrently, high-throughput approaches are lacking in the isolation of antibodies with functional readouts beyond simple binding.
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