Generation and Screening of Antigen-Specific Nanobodies from Mammalian Cells Expressing the BCR Repertoire Library Using Droplet-Based Microfluidics

Abstract

Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.

Single-Cell Transcriptome Analysis of H5N1-HA-Stimulated Alpaca PBMCs

“…This is helpful for alpaca plasma cell enrichment during nanobody development. The proportion of plasma B cells during antigen immunization determines the effectiveness of antigen-specific antibody generation [78][79][80]. In this study, we found that the proportions of plasma B cells dramatically increased by day 17 but were significantly reduced by day 19 (day 5 after the second HA immunization), suggesting that the proportion of plasma B cells would reach a peak at three days after the second immunization.…”

“…In this study, we found that the proportions of plasma B cells dramatically increased by day 17 but were significantly reduced by day 19 (day 5 after the second HA immunization), suggesting that the proportion of plasma B cells would reach a peak at three days after the second immunization. Plasma cell dynamics during the specific antigen immunization should offer valuable references for nanobody discovery in immunized camelids [79,80]. However, the sampling periods between the first and second immuniza-tions were not studied here.…”

Single Cell Microgels for High‐Throughput Magnetic Sorting and Sequencing of Antigen‐Specific Antibodies

“…[4] IgG + memory B cells and plasmablasts have been stained with fluorescently labeled antibodies or fluorochrome conjugated antigens for FACS isolation, but antigen-specific plasma cells have not, given that they lack surface IgG expression. [5] New formats have extended the application scenario to phenotypic analysis of antibody secreting cells based on structured hydrogel-based microcontainers, [6,7] microwells, [8,9] or waterin-oil microdroplets [10,11] to isolate cells, accumulate secreted molecules, and perform sorting. For instance, a droplet microfluidics system CelliGO was developed for mAb discovery, where IgG activity was screened by fluorescence-based bioassays and paired antibody V genes were sequenced by barcoded reverse transcription with an in-droplet single-cell workflow.…”

Smart Droplet Microfluidic System for Single-Cell Selective Lysis and Real-Time Sorting Based on Microinjection and Image Recognition

“…Due to its merits of precision, throughput, compatibility, independence, and lack of contamination, microfluidics, especially droplet microfluidics, − is becoming a powerful tool for single-cell lysis . According to the detailed principle, single-cell lysis methods based on microfluidics can be classified as physical and chemical protocols .…”