Rapid single B cell antibody discovery using nanopens and structured light

Abstract

Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.

Keywords: ASC; ELISA; Monoclonal antibody; NanOBlast; OEP; OptoElectro Positioning; antibody discovery; antibody generation; nanofluidics; structured light.

Figure 1.

NanOBlast workflow overview. (a). Antigen-experienced immune cells were harvested from wild type mice and enriched using a combination of magnetic negative selection and/or multiparameter FACS sorting. ASCs are loaded into the nanopens of an OptoSelect OS3500 culturing chip via OEP or by inverting the chip and allowing gravity to pull the ASCs downward from the channel into the nanopen. Nanopens are screened for both IgG secretion and antigen specificity using an in-channel multiplex bead-based assay. Immediately after screening, the beads are flushed to waste and the ASC in the selected nanopens is manipulated out of the nanopen using OEP and exported directly into 96-well plates containing lysis buffer using the onboard fluidic system. Single-cell reverse transcriptase (RT) PCR, a modified 5’ RACE amplicon generation step for VH and VL sequence recovery, Golden Gate cloning and recombinant expression using human embryonic kidney 293T cells generate material for binding characterization studies. (b). OS3500 chip from Berkeley Lights containing 3513 polydimethylsiloxane nanopens. The optical train on the Beacon platform subdivides the imaging of the chip into 21 specific FOV. (c). FOV 17 brightfield image showing ASCs sequestered in nanopens after loading overlaid with results of the on-board cell counting algorithm. (d). OEP manipulation of a single ASC out of a nanopen into the channel space of the chip in preparation for export.

Figure 2.

NanOBlast on-chip screening. (a). Cartoon schematic and representative brightfield image of the channel and nanopen space depicting the homogenous bead-based assay design for detection of IgG secretion and antigen specificity in the channel of the OS3500 chip. 3.2-micron polystyrene beads precoated with goat anti-murine IgG, Fc-specific polyclonal antibodies were mixed with AF 568–labelled, goat, anti-murine IgG H+L–specific, polyclonal antibodies; biotin-labelled therapeutic human IgG; streptavidin AF 647; and 10% human serum were imported into the channel space of the chip via onboard fluidics. (b). 45-min (final in the assay series) images of all 21 FOV stitched together depicting the blooms generated for the IgG secretion AF 568 signal as captured in the TRED filter cube of the Beacon. Magnification view of 60 individual nanopens on the chip demonstrating fluorescent bloom formation at the mouth of the nanopen. Time course of bloom formation for nanopen 928 (clone 1A3) demonstrating the change in intensity and size over the duration of the screen. (c). 45-min (final in the assay series) images of all 21 FOV stitched together depicting the blooms generated for the antigen-specific secretion AF 647 signal as captured in the Cy5 filter cube of the Beacon. Magnification view of 60 individual nanopens on the chip demonstrating fluorescent “bloom” formation at the mouth of the nanopen. Time course of bloom formation for nanopen 928 (clone 1A3) demonstrating the change in intensity and size over the duration of the screen. (d). 45-min timepoint images for antigen specificity and IgG secretion of the 13 nanopens from recovered mouse IgGs after constraining the panel to only single exported ASCs.

Figure 2.

Continued.

Figure 3.

Recombinant Mouse IgG Characterization. (a). VH and VL V(D)J dendrogram illustrating the sequence diversity of the single-cell, recovered, antigen-specific clones. The yellow box indicates a chosen maximum distance between two antibodies to allow grouping and is based on a calculation of similarity equal to the number of mismatches divided by the sequence length. The tree length is on the x-axis and antibodies with a distance less than 0.1 from each other were grouped together resulting in 10 separate antibody clades. (b). Plate-based immunoassay design for verification of antigen specificity and relative affinity of the recombinantly expressed antitherapeutic murine IgGs. (c). Dose response, antigen-specific immunoassay binding curves using a limiting concentration of 66 pM of the therapeutic IgG. (d). Design of the completed LBA for evaluation of free therapeutic IgG in human serum. E. LBA with lower limit of quantification < 1ng/ml using 1A3 as the capture antibody on the plate and either 1B4, 1B9, or 1C1 as the detection antibody in a matrix of human serum.