This case study was made in collaboration with Miltenyi Biotec.
AbTheneum™ uses antibody-secreting cells. For mice, there are commercially available kits to isolate plasma cells, and they work pretty well. For human cells, there are many other commercially available kits, which we used some in combination with other in-house methods to get to our goal – antibody-secreting cells from humans that we can enrich for target-specific ones.
Our collaborator, Miltenyi Biotec, sourced peripheral mononuclear blood cells (PBMCs) from 3 donors that had received their seasonal influenza vaccine within 6 weeks. We thawed the PBMCs several days before the scheduled AbTheneum screen and isolated Class-Switched Memory B Cells (CSMBCs) using a kit by Miltenyi (catalog # 130-093-617). The kit yields “untouched” cells, which is perfect for this workflow as we want to apply positive selection on the CSMBCs to enrich for anti-influenza antibody-secreting cells. The CSMBCs have antibodies expressed on the surface but are not secreting. These cells can be activated to secrete using different cytokines and cofactors, similar to the B Cell Expansion Kit available from Miltenyi (catalog # 130-106-196). Single Cell used an in-house cytokine cocktail to activate cells over 5 days. On Day 3, cells were enriched for target-specific cells using the MACSQuant Tyto cell sorter.
On Day 5, the activated memory B cells were enriched by MACSQuant Tyto by staining cells with fluorescently-labeled anti-CD38, 7-AAD, and 4 influenza HA proteins. Cells were sorted for CD38+/7-AAD-/HA+ to isolate antibody-secreting, antigen-specific cells. Fig 1 shows the sort gate and frequency of events in this cell isolation.
Figure 1
We selected the 4 HA proteins (Table 1) used for cell isolation because they cover the 2 groups within the influenza A family and are therefore highly diverse compared to each other. Our goal is to mine the antibody repertoire from these donor samples to find antibodies that have ability to bind to diverse strains of influenza protein, in order to be useful in a pandemic influenza event.
Table 1
Cells sorted from Tyto were deposited onto picoliter devices. Antibodies secreted from the cells were captured onto 3 capture slides and screened according to the Screening Plan (Fig 2). The Screening Plan was designed to allow assigning antibodies to categories (e.g. highly specific to a single HA, or cross-reactive to 2, 3, or 4 HAs). The Screening Plan also includes assessing all captured antibodies for their ability to compete with a benchmark antibody, MEDI8852, which is currently being evaluated in clinical trials as a broadly-neutralizing anti-influenza therapy. After staining each slide with the stain condition in the Screening Plan, the slides are scanned in a fluorescent scanner and images are generated. This project has sequential stains on 3 slides, generating 8 images for each picoliter device. A small region of the images is cropped and shown for all the stain conditions in Fig 3.
Figure 2
Figure 3
After antibody capture and profiling, antibodies were sequenced in parallel by lysing all cells, capturing encoding mRNA, and sequencing by NGS. The output from 2,593 antigen-specific antibodies profiled is displayed in the heat map chart (Fig 4).
Figure 4
After recovering sequences from the screened antibodies, Single Cell selected 5 antibodies with varied binding profiles as a first round to validate the screening result. ELISA against the 4 HA proteins confirmed the SCT screening results for all 5 antibodies (Fig 5).
Figure 5
This case study demonstrates 3 different abilities that AbTheneum can address: