Bin City

The Challenge 

• Discover high diversity set of antibodies that bind to a large protein target
• Characterize the epitope bin of the hits

The Single Cell Solution

• Develop an epitope binning screen using segments of the full-length protein that include separate domains

Highlights

• >1,000 antibody hits to the target, comprising >100 sequence clonotypes
• All antibodies assigned to a bin, where they bind to certain domains of the target

Problem: Taking on the Challenge

Large protein targets can yield a large diversity of antibodies, but further profiling and down-selecting those antibodies can be a challenge. Further segmenting the output by binding site or epitope bins helps to focus downstream validation efforts and triage.

Solution: Finding a Better Way

To run the epitope binning screen, 3 proteins were generated for screening: a full-length extra-cellular domain (ECD), a protein fragment of Domains 4 through 7 (D4-7), and a protein fragment of Domains 6 and 7 (D6-7), as outlined in Figure 1. 

Figure 1. A schematic of the target structure and screening molecules used to screen for Full-length ECD (yellow), Domain 4-7 (pink), and Domain 6-7 (green).

Transgenic mice were immunized with the full-length ECD using a Rapid Protocol and lymph node cells were harvested. Antibody-secreting cells were isolated from lymph node cells and deposited onto a picowell device.

Antibodies were captured onto an antibody capture slide by creating a leakproof seal, generating an array
of all secreted antibodies. The array was screened with the screening molecules in order from shortest to longest, as outlined in Figure 2.

Figure 2. One antibody capture slide was generated and screened with fluorescent labeled D6-7 first, D4-7 second, and FL last and scanned after each screen. The same region from all the scans is displayed and new hits from each screen colored in corresponding colors (D6-7=green, D4-7=pink, FL=yellow). The final screen used labeled secondary antibody to detect all IgGs captured from secreting cells.

Ready for the condensed cut? 

All cells are lysed and mRNA encoding for IgG is captured onto a DNA microarray, and a DNA barcode
is incorporated into the cDNA at each capture location. A total of 1,336 antigen-specific hits were delivered with full-length sequences, broken into >100 clonotypes. All hits were profiled and assigned a bin according to the screen. Figure 3 shows 20 clonotypes assigned to 1 of 3 bins based on the screening data. Antibodies within a clonotype are assigned to the same bin.

Figure 3. Twenty clonotypes from >100 total clonotypes are grouped on the left and their assigned bin shown on the right. 

 
This screen can be combined with others in an AbTheneum discovery campaign to enrich the data further.

Impact: What We Achieved

The epitope binning screen was able to characterize all the antibodies in a campaign and assign to their epitope bin using only one antibody capture slide. Additional screens could be combined to find more differentiated antibodies. Large proteins with stable domains could be used for the epitope screen to target antibodies that bind to functional epitopes.

Get in touch to find out more