Single-Cell Screening Combined with Repertoire Sequencing from Humanized Mice
Probing and searching natural immune diversity for therapeutic antibodies is a daunting task. Nature produces an immense amount of diversity in response to any given antigen, and from an antibody discovery perspective, the nature of the response determines the overall quality and diversity of the antibodies that can be identified for therapeutic applications.
In recent years, humanized transgenic mice have become the preferred starting source of natural diversity given their ability to generate fully human antibodies in a timely manner and under constraints of in vivo selection. However, to fully utilize the potential of these animals, deep screening of the immune response is required. Frequently employed outdated technologies, such as hybridoma and display-based approaches, are unable to optimally mine the natural diversity in these animals.
Next-generation antibody discovery platforms, such as AbCellera’s high-throughput single-cell screening systems, enable an enhanced level of sensitivity to search and identify rare and valuable antibodies. This is in large part due to unbiased deep screening of millions of antibody-producing cells in any given day, as opposed to only a few tens of thousands of cells with legacy systems, which capture only a small fraction of the natural diversity in any immune system. When combined with humanized mice, this deep screening approach generates large panels of paired heavy/light chain antibody sequences that bypass the need for additional humanization.
To mine the immune response even deeper, the process of immune repertoire sequencing (RepSeq) has recently emerged. RepSeq uses high-throughput sequencing to perform near-comprehensive profiling of the repertoire of heavy and light chain antibody genes present in a pooled or single sample. In its highest-throughput implementation, a single RepSeq sequencing run generates approximately 800 million antibody sequences. This vast diversity is often used to provide a high level profile of an immune response but lacks (1) contextual information about the binding properties of the corresponding antibodies and, (2) is not as sensitive to rare antibodies that might be present in the initial sample.
To combine the best of both worlds, we have integrated high throughput single-cell screening with RepSeq. With this approach, we can generate very large panels of antibodies with known properties and expand diversity around single cell data with RepSeq.
The Challenge: A discovery program to identify an antibody against a validated human target, with 100% homology across mice, rats and humans. Accessing a diverse set of antibodies was a must. In addition, the human target had two related protein homologs, against which antibodies had to be carefully screened.
Our Approach: In this campaign we deployed our optimized immunization protocols to generate a robust immune response in humanized rodents. Next, we developed a high throughput single-cell screening strategy that used multiplexed fluorescence detection to find antibodies specific to the target, but also with binding profiles that would cross-react with the closely related targets.
The Result: We screened four million single cells from immunized rodents and identified more than 1,900 target-specific antibodies, a hit-frequency that demonstrates a robust immune response from breaking tolerance against a 100% homologous target. Of these hits, we identified 428 unique antibody leads with a degree of somatic hypermutation that indicates a mature and directed immune response against a difficult target. We also performed immune repertoire sequencing in parallel and identified clonal lineages in Celium™.