High throughput antibody discovery against cell membrane bound target proteins using innovative MOD technology for direct screening in single-cell assays

ABSTRACT Scribe Biosciences are leading experts in the field of droplet microfluidics and have developed a best-in-class droplet manipulation platform, Microenvironment on Demand (MOD), that can currently assemble >100k paired-cell assays in <3 hours, with proven proof of concept. Using this innovative technology, this SBIR Phase 1 project proposes the development and quantification of assay methods to be used for single-cell functional screening workflows to enable large scale screening of therapeutic antibody (Ab) candidates. The development of such a workflow to reliably, consistently, and repeatably identify large and diverse pools of B cell hits would offer a significant advantage over the classical but inefficient hybridoma method. Porting direct B-cell assays to microfluidics is a natural fit because short lived B-cells can rapidly generate significant secreted Ab concentrations when incubated in appropriately small volumes; current attempts are limited by cost and scalability, and none offer high throughput (HT) assays against target cells, sensitive assays, or integrated HT sequencing. MOD represents an evolutionary advancement in the capability to build droplet-based cell assays with precision and scale, effectively integrating assay construction, readouts, hit selection, and sample prep into a single workflow and instrument. MOD co-encapsulates Ab-secreting and target cells in the same microfluidic droplet, which enables building an assay based on the target cell, since it will carry along the Ab-secreting cell and therefore the RNA that is available to identify the Ab in a subsequent sequencing step. MOD utilizes flow cytometry-style detection and sorting, so it is readily scalable for HT. The approach for this project has been informed by previous work developing assays on the MOD platform. In the first aim, two assays will be developed to detect Ab binding against membrane protein targets. The first will adopt an existing bead-based no wash assay scheme for use with high copy number targets, and the second will develop a more sensitive assay for low copy number targets with a wash step, and will explore the appropriate method for creating a durable physical linkage between the cells that will last through FACS sorting or re-encapsulation. The second aim will test and quantify the system with B-cells from immunized mice for a real-world demonstration of Ab discovery. B-cells will be sourced from standard 4-week immunization protocols on groups of 3 mice using SARS-CoV-2 as the antigen, and will be used to explore the parameters of primary B-cell culture in droplets and other factors associated with porting B-cell biology on to the MOD platform. A small batch (50-100k) of B-cell/target cell assays will be tested, and assuming that the HT of the platform will correlate with a high number of hits (~1000 positive assays), a small number (~10) of Ab candidates will be bioinformatically selected for subsequent re-cloning and hit validation. Successful MOD-enabled antibody screening would introduce a new paradigm in the capabilities of researchers to identify a larger and more diverse field of Ab candidates, bypassing current limitations of cost, scalability, commercial availability, or technical complexity, and ultimately leading to better therapies.