An Ultra High Throughput Screening Platform for Discovery and Engineering of Full-Length Antibodies in Soluble and Secreted Forms

Traditional display techniques for antibody engineering have two major limitations. First, the antigen binding affinities are normally detected with single-chain variable fragment (scFv) or fragment antigen-binding (Fab). However, the physicochemical properties and expressibilities of those fragments may not correlate with their cognate full-length formats. Second, high throughput screening or selections require high quality, soluble recombinant antigen, which can be challenging or even intractable with membrane proteins such as G-protein-coupled receptors (GPCRs) and ion-channel receptors.

To overcome these limitations, we have developed a novel high throughput screening platform for the discovery and engineering of full-length monoclonal antibodies (mAbs) in soluble, secreted forms. This state-of-the-art technology utilizes hydrogel microdroplets (GMDs) coupled with fluorescence-activated cell sorting (FACS). The GMDs provide a linkage between phenotype and genotype. Both bulk emulsion and microfluidic compartmentalization have been used to co-encapsulate antibody secreting cells (Pichia pastoris or Saccharomyces cerevisiae) and antigen bearing cells (cancer cell lines) in picoliter-volume hydrogels. Full-length antibodies secreted by recombinant yeast will bind with the cognate antigens on the surface of mammalian target cells within the same droplet, which can then be labeled with fluorophore conjugated secondary antibodies. Fluorescently stained GMDs can be sorted by FACS at rates of 2,000 events per second or more. It has enabled us to easily screen millions of clones per day, which is impossible with the conventional microtiter well plate- based method. Furthermore, this technology is very cost-efficient. The expenses for the entire setup (except for the FACS system) were estimated to be only several thousand dollars, while each round of GMDs production costs around 50 USD.

We successfully validated our approach by the enrichment and one-step isolation of anti-EGFR IgG secreting Pichia pastoris from a 1:10,000 mixture of Pichia cells secreting negative control anti-CCR5 IgG antibodies. We also applied the GMD-FACS screening platform to distinguish cancer cells with different surface expression levels of EGFR.

In general, we have presented an innovative high throughput screening platform which is efficient, robust, and cheap for discovery and engineering of secreted, full-length mAbs. The broad utility of this methodology has been demonstrated through the screening of a spike-in library and identification of different cancer cells. In addition, this platform can also be utilized to isolate and engineer affinity proteins such as F(ab’)2, nanobodies, and various non-immunoglobulin scaffold proteins. Our GMD-FACS technique can be a valuable tool to facilitate antibody discovery in both academic and industrial settings.