Rapid mRNA‐Display Selection of an IL‐6 Inhibitor Using Continuous‐Flow Magnetic Separation

Abstract

Since the invention of hybridoma technology, methods for generating affinity reagents that bind specific target molecules have revolutionized biology and medicine.[1] In the postgenomic era, there is a pressing need to accelerate the pace of ligand discovery to elucidate the functions of a rapidly growing number of newly characterized molecules and their modified states.[2] Nonimmunoglobulin-based proteins such as DARPins, affibodies, and monobodies represent attractive alternatives to traditional antibodies as these are small, soluble, disulfide-free, single-domain scaffolds that can be selected from combinatorial libraries and expressed in bacteria.[3] For example, monobodies—highly stable scaffolds based on the immunoglobulin VH-like 10th fibronectin type III (10Fn3) domain of human fibronectin[4]—have yielded antibody mimetics that bind to numerous targets for applications including intracellular inhibition,[5,6] therapeutics,[7] and biosensors.[6,8] These 10Fn3-based ligands can be derived from highly diverse libraries using techniques such as phage, ribosome, mRNA, bacterial, and yeast displays.[9]

Single‐Round, Multiplexed Antibody Mimetic Design through mRNA Display

“…[1] Hybridoma-based monoclonal antibodies, [2] the standard for protein reagents, are undesirable for this task because of the number of animals, amount of target, time, and effort required to generate each reagent. Here, we developed a unified approach to solve this problem by integrating four distinct technologies: 1) a combinatorial protein library based on the 10th fibronectin type III domain of human fibronectin (10Fn3), [3] 2) protein library display by mRNA display, [4] 3) selection by continuous flow magnetic separation (CFMS), [5] and 4) sequence analysis by high throughput sequencing (HTS). [6] Next generation sequencing has revolutionized many fields of biology, and is increasingly being utilized to improve ligand design efforts.…”

“…Previously, we had designed, created and optimized a high-complexity library termed e10Fn3 [5,8] —based on the 10Fn3 scaffold developed by Koide and coworkers. [3] This scaffold contains only two random sequence regions, the BC loop (7 residues) and the FG loop (10 residues) (Figure 1C), which can be read by paired end sequencing using customized primers (Figure 1D; Figure S1).…”

“…[5,8-10] In order to achieve a single round selection, we needed to balance the input diversity with three factors: 1) the number of clones we could sequence, 2) the fold enrichment in a single round of CFMS, and 3) the inherent frequency of functional clones in our library. One lane of an Illumina GAIIx yields 20-30 million sequences, thus we reasoned that clones enriched to greater than 1 in 1 million would be identified with 20-30 copies and therefore would be identifiable above the statistical background.…”

“…One lane of an Illumina GAIIx yields 20-30 million sequences, thus we reasoned that clones enriched to greater than 1 in 1 million would be identified with 20-30 copies and therefore would be identifiable above the statistical background. Our previous work indicated that CFMS enrichment was >1,000-fold per round, [5] thus enabling us to identify functional clones occurring at a frequency of ~1 in every 1 billion sequences in the naïve pool (Figure 1A). Prior yeast display work used much smaller libraries to isolate functional clones [11] supporting a complexity of ~10 9 .…”

“…In conventional mRNA display selections with low copy numbers, 1-2 copies of each functional clone are retained by the first enrichment (Figure S2A). However, even if only ~0.01% of the nonfunctional clones are retained due to our CFMS protocol, [5] the individual copy number of these clones (1 copy for each random, sequence-independent carryover) would be indistinguishable from the functional clones. Therefore, here we aimed to achieve a copy number of 1,000 for each sequence, allowing adequate separation of functional from non-functional clones (Figure S2B).…”