GPCRs are ubiquitous in human biology, participating in diverse processes such as metabolism, inflammation, neurotransmission, and carcinogenesis. The Twist GPCR Library Series leverages all known GPCR-ligand interactions and existing GPCR antibody diversities to enable antibody discovery beyond existing GPCR-targeting small molecules and antibodies, which cover only 13% of the human GPCRome. This fully human synthetic library series includes the GPCR 2.0 scFv, GPCR 3.0 scFv, and VHH hShuffle GPCR libraries.

Using Twist’s high-throughput DNA synthesis platform, multiple high diversity synthetic antibody libraries have been built to serve as powerful tools for antibody discovery. Leveraging these highly diverse libraries, we have been able to identify high-affinity antibody candidates for a variety of targets. Here we demonstrate the antibody discovery process for 6 cytokine or immunomodulatory protein targets. This process spans constructing synthetic antibody libraries, using phage display to pan libraries against biotinylated protein targets, and screening for lead candidates through ELISA binding assays and NGS enrichment tracking. Utilizing a high-throughput approach, candidates are reformatted into full length IgG or VHH-Fc; the resulting monoclonal antibodies are then validated through SPR and cell-based assays. Many high affinity candidates with ranges of picomolar to nanomolar binding were identified for the six targets, demonstrating the efficacy of this discovery process.

Antibodies must bind their targets with high avidity to effect therapeutic or diagnostic outcomes. The mammalian immune system can produce antibodies with micro- to nanomolar affinity through in vivo affinity maturation. In vitro display technologies have surpassed this affinity ceiling, enabling discovery of antibodies with picomolar and femtomolar affinities. In vitro display technologies offer immediate access to the antibody fragment genes, which can subsequently be multimerized to generate high-avidity antibody fusions. Among the available antibody formats, single-domain antibodies (VHH) are one of the most engineerable. VHH antibodies have many advantages including their increased thermostability, smaller- molecular weight, lower production cost, and modular format for building multivalent biologics. These advantages have encouraged many to prioritize VHHs when developing diagnostic and therapeutic antibodies against severe acute respiratory coronavirus 2 (SARS-CoV- 2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic. Multivalent SARS-CoV-2 antibodies have been engineered more recently. VHH antibodies can be sourced from immune, naïve, or synthetic antibody libraries. Immune and naive libraries require amplification from animal B cells, and are prone to redundancy and bias, which can limit library diversity. Synthetic libraries are based on computational in silico design and use synthetic oligos to introduce diversity. Using Twist’s unique silicon-based DNA synthesis platform, we have generated VHH libraries with high diversity (>1010), accuracy (1 error/2 kb), and productivity (> 80%). Recently, we reported the discovery of several high-affinity VHH-Fc antibodies against the SARS-CoV-2 S1 protein subunit from these libraries. Unlike methods for the construction of synthetic antibody libraries, methods for screening multivalents are comparatively low throughput and ineffective for higher-order multivalent constructs where the number of possible permutations increases exponentially. Beginning with 13 anti- S1 VHH candidates, we constructed multivalent libraries of tandem-assembled VHHs, screened them by phage display, and sequenced the enriched libraries to determine the positional frequencies of each individual VHH fragment Using this pipeline, we engineered mono- and bispecific multivalent VHH-Fc constructs with higher affinity for S1 than their monovalent counterparts.