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Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) possess mutations that prevent antibody therapeutics from maintaining antiviral binding and neutralizing efficacy. Monoclonal antibodies (mAbs) shown to neutralize Wuhan-Hu-1 SARS-CoV-2 (ancestral) strain have reduced potency against newer variants. Plasma-derived polyclonal hyperimmune drugs have improved neutralization breadth compared with mAbs, but lower titers against SARS-CoV-2 require higher dosages for treatment. We previously developed a highly diverse, recombinant polyclonal antibody therapeutic anti-SARS-CoV-2 immunoglobulin hyperimmune (rCIG). rCIG was compared with plasma-derived or mAb standards and showed improved neutralization of SARS-CoV-2 across World Health Organization variants; however, its potency was reduced against some variants relative to ancestral, particularly omicron. Omicron-specific antibody sequences were enriched from yeast expressing rCIG-scFv and exhibited increased binding and neutralization to omicron BA.2 while maintaining ancestral strain binding and neutralization. Polyclonal antibody libraries such as rCIG can be utilized to develop antibody therapeutics against present and future SARS-CoV-2 threats.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply, and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates thousands-diverse mixtures of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors, or immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in under three months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin (ATG), we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense–response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense–response applications.

Generating combinatorial libraries of specific sets of mutations are essential for addressing protein engineering questions involving contingency in molecular evolution, epistatic relationships between mutations, as well as functional antibody and enzyme engineering. Here we present optimization of a combinatorial mutagenesis method involving template-based nicking mutagenesis, which allows for the generation of libraries with >99% coverage for tens of thousands of user-defined variants. The non-optimized method resulted in low library coverage, which could be rationalized by a model of oligonucleotide annealing bias resulting from the nucleotide mismatch free-energy difference between mutagenic oligo and template. The optimized method mitigated this thermodynamic bias using longer primer sets and faster annealing conditions. Our updated method, applied to two antibody fragments, delivered between 99.0% (32451/32768 library members) to >99.9% coverage (32757/32768) for our desired libraries in 2 days and at an approximate 140-fold sequencing depth of coverage.

Plasma-derived polyclonal antibodies are polyvalent drugs used for many important clinical indications that require modulation of multiple drug targets simultaneously, including emerging infectious disease and transplantation. However, plasma-derived drugs suffer many problems, including low potency, impurities, constraints on supply, and batch-to-batch variation. In this study, we demonstrated proofs-of-concept for a technology that uses microfluidics and molecular genomics to capture diverse mammalian antibody repertoires as multivalent recombinant drugs. These “recombinant hyperimmune” drugs comprised thousands to tens of thousands of antibodies and were derived from convalescent human donors, or vaccinated human donors or immunized mice. Here we used our technology to build a highly potent recombinant hyperimmune for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2) in less than three months. We also validated a recombinant hyperimmune for Zika virus disease that abrogates antibody-dependent enhancement (ADE) through Fc engineering. For patients with primary immune deficiency (PID), we built high potency polyvalent recombinant hyperimmunes against pathogens that commonly cause serious lung infections. Finally, to address the limitations of rabbit-derived anti-thymocyte globulin (ATG), we generated a recombinant human version and demonstrated in vivo function against graft-versus-host disease (GVHD). Recombinant hyperimmunes are a novel class of drugs that could be used to target a wide variety of other clinical applications, including cancer and autoimmunity.

Interleukin-31 (IL-31) is a major protein involved in severe inflammatory skin disorders. Its signaling pathway is mediated through two type I cytokine receptors, IL-31RA (also known as the gp130-like receptor) and the oncostatin M receptor (OSMR). Understanding molecular details in these interactions would be helpful for developing antagonist anti-IL-31 monoclonal antibodies (mAbs) as potential therapies. Previous studies suggest that human IL-31 binds to IL-31RA and then recruits OSMR to form a ternary complex. In this model, OSMR cannot interact with IL-31 in the absence of IL-31RA. In this work, we show that feline IL-31 (fIL-31) binds independently with feline OSMR using surface plasmon resonance, an enzyme-linked immunosorbent assay, and yeast surface display. Moreover, competition experiments suggest that OSMR shares a partially overlapping epitope with IL-31RA. We then used deep mutational scanning to map the binding sites of both receptors on fIL-31. In agreement with previous studies of the human homologue, the binding site for IL31-RA contains fIL-31 positions E20 and K82, while the binding site for OSMR comprises the "PADNFERK" motif (P103-K110) and position G38. However, our results also revealed a new overlapping site, composed of positions R69, R72, P73, D76, D81, and E97, between both receptors that we called the "shared site". The conformational epitope of an anti-feline IL-31 mAb that inhibits both OSMR and IL-31RA also mapped to this shared site. Combined, our results show that fIL-31 binds IL-31RA and OSMR independently through a partially shared epitope. These results suggest reexamination of the putative canonical mechanisms for IL-31 signaling in higher animals.

User-defined mutagenic libraries are fundamental for applied protein engineering workflows. Here we show that unamplified oligo pools can be used to prepare site saturation mutagenesis libraries from plasmid DNA with near-complete coverage of desired mutations and few off-target mutations. We find that oligo pools yield higher quality libraries when compared to individually synthesized degenerate oligos. We also show that multiple libraries can be multiplexed into a single oligo pool, making preparation of multiple libraries less expensive and more convenient. We provide software for automatic oligo pool design that can generate mutagenic oligos for saturating or focused libraries.

Nerve growth factor (NGF) plays a central role in multiple chronic pain conditions. As such, anti-NGF monoclonal antibodies (mAbs) that function by antagonizing NGF downstream signaling are leading drug candidates for non-opioid pain relief. To evaluate anti-canine NGF (cNGF) mAbs we sought a yeast surface display platform of cNGF. Both mature cNGF and pro-cNGF displayed on the yeast surface but bound conformationally sensitive mAbs at most 2.5-fold in mean fluorescence intensity above background, suggesting that cNGF was mostly misfolded. To improve the amount of folded, displayed cNGF, we used comprehensive mutagenesis, FACS, and deep sequencing to identify point mutants in the pro-region of canine NGF that properly enhance the folded protein displayed on the yeast surface. Out of 1,737 tested single point mutants in the pro region, 49 increased the amount of NGF recognized by conformationally sensitive mAbs. These gain-of-function mutations cluster around residues A-61-P-26. Gain-of-function mutants were additive, and a construct containing three mutations increased amount of folded cNGF to 23-fold above background. Using this new cNGF construct, fine conformational epitopes for tanezumab and three anti-cNGF mAbs were evaluated. The epitope revealed by the yeast experiments largely overlapped with the tanezumab epitope previously determined by X-ray crystallography. The other mAbs showed site-specific differences with tanezumab. As the number of binding epitopes of functionally neutralizing anti-NGF mAbs on NGF are limited, subtle differences in the individual interacting residues on NGF that bind each mAb contribute to the understanding of each antibody and variations in its neutralizing activity. These results demonstrate the potential of deep sequencing-guided protein engineering to improve the production of folded surface-displayed protein, and the resulting cNGF construct provides a platform to map conformational epitopes for other anti-neurotrophin mAbs.

In this chapter, we discuss a method to determine the affinity and specificity of nearly all single-point mutants for a full-length protein binder. This method combines deep sequencing, comprehensive mutagenesis, yeast surface display, and fluorescence-activated cell sorting. This approach has been used to study sequence-function relationships for protein-protein interactions. The data can be used to determine the fine conformational epitope on the protein binder.