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Monoclonal antibodies are being used to treat a remarkable breadth of human disorders. Nevertheless, there are several key challenges at the earliest stages of antibody drug development that need to be addressed using simple and widely accessible methods, especially related to generating antibodies against membrane proteins and identifying antibody candidates with drug-like biophysical properties (high solubility and low viscosity). Here we highlight key bionanotechnologies for preparing functional and stable membrane proteins in diverse types of lipoparticles that are being used to improve antibody discovery and engineering efforts. We also highlight key bionanotechnologies for high-throughput and ultra-dilute screening of antibody biophysical properties during antibody discovery and optimization that are being used for identifying antibodies with superior combinations of in vitro (formulation) and in vivo (half-life) properties.

Neutralizing monoclonal antibodies and nanobodies have shown promising results as potential therapeutic agents for COVID-19. Identifying such antibodies and nanobodies requires evaluating the neutralization activity of a large number of lead molecules via biological assays, such as the virus neutralization test (VNT). These assays are typically time-consuming and demanding on lab facilities. Here, we present a rapid and quantitative assay that evaluates the neutralizing efficacy of an antibody or nanobody within 1.5 hours, does not require BSL-2 facilities, and consumes only 8 μL of low concentration (ng/mL) sample for each assay run. We tested the human angiotensin-converting enzyme 2 (ACE2) binding inhibition efficacy of seven antibodies and eight nanobodies and verified that the IC(50) values of our assay are comparable with those from SARS-CoV-2 pseudovirus neutralization tests. We also found that our assay could evaluate the neutralizing efficacy against three widespread SARS-CoV-2 variants. We observed increased affinity of these variants for ACE2, including the Beta and Gamma variants. Finally, we demonstrated that our assay enables rapid identification of an immune-evasive mutation of the SARS-CoV-2 spike protein utilizing a set of nanobodies with known binding epitopes.

Agonist antibodies that activate cellular signaling have emerged as promising therapeutics for treating myriad pathologies. Unfortunately, the discovery of rare antibodies with the desired agonist functions is a major bottleneck during drug development. Nevertheless, there has been important recent progress in discovering and optimizing agonist antibodies against a variety of therapeutic targets that are activated by diverse signaling mechanisms. Herein, we review emerging high-throughput experimental and computational methods for agonist antibody discovery as well as rational molecular engineering methods for optimizing their agonist activity.

SARS-CoV-2 variants with enhanced transmissibility represent a serious threat to global health. Here we report machine learning models that can predict the impact of receptor-binding domain (RBD) mutations on receptor (ACE2) affinity, which is linked to infectivity, and escape from human serum antibodies, which is linked to viral neutralization. Importantly, the models predict many of the known impacts of RBD mutations in current and former Variants of Concern on receptor affinity and antibody escape as well as novel sets of mutations that strongly modulate both properties. Moreover, these models reveal key opposing impacts of RBD mutations on transmissibility, as many sets of RBD mutations predicted to increase antibody escape are also predicted to reduce receptor affinity and vice versa. These models, when used in concert, capture the complex impacts of SARS-CoV-2 mutations on properties linked to transmissibility and are expected to improve the development of next-generation vaccines and biotherapeutics.

The COVID‐19 pandemic continues to be a severe threat to human health, especially due to current and emerging SARS‐CoV‐2 variants with potential to escape humoral immunity developed after vaccination or infection. The development of broadly neutralizing antibodies that engage evolutionarily conserved epitopes on coronavirus spike proteins represents a promising strategy to improve therapy and prophylaxis against SARS‐CoV‐2 and variants thereof. Herein, a facile multivalent engineering approach is employed to achieve large synergistic improvements in the neutralizing activity of a SARS‐CoV‐2 cross‐reactive nanobody (VHH‐72) initially generated against SARS‐CoV. This synergy is epitope specific and is not observed for a second high‐affinity nanobody against a non‐conserved epitope in the receptor‐binding domain. Importantly, a hexavalent VHH‐72 nanobody retains binding to spike proteins from multiple highly transmissible SARS‐CoV‐2 variants (B.1.1.7 and B.1.351) and potently neutralizes them. Multivalent VHH‐72 nanobodies also display drug‐like biophysical properties, including high stability, high solubility, and low levels of non‐specific binding. The unique neutralizing and biophysical properties of VHH‐72 multivalent nanobodies make them attractive as therapeutics against SARS‐CoV‐2 variants., This work reports a multivalent engineering strategy to synergistically increase the neutralization potency of nanobodies against SARS‐CoV‐2. An engineered hexavalent nanobody directed against an evolutionarily conserved epitope potently neutralizes SARS‐CoV‐2 and two key variants of concern (B.1.1.7 and B.1.351). This facile approach significantly improves the neutralization breadth and potency of promising nanobodies for potential use in therapeutic applications.

We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.

There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses., Graphical abstract, Zupancic et al. report potent neutralizing nanobodies against SARS-CoV-2. They demonstrate an approach that involves swapping the complementarity-determining regions of low-affinity clones to generate matured nanobodies with large increases in affinity and neutralization activity.

Expression of the receptor tyrosine kinase HER3 is negatively correlated with survival in ovarian cancer, and HER3 overexpression is associated with cancer progression and therapeutic resistance. Thus, improvements in HER3-targeted therapy could lead to significant clinical impact for ovarian cancer patients. Previous work from our group established multivalency as a potential strategy to improve the therapeutic efficacy of HER3-targeted ligands, including affibodies. Others have established HER3 affibodies as viable and potentially superior alternatives to monoclonal antibodies for cancer therapy. Here, bivalent HER3 affibodies were engineered for optimized production, specificity, and function as evaluated in an ovarian cancer xenograft model. Enhanced inhibition of HER3-mediated signaling and increased HER3 downregulation associated with multivalency could be achieved with a simplified construct, potentially increasing translational potential. Additionally, functional effects of affibodies due to multivalency were found to be specific to HER3 targeting, suggesting a unique molecular mechanism. Further, HER3 affibodies demonstrated efficacy in ovarian cancer xenograft mouse models, both as single agents and in combination with carboplatin. Overall, these results reinforce the potential of HER3-targeted affibodies for cancer therapy and establish treatment of ovarian cancer as an application where multivalent HER3 ligands may be useful. Further, this work introduces the potential of HER3 affibodies to be utilized as part of clinically relevant combination therapies (e.g., with carboplatin).

Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.

For therapies targeting diseases of the gastrointestinal tract, we and others envision probiotic bacteria that synthesize and excrete biotherapeutics at disease sites. Toward this goal, we have engineered commensal E. coli that selectively synthesize and secrete a model biotherapeutic in the presence of nitric oxide (NO), an intestinal biomarker for Crohn's disease (CD). This is accomplished by co‐expressing the pore forming protein TolAIII with the biologic, granulocyte macrophage‐colony stimulating factor (GM‐CSF). We have additionally engineered these bacteria to accumulate at sites of elevated NO by engineering their motility circuits and controlling pseudotaxis. Importantly, because we have focused on in vitro test beds, motility and biotherapeutics production are spatiotemporally characterized. Together, the targeted recognition, synthesis, and biomolecule delivery comprises a “smart” probiotics platform that may have utility in the treatment of CD. Further, this platform could be modified to accommodate other pursuits by swapping the promoter and therapeutic gene to reflect other disease biomarkers and treatments, respectively.

Extracellular vesicles (EVs)—comprising a heterogeneous population of cell-derived lipid vesicles including exosomes, microvesicles, and others—have recently emerged as both mediators of intercellular information transfer in numerous biological systems and vehicles for drug delivery. In both roles, EVs have immense potential to impact tissue engineering and regenerative medicine applications. For example, the therapeutic effects of several progenitor and stem cell-based therapies have been attributed primarily to EVs secreted by these cells, and EVs have been recently reported to play direct roles in injury-induced tissue regeneration processes in multiple physiological systems. In addition, EVs have been utilized for targeted drug delivery in regenerative applications and possess unique potential to be harnessed as patient-derived drug delivery vehicles for personalized medicine. This review discusses EVs in the context of tissue repair and regeneration, including their utilization as drug carriers and their crucial role in cell-based therapies. Furthermore, the article highlights the growing need for bioengineers to understand, consider, and ultimately design and specifically control the activity of EVs to maximize the efficacy of tissue engineering and regenerative therapies.

The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic investigations revealed that engineered multivalency enhanced affibody-mediated HER3 downregulation in multiple cancer cell types. Overall, these results highlight the promise of engineered multivalency as a general strategy for enhanced efficacy of HER3-targeted therapeutics against a variety of cancers.