IL-2-armored peptide-major histocompatibility class I bispecific antibodies redirect antiviral effector memory CD8+ T cells to induce potent anti-cancer cytotoxic activity with limited cytokine release.

T cell engagers (TCEs) are becoming an integral class of biological therapeutic owing to their highly potent ability to eradicate cancer cells. Nevertheless, the widespread utility of classical CD3-targeted TCEs has been limited by narrow therapeutic index (TI) linked to systemic CD4+ T cell activation and aberrant cytokine release. One attractive approach to circumvent the systemic activation of pan CD3+ T cells and reduce the risk of cytokine release syndrome is to redirect specific subsets of T cells.

Monoclonal Antibody Generation Using Single B Cell Screening for Treating Infectious Diseases.

The screening of antigen-specific B cells has been pivotal for biotherapeutic development for over four decades. Conventional antibody discovery strategies, including hybridoma technology and single B cell screening, remain widely used based on their simplicity, accessibility, and proven track record. Technological advances and the urgent demand for infectious disease applications have shifted paradigms in single B cell screening, resulting in increased throughput and decreased time and labor, ultimately enabling the rapid identification of monoclonal antibodies with desired biological and biophysical properties.

Facile generation of biepitopic antibodies with intrinsic agonism for activating tumor necrosis factor receptors.

Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.

Facile generation of biepitopic antibodies with intrinsic agonism for activating receptors in the tumor necrosis factor superfamily.

Agonist antibodies that activate cellular receptors are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering.

Optimization of therapeutic antibodies for reduced self-association and non-specific binding via interpretable machine learning.

Antibody development, delivery, and efficacy are influenced by antibody-antigen affinity interactions, off-target interactions that reduce antibody bioavailability and pharmacokinetics, and repulsive self-interactions that increase the stability of concentrated antibody formulations and reduce their corresponding viscosity. Yet identifying antibody variants with optimal combinations of these three types of interactions is challenging. Here we show that interpretable machine-learning classifiers, leveraging antibody structural features descriptive of their variable regions and trained on experimental data for a panel of 80 clinical-stage monoclonal antibodies, can identify antibodies with optimal combinations of low off-target binding in a common physiological-solution condition and low self-association in a common antibody-formulation condition.

Co-optimization of therapeutic antibody affinity and specificity using machine learning models that generalize to novel mutational space.

Therapeutic antibody development requires selection and engineering of molecules with high affinity and other drug-like biophysical properties. Co-optimization of multiple antibody properties remains a difficult and time-consuming process that impedes drug development. Here we evaluate the use of machine learning to simplify antibody co-optimization for a clinical-stage antibody (emibetuzumab) that displays high levels of both on-target (antigen) and off-target (non-specific) binding.

Mutational analysis of SARS-CoV-2 variants of concern reveals key tradeoffs between receptor affinity and antibody escape.

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.

Rapid and Quantitative Evaluation of SARS-CoV-2 Neutralizing Antibodies and Nanobodies.

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 biological assays, such as the virus neutralization test (VNT). These assays are typically time-consuming and demanding on-lab facilities.

Improving antibody drug development using bionanotechnology.

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.

Discovery and characterization of high-affinity, potent SARS-CoV-2 neutralizing antibodies via single B cell screening.

Monoclonal antibodies that target SARS-CoV-2 with high affinity are valuable for a wide range of biomedical applications involving novel coronavirus disease (COVID-19) diagnosis, treatment, and prophylactic intervention. Strategies for the rapid and reliable isolation of these antibodies, especially potent neutralizing antibodies, are critical toward improved COVID-19 response and informed future response to emergent infectious diseases. In this study, single B cell screening was used to interrogate antibody repertoires of immunized mice and isolate antigen-specific IgG1 memory B cells.

Agonist antibody discovery: Experimental, computational, and rational engineering approaches.

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.

Engineered Multivalent Nanobodies Potently and Broadly Neutralize SARS-CoV-2 Variants.

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.

Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis.

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.

Homologous Quorum Sensing Regulatory Circuit: A Dual-Input Genetic Controller for Modulating Quorum Sensing-Mediated Protein Expression in .

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 .

A platform of genetically engineered bacteria as vehicles for localized delivery of therapeutics: Toward applications for Crohn's disease.

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).

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

Objective: Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation.

Approach And Results: AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice.

Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells.

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.

Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication.

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.

Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine.

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.