A Novel Antigen Design Strategy to Isolate Single-Domain Antibodies that Target Human Nav1.7 and Reduce Pain in Animal Models.

Genetic studies have identified the voltage-gated sodium channel 1.7 (Na1.7) as pain target.

Effects of Two Soluble ACE2-Fc Variants on Blood Pressure and Albuminuria in Hypertensive Mice: Research Letter.

Background: Angiotensin-converting enzyme 2 (ACE2) hydrolyzes angiotensin (Ang) II to Ang-(1-7), promoting vasodilatation, and inhibiting oxidative stress and inflammation. Plasma membrane ACE2 is the receptor for all known SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral variants. In COVID-19 infection, soluble ACE2 variants may act as decoys to bind and neutralize the coronavirus, reducing its tissue infectivity.

Enhanced antibody-antigen structure prediction from molecular docking using AlphaFold2.

Predicting the structure of antibody-antigen complexes has tremendous value in biomedical research but unfortunately suffers from a poor performance in real-life applications. AlphaFold2 (AF2) has provided renewed hope for improvements in the field of protein-protein docking but has shown limited success against antibody-antigen complexes due to the lack of co-evolutionary constraints. In this study, we used physics-based protein docking methods for building decoy sets consisting of low-energy docking solutions that were either geometrically close to the native structure (positives) or not (negatives).

Exploring rigid-backbone protein docking in biologics discovery: a test using the DARPin scaffold.

Accurate protein-protein docking remains challenging, especially for artificial biologics not coevolved naturally against their protein targets, like antibodies and other engineered scaffolds. We previously developed ProPOSE, an exhaustive docker with full atomistic details, which delivers cutting-edge performance by allowing side-chain rearrangements upon docking. However, extensive protein backbone flexibility limits its practical applicability as indicated by unbound docking tests.

Comparative Performance of High-Throughput Methods for Protein p Predictions.

The medically relevant field of protein-based therapeutics has triggered a demand for protein engineering in different pH environments of biological relevance. engineering workflows typically employ high-throughput screening campaigns that require evaluating large sets of protein residues and point mutations by fast yet accurate computational algorithms. While several high-throughput p prediction methods exist, their accuracies are unclear due to the lack of a current comprehensive benchmarking.

Solvated interaction energy: from small-molecule to antibody drug design.

Scoring functions are ubiquitous in structure-based drug design as an aid to predicting binding modes and estimating binding affinities. Ideally, a scoring function should be broadly applicable, obviating the need to recalibrate and refit its parameters for every new target and class of ligands. Traditionally, drugs have been small molecules, but in recent years biologics, particularly antibodies, have become an increasingly important if not dominant class of therapeutics.

A glyco-engineering approach for site-specific conjugation to Fab glycans.

Effective processes for synthesizing antibody-drug conjugates (ADCs) require: 1) site-specific incorporation of the payload to avoid interference with binding to the target epitope, 2) optimal drug/antibody ratio to achieve sufficient potency while avoiding aggregation or solubility problems, and 3) a homogeneous product to facilitate approval by regulatory agencies. In conventional ADCs, the drug molecules are chemically attached randomly to antibody surface residues (typically Lys or Cys), which can interfere with epitope binding and targeting, and lead to overall product heterogeneity, long-term colloidal instability and unfavorable pharmacokinetics. Here, we present a more controlled process for generating ADCs where drug is specifically conjugated to only Fab -linked glycans in a narrow ratio range through functionalized sialic acids.

Optimizing Antibody-Antigen Binding Affinities with the ADAPT Platform.

The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform guides the selection of mutants that improve/modulate the affinity of antibodies and other biologics. Predicted affinities are based on a consensus z-score from three scoring functions. Computational predictions are interleaved with experimental validation, significantly enhancing the robustness of the design and selection of mutants.

Programmable Attenuation of Antigenic Sensitivity for a Nanobody-Based EGFR Chimeric Antigen Receptor Through Hinge Domain Truncation.

Epidermal growth factor family receptor (EGFR) is commonly overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR)-T therapy, but as EGFR is also expressed at lower levels in healthy tissues a therapeutic strategy must balance antigenic responsiveness against the risk of on-target off-tumor toxicity. Herein, we identify several camelid single-domain antibodies (also known as nanobodies) that are effective EGFR targeting moieties for CARs (EGFR-sdCARs) with very strong reactivity to EGFR-high and EGFR-low target cells. As a strategy to attenuate their potent antigenic sensitivity, we performed progressive truncation of the human CD8 hinge commonly used as a spacer domain in many CAR constructs.

Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor.

The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood-brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB.

Coevolved Canonical Loops Conformations of Single-Domain Antibodies: A Tale of Three Pockets Playing Musical Chairs.

Single-domain antibodies (sdAbs) are a promising class of biotherapeutics with unique structural traits within their paratope region. The distribution of canonical conformations explored by their complementarity determining region (CDR) loops differs to some extent from conventional two-chain Fv fragments of monoclonal antibodies (mAbs). In this study, we explored in detail the canonical structures of sdAb CDR-H1 and CDR-H2 loops and compared those with mAbs from the IGHV3 and IGHV1 gene families.

Antibody mutations favoring pH-dependent binding in solid tumor microenvironments: Insights from large-scale structure-based calculations.

Antibody-based therapeutics for treatment of various tumors have grown rapidly in recent years. Unfortunately, safety issues, attributed to off-tumor effects and cytotoxicity, are still a significant concern with the standard of care. Improvements to ensure targeted delivery of antitumor pharmaceuticals are desperately needed.

Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody.

The SARS coronavirus 2 (SARS-CoV-2) spike (S) protein binding to the human ACE2 receptor is the molecular event that initiates viral entry into host cells and leads to infection and virus replication. There is a need for agents blocking viral entry into host cells that are cross-reactive with emerging virus variants. VHH-72 is an anti-SARS-CoV-1 single-domain antibody that also exhibits cross-specificity with SARS-CoV-2 but with decreased binding affinity.

Humanization of Camelid Single-Domain Antibodies.

Humanization of therapeutic antibodies derived from animal immunizations is often required to minimize immunogenicity risks in humans, which can cause potentially harmful and serious side effects and reduce antibody efficacy. Humanization is typically applied to conventional monoclonal antibodies derived in rodents as well as single-domain antibodies isolated from camelids and sharks (VHHs and VNARs). A streamlined protocol is described here for sequence humanization of camelid VHHs, which represent a promising biotherapeutic format with many desirable attributes.

Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors.

Redesigning an antibody H3 loop by virtual screening of a small library of human germline-derived sequences.

The design of superior biologic therapeutics, including antibodies and engineered proteins, involves optimizing their specific ability to bind to disease-related molecular targets. Previously, we developed and applied the Assisted Design of Antibody and Protein Therapeutics (ADAPT) platform for virtual affinity maturation of antibodies (Vivcharuk et al. in PLoS One 12(7):e0181490, https://doi.

Binding mechanism of a de novo coiled coil complex elucidated from surface forces measurements.

We used the Surface Forces Apparatus to elucidate the interaction mechanism between grafted 5 heptad-long peptides engineered to spontaneously form a heterodimeric coiled-coil complex. The results demonstrated that when intimate contact between peptides is reached, binding occurs first via weakly interacting but more mobile distal heptads, suggesting an induced-fit association process. Precise control of the distance between peptide-coated surfaces allowed to quantitatively monitor the evolution of their biding energy.

Serum albumin-binding V Hs with variable pH sensitivities enable tailored half-life extension of biologics.

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (V Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days.

Binding and functional profiling of antibody mutants guides selection of optimal candidates as antibody drug conjugates.

An increasingly appreciated conundrum in the discovery of antibody drug conjugates (ADCs) is that an antibody that was selected primarily for strong binding to its cancer target may not serve as an optimal ADC. In this study, we performed mechanistic cell-based experiments to determine the correlation between antibody affinity, avidity, internalization and ADC efficacy. We used structure-guided design to assemble a panel of antibody mutants with predicted Her2 affinities ranging from higher to lower relative to the parent antibody, Herceptin.

Structure-based engineering of pH-dependent antibody binding for selective targeting of solid-tumor microenvironment.

Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues.

Role of the non-hypervariable FR3 D-E loop in single-domain antibody recognition of haptens and carbohydrates.

Single-domain antibodies (sdAbs), the variable domains of camelid heavy chain-only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti-methotrexate, anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop, formed between β-strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15-acetyl-deoxynivalenol (15-AcDON), and to carbohydrates.

Binding symmetry and surface flexibility mediate antibody self-association.

Solution stability is an important factor in the optimization of engineered biotherapeutic candidates such as monoclonal antibodies because of its possible effects on manufacturability, pharmacology, efficacy and safety. A detailed atomic understanding of the mechanisms governing self-association of natively folded protein monomers is required to devise predictive tools to guide screening and re-engineering along the drug development pipeline. We investigated pairs of affinity-matured full-size antibodies and observed drastically different propensities to aggregate from variants differing by a single amino-acid.

The adaptor protein Ste50 directly modulates yeast MAPK signaling specificity through differential connections of its RA domain.

Discriminating among diverse environmental stimuli is critical for organisms to ensure their proper development, homeostasis, and survival. Saccharomyces cerevisiae regulates mating, osmoregulation, and filamentous growth using three different MAPK signaling pathways that share common components and therefore must ensure specificity. The adaptor protein Ste50 activates Ste11p, the MAP3K of all three modules.