Rapid engineering of SARS-CoV-2 therapeutic antibodies to increase breadth of neutralization including BQ.1.1, CA.3.1, CH.1.1, XBB.1.16, and XBB.1.5.

SARS-CoV-2 Omicron variant XBB.1.5 has shown extraordinary immune escape even for fully vaccinated individuals.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike.

A New Method to Characterize Conformation-Specific Antibody by a Combination of Agarose Native Gel Electrophoresis and Contact Blotting.

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein.

Western blotting of native proteins from agarose gels.

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes.

Ion Binding Properties of a Naturally Occurring Metalloantibody.

LT1009 is a humanized version of murine LT1002 IgG1 that employs two bridging Ca ions to bind its antigen, the biologically active lipid sphingosine-1-phosphate (S1P). We crystallized and determined the X-ray crystal structure of the LT1009 Fab fragment in 10 mM CaCl and found that it binds two Ca in a manner similar to its antigen-bound state. Flame atomic absorption spectroscopy (FAAS) confirmed that murine LT1002 also binds Ca in solution and inductively-coupled plasma-mass spectrometry (ICP-MS) revealed that, although Ca is preferred, LT1002 can bind Mg and, to much lesser extent, Ba.

High-affinity pan-specific monoclonal antibodies that target cysteinyl leukotrienes and show efficacy in an acute model of colitis.

Cysteinyl leukotrienes (CysLTs) are a small family of biological signaling lipids produced by active leukocytes that contribute to diverse inflammatory disease states as a consequence of their engagement with dedicated G protein-coupled receptors. Immunization of mice with a CysLT-modified hapten carrier protein yielded novel monoclonal antibodies that display variable binding affinity to CysLTs. Solution binding assays indicated differing specificities among the antibodies tested, with antibody 10G4 displaying a preference for leukotriene C (LTC).

Measuring Sphingosine-1-Phosphate/Protein Interactions with the Kinetic Exclusion Assay.

By directly detecting the ligand-free binding sites in a sample, the kinetic exclusion assay (KinExA) provides a compelling alternative to SPR-based techniques for determining equilibrium dissociation constants of protein-ligand interactions. It is especially useful for observing protein-lipid interactions, as binding of native lipids occurs entirely in solution, and monoclonal antibodies can be used to directly compete with a protein of interest for lipid binding. By measuring the antigen-free binding sites on the antibody and using competition affinity analysis, the K for the lipid binding the protein and the antibody can be determined simultaneously.

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay.

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species.

Anti-lysophosphatidic acid antibodies improve traumatic brain injury outcomes.

Background: Lysophosphatidic acid (LPA) is a bioactive phospholipid with a potentially causative role in neurotrauma. Blocking LPA signaling with the LPA-directed monoclonal antibody B3/Lpathomab is neuroprotective in the mouse spinal cord following injury.

Findings: Here we investigated the use of this agent in treatment of secondary brain damage consequent to traumatic brain injury (TBI).

X-ray crystal structure of the UCS domain-containing UNC-45 myosin chaperone from Drosophila melanogaster.

UCS proteins, such as UNC-45, influence muscle contraction and other myosin-dependent motile processes. We report the first X-ray crystal structure of a UCS domain-containing protein, the UNC-45 myosin chaperone from Drosophila melanogaster (DmUNC-45). The structure reveals that the central and UCS domains form a contiguous arrangement of 17 consecutive helical layers that arrange themselves into five discrete armadillo repeat subdomains.

Biochemical and structural characterization of lysophosphatidic Acid binding by a humanized monoclonal antibody.

Lysophosphatidic acid (LPA) is a common product of glycerophospholipid metabolism and an important mediator of signal transduction. Aberrantly high LPA concentrations accompany multiple disease states. One potential approach for treatment of these diseases, therefore, is the therapeutic application of antibodies that recognize and bind LPA as their antigen.