A Tetravalent Bispecific Antibody Selectively Inhibits Diverse FGFR3 Oncogenic Variants.

The receptor tyrosine kinase FGFR3 is frequently mutated in bladder cancer and is a validated therapeutic target. Although pan-FGFR tyrosine kinase inhibitors (TKI) have shown clinical efficacy, toxicity and acquired resistance limit the benefit of these agents. While antibody-based therapeutics can offer superior selectivity than TKIs, conventional ligand-blocking antibodies are usually ineffective inhibitors of constitutively active receptor tyrosine kinases. Furthermore, the existence of multiple oncogenic variants of FGFR3 presents an additional challenge for antibody-mediated blockade. Here, we developed a tetravalent FGFR3×FGFR3 bispecific antibody that inhibited FGFR3 point mutants and fusion proteins more effectively than any of the conventional FGFR3 antibodies that we produced. Each arm of the bispecific antibody contacted two distinct epitopes of FGFR3 through a cis mode of binding. The antibody blocked dimerization of the most common FGFR3 oncogenic variant (S249C extracellular domain mutation) and inhibited the function of FGFR3 variants that are resistant to pan-FGFR TKIs. The antibody was highly effective in suppressing growth of FGFR3-driven tumor models, providing efficacy comparable to that of the FDA-approved TKI erdafitinib. Thus, this bispecific antibody may provide an effective approach for broad and highly selective inhibition of oncogenic FGFR3 variants. Significance: Development of a bispecific antibody that broadly inhibits gain-of-function FGFR3 variants provides a therapeutic strategy to target tumors with oncogenic FGFR3 point mutations and fusions, a particularly difficult case for antibody blockade.

Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail.

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.

The Cap-Snatching SFTSV Endonuclease Domain Is an Antiviral Target.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%-30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (IC50) of ∼100 nM in enzyme inhibition and an EC50 value of ∼250 nM against SFTSV and HRTV in plaque assays. Together, our data support sNSV endonucleases as an antiviral target.

Hydroxyl-Radical Reaction Pathways for the Fast Photochemical Oxidation of Proteins Platform As Revealed by 18O Isotopic Labeling.

Fast photochemical oxidation of protein (FPOP) has become an important mass spectrometry-based protein footprinting approach. Although the hydroxyl radical (•OH) generated by photolysis of hydrogen peroxide (H2O2) is most commonly used, the pathways for its reaction with amino-acid side chains remain unclear. Here, we report a systematic study of •OH oxidative modification of 13 amino acid residues by using 18O isotopic labeling. The results differentiate three classes of residues on the basis of their oxygen uptake preference toward different oxygen sources. Histidine, arginine, tyrosine, and phenylalanine residues preferentially take oxygen from H2O2. Methionine residues competitively take oxygen from H2O2 and dissolved oxygen (O2), whereas the remaining residues take oxygen exclusively from O2. Results reported in this work deepen the understanding of •OH labeling pathway on a FPOP platform, opening new possibilities for tailoring FPOP conditions in addressing many biological questions in a profound way.

Phycobilisomes Harbor FNRL in Cyanobacteria.

Cyanobacterial phycobilisomes (PBSs) are photosynthetic antenna complexes that harvest light energy and supply it to two reaction centers (RCs) where photochemistry starts. PBSs can be classified into two types, depending on the presence of allophycocyanin (APC): CpcG-PBS and CpcL-PBS. Because the accurate protein composition of CpcL-PBS remains unclear, we describe here its isolation and characterization from the cyanobacterium Synechocystis sp. strain 6803. We found that ferredoxin-NADP+ oxidoreductase (or FNRL), an enzyme involved in both cyclic electron transport and the terminal step of the electron transport chain in oxygenic photosynthesis, is tightly associated with CpcL-PBS as well as with CpcG-PBS. Room temperature and low-temperature fluorescence analyses show a red-shifted emission at 669 nm in CpcL-PBS as a terminal energy emitter without APC. SDS-PAGE and quantitative mass spectrometry reveal an increased content of FNRL and CpcC2, a rod linker protein, in CpcL-PBS compared to that of CpcG-PBS rods, indicative of an elongated CpcL-PBS rod length and its potential functional differences from CpcG-PBS. Furthermore, we combined isotope-encoded cross-linking mass spectrometry with computational protein structure predictions and structural modeling to produce an FNRL-PBS binding model that is supported by two cross-links between K69 of FNRL and the N terminus of CpcB, one component in PBS, in both CpcG-PBS and CpcL-PBS (cross-link 1), and between the N termini of FNRL and CpcB (cross-link 2). Our data provide a novel functional assembly form of phycobiliproteins and a molecular-level description of the close association of FNRL with phycocyanin in both CpcG-PBS and CpcL-PBS.IMPORTANCE Cyanobacterial light-harvesting complex PBSs are essential for photochemistry in light reactions and for balancing energy flow to carbon fixation in the form of ATP and NADPH. We isolated a new type of PBS without an allophycocyanin core (i.e., CpcL-PBS). CpcL-PBS contains both a spectral red-shifted chromophore, enabling efficient energy transfer to chlorophyll molecules in the reaction centers, and an increased FNRL content with various rod lengths. Identification of a close association of FNRL with both CpcG-PBS and CpcL-PBS brings new insight to its regulatory role for fine-tuning light energy transfer and carbon fixation through both noncyclic and cyclic electron transport.

Potent Neutralization of Staphylococcal Enterotoxin B In Vivo by Antibodies that Block Binding to the T-Cell Receptor.

To develop an antibody (Ab) therapeutic against staphylococcal enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a "class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed "class M" Abs. A systematic engineering approach was applied to affinity-mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen-deuterium exchange coupled to mass spectrometry revealed that the class T epitope on SEB overlapped with the T-cell receptor binding site, whereas other evidence suggested that the class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ∼50-fold more potent toxin-neutralizing efficacy than the best class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two class M binding sites and two class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bsAb engineering.

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology.

Fast photochemical oxidation of proteins (FPOP) is a footprinting technique used in mass spectrometry-based structural proteomics. It has been applied to solve a variety of problems in different areas of biology. A FPOP platform requires a laser, optics, and sample flow path properly assembled to enable fast footprinting. Sample preparation, buffer conditions, and reagent concentrations are essential to obtain reasonable oxidations on proteins. FPOP samples can be analyzed by LC-MS methods to measure the modification extent, which is a function of the solvent-accessible surface area of the protein. The platform can be expanded to accommodate several new approaches, including dose-response studies, new footprinting reagents, and two-laser pump-probe experiments. Here, we briefly review FPOP applications and in a detailed manner describe the procedures to set up an FPOP protein footprinting platform.

Burkholderia bacteria use chemotaxis to find social amoeba Dictyostelium discoideum hosts.

A key question in cooperation is how to find the right partners and maintain cooperative relationships. This is especially challenging for horizontally transferred bacterial symbionts where relationships must be repeatedly established anew. In the social amoeba Dictyostelium discoideum farming symbiosis, two species of inedible Burkholderia bacteria (Burkholderia agricolaris and Burkholderia hayleyella) initiate stable associations with naive D. discoideum hosts and cause carriage of additional bacterial species. However, it is not clear how the association between D. discoideum and its carried Burkholderia is formed and maintained. Here, we look at precisely how Burkholderia finds its hosts. We found that both species of Burkholderia clones isolated from D. discoideum, but not other tested Burkholderia species, are attracted to D. discoideum supernatant, showing that the association is not simply the result of haphazard engulfment by the amoebas. The chemotactic responses are affected by both partners. We find evidence that B. hayleyella prefers D. discoideum clones that currently or previously carried Burkholderia, while B. agricolaris does not show this preference. However, we find no evidence of Burkholderia preference for their own host clone or for other hosts of their own species. We further investigate the chemical differences of D. discoideum supernatants that might explain the patterns shown above using a mass spectrometry based metabolomics approach. These results show that these bacterial symbionts are able to preferentially find and to some extent choose their unicellular partners. In addition, this study also suggests that bacteria can actively search for and target phagocytic cells, which may help us better understand how bacteria interact with immune systems.

Laser-Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass-Spectrometry-Based Protein Footprinting.

Described is a novel, laser-initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. . CF3 reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to . OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chemistry and structural biology and biotechnology. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility.

Protein Footprinting by Carbenes on a Fast Photochemical Oxidation of Proteins (FPOP) Platform.

Protein footprinting combined with mass spectrometry provides a method to study protein structures and interactions. To improve further current protein footprinting methods, we adapted the fast photochemical oxidation of proteins (FPOP) platform to utilize carbenes as the footprinting reagent. A Nd-YAG laser provides 355 nm laser for carbene generation in situ from photoleucine as the carbene precursor in a flow system with calmodulin as the test protein. Reversed-phase liquid chromatography coupled with mass spectrometry is appropriate to analyze the modifications produced in this footprinting. By comparing the modification extent of apo and holo calmodulin on the peptide level, we can resolve different structural domains of the protein. Carbene footprinting in a flow system is promising.