Characterization of Odorants in a Commercial Culinary Sage (Salvia officinalis L.) and Several Cultivars.

Culinary sage, Salvia officinalis L., is a popular spice plant commonly used throughout the world. In this study, 35 odorants were identified in dried sage via solvent-assisted flavor evaporation (SAFE) and aroma extract dilution analysis (AEDA), including 9 that were identified in sage for the first time. Fifteen odorants were quantitated by stable isotope dilution analysis (SIDA), and their odor activity values (OAVs) were determined. Odorants with high OAVs included (2E,6Z)-nona-2,6-dienal, 1,8-cineole, and ß-myrcene. A formulated aroma simulation model closely matched the aroma profile of an aqueous infusion of dried sage. Enantiomeric proportions of selected odorants were determined by chiral gas chromatography. Furthermore, 6 different sage cultivars were grown in the greenhouse, dried under the same conditions, and analyzed. Sensory analysis determined that all cultivars were dominated by an herbaceous sensory attribute and had varying intensities of eucalyptus, mint, clove, pine, green, earthy, floral, and citrus notes. Cultivars with varying intensities of herbaceous, eucalyptus, pine, and green sensory notes correlated with the OAVs of α-thujone/ß-thujone, 1,8-cineole, α-pinene, and (2E,6Z)-nona-2,6-dienal, respectively. This study identified the odorants driving the sensory profiles of different sage cultivars and serves as a foundation for future studies on the aroma chemistry of culinary sage.

Modulation of Brain-Derived Neurotrophic Factor (BDNF) Signaling Pathway by Culinary Sage (Salvia officinalis L.).

Culinary sage (Salvia officinalis L.) is a common spice plant in the mint family (Lamiaceae) well known for its distinctive culinary and traditional medicinal uses. Sage tea has been used traditionally as a brain-enhancing tonic and extracts from sage have been reported to have both cognitive and memory enhancing effects. Brain-derived neurotrophic factor (BDNF) is an endogenous signaling molecule involved in cognition and memory function. In this study, activity-guided fractionation employing preparative reverse-phase high performance liquid chromatography (RP-HPLC) of culinary sage extracts led to the discovery of benzyl 6-O-ß-D-apiofuranosyl-ß-D-glucoside (B6AG) as a natural product that upregulates transcription of neurotrophic factors in C6 glioma cells. Purified B6AG showed a moderate dose response, with upregulation of BDNF and with EC50 at 6.46 µM. To better understand the natural variation in culinary sage, B6AG was quantitated in the leaves of several commercial varieties by liquid chromatography-mass spectrometry (LC-MS). The level of B6AG in dried culinary sage was found to range from 334 ± 14 to 698 ± 65 µg/g. This study provided a foundation for future investigations, including quantitative inquiries on the distribution of B6AG within the different plant organs, explorations in optimizing post-harvest practices, and aid in the development of sage varieties with elevated levels of B6AG.

Potent Killing of Pseudomonas aeruginosa by an Antibody-Antibiotic Conjugate.

Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.

Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.

Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.

Massive antibody discovery used to probe structure-function relationships of the essential outer membrane protein LptD.

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.

A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies.

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

Monoclonal antibody targeting the ß-barrel assembly machine of Escherichia coli is bactericidal.

The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

Automated high throughput microscale antibody purification workflows for accelerating antibody discovery.

To rapidly find "best-in-class" antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification.

Molecular characterization of human anti-hinge antibodies derived from single-cell cloning of normal human B cells.

Anti-hinge antibodies (AHAs) are an autoantibody subclass that, following proteolytic cleavage, recognize cryptic epitopes exposed in the hinge regions of immunoglobulins (Igs) and do not bind to the intact Ig counterpart. AHAs have been postulated to exacerbate chronic inflammatory disorders such as inflammatory bowel disease and rheumatoid arthritis. On the other hand, AHAs may protect against invasive microbial pathogens and cancer. However, despite more than 50 years of study, the origin and specific B cell compartments that express AHAs remain elusive. Recent research on serum AHAs suggests that they arise during an active immune response, in contrast to previous proposals that they derive from the preexisting immune repertoire in the absence of antigenic stimuli. We report here the isolation and characterization of AHAs from memory B cells, although anti-hinge-reactive B cells were also detected in the naive B cell compartment. IgG AHAs cloned from a single human donor exhibited restricted specificity for protease-cleaved F(ab')2 fragments and did not bind the intact IgG counterpart. The cloned IgG-specific AHA-variable regions were mutated from germ line-derived sequences and displayed a high sequence variability, confirming that these AHAs underwent class-switch recombination and somatic hypermutation. Consistent with previous studies of serum AHAs, several of these clones recognized a linear, peptide-like epitope, but one clone was unique in recognizing a conformational epitope. All cloned AHAs could restore immune effector functions to proteolytically generated F(ab')2 fragments. Our results confirm that a diverse set of epitope-specific AHAs can be isolated from a single human donor.

Corrigendum: A broadly protective therapeutic antibody against influenza B virus with two mechanisms of action.

This corrects the article DOI: 10.1038/ncomms14234.

A broadly protective therapeutic antibody against influenza B virus with two mechanisms of action.

Influenza B virus (IBV) causes annual influenza epidemics around the world. Here we use an in vivo plasmablast enrichment technique to isolate a human monoclonal antibody, 46B8 that neutralizes all IBVs tested in vitro and protects mice against lethal challenge of all IBVs tested when administered 72 h post infection. 46B8 demonstrates a superior therapeutic benefit over Tamiflu and has an additive antiviral effect in combination with Tamiflu. 46B8 binds to a conserved epitope in the vestigial esterase domain of hemagglutinin (HA) and blocks HA-mediated membrane fusion. After passage of the B/Brisbane/60/2008 virus in the presence of 46B8, we isolated three resistant clones, all harbouring the same mutation (Ser301Phe) in HA that abolishes 46B8 binding to HA at low pH. Interestingly, 46B8 is still able to protect mice against lethal challenge of the mutant viruses, possibly owing to its ability to mediate antibody-dependent cellular cytotoxicity (ADCC).

In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells.

The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/beige mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.

Development and preclinical characterization of a humanized antibody targeting CXCL12.

PURPOSE: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities. EXPERIMENTAL DESIGN: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities. RESULTS: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12ß, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. CONCLUSION: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.

An in vivo human-plasmablast enrichment technique allows rapid identification of therapeutic influenza A antibodies.

Recent advances enabling the cloning of human immunoglobulin G genes have proven effective for discovering monoclonal antibodies with therapeutic potential. However, these antibody-discovery methods are often arduous and identify only a few candidates from numerous antibody-secreting plasma cells or plasmablasts. We describe an in vivo enrichment technique that identifies broadly neutralizing human antibodies with high frequency. For this technique, human peripheral blood mononuclear cells from vaccinated donors are activated and enriched in an antigen-specific manner for the production of numerous antigen-specific plasmablasts. Using this technology, we identified four broadly neutralizing influenza A antibodies by screening only 840 human antibodies. Two of these antibodies neutralize every influenza A human isolate tested and perform better than the current anti-influenza A therapeutic, oseltamivir, in treating severe influenza infection in mice and ferrets. Furthermore, these antibodies elicit robust in vivo synergism when combined with oseltamivir, thus highlighting treatment strategies that could benefit influenza-infected patients.

Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4 (IL-4) and interleukin-13 (IL-13) cytokines.

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Identification of IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn: comparison of soluble receptor-based and cell-based binding assays.

Clinical response to the anti-CD20 antibody rituximab has been demonstrated to correlate with the polymorphism in the FcγRIIIa receptor where patients homozygous for the higher affinity V158 allotype showed a better response rate. This finding suggests that engineering of anti-CD20 for increased FcγRIIIa affinity could result in improved clinical outcome. To identify variants with increased affinity to FcγRIIIa, we developed quantitative assays using soluble receptors as well as engineered cell lines expressing FcγRI or FcγRIIIa on the cell surface. We assayed a set of anti-CD20 IgG(1) variants that had identical Fab regions, but alterations in the Fc regions, in both the soluble receptor-based and cell-based FcγRIIIa binding assays. We obtained similar relative binding affinity increases and assay precisions. The increase in affinity for FcγRIIIa correlated with the increase in activity in the antibody-dependent cellular cytotoxicity assay. These variants had unaltered FcγRI binding. In addition to Fcγ receptors, IgG also binds to FcRn, the receptor responsible for the long circulating half-life of IgG. The mutations in the anti-CD20 variants were previously found not to affect FcRn binding in the soluble receptor-based assays; consequently, we used anti-Her2 variants with different binding affinities to FcRn to study FcRn binding assays. We generated a cell line expressing FcRn on the cell surface to measure IgG binding and obtained similar ranking of these anti-Her2 variants in the cell-based and the soluble receptor-based FcRn binding assays. In conclusion, both the soluble receptor-based and cell-based binding assays can be used to identify IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn.

Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling.

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor FcepsilonRI on mast cells and basophils by cross-linking FcepsilonRI with the inhibitory receptor FcgammaRIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma.

Cutting edge: novel human dendritic cell- and monocyte-attracting chemokine-like protein identified by fold recognition methods.

Chemokines play an important role in the immune system by regulating cell trafficking in homeostasis and inflammation. In this study, we report the identification and characterization of a novel cytokine-like protein, DMC (dendritic cell and monocyte chemokine-like protein), which attracts dendritic cells and monocytes. The key to the identification of this putative new chemokine was the application of threading techniques to its uncharacterized sequence. Based on our studies, DMC is predicted to have an IL-8-like chemokine fold and to be structurally and functionally related to CXCL8 and CXCL14. Consistent with our predictions, DMC induces migration of monocytes and immature dendritic cells. Expression studies show that DMC is constitutively expressed in lung, suggesting a potential role for DMC in recruiting monocytes and dendritic cells from blood into lung parenchyma.