Fab Fragment Of Antibody Research Articles

Investigation of SARS-CoV-2 nucleocapsid protein interaction with a specific antibody by combined spectroscopic ellipsometry and quartz crystal microbalance with dissipation

Detailed evaluations of the antigen and antibody interaction rate and strength of the immune complex formed are very important for medical and bioanalytical applications. These data are crucial for the development of sensitive and fast immunosensors suitable for continuous measurements. Therefore, combined spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) technique (SE/QCM-D) was used for the evaluation: (i)of covalent immobilization of SARS-CoV-2 nucleocapsid protein (SCoV2-N) on QCM-D sensor disc modified by self-assembled monolayer based on 11-mercaptoundecanoic acid and (ii)interaction of immobilized SCoV2-N with specific polyclonal anti-SCoV2-N antibodies followed by immune complex formation process. The results show that the SCoV2-N monolayer is rigid due to the low energy dissipation registered during the QCM-D measurement. In contrast, the anti-SCoV2-N layer produced after interaction with the immobilized SCoV2-N formed a soft and viscous layer. It was determined, that the sparse distribution of SCoV2-N on the surface affected the spatial arrangement of the antibody during the formation of immune complexes. The hinge-mediated flexibility of the antibody Fab fragments allows them to reach the more distantly located SCoV2-N and establish a bivalent binding between proteins in the formed SCoV2-N/anti-SCoV2-N complex. It was noted that the SE/QCM-D method can provide more precise quantitative information about the flexibility and conformational changes of antibody during the formation of the immune complex on the surface over time.

Interactions of epitopic variants of epidermal growth factor receptor with therapeutic anti- EGFR antibodies

Epidermal growth factor receptor (EGFR) plays important roles in cancerous transformation of epithelial cells in many solid cancers. Due to the pivotal role of EGFR in cellular proliferation and metastasis, it is a promising molecular target for the treatment of various cancers. One of the major treatment approaches uses anti-EGFR monoclonal antibodies (mAbs) targeted to the extracellular domain of the receptor to competitively block the binding of its ligands. Cetuximab, necitumumab, nimotuzumab, and panitumumab are such approved mAbs which are commercially available and used to treat multiple types of cancers. The response rates to these expensive therapeutics in various cancers range from nearly 9% to 91%. Hence, the objective of this study was to indentify whether any of the missense single nucleotide polymorphisms (SNPs) in the EGFR gene impart any structural and functional impact on the receptor’s interaction with these antibodies. We used X-ray crystallographic structures (from Protein Data Bank) of the Fab fragments of these therapeutic antibodies in complex with EGFR to analyze the effects of the missense mutations on the antigen-antibody interactions. We also assessed the potential association of the destabilizing variants with pathogenicity and disease susceptibility. EGFR H433Q (rs1171743336), S464T (rs746763556), S492G (rs1057519760) and S492R (rs1057519860) variants appear to weaken interactions between EGFR and cetuximab, which is the most widely used anti-EGFR therapeutic antibody. Other epitopic variants do not appear to affect interactions between EGFR and relevant mAbs (necitumumab, nimotuzumab, and panitumumab). Prior to treatment of the EGFR mediated conditions with cetuximab, screening of variants that destabilize antibody-EGFR interaction may be considered as a companion diagnostic test for avoiding unresponsiveness and improving therapeutic outcomes. Dhaka Univ. J. Biol. Sci. 30(3 CSI): 393-403, 2022 (June)

Uptake-leak balance of SR Ca2+ determines arrhythmogenic potential of RyR2R420Q+/− cardiomyocytes

Mutations of the RyR2 are channelopathies that can predispose to life threatening catecholaminergic polymorphic ventricular tachycardias (CPVTs) during exercise or stress. However, the cellular and molecular mechanisms that are causal for the arrhythmias downstream of the β-adrenergic receptor (β-AR) activation are not defined. They may be specific and different for each particular RyR2 mutation. Obvious possibilities are the phosphorylation of the mutated RyR2s or the stimulation of the SR Ca2+ pump (SERCA), which could increase SR Ca2+ loading. Potentially arrhythmogenic Ca2+ signals, such as Ca2+ waves, were recorded and analyzed from WT and RyR2R420Q+/− mouse cardiomyocytes with confocal microscopy after field stimulation at 1 Hz. In RyR2R420Q+/− cardiomyocytes we found a higher occurrence and frequency of Ca2+ waves, particularly upon β-AR stimulation with isoproterenol. This was accompanied by a shorter latency to the first spontaneous wave. Wave velocity from raw traces, as well as amplitude and decay time constant (τ) analyzed in de-skewed traces were comparable in both cell types. To obtain further insight into the role of the SERCA we selectively stimulated SERCA in permeabilized myocytes using Fab fragments of a PLB antibody (2D12). Surprisingly, SERCA stimulation alone resulted in considerably higher wave frequencies than when mimicking β-AR stimulation with cAMP, particularly in RyR2R420Q+/− cardiomyocytes. This may be a consequence of some protective SR Ca2+ unloading resulting from the SR Ca2+ leak via phosphorylated RyR2s in cAMP. Spark-to-spark recovery analysis suggested a remarkably higher Ca2+ release sensitivity in RyR2R420Q+/− cells, both in control and upon β-AR stimulation. Together these findings suggest that the fine balance between SR Ca2+ loading via SERCA and the Ca2+ leak via mutated and phosphorylated RyR2s is an important determinant for the overall cellular arrhythmogenicity prevailing in the RyR2R420Q+/− myocytes.

Direct Affinity Purification of Long-Acting PASylated Proteins with Therapeutic Potential Using L-Prolinamide for Mild Elution.

Both insufficient plasma half‐life (circulation for only few hours or less) and laborious downstream purification can be bottleneck for biological drug development. We report a novel strategy for the efficient and gentle affinity purification of pharmacologically relevant proteins modified by PASylation for prolonged action in vivo. We previously described antibodies specific for Pro/Ala‐rich sequences (PAS) covering a range of binding characteristics. Our present approach relies on a chromatography matrix functionalized with a low‐affinity PAS‐specific antibody Fab fragment for specific adsorption of the PASylated protein from a macromolecular mixture. With the complete absence of hydrophobic/aromatic or ionic groups in the PAS sequence epitope, binding is mediated by Van der Waals contacts and distinct hydrogen bonds only. Surprisingly, selective competitive elution is achieved by application of the highly soluble and biologically inactive imino acid derivative L‐prolinamide. Based on the specific but strongly dynamic biomolecular interaction, our procedure allows the direct one‐step purification of PASylated proteins from a cell extract or culture supernatant while avoiding harsh elution conditions as they are often needed for conventional affinity chromatography.

Direkte Affinitätsreinigung lang wirksamer PASylierter Proteine mit therapeutischem Anwendungspotential unter Nutzung von L‐Prolinamid zur milden Elution

AbstractSowohl unzureichende Plasma‐Halbwertszeit (Zirkulation für nur wenige Stunden oder kürzer) als auch ein aufwendiger Reinigungsprozeß können Flaschenhals sein für die biologische Wirkstoffentwicklung. Wir berichten über eine neuartige Strategie zur effizienten und schonenden Afinitätsreinigung pharmakologisch relevanter Proteine, die durch PASylierung hinsichtlich verlängerter Aktivität in vivo modifiziert wurden. Zuvor hatten wir Antikörper mit Spezifität für Pro/Ala‐reiche Sequenzen (PAS) beschrieben, die ein Spektrum an Bindungseigenschaften abdecken. Unser Ansatz hier beruht auf einer Chromatographiematrix, die mit einem niedrigaffinen PAS‐spezifischen Fab‐Fragment zum Zweck der spezifischen Adsorption des PASylierten Proteins aus einer makromolekularen Mischung funktionalisiert ist. Aufgrund vollständiger Abwesenheit hydrophober/aromatischer oder ionischer Gruppen in dem PAS‐Sequenzepitop wird die Bindung allein durch Van‐der‐Waals‐Kontakte und charakteristische Wasserstoffbrückenbindungen vermittelt. Überraschenderweise kann die selektive kompetitive Elution durch Anwendung des hochlöslichen und biologisch inaktiven Iminosäurederivats L‐Prolinamid erreicht werden. Basierend auf der spezifischen, aber hochdynamischen biomolekularen Wechselwirkung gestattet unser Verfahren die direkte Einschrittreinigung PASylierter Proteine aus einem Zellextrakt oder Kulturüberstand, wobei grobe Elutionsbedingungen, wie sie häufig für die konventionelle Affinitätschromatographie erforderlich sind, vermieden werden.

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments.

Purpose: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. Experimental design: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts. Results: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts. Conclusions: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.

Safety and Efficacy of Systemic Anti-Scg3 Therapy to Treat Oxygen-Induced Retinopathy.

To circumvent possible systemic side effects, anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) for ocular neovascular diseases in adults are approved only for intravitreal administration. However, intravitreal injection itself can elicit injection-related adverse effects, and premature eyes of infants with retinopathy of prematurity (ROP) may be particularly susceptible to intravitreal injection. Therefore, an unmet clinical need is to develop safe systemic anti-angiogenic therapies for ROP. We recently reported that secretogranin III (Scg3) is a disease-restricted angiogenic factor and that systemic anti-Scg3 mAb alleviates ROP in animal models with minimal side effects on developing eyes and organs. The aim of this study is to investigate the safety and efficacy of a humanized anti-Scg3 antibody via systemic administration. We analyzed the safety and efficacy of a humanized anti-Scg3 antibody Fab fragment (hFab) delivered by intraperitoneal injection in oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP. The results showed that systemic anti-Scg3 hFab effectively alleviated pathological retinal neovascularization in OIR mice with similar efficacy to the anti-VEGF drug aflibercept. Systemic aflibercept conferred significant adverse side effects in neonatal mice, including reduced body weight, abnormalities in retinal and renal development, and retarded physiological neovascularization, whereas systemic anti-Scg3 hFab elicited no such side effects. The findings suggest that systemic anti-Scg3 hFab is a safe and effective therapy for OIR and support further development for ROP treatment.

Structures of Omicron spike complexes and implications for neutralizing antibody development.

The emergence of the SARS-CoV-2 Omicron variant is dominant in many countries worldwide. The high number of spike mutations is responsible for the broad immune evasion from existing vaccines and antibody drugs. To understand this, we first present the cryo-electron microscopy structure of ACE2-bound SARS-CoV-2 Omicron spike. Comparison to previous spike antibody structures explains how Omicron escapes these therapeutics. Secondly, we report structures of Omicron, Delta, and wild-type spikes bound to a patient-derived Fab antibody fragment (510A5), which provides direct evidence where antibody binding is greatly attenuated by the Omicron mutations, freeing spike to bind ACE2. Together with biochemical binding and 510A5 neutralization assays, our work establishes principles of binding required for neutralization and clearly illustrates how the mutations lead to antibody evasion yet retain strong ACE2 interactions. Structural information on spike with both bound and unbound antibodies collectively elucidates potential strategies for generation of therapeutic antibodies.

Generation of a Porcine Antibody Fab Fragment Using Protein Engineering to Facilitate the Evaluation of Ocular Sustained Delivery Technology.

Treatment of age-related macular degeneration (AMD) with anti-vascular endothelial growth factor (VEGF) biologic agents has been shown to restore and maintain visual acuity for many patients afflicted with wet AMD. These agents are usually administered via intravitreal injection at a dosing interval of 4-8 weeks. Employment of long-acting delivery (LAD) technologies could improve the therapeutic outcome, ensure timely treatment, and reduce burden on patients, caregivers, and the health care system. Development of LAD approaches requires thorough testing in pre-clinical species; however, therapeutic proteins of human origin may not be well tolerated during testing in non-human species due to immunogenicity. Here, we have engineered a surrogate porcine antibody Fab fragment (pigG6.31) from a human antibody for testing ocular LAD technologies in a porcine model. The engineered Fab retains the VEGF-A-binding and inhibition properties of the parental human Fab and has stability properties suitable for LAD evaluation. Upon intravitreal injection in minipigs, pigG6.31 showed first-order clearance from the ocular compartments with vitreal elimination rates consistent with other molecules of this size. Application of the surrogate molecule in an in vivo evaluation in minipigs of a prototype of the port delivery (PD) platform indicated continuous ocular delivery from the implant, with release kinetics consistent with both the results from in vitro release studies and the efficacy observed in human clinical studies of the PD system with ranibizumab (PDS). Anti-drug antibodies in the serum against pigG6.31 were not detected over exposure durations up to 16 weeks, suggesting that this molecule has low porcine immunogenicity.

Architecture and antigenicity of the Nipah virus attachment glycoprotein

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness. The entry of HNVs into host cells requires the attachment (G) and fusion (F) glycoproteins, which are the main targets of antibody responses. To understand viral infection and host immunity, we determined a cryo-electron microscopy structure of the NiV G homotetrameric ectodomain in complex with the nAH1.3 broadly neutralizing antibody Fab fragment. We show that a cocktail of two nonoverlapping G-specific antibodies neutralizes NiV and HeV synergistically and limits the emergence of escape mutants. Analysis of polyclonal serum antibody responses elicited by vaccination of macaques with NiV G indicates that the receptor binding head domain is immunodominant. These results pave the way for implementing multipronged therapeutic strategies against these deadly pathogens.

Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase.

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo–enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo–enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels–Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar KD values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts.

Crystal structures of human neuropeptide Y (NPY) and peptide YY (PYY)

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) form the evolutionarily conserved pancreatic polypeptide family. While the fold is widely utilized in nature, crystal structures remain elusive, particularly for the human forms, with only the structure of a distant avian form of PP reported. Here we utilize a crystallization chaperone (antibody Fab fragment), specifically recognizing the amidated peptide termini, to solve the structures of human NPY and human PYY. Intriguingly, and despite limited sequence identity (~50%), the structure of human PYY closely resembles that of avian PP, highlighting the broad structural conservation of the fold throughout evolution. Specifically, the PYY structure is characterized by a C-terminal amidated α-helix, preceded by a backfolded poly-proline N-terminus, with the termini in close proximity to each other. In contrast, in the structure of human NPY the N-terminal component is disordered, while the helical component of the peptide is observed in a four-helix bundle type arrangement, consistent with a propensity for multimerization suggested by NMR studies.

Comparative ligandomics implicates secretogranin III as a disease-restricted angiogenic factor in laser-induced choroidal neovascularization.

Choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. All approved anti-angiogenic drug therapies for CNV target vascular endothelial growth factor (VEGF) but confer limited efficacy. Identification of other CNV-related angiogenic factors will facilitate the development of VEGF-independent alternative therapies. Here, we applied comparative ligandomics to live mice with or without laser-induced CNV for global mapping of CNV-selective endothelial ligands. Secretogranin III (Scg3) previously identified by the same approach as a diabetes-restricted angiogenic factor was mapped with a more than 935-fold increase in binding to CNV vessels compared to healthy choriocapillaris. A novel in vivo ligand binding assay independently confirmed a marked increase in Scg3 binding to CNV vessels, whereas VEGF showed no increase in CNV-selective binding. A new technique of functional immunohistochemistry allowed the visualization and confirmed the increase in in vivo Scg3 binding to CNV vasculatures, including CNV microcapillaries with detailed vascular structures, which was blocked by anti-Scg3 humanized antibody Fab fragment (hFab). The hFab effectively alleviated laser-induced CNV with an efficacy similar to the anti-VEGF drug aflibercept. Homozygous deletion of the Scg3 gene in mice significantly reduced the severity of CNV. Furthermore, the therapeutic activity of anti-Scg3 hFab, but not aflibercept, was abolished in Scg3-/- mice, suggesting the Scg3-dependent nature of the hFab-mediated therapy. These findings suggest that Scg3 plays an important role in CNV pathogenesis and is a promising disease-restricted angiogenic factor for ligand-guided disease-targeted anti-angiogenic therapy of CNV.

Aerosol Biologics for the Treatment of Eosinophilic Asthma

Asthma is a heterogeneous chronic airway disease comprising of distinct phenotypes characterized by different immunopathophysiologic pathways, clinical features, disease severity, and response to treatment. The phenotypes of asthma include eosinophilic, neutrophilic, mixed cellularity, and paucigranulocytic asthma. Eosinophilic asthma is principally a T helper type 2 (Th2)-mediated airway disease. However, several other immune and structural cells secrete the cytokines implicated in the pathogenesis of eosinophilic asthma. Innate type 2 lymphoid cells, mast cells, basophils, and eosinophils secrete Th2 cytokines, such as interleukin-4 (IL-4), IL-13, and IL-5. Additionally, airway epithelial cells produce alarmin cytokines, including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). Alarmins are the key initiators of allergic inflammation at the sentinel mucosal surfaces. Innovative biotherapeutic research has led to the discovery of monoclonal antibodies which target and inhibit the immunopathological effects of the cytokines involved in the pathogenesis of eosinophilic asthma. Parenteral biologics targeting the inciting interleukins, include mepolizumab and reslizumab (anti-IL-5), benralizumab (anti-IL-5Rα), dupilumab (anti-4Rα), and tezelizumab (anti-TSLP). They have been shown to significantly reduce annualized exacerbation rates, improve asthma control, lung function, and quality of life. Currently, there are no pulmonary delivered aerosol biologics for topical treatment of asthma. CSJ117 is a potent neutralizing antibody Fab fragment against TSLP, formulated as a PulmoSol TM engineered powder, and is delivered to the lungs by a dry powder inhaler. Phase 2 placebo-controlled clinical trial evaluated the efficacy and safety of CSJ117. CSJ117 delivered as an inhaler attenuated the late asthmatic response (LAR), and the early asthmatic response (EAR) after allergen inhalation challenge (AIC) at day 84 of treatment. The maximum decrease in FVE1 from pre-AIC were significantly lower in the CSJ117 group compared to placebo (P = 029), during LAR. CSJ117 also significantly reduced fractional exhaled nitric oxide before AIC at day 83; and significantly reduced the allergen-induced increase in % sputum eosinophil count. Pulmonary delivery of biologics directly to the airway mucosal surface has several advantages over parenteral routes, particularly in treating airway diseases such as asthma. Inhaler delivered biologics, such as CSJ117 are innovative and attractive methods of future precision treatment of asthma, and other respiratory diseases.

Tryptophan association in water driven by charge-transfer interactions with electron-deficient aromatic haptens.

The ability of a series of electron-deficient aromatic compounds to form charge-transfer complexes with tryptophan in water has been evaluated by X-ray diffraction studies, UV-vis spectra and NMR. As dinitrophenyl (DNP) ligands are well-known to generate antibody-mediated responses and the π-π stacking interactions with tryptophan residues of the antibody Fab fragment have been reported, most of the aromatic receptors studied here are nitro derivatives. Charge-transfer interactions between the rich indole ring of tryptophan and the electron-deficient aromatic receptors have been observed in the solid state, as four crystal structures of the complexes were obtained. The aromatic donor-acceptor interactions in solution were also verified by UV-vis and NMR spectroscopy. The association of the tripeptide Trp-Gly-Trp, a motif found in antigen Ag43, with the electron-deficient aromatic diimide was also studied by UV-vis and NMR spectroscopy. Our results show that these simple electron-deficient molecules could potentially behave as novel haptens and be incorporated in more elaborated drugs targeting protein-protein interactions, due to the synergistic effect of multiple non-covalent interactions.

Safety, Tolerability, and Clinical Pharmacology of ANX009, an Inhibitory Antibody Fab Fragment Against C1q, Administered Subcutaneously to Healthy Volunteers

Certain autoantibodies that bind to tissue antigens or that deposit in tissues as a component of immune complexes can activate the classical complement cascade, leading to inflammation and tissue damage. As the initiating molecule of the classical complement cascade, C1q is an attractive target for preventing complement activation and its multiple tissue-damaging effects. ANX009 is an antigen binding fragment (Fab) of a humanized antibody against C1q that inhibits C1q substrate interactions and fully blocks activation of all downstream classical complement components. While inhibiting the classical cascade, ANX009 leaves the lectin and alternative complement pathways intact for their normal immune functions. ANX009 is formulated for subcutaneous (SC) administration and is designed for treatment of blood-based and vascular antibody-mediated autoimmune diseases, such as autoimmune hemolytic anemia (AIHA) and lupus nephritis, where complement activation is a key component of disease pathology.A phase 1 first-in-human single ascending dose (SAD) and multiple ascending dose (MAD) study of ANX009 with subcutaneous administration was conducted in 48 healthy volunteers (NCT04535752). Four SAD cohorts were enrolled followed by two MAD cohorts evaluating daily dosing for 7 days or twice weekly dosing x 4 doses. Each cohort had eight participants randomized in a 6:2 active:placebo ratio. Safety and tolerability were assessed, along with serum pharmacokinetics (unbound drug), pharmacodynamics (unbound C1q target), and an ex vivo measure of C1q activity (CH 50 hemolysis of antibody-sensitized sheep red blood cells).All dose levels were well-tolerated. No drug-related safety signals, dose-limiting toxicities, serious adverse events, or adverse events leading to discontinuations were observed. Mild, transient, local injection site reactions were observed.A clear dose-response relationship was observed in SAD cohorts. Negligible reduction in free C1q was observed in the two lowest dose cohorts. A maximum mean reduction in free C1q of 80% was observed at 48 hours post-dose at the third dose level, and full reduction of free C1q through 72 hours was observed at the highest dose level. Similarly, full reduction of free C1q was observed in the MAD cohort with daily dosing as well as in the second MAD cohort with twice weekly dosing. Full reduction of C1q was maintained for 4 days following the last dose in the second MAD cohort. Ex vivo functional activity of C1q was completely inhibited in close correspondence with free C1q levels.Combined safety, tolerability, and clinical pharmacology results from this phase 1 study support advancement of ANX009 to studies in patients with complement-mediated autoimmune disorders. DisclosuresGrover: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Teigler: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Radomile: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Rose: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Yednock: Annexon Inc: Current Employment, Current equity holder in publicly-traded company. Keswani: Annexon Inc: Current Employment, Current equity holder in publicly-traded company.

Crystal structure of the Tspan15 LEL domain reveals a conserved ADAM10 binding site

Tetraspanins are four-pass transmembrane proteins that function by regulating trafficking of partner proteins and organizing signaling complexes in the membrane. Tspan15, one of a six-member TspanC8 subfamily, forms a complex that regulates the trafficking, maturation, and substrate selectivity of the transmembrane protease ADAM10, an essential enzyme in mammalian physiology that cleaves a wide variety of membrane-anchored substrates, including Notch receptors, amyloid precursor protein, cadherins, and growth factors. We present here crystal structures of the Tspan15 large extracellular loop (LEL) required for functional association with ADAM10 both in isolation and in complex with the Fab fragment of an anti-Tspan15 antibody. Comparison of the Tspan15 LEL with other tetraspanin LEL structures shows that a core helical framework buttresses a variable region that structurally diverges among LELs. Using co-immunoprecipitation and a cellular N-cadherin cleavage assay, we identify a site on Tspan15 required for both ADAM10 binding and promoting substrate cleavage.

Elucidating the Structural and Minimal Protective Epitope of the Serogroup X Meningococcal Capsular Polysaccharide.

Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the in silico docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide via hydrogen bonding, salt bridges, and hydrophobic interactions. In vivo studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design.

GPVI inhibition by glenzocimab synergistically inhibits atherosclerotic plaque-induced platelet activation when combined with conventional dual antiplatelet therapy

Abstract Introduction Aspirin and a potent platelet P2Y12 inhibitor, such as prasugrel or ticagrelor, are not always sufficient to prevent thrombus formation in patients with ST-elevation MI (STEMI), leading to “slow flow” or “no reflow” effects after stenting. GPIIb/IIIa inhibitors, such as eptifibatide, may help in this setting, but are not used routinely due to their bleeding risk. GPVI has critical roles in thrombosis and a minimal role in haemostasis. Here we tested whether depletion of GPVI has effects on thrombus formation after MI in an animal model and investigated the effects of a novel platelet GPVI inhibitor, glenzocimab (a Fab fragment of a monoclonal antibody), on platelet activation and thrombus formation when combined with aspirin and ticagrelor. Methods We used intravital microscopy in a murine model of ST-elevation myocardial infarction and ischaemia-reperfusion injury to investigate microvascular thrombosis. We investigated the antithrombotic effects of adding glenzocimab (previously known as ACT017) to blood from healthy donors and 20 patients with ACS treated with aspirin and ticagrelor. We compared the effect of glenzocimab with the GPIIb/IIIa inhibitor eptifibatide ex-vivo. We stimulated platelets with collagen and atherosclerotic plaque material that was sourced from patients undergoing carotid endarterectomy. We investigated effects on platelet aggregation, spreading, signalling, adhesion, thrombin generation, thrombus formation and clot stability ex vivo. Results Genetic depletion of GPVI in an animal model of myocardial infarction reduced microvascular thrombosis. Ex vivo, aspirin and ticagrelor partially inhibited atherosclerotic plaque-induced platelet aggregation (assessed by multiple electrode aggregometry) by 48% compared to control (34±3 vs. 65±4 U; P<0.001; Figure 1). Atherosclerotic plaque-induced platelet aggregation, adhesion, secretion and activation were critically dependent on platelet GPVI activation and were potently inhibited by glenzocimab. Glenzocimab alone reduced atherosclerotic plaque-induced platelet aggregation by 75% compared to control (16±4 vs. 65±4 U; P<0.001; Figure 1) and by over 95% when combined with aspirin and ticagrelor (3±1 vs 65±4 U; P<0.001; Figure 1). Furthermore, glenzocimab provided multiple synergistic antithrombotic effects when added to the blood of aspirin and ticagrelor-treated patients with ACS ex vivo. Glenzocimab and the GPIIb/IIIa inhibitor, eptifibatide, had many similar antithrombotic effects but glenzocimab had less effect on mechanisms of general haemostasis compared to eptifibatide, as assessed by ROTEM (Figure 2). Conclusions The addition of glenzocimab to aspirin and ticagrelor provides synergistic inhibition of multiple critical mechanisms of atherothrombosis. Glenzocimab and the GPIIb/IIIa inhibitor, eptifibatide, share many similar antithrombotic effects, although glenzocimab has less impact on mechanisms involved in haemostasis compared to eptifibatide. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Academy of Medical Sciences UK Clinical Lecturer Starter GrantRoyal Embassy of Saudi Arabia

Pharmacological aspects of acute treatment glycoside intoxication

Despite the active introduction of surgical treatments for cardiac pathology and new synthetic cardiotonic drugs, cardiac glycosides (CG) remain an important component of the pharmacological treatment of heart diseases. Moreover, interest in this group of drugs is only increasing with the discovery of new pharmacological effects, such as antitumor and antiviral. However, despite many years of experience, the issue of managing patients with symptoms of both acute and chronic poisoning with cardiac glycosides remains one of the leading ones. The literature review provides a brief historical outline of cardiac glycoside poisoning and comprehensively describes the pharmacokinetics and pharmacodynamics of this group of drugs. The clinical picture of intoxication is described. We analyzed modern ideas and limitations of using antidote therapy such as digoxin immune Fab-antibody fragments, as well as the features of pathogenetic and symptomatic treatment of patients with acute glycoside poisoning.