Extremely potent human monoclonal antibodies from COVID-19 convalescent patients.

Human monoclonal antibodies are safe, preventive, and therapeutic tools that can be rapidly developed to help restore the massive health and economic disruption caused by the coronavirus disease 2019 (COVID-19) pandemic. By single-cell sorting 4,277 SARS-CoV-2 spike protein-specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor-binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer, and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody-dependent enhancement and prolong half-life, neutralized the authentic wild-type virus and emerging variants containing D614G, E484K, and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc functions.

The Bardet-Biedl syndrome complex component BBS1 controls T cell polarity during immune synapse assembly.

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here, we investigated the potential role of Bardet-Biedl syndrome 1 protein (BBS1), an essential core component of the BBS complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrated that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH1 (also known as WASHC1), a process that we demonstrated to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that functions upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling. This article has an associated First Person interview with the first author of the paper.

In Vivo Efficacy and Toxicity of an Antimicrobial Peptide in a Model of Endotoxin-Induced Pulmonary Inflammation.

SET-M33 is a synthetic peptide that is being developed as a new antibiotic against major Gram-negative bacteria. Here we report two in vivo studies to assess the toxicity and efficacy of the peptide in a murine model of pulmonary inflammation. First, we present the toxicity study in which SET-M33 was administered to CD-1 mice by snout inhalation exposure for 1 h/day for 7 days at doses of 5 and 20 mg/kg/day. The results showed adverse clinical signs and effects on body weight at the higher dose, as well as some treatment-related histopathology findings (lungs and bronchi, nose/turbinates, larynx and tracheal bifurcation). On this basis, the no observable adverse effect level (NOAEL) was considered to be 5 mg/kg/day. We then report an efficacy study of the peptide in an endotoxin (LPS)-induced pulmonary inflammation model. Intratracheal administration of SET-M33 at 0.5, 2 and 5 mg/kg significantly inhibited BAL neutrophil cell counts after an LPS challenge. A significant reduction in pro-inflammatory cytokines, KC, MIP-1α, IP-10, MCP-1 and TNF-α was also recorded after SET-M33 administration.

An antimicrobial molecule mitigates signs of sepsis in vivo and eradicates infections from lung tissue.

The peptide sequence KKIRVRLSA was synthesized in a dimeric structure (SET-M33DIM) and evaluated as a candidate drug for infections due to multidrug-resistant (MDR) Gram-negative pathogens. SET-M33DIM showed significant antibacterial activity against MDR strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli (Minimal Inhibitory Concentration [MICs], 1.5-11 µM), and less activity against Pseudomonas aeruginosa (MICs, 11-22 µM). It showed very low toxicity in vitro, ex vivo, and in vivo; in cytotoxicity tests, its EC50 was as much as 22 times better than that of SET-M33, a peptide with the same amino-acid sequence, but synthesized in tetra-branched form (638 vs 28 µM). In in vivo and ex vivo experiments, SET-M33DIM cleared P. aeruginosa infection, significantly reducing signs of sepsis in animals, and restoring cell viability in lung tissue after bacterial challenge. It also quelled inflammation triggered by LPS and live bacterial cells, inhibiting expression of inflammatory mediators in lung tissue, cultured macrophages, and bronchial cells from a cystic fibrosis patient.

Molecular definition of the interaction between a tumor-specific tetrabranched peptide and LRP6 receptor.

The tumor-specific tetrabranched peptide NT4 binds membrane sulfate glycosaminoglycans and receptors belonging to the low density lipoprotein receptor-related protein (LRP) family, like LRP6, which are overexpressed in cancer. The binding occurs through a multimeric positively-charged motif of NT4 that interacts with negatively charged motives in both glycosaminoglycans and LRP receptors. LRP6 has an essential function in canonical Wnt signaling, acting together with receptors of the Frizzled family as coreceptor for Wnt ligands. The extracellular domain of LRP6 contains four YWTD ß-propellers, which are fundamental for interactions with ligands, such as Wnt and Wnt inhibitors. To investigate the molecular interactions between the NT4 peptide and LRP6 receptor, we synthesized a library of epitope mapping peptides reproducing the YWTD ß-propeller 3 and 4 of LRP6. The peptides that showed to bind NT4 represented the portion of LRP6 located on the top face of ß-propeller 3 and contained negatively charged residues, including glutamic acid-708 which is known to be involved in Wnt3a interaction. The results pave the way for a possible development of peptide inhibitors of Wnt3a pathway to be used as drugs in oncology.

Endocytosis and Trafficking of Heparan Sulfate Proteoglycans in Triple-Negative Breast Cancer Cells Unraveled with a Polycationic Peptide.

The process of heparan sulfate proteoglycan (HSPG) internalization has been described as following different pathways. The tumor-specific branched NT4 peptide has been demonstrated to bind HSPGs on the plasma membrane and to be internalized in tumor cell lines. The polycationic peptide has been also shown to impair migration of different cancer cell lines in 2D and 3D models. Our hypothesis was that HSPG endocytosis could affect two important phenomena of cancer development: cell migration and nourishment. Using NT4 as an experimental tool mimicking heparin-binding ligands, we studied endocytosis and trafficking of HSPGs in a triple-negative human breast cancer cell line, MDA-MB-231. The peptide entered cells employing caveolin- or clathrin-dependent endocytosis and macropinocytosis, in line with what is already known about HSPGs. NT4 then localized in early and late endosomes in a time-dependent manner. The peptide had a negative effect on CDC42-activation triggered by EGF. The effect can be explained if we consider NT4 a competitive inhibitor of EGF on HS that impairs the co-receptor activity of the proteoglycan, reducing EGFR activation. Reduction of the invasive migratory phenotype of MDA-MB-231 induced by NT4 can be ascribed to this effect. RhoA activation was damped by EGF in MDA-MB-231. Indeed, EGF reduced RhoA-GTP and NT4 did not interfere with this receptor-mediated signaling. On the other hand, the peptide alone determined a small but solid reduction in active RhoA in breast cancer cells. This result supports the observation of few other studies, showing direct activation of the GTPase through HSPG, not mediated by EGF/EGFR.

A New NT4 Peptide-Based Drug Delivery System for Cancer Treatment.

The development of selective tumor targeting agents to deliver multiple units of chemotherapy drugs to cancer tissue would improve treatment efficacy and greatly advance progress in cancer therapy. Here we report a new drug delivery system based on a tetrabranched peptide known as NT4, which is a promising cancer theranostic by virtue of its high cancer selectivity. We developed NT4 directly conjugated with one, two, or three units of paclitaxel and an NT4-based nanosystem, using NIR-emitting quantum dots, loaded with the NT4 tumor-targeting agent and conjugated with paclitaxel, to obtain a NT4-QD-PTX nanodevice designed to simultaneously detect and kill tumor cells. The selective binding and in vitro cytotoxicity of NT4-QD-PTX were higher than for unlabeled QD-PTX when tested on the human colon adenocarcinoma cell line HT-29. NT4-QD-PTX tumor-targeted nanoparticles can be considered promising for early tumor detection and for the development of effective treatments combining simultaneous therapy and diagnosis.

NMR Study of the Secondary Structure and Biopharmaceutical Formulation of an Active Branched Antimicrobial Peptide.

The synthetic antimicrobial peptide SET-M33 is being developed as a possible new antibacterial candidate for the treatment of multi-drug resistant bacteria. SET-M33 is a branched peptide featuring higher resistance and bioavailability than its linear analogues. SET-M33 shows antimicrobial activity against different species of multi-resistant Gram-negative bacteria, including clinically isolated strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and Escherichia coli. The secondary structure of this 40 amino acid peptide was investigated by NMR to fully characterize the product in the framework of preclinical studies. The possible presence of helixes or ß-sheets in the structure had to be explored to predict the behavior of the branched peptide in solution, with a view to designing a formulation for parenteral administration. Since the final formulation of SET-M33 will be strictly defined in terms of counter-ions and additives, we also report the studies on a new salt form, SET-M33 chloride, that retains its activity against Gram-negative bacteria and gains in solubility, with a possible improvement in the pharmacokinetic profile. The opportunity of using a chloride counter-ion is very convenient from a process development point of view and did not increase the toxicity of the antimicrobial drug.

Near-infrared quantum dots labelled with a tumor selective tetrabranched peptide for in vivo imaging.

BACKGROUND: Near-infrared quantum dots (NIR QDs) are a new class of fluorescent labels with excellent bioimaging features, such as high fluorescence intensity, good fluorescence stability, sufficient electron density, and strong tissue-penetrating ability. For all such features, NIR QDs have great potential for early cancer diagnosis, in vivo tumor imaging and high resolution electron microscopy studies on cancer cells. RESULTS: In the present study we constructed NIR QDs functionalized with the NT4 cancer-selective tetrabranched peptides (NT4-QDs). We observed specific uptake of NT4-QDs in human cancer cells in in vitro experiments and a much higher selective accumulation and retention of targeted QDs at the tumor site, compared to not targeted QDs, in a colon cancer mouse model. CONCLUSIONS: NIR QDs labelled with the tetrabranched NT4 peptide have very promising performance for selective addressing of tumor cells in vitro and in vivo, proving rising features of NT4-QDs as theranostics.

Immunomodulatory and Anti-inflammatory Activity in Vitro and in Vivo of a Novel Antimicrobial Candidate.

The synthetic antimicrobial peptide SET-M33 has strong activity against bacterial infections caused by Gram-negative bacteria. It is currently in preclinical development as a new drug to treat lung infections caused by Gram-negative bacteria. Here we report its strong anti-inflammatory activity in terms of reduced expression of a number of cytokines, enzymes, and signal transduction factors involved in inflammation triggered by LPS from Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli Sixteen cytokines and other major agents involved in inflammation were analyzed in macrophages and bronchial cells after stimulation with LPS and incubation with SET-M33. The bronchial cells were obtained from a cystic fibrosis patient. A number of these proteins showed up to 100% reduction in expression as measured by RT-PCR, Western blotting, or Luminex technology. LPS neutralization was also demonstrated in vivo by challenging bronchoalveolar lavage of SET-M33-treated mice with LPS, which led to a sharp reduction in TNF-α with respect to non-SET-M33-treated animals. We also describe a strong activity of SET-M33 in stimulating cell migration of keratinocytes in wound healing experiments in vitro, demonstrating a powerful immunomodulatory action generally characteristic of molecules taking part in innate immunity.

Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. pneumon iae and P. aeruginosa.

SET-M33 is a multimeric antimicrobial peptide active against Gram-negative bacteria in vitro and in vivo. Insights into its killing mechanism could elucidate correlations with selectivity. SET-M33 showed concentration-dependent bactericidal activity against colistin-susceptible and resistant isolates of P. aeruginosa and K. pneumoniae. Scanning and transmission microscopy studies showed that SET-M33 generated cell blisters, blebs, membrane stacks and deep craters in K. pneumoniae and P. aeruginosa cells. NMR analysis and CD spectra in the presence of sodium dodecyl sulfate micelles showed a transition from an unstructured state to a stable α-helix, driving the peptide to arrange itself on the surface of micelles. SET-M33 kills Gram-negative bacteria after an initial interaction with bacterial LPS. The molecule becomes then embedded in the outer membrane surface, thereby impairing cell function. This activity of SET-M33, in contrast to other similar antimicrobial peptides such as colistin, does not generate resistant mutants after 24h of exposure, non-specific interactions or toxicity against eukaryotic cell membranes, suggesting that SET-M33 is a promising new option for the treatment of Gram-negative antibiotic-resistant infections.

Synergistic activity profile of an antimicrobial peptide against multidrug-resistant and extensively drug-resistant strains of Gram-negative bacterial pathogens.

Infection sustained by multidrug-resistant and extensively drug-resistant bacterial pathogens is often untreatable with the standard of care antibiotics, and the combination of anti-infective compounds often represents the only therapeutic strategy to face this major clinical treat. SET-M33 is a novel antimicrobial peptide (AMP) that has demonstrated in vitro and in vivo antimicrobial activity against Gram-negative bacteria and has shown interesting features in preclinical evaluations. Particularly, it showed efficacy against a number of multidrug-resistant and extensively drug-resistant clinical strains of Gram-negative pathogens, in in vitro and in vivo assessments. Here, we explored the potential synergistic activity of SET-M33 in combination with different standard of care antibiotics by the checkerboard method against a panel of six strains of Gram-negative pathogens including multidrug-resistant and extensively drug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. SET-M33 showed synergistic activity with antibiotics of different families against these clinically relevant strains. A synergistic effect was observed for SET-M33 in combination with rifampin, meropenem, aztreonam, and tobramycin mostly on K. pneumoniae and A. baumannii strains, while the SET-M33 plus ciprofloxacin combination was additive with all tested strains. Synergy was not apparently linked to the bacterial species or phenotype but was rather strain-specific, highlighting the need for individual strain testing for synergistic antimicrobial combinations. These findings extend current knowledge on synergistic activity of AMPs in combination with conventional agents and support the potential role of SET-M33 as a novel therapeutic agent against antibiotic-resistant Gram-negative pathogens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Exploiting Free-Energy Minima to Design Novel EphA2 Protein-Protein Antagonists: From Simulation to Experiment and Return.

The free-energy surface (FES) of protein-ligand binding contains information useful for drug design. Here we show how to exploit a free-energy minimum of a protein-ligand complex identified by metadynamics simulations to design a new EphA2 antagonist with improved inhibitory potency.

Site-specific pegylation of an antimicrobial peptide increases resistance to Pseudomonas aeruginosa elastase.

M33 is a branched peptide currently under preclinical characterization for the development of a new antibacterial drug against gram-negative bacteria. Here, we report its pegylation at the C-terminus of the three-lysine-branching core and the resulting increase in stability to Pseudomonas aeruginosa elastase. This protease is a virulence factor that acts by destroying peptides of the native immune system. Peptide resistance to this protease is an important feature for M33-Peg activity against Pseudomonas.

A novel phage-library-selected peptide inhibits human TNF-α binding to its receptors.

We report the identification of a new human tumor necrosis factor-alpha (TNF-α) specific peptide selected by competitive panning of a phage library. Competitive elution of phages was obtained using the monoclonal antibody adalimumab, which neutralizes pro-inflammatory processes caused by over-production of TNF-α in vivo, and is used to treat severe symptoms of rheumatoid arthritis. The selected peptide was synthesized in monomeric and branched form and analyzed for binding to TNF-α and competition with adalimumab and TNF-α receptors. Results of competition with TNF-α receptors in surface plasmon resonance and melanoma cells expressing both TNF receptors make the peptide a candidate compound for the development of a novel anti-TNF-α drug.

Nanoparticles exposing neurotensin tumor-specific drivers.

Nanoparticles have attracted much attention for their potential application as in vivo carriers of drugs. Labeling of nanoparticles with bioactive markers that are able to direct them toward specific biological target receptors has led to a new generation of drug delivery systems. In particular, low molecular weight peptides that remain stable in vivo could be promising tools to selectively drive nanoparticles loaded with active components to tumor cells. We reported, recently, that tetrabranched neurotensin peptides (NT4) may be used to selectively target tumor cells with liposomes. Liposomes functionalized with tetrabranched neurotensin peptide, NT4, and loaded with doxorubicin showed clear advantages in cell binding, anthracyclin internalization, and cytotoxicity in respect of not functionalized liposomes. In this study, we compare branched (NT4) versus linear (NT) peptides in the ability to drive liposomes to target cells and deliver their toxic cargo. We showed here that the more densely decorated liposomes had a better activity profile in terms of drug delivery. Presentation of peptides to the cell membranes in the grouped shape provided by branched structure facilitates liposome cell binding and fusion.

Modeling PCDH19 clustering epilepsy by Neurogenin 2 induction of patient-derived induced pluripotent stem cells.

BACKGROUND: Loss of function mutations in PCDH19 gene causes an X-linked, infant-onset clustering epilepsy, associated with intellectual disability and autistic features. The unique pattern of inheritance includes random X-chromosome inactivation, which leads to pathological tissue mosaicism. Females carrying PCDH19 mutations are affected, while males have a normal phenotype. No cure is presently available for this disease. METHODS: Fibroblasts from a female patient carrying frameshift mutation were reprogrammed into human induced pluripotent stem cells (hiPSCs). To create a cell model of PCDH19-clustering epilepsy (PCDH19-CE) where both cell populations co-exist, we created mosaic neurons by mixing wild-type (WT) and mutated (mut) hiPSC clones, and differentiated them into mature neurons with overexpression of the transcriptional factor Neurogenin 2. RESULTS: We generated functional neurons from patient-derived iPSC using a rapid and efficient method of differentiation through overexpression of Neurogenin 2. Was revealed an accelerated maturation and higher arborisation in the mutated neurons, while the mosaic neurons showed the highest frequency of action potential firing and hyperexcitability features, compared to mutated and WT neurons. CONCLUSIONS: Our findings provide evidence that PCDH19 c.2133delG mutation affects proper metaphases with increased numbers of centrosomes in stem cells and accelerates neuronal maturation in premature cells. PCDH19 mosaic neurons showed elevated excitability, representing the situation in PCDH19-CE brain. We suggest Ngn2 hiPSC-derived PCDH19 neurons as an informative experimental tool for understanding the pathogenesis of PCDH19-CE and a suitable approach for use in targeted drug screening strategies.

Efficacy and toxicity of the antimicrobial peptide M33 produced with different counter-ions.

The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015-1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5-30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.

Safety evaluations of a synthetic antimicrobial peptide administered intravenously in rats and dogs.

The antimicrobial peptide SET-M33 is under study for the development of a new antibiotic against major Gram-negative pathogens. Here we report the toxicological evaluation of SET-M33 administered intravenously to rats and dogs. Dose range finding experiments determined the doses to use in toxicokinetic evaluation, clinical biochemistry analysis, necroscopy and in neurological and respiratory measurements. Clinical laboratory investigations in dogs and rats showed a dose-related increase in creatinine and urea levels, indicating that the kidneys are the target organ. This was also confirmed by necroscopy studies of animal tissues, where signs of degeneration and regeneration were found in kidney when SET-M33 was administered at the highest doses in the two animal species. Neurological toxicity measurements by the Irwin method and respiratory function evaluation in rats did not reveal any toxic effect even at the highest dose. Finally, repeated administration of SET-M33 by short infusion in dogs revealed a no-observed-adverse-effect-level of 0.5 mg/kg/day.

Inhalable Polymeric Nanoparticles for Pulmonary Delivery of Antimicrobial Peptide SET-M33: Antibacterial Activity and Toxicity In Vitro and In Vivo.

Development of inhalable formulations for delivering peptides to the conductive airways and shielding their interactions with airway barriers, thus enhancing peptide/bacteria interactions, is an important part of peptide-based drug development for lung applications. Here, we report the construction of a biocompatible nanosystem where the antimicrobial peptide SET-M33 is encapsulated within polymeric nanoparticles of poly(lactide-co-glycolide) (PLGA) conjugated with polyethylene glycol (PEG). This system was conceived for better delivery of the peptide to the lungs by aerosol. The encapsulated peptide showed prolonged antibacterial activity, due to its controlled release, and much lower toxicity than the free molecule. The peptide-based nanosystem killed Pseudomonas aeruginosa in planktonic and sessile forms in a dose-dependent manner, remaining active up to 72 h after application. The encapsulated peptide showed no cytotoxicity when incubated with human bronchial epithelial cells from healthy individuals and from cystic fibrosis patients, unlike the free peptide, which showed an EC50 of about 22 µM. In vivo acute toxicity studies in experimental animals showed that the peptide nanosystem did not cause any appreciable side effects, and confirmed its ability to mitigate the toxic and lethal effects of free SET-M33.