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.

In previous papers, we observed that dendrimers of peptide mimotopes of the nicotinic receptor ligand site are strong antidotes against the lethality of the nicotinic receptor ligand ␣-bungarotoxin. Although their in vitro activity is identical to that of dendrimers, the corresponding monomeric peptide mimotopes are not effective in vivo. Because the higher in vivo efficiency of dendrimers could not in this case be related to polyvalent interaction, the stability to blood protease activity of monomeric versus tetrabranched dendrimeric mimotope peptides was compared here by incubating three different mimotopes with human plasma and serum. Unmodified peptides and cleaved sequences were followed by high pressure liquid chromatography and mass spectrometry. Tetrabranched peptides were shown to be much more stable in plasma and also in serum. To assess the notable stability of multimeric peptides, different bioactive neuropeptides, including enkephalins, neurotensin and nociceptin, were synthesized in monomeric and tetrabranched forms and incubated with human plasma and serum and with rat brain membrane extracts. All the tetrabranched neuropeptides fully retained biological activity and generally showed much greater stability to blood and brain protease activity. Some tetrabranched peptides were also resistant to trypsin and chymotrypsin. Our findings provide new insights into the possible therapeutic use of bioactive peptides.Hundreds of peptides with potential therapeutic activities have been identified. These include naturally occurring peptide hormones and neurotransmitters, which influence and control series of vital functions, such as cell proliferation, tissue development, metabolism, immune defense, perception of pain, reproduction, behavior, and blood pressure. Selective agonists or antagonists of these natural peptides are extremely useful for the investigation of peptidergic systems and are also potential therapeutic agents (1). Moreover, several peptide fragments or mimotopes derived from potential therapeutic proteins show promising biological activity (2).However, the use of peptides as therapeutic drugs has largely been limited by their short half-life in vivo. Because peptides are mainly broken down by proteases and peptidases, peptide delivery is the bottleneck in the development of new peptide drugs. To increase peptide half-life, many strategies involving different levels of chemical modification are possible (3, 4). The introduction of D-amino acids, or pseudo amino acids, and peptide cyclization are the most common strategies to increase peptide stability. However, these modifications may profoundly alter peptide activity. Alternatively, peptidomimetic molecules can be developed by the synthesis of conformationally restricted compounds, in which the peptide is locally or globally constrained in order to reproduce the active conformation. The resulting structures are mostly non-peptide molecules, more resistant to degrading enzymes.In general, peptide molecules have the advantage of good specifici...

Long-pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 comprises a pentraxin-like C-terminal domain involved in complement activation via C1q interaction and an N-terminal extension with unknown functions. PTX3 binds fibroblast growth factor-2 (FGF2), inhibiting its pro-angiogenic and pro-restenotic activity. Here, retroviral transduced endothelial cells (ECs) overexpressing the N-terminal fragment PTX3-(1-178) showed reduced mitogenic activity in response to FGF2. Accordingly, purified recombinant PTX3-(1-178) binds FGF2, prevents PTX3/FGF2 interaction, and inhibits FGF2 mitogenic activity in ECs. Also, the monoclonal antibody mAb-MNB4, which recognizes the PTX3-(87-99) epitope, prevents FGF2/PTX3 interaction and abolishes the FGF2 antagonist activity of PTX3. Consistently, the synthetic peptides PTX3-(82-110) and PTX3-(97-110) bind FGF2 and inhibit the interaction of FGF2 with PTX3 immobilized to a BIAcore sensor chip, FGF2-dependent EC proliferation, and angiogenesis in vivo. Thus, the data identify a FGF2-binding domain in the N-terminal extension of PTX3 spanning the PTX3-(97-110) region, pointing to a novel function for the N-terminal extension of PTX3 and underlining the complexity of the PTX3 molecule for modular humoral pattern recognition.Pentraxins are a superfamily of proteins characterized by a pentameric structure (1). The classical short-pentraxins C-reactive protein and serum amyloid P component are acute phase proteins in man and mouse, respectively (2). Pentraxins bind various ligands and are involved in the innate resistance to microbes and scavenging of cellular debris and extracellular matrix components (1, 3).Long-pentraxins are characterized by an unrelated N-terminal domain coupled to a pentraxin-like C-terminal domain (4). The prototypic long-pentraxin PTX3 (5, 6) is a 45-kDa glycosylated protein produced locally by mononuclear phagocytes, dendritic cells, and endothelial cells (ECs) 2 in response to primary inflammatory signals (7). Studies in ptx3 Ϫ/Ϫ mice have shown that PTX3 plays complex non-redundant functions, ranging from the assembly of a hyaluronic acid-rich extracellular matrix and female fertility to innate immunity against diverse microorganisms (8,9). This is related, at least in part, to the capacity of PTX3 to bind the complement component C1q, the extracellular matrix protein TSG6, and selected microorganisms, activating complement activation and facilitating pathogen recognition by macrophages and dendritic cells (1, 10). Thus, PTX3 is a soluble pattern recognition receptor with unique non-redundant functions in various pathophysiological conditions (1, 10).Fibroblast growth factor-2 (FGF2) is a potent angiogenic growth factor (11). FGF2 modulates neovascularization during wound healing, inflammation, atherosclerosis, and tumor growth (12). Several molecules sequester FGF2 in the extracellular environment and inhibit its angiogenic activity (reviewed in Ref. 12). Many of these inhibitors...

Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exerted a strong candidacidal activity in vitro, with a 50% inhibitory concentration of 0.056 M, and was therefore designated killer peptide (KP). Its activity was neutralized by laminarin, a ␤1-3 glucan molecule, but not by pustulan, a ␤1-6 glucan molecule. KP also competed with the binding of a KT-like monoclonal IdAb to germinating cells of the fungus. In a rat model of vaginal candidiasis, local, postchallenge administration of KP was efficacious in rapidly abating infections caused by fluconazole-susceptible or -resistant C. albicans strains. In systemic infection of BALB/c or SCID mice preinfected intravenously with a lethal fungal load, KP caused a highly significant prolongation of the median survival time, with >80% of the animals still surviving after >60 days, whereas >90% of control mice died within 3 to 5 days. KP is therefore the first engineered peptide derived from a recombinant IdAb retaining KT microbicidal activity, probably through the interaction with the ␤-glucan KT receptor on target microbial cells.

A large 10-mer phage peptide library was panned against whole Escherichia coli cells, and an antimicrobial peptide (QEKIRVRLSA) was selected. The peptide was synthesized in monomeric and dendrimeric tetrabranched form (multiple antigen peptide [MAP]), which generally allows a dramatic increase of peptide stability to peptidases and proteases. The antibacterial activity of the dendrimeric peptide against E. coli was much higher than that of the monomeric form. Modification of the original sequence, by residue substitution or sequence shortening, produced three different MAPs, M4 (QAKIRVRLSA), M5 (KIRVRLSA), and M6 (QKKIRVRLSA) with enhanced stability to natural degradation and antimicrobial activity against a large panel of gram-negative bacteria. The MICs of the most potent peptide, M6, were as low as 4 to 8 g/ml against recent clinical isolates of multidrug-resistant Pseudomonas aeruginosa and members of the Enterobacteriaceae. The same dendrimeric peptides showed high stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells, making them promising candidates for the development of new antibacterial drugs.

A synthetic antimicrobial peptide was identified as a possible candidate for the development of a new antibacterial drug. The peptide, SET-M33L, showed a MIC90 below 1.5 μM and 3 μM for Pseudomonas aeruginosa and Klebsiella pneumoniae, respectively. In in vivo models of P. aeruginosa infections, the peptide and its pegylated form (SET-M33L-PEG) enabled a survival percentage of 60–80% in sepsis and lung infections when injected twice i.v. at 5 mg/Kg, and completely healed skin infections when administered topically. Plasma clearance showed different kinetics for SET-M33L and SET-M33L-PEG, the latter having greater persistence two hours after injection. Bio-distribution in organs did not show significant differences in uptake of the two peptides. Unlike colistin, SET-M33L did not select resistant mutants in bacterial cultures and also proved non genotoxic and to have much lower in vivo toxicity than antimicrobial peptides already used in clinical practice. The characterizations reported here are part of a preclinical development plan that should bring the molecule to clinical trial in the next few years.

In human brain the flavoprotein D-amino acid oxidase (hDAAO) is responsible for the degradation of the neuromodulator D-serine, an important effector of NMDA-receptor mediated neurotransmission. Experimental evidence supports the concept that D-serine concentration increase by hDAAO inhibition may represent a valuable therapeutic approach to improve the symptoms in schizophrenia patients. This study investigated the effects on hDAAO conformation and stability of the substrate D-serine (or of the pseudo-substrate trifluoro-D-alanine), the FAD cofactor, and two inhibitors (benzoate, a classical substrate-competitive inhibitor and the drug chlorpromazine (CPZ), which competes with the cofactor). We demonstrated that all these compounds do not alter the interaction of hDAAO with its physiological partner pLG72. The ligands used affect the tertiary structure of hDAAO differently: benzoate or trifluoro-D-alanine binding increases the amount of the holoenzyme form in solution and stabilizes the flavoprotein, while CPZ binding favors a protein conformation resembling that of the apoprotein, which is more sensitive to degradation. Interestingly, the apoprotein form of hDAAO binds the substrate D-serine: this interaction increases FAD binding thus increasing the amount of active holoenzyme in solution. Benzoate and CPZ similarly modify the short-term cellular D-serine concentration but affect the cellular concentration of hDAAO differently. In conclusion, the different alteration of hDAAO conformation and stability by the ligands used represents a further parameter to take into consideration during the development of new drugs to cope schizophrenia.

We found that synthetic peptides in the form of dendrimers become resistant to proteolysis. To determine the molecular basis of this resistance, different bioactive peptides were synthesized in monomeric, two-branched and tetra-branched form and incubated with human plasma and serum. Proteolytic resistance of branched multimeric sequences was compared to that of the same peptides synthesized as multimeric linear molecules. Unmodified peptides and cleaved sequences were detected by high pressure liquid chromatography and mass spectrometry. An increase in peptide copies did not increase peptide resistance in linear multimeric sequences, whereas multimericity progressively enhanced proteolytic stability of branched multimeric peptides. A structure-based hypothesis of branched peptide resistance to proteolysis by metallopeptidases is presented.