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Plasma-derived proteins are a subset of relevant biotherapeutics also known as “well-characterized biologicals”. They are enriched from plasma through several steps of physical and biochemical methodologies, reaching the regulatory accepted standards of safety, levels of impurities, activity and lot-to-lot consistency. Final products accepted for commercialization are submitted to tight analytical, functional and safety controls by a number of different approaches that fulfill the requirements of sensitivity and reliability. We report here the use of a multianalytical approach for the comparative evaluation of different lots of Factor IX isolated from plasma preparations and submitted or not to a step of nanofiltration. The approach include, among the other, proteomic techniques based on both MALDI-TOF and LC-MS Orbitrap mass spectrometry, circular dichroism for structural characterization, chromatographic and electrophoretic techniques, ELISA and functional assays based on clotting activity and binding to known anticoagulants. Comparative data obtained on two sets of nanofiltered and non-nanofiltered lots with different final activity show that the products have substantially overlapping profiles in terms of activity, contaminants, structural properties and protein content, suggesting that the proposed multianalytical approach is robust enough to be used for the routine validation of clinical lots.

The synthesis of the O-3 triazole-linked galactosyl arylsulfonamides 1–7 as potential inhibitors of Trypanosoma cruzi cell invasion is described. These target compounds were synthesized by Cu(I)-catalysed azide-alkyne cycloaddition reaction (‘click chemistry’) between different azide arylsulfonamides and the alkyne-based sugar 3-O-propynyl-βGalOMe. Inhibition assays of T. cruzi cell invasion with compounds 1–7 showed reduced values of infection index (∼20) for compounds 3 and 5, bearing the corresponding 5-methylisoxazole and 2,4-dimethoxypyrimidine groups, which also presented higher binding affinities to galectin-3 (EC50 17–18 μM) in Corning Epic label-free assays. In agreement with experimental results, the assessment of the theoretical binding of compounds 1–7 to galectin-3 by MM/PBSA method displayed higher affinities for compounds 3 (−9.7 kcal/mol) and 5 (−11.1 kcal/mol). Overall, these achievements highlight compounds 3 and 5 as potential T. cruzi cell invasion blockers by means of a galectin-3 binding-related mechanism, revealing galectin-3 as an important host target for design of novel anti-trypanosomal agents.

Using a combined approach based on MS, enzyme digestion and advanced MD studies we have determined the sequential order of formation of the three disulfide bridges of the Cripto-1 CFC domain. The domain has a rare pattern of bridges and is involved in the recognition of several receptors. The bridge formation order is C1-C4, C3-C5, C2-C6, however formation of C1-C4 plays no roles for the formation of the others. Folding is driven by formation of the C3-C5 bridge and is supported by residues lying within the segment delimited by these cysteines. We indeed observe that variants CFC-W123A and CFC-ΔC1/C4, where C1 and C4 are replaced by serines, are able to refold in the same time window as the wild type, while CFC-K132A and CFC-W134A are not. A variant where cysteines of the second and third bridge are mutated to serine, convert slowly to the monocyclic molecule. Data altogether support a mechanism whereby the Cripto-1 CFC domain refolds by virtue of long-range intramolecular interactions that involve residues close to cysteines of the second and third bridge. These findings are supported by the in silico study that shows how distant parts of the molecules come into contact on a long time scale. Using a combined approach based on MS, enzyme digestion and advanced MD studies we have determined the sequential order of formation of the three disulfide bridges of the Cripto-1 CFC domain. The domain has a rare pattern of bridges and is involved in the recognition of several receptors. The bridge formation order is C1-C4, C3-05, C2-C6, however formation of C1-C4 plays no roles for the formation of the others. Folding is driven by formation of the C3-05 bridge and is supported by residues lying within the segment delimited by these cysteines. We indeed observe that variants CFC-W123A and CFC-Delta C1/C4, where C1 and C4 are replaced by serines, are able to refold in the same time window as the wild type, while CFC-K132A and CFC-W134A are not. A variant where cysteines of the second and third bridge are mutated to serine, convert slowly to the monocyclic molecule. Data altogether support a mechanism whereby the Cripto-1 CFC domain refolds by virtue of long-range intramolecular interactions that involve residues close to cysteines of the second and third bridge. These findings are supported by the in silica study that shows how distant parts of the molecules come into contact on a long time scale. (C) 2019 Elsevier B.V. All rights reserved.

The placental growth factor (PlGF), a member of VEGF family, plays a crucial role in pathological angiogenesis, especially ischemia, inflammation, and cancer. This activity is mediated by its selective binding to VEGF receptor 1 (VEGFR-1), which occurs predominantly through receptor domains 2 and 3. The PlGF β-hairpin region spanning residues Q87 to V100 is one of the key binding elements on the protein side. We have undertaken a study on the design, preparation, and functional characterization of the peptide reproducing this region and of a set of analogues where glycine 94, occurring at the corner of the hairpin in the native protein, is replaced by charged as well as hydrophobic residues. Also, some peptides with arginine 96 replaced by other residues have been studied. We find that the parent peptide weakly binds VEGFR-1, but replacement of G94 with residues bearing H-bond donating residues significantly improves the affinity. Replacement of R96 instead blocks the interaction between the peptide and the domain. The strongest affinity is observed with the G94H (peptide PlGF-2) and G94W (peptide PlGF-10) mutants, while the peptide PlGF-8, bearing the R96G mutation, is totally inactive. The PlGF-1 and PlGF-2 peptides also bind the VEGFR-2 receptors, though with a reduced affinity, and are able to interfere with the VEGF-induced receptor signaling on endothelial cells. The peptides also bind VEGFR-2 on the surface of cells, while PlGF-8 is inactive. Data suggest that these peptides have potential applications as PlGF/VEGF mimic in various experimental settings.

Human Cripto-1 (Cripto-1), the founding member of the EGF-CFC superfamily, is a key regulator of many processes during embryonic development and oncogenesis. Cripto-1 is barely present or even absent in normal adult tissues while it is aberrantly re-expressed in various tumors. Blockade of the CFC domain-mediated Cripto-1 functions is acknowledged as a promising therapeutic intervention point to inhibit the tumorigenic activity of the protein. In this work, we report the generation and characterization of murine monoclonal antibodies raised against the synthetic folded CFC [112−150] domain of the human protein. Through subtractive ELISA assays clones were screened for the ability to specifically recognize “hot spot” residues on the CFC domain, which are crucial for the interaction with Activin Type I receptor (ALK4) and GRP78. On selected antibodies, SPR and epitope mapping studies have confirmed their specificity and have revealed that recognition occurs only on a conformational epitope. Furthermore, FACS analyses have confirmed the ability of 1B4 antibody to recognize the membrane-anchored and soluble native Cripto-1 protein in a panel of human cancer cells. Finally, we have evaluated its functional effects through in vitro cellular signaling assays and cell cycle analysis. These findings suggest that the selected anti-CFC mAbs have the potential to neutralize the protein oncogenic activity and may be used as theranostic molecules suitable as tumor homing agents for Cripto-1-overexpressing cancer cells and tissues and to overcome drug-resistance in routine cancer therapies.

Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them 'mini-factors'. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different specificity and affinity VEGF receptors but none blocks receptor activity. Following derivatization with suitable tracers they have been employed as molecular probes for sensing receptors on cell surface without affecting their activity as is usually observed with other binders having neutralizing activity.

Nodal is a potent embryonic morphogen belonging to the TGF-beta superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44-67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (KD 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.

Nodal, a member of the TGF-! superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF-! s, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co-receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4and ALK7) as well as with Cripto, as obtained by homologymodelingand dockingsimulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44‐67 of the Nodal protein, corresponding to the prehelix loop and the H3 helix, and reproduce the wild-type sequence or bear some modifications to evaluate the hot-spot role of modified residues in the receptor binding. Here,weshowthestructuralcharacterizationinsolutionbyCDandNMRoftheNodalpeptidesandthemeasurementofbinding affinity toward Cripto by surface plasmon resonance. Data collected by both conformational analyses and binding measurements suggest a role for Y58 of Nodal in the recognition with Cripto and confirm that previously reported for E49 and E50. Surface plasmon resonance binding assays with recombinant proteins show that Nodal interacts in vitro also with ALK7 and ALK4 and preliminary data, generated using the Nodal synthetic fragments, suggest that Y58 of Nodal may also be involved in the recognition with these protein partners. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site.

Nodal is a growth factor expressed during early embryonic development, but reactivated in several advanced-stage cancers. Targeting of Nodal signaling, which occurs via the binding to Cripto-1 co-receptor, results in inhibition of cell aggressiveness and reduced tumor growth. The Nodal binding region to Cripto-1 was identified and targeted with a high affinity monoclonal antibody (3D1). By STD-NMR technique, we investigated the interaction of Nodal fragments with 3D1 with the aim to elucidate at atomic level the interaction surface. Data indicate with high accuracy the antibody-antigen contact atoms and confirm the information previously obtained by immune-enzymatic methods. Main residues contacted by 3D1 are P46, V47, E49 and E50, which belong to the Nodal loop involved in the interaction with the co-receptor.

PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.

Nodal, a member of the TGF-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF-βs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co-receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homology modeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44-67 of the Nodal protein, corresponding to the pre-helix loop and the H3 helix, and reproduce the wild-type sequence or bear some modifications to evaluate the hot-spot role of modified residues in the receptor binding. Here, we show the structural characterization in solution by CD and NMR of the Nodal peptides and the measurement of binding affinity toward Cripto by surface plasmon resonance. Data collected by both conformational analyses and binding measurements suggest a role for Y58 of Nodal in the recognition with Cripto and confirm that previously reported for E49 and E50. Surface plasmon resonance binding assays with recombinant proteins show that Nodal interacts in vitro also with ALK7 and ALK4 and preliminary data, generated using the Nodal synthetic fragments, suggest that Y58 of Nodal may also be involved in the recognition with these protein partners.

Intravenous immunoglobulin are important bio-therapeutics used in the replacement therapy for primary and secondary immunodeficiencies, chronic inflammatory disorders and several autoimmune haematologic disorders. Currently, a number of immunoglobulin intravenous (IVIG) products have been approved by the Food and Drug Administration (FDA) and are available commercially. It is known that small differences in the manufacturing processes as well as in the formulations may affect their clinical efficacy and tolerability. Therefore, given the complexity of the multi-step process required for the isolation of IVIG from human plasma, it is necessary to ensure a rigorous quality control of final products. We show here that a set of different bioanalytical techniques can be conveniently used to comparatively characterize, at a quantitative and qualitative level, different lots of IVIG preparations and to unveil randomly occurring impurities which can also affect the overall product stability. We have used circular dichroism, surface plasmon resonance and two-dimensional electrophoresis (2DE), and have demonstrated that this combination of bioanalytical approaches is very useful to improve the quality control of antibodies and to monitor the reliability of the IVIG manufacturing process. © 2013 Springer Science+Business Media New York.