Publications by authors named "Giancarlo Tonon"

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E.

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Microbial transglutaminases (MTGs) catalyzes the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and in biotechnological applications for bioconjugation reactions. In view of its practical utility, a comparative study of the catalytic activity and stability of the enzyme in a wide range of denaturing conditions has been performed through Circular Dichroism (CD), fluorescence and activity assays performed with model substrates. In agreement with previous results, we show that MTG has a significant structural and functional tolerance to pH changes, whereas the enzyme stability and activity decrease in presence of increasing amounts of denaturing agents, such as urea and guanidinium chloride (GdnHCl).

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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.

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Most GastroIntestinal Stromal Tumors (GISTs) are characterized by KIT gene overexpression, which in turn is regulated by levels of microRNA 221 and microRNA 222. GISTs can also be distinguished by their miRNAs expression profile in which miRNAs 221/222 result reduced in comparison with GI normal tissues. In this paper, to restore normal miRNAs levels and to improve the silencing performances of miRNAs 221/222, new miRNA mimics in which guide strands are modified by Phosphorothioate (PS) and/or 2'-O-methyl RNA (2'-OMe) inside and outside the seed region, were synthesized and tested in GIST48 cells.

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Background: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter.

Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ1-8PNP1-20 or LacZ1-8 fusion protein under different promoters.

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Background And Objectives: The new filgrastim formulation, BK0023, whose synthesis method is patented, was tested in a phase I clinical study that was aimed at investigating the pharmacodynamic and pharmacokinetic equivalence and the safety of BK0023 in healthy male subjects.

Methods: Single and multiple escalating doses were administered to healthy male volunteers according to a double-blind, randomised, two-way crossover design. Thirty-two subjects received subcutaneous filgrastim 2.

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Transglutaminases catalyze transglutamination reactions on glutamines. Transglutaminases are largely exploited for modifying proteins in pharmaceutical, food, and other biotechnological applications. A library of synthetic peptides has been designed, prepared, and screened to identify new peptide substrates.

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Background: Filgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34+) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E.

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The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation.

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We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH(2)-terminal sequence and peptide mapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim.

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PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane self-assembling polymers have been synthesised by the end-functionalisation of 5, 10 and 20 kDa linear amino-terminating monomethoxy-poly(ethylene glycol) (PEG-NH(2)) with 5β-cholanic acid. Spectroscopic studies and isothermal titration calorimetry showed that the CMC of the PEG-cholane derivatives increased from 23.5 ± 1.

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Human glucagon-like peptide-1 (GLP-1) is a physiological gastrointestinal peptide with glucose-dependent insulinotropic effects which is therefore considered an interesting antidiabetic agent. However, after in vivo administration, exogenous GLP-1 does not exert its physiological action due to the combination of rapid proteolytic degradation by ubiquitous dipeptidyldipeptidase IV (DPP IV) enzyme and renal clearance resulting in an extremely short circulating half-life. In this work we describe the conjugation of GLP-1-(7-36)-amide derivatives with polyethylene glycol (PEG) by enzymatic site-specific transglutamination reaction as an approach to reduce both the proteolysis and the renal clearance rates.

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Therapeutic vaccination against idiotype is a promising strategy for immunotherapy of B-cell malignancies. We have previously shown that CDR3-based DNA immunization can induce immune response against lymphoma and explored this strategy to provide protection in a murine B-cell lymphoma model. Here we performed vaccination employing as immunogen a naked DNA fusion product.

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After more than 15 years of experimentation, DNA vaccines have become a promising perspective for tumour diseases, and animal models are widely used to study the biological features of human cancer progression and to test the efficacy of vaccination protocols. In recent years, immunisation with naked plasmid DNA encoding tumour-associated antigens or tumour-specific antigens has revealed a number of advantages: antigen-specific DNA vaccination stimulates both cellular and humoral immune responses; multiple or multi-gene vectors encoding several antigens/determinants and immune-modulatory molecules can be delivered as single administration; DNA vaccination does not induce autoimmune disease in normal animals; DNA vaccines based on plasmid vectors can be produced and tested rapidly and economically. However, DNA vaccines have shown low immunogenicity when tested in human clinical trials, and compared with traditional vaccines, they induce weak immune responses.

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Poly(ethylene glycol) (PEG) is a widely used polymer employed to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. PEG attaches to free amines, typically at lysine residues or at the N-terminal amino acid. This lack of selectivity can present problems when a PEGylated protein therapeutic is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval.

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A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation was synthesized according to a three-step procedure: (1) linear 20 kDa PEG-NH(2) was conjugated to 12-(Boc-amino)dodecanoic acid; (2) PEG-NHCO(CH(2))(11)-Boc was deprotected by TFA treatment; (3) PEG-NHCO(CH(2))(11)-NH(2) was conjugated to 6-maleimidohexanoic acid to yield PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal (PEG-C(18)-Mal). The chemical intermediates as well as the final product were purified by solvent precipitation/extraction and characterized by (1)H NMR spectroscopy and colorimetric analysis. The synthesis procedure yielded over 90% activated product [PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal/PEG-NH(2) molar ratio, %].

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A secreted, soluble variant of the Kex-1 endopeptidase from Kluyveromyces lactis has been produced and studied as a novel cleavage enzyme exhibiting high specificity for the Lys-Arg peptide. This highly selective, efficient enzyme is particularly adapted for use in manufacturing when a recombinant therapeutic protein, possessing its native N-terminus, has to be released in vitro from a bacterially-expressed fusion protein. In this paper, we describe the preparation of a Kex-1 variant using Saccharomyces cerevisiae and its application in the production of important therapeutic recombinant proteins such as human growth hormone, granulocyte colony-stimulating factor and interferon-alpha-2b.

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The idiotypic structure present on B-cell neoplasms is a tumor-specific antigen and an attractive target for immunotherapy. Here, the tumor protective effects recruited by CDR3-based DNA vaccines in the poorly immunogenic, highly aggressive 38C13 murine B-cell lymphoma model were evaluated. The regions belonging to the idiotypic V(H) and V(L) CDR3 sequences were chosen for the design of two synthetic mini-genes and arranged in high-level expression plasmids.

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The protein delivery properties of polymer supramolecular assemblies were investigated by using recombinant human growth hormone (rh-GH) and two polyhydroxyethylaspartamide (PHEA) derivatives: (a) PHEA-C16 obtained by PHEA random grafting with hexadecylalkylamine; (b) PHEA-PEG 5000-C16 obtained by PHEA random co-grafting with hexadecylalkylamine and 5 kDa poly(ethylene glycol). The two polymers possessed similar self-assembling properties: critical micelle concentration (CMC) and particle size. The protein loading (protein/polymer, w/w, %) was 12.

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Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts.

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The expression of recombinant human growth hormone (h-GH) and human interferon-alpha-2b (IFN-alpha-2b) in E. coli leads to the formation of insoluble protein aggregates or inclusion bodies (IBs). The secondary structure of these IBs, their corresponding native forms and thermal aggregates were studied by Fourier Transform Infrared (FT-IR) spectroscopy and microspectroscopy.

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The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. The X-ray structure of E. coli UP resolved by two different groups produced conflicting results.

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Two recombinant Escherichia coli strains expressing different levels of an interferon fusion protein as inclusion bodies have been studied by Fourier transform infrared (FT-IR) microspectroscopy. A marker band at 1628 cm(-1) allowed monitoring of the protein expression by direct analysis of cell pellets in a rapid, non-invasive and quantitative way. The results demonstrate that FT-IR microspectroscopy is a technique of potential biotechnological interest for studying inclusion body formation.

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