Biochemical and biological characterization of a new oxidized avidin with enhanced tissue binding properties.

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff's base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected (111)In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.

The angiogenic inhibitor long pentraxin PTX3 forms an asymmetric octamer with two binding sites for FGF2.

The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.

Efficacy of PTX3 in a rat model of invasive aspergillosis.

Pentraxin 3 (PTX3) is an acute-phase glycoprotein with a nonredundant function in the host resistance to Aspergillus fumigatus. PTX3 activity was evaluated against pulmonary aspergillosis in rats immunosuppressed with cortisone acetate. PTX3 enhanced the survival rate and reduced the lung fungal burden of infected rats in both therapeutic and prophylactic modalities. Thus, we extended the protective activity of PTX3 in pulmonary aspergillosis to corticosteroid-induced immunodeficiency, which is a relevant clinical condition in graft-versus-host disease and in solid organ transplant.

Simplified ß-amyloid peptides for safer Alzheimer's vaccines development.

Over the past few years, new ways of fighting Alzheimer's disease have emerged based on stimulating the immunitary defence system of the patients. To avoid toxicity and autoimmune response related to the Aß[1-42] peptide immunotherapy, in the last decade a large number of works aimed at identifying new classes of safe Aß derivatives by modifying the full length ß-amyloid form. In strict agreement with the purposes of the sequence-simplification technology, Aß[1-16], Aß[13-28] and Aß[25-42] fragments were selected in order to retain the major immunogenic sites of the Aß[1-42] peptide, and corresponding simplified forms were designed and synthesized. All glycinated Aß derivatives showed immunogenic and antigenic properties similar to the parent Aß[1-42] peptide, and raised antibodies were all able to cross-recognize both Aß[1-42] and Aß[1-40] synthetic structures. All Aß simplified forms showed reduced fibrillogenic and inflammatory properties. In particular, the Aß[13-28]+G form failed to induce IFN-γ production thus suggesting that this molecule could represent a good candidate for potentially safer AD vaccine therapy.

OXavidin for tissue targeting biotinylated therapeutics.

Avidin is a glycoprotein from hen egg white that binds biotin with very high affinity. Here we describe OXavidin, a product containing aldehyde groups, obtained by ligand-assisted sugar oxidation of avidin by sodium periodate. OXavidin chemically reacts with cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks while preserving the biotin binding capacity. The long tissue residence of OXavidin as well as that of OXavidin/biotinylated agent complex occurs in normal and neoplastic tissues and immunohistochemistry shows a strong and homogenous stromal localization. Once localized in tissue/tumor, OXavidin becomes an "artificial receptor" for intravenous injected biotin allowing tumor targeting with biotinylated therapeutics like radioisotopes or toxins. Moreover, present data also suggest that OXavidin might be useful for the homing of biotinylated cells. Overall, OXavidin exhibits a remarkable potential for many different therapeutic applications.

Anti-allergic properties of a new all-D synthetic immunoglobulin-binding peptide.

Using a combinatorial chemistry approach, we identified a tetrameric tripeptide, denoted Protein A Mimetic (PAM) or TG19318, able to bind to immunoglobulins of different classes and species. The inverso variant, with the tripeptide in the all-D configuration (D-PAM or TG19320), is described as retaining binding properties to Ig. This peptide has now been assayed as a binder for E class immunoglobulins, in linear and competitive ELISA experiments, dot-blot and surface plasmon resonance (SPR) analyses. We show that D-PAM binds IgE with high specificity and selectivity, the interaction being sufficient to inhibit anaphylactic release of beta-hexosaminidase from RBL 2H3 cells, with an IC50 value of 10 microg/mL. Intradermal administration of D-PAM suppresses PCA in the rat, with an IC50 of 1.25 microg/kg dose of peptide, while its intraperitoneal injection inhibits mouse PCA with an IC50 of about 7 mg/kg and an efficacy comparable to that of ketotifen. Similarly, D-PAM inhibits ACA in the mouse, with 50% suppression at 10 mg/kg. The results presented here show that the peptide is active on the studied models, with effective doses below toxicity level, hence the molecule is a promising candidate for development of a new class of anti-allergic drugs.

Affinity purification of IgG monoclonal antibodies using the D-PAM synthetic ligand: chromatographic comparison with protein A and thermodynamic investigation of the D-PAM/IgG interaction.

This study investigates the applicability of D-PAM, the inverso form of the Protein A Mimetic synthetic peptide affinity ligand (PAM) obtained from the screening of a multimeric combinatorial peptide library, in monoclonal IgG isolation from ascitic fluids and cellular supernatants. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture monoclonal antibodies in a single chromatographic step, with a recovery yield and purity degree above 90% and full recovery of antibody activity. D-PAM/Emphaze resin showed a host cell protein (HCP) and DNA reduction similar to protein A sorbent. Indeed, column capacity, determined by applying a large excess of purified antibodies to 1 mL of column bed volume, was always higher than 50 mg/mL. D-PAM/IgG interaction was characterized by isothermal titration calorimetry (ITC) and an analysis of binding isotherms, obtained for titration of ST2146, ST1485 and 7H3 IgG monoclonal antibodies, suggested that two peptides bind simultaneously to the IgG molecule, with a K(A) (equilibrium association constant) of 3.4, 6.2 and 3.4 x 10(4) M(-1), and a DeltaH (change in enthalpy) of -1.3, -4.2 and -4.1 kcal mol(-1), respectively.

HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein.

BACKGROUND: The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN) (IN inhibitors, IINs) has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp) thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA) derivatives active on the HIV-1 IN strand transfer (ST) step and with EC50 ranging from 1.83 to >50 mum in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR) reversing ability. RESULTS: The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells. CONCLUSION: To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.

Low and high tenascin-expressing tumors are efficiently targeted by ST2146 monoclonal antibody.

ST2146biot is a biotinylated anti-tenascin monoclonal antibody (mAb) to be used for Pretargeted Antibody Guided Radioimmunotherapy (PAGRIT) of solid tumors. In vivo biodistribution studies of (125)I-labeled ST2146biot were done in nude mice transplanted with human HT-29 colon carcinoma and/or human U-118MG glioblastoma cells characterized for low and high tenascin expression, respectively. In vitro results show that ST2146 retains immunoreactivity upon biotinylation, in contrast to other anti-tenascin mAbs. In vivo biodistribution of ST2146 shows specific tumor accumulation up to 10 days after the i.v. injection, with no relevant differences between biotinylated and nonbiotinylated ST2146. A dose of 4 microg/mouse saturates the low tenascin-expressing human colon carcinoma HT-29, whereas the high tenascin-expressing human glioblastoma U-118MG seems to be saturated at a ST2146biot dose between 320 and 640 microg/mouse. The percentage of injected dose per gram of tumor ranges from 10% to 30%, corresponding to an amount of ST2146biot/g of tumor of approximately 400 ng/g and >200 microg/g for HT-29 and U-118MG, respectively. Tumor to normal organs uptake ratios are between 15 and 60, confirming high tumor selectivity of ST2146biot despite its cross-reactivity with the tenascin expressed at low level in the normal mouse organs. The ST2146biot localization data are substantially confirmed even when both low and high tenascin-expressing tumors are implanted in the same animal. To our knowledge, the absolute amount of ST2146biot, specifically localized in xenotransplanted human tumors, is the highest thus far described and supports the clinical use of this mAb in PAGRIT(R).

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library.

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.

Sequence-simplification and chimeric assembly: new models of peptide antigen modification.

Sequence-simplified variants of a 15-mer peptide antigen, identified by amino acid side chains in alternating positions were synthesized introducing glycine residues alternatively in the parent peptide sequence and used to induce antibodies in rabbit. They reacted to a significant extent with anti-parent peptide antibodies, and in addition, affinity purified antibodies against these halved forms recognized with similar affinity and specificity, the starting peptide in affinity chromatography, optical biosensor and enzyme linked immunosorbent assay (ELISA) experiments, while no cross-reactivity was detected between reduced antigens. These findings suggest that a peptide antigen can display two molecular surfaces of recognition, identified by side chains of residues in alternating positions. Each surface can even take part in antigen/antibody interaction independently, thus indicating the possibility to select and assembly sequence-simplified forms belonging to different epitopes, also deriving from different molecules, to generate new structures incorporating a two-fold antigen/antibody specificity. Two "chimeric" forms were then synthesized starting from the P15 and P13 complementary peptides, both able to bind interleukin 2. These structures, showing simultaneously trans-surfaces of recognition belonging to both parent forms, have been found to retain antigenic properties against antibodies of simplified P15 derivatives showing the same molecular surface of recognition. In addition, anti-chimeric antibodies recognized both P15 and P13 starting peptides, while no cross-antibody recognition was observed between chimeric antigens.

Affinity purification of polyclonal antibodies using a new all-D synthetic peptide ligand: comparison with protein A and protein G.

This study investigates the applicability of the protein A Mimetic (PAM) affinity ligand, obtained from the screening of a multimeric combinatorial peptide library, in immunoglobulin isolation from serum. To avoid protease degradation, the ligand has been substituted by its inverso form, named D-PAM, synthesized by replacing all amino acids with the corresponding D derivatives. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture IgG directly from the serum in a single chromatographic step, with a recovery yield ranging from 60% to 90%, a purity degree higher than 90%, and with a full recovery of antibody activity. Column capacity, determined by applying a large excess of purified IgG to 1 ml bed volume column, was close to 52 mg/ml for bovine IgG, 58 mg/ml for goat IgG, 66 mg/ml for horse IgG, 50 mg/ml for human IgG, 52 mg/ml for mouse IgG, 36 mg/ml for rabbit IgG and 48 mg/ml for sheep IgG. D-PAM peptide was found to be very stable to protease activity, and after prolonged incubation with mouse serum. Similarly, the corresponding derivatized matrix tested before and after various treatments, including sanitization and autoclaving procedures maintained its IgG binding properties, thus indicating a very high stability in terms of ligand leakage and degradation.

Chemical linkage to injected tissues is a distinctive property of oxidized avidin.

We recently reported that the oxidized avidin, named AvidinOX®, resides for weeks within injected tissues as a consequence of the formation of Schiff's bases between its aldehyde groups and tissue protein amino groups. We also showed, in a mouse pre-clinical model, the usefulness of AvidinOX for the delivery of radiolabeled biotin to inoperable tumors. Taking into account that AvidinOX is the first oxidized glycoprotein known to chemically link to injected tissues, we tested in the mouse a panel of additional oxidized glycoproteins, with the aim of investigating the phenomenon. We produced oxidized ovalbumin and mannosylated streptavidin which share with avidin glycosylation pattern and tetrameric structure, respectively and found that neither of them linked significantly to cells in vitro nor to injected tissues in vivo, despite the presence of functional aldehyde groups. The study, extended to additional oxidized glycoproteins, showed that the in vivo chemical conjugation is a distinctive property of the oxidized avidin. Relevance of the high cationic charge of avidin into the stable linkage of AvidinOX to tissues is demonstrated as the oxidized acetylated avidin lost the property. Plasmon resonance on matrix proteins and cellular impedance analyses showed in vitro that avidin exhibits a peculiar interaction with proteins and cells that allows the formation of highly stable Schiff's bases, after oxidation.

Biotin derivatives carrying two chelating DOTA units. Synthesis, in vitro evaluation of biotinidases resistance, avidin binding, and radiolabeling tests.

The synthesis of four biotin derivatives carrying two DOTA moieties for each ligand (BisDOTA set) is reported, for increasing radiation/dose ratio and improving efficiency in the pretargeted avidin-biotin radioimmunotherapy. The biotin-containing scaffold of two BisDOTA was similar to the mono-DOTA derivative previously described. Then the scaffold was elongated by trifunctionalized spacers of different length and conjugated with one of the COOH groups of two DOTA. Two others were prepared starting from a on-resin lysine residue. The lysine alpha-NH2 was bonded to biotin, and then spacers were appended to the epsilon-NH2 and conjugated with two DOTA molecules. One compound contained a p-aminobenzoic acid spacer, which ensured higher head-to-tail distance and increased rigidity of the chain. These last two compounds had a very high ability to bond avidin and were labeled with 90Y at high specific activity. All the compounds were resistant to the action of serum biotinidases.

Immunogenic, antigenic, fibrillogenic and inflammatory properties of new simplified beta-amyloid peptides.

The most promising approach in Alzheimer disease immunotherapy is represented by amyloid beta derivatives with low intrinsic neurotoxicity and minimal overall T cell responses. To avoid toxicity and autoimmune response, we have designed a new class of Abeta derivatives through segmentation of the original Abeta[1-42] peptide and application of the glycine substitution modification technology. Abeta[1-16], Abeta[13-28] and Abeta[25-42] fragments were selected in order to retain the major immunogenic sites of the Abeta[1-42] peptide. All peptides showed comparable immunogenicity, and raised antibodies were all able to cross-recognize both Abeta[1-42] and Abeta[1-40] synthetic amyloid forms. Polyclonal antibodies produced against the simplified variants were able to recognize the parent peptide, but not the opposite simplified forms, in strict agreement with the model of independent surfaces of recognition. All Abeta simplified derivatives showed reduced fibrillogenic properties, thus underlining that the introduction of glycine residues in alternating positions allows to obtain modified peptides maintaining the main immunogenic properties of the parent peptides, but with reduced ability to adopt a beta-sheet conformation and therefore a much lower risk of toxicity in humans. In addition, in vitro studies on peripheral blood mononuclear cells (PBMCs) from healthy donors showed that only the Abeta[13-28]+G peptide failed to induce IFN-gamma production, thus suggesting that this molecule could represent a good candidate for potentially safer vaccine therapy to reduce amyloid burden in Alzheimer's disease instead of using toxic Abeta[1-42].

Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization.

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells.

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.

A new ligand for immunoglobulin g subdomains by screening of a synthetic peptide library.

By screening a synthetic peptide library of general formula (NH(2)-Cys1-X2-X3-X4)(2)-Lys-Gly-OH, a disulfide-bridged cyclic peptide, where X2-X3-X4 is the tripeptide Phe-His-His, has been selected as a ligand for immunoglobulin G (IgG). The peptide, after a preliminary chromatographic characterization, has proved useful as a new affinity ligand for the purification of polyclonal as well as monoclonal antibodies from biological fluids, with recovery yields of up to 90% (90% purity). The ligand is able to bind antibody fragments containing both Fab and Fc from different antibody isotypes, a fact suggesting the presence of at least two different antibody-binding sites. While the recognition site on Fab is unknown, comparative binding studies with Fc, in association with the striking similarities of the peptide (named Fc-receptor mimetic, FcRM) with a region of the human FcgammaRIII receptor, strongly indicate that the peptide could recognize a short amino acid stretch of the lower hinge region, which has a key role in autoimmune disease triggering. The unique properties make the ligand attractive for both the purification of antibody fragments and as a lead for the generation of Fc-receptor antagonists.