Generation and brain delivery of anti-aggregating antibodies against beta-amyloid plaques using phage display technology

Antibodies towards the N-terminal region of the beta-amyloid peptide bind to beta-amyloid fibrils, leading to their disaggregation. We generated anti-aggregating beta-amyloid antibodies using filamentous phages displaying the only four amino acids EFRH found to be the main regulatory site for beta-amyloid formation. In order to overcome the low permeability of the blood brain barrier for targeting 'anti-aggregating' mAbs to the betaA plaques in the brain, we applied antibody engineering methods to minimize the size of the mAbs while maintaining their biological activity. We found that single-chain antibodies displayed on the surface of the phage are capable of entering the central nervous system (CNS). The feasibility of these novel strategies for the production and targeting of anti-aggregating antibodies against beta-amyloid plaques to disease affected regions in the CNS may have clinical potential for treatment of Alzheimer's disease.     

Nicergoline, a drug used for age-dependent cognitive impairment, protects cultured neurons against beta-amyloid toxicity

Nicergoline, a drug used for the treatment of Alzheimer's disease and other types of dementia, was tested for its ability to protect neurons against beta-amyloid toxicity. Pure cultures of rat cortical neurons were challenged with a toxic fragment of beta-amyloid peptide (betaAP(25-35)) and toxicity was assessed after 24 h. Micromolar concentrations of nicergoline or its metabolite, MDL, attenuated betaAP(25-35)-induced neuronal death, whereas MMDL (another metabolite of nicergoline), the alpha1-adrenergic receptor antagonist, prazosin, or the serotonin 5HT-2 receptor antagonist, methysergide, were inactive. Nicergoline increased the basal levels of Bcl-2 and reduced the increase in Bax levels induced by beta-amyloid, indicating that the drug inhibits the execution of an apoptotic program in cortical neurons. In mixed cultures of rat cortical cells containing both neurons and astrocytes, nicergoline and MDL were more efficacious than in pure neuronal cultures in reducing beta-amyloid neurotoxicity. Experiments carried out in pure cultures of astrocytes showed that a component of neuroprotection was mediated by a mechanism of glial-neuronal interaction. The conditioned medium of cultured astrocytes treated with nicergoline or MDL for 72-96 h (collected 24 h after drug withdrawal) was neuroprotective when transferred to pure neuronal cultures challenged with beta-amyloid. In cultured astrocytes, nicergoline increased the intracellular levels of transforming-growth factor-beta and glial-derived neurotrophic factor, two trophic factors that are known to protect neurons against beta-amyloid toxicity. These results raise the possibility that nicergoline reduces neurodegeneration in the Alzheimer's brain.     

Beta-amyloid-activated cell cycle in SH-SY5Y neuroblastoma cells: correlation with the MAP kinase pathway

Primary cultures of rat cortical neurons exposed to toxic concentrations of beta-amyloid peptide (betaAP) begin an unscheduled mitotic cell cycle that does not progress beyond the S phase. To analyze possible signal transduction pathways involved in this effect, the action of betaAP has been studied in SH-SY5Y neuroblastoma cells differentiated by a 7-d exposure to 10 microM retinoic acid. Treatment with the betaAP fragment, betaAP(25-35), (25 microM) for 24, 48, or 72 h caused apoptotic cell death, detected by flow cytometry as a prediploid cell population. Cell cycle analysis showed that betaAP(25-35) modified cell cycle profiles by markedly increasing the number of cells in the S phase and reducing the population of the G2/M area. These effects seem to involve activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2). Inhibition of this pathway by the specific inhibitor PD98059 (2 microM) completely prevented changes of cell cycle distribution induced by betaAP and significantly reduced neuronal death. The data suggest that MAPK cascade can mediate the induction of cell cycle induced by betaAP, thus contributing to the toxicity of the peptide.     

Beta-amyloid-induced synthesis of the ganglioside GD3 is a requisite for cell cycle reactivation and apoptosis in neurons

We have shown that cortical neurons challenged with toxic concentrations of beta-amyloid peptide (betaAP) enter the S phase of the cell cycle before apoptotic death. Searching for a signaling molecule that lies at the border between cell proliferation and apoptotic death, we focused on the disialoganglioside GD3. Exposure of rat cultured cortical neurons to 25 microm betaAP(25-35) induced a substantial increase in the intracellular levels of GD3 after 4 hr, a time that precedes neuronal entry into S phase. GD3 levels decreased but still remained higher than in the control cultures after 16 hr of exposure to betaAP(25-35). Confocal microscopy analysis showed that the GD3 synthesized in response to betaAP colocalized with nuclear chromatin. The increase in GD3 was associated with a reduction of sphingomyelin (the main source of the ganglioside precursor ceramide) and with the induction of alpha-2,8-sialyltransferase (GD3 synthase), the enzyme that forms GD3 from the monosialoganglioside GM3. A causal relationship between GD3, cell-cycle activation, and apoptosis was demonstrated by treating the cultures with antisense oligonucleotides directed against GD3 synthase. This treatment, which reduced betaAP(25-35)-stimulated GD3 formation by approximately 50%, abolished the neuronal entry into the S phase and was protective against betaAP(25-35)-induced apoptosis.     

Effect of beta-amyloid peptide on behavior and synaptic plasticity in terrestrial snail

A large body of evidence implicates beta-amyloid peptide (betaAP) and other derivatives of the evolutionarily highly conserved amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease. However, the functional relationship of APP and its proteolytic derivatives to synaptic plasticity is not well known. We demonstrate that 30 min exposure to the 25-35 fragment of betaAP do not markedly change the dynamics of synaptic responses in identified neurons of terrestrial snail while a significant decrease of long-term sensitization was observed after 180 min betaAP bath application. In the behavioral experiments, a significant reduction of sensitization, and decreased ability to develop food-aversion conditioning was observed after betaAP injection. Our results clearly demonstrate that the neurotoxic 25-35 fragment of betaAP may play a significant role in behavioral plasticity by chronically eliminating certain underlying forms of synaptic plasticity. The study also proposes a novel invertebrate model to Alzheimer's disease.     

Filamentous bacteriophage as a novel therapeutic tool for Alzheimer's disease treatment

Antibodies towards the N-terminal region of the amyloid-beta peptide (AbetaP) bind to Abeta fibrils, leading to their disaggregation. We developed an immunization procedure using filamentous phages displaying the only four amino acids EFRH encompassing amino acids 3-6 of the 42 residues of AbetaP, found to be the main regulatory site for Abeta formation. Phages displaying EFRH epitope are effective in eliciting humoral response against AbetaP which, in turn, relieves amyloid burden in brains of amyloid-beta protein precursor transgenic mice, improving their ability to perform cognitive tasks. In order to overcome the low permeability of the blood brain barrier for targeting 'anti-aggregating' monoclonal antibodies (mAbs) to Abeta plaques in the brain, we applied antibody engineering methods to minimize the size of mAbs while maintaining their biological activity. Single-chain antibodies displayed on the surface of filamentous phage showed the ability to enter the central nervous system (CNS). The genetically engineered filamentous bacteriophage proved to be an efficient, nontoxic viral delivery vector to the brain, offering an obvious advantage over other mammalian vectors. The feasibility of these novel strategies for production and targeting of anti-aggregating antibodies against Abeta plaques to disease affected regions in the CNS may have clinical potential for treatment of Alzheimer's disease.     

Involvement of beta(1) integrin in betaAP-induced apoptosis in human neuroblastoma cells

Integrin-mediated cell adhesion is required for cell survival and differentiation. Recently, integrins have been proposed as a target for beta-amyloid peptide (betaAP) neurotoxicity. We report here that treatment with betaAP (1-42) or with the active betaAP fragment (25-35) induced a great deal of apoptosis in SK-N-BE and SH-SY5Y cell lines. In the presence of either collagen I degrees, fibronectin, or laminin, betaAP toxicity was severely reduced. This protective effect seems to be mediated by integrins, because preincubation of neuroblastoma cells with antibodies directed against beta(1) and alpha(1) integrin subunits greatly enhanced betaAP-induced apoptosis. In addition, treatment with betaAP induced a strong reduction of beta(1) and alpha(1) integrin subunits expressed in plasma membrane, which occurred 3 h after treatment, before the appearance of the apoptotic morphology. The rapid downregulation of the alpha(1)beta(1) integrin was almost completely recovered 15-24 h after betaAP treatment and was not prevented by cycloheximide. In conclusion, our data indicate a relationship between betaAP neurotoxicity and modulation of alpha(1)beta(1) integrin expression, and support the hypothesis that aberrant integrin function may play a significant role in betaAP-mediated neurotoxicity.     

Preparation of monoclonal antibodies to the thrombin/hirudin complex

Monoclonal antibodies (mAbs) binding to the thrombin/hirudin (T.H) complex were prepared by either immunizing mice with hirudin and by screening for the mAbs cross-reacting with the T.H complex (group I), or by immunizing the animals directly with the T.H complex (group II). Epitope mapping of the mAbs of group I indicated that all the mAbs were binding to the hirudin N-terminal core domain only (residues 1-43). Among the mAbs raised against the T.H complex (group II), one mAb recognized an antigenic determinant expressed selectively upon binding of hirudin to thrombin. A double antibody sandwich type ELISA combining the mAb of group II with a mAb of group I was developed, allowing the determination of the T.H complex in human citrated plasma down to nanogram concentration levels.     

Inhibition of conditioned media-mediated neuritogenesis of sensory ganglia by monoclonal antibodies to GM1 ganglioside

The monosialoganglioside GM1 can potentiate the neuritogenic activity of media conditioned by several cell types: neonatal glia, C6 glioma, embryonic chick heart or skeletal muscle and the rat myogenic line L6. To probe further the neuritogenic activity of conditioned media (CM), 5 mouse monoclonal antibodies (mAbs) against GM1, designated B6, C3, C4h2, D1 and D3 were incorporated individually into nutrient medium (NM) supplemented with CM prior to incubation with sensory ganglia. Nine-day embryonic chick dorsal root ganglia were explanted onto collagen-coated coverslips and incubated at 35 degrees C for 5 h in NM supplemented with 150 micrograms/ml GM1. After washing with NM, the explants were re-fed with NM + CM containing 20% mAb and cultured for an additional 43 h. The resultant neuritogenesis was evaluated microscopically by determining mean neurite number and length of randomly mixed cultures. The 5 antibodies differed in their capacities to inhibit CM-mediated neuritogenesis of these primed target cells. D1 and D3 were most effective in reducing neurite length and number produced by all sources of the CM, while C3 and C4h2 were intermediate in their inhibition of neurite initiation (number). The effect of B6 on neurite initiation and elongation was the least. The ability of these mAbs to inhibit neuritogenic activity of CM derived from both glial and myogenic tissue suggests that gangliosides play a basic role in neuronal development. The differing responses elicited by the individual mAbs may reflect a relationship between the structural complexity of the GM1 molecule and the neuritogenic mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-amyloid-induced increase in the resting intracellular calcium concentration gives support to tell Alzheimer lymphocytes from control ones

Senile plaques containing beta-amyloid peptide (betaAP) comprise the major neuropathological lesions in Alzheimer's disease (AD). In line with ongoing studies investigating alterations of various biochemical processes of cells of peripheral tissues, the authors demonstrate differences in resting intracellular free calcium levels of lymphocytes harvested from sporadic Alzheimer patients and from age-matched controls. Resting intracellular calcium concentration was measured in Fura-2AM-loaded human lymphocytes by dual wavelength spectrofluorimetry. Resting calcium level appeared to be higher in Alzheimer cells when compared to control lymphocytes. After incubating cells in 10(-7)M of beta-amyloid, the resting calcium concentration of the control cells elevated, while that of Alzheimer lymphocytes did not differ considerably.     

Alzheimer's disease and immunotherapy

Site-directed antibodies which modulate conformation of beta-amyloid peptide became the theoretical basis of the immunological approach for treatment of Alzheimer's disease (AD). Indeed, antibodies towards the EFRH sequence, located between amino acids 3-6 of the N-terminal region of Alzheimer's AbetaP, found to be a key position in protein conformation modulation, suppress formation of beta-amyloid and dissolve already formed fibrillar amyloid. The performance of anti-beta-amyloid antibodies in transgenic mice models of AD showed they are delivered to the central nervous system (CNS), preventing and dissolving beta-amyloid plaques. Moreover, these antibodies protected the mice from learning and age-related memory deficits. Naturally occurring anti-AbetaP antibodies have been found in human CSF and in the plasma of healthy individuals, but were significantly lower in AD patients, suggesting that AD may be an immunodeficient disorder. Active and/or passive immunization against beta-amyloid peptide has been proposed as a method for preventing and/or treating Alzheimer's disease. Experimental active immunization with Abeta 1-42 in humans was stopped in phase II clinical trials due to unexpected neuroinflammatory manifestations. Antibodies generated with this first-generation vaccine might not have the desired therapeutic properties to target the "correct" mechanism, however, new clinical approaches are now under consideration. Immunotherapy represents fascinating ways to test the amyloid hypothesis and offers genuine opportunities for AD treatment, but requires careful antigen and antibody selection to maximize efficacy and minimize adverse events.     

Monoclonal antibodies and conventional antisera to the GABAA receptor/benzodiazepine receptor/Cl- channel complex

Monoclonal antibodies (mAbs) and conventional antisera were raised to the affinity-purified GABAA receptor/benzodiazepine receptor/Cl- channel complex. The antibodies immunoprecipitated the affinity-purified complex in Triton X-100 and also reacted with the complex in a solid-phase radioimmunoassay. Immunoblots indicated that the mAb 62-3G1 reacted with the 57,000 Mr peptide subunit of the affinity-purified complex, while the antisera mainly reacted with the 51,000 Mr peptide subunit. The mAbs and the antisera also immunoprecipitated the GABAA receptor/benzodiazepine receptor/Cl- channel complex after being solubilized from cerebral cortex membranes by the zwitterionic detergent CHAPS. The immunoprecipitated complex bound 3H-muscimol, 3H-flunitrazepam (FNZ) and 35S-t-butylbicyclophosphorothionate (TBPS). The 3H-FNZ binding was stimulated by GABA, indicating that the functional interactions among the immunoprecipitated components of the complex were preserved. The mAb 62-3G1 also recognized the 57,000 Mr peptide in immunoblots with crude brain membranes. Immunocytochemistry experiments showed that the binding of both the mAb 62-3G1 and 3H-muscimol colocalized throughout the brain. The results suggest that (1) the 57,000 Mr peptide is the muscimol (GABAA receptor agonist) binding peptide of the complex, and (2) in the cerebral cortex, most of the GABAA receptors (GABARs), benzodiazepine receptors (BDZRs), and Cl- channels are physically coupled to one another.     

Complement-receptor-3 and scavenger-receptor-AI/II mediated myelin phagocytosis in microglia and macrophages

Microglia and macrophages express the alpha(M)/beta(2) integrin complement-receptor-3 (CR3/MAC-1; CD11b/CD18) and scavenger-receptor-AI/II (SRAI/II). Both can mediate myelin phagocytosis. We document that CR3/MAC-1 mediated myelin phagocytosis in microglia is modulated by complement and anti-CR3/MAC-1 mAbs. Complement augmented phagocytosis twofold. Anti-alpha(M) mAbs M1/70 and 5C6 inhibited and anti-beta(2) mAb M18/2 augmented myelin phagocytosis in the presence and absence of active complement. Active complement modulated phagocytosis inhibition by M1/70 and 5C6 and phagocytosis augmentation by M18/2. CR3/MAC-1 mediated myelin phagocytosis may thus be, at least partially, independent of but modulated by complement. Anti-beta(2) mAb Game-46 did not affect phagocytosis. However, combining M18/2 with Game-46 resulted in phagocytosis augmentation that was larger in magnitude than that induced by M18/2 alone. Thus, phagocytosis augmentation induced by one anti-beta(2) mAb was potentiated by another anti-beta(2) mAb. Combining M1/70 or 5C6 with M18/2 inhibited M18/2-induced augmentation. Overall, mAbs-induced phagocytosis modulation ranged three- to sevenfold from inhibition to augmentation. Anti-CR3/MAC-1 mAbs may reveal a mechanism by which native extracellular molecules bind to and modulate CR3/MAC-1 mediated myelin phagocytosis in microglia and macrophages. We further document SRAI/II mediated myelin phagocytosis in microglia and CR3/MAC-1 contributing to myelin phagocytosis two- to threefold more than SRAI/II when the two receptors function together.     

Differential expression of a ligand induced binding site (LIBS) by GPIIb-IIIa ligand recognition peptides and parenteral antagonists

The glycoprotein (GP) IIb-IIIa complex is an attractive anti-platelet target for the prevention of thrombotic events associated with coronary artery disease. Although GPIIb-IIIa antagonists inhibit GPIIb-IIIa binding to its ligands, the interactions have not been fully clarified, particularly with respect to their ability to induce structural changes in the complex that lead to exposure of neoantigenic epitopes or ligand-induced binding sites (LIBS). In this study we used the anti-LIBS monoclonal antibody (mAb) D3 to further define the activation states of purified active and inactive GPIIb-IIIa. We also compared the data obtained in the purified system to that observed with intact human platelets. Active GPIIb-IIIa expressed significantly greater high-affinity D3 LIBS sites compared to the inactive form. In addition, the ligand recognition peptides RGDS and H12 caused increased expression of the D3 epitope, with RGDS eliciting a much more potent response. The response of the purified GPIIb-IIIa to these peptides paralleled that observed with human platelets. To explore whether the platelet antagonists abciximab, eptifibatide and tirofiban induced expression of the D3 LIBS site, a modified competitive ELISA was developed. Our data indicate that the use of purified GPIIb-IIIa with this ELISA system provides a reproducible approach for exploring the interactions between GPIIb-IIIa and its antagonists. Whereas abciximab caused no detectable increase in the expression of the D3 epitope on purified GPIIb-IIIa, eptifibatide, tirofiban, RGDS, and H12 induced differential expression of the high-affinity LIBS. Studies with intact platelets suggested that abciximab blocked the binding of the D3 and LIBS6 mAbs, and that the pre bound anti-LIBS D3 sterically hindered abciximab binding.     

Use of peptide ligands to analyze the fine specificity of antibodies against asialo GM1

We recently described clone 10, a monoclonal Fab fragment that binds to asialo GM1 (GA1), and three mutated Abs derived from it that also bind GA1 and have a three to four times increase in avidity. We selected a phage display linear heptapeptide library with these four Abs, and an IgM mAb, 156, which binds to GM1 and GD1b, but not to GA1. Peptides with the same motif, KL/VWQXXX, were selected by clones 10 and the two heavy chain mutants 227 and 109. In contrast, the light chain mutant L3 58 selected an entirely different peptide motif, TFGLQSL. Moreover, a different motif, K/SWTNL/MPP, was selected by mAb 156. Although mAbs clone 10 and its mutants 109, 227 and L3 58 all bind only to GA1, differences in their fine specificity were revealed by binding to peptide ligands.     

Mimotopes of conformational epitopes in fibrillar beta-amyloid

In Alzheimer's disease (AD) beta-amyloid peptide accumulates in the brain in different forms including fibrils. Amyloid fibrils could be recognized as foreign by the mature immune system since they are not present during its development. Thus, using mouse antisera raised against the fibrillar form of Abeta42, we have screened two phage peptide libraries for the presence of foreign conformational mimotopes of Abeta. Antisera from wild type animals recognized predominately peptides with the EFRH motif from Abeta42 sequence, whereas amyloid precursor protein (APP) transgenic mice recognized mainly phage clones that mimic epitopes (mimotopes) within the fibrillar Abeta42 but lack sequence homology with this peptide.     

Structure of epitopes recognized by the antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptors: extrapolation to the structure of acetylcholine-binding domain.

Using the alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor (nAChR) and Ala-substituted peptide analogues, amino acid residues critical for specific monoclonal antibody (mAb) binding were identified. By means of 2D nuclear magnetic resonance (2D-NMR) analysis followed by molecular modeling, it was found that mAb binding resulted in stabilization of the free alpha3(181-192) peptide flexible conformation yielding an extended structure with residues 6-11 of the peptide being in direct contact with the Ab. Since the Ab binds the native AChR as well, it is suggested that the corresponding fragment of AChR alpha3 subunit is exposed to solution and also appears in extended conformation.     

Molecular characterization of human B domain-specific anti-factor VIII monoclonal antibodies generated in transgenic mice

The development of antibodies directed against factor VIII (FVIII) represents a major hurdle in the treatment of hemophilia A. Most anti-FVIII antibodies are identified through their ability to inhibit the FVIII procoagulant activity. Many of them, however, do not interfere with the functional properties of FVIII. Antibodies directed against the B domain belong to this latter category. Here, we characterized B domain-specific human monoclonal Abs (mAbs) at the molecular level. A series of human mAbs directed against FVIII was produced upon immunization of transgenic XenoMouse mice with human recombinant FVIII (rFVIII). Selection of the hybridoma with epitope specificity for the B domain was performed by differential recognition of full-length and B domain-deleted rFVIII. None of the anti-B domain mAbs demonstrated inhibitory activity against FVIII. Three of the mAbs recognized linear epitopes: mAb 25H3 bound to the (1014)HIDGPSLLIEN(1024) sequence; mAbs 8E3 and 22B6 shared the same epitope, composed of residues (1534)KWNEANR(1540). The corresponding soluble peptides inhibited the binding of their respective mAbs to FVIII. mAbs 8E3 and 22B6 displaced the binding of FVIII to vonWillebrand factor. Moreover, some of them (in particular mAbs 4G6 and 8E3) were able to compete for binding to the B domain with the anti-FVIII Abs from hemophilia A patients without inhibitor or with low Bethesda titers. Further investigation will allow to better characterize their clinical relevance.     

Two monoclonal antibodies to D-dimer-specific inhibitors of fibrin polymerization

D-dimer of human fibrin was used as antigen to obtain monoclonal antibodies (mAbs). We have obtained 16 hybridomas producing mAbs of different specificity. Only two of these mAbs inhibited fibrin polymerization. They are of the IgG-class. One mAb (II-4d) inhibited fibrin polymerization to 100% and another (II-3b) to 60% at a molar ratio mAb/fibrin=1.0. Fab-fragments of these mAbs inhibited fibrin polymerization completely at the same molar ratio. The epitopes for the mAbs studied are situated in the NH2-terminal part of the gamma-chain in fibrin D-domain. Electron microscopy showed that fibrin was in monomeric form in the presence of these mAbs or their Fab-fragments. Thus, these mAbs stop the initial step of fibrin polymerization, i.e. protofibril formation. Only one site of protofibril formation is known now in COOH-terminal half of the D-domain gamma chain named "a" site, which is complementary to the "A" site in the central E-domain of fibrin molecule. Our experiment with immobilized GPRP showed that the "a" site in fibrin D-fragment preserved its binding activity to GPRP when the D-fragment was complexed with mAbs-inhibitors of fibrin polymerization. Thus, these two mAbs inhibit fibrin polymerization not by blocking the sites "a" but either by blocking another (not "a") specific site in D-domain or by steric hindrance of highly organized fibrin polymerization process.     

Mechanism of action of a potent antiplatelet peptide, triflavin from Trimeresurus flavoviridis snake venom.

Triflavin, an antiplatelet peptide from Trimeresurus flavoviridis snake venom, inhibits aggregation of human platelets stimulated by a variety of agonists. However, triflavin does not affect the shape change and release reaction of platelets stimulated by thrombin and collagen. In this paper, we further investigate its effect on the intracellular events occurring after the activation of platelets. Triflavin does not inhibit the intracellular free calcium rise of Quin 2-AM loaded platelets stimulated by thrombin and it also has no significant effect on thromboxane B2 formation of platelets stimulated by thrombin. Triflavin does not affect the 3(H)-inositol monophosphate formation of the 3(H)-myoinositol loaded platelets. However, triflavin dose-dependently inhibits fibrinogen-induced aggregation and 125I-fibrinogen binding of ADP-stimulated platelets. In addition, triflavin dose-dependently blocks fibrinogen-induced aggregation of elastase-treated platelets. It is concluded that triflavin specifically inhibits fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex on platelet membrane surface without any inhibitory effect on the platelet-activation process.