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Amyloid beta (Aβ) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Aβ oligomers (amyloid β-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Aβ oligomers and fibrils but not the physiologically prevalent Aβ monomer. Discrimination derived from an epitope found in assemblies of Aβ1–28 and ADDLs but not in other sequences, including Aβ1–40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL-induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer-dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Aβ assemblies, these antibodies provide tools by which pathological Aβ assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.
Immunization of transgenic (tg) mice models of Alzheimer's disease with fibrillar amyloid beta protein (Aβ) results in reduction of Aβ deposits in the brain and prevents the accumulation of this pathology when administered before its formation (Schenk et al. 1999; Schenk 2002). Learning and memory deficits produced in these mice are also reduced or prevented by similar active vaccination with preparations containing fibrillar Aβ (Janus et al. 2000; Morgan et al. 2000). Based on results from animal models, clinical trials were initiated and showed few adverse reactions in Phase 1. However, Phase 2 trials were halted when 6% of the patients developed meningoencephalitis (Birmingham and Frantz 2002; Schenk 2002; Hock et al. 2003; Orgogozo et al. 2003; Schenk et al. 2004). Reports of the clinical outcome of these trials revealed that after 1 year patients producing antibodies that targeted plaques had a slower rate of cognitive decline than those patients who did not produce antibodies (Hock et al. 2003). Postmortem results on three patients showed absent or sparse plaques in the neocortex, with reactive microglia suggesting an effective immune response (Nicoll et al. 2003; Ferrer et al. 2004; Masliah et al. 2005).
Alternative approaches to avoid inflammatory responses through the use of therapeutic antibodies are now under development (Gelinas et al. 2004; Morgan and Gitter 2004; Schenk et al. 2004; Agadjanyan et al. 2005). Most strikingly, it has been established that injections with Aβ-generated monoclonal antibodies produce cognitive improvement in tg mice models of Alzheimer's disease. Using an antibody whose epitope targets the center of the Aβ peptide, Dodart et al. (2002) showed that memory deficits can be reversed in PDAPP mice within 24 h after treatment. Reversal of memory deficits by monoclonal antibody injection has been confirmed independently in other laboratories (Kotilinek et al. 2002; Lee et al. 2006). Although passive vaccination previously had been shown to clear plaques from PDAPP and other tg mice models (Bard et al. 2000,2003; Bacskai et al. 2002; McLaurin et al. 2002), in some studies showing recovery from memory deficits, Aβ plaque burden was not decreased. A likely explanation for cognitive improvement without change in plaque burden is that these therapeutic antibodies immunoneutralize small, soluble oligomers of Aβ that have been implicated in Alzheimer's disease synapse failure (Lambert et al. 1998; Hardy and Selkoe 2002). Aβ oligomers form at low doses of Aβ1–42, block long-term potentiation (LTP), and specifically attach to synaptic terminals (Lambert et al. 1998; Wang et al. 2002; Lacor et al. 2004; Wang et al. 2004). These oligomers (referred to as amyloid β-derived diffusible ligands, ADDLs) are elevated in Alzheimer's disease brain and CSF and in tg mouse models (Chang et al. 2003; Gong et al. 2003; Georganopoulou et al. 2005). It previously had been predicted that if ADDLs were an important part of early Alzheimer's disease pathogenesis, then memory deficits might prove to be reversible (Lambert et al. 1998; Klein 2000), as subsequently proven by Dodart et al. (2002).
Given these considerations, oligomers may provide an optimum target for therapeutic antibodies. The current work therefore addresses the need to obtain monoclonal antibodies selective for oligomers. Our approach has used as antigen soluble Aβ oligomers because of their previously demonstrated utility in providing epitopes dependent on quaternary structure in the generation of polyclonal antibodies. This strategy has generated monoclonal antibodies that distinguish between Alzheimer's disease and control brains and that neutralize oligomers in solution, characteristics that will ultimately be essential for therapeutically useful antibodies.
- Top of page
- Materials and methods
- Conflict of interest statement
ADDLs were used to generate monoclonal antibodies that targeted pathological Aβ assemblies. Three types of antibodies of interest were generated: those capable of binding oligomers and fibrils; those capable of binding fibrils but not oligomers; and those capable of binding oligomers but not fibrils. The first class was the most common, and several clones of this class were expanded and characterized in depth. These clones distinguished Alzheimer's disease from control brains in extracts and in tissue sections. In some cases, discrimination derived from a 3D epitope common to ADDLs and assemblies of Aβ1–28 that were absent from Aβ1–40 and other linear sequences. The antibodies blocked binding of ADDLs to synapses in hippocampal cultures, inhibited ADDL-induced increases in ROS, and prevented ADDL toxicity in the MTT assay. This selective immunoneutralization of pathological Aβ assemblies indicates these antibodies provide a useful prototype for therapeutic strategies based on monoclonal antibodies.
Immunization with various forms of Aβ fragments and assemblies has previously generated antibodies with a variety of useful properties. Short N-terminal peptides, for example, were used to generate the 6E10 antibody (Kim et al. 1990) used in the current study which binds to virtually all forms of Aβ. C-terminal antigens produced monoclonals that distinguish Aβ1–40 from Aβ1–42, making them useful for selective ELISAs (Savage et al. 1998; Jensen et al. 2000). Kayed et al. (2003) recently used Aβ1–40 coupled to gold colloids to generate antibodies that bind oligomers (but not monomers or fibrils) of several amyloidogenic proteins, including amyloid beta. In human brain sections the antibodies react with diffuse, early stage plaques but not thioflavin-positive dense-core plaques. Polyclonal antibodies generated by vaccination with ADDL preparations (Lambert et al. 2001) have been used to distinguish Alzheimer's disease brain tissue from control in both slices and dot immunoblots, to identify the 12mer found in Alzheimer's disease brain, to recognize ADDLs bound to synapses on cultured cells, and to identify lead compounds for anti-ADDL drug discovery (Lambert et al. 2001; Chang et al. 2003; Gong et al. 2003; Lacor et al. 2004).
The NU-1 and NU-4 monoclonals resemble ADDL-generated polyclonal antibodies in recognizing pathogenic assemblies but not monomers in Western and dot immunoblot paradigms. In Alzheimer's disease brain sections, the expected Alzheimer's disease-type immunoreactivity was present, coupled with an intriguing pericellular immunoreactivity. The pericellular immunoreactivity has been attributed hypothetically to oligomer attachment to synapses (Lacor et al. 2004), thought to occur early in Alzheimer's disease or even preclinically. With NU-6, which also was generated using ADDLs as antigen, the staining was strikingly different. Immunoreactivity was intracellular and present in cells whose morphology and GFAP-staining were compatible with reactive astrocytes. Appearance of reactive astrocytes and activated microglia in Alzheimer's disease pathology has been extensively described (Itagaki et al. 1989). The pattern seen here is interesting because, while NU-6 binds human aggregates but not oligomeric Aβ in western blots, it does not stain compact plaques. Thus the intracellular astrocyte species might represent a novel intermediate in fibril pathology. How these species originate is unknown, but astrocytes have been shown to participate in clearance and degradation of Aβ in tg mice (Wyss-Coray et al. 2003). Hypothetically, this capacity in Alzheimer's disease becomes overwhelmed, perhaps in a manner related to the intracellular accumulation and processing of ADDLs.
The species recognized by NU-4 in western blots included prominent higher order oligomers (12–24mers), even though no material was detected by silver stain. Aβ1–40 monomer was not recognized in immunoblots by NU-4, nor was Aβ1–42 monomer recognized when SDS was present in transfer buffer. Without SDS, however, NU-4 showed immunoreactivity at the Aβ 1–42 monomer position. Transfer of monomer was unaffected by SDS, verified in streptavidin-HRP assays for biotinylated Aβ1–42 (data not shown). We hypothesize that without SDS, monomers combine during transfer and produce oligomers, consistent with the rapid oligomerization seen even at very low Aβ concentrations (Fig. 4). Competition experiments verified that NU-4 had minimal affinity for monomers in solution, circumventing the ambiguities associated with SDS-PAGE analysis.
Competition experiments (Fig. 6) further showed that binding of ADDLs to some antibodies depended on conformational determinants, consistent with previous suggestions of Aβ1–42 antigenicity (Cribbs et al. 2003). Preincubation of NU-1 and NU-4 with solutions containing ADDLs or Aβ1–28 blocked subsequent binding to ADDLs in dot immunoblots. NU-2 was blocked only by ADDLs. Other peptides, including Aβ1–40, were without effect. Analysis of Aβ1–28 by electrospray mass spectrometry indicates it forms dimers and possibly other higher order oligomers in the gas phase (Li and Fenselau 2004). Two dimer structures were hypothesized to fit the data, with core overlaps of either Aβ17–23 or Aβ17–28. We therefore suggest that the NU-1 and NU-4 epitope is three-dimensional, in which aggregation of Aβ1–28 to form a dimer is necessary to align correct amino acids in the binding pocket, perhaps several from each single peptide. Denatured Aβ1–28 migrates at a position consistent with dimer, as seen by silver stain (Fig. 6). Alternatively, the single peptide itself could fold to align amino acids in the proper sequence, whereas the shorter peptides, as well as full-length Aβ1–40, could not.
Earlier, antibodies with epitopes against the N-terminal of Aβ were suggested to be more effective than C-terminal or central epitopes at plaque clearance in tg mice (Bard et al. 2003) and at inhibition of fibrillogenesis and cytotoxicity (McLaurin et al. 2002). For example, a substituted single chain antibody that relies on the Aβ3–6 sequence (EFRH) suppressed in vitro formation of Aβ aggregates and caused a loss of brain amyloid burden in tg mice (Frenkel et al. 2000; Solomon 2003). However, more recent results have shown that antibodies developed against other parts of the Aβ sequence, as well as against cross-linked Aβ1–40 oligomers (Lee et al. 2006), can be effective at reversing memory deficits. In most cases, this reversal is seen without change in amyloid burden. One antibody, M266, generated against the central domain Aβ13–28, caused reversal of memory deficits after only 24 h with no change in brain Aβ plaques (Dodart et al. 2002). As this antibody alters clearance of CNS and plasma Aβ (Seubert et al. 1992), the authors suggest that the antibody is acting as a sink for Aβ, leading to CNS clearance (DeMattos et al. 2001). Another N-terminal antibody, BAM-10, caused reversal of memory impairment in tg mice with no decrease in amyloid plaques (Kotilinek et al. 2002). The antibody was hypothesized to act by neutralizing soluble Aβ assemblies in the brain that cause cognitive impairment. Soluble Aβ assemblies accumulate in tg mice (Chang et al. 2003) as well as in Alzheimer's disease brain, where the major species is a 12mer (Gong et al. 2003). Confirmation that the 12mer is associated with learning deficits has recently been reported in Tg 2576 mice by Lesne et al. (2006). In other studies with tg mice, oligomer-selective antibodies have recently been shown to reduce levels of oligomers, plaques, and Alzheimer's disease-type tau phosphorylation (Ma et al. 2006; Oddo et al. 2006).
Success of active and passive vaccinations in mouse Alzheimer's disease models led to clinical trials for an active vaccine in Alzheimer's disease patients (Schenk et al. 1999, 2004). Phase 2 trials were stopped due to the appearance of meningoencephalitis in 6% of the patients (Hock et al. 2003; Orgogozo et al. 2003), but early clinical data from immunotherapy have been encouraging and efforts are underway to develop alternative Aβ immunotherapies (Gelinas et al. 2004). Active vaccines are being developed using immunoconjugates composed of a fragment of Aβ, usually from the N-terminal or central region, linked to a carrier protein (Schenk et al. 2004). These antigens contain no epitopes from Aβ1–42 that will initiate a T-cell response, which may eliminate autoimmune reactions. Passive vaccines are also being developed, which present advantages in avoiding poor immune responses in older patients as well as eliminating the T-cell response to Aβ. Oral immunization in tg animals has also been reported (Youm et al. 2005). We previously suggested that development of therapeutic antibodies that target ADDLs may optimize the efficacy of this approach (Klein 2000). The current study establishes that relatively potent monoclonal antibodies can be generated using synthetic ADDLs as immunogens, and that the resultant antibodies are selective for pathological assemblies of Aβ. The antibodies, moreover, immunoneutralize ADDLs obtained from Alzheimer's disease brain, blocking their toxicity and attachment to synapses in cell culture assays and blocking ADDL-induced generation of ROS. The antibodies thus hold promise for use in future behavioral studies of ADDL neutralization in tg-mice Alzheimer's disease models and provide a step toward antibodies of clinical value.
ADDL-selective monoclonal antibodies are also expected to be of value for Alzheimer's disease diagnostics. In recent studies, ADDL-selective antibodies were combined with developments in nanotechnology to develop assays capable of detecting ADDLs in human CSF (Georganopoulou et al. 2005; Haes et al. 2005). The new assays are orders of magnitude more sensitive than the best available ELISAs, capable of detecting atomolar ADDL concentrations. Results showing a 10-fold difference in CSF ADDL levels between Alzheimer's disease and control subjects (Georganopoulou et al. 2005) suggest that nanotechnology-based assays in combination with ADDL-selective monoclonal antibodies could provide the first clinical diagnostics for Alzheimer's disease.