Contents
1. Alzheimer's Disease
As people age they lose a small percentage of neurons in the brain without any clinical manifestation of memory loss. Accelerated neuronal loss in aging brains, however, leads to neurodegenerative diseases like Alzheimer’s disease (AD). Epidemiological studies have unequivocally demonstrated that aging is the single most significant risk factor contributing to the development of AD. AD accounts for more than 50% of cases of dementia in the elderly and has a prevalence estimated at 15-20 million patients worldwide. It is associated with progressive neuronal loss that leads to profound dementia and eventually death (Selkoe 2004).
Amyloid plaques and neurofibrillary tangles are characteristic of AD brains. Neurofibrillary tangles are found in select neuronal cell bodies, and these paired helical filaments are composed of hyperphosphorylated forms of tau protein. Amyloid plaques, both diffuse plaques and neuritic plaques, are extracellular fibrils composed of amyloid β protein (Aβ). The neuritic plaques are closely associated with dystrophic dendrites and axons.
While the majority AD cases are sporadic, in a subset of cases (~5%), familial AD (FAD) occurs as an inherited autosomal dominant disease. Missense mutations in three different genes are associated with FAD (Selkoe and Podlisny 2002). The first is the β-amyloid precursor protein (APP) (Goate et al. 1991), the precursor to Aβ (Selkoe 2000; Selkoe and Podlisny 2002). Several FAD-linked mutations in APP cause increased generation of Aβ. The other genes, presenilin 1 (PS1) on chromosome 14 (Sherrington et al. 1995) and presenilin 2 (PS2) on chromosome 1 (Levy-Lahad et al. 1995; Rogaev et al. 1995), account for the majority of early onset cases of FAD. The age of onset in families with PS1 mutations is very early, and some patients carrying mutant PS1 (e.g., P117L or L166P) died in their 20’s and 30’s (Wisniewski et al. 1998; Moehlmann et al. 2002). PS1 and PS2 are 467 and 448 amino acid polypeptides with ~60% homology, and over a hundred point mutations linked to FAD have been identified in the PS1 gene. In brain tissue (Lemere et al. 1996) and plasma (Scheuner et al. 1996) of FAD mutant PS gene carriers, a significant increase of 42 residues of Aβ peptide (Aβ42) has been detected. The elevation of Aβ42 appears to be a direct result of the expression of mutant PS genes, as all FAD mutations in PS1 and PS2 studied to date selectively enhance the production of Aβ42 both in transfected cells and in the brains of transgenic mice (Borchelt et al. 1996; Duff et al. 1996; Lemere et al. 1996; Borchelt et al. 1997; Citron et al. 1997; Xia et al. 1997; Petanceska et al. 2000; Xia 2000). The findings that FAD-linked mutations in three genes all lead to an increase of Aβ generation strongly support the hypothesis that gradual accumulation of Aβ in brains is one of the main contributors to the neuropathogenesis of AD.
Abcam's full catalogue of Alzheimer's antibodies.
Aβ is derived from sequential cleavage of its precursor protein APP (Xia and Xu 2004; Figure 1). APP occurs in three alternatively spliced forms of 695-, 751- and 770-residues, and these proteins undergo N- and N+O-glycosylation as well as phosphorylation. Full length APP can be recognized by many antibodies, such as 22C11 and 8E5. APP is proteolytically processed by at least two broad alternative pathways (Figure 1) (Xia 2001).
Figure 1: Amyloid Precursor Protein processing |
| Adapted from Xia, Xu, Amyloid Precursor Protein, A Practical Approach, CRC Press, 2004 |
First, cleavage of APP by the β-secretase (called BACE or memapsin 2) generates a soluble N-terminal fragment (β-APPs), and a ~12 kDa C-terminal stub of APP (C99), which can be recognized by antibody 192 and antibody C7. Alternative cleavage slightly N-terminal to the beginning of the APP transmembrane domain (at residues 16-17 of the Aβ region) by an "α-secretase" protease generates the major secreted derivative, β-APPs and a shorter ~10 kDa C-terminal stub of APP (C83), precluding Aβ formation ("non-amyloidogenic”). α-APPs and C83 can be recognized by 1736 and C7, respectively. The longer C-terminal stub C99 can be further cleaved by a protease called γ-secretase to yield two major species of Aβ ending at residue 40 (Aβ40) or 42 (Aβ42), while the shorter C83 can be cleaved by γ-secretase to generate p3. Both Aβ and p3 can be recognized by antibody 1282. Both β-secretase and γ-secretase have been studied as targets to reduce Aβ generation (Xia 2003a), and a variety of inhibitors have been tested for their potential therapeutic applications (Xia 2003b).
Browse the full APP processing pathway and download a pdf.
2 Antibody Drugs
While the tale of AD began with Alois Alzheimer's characterization of his patient at the beginning of last century, 1906, the tale of Antibody Drugs only started at the end of last century, 1999. Initial effort was focused on Aβ antigen, a synthetic, aggregated form of Aβ42, named AN-1792, was used to immunize transgenic mice over-expressing APP (Schenk et al. 1999). Like a magic bullet, synthetic peptide Aβ42 effectively prevented Aβ plaque formation, neuritic dystrophy and astrogliosis in these immunized mice. Later studies indicate that memory loss in the APP transgenic mice vaccinated with Aβ was clearly reduced (Janus et al. 2000; Morgan et al. 2000). Nasal administration of Aβ can similarly reduce AD-like neuropathologies in APP transgenic mice (Lemere et al. 2000; Weiner et al. 2000). Unfortunately, basic research and clinical activities for Aβ vaccination changed dramatically in the following three years, and the phase II trial of AN-1792 was discontinued, because several patients developed clinical signs of post-vaccination syndromes, including aseptic meningoencephalitis (Check 2002). Analysis of neuropathology of post mortem brain of a patient who underwent immunization with AN-1792 suggests similar changes found in brains of immunized APP transgenic mice. Few Aβ plaques were detected in a large area of neocortex, and these areas lack plaque associated dystrophic neurites and astrocytes, which are usually found in AD brains (Nicoll et al. 2003). On the other hand, cognitive test of immunized AD patients has shown a beneficial effect of AN-1792. Twenty patients, including two of three patients who transiently developed aseptic meningoencephalitis, generated antibodies against Aβ and showed reduced decline of cognitive functions, while this improvement was not observed in patients who failed to generate antibodies (Hock et al. 2003). As predicted, antibody was the key for improvement.
While research activities are still carried on to use different forms/lengths of Aβ peptides for immunization, much effort has been directed toward passive immunization, using antibodies against Aβ. Antibodies generated in mice through immunization with aggregated Aβ42 seem to recognize residues four to ten of Aβ, and these antibodies can inhibit Aβ fibril formation without causing an inflammatory response (McLaurin et al. 2002). In humans, antibodies produced in subjects injected with aggregated Aβ42 (enrolled in the phase II trial of AN-1792) recognize Aβ plaques, diffuse deposits and vascular Aβ deposit in blood vessels, but not soluble Aβ42 or full-length APP (Hock et al. 2002). Antibodies in the cerebrospinal fluid from one of the patients who developed aseptic meningoencephalitis showed similar specificity against Aβ plaques, diffuse deposits and vascular Aβ deposit, indicating that the symptom was unlikely to be related to an antibody response against cellular brain structures other than Aβ deposits (Hock et al. 2002). To avoid complication with potential meningoencephalitis, antibodies against Aβ seem to be an alternative approach for a safer vaccine.
A large battery of antibodies have been raised against Aβ during the past twenty years, and some of them have been widely used for immunohistochemical staining, ELISA, Western blotting, immunoprecipitation, e.g., polyclonal antibodies 1280/1282, monoclonal antibodies 3D6, 2G3, 21F12, 266, 4G8, 6E10, W0-2, G2-10, G2-11 (Xia and Xu 2004). Peripheral administration of antibodies against Aβ has been shown to reduce Aβ plaque formation in several lines of APP transgenic mice (Bard et al. 2000; DeMattos et al. 2001). An ex vivo assay with sections of brain tissue indicated that antibodies may trigger microglial cells to clear plaques through Fc receptor-mediated phagocytosis (Bard et al. 2000). While complement activation is not required for plaque clearance, Aβ antibodies with high affinity for Fc receptor on microglial cells are more effective than those with high affinity for Aβ itself (Bard et al. 2003). Murine phagocytotic effector cells (e.g., microglia) express three classes of IgG-specific Fc receptors, a high affinity FcγRI, and two low affinity FcγRII and FcγRIII (Ravetch and Kinet 1991), and FcγRI exhibits a higher affinity for isotype IgG2a than for IgG1 or IgG2b (Fossati-Jimack et al. 2000). Therefore, in these APP transgenic mice, it is not surprising to find that IgG2a antibodies against Aβ are more efficient in reducing plaques than IgG1 and IgG2b antibodies (Bard et al. 2003). Importantly, both ex vivo and in vivo assays have demonstrated that antibodies raised against N-terminal regions of Aβ are able to clear plaques, and this clearance response correlates with the plaque binding capability of Aβ antibodies. Although the capacity of antibodies to bind soluble Aβ is not critical (Bard et al. 2003), another study suggests that soluble Aβ may play an equally important role in clearing plaques. A second mechanism for clearing plaque by Aβ antibodies in mouse brains could be the drainage of brain soluble Aβ to plasma (DeMattos et al. 2001). Disequilibrium between central nervous system and plasma Aβ levels (rapid increase in plasma Aβ) could be introduced after peripheral administration of anti-Aβ antibody to mice.
Nevertheless, the antibody against Aβ as a drug has not reached prime time yet. In a different line of APP transgenic mice, passive immunization caused an increase in small hemorrhages in brain areas with amyloid deposits in blood vessels (Pfeifer et al. 2002). This particular APP transgenic line, APP23, was used as a spontaneous hemorrhagic stroke mouse model (Winkler et al. 2001), and the phenotype of small hemorrhages was not observed in young APP23 mice or other lines of APP transgenic mice. In addition, the effect could be caused by the particular anti-Aβ monoclonal antibody used in this study. Apparently, concerns have been raised about passive immunization for AD patients with cerebral amyloid angiopathy, and much effort is needed to elucidate the molecular mechanism for Aβ antibody immunization.
The tale of Alzheimer's Disease lasts more than a century, but it should take less than a century to end it with Antibody Drugs.
3. References
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