Source: http://www.google.com/patents/US8106164?dq=4740761
Timestamp: 2017-04-23 20:37:10
Document Index: 268671368

Matched Legal Cases: ['§371', 'Application No. 01945896', 'Application No. 07747965', 'Application No. 05753672', 'Application No. 07747965', 'Application No. 07747965', 'Application No. 08019830', 'Application No. 07747965', 'Application No. 07747965', 'Application No. 01945896', 'Application No. 01945896', 'Application No. 01945896', 'Application No. 01945896', 'Application No. 01945896', 'Application No. 07747965', 'Application No. 07747965', 'Application No. 200803655', 'Application No. 01945896']

Patent US8106164 - Antibodies specific for soluble amyloid beta peptide protofibrils and uses ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsThe invention pertains to the development of antibodies that specifically bind amyloid beta protein (Abeta) in its protofibril conformation. The invention also comprises methods of using anti-Abeta protofibril antibodies to diagnose or treat Alzheimer's disease, Down's syndrome Lewybody dementia, vascular...http://www.google.com/patents/US8106164?utm_source=gb-gplus-sharePatent US8106164 - Antibodies specific for soluble amyloid beta peptide protofibrils and uses thereofAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS8106164 B2Publication typeGrantApplication numberUS 11/570,995PCT numberPCT/SE2005/000993Publication dateJan 31, 2012Filing dateJun 21, 2005Priority dateJun 21, 2004Fee statusPaidAlso published asCA2570130A1, CA2570130C, EP1781703A1, EP1781703B1, US8404459, US8999936, US20090155246, US20120230912, US20130236452, US20150307601, WO2005123775A1Publication number11570995, 570995, PCT/2005/993, PCT/SE/2005/000993, PCT/SE/2005/00993, PCT/SE/5/000993, PCT/SE/5/00993, PCT/SE2005/000993, PCT/SE2005/00993, PCT/SE2005000993, PCT/SE200500993, PCT/SE5/000993, PCT/SE5/00993, PCT/SE5000993, PCT/SE500993, US 8106164 B2, US 8106164B2, US-B2-8106164, US8106164 B2, US8106164B2InventorsPär Gellerfors, Lars LannfeltOriginal AssigneeBioarctic Neuroscience AbExport CitationBiBTeX, EndNote, RefManPatent Citations (84), Non-Patent Citations (290), Referenced by (19), Classifications (21), Legal Events (4) External Links: USPTO, USPTO Assignment, EspacenetAntibodies specific for soluble amyloid beta peptide protofibrils and uses thereof
US 8106164 B2Abstract
The invention pertains to the development of antibodies that specifically bind amyloid beta protein (Abeta) in its protofibril conformation. The invention also comprises methods of using anti-Abeta protofibril antibodies to diagnose or treat Alzheimer's disease, Down's syndrome Lewybody dementia, vascular dementia, and other neurodegenerative disorders. Furthermore, the invention pertains to the use of anti-Abeta protofibril antibodies to screen and identify substances that will modulate protofibril activity or formation in vitro or in vivo. The invention also pertains to methods for synthesising pure Abeta protofibril antigens as well as to a method for stabilising Abeta protofibrils antigens as well as to a method for stabilising Abeta protofibrils.
1. An isolated monoclonal antibody or antigen-binding fragment thereof that binds wild-type Aβ42/40 protofibril and Aβ42/40arc protofibril, and has no or little cross-reactivity to Aβ42/40 monomers.
2. The antibody of claim 1, wherein said antibody is an antigen-binding fragment.
3. The antibody of claim 1, wherein said antibody is humanized.
4. The antibody of claim 1, wherein said antibody is produced by immunizing and screening with at least >95% pure Aβ42/40arc protofibrils.
5. The antibody of claim 1, wherein said antibody is produced by immunizing and screening with at least >95% pure Aβ42/40 protofibrils.
6. The antibody of claim 1, wherein said antibody is a protofibril conformation specific antibody or antibody fragment.
7. A composition comprising one or more antibodies according to any of claims 1-2, 3, and 4-6.
8. A method of treating Alzheimer's disease comprising the step of administering to a patient having or suspected of having Alzheimer's disease, the antibody according to claim 1.
9. A method of treating Alzheimer's disease comprising the step of administering to a patient having or suspected of having Alzheimer's disease a composition comprising one or more antibodies according to any of claims 1-2, 3, and 4-6. Description
This application is the U.S. national stage filing under 35 U.S.C. §371 of international application PCT/SE2005/000993, filed Jun. 21, 2005, which claims benefit of Swedish Patent Application 0401601-0, filed Jun. 21, 2004.
This invention pertains to the diagnosis, prevention and treatment of neurodegenerative diseases, in particular Alzheimer's disease, and other similar disease. More precisely, to antibodies that specifically bind amyloid beta protein (Aβ) in its protofibril conformation.
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder causing cognitive, memory and behavioural impairments. It is the most common cause of dementia in the elderly population affecting roughly 5% of the population above 65 years and 20% above 80 years of age. AD is characterized by an insidious onset and progressive deterioration in multiple cognitive functions. The neuropathology involves both extracellular and intracellular argyrophillic proteineous deposits. The extracellular deposits, referred to as neuritic plaques, mainly consist in amyloid beta protein (Aβ) surrounded by dystrophic neurites (swollen, distorted neuronal processes). Aβ within these extracellular deposits are fibrillar in their character with a β-pleated sheet structure. Aβ in these deposits can be stained with certain dyes e.g. Congo Red and display a fibrillar ultrastructure. These characteristics, adopted by Aβ in its fibrillar structure of neuritic plaques, are the definition of the generic term amyloid. The classic intracellular AD pathologic lesion is the neurofibrillary tangle (NFT) which consists of filamentous structures called paired helical filaments (PHFs) composed of twisted strands of hyperphosphorylated microtubule-associated protein tau. Frequent neuritic plaques and neurofibrillary tangle deposits in the brain are diagnostic criteria for AD, as carried out post mortem. AD brains also display macroscopic brain atrophy, nerve cell loss, local inflammation (microgliosis and astrocytosis) and often congophilic amyloid angiopathy (CAA) in cerebral vessel walls.
Two forms of Aβ peptides, Aβ40 and Aβ42, are the dominant species in AD neuritic plaques (Masters 1985), while Aβ40 is the prominent species in cerebrovascular amyloid associated with AD (Glenner 1984). Enzymatic activities allow Aβ to be continuously formed from a larger protein called the amyloid precursor protein (APP) in both healthy and AD afflicted subjects in all cells of the body. Two major APP processing events through β- and γ-secretase activities enables Aβ production, while a third enzyme called o-secretase activities prevents Aβ generation by cleavage inside the Aβ sequence (Selkoe, 1994; U.S. Pat. No. 5,604,102). The Aβ42 is a forty two amino acid long peptide i.e. two amino acids longer at the C-terminus, as compared to Aβ40. Aβ42 is more hydrophobic, and does more easily aggregate into larger structures of Aβ peptides such as Aβ dimers, Aβ tetramers, Aβ oligomers, Aβ protofibrils or Aβ fibrils. Aβ fibrils are hydrophobic and insoluble, while the other structures are all less hydrophobic and soluble. All these higher molecular structures of Aβ peptides are individually defined based on their biophysical and structural appearance e.g. in electron microscopy, and their biochemical characteristics e.g. by analysis with size-exclusion chromatography/western blot. These Aβ peptides, particularly Aβ42, will gradually assemble into a various higher molecular structures of Aβ during the life span. AD, which is a strongly age-dependent disorder, will occur earlier in life if this assembly process occurs more rapidly. This is the core of the “amyloid cascade hypothesis” of AD which claims that APP processing, the Aβ42 levels and their assembly into higher molecular structures is a central cause of AD. All other neuropathology of AD brain and the symptoms of AD such as dementia are somehow caused by Aβ or assembled forms thereof.
Aβ can exist in different lengths i.e. 1-39, 1-40, 1-42 and 1-43 and fragments sizes i.e. 1-28, 3-40/42, 11-40/42, 17-40/42 and 25-35. All these peptides can aggregate and form soluble intermediates and insoluble fibrils, each molecular form having a unique structural conformation and biophysical property. Monomeric Aβ1-42 for example, is a 42 amino acid long soluble and non-toxic peptide, that is suggested to be involved in normal synapse functions. Under certain conditions, the Aβ1-42 can aggregate into dimers, trimers, tetramers, pentamers up to 12-mer and higher oligomeric forms, all with its distinct physicochemical property such as molecular size, EM structure and AFM (atomic force microscopy) molecular shape. An example of a higher molecular weight soluble oligomeric Aβ form is the protofibril (Hartley 1999, Walsh 1999), which has an apparent molecular weight>100 kDa and a curvelinear structure of 4-11 nm in diameter and <200 nm in length. It has recently been demonstrated that soluble oligomeric Aβ peptides such as Aβ protofibrils impair long-term potentiation (LTP) (Hartley, 1999), a measure of synaptic plasticity that is thought to reflect memory formation in the hippocampus (Walsh 2001). Furthermore, oligomeric Arctic Aβ peptides display much more profound inhibitory effect than wtAβ on LTP in the brain, likely due to their strong propensity to form Aβ protofibrils (Klyubin 2003).
There are also other soluble oligomeric forms described in the literature that are distinctly different from protofibrils. One such oligomeric form is ADDL (Amyloid Derived Diffusible Ligand) (Lambert 1998). AFM analysis of ADDL revealed predominantly small globular species of 4.7-6.2 nm along the z-axis with molecular weights of 17-42 kDa. (Stine 1996). Another form is called ASPD (Amyloidspheroids). ASPD are spherical oligomers of Aβ1-40. Toxicity studies showed that spherical ASPD>10 nm were more toxic than lower molecular forms (Hoshi 2003). The Aβ fibril as the main neurotoxic species is inconsistent with the poor correlation between neuritic plaque density and AD dementia score and also with the modest signs of neurodegeneration in current APP transgenic mice. Soluble neurotoxic Aβ-intermediate species and their appropriate subcellular site of formation and distribution could be the missing link that will better explain the amyloid hypothesis. This idea has gained support from recent discovery of the Arctic (E693) APP mutation, which causes early-onset AD (US 2002/0162129 A1; Nilsberth et al., 2001). The mutation is located inside the Aβ peptide sequence. Mutation carriers will thereby generate variants of Aβ peptides e.g. Arctic Aβ40 and Arctic Aβ42. Both Arctic Aβ40 and Arctic Aβ42 will much more easily assemble into higher molecular structures (protofibrils) that are soluble and non-fibrillar. Thus the pathogenic mechanism of the Arctic mutation suggests that the soluble higher molecular protofibrils are causing AD.
2.1 Diagnosis of Alzheimer's Disease
2.1.1 Clinical Diagnosis
The clinical diagnosis of Alzheimer's disease (AD) is difficult to make, especially in early stages of the disease. Today, the diagnosis is based on a typical medical history combined with the exclusion of other causes of dementia. Clinical centres with high specialization can have a diagnostic accuracy of 85-90% compared with the neuropathological diagnosis. In the early stages of the disease the clinical picture is vague and definite diagnostic markers have not yet been identified (McKhann 1984). The development of biochemical diagnostic markers is important for a number of reasons: to support the clinical diagnosis, to allow clinicians to give adequate information to patients and their relatives, to initiate pharmacological treatment and care-giving, and in various aspects of clinical research.
2.1.2 Amyloid β-Peptide
Pathogenic mutations in the APP and presenilin (PS) genes have been discovered in families with early-onset AD inherited as a dominant trait (Hardy 1992). The effects of some of these mutations are now fairly well understood. The Swedish AD mutation (Mullan 1992; Axelman 1994; Lannfelt 1994) has revealed one pathogenic mechanism for the development of AD. When a cDNA construct with this mutation was transfected into human cell-lines it gave rise to approximately six times higher release of soluble Aβ (Citron 1992, Cai 1993). Furthermore, fibroblasts from individuals with the Swedish mutation secreted three times more Aβ into the media compared to fibroblasts from non-carriers (Johnston 1994). Overproduction of Aβ therefore seemed to be an important factor in the disease pathogenesis in this Swedish family. Thus, it was expected that Aβ levels measured in cerebrospinal fluid (CSF) from family members would differentiate carriers from non-carriers of the mutation. However, no difference was found in levels of total Aβ between the groups (14.5±3.3 ng/ml versus 14.9±2.3 ng/ml) (Lannfelt 1995). One explanation for this result may be that Aβ is cleared from CSF by aggregating to amyloid in the brain. However, there was a strong correlation between duration of dementia and decreasing Aβ levels. These measurements were done with antibodies recognizing soluble monomeric Aβ. With protofibril specific monoclonal antibodies more accurate measurements of the toxic species will be possible.
2.1.3 Aβ in Plasma
Aβ is found in a soluble form in plasma and other tissues (Seubert 1992), and not as previously presumed, only in the brains of AD cases. Aβ plasma levels in members of the Swedish mutation family revealed that both Aβ40 and Aβ42 were 2-3 times increased in mutation-carriers (Scheuner 1996). The proportion of Aβ42 of total Aβ was approximately 10% in both groups, which is in agreement with experiments performed in cell cultures with the Swedish mutation. Mutation-carriers below the age of expected onset of the disease had the same levels of Aβ as already affected cases. This indicates that APP mismetabolism may play an important role early in the pathogenesis of the disease.
2.1.4 Aβ42 in CSF in Alzheimer's Disease
ELISAs specifically measuring Aβ40 and Aβ42 in CSF in AD cases have given different results. Some researchers (Pirttilä 1994; Motter1995) have found decreased Aβ42 in AD, while one group have found elevated levels in cases early in the disease progression. The most demented cases in one study had all very low levels of Aβ42. In conclusion, Aβ42 is most likely increased early in the disease process and levels of Aβ42 and Aβ40 decreased during progression of the disease. The development of accurate biochemical markers of early AD is important especially when efficient pharmacological treatments will be available in the future. Pharmacological therapy should most likely be initiated at an early stage of disease, before severe brain damage has occurred. A therapy making it possible to prevent the progression of the disease to its later stages would therefore be much desired.
2.2 Prevention and Treatment of Alzheimer's Disease
Antibodies that are specific towards different conformations of Aβ, such as Aβ fibrils (O'Nuallain 2002), micellar Aβ (Kayed 2003), ADDL (M93, M94) (Lambert 2001) have also been described.
Several pre-clinical studies in transgenic animal models have shown decreased plaque burden and improvements in memory function after active or passive immunization with antibodies raised against fibrils (Shenk 1999, Janus 2000, Morgan 2000, Weiner 2000, Sigurdsen 2001). Since fibrils are present in pathological deposits occurring late in the Alzheimer disease process, the Shenk antibodies may only be used to slow the progression of Alzheimer's disease when it has already reached its later stages.
Recently, a phase II clinical trial in Alzheimer patients with mild to moderate dementia was performed by ELAN Pharmaceuticals with their vaccine AN 1792, which is an aggregated preparation of human wtAβ42. The study had to be stopped due to side effects in 5% of the patients. The side effect was considered to be due to T-lymphocyte-induced meningioencephalitis (Nicholl 2003). The drug targets of the ELAN vaccine were insoluble fibrils found in plaques inside the brain and deposits on the brain blood vessel walls (Congophilic Amyloid Angiopathy, CAA), which are common features of Alzheimer's disease. Thus, an immune response towards insoluble fibrils could be responsible for the invasive inflammation in the brain blood vessel walls leading to meningioencephalitis.
WO02/203911 disclose the discovery of the Arctic mutation in a Swedish family leading to early onset of Alzheimer's disease (55.6 years). The Arctic mutation (Glu>Gly), which is located at position 22 in the beta amyloid peptide (Aβ), in combination with various experiments led to the insight that the Aβ peptide was much more prone to oligomerize and form protofibrils compared to wild type Aβ40. The discovery indicated for the first time that the protofibril is a central component in the disease process, and that AD could be treated by reducing the amount of protofibrils in the brain. This unique property of the Arctic mutation could then be used to generate protofibrils. WO02/203911 then suggested that said protofibrils could be used to immunize a mouse or other animal, in order to generate antibodies, after which any protofibril-specific monoclonal antibody could identified be screening. Said antibodies should then be specific towards an Aβ peptide of SEQ ID No 1 (page 7, third paragraph), i.e. a peptide carrying the Arctic mutation and having a protofibril conformation.
Thus, in view of the prior art techniques for preventing and treating Alzheimer's disease, there is a need for a technique that enables earlier detection of markers of Alzheimer's disease. If said markers could be prevented without causing negative side-effects, this would be a means to prevent and treat Alzheimer's disease at an early stage. Any treatment of Alzheimer's disease that would reduce the amount of protofibrils in the brain of AD patients, would be of significant therapeutic value.
Protofibrils occur early in Alzheimer's disease, when little brain damage has occurred. An antibody treatment that reduce protofibril levels in the brain will save the brain from neuronal destruction and hence be more advantageous for Alzheimer patients. The present invention describes the development of such therapeutic protofibril specific antibodies.
According to one aspect, the present invention relates to antibodies that have the property to bind both wild type Aβ42/40 and Aβ42/40arc protofibrils (conformation-specific antibodies) and which are suitable for development into pharmaceuticals for Alzheimer's disease, targeting wild type Aβ42/40 protofibrils. Current ideas in the field of Alzheimer's disease research is that Aβ fibrils are the main cause of the disease. Hence, the approach of the present invention contradicts the general opinion within the field.
According to another aspect, the present invention also relates to a composition comprising said antibody and optionally a carrier or excipient.
To immunise and screen for conformation-specific anti-protofibril antibodies, it is necessary to produce pure Aβ42arc and Aβ42 protofibrils (>95% degree of purity). In order to obtain said pure protofibril preparation, it is also necessary to be able to stabilise the protofibrils, such that they stay protofibrils and do not separate into monomers or aggregate into fibrils. It is also necessary to find a solvent that made it possible to separate the protofibrils by column chromatography. Another necessity is the ability to test said purity before immunisation. The above-mentioned abilities are essential, since the degree of purity of the antigen (protofibrils) decide the possibility to produce conformation-specific anti-protofibril antibodies, which in turn decide the possibility to screen for antibodies with useful specificity. Should the preparation for example contain 50% Aβ monomer and 50% Aβ protofibril, it is only possible to claim that the antibody binding said preparation may bind both forms or only one. All of said necessary features are provided by the present invention.
The above-mentioned features were not possible when WO02/203911 was filed. The HPLC analysis method disclosed it the present application or any other analysis method had not been developed, wherefore it was not possible to determine whether the Aβ42arc peptide actually formed protofibrils, probably due to the lack of binding to protofibrils. It was only a hypothesis at the time. The major reasons why WO02/203911 could not analyse and purify protofibril preparation to a degree of purity of >95%. Also, to the fact that they did not know how to stabilise protofibrils. In that connection, it should also be mentioned that protofibrils are very difficult to handle in comparison with for example Aβ40 peptides. Aβ42, but in particular the Aβ42βrc peptide, is extremely difficult to handle, since they stick to test tubes and columns and aggregate into fibrils. The latter problems has been solved by the present invention by the provision of methods to stabilise the protofibrils involving low temperatures and selected solvents. WO02/203911 also had no means for testing the purity of the protofibrils before immunisation. This is possible with the present invention by the provision of a new HPLC (size exclusion) method. In addition to this, WO02/203911 did not provide any means to check the protofibril specificity of the screened antibodies. The present invention makes this possible by the provision of a new Elisa method. WO02/203911 could only produce impure and transient Aβ40arc protofibrils, not useful for monoclonal development of protofibril specific antibodies (WO02/203911 only describes analysis of Aβ40 and Aβ40Arc peptides, see Example 3).
The antibodies according to the present invention are particularly suitable for treating Alzheimer's disease since they will: i) target and eliminate the most toxic AZ form (protofibril), ii) provide an opportunity to treat the disease early since protofibrils develops early in the disease process, iii) avoid side-effects since the will not significantly cross-react with Aβ fibrils present in the blood vessel wall of the brain (CAA=Congophilic Amyloidogenic Angiopathy).
The antibodies according to the present invention may be human or humanised, monoclonal or polyclonal. The present invention also relates to biologically active fragments of said antibodies, with the proviso that they still have the claimed properties.
The Aβ protofibril specific antibodies according to the present invention can also be used to quantitate wild type Aβ42/40 protofibrils in biological fluids, thus the antibodies will be suitable for clinical diagnosis at an early stage of the disease and suitable as a biomarker to monitor efficacy in clinical studies.
Furthermore, the invention describes procedures to generate wild type Aβ42 and Aβ42arc protofibrils as antigens for immunization and for reagents to screen for antibodies that binds Aβ42arc and wild type Aβ42 protofibrils. Antigen (protofibril) purity is very important to determine in antibody screening experiments, since it affects antibody specificity determinations by ELISA.
To assess the purity of antigen (Aβ42arc and wild type Aβ42 protofibrils) preparation, a size exclusion HPLC method has been developed. The HPLC method invented uses a detergent, with low optical interference in chromatography. The detergent has the advantage that it eliminates interactions of Aβ42arc and wild type Aβ42 protofibrils with the column matrix, preventing loss of material and material from adhering to the column, and erroneous estimations of protofibril purity. Furthermore, the detergent is compatible with Aβ protofibrils and does not solubilize them into Aβ monomers or alter their biological profile. Polysorbate (Tween) has been found to be a suitable detergent, in particular Polysorbate-80 (Tween-80). Other detergents with similar properties are also useful.
The antibody according to the present cross-reacts less than 50% with Aβ fibrils. Said antibody can detect both wild type Aβ protofibrils and Arctic Aβ protofibrils in an ELISA in a concentration range of 1000-10 ng/ml (see Example 5 and FIGS. 4A-C). In an optimised ELISA or by other detection systems with higher sensitivity, for example proximity ligation, said detection level could probably be brought down to a detection level of 1-0.1 ng/ml.
The present invention provides a method to raise antibodies that are specific to amyloid β (Aβ) protofibrils (high molecular weight soluble non-fibrillar Aβ oligomers). The antibodies are raised against Aβ in its protofibril conformation. These antibodies will be administered to AD patients to reduce protofibril levels in the brain, which will be of significant therapeutic value.
The lowering of protofibril levels might occur through the elimination of the antigen when bound to an antibody, through microglia-mediated phagocytosis. This is a well documented biological process, which occurs through the binding of the antibody's constant region (Fc) to an Fc receptor on microglial cells. Binding induces phagocytosis (internalisation and destruction) of the antibody and its bound antigen (protofibril) leading to reduced levels of protofibrils in the brain. Other non-Fc receptor mediated processes might also occur to eliminate the antigen (protofibril).
Another aspect of the invention pertains to a method to synthesize Aβ protofibrils which are to be used as antigen for immunization. The synthesis can be made from wild type Aβ (wtAβ) or alternatively, from non-wild type, mutated or modified forms of Aβ. In a preferred embodiment of the invention, but not limited to said embodiment, the synthesis is initiated by dissolving wtAβ1-42 in a dissociating agent, for example NaOH, to achieve a homogenous Aβ solution, of about 50-500 uM in peptide concentration, but not limited to this concentration. Alternative agents with dissociating capacity are for example dimethylsulfoxide, DMSO; hexafluoroisopropanol, HFIP; trifluoroacetic acid, TFA. Wild type protofibrils can also be made from wtAβ peptides of different lengths i.e. wtAβ1-39, wtAβ1-40, wtAβ1-43 or N-terminal truncated forms of these peptides. The truncation can be 1-10 amino acids, giving wtAβ forms such as: wtAβ2-42, wtAβ3-42, wtAβ4-42, wtAβ5-42 and so on. The dissolved wtAβ1-42 peptide is subsequently neutralized by PBS or similar physiological buffer and incubated at higher temperature, preferably 37° C., for a period of time sufficient for protofibril-formation to occur, for example over night. This will yield wild type Aβ protofibrils. The invention also provide a molecular size (molecular weight) determination method to assess protofibril formation and purity, preferably, but not limited to, a size-exclusion chromatographic method (SEC).
The method of synthesizing pure wild type (wtAβ42) protofibril antigen, comprises the steps of dissolving an wtAβ42 by using a dissociating agent, such as NaOH (pH>10), dimethylsulfoxide, DMSO; hexafluoroisopropanol, HFIP; trifluoroacetic acid and TFA, to achieve a monodisperse solution, neutralizing the solution by PBS (pH 7-8) or similar biocompatible buffers, to achieve a physiological solution, incubating the neutralized wtAβ42 peptide solution at an elevated protein concentration between 1-1000 uM, preferably 440 uM, for 6 hours or longer at 20-40° C., preferably 37° C., to form Aβ42wt protofibrils, diluting the protofibrils to approximately 1-500 uM, preferably 50 uM centrifuging at sufficient speed and time to sediment wtAβ42 fibrils, which normally takes 5 minutes at 17.000×g at +4° C., assessing the purity of the protofibril preparation by HPLC to control that the purity is >95%, using for example size exclusion HPLC, using a physiological buffer such as PBS at neutral pH as running buffer, including a detergent, such as Polysorbate (Tween), or similar detergent, to avoid sticking of the protofibrils to the column matrix and dissociation.
Another aspect of the invention pertains to the synthesis of non-wild type Aβ protofibrils using Aβ1-42, Aβ1-41, Aβ1-40 or Aβ1-39 or N-terminal truncated forms (1-10 truncations) with either the Arctic (G22E) mutation (U.S. Ser. No. 09/899,815), the Dutch mutation (E22Q), the Flemish (A21G) mutation, the Italian (E22K) mutation, the Iowa (D23N) mutation, and combinations thereof. In a preferred embodiment of the invention Aβ42arc peptide (i.e., comprising the arctic mutation) is used. The method to make Aβ42arc protofibrils is similar to that for wtAβ42 protofibrils except that Aβ42arc protofibrils are not incubated at 37° C. over night since they are spontaneously formed after the neutralization step.
The method of synthesizing pure Aβ42arc protofibril antigen comprises the steps of dissolving an Aβ42arc peptide by using a dissociating agent, such as NaOH (pH>10), dimethylsulfoxide, DMSO; hexafluoroisopropanol, HFIP; trifluoroacetic acid and TFA to achieve a monodisperse solution, neutralizing the solution by PBS (pH 7-8) or similar biocompatible buffers, to achieve a physiological solution, stabilizing the spontaneously formed protofibrils by keeping them at below 20° C., preferably at 0-5° C., centrifuging the protofibrils, at the same temperature as in the stabilising step, at a sufficient speed and time to sediment Aβ42arc fibrils, which normally takes 5 minutes at 17.000×g at +4° C., assessing the purity of the protofibril preparation by HPLC to control that the purity is >95%, using for example size exclusion HPLC, using a physiological buffer such as PBS at neutral pH as running buffer, including a detergent, such as Polysorbate (Tween), or similar detergent, to avoid sticking of the protofibrils to the column matrix and dissociation.
Another aspect of the invention pertains to the development of antibodies that cross-react with wild type Aβ42 protofibrils after immunization with Aβ42arc protofibrils.
Another aspect of the invention pertains to a method to stabilize Aβ protofibril and where stabilization can be assessed by size-exclusion chromatography (SEC). Aβ protofibrils elutes after 12-13 minutes in a uniform peak on a Superdex 75 or similar size-exclusion column. Conformation stability can also be assessed by staining with Congo Red and (electron microscopy), where protofibrils attain a curve-linear structure of 6-10 nm in diameter and <200 nm in length. Lowered temperature has a significant effect on Aβ protofibril conformational stability, Samples should preferably be kept below 20° C., preferably below 5° C., and most between 0° C. and 5° C. Furthermore, agents that decrease the polarity or surface tension or increase viscosity have stabilizing effects on Aβ protofibril conformation. For example, 10-50% glycerol or 0.6-5% Polysorbate (Tween), have stabilizing effects on Aβ protofibrils. These agents and treatments can be added to the Aβ protofibril preparation preferably after the neutralization step in the method to synthesize Aβ protofibrils described above. Addition of these agents before this step is also possible. Increased stability of protofibrils is advantageous when developing monoclonal antibodies and when Aβ protofibrils are used as reagents in immunoassays, such as ELISA, radio-immunoassay (RIA), Western blotting or dot blotting.
The method of stabilizing the Aβ protofibrils includes mixing them with an agent that decreases the solvent polarity or one that lowers the surface tension, such as glycerol or Polysorbate (Tween), or a combination of said agents. The stabilisation can also be achieved by keeping the protofibrils at a temperature below 20° C., preferably 0-5° C. Said methods can also be combined.
The invention further pertains to the use of anti-Aβ protofibril specific antibodies for determinations of Aβ protofibrils in human and animal tissues, for example, cerebrospinal fluid, blood, serum, urine and brain tissue, but is not limited to these tissues, providing for a possible diagnostic method for Alzheimer's disease. Suitable methods for assaying Aβ protofibrils in these tissues as well as in cell cultures using an anti-Aβ protofibril antibody are immunoassays such as ELISA, RIA, Western blotting dot blotting or proximity ligation. The method would be suitable to follow treatment efficacy (protofibril reduction) in clinical trials and suitable as a diagnostic test for Alzheimer's disease or Down's syndrome.
The method of diagnosing or monitoring Alzheimer's disease (AD) or Down's syndrome comprises the steps of labelling the antibody according to the present invention with an agent that can generate a measurable signal, administering said antibody according to a subject having or suspected of having AD or Down's syndrome, measuring the amount of protofibrils bound to the antibody by measuring the signal generated by the agent.
The invention further pertains to the use of an anti-Aβ protofibril antibody in imaging for detection, localization and quantitation of Aβ protofibrils in human and animal tissues. The anti-Aβ protofibril antibody could be label with a radioactive ligand such as I131, C14, H3 or Gallium68, but it is not limited to these radioisotopes, for detection purposes. In addition to labelling with radioactive markers, DNA, fluorescent molecules, enzymes which converts a substrate such that its absorbance can be measured, could also be used. The method will be suitable as a diagnostic tool for Alzheimer's disease or Down's syndrome, Lewybody dementia, vascular dementia, and other neurodegenerative disorders.
A further aspect of the invention pertains to the use of an anti-Aβ protofibril antibody for the prevention or treatment of Alzheimer's disease. Pre-clinical studies in transgenic animal models have demonstrated effects on plaque burden (Bard 2000), reversal of memory deficits (Dodard 2002) and drainage of Aβ from CNS after anti-Aβ antibody treatment. However, the antibodies used in these studies have not been specific to Aβ protofibrils. Administration of an anti-Aβ protofibril antibody, with no or little cross-reactivity towards Aβ monomers and fibrils would be particular suitable for treatment and prevention of Alzheimer's disease. Firstly, it would not bind significantly to fibrillar forms of Aβ and avoid interaction with fibrillar deposits which are prevalent in blood vessel walls, avoiding sever immunoreactions with concomitant serious brain inflammation and encephalitis, which was encountered in the ELAN vaccination study (see at the end of the Background) with their vaccine AN-1792 (Nicoll 2004). Secondly, an anti-Aβ protofibril antibody with low Aβ1-40 and Aβ1-42 monomer cross-reactivity would not bind and interfere with their normal biological functions, thus avoiding side effects.
In said method of prevention or treatment of Alzheimer's disease or Down's a subject having or suspected of having Alzheimer's disease, Down's syndrome, Lewybody dementia, vascular dementia, and other neurodegenerative disorders, is administered the antibody or composition according to the present invention.
The invention also provides a technique whereby anti-Aβ protofibril antibodies can be used to identify and select for epitopes present on Aβ protofibrils but less on Aβ monomers and Aβ fibrils or other Aβ conformational forms. The method is general and is applicable to other amyloids forming protofibrils such as, but not limited to, islet amyloid protein peptide (IAPP, amylin) associated with Type-2 diabetes, prion protein (PrP), alpha-synuclein (Parkinson). Considering the central role of amyloid beta (Aβ) protofibrils in Alzheimer's disease, these antibodies can be used to diagnose or treat Alzheimer's disease. There is a need for a method that specifically can determine protofibrils in different human and animal tissues such as cerebral spinal fluid (CSF), plasma, blood, urine and brain tissue, but is not limited to these tissues. Such a method could be used as a diagnostic method for Alzheimer's disease but also for similar diseases that forms amyloid protofibrils including Parkinson (alpha-synuclein) (Sthilerman 2002), Type-2 diabetes (Islet amyloid polypeptide, IAPP) (WO03/092619 A2), and Creutzfeldt-Jacob Disease and the corresponding animal disease called mad cow disease (Prion protein) (DeMarco 2004), but is not limited to these diseases.
According to another aspect, the present invention relates to the use of anti-Aβ protofibril antibodies for in vitro or in vivo screening of substances that inhibit or modulate Aβ protofibril levels and/or activity in cell cultures or animal models, being potential Alzheimer drugs. Suitable screening systems would be, but are not limited to, cell cultures (HEC or neuroblastoma cells) or for example the Thy-1 APPSweArc transgenic mouse model, (Nilsson L. et al. Swedish patent application 0400707-6, 2004) that expresses human Amyloid Precursor Protein (APP) with the Arctic mutation (E693G) providing increased production of Aβ protofibrils. Alzheimer drug candidates can be administered to these cell cultures or transgenic animal models and their effects on Aβ protofibril levels measured by an immunoassay, using an anti-Aβ protofibril antibody as reagent. Such method would be ideally suitable for identifying potent Alzheimer drug candidates.
Said method for in vitro or in vivo screening of substances that inhibit or modulate Aβ protofibril levels and/or activity in cell cultures or animal models, comprises the steps of administering potential drug candidates to a cell culture or an animal model, administering the antibody according to any the present invention, labelled with an agent that can generate a measurable signal, to said cell culture or animal model, evaluating the effect of said drug candidates by measuring the amount of protofibrils bound to the antibody by measuring the signal generated by the agent.
According to another aspect, the present invention relates to a method of detecting Aβ protofibrils in vitro, comprising labelling the according to the present invention with an agent that can generate a measurable signal, contacting said antibody or the composition comprising the antibody with a biological sample suspected of containing soluble protofibrils, measuring the amount of protofibrils bound to the antibody by measuring the signal generated by the agent. Said method may be an immunoassay. The biological samples tested may be selected from plasma, CSF, brain and other tissues of animal or human origin. The labelling includes labelling with radioactive markers, DNA, fluorescent molecules, enzymes which converts a substrate such that its absorbance can be measured.
All percentages in the description relates to percent volume, unless stated otherwise.
FIG. 1. Size Exclusion Chromatography (SEC) of Human Monomer Aβ40, Protofibril Aβ42Arc, Protofibril wtAβ42 and Fibril wtAβ42 Preparations
Human Aβ monomers and Aβ protofibrils are eluting in 19-20 minutes and at 12-13 minutes, respectively. The protofibril preparations (B and C) are essentially free (<3%) from contamination of other conformational Aβ forms. The analysis of the fibril preparation (D) was done after centrifugation at 17.900×g for 5 minutes at room temp. The supernatant was analysed demonstrating the almost complete absence of any soluble Aβ forms in the fibril preparation.
FIG. 2. Stability Study of Protofibrils
The stability of Protofibril Aβ42Arc and Protofibril wt42 preparations was measured by SEC. A conversion of protofibrils to fibrils was assessed by a decrease in the protofibril peak area (decreased absorbance (AU) at 214 nm elution time 12-13 minutes). The addition of 10-50% glycerol, 0.6% Tween-20 or storage at 0-5° C., all increased the protofibril stability significantly.
FIG. 3. Titration of Mouse Serum after Immunization with Protofibril wtAβ42
Mice were immunized with a protofibril wtAβ42 preparation in Freund complete adjuvant (first injection) and incomplete Freund adjuvant (5-6 boosters). Blood was collected and the antibody titers against Aβ protofibrils were determined. Mouse #2 and #4 showed the highest titers.
FIG. 4. Sandwich-ELISA Method using Anti-mAb 258 for Aβ Protofibril Determination
mAb 258 was coated over night in a microtiterplate. Preparations of wtAβ42 protofibrils, Aβ42Arc protofibrils, wtAβ40 monomers and wtAβ42 fibrils were added in decreasing concentrations and incubated for 1 hour at +4° C. Secondary anti-Aβ antibody (6E10) was added. Finally, a detection ALP (alkaline phosphatase) conjugated antibody (anti-IgG) was added to each well. Detection was achieved by adding ALP substrate. The colour formation was measured in at 405 nm and 492 nm.
mAb 258 bound wtAβ42 protofibrils and slightly less Aβ42Arc protofibrils. No binding was observed to wtAβ40 monomers. The slight binding to wtAβ42 fibrils is probably due to a slight contamination with wtAβ342 protofibrils (see FIG. 1 D).
The following examples are provided for illustration and are not intended to limit the invention to the specific examples provided.
Synthesis of Human Amyloid Beta Peptide (Aβ) of Different Conformations
Synthetic human wild type or mutant forms of Aβ1-42 or Aβ1-40 peptides were purchased from Polypeptide Laboratories GmbH. The peptides had been synthesized by a standard solid phase peptide synthesis procedure. The peptides were subsequently purified with RP-HPLC to a purity in the range of 90-95%. Alternative vendors producing Aβ peptides with similar purity are possible to use as well.
Synthesis of Human Wild Type Aβ1-40 Monomers
A synthetic wtAβ31-40 peptide was dissolved in 1 volumes of 10 mM NaOH pH>10, and 1 volume of cold 2×PBS (pH 7-8) to a concentration of 50 uM. The wtAβ1-40 monomer preparation was centrifuged at 17.900×g at +4° C. for 5 minutes prior to analysis (FIG. 1).
Synthesis of Human Wild Type Aβ1-42 Protofibrils
A synthetic wtAβ1-42 peptide was dissolved in 9 volumes of 10 mM NaOH pH>10, vortexed for 2 minutes and diluted with 1 volume of 10×PBS (pH 7-8). The final peptide concentration was at this point 443 uM. The peptide was further incubated over night at 37° C. After the overnight incubation, the peptide was further diluted with PBS to 50 uM. The sample was centrifuged for 17.900×g for 5 minutes prior to analysis and immunisation.
Synthesis of Human Aβ1-42Arc Protofibrils
A synthetic Aβ1-42Arc (E22G) peptide was dissolved in 1 volumes of 10 mM NaOH pH>10 and 1 volume cold 2×PBS (pH 7-8) to a final peptide concentration of 50 uM. The Aβ1-42Arc protofibrils were formed immediately and the Aβ1-42Arc protofibrils were stabilized by keeping the solution at 0-4° C. before analysis or immunization. The sample was centrifuged at 17.000×g for 5 minutes at +4° C. prior to analysis. Alternative ways to stabilize the Aβ1-42Arc protofibrils were to add glycerol to a final concentration of 5-50% or to add Tween-20 to a final concentration of 0.6%.
Synthesis of Human Wild Type Aβ1-42 Fibrils
A synthetic wtAβ1-42 peptide was dissolved in 1 volumes of double distilled water, vortexed for 2 minutes and diluted with 1 volume of 2×PBS (pH 7-8) and vortexed again for 2 minutes. The final peptide concentration at this point was 50 uM. The wtAβ1-42 protofibril preparation was incubated at 37° C., for 48 hours before analysis. The sample was centrifuged at 17.900×g for 5 minutes prior to SEC analysis. The supernatant was analysed after centrifugation. No centrifugation was performed when Aβ1-42 fibril preparations were analysed by ELISA or dot blotting.
Analysis of Aβ Preparation by Size Exclusion Chromatography (SEC)
A Merck Hitachi D-7000 HPLC LaChrom system, having a diode array detector (DAD) model L-7455 and a model L-7100 pump, was used for the chromatographic analysis of protofibril preparations in combination with a Superdex 75 PC3.2/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden). The chromatographic system separates Aβ monomers from protofibrils, which are eluting at the void volume of the column. The column was equilibrated with 50 mM Na2HPO4.NaH2PO4 (pH 7.4) with 0.15 M NaCl (PBS) and 0.6% Tween 20 and eluted with the same buffer at a flow rate of 0.08 ml/min (pressure was 5-6 bars) at ambient temperature (22° C.). Ten (10 ul) of a 50 uM-100 uM Aβ sample was subjected analysis using a wavelength scan between 200-400 nm. Tween-20 was added to the sample to give a final concentration of 0.6% prior to chromatography. Data was extracted from measurements at 214 and 280 nm. Peak areas were integrated using Merck Hitachi model D-7000 Chromatography Data Station Software. FIG. 1 shows chromatograms of wtAβ1-40 monomer, wtAβ1-42 protofibril, Aβ1-42Arc protofibril and wtAβ1-42 fibril preparations.
Each preparation was essentially free (<3%) of other contaminating conformational Aβ forms except the wtAβ1-40 monomer preparation which contained 6.4% protofibrils (elution time 12-13 minutes).
Protofibrils Specific Monoclonal Antibody Development
A standard procedure was used for monoclonal development. Mice were injected with wtAβ1-42 protofibril preparation. Alternatively, Aβ1-42Arc protofibrils could be used. Every wtAβ1-42 protofibril batch was assayed by SEC and Congo Red (binds β-sheet structures in proteins) before immunisation to ascertain protofibril purity and that the preparation contained β-sheet structure. The procedure used to immunise mice was a standard protocol involving s.c injections in the presence of Freund adjuvant. Each of six mice were immunized with 10 ug wtAβ1-42 protofibrils and subsequently boostered with 30 ug. The sample was mixed 1:1 with Freund complete adjuvant prior to the first injection. For subsequent 5 booster injections incomplete Freund adjuvant was used. Mouse n was not immunized (control). Mice were bleed and blood collected for antibody titer determinations (FIG. 3). Mice were given one more booster (30 ug) and then sacrificed and the spleen collected for hybridom development. The hybridom preparation method used was according to standard procedure (Harlow 1988).
Screening for wtAβ1-42 Protofibril Specific Antibodies
An ELISA method was developed where by hybridom supernatants were screened for antibodies that bind Aβ1-42 protofibrils. Hybridom supernatant #258 showed high protofibril specificity (FIG. 3). The hybridom #258 was reseeded and screened again to ascertain a homogenous cell line. The monoclonal antibody that was produced from the #258 hybridom was defined as monoclonal antibody 258 (mAb258). Alternatively, screening (binding to Aβ protofibrils but not to Aβ monomers or fibrils) can be performed against an antibody phage display library, where the library has been made from RNA isolated from animals immunized with protofibril.
Characterization of mAb 258 by Western Blot Analysis
The aim of the experiment was to determine if mAb 258 cross-reacts with wild type human amyloid precursor protein (wtAPP) or mutated human APP, APPswe, APP swe-arc (Nilsberth 2001, Mullan 1992) all of which contains uncleaved Aβ1-42. Cells were transfected with plasmids expressing human wtAPP, APPswe and APPswe-arc. Cells were subsequently harvested and solubilized and cellular proteins separated by SDS-PAGE and transferred to nitrocellulose filter papers and subsequently incubated with either mAb 258 or mAb 6E10. Specific binding of these antibodies to separated cellular proteins was detected by incubating the filter papers in a secondary anti-mouse IgG/IgM antibody solution and subsequent developed by incubation with Pierce super signal (art nr 34080-P) and a light sensitive film.
mAb 258 showed no binding to wtAPP, APPswe or APPswe-arc nor to wtAβ40 monomer, The commercial mAb 6E10 bound all these forms.
Sandwich ELISA for Protofibril Characterization and Determinations
The specificity of mAb 258 was determined by a sandwich-ELISA. An 96-well ELISA plate was coated with mAb 258 over night at +4° C. After coating, wells were blocked with BSA for 1 hour at room temp. Aβ samples i.e wtAβ40 monomers, wtAβ42 protofibrils, wtAβ42-Arc protofibrils wtAβ42 fibrils, were added to the microtiterplate in 5× dilutions starting with 10 ug/ml. Samples were incubated for 1 hour at +4° C., after which 10 ng/well of a commercial secondary antibody, 6E10 (Signet) was added and incubated for 1 hour at room temp. Detection was achieved by incubation with a commercial ALP-conjugated anti-IgG antibody for 1 hour at room temp. and subsequent incubation with the substrate at room temp. for one hour according to the manufacturer's procedure. Samples were read in a microtiterplate reader (Spectra max 190 Molecular Devices, Sunyvale, USA) at 405 nm and 492 nm. FIG. 4A.
mAb 258 showed little or no cross-reactivity towards wtAβ40 monomers or wtAβ42 fibrils. Concentrations of wtAβ42 protofibrils down to 10 ng/ml were measurable.
Binding of an anti-Aβ42arc protofibril specific antibody (7E4) (Aβ42arc in protofibril conformation was used as a antigen during immunization) was measured in the sandwich-ELISA coated with increasing concentrations of wtAβ42 protofibrils. Detection was achieved by either an Aβ-specific mAb (1C3) as a secondary detection antibody (FIG. 4B) or by the commercial mAb 6E10, as secondary antibody (FIG. 4C). Strong binding of mAb 7E4 was achieved to wtAβ42 protofibrils. Concentration levels as low as 2-5 ng/ml of wtAβ42 protofibrils were measurable. The monoclonal antibodies 4E11 and 10F7 show less strong binding to wtAβ42 protofibrils (FIGS. 4B and 4C).
Axelman K., et. al., Neurol., 51, 1193-1197, 1994
Bard F. et. al., Nature medicine, 6, 916-919, 2000
Cai, X-D. et. al., Science, 259, 514-516, 1993
Citron, M. et. al., Nature, 360, 672-674, 1992
DeMarco et. al., Proc. Natl. Acad. Sci., 101, 2293-2298, 2004
Dodard J-C. et. al. Nature medicine, 5 452-457, 2002
Glenner et. al., Biochem. Biophys. Res. Commun. 120, 885-890, 1984
Hardy, J. et. al., Nature Genet., 1, 233-234, 1992
Hartley D. M. et. al., J. Neuroscience 19, 8876-8884, 1999
Harlow E. et. al., Antibodies: A Laboratory Manual (Cold Spring Harbor) Lab. Press, Plainview, N.Y., 1988
Hoshi M. et. al., Proc. Natl. Acad. Sci., 100, 6370-6375, 2003
Janus C. V. et. al., Nature, 408, 979-982, 2000
Johnston, J. et. al., FEBS Lett., 354, 274-278, 1994
Kayed R. et. al., Science 300, 486-489, 2003
Klyubin et al., J. Physiol 551P, C32, commun., 2003
Lambert M. P. et. al., Proc. Natl. Acad. Sci., 95, 6448-6453, 1998
Lambert M. P. et. al., J. Neurochemistry, 79, 595-605, 2001
Lannfelt, L., et. al., Neurosci. Lett., 168, 254-256, 1994
Lannfelt L. et. al., Neurosci Lett., 199, 203-206, 1995
Masters et. al., Proc. Natl. Acad. Sci., 82, 4245-4249, 1985
McKhann, G., et. al., Neurology, 34, 939-944, 1984
Morgan D. et. al., Nature, 408, 982-985, 2000
Motter R. Et. al., Ann. Neurol., 38, 643-648, 1995
Mullan M. et. al., Nature Genet. 1, 345-347, 1992
Nicoll J. A. R. et. al., Nature medicine, 1-5, on line publ. 17 mars, 2003
Nilsberth C. et. al., Nat. Neurosci. 4, 887-893, 2001
Nilsson L. et. al., Swedish patent application 0400707-6, 2004
O'Nuallain B. et. al., PNAS 99, 1485-1490, 2002
Pirttilä et. al., J. Neurol. Sci., 127, 90-95, 1994
Scheuner D. et. al., Nature Med., 2, 864-869, 1996
Selkoe D. J., Ann. Rev. Cell Biol. 10, 373-403, 1994
Selkoe D. J., Annu. Rev. Neurosci. 17, 489-517, 1994
Seubert P. et. al., Nature, 359, 325-327, 1992
Sigurdsen E. M. et. al., Am. J. Pathol., 105, 439-447, 2001
Sthilerman M. et. al., Biochemistry 41, 3855-3860, 2002
Stine et. al., J. Protein Chem. 15, 193-203, 1996
Walsh D. M. et. al., J. Biol. Chem. 274, 25945-25952, 1999
Walsh D. M. et. al., Nature 416, 535-539, 2001
Weiner H. L. et. al., Ann. Neurol. 48, 567-579, 2000
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS5604102Oct 27, 1993Feb 18, 1997Athena Neurosciences, Inc.Methods of screening for β-amyloid peptide production inhibitorsUS5612486Nov 1, 1993Mar 18, 1997Athena Neurosciences, Inc.Transgenic animals harboring APP allele having swedish mutationUS5679531Jun 7, 1995Oct 21, 1997Bayer CorporationMethods of detecting βA4 peptide species ending at carboxy-terminus residue 42 using monoclonal antibody 369.2BUS5753624Aug 8, 1996May 19, 1998Milkhaus Laboratory, Inc.Materials and methods for treatment of plaquing diseaseUS5817626Mar 14, 1995Oct 6, 1998Praecis Pharmaceuticals IncorporatedModulators of beta-amyloid peptide aggregationUS5850003Jan 22, 1997Dec 15, 1998Athena NeurosciencesTransgenic rodents harboring APP allele having swedish mutationUS5851996Feb 13, 1998Dec 22, 1998Milkhaus Laboratory, Inc.Materials and methods for treatment of plaquing diseasesUS5854204Mar 14, 1996Dec 29, 1998Praecis Pharmaceuticals, Inc.Aβ peptides that modulate β-amyloid aggregationUS5854215Jun 7, 1995Dec 29, 1998Praecis Pharmaceuticals IncorporatedModulators of β-amyloid peptide aggregationUS5985242Aug 27, 1997Nov 16, 1999Praecis Pharmaceuticals, Inc.Modulators of β-amyloid peptide aggregation comprising D-amino acidsUS6054114May 7, 1997Apr 25, 2000Massachusetts Institute Of TechnologyOrganometallic ligands for the localization and quantification of amyloid in vivo and in vitroUS6114133Nov 14, 1994Sep 5, 2000Elan Pharmaceuticals, Inc.Methods for aiding in the diagnosis of Alzheimer's disease by measuring amyloid-β peptide (x-≧41)US6174916Aug 8, 1997Jan 16, 2001Milkhaus Laboratory, Ltd.Methods for treating herpes virus infectionsUS6218506Feb 5, 1997Apr 17, 2001Northwestern UniversityAmyloid β protein (globular assembly and uses thereof)US6245964Dec 10, 1998Jun 12, 2001Elan Pharmaceuticals, Inc.Transgenic rodent comprising APP-SwedishUS6303567Aug 27, 1996Oct 16, 2001Praecis Pharmaceuticals, Inc .Modulators of β-amyloid peptide aggregation comprising D-amino acidsUS6319498Mar 14, 1996Nov 20, 2001Praecis Pharmaceuticals IncorporatedModulators of amyloid aggregationUS7179463Jul 9, 2001Feb 20, 2007Lars LannfeltTreatment of alzheimer's diseaseUS7427392Jun 6, 1995Sep 23, 2008Elan Pharmaceuticals, Inc.Methods for aiding in the diagnosis of alzheimer's disease by measuring amyloid-β peptide (x-≧41) and tauUS20020162129Jul 9, 2001Oct 31, 2002Lars LannfeltPrevention and treatment of alzheimer's diseaseUS20030068316Jun 11, 2002Apr 10, 2003Klein William L.Anti-ADDL antibodies and uses thereofUS20030187011Dec 17, 2002Oct 2, 2003Lashuel Hilal A.Apomorphine inhibitors of amyloid-beta (Abeta) fibril formation and their use in amyloidosis based diseaseUS20030232758Apr 10, 2003Dec 18, 2003Hospital For Sick Children And University Of TorontoImmunological methods and compositions for the treatment of Alzheimer's diseaseUS20040049134Jul 1, 2003Mar 11, 2004Tosaya Carol A.System and methods for treatment of alzheimer's and other deposition-related disorders of the brainUS20040170641Mar 31, 2004Sep 2, 2004Neuralab LimitedPrevention and treatment of amyloidogenic diseaseUS20040171815Nov 7, 2003Sep 2, 2004Schenk Dale B.Humanized antibodies that recognize beta amyloid peptideUS20040171816Nov 7, 2003Sep 2, 2004Schenk Dale B.Humanized antibodies that recognize beta amyloid peptideUS20050031629Mar 31, 2004Feb 10, 2005Neuralab LimitedPrevention and treatment of amyloidogenic diseaseUS20050124016Aug 2, 2004Jun 9, 2005Enh Research InstituteAntibodies specific for toxic amyloid beta protein oligomersUS20050142132Feb 14, 2005Jun 30, 2005Neuralab LimitedPrevention and treatment of amyloidogenic diseaseUS20050191314Apr 15, 2005Sep 1, 2005Neuralab LimitedPrevention and treatment of amyloidogenic diseaseUS20050249725Feb 27, 2004Nov 10, 2005Schenk Dale BHumanized antibodies that recognize beta amyloid peptideUS20060079447Oct 7, 2005Apr 13, 2006Wetzel Ronald BStabilized A-beta protofibrillar aggregatesUS20060166275May 16, 2005Jul 27, 2006Northwestern University & University Of Southern CaliforniaAmyloid beta protein (globular assembly and uses thereof)US20060178302Apr 6, 2005Aug 10, 2006Northwestern University & The University Of Southern CaliforniaAmyloid beta protein (globular assembly and uses thereof)US20060193850Jan 27, 2006Aug 31, 2006Warne Nicholas WAnti a beta antibody formulationUS20060228349Oct 21, 2005Oct 12, 2006Paul ActonAnti-ADDL antibodies and uses thereofUS20060240486Dec 15, 2005Oct 26, 2006Johnson-Wood Kelly LImmunoprecipitation-based assay for predicting in vivo efficacy of beta-amyloid antibodiesUS20060280733Sep 12, 2003Dec 14, 2006The Regents Of The University Of CaliforniaImmunogens and corresponding antibodies specific for high molecular weight aggregation intermediates common to amyloids formed from proteins of differing sequenceUS20070048312Jul 31, 2006Mar 1, 2007Klein William LAnti-ADDL antibodies and uses thereofUS20070081998Oct 17, 2006Apr 12, 2007Gene KinneyAnti-ADDL monoclonal antibody and use thereofUS20070098721Feb 2, 2004May 3, 2007Heinz HillenAmyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereofUS20070099185Nov 4, 2003May 3, 2007Bioarctic Neuroscience AbMethods for the identification of agents that modulate the structure and processing of beta-amyloid precursor proteinUS20070110750Sep 13, 2004May 17, 2007The Regents Of The University Of CaliforniaMonoclonal antibodies specific for high molecular weight aggregation intermediates common to amyloids formed from proteins of differing sequenceUS20070148167Oct 17, 2006Jun 28, 2007Strohl William RNon-immunostimulatory antibody and compositions containing the sameUS20080181902Mar 19, 2008Jul 31, 2008Lars LannfeltPrevention and treatment of alzheimer's diseaseEP0783104A1Dec 17, 1996Jul 9, 1997Oriental Yeast Co., Ltd.Method for assaying soluble amyloid precursor proteinWO1991016819A1Mar 26, 1991Nov 14, 1991Molecular Rx., Inc.Method and composition for treatment of central nervous systems disease states associated with abnormal amyloid beta proteinWO1995011994A1Oct 17, 1994May 4, 1995Athena Neurosciences, Inc.Methods of screening for beta-amyloid peptide production inhibitorsWO1995031996A1May 25, 1995Nov 30, 1995Milkhaus LabMaterials and methods for treatment of plaquing diseasesWO1996015452A1Nov 13, 1995May 23, 1996Athena Neurosciences, Inc.METHODS FOR AIDING IN THE DIAGNOSIS OF ALZHEIMER'S DISEASE BY MEASURING AMYLOID-β PEPTIDE (X- ≥41) AND TAUWO1997041856A1May 7, 1997Nov 13, 1997Massachusetts Institute Of TechnologyORGANOMETALLIC LIGANDS FOR THE LOCALIZATION AND QUANTIFICATION OF AMYLOID IN VIVO AND $i(IN VITRO)WO1998033815A1Feb 5, 1998Aug 6, 1998Northwestern UniversityAmyloid beta protein (globular assembly and uses thereof)WO1999027944A1Nov 30, 1998Jun 10, 1999Neuralab LimitedPrevention and treatment of amyloidogenic diseaseWO1999027949A1Dec 3, 1998Jun 10, 1999Brigham And Women's HospitalMETHOD OF SUPPRESSING β-AMYLOID-RELATED CHANGES IN ALZHEIMER'S DISEASEWO2000039310A1Dec 29, 1999Jul 6, 2000The University Of Georgia Research Foundation, Inc.Rubredoxin fusion proteins, protein expression system and methodsWO2000071671A2May 26, 2000Nov 30, 2000New York UniversityNew mutant genes in familial british dementia and familial danish dementiaWO2000072870A1Jun 1, 2000Dec 7, 2000Neuralab, Ltd.Compositions of a-beta peptide and processes for producing sameWO2000072876A2Jun 1, 2000Dec 7, 2000Neuralab LimitedPrevention and treatment of amyloidogenic diseaseWO2000072880A2May 26, 2000Dec 7, 2000Neuralab LimitedPrevention and treatment of amyloidogenic diseaseWO2001010900A2Aug 4, 2000Feb 15, 2001University Of Southern CaliforniaGlobular assembly of amyloid beta protein and uses thereofWO2001039796A2Nov 29, 2000Jun 7, 2001Neurochem Inc.Vaccine for the prevention and treatment of alzheimer's and amyloid related diseasesWO2001090182A2May 22, 2001Nov 29, 2001New York UniversitySynthetic immunogenic but non-amyloidogenic peptides homologous to amyloid beta for induction of an immune response to amyloid beta and amyloid depositsWO2002003911A2Jul 5, 2001Jan 17, 2002Lars LannfeltPrevention and treatment of alzheimer's diseaseWO2003089460A1Apr 7, 2003Oct 30, 2003The Governing Council Of The University Of TorontoImmunological methods and compositions for the treatment of alzheimer's diseaseWO2003104437A2Jun 11, 2003Dec 18, 2003Northwestern UniversityAnti-addl antibodies and uses thereofWO2004024090A2Sep 12, 2003Mar 25, 2004The Regents Of The University Of CaliforniaImmunogens and corresponding antibodies specific for high molecular weight aggregation intermediates common to amyloids formed from proteins of differing sequenceWO2004031400A2Oct 1, 2003Apr 15, 2004Northwestern UniversityAmyloid beta-derived diffusible ligands (addls), addl-surrogates, addl-binding molecules, and uses thereofWO2005019828A1Aug 20, 2004Mar 3, 2005Amorfix Life Sciences Ltd.Epitope protection assay and method for detecting protein conformationsWO2005025516A2Sep 13, 2004Mar 24, 2005The Regents Of The University Of CaliforniaMonoclonal antibodies specific for conformational epitopes of prefibrillar aggregatesWO2005089539A1Mar 17, 2005Sep 29, 2005Bioarctic Neuroscience AbTransgenic model for alzheimer’s diseaseWO2005123775A1Jun 21, 2005Dec 29, 2005Bioarctic Neuroscience AbAntibodies specific for soluble amyloid beta peptide protofibrils and uses thereofWO2006014478A1Jul 5, 2005Feb 9, 2006Northwestern UniversityMONOLOCAL ANTIBODIES THAT TARGET PATHOLOGICAL ASSEMBLIES OF AMYLOID β (ABETA)WO2006047254A1Oct 21, 2005May 4, 2006Regents Of The University Of MinnesotaAssemblies of oligomeric amyloid beta protein and uses thereofWO2006055178A2Oct 21, 2005May 26, 2006Merck & Co., Inc.Anti-addl antibodies and uses thereofWO2006066233A1Dec 15, 2005Jun 22, 2006Neuralab LimitedAn immunoprecipitation-based assay for predicting in vivo efficacy of beta-amyloid antibodiesWO2006083533A2Jan 17, 2006Aug 10, 2006The Regents Of The University Of CaliforniaCompositions and methods for inhibiting drusen formation and for diagnosing or treating drusen-related disordersWO2006094724A2Mar 3, 2006Sep 14, 2006Abbott Gmbh & Co. KgScreening method, process for purifying of non-diffusible a-beta oligomers, selective antibodies against said non-diffusible a-beta oligomers and a process for manufacturing of said antibodiesWO2006137354A1Jun 19, 2006Dec 28, 2006Medical & Biological Laboratories Co., Ltd.Antibody having inhibitory effect on amyloid fibril formationWO2007005358A2Jun 26, 2006Jan 11, 2007Merck & Co., Inc.Method for preparing a covalently cross linked oligomer of amyloid beta peptidesWO2007005359A1Jun 26, 2006Jan 11, 2007Merck & Co., Inc.Composition and method for producing stable amyloid beta oligomers of high molecular weightWO2007050359A2Oct 17, 2006May 3, 2007Merck & Co., Inc.Anti-addl monoclonal antibody and use thereofWO2007062088A1Nov 21, 2006May 31, 2007The Trustees Of The University Of PennsylvaniaAntibody treatment of alzheimer's and related diseasesWO2007108756A1Mar 23, 2007Sep 27, 2007Bioarctic Neuroscience AbImproved protofibril selective antibodies and the use thereofNon-Patent CitationsReference1Advisory Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Feb. 20, 2004.2Andreasen and Blennow, "Beta-amyloid (Abeta) Protein in Cerebrospinal Fluid as a Biomarker for Alzheimer's Disease," Peptides 23:1205-1214, 2002.3Appellant's Statement of Grounds of Appeal together with a Main Request, First Auxiliary Request and Second Auxiliary Request for consideration by the Appeal Board, and a signed Declaration by Professor Dominic Walsh of the Conway Institute, Dublin, IE as filed in European Patent Application No. 01945896.7, Jan. 15, 2010.4Axelman et al., "A Large Swedish Family with Alzheimer's Disease with a Codon 670/671 Amyloid Precursor Protein Mutation," Arch. Neurol. 51:1193-1197, 1994.5Axelman, et al., "A Large Swedish Family with Alzheimer's Disease with a Codon 670/671 Amyloid Precursor Protein Mutation", Arch. Neurol., 51:1193-1197 (1994).6Bacskai et al., "Imaging of Amyloid-beta Deposits in Brains of Living Mice Permits Direct Observation of Clearance of Plaques with Immunotherapy," Nature Med. 7(3):369-372, 2001.7Bacskai et al., "Imaging of Amyloid-β Deposits in Brains of Living Mice Permits Direct Observation of Clearance of Plaques with Immunotherapy," Nature Med. 7(3):369-372, 2001.8Bard et al., "Peripherally Administered Antibodies Against Amyloid beta-Peptide Enter the Central Nervous System and Reduce Pathology in a Mouse Model of Alzheimer Disease," Nature Med. 6(8):916-919, 2000.9Bard et al., "Peripherally Administered Antibodies Against Amyloid β-Peptide Enter the Central Nervous System and Reduce Pathology in a Mouse Model of Alzheimer Disease," Nature Med. 6(8):916-919, 2000.10Bard, et al., "Peripherally administered antibodies against amyloid B-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease", Nature Medicine 6(8):916-919 (Aug. 2000).11Barghorn et al., "Globular Amyloid beta-Peptide Oligomer: A Homogenous and Stable Neuropathological Protein in Alzheimer's Disease," J. Neurochem. 95:834-47, 2005.12Barghorn et al., "Globular Amyloid β-Peptide Oligomer: A Homogenous and Stable Neuropathological Protein in Alzheimer's Disease," J. Neurochem. 95:834-47, 2005.13Bayer et al., "Evaluation of the Safety and Immunogenicity of Synthetic Abeta42 (AN1792) in Patients with AD," Neurology 64:94-101, 2005.14Bayer et al., "Evaluation of the Safety and Immunogenicity of Synthetic Aβ42 (AN1792) in Patients with AD," Neurology 64:94-101, 2005.15Bitan et al., "Amyloid beta-Protein (Abeta) Assembly: Abeta40 and Abeta42 Oligomerize Through Distinct Pathways," Proc. Natl. Acad. Sci. U.S.A. 100:330-5, 2003.16Bitan et al., "Amyloid β-Protein (Aβ) Assembly: Aβ40 and Aβ42 Oligomerize Through Distinct Pathways," Proc. Natl. Acad. Sci. U.S.A. 100:330-5, 2003.17Blanchard et al., "Efficient Reversal of Alzheimer's Disease Fibril Formation and Elimination of Neurotoxicity by a Small Molecule," Proc. Natl. Acad. Sci. U.S.A. 101(40):14326-32, 2004.18Cai et al., "Release of Excess Amyloid beta-Protein from a Mutant Amyloid beta-Protein Precursor," Science 259:514-516, 1993.19Cai et al., "Release of Excess Amyloid β-Protein from a Mutant Amyloid β-Protein Precursor," Science 259:514-516, 1993.20Cai, et al., "Release of Excess Amyloid b Protein from a Mutant Amyloid b Protein Precursor", Science, New Series, 259:514-516 (Jan. 22, 1993).21Caughey and Lansbury, "Protofibrils, Pores, Fibils, and Neurodegeneration: Separating the Responsible Protein Aggregates from the Innocent Bystanders," Ann. Rev. Neurosci. 26:267-98, 2003.22Chen et al., "A Learning Deficit Related to Age and beta-Amyloid Plaques in a Mouse Model of Alzheimer's Disease," Nature 408:975-978, 2000.23Chen et al., "A Learning Deficit Related to Age and β-Amyloid Plaques in a Mouse Model of Alzheimer's Disease," Nature 408:975-978, 2000.24Chromy et al., "Self-Assembly of Abeta1-42 Into Globular Neurotoxins," Biochemistry 42:12749-12760, 2003.25Chromy et al., "Self-Assembly of Aβ1-42 Into Globular Neurotoxins," Biochemistry 42:12749-12760, 2003.26Chromy et al., "Stability of Small Oligomers of Abeta1-42(ADDLs)," Society for Neuroscience 25:2129; 852.5, 1999.27Chromy et al., "Stability of Small Oligomers of Aβ1-42(ADDLs)," Society for Neuroscience 25:2129; 852.5, 1999.28Chromy, et al., "Self-Assembly of Aβ1-42 into Globular Neurotoxins", Biochemistry, 42:12749-12760 (2003).29Citron et al., "Mutant Presenilins of Alzheimer's Disease Increase Production of 42-Residue Amyloid beta-Protein in Both Transfected Cells and Transgenic Mice," Nature Med. 3(1):67-72, 1997.30Citron et al., "Mutant Presenilins of Alzheimer's Disease Increase Production of 42-Residue Amyloid β-Protein in Both Transfected Cells and Transgenic Mice," Nature Med. 3(1):67-72, 1997.31Citron et al., "Mutation of the beta-Amyloid Precursor Protein in Familial Alzheimer's Disease Increases beta-Protein Production," Nature 360:672-674, 1992.32Citron et al., "Mutation of the β-Amyloid Precursor Protein in Familial Alzheimer's Disease Increases β-Protein Production," Nature 360:672-674, 1992.33Citron, et al., "Mutation of the β-amyloid precursor protein in familial Alzheimer's disease increases β-protein production", Nature, 360:672-674 (Dec. 17, 1992).34Communication as issued by European Patent Office regarding extended European Search Report for European Patent Application No. 07747965.7, dated May 13, 2009.35Communication from European Patent Application No. 05753672.4-2402, mailed Jul. 6, 2009.36Communication from European Patent Application No. 07747965.7, Nov. 17, 2009.37Communication of Notice of Opposition for European Patent Application No. 07747965.7, dated Oct. 13, 2011.38Conway et al., "Acceleration of Oligomerization, Not Fibrillization, is a Shared Property of Both alpha-Synuclein Mutations Linked to Early-Onset Parkinson's Disease: Implications for Pathogenesis and Therapy," Proc. Natl. Acad. Sci. U.S.A. 97(2):571-576, 2000.39Conway et al., "Acceleration of Oligomerization, Not Fibrillization, is a Shared Property of Both α-Synuclein Mutations Linked to Early-Onset Parkinson's Disease: Implications for Pathogenesis and Therapy," Proc. Natl. Acad. Sci. U.S.A. 97(2):571-576, 2000.40Dahlgren et al., "Oligomeric and Fibrillar Species of Amyloid-beta Peptides Differentially Affect Neuronal Viability," J. Biol. Chem. 277:32046-53, 2002.41Dahlgren et al., "Oligomeric and Fibrillar Species of Amyloid-β Peptides Differentially Affect Neuronal Viability," J. Biol. Chem. 277:32046-53, 2002.42Dalfo et al., "Evidence of Oxidative Stress in the Neocortex in Incidental Lewy Body Disease," J. Neuropathol. Exp. Neurol. 64(9):816-830, 2005.43De Jonghe et al., "Flemish and Dutch Mutations in Amyloid beta Precursor Protein Have Different Effects on Amyloid beta Secretion," Neurobiol. Dis. 5:281-286, 1998.44De Jonghe et al., "Flemish and Dutch Mutations in Amyloid β Precursor Protein Have Different Effects on Amyloid β Secretion," Neurobiol. Dis. 5:281-286, 1998.45Declaration of Pär Gellerfors, dated Nov. 28, 2010.46Declaration Under Rule 132 (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Mar. 9, 2006.47DeMarco et al., "From Conversion to Aggregation: Protofibril Formation of the Prion Protein," Proc. Natl. Acad. Sci. U.S.A. 101:2293-2298, 2004.48DeMarco, et al., "From conversion to aggregation: Protofibril formation of the prion protein", PNAS., 101:2293-2298 (Feb. 24, 2004).49Dennis J. Selkoe, "Cell Biology of the Amyloid B-Protein Precursor and the Mechanism of Alzheimer's Disease", Annu. Rev. Cell Biol., 10:373-403 (1994).50Dennis J. Selkoe, "Normal and Abnormal Biology of the B-Amyloid Precursor Protein", Annu. Rev. Neurosci., 17:489-517 (1994).51Dodart et al., "Immunization Reverses Memory Deficits Without Reducing Brain Abeta Burden in Alzheimer's Disease Model," Nature Neurosci. 5:452-457, 2002.52Dodart et al., "Immunization Reverses Memory Deficits Without Reducing Brain Aβ Burden in Alzheimer's Disease Model," Nature Neurosci. 5:452-457, 2002.53Dodart, et al., "Immunization reverses memory deficits without reducing brain AB burden in alzheimer's disease model", Nature Neuroscience, 5:452-457 (May 2002).54El-Agnaf et al., "Oligomerization and Toxicity of β-Amyloid-42 Implicated in Alzheimer's Disease," Biochem. Biophys. Res. Comm. 273:1003-1007, 2000.55Enya et al., "Appearance of Sodium Dodecyl Sulfate-Stable Amyloid β-Protein (Aβ) Dimer in the Cortex During Aging," Am. J. Pathol. 154:271-279, 1999.56European Examination Report (EP 01 945 896.7), dated Apr. 24, 2007.57European Examination Report (EP 01 945 896.7), dated May 22, 2006.58European Examination Report (EP 01 945 896.7), dated Sep. 30, 2005.59Evidence D1-D6 included in Opposition Against European Patent No. 2,004,688 as filed by Acumen Pharmaceuticals dated Sep. 22, 2011.60Evidence D7-D11 included in Opposition Against European Patent No. 2,004,688 as filed by Acumen Pharmaceuticals dated Sep. 22, 2011.61Extended European Search Report for European Patent Application No. 08019830.2, May 31, 2010.62Extended European Search Report from European Patent Application No. 07747965.7, dated May 13, 2009.63Finder and Glockshuber, "Amyloid-β Aggregation," Neurodegener. Dis. 4(1):13-27, 2007.64Forsell et al., "Amyloid Precursor Protein Mutation at Codon 713 (Ala ->Val) Does Not Cause Schizophrenia: Non-Pathogenic Variant Found at Codon (Silent)," Neurosci. Lett. 184:90-93, 1995.65Forsell et al., "Amyloid Precursor Protein Mutation at Codon 713 (Ala →Val) Does Not Cause Schizophrenia: Non-Pathogenic Variant Found at Codon (Silent)," Neurosci. Lett. 184:90-93, 1995.66Frackowiak et al., "Non-Fibrillar β-Amyloid Protein is Associated with Smooth Muscle Cells of Vessel Walls in Alzheimer Disease," J. Neuropathol. Exp. Neurol. 53:637-645, 1994.67Frenkel et al, "Modulation of Alzheimer's Beta-amyloid Neurotoxicity by Site-directed Single-chain Antibody," J. Neuroimmunol. 106:23-31, 2000.68Frenkel et al, "Modulation of Alzheimer's Beta-amyloid Neurotoxicity by Site-directed Single-chain Antibody," Neuroimmunomodulation 6:444, 1999.69Frenkel et al., "Immunization Against Alzheimer's β-Amyloid Plaques Via EFRH Phage Administration," Proc. Natl. Acad. Sci. U.S.A. 97(21):11455-11459, 2000.70Giasson et al., "A Panel of Epitope-Specific Antibodies Detects Protein Domains Distributed Throughout Human Alpha-Synuclein in Lewy Bodies of Parkinson's Disease," J. Neurosci. Res. 59:528-533, 2000.71Giulian et al., "The HHQK Domain of β-Amyloid Provides a Structural Basis for the Immunopathology of Alzheimer's Disease," J. Biol. Chem. 273(45):29719-29726, 1998.72Glenner et al., "Alzheimer's Disease: Initial Report of the Purification and Characterization of a Novel Cerebrovascular Amyloid Protein," Biochem. Biophys. Res. Comm. 120(3):885-890, 1984.73Glenner, et al., "Alzheimer's Disease: Initial Report of the Purification and Characterization of a Novel Cerebrovascular Amyloid Protein", Biochemical and Biophysical Research Communications, 120(3):885-890 (1984).74Golabek et al., "The Interaction between Apolipoprotein E and Alzheimer's Amyloid β-Peptide Is Dependent on β-Peptide Conformation," J. Biol. Chem. 271(18):10602-10606, 1996.75Grabowski et al., "Novel Amyloid Precursor Protein Mutation in an Iowa Family with Dementia and Severe Cerebral Amyloid Angiopathy," Ann. Neurol. 49:697-705, 2001.76Guerette et al., "Oligomeric Aβ in PBS-Soluble Extracts of Human Alzheimer Brain," Society for Neuroscience 25:2129; 852.1, 1999.77Hardy, "Amyloid, the Presenilins and Alzheimer's Disease," Trends Neurosci. 20(4):154-159, 1997.78Hardy, "Framing β-Amyloid," Nature Genet. 1:233-234, 1992.79Harley, et al., "Protofibrillar Intermediates of Amyloid β-Protein Induce Acute Electrophysiological Changes and Progressive Neurotoxicity in Cortical Neurons", The Journal of Neuroscience, 19(20):8876-8884 (Oct. 15, 1999).80Harper et al., "Assembly of Aβ Amyloid Protofibrils: An In Vitro Model for a Possible Early Event in Alzheimer's Disease," Biochemistry 38:8972-8980, 1999.81Harper et al., "Observation of Metastable Aβ Amyloid Protofibrils by Atomic Force Microscopy," Chem. Biol. 4:119-125, 1997.82Hartley et al., "Protofibrillar Intermediates of Amyloid β-Protein Induce Acute Electrophysiological Changes and Progressive Neurotoxicity in Cortical Neurons," J. Neurosci. 19(20):8876-8884, 1999.83Hendriks et al., "Presenile Dementia and Cerebral Hemorrhage Linked to a Mutation at Codon 692 of the β-Amyloid Precursor Protein Gene," Nature Genet. 1:218-221, 1992.84Hock and Nitsch, "Clinical Observations with AN-1792 Using TAPIR Analyses," Neurodegener Dis. 2:273-276, 2005.85Hoshi et al., "Spherical Aggregates of β-Amyloid (Amylospheroid) Show High Neurotoxicity and Activate Tau Protein Kinase I/glycogen Synthase Kinase-3β," Proc. Natl. Acad. Sci. U.S.A. 100(11):6370-6375, 2003.86Hoshi, et al., "Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3β", PNAS, 100(11):6370-6375 (May 27, 2003).87International Preliminary Report (PCT/US2003/30930), completed Feb. 6, 2006.88International Preliminary Report on Patentability (PCT/SE01/01553), completed Oct. 23, 2002.89International Preliminary Report on Patentability (PCT/SE05/000993), issued Dec. 28, 2006.90International Preliminary Report on Patentability (PCT/SE07/000292), issued Sep. 23, 2008.91International Preliminary Report on Patentability (PCT/US2003/19640), completed Aug. 7, 2006.92International Search Report (PCT/SE01/01553), mailed Feb. 4, 2002.93International Search Report (PCT/SE05/000993), mailed Oct. 4, 2005.94International Search Report (PCT/SE07/000292), mailed Jul. 20, 2007.95Interview Summary (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), dated Oct. 21, 2003.96Invitation Pursuant to Article 94(3) and Rule 71(1) EPC from European Patent Application No. 07747965.7-1222, Mar. 26, 2010.97Isaacs et al., "Acceleration of Amyloid β-Peptide Aggregation by Physiological Concentrations of Calcium," J. Biol. Chem. 281(38):27916-23, 2006.98Janus et al., "A β Peptide Immunization Reduces Behavioral Impairment and Plaques in a Model of Alzheimer's Disease," Nature 408:979-982, 2000.99Janus, et al., "Aβ peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease", Nature, 408:979-982 (Dec. 2000).100Jensen et al., "Quantification of Alzheimer Amyloid beta Peptides Ending at Residues 40 and 42 by Novel ELISA Systems," Mol. Med. 6:291-302, 2000.101Jensen et al., "Quantification of Alzheimer Amyloid β Peptides Ending at Residues 40 and 42 by Novel ELISA Systems," Mol. Med. 6:291-302, 2000.102Johansson et al., "Physiochemical Characterization of the Alzheimer's Disease-Related Peptides Abeta1-42 Arctic and Abeta1-42 wt" FEBS J. 273:2618-2630, 2006.103Johansson et al., "Physiochemical Characterization of the Alzheimer's Disease-Related Peptides Aβ1-42 Arctic and Aβ1-42 wt" FEBS J. 273:2618-2630, 2006.104John Hardy, "Framing β-amyloid", Nature Genetics, 1:233-234 (Jul. 1992).105Johnston et al., "Increased beta-Amyloid Release and Levels of Amyloid Precursor Protein (APP) in Fibroblast Cell Lines From Family Members With the Swedish Alzheimer's Disease APP670/671 Mutation," FEBS Lett. 354:274-278, 1994.106Johnston et al., "Increased β-Amyloid Release and Levels of Amyloid Precursor Protein (APP) in Fibroblast Cell Lines From Family Members With the Swedish Alzheimer's Disease APP670/671 Mutation," FEBS Lett. 354:274-278, 1994.107Johnston, et al., "Increased β-amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimer's disease APP670/671 mutation", FEBS Letters, 354:274-278 (1994).108Kamino et al., "Linkage and Mutational Analysis of Familial Alzheimer Disease Kindreds for the APP Gene Region," Am. J. Hum. Genet. 51:998-1014, 1992.109Kang et al., "The Precursor of Alzheimer's Disease Amyloid A4 Protein Resembles a Cell-surface Receptor," Nature 325:733-736, 1987.110Kayed et al., "Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis," Science 300:486-489, 2003.111Kayed et al., "Immunization With a Molecular Mimic of a Toxic Aggregates Generates a Conformation-Dependent Antibody Specific for High Molecular Weight A Aggregates (Micelles and Protofibrils)," 32nd Annual Meeting of the Society for Neuroscience, Orlando, Florida (Society for Neuroscience Abstract Viewer and Itinerary Planner, Abstract No. 685.3, 2002).112Kayed, et al., "Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis", Science, 300:486-489 (Apr. 18, 2003).113Kirkitadze et al., "Paradigm Shifts in Alzheimer's Disease and Other Neurodegenerative Disorders: The Emerging Role of Oligomeric Assemblies," J. Neurosci. Res. 1, 69:567-77, 2002.114Klafki et al., "Therapeutic Approaches to Alzheimer's Disease," Brain 129:2840-2855, 2006.115Klein et al., "Oligemia-Induced Expression of c-fos and Oxidative Stress-Related Protein in the Murine Brain," 30th Annual Meeting of the Society of Neuroscience, New Orleans, LA, Nov. 4-9, 2000, Soc. Neurosci. Abstracts 26(1-2), Abstract 383.15, 2000.116Klein et al., "Oligomer/Conformation-Dependent Aβ Antibodies," Soc. Neurosci. Abstr. Presentation No. 475.11, Tuesday Nov. 7, 2000.117Klein et al., "Targeting Small Aβ Oligomers: The Solution to an Alzheimer's Disease Conundrum?" Trends Neurosci. 24:219-224, 2001.118Klein, "Aβ Toxicity in Alzheimer's Disease," Contemporary Clinical Neuroscience: Molecular Mechanisms of Neurodegenerative Diseases 1.1 Introduction, 2001.119Klyubin et al., "Inhibitory Effect of Amyloid-beta Peptide with the Arctic Mutation on Long-term Potentiation in Area CA1 of Rat Hippocampus In Vivo," J. Physiol. 551P, C32, 2003.120Klyubin et al., "Inhibitory Effect of Amyloid-β Peptide with the Arctic Mutation on Long-term Potentiation in Area CA1 of Rat Hippocampus In Vivo," J. Physiol. 551P, C32, 2003.121Klyubin et al., "Soluble Arctic Amyloid β Protein Inhibits Hippocampal Long-Term Potentiation In Vivo," Eur. J. Neurosci. 19:2839-2846, 2004.122Klyubin, et al., "Soluble Arctic amyloid β protein inhibits hippocampal long-term potentiation in vivo", European Journal of Neuroscience, 19:2839-2846 (2004).123Kuo et al., "Water-soluble Aβ (N-40, N-42) Oligomers in Normal and Alzheimer Disease Brains," J. Biol. Chem. 271:4077-4081, 1996.124Lambert et al., "Diffusible, Nonfibrillar Ligands Derived From Aβ1-42 Are Potent Central Nervous System Neurotoxins," Proc. Natl. Acad. Sci. U.S.A. 95:6448-6453, 1998.125Lambert et al., "Monoclonal Antibodies that Target Pathological Assemblies of Aβ," J. Neurochem. 100:23-35, 2007.126Lambert et al., "Neuron Dysfunction and Death Caused by Small Aβ Oligomers: Role of Signal Transduction," Society for Neuroscience 25:2129, 1999.127Lambert et al., "Vaccination With Soluble Aβ Oligomers Generates Toxicity-Neutralizing Antibodies," J. Neurochem. 79:595-605, 2001.128Lambert, et al., "Diffusible, nonfibrillar ligands derived from Aβ1-42 are potent central nervous system neurotoxins", Proc. Natl. Acad. Sci. USA, 95:6448-6453 (May 1998).129Lambert, et al., "Vaccination with soluble Aβ oligomers generates toxicity-neutralizing antibodies", Journal of Neurochemistry, 79:595-605 (2001).130Lannfelt et al., "Amyloid beta-Peptide in Cerebrospinal Fluid in Individuals with the Swedish Alzheimer Amyloid Precursor Protein Mutation," Neurosci. Lett. 199:203-206, 1995.131Lannfelt et al., "Amyloid Precursor Protein Mutation Causes Alzheimer's Disease in a Swedish Family," Neurosci. Lett. 168:254-256, 1994.132Lannfelt et al., "Amyloid β-Peptide in Cerebrospinal Fluid in Individuals with the Swedish Alzheimer Amyloid Precursor Protein Mutation," Neurosci. Lett. 199:203-206, 1995.133Lannfelt et al., "Genetics of Alzheimer's Disease-Routes to the Pathophysiology," J. Neural. Transm. [Suppl.] 59:155-161, 2000.134Lannfelt et al., "Genetics of Alzheimer's Disease—Routes to the Pathophysiology," J. Neural. Transm. [Suppl.] 59:155-161, 2000.135Lannfelt et al., "Genetics, Pathophysiology and Abeta Protofibril Formation in Alzheimer's Disease," Neurobiol. Aging 25(Suppl. 2): Poster Session P2: Epidemiology and Risk Factors of Alzheimer's Disease P2-268; S308, 2004.136Lannfelt et al., "Genetics, Pathophysiology and Aβ Protofibril Formation in Alzheimer's Disease," Neurobiol. Aging 25(Suppl. 2): Poster Session P2: Epidemiology and Risk Factors of Alzheimer's Disease P2-268; S308, 2004.137Lannfelt et al., "Monoclonal Antibodies Selective for Abeta Protofibrils Reduce Plaque Sensitive Detection of Alzheimer Abeta Protofibrils by Burden in Transgenic Mice Models of Alzheimer's Disease Conformation Specific ELISA," ICAD meeting, Uppsala University, Sweden, Jul. 16, 2006.138Lannfelt et al., "Monoclonal Antibodies Selective for Abeta Protofibrils: Detection of Protofibrils and Reduction of Plaque Burden in Tg-mice Models of Alzheimer's Disease," SfN meeting, Uppsala University, Sweden, Oct. 17, 2006.139Lannfelt et al., "Monoclonal Antibodies Selective for Aβ Protofibrils Reduce Plaque Sensitive Detection of Alzheimer Aβ Protofibrils by Burden in Transgenic Mice Models of Alzheimer's Disease Conformation Specific ELISA," ICAD meeting, Uppsala University, Sweden, Jul. 16, 2006.140Lannfelt et al., "Monoclonal Antibodies Selective for Aβ Protofibrils: Detection of Protofibrils and Reduction of Plaque Burden in Tg-mice Models of Alzheimer's Disease," SfN meeting, Uppsala University, Sweden, Oct. 17, 2006.141Lannfelt, et al., "Amyloid Precursor Protein Mutation Causes Alzheimer's Disease in a Swedish Family", Neuroscience Letters, 168:254-256 (1994).142Lannfelt, et al., "Amyloid β-peptide in Cerebrospinal Fluid in Individuals with the Swedish Alzheimer Amyloid Precursor Protein Mutation", Neuroscience Letters, 199:203-206 (1995).143Lashuel et al., "Mixtures of Wild-Type and a Pathogenic (E22G) Form of Aβ40 In Vitro Accumulate Protofibrils, Including Amyloid Pores," J. Mol. Biol. 332:795-808, 2003.144Lashuel, et al., "Mixtures of Wild-type and a Pathogenic (E22G) Form of Aβ40 in Vitro Accumulate Protofibrils, Including Amyloid Pores", J. Mol. Biol., 332:795-808 (2003).145Lee et al., "Targeting Amyloid-β Peptide (Aβ) Oligomers by Passive Immunization with a Conformation-selective Monoclonal Antibody Improves Learning and Memory in Aβ Precursor Protein (APP) Transgenic Mice," J. Biol. Chem. 2006 281:4292-4299.146Levy et al., "Mutation of the Alzheimer's Disease Amyloid Gene in Hereditary Cerebral Hemorrhage, Dutch Type," Science 248:1124-1126, 1990.147Liu et al., "Residues 17-20 and 30-35 of β-Amyloid Play Critical Roles in Aggregation," J. Neurosci. Res. 75(2):162-71, 2004.148Longo and Finch, "Nonfibrillar Aβ 1-42 (ADDL) Causes Aconitase Inactivation and Iron-dependent Neurotoxicity," Society for Neuroscience 25: 2129, 1999.149Masters et al., "Amyloid Plaque Core Protein in Alzheimer's Disease and Down Syndrome," Proc. Natl. Acad. Sci. U.S.A. 82:4245-4249, 1985.150Masters, et al., "Amyloid Plaque Core Protein in Alzheimer Disease and Down Syndrome", PNAS, 82:4245-4249 (1985).151McKhann et al., "Clinical Diagnosis of Alzheimer's Disease: Report of the NINCHS-ADRDA Work Group Under the Auspices of Department of Health and Human Services Task Force on Alzheimer's Disease," Neurology 34:939-944, 1994.152McKhann, et al., "Clinical diagnosis of Alzheimer's disease: Report of the NINCDS-ADRDA Work Group* under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease", Neurology, 34:939-944 (Jul. 1994).153Minutes from Oral Proceedings for European Patent Application No. 01945896.7-2402, dated Dec. 17, 2008.154Miravelle et al., "Substitutions at Codon 22 of Alzheimer's Aβ Peptide Induce Diverse Conformational Changes and Apoptotic Effects in Human Cerebral Endothelial Cells," J. Biol. Chem. 275:27110-27116, 2000.155Morgan et al., "Aβ Peptide Vaccination Prevents Memory Loss in an Animal Model of Alzheimer's Disease," Nature 408:982-985, 2000.156Morgan, et al., "AB peptide vaccination prevents memory loss in an animal model of Alzheimer's disease", Nature, 408:982-985 (Dec. 2000).157Moss et al., "The Peptide KLVFF-K6 Promotes β-Amyloid(1-40) Protofibril Growth by Association but Does Not Alter Protofibril Effects on Cellular Reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT)," Mol. Pharmacol. 64(5):1160-8, 2003.158Motter et al., "Reduction of β-Amyloid Peptide42 in the Cerebrospinal Fluid of Patients with Alzheimer's Disease," Ann. Neurol. 38:643-648, 1995.159Motter, et al., "Reduction of B-Amyloid Peptide42 in the Cerebrospinal Fluid of Patients with Alzheimer's Disease", Ann. Neurol., 38:643-648 (1995).160Mullan et al., "A Pathogenic Mutation for Probable Alzheimer's Disease in the APP Gene at the N-terminus of beta-Amyloid," Nature Genet. 1:345-347, 1992.161Mullan et al., "A Pathogenic Mutation for Probable Alzheimer's Disease in the APP Gene at the N-terminus of β-Amyloid," Nature Genet. 1:345-347, 1992.162Mullen, et al., "A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of β-amyloid", Nature Genetics, 1:345-347 (Aug. 1992).163Nichols et al., "Amyloid-β Aggregates Formed at Polar-Nonpolar Interfaces Differ From Amyloid-β Protofibrils Produced in Aqueous Buffers," Microsc. Res. Tech. 67(3-4):164-74, 2005.164Nichols et al., "Growth of beta-amyloid(1-40) Protofibrils by Monomer Elongation and Lateral Association. Characterization of Distinct Products by Light Scattering and Atomic Force Microscopy," Biochemistry 41(19):6115-6127, 2002.165Nichols et al., "Growth of β-amyloid(1-40) Protofibrils by Monomer Elongation and Lateral Association. Characterization of Distinct Products by Light Scattering and Atomic Force Microscopy," Biochemistry 41(19):6115-6127, 2002.166Nicoll et al., "Neuropathology of Human Alzheimer Disease After Immunization With Amyloid-β Peptide: A Case Report," Nature Med. 9(4):448-452, 2003.167Nicoll, et al., "Neuropathology of human Alzheimer disease after immunization with amyloid-β peptide: a case report", Nature Medicine, 9:4:448-452 (Apr. 2003).168Nilsberth et al., "A Novel APP Mutation (E693G)—The Arctic Mutation Causing Alzheimer's Disease with Vascular Symptoms," Soc. Neurosci. Abstr. 25:297; 1999.169Nilsberth et al., "A Novel APP Mutation (E693G)-The Arctic Mutation, Causing, Alzheimer's Disease with Vascular Symptoms," Society for Neuroscience Annual Meeting, Miami Beach, Abstract, 120.4; Nov. 1999.170Nilsberth et al., "A Novel APP Mutation (E693G)—The Arctic Mutation, Causing, Alzheimer's Disease with Vascular Symptoms," Society for Neuroscience Annual Meeting, Miami Beach, Abstract, 120.4; Nov. 1999.171Nilsberth et al., "The ‘Arctic’ APP Mutation (E693G) Causes Alzheimer's Disease by Enhanced Aβ Protofibril Formation," Nature Neurosci. 4(9):887-893, 2001.172Nilsberth et al., "The ‘Arctic’ APP Mutation (E693G) Causes Alzheimer's Disease by Enhanced Aβ Protofibril Formation," Neurobiology of Aging, May-Jun. 2000, 21, Abstract 265, Supplement 1, 1-304.173Nilsberth et al., "The 'Arctic' APP Mutation (E693G) Causes Alzheimer's Disease by Enhanced Abeta Protofibril Formation," Nature Neurosci. 4(9):887-893, 2001.174Nilsberth et al., "The 'Arctic' APP Mutation (E693G) Causes Alzheimer's Disease by Enhanced Abeta Protofibril Formation," Neurobiology of Aging, May-Jun. 2000, 21, Abstract 265, Supplement 1, 1-304.175Nilsberth et al., "The Attic APP Mutation (E693G) Causes Alzheimer's Disease Through a Novel Mechanism: Increased Amyloid beta Protofibril Formation and Decreased Amyloid beta Levels in Plasma and Conditioned Media," Neurobiol. Aging 21:S58, 2000.176Nilsberth et al., "The Attic APP Mutation (E693G) Causes Alzheimer's Disease Through a Novel Mechanism: Increased Amyloid β Protofibril Formation and Decreased Amyloid β Levels in Plasma and Conditioned Media," Neurobiol. Aging 21:S58, 2000.177Nilsberth, et al., "The ‘Arctic’ APP mutation (E693G) causes Alzheimer's disease by enhanced Aβ protofibril formation", Nature Neuroscience, 4(9):887-893 (2001).178Notice of Allowance (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Oct. 16, 2006.179Notice of Appeal (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Nov. 25, 2003.180Notice of intent to grant a European patent and Annex (Reasons for Decision) (EP 01 945 896.7), dated Mar. 18, 2009.181Notice of Opposition to a European Patent for European Patent No. 2,004,688 as filed by Acumen Pharmaceuticals, dated Sep. 22, 2011.182Oda et al., "Clusterin (apoJ) Alters the Aggregation of Amyloid β-Peptide (Aβ 1-42) and Forms Slowly Sedimenting Aβ Complexes that Cause Oxidative Stress," Exp. Neurol. 136:22-31, 1995.183Oda et al., "Purification and Characterization of Brain Clusterin," Biochem. Biophys. Res. Comm. 204:1131-6, 1994.184Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463) mailed May 8, 2006.185Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Jul. 7, 2005.186Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Jun. 25, 2003.187Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Nov. 19, 2002.188Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Nov. 24, 2004.189Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Nov. 9, 2005.190O'Nuallain et al., "Conformational Abs Recognizes a Generic Amyloid Fibril Epitope," Proc. Natl. Acad. Sci. U.S.A. 99:1485-1490, 2002.191O'Nuallain, et al., "Conformational Abs recognizing a generic amyloid fibril epitope", PNAS., 99:1485-1490 (Feb. 5, 2002).192Opposition Against European Patent No. 2,004,688 as filed by Acumen Pharmaceuticals dated Sep. 22, 2011, including (1) Facts and arguments in support of opposition: Annex 1; (2) Annex 2.193Päiviö et al., "Unique Physicochemical Profile of beta-Amyloid Peptide Variant Abeta1-40E22G Protofibrils: Conceivable Neuropathogen in Arctic Mutant Carriers," J. Med. Biol. 339:145-159, 2004.194Päiviö et al., "Unique Physicochemical Profile of β-Amyloid Peptide Variant Aβ1-40E22G Protofibrils: Conceivable Neuropathogen in Arctic Mutant Carriers," J. Med. Biol. 339:145-159, 2004.195Palmert et al., "The β-Amyloid Protein Precursor of Alzheimer Disease Has Soluble Derivatives Found in Human Brain and Cerebrospinal Fluid," Proc. Natl. Acad. Sci. U.S.A. 86:6338-6342, 1989.196Pirttilä et al., "Soluble Amyloid β-Protein in the Cerebrospinal Fluid From Patients with Alzheimer's Disease, Vascular Dementia and Controls," J. Neurol. Sci. 127:90-95, 1994.197Pirttilä, et al., "Soluble amyloid β-protein in the cerebrospinal fluid from patients with Alzheimer's disease, vascular dementia and controls", Journal of the Neurological Science, 127:90-95 (1994).198Ponte et al., "A New A4 Amyloid mRNA Contains a Domain Homologous to Serine Proteinase Inhibitors," Nature 331:525-527, 1988.199Qin et al., "Effect of 4-Hydroxy-2-Nonenal Modification on Alpha-Synuclein Aggregation," J. Biol. Chem. 282(8):5862-5870, 2007.200Reply to Examination Report (European Patent Application No. 01945896.7-2402), mailed Apr. 7, 2006.201Reply to Examination Report (European Patent Application No. 01945896.7-2402), mailed Nov. 5, 2007.202Reply to Examination Report (European Patent Application No. 01945896.7-2402), mailed Sep. 22, 2006.203Reply to Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Apr. 21, 2003.204Reply to Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Aug. 8, 2006.205Reply to Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Dec. 18, 2003.206Reply to Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Mar. 24, 2005.207Reply to Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Mar. 9, 2006.208Reply to Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Oct. 7, 2005.209Reply to Restriction Requirement (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Sep. 10, 2004.210Reply to Restriction Requirement (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Sep. 3, 2002.211Request for Continued Examination (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Mar. 25, 2004.212Request Pursuant to Oral Proceedings (European Patent Application No. 01945896.7-2402), mailed Oct. 10, 2008.213Response to Communication in European Patent Application No. 07747965.7-1222, Jan. 29, 2010.214Response to Communication in European Patent Application No. 07747965.7-1222, Sep. 11, 2009.215Restriction Requirement (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Jul. 3, 2002.216Restriction Requirement (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), mailed Jun. 10, 2004.217Resubmission of Reply to Office Action (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Feb. 3, 2004.218Roher et al., "Morphology and Toxicity of Aβ-(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of Alzheimer's Disease," J. Biol. Chem. 271:20631-20635, 1996.219Russo et al., "Presenilin-1 Mutations in Alzheimer's Disease," Nature 405:531-532, 2000.220Rzepecki et al., "Prevention of Alzheimer's Disease-Associated Aβ Aggregation by Rationally Designed Nonpeptitdic β-Sheet Ligands," J. Biol. Chem. 279:47497-47505, 2004.221Sahlin et al., "The Arctic Alzheimer Mutation Favors Intracellular Amyloid-beta Production by Making Amyloid Precursor Protein Less Available to alpha -secretase," J. Neurochem. 101:854-862, 2007.222Sahlin et al., "The Arctic Alzheimer Mutation Favors Intracellular Amyloid-β Production by Making Amyloid Precursor Protein Less Available to α -secretase," J. Neurochem. 101:854-862, 2007.223Schenk et al., "Immunization with Amyloid-β Attenuates Alzheimer-Disease-Like Pathology in the PDAPP Mouse," Nature 400:173-177, 1999.224Schenk et al., "Immunization with Amyloid-β Attenuates Alzheimer-Disease-Like Pathology in the PDAPP," Nature 400:173-177, 1999.225Scheuner et al., "Secreted Amyloid beta-Protein Similar to That in the Senile Plaques of Alzheimer's Disease is Increased In Vivo by the Presenilin 1 and 2 and APP Mutations Linked to Familial Alzheimer's Disease," Nature Med. 2(8):864-870, 1996.226Scheuner et al., "Secreted Amyloid β-Protein Similar to That in the Senile Plaques of Alzheimer's Disease is Increased In Vivo by the Presenilin 1 and 2 and APP Mutations Linked to Familial Alzheimer's Disease," Nature Med. 2(8):864-870, 1996.227Scheuner, et al., "Secreted amyloid β-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease", Nature Medicine, 2(8):864-870 (Aug. 1996).228Search Report and Written Opinion as issued by Intellectual Property Office of Singapore regarding Singapore Patent Application No. 200803655-0, dated Oct. 8, 2009.229Selkoe, "Cell Biology of the Amyloid β-Protein Precursor and the Mechanism of Alzheimer's Disease," Ann. Rev. Cell Biol. 10:373-403, 1994.230Selkoe, "Normal and Abnormal Biology of the β-Amyloid Precursor Protein," Ann. Rev. Neurosci. 17:489-517, 1994.231Serpell, "Alzheimer's Amyloid Fibrils: Structure and Assembly," Biochim. Biophys. Acta 1502:16-30, 2000.232Seubert et al., "Isolation and Quantification of Soluble Alzheimer's β-Peptide From Biological Fluids," Nature 359:325-327, 1992.233Seubert, et al., "Isolation and quantification of soluble Alzheimer's β-peptide from biological fluids", Nature, 359:325-327 (Sep. 1992).234Sherrington et al., "Cloning of a Gene Bearing Missense Mutations in Early-Onset Familial Alzheimer's Disease," Nature 375:754-760, 1995.235Shtilerman et al., "Molecular Crowding Accelerates Fibrillization of α-Synuclein: Could an Increase in the Cytoplasmic Protein Concentration Induce Parkinson's Disease?" Biochemistry 41:3855-3860, 2002.236Shtilerman, et al., "Molecular Crowding Accelerates Fibrillization of a-Synuclein: Could an Increase in the Cytoplasmic Protein Concentration Induce Parkinson's Disease?", Biochemistry, 41:3855-3860 (2002).237Sigurdsson et al., "Immunization With a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer's Disease-Associated Pathology in Transgenic Mice," Am. J. Pathol. 159(2):439-447, 2001.238Sigurdsson, et al., "Immunization with a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer's Disease-Associated Pathology in Transgenic Mice", American Journal of Pathology, 159:2:439-447 (Aug. 2001).239Solomon et al., "Disaggregation of Alzheimer β-amyloid by Site-directed mAb," Proc. Natl. Acad. Sci. U.S.A. 94:4109-4112, 1997.240Solomon et al., "Monoclonal Antibodies Inhibit In Vitro Fibrillar Aggregation of the Alzheimer Beta-amyloid Peptide," Proc. Natl. Acad. Sci. U.S.A. 93:452-455, 1996.241Solomon et al., "Monoclonal Antibodies Restore and Maintain the Soluble Conformation of β-amyloid Peptide," Neurobiol. Aging, vol. 17, No. 4, Suppl. 152, 1996.242Soto et al., "The Conformation of Alzheimer's β Peptide Determines the Rate of Amyloid Formation and Its Resistance to Proteolysis," Biochem. J. 1:314:701-7, 1996.243Soto et al., "The Conformation of Alzheimer's β Peptide Determines the Rate of Amyloid Formation and Its Resistance to Proteolysis," Biochem. J. 1:314:701-707, 1996.244Srinivasan et al., "ABri Peptide Associated with Familial British Dementia Forms Annular and Ring-Like Protofibrillar Structures," Amyloid 11(1):10-3, 2004.245St. George-Hyslop et al., "Genetic Linkage Studies Suggest that Alzheimer's Disease is Not a Single Homogeneous Disorder," Nature 347:194-197, 1990.246St. George-Hyslop et al., "The Genetic Defect Causing Familial Alzheimer's Disease Maps on Chromosome 21," Science 235:885-890, 1987.247Stenh et al., "Amyloid-beta Oligomers are Inefficiently Measured by Enzyme-Linked Immunosorbent Assay," Ann. Neurol. 58: 147-150, 2005.248Stenh et al., "Amyloid-β Oligomers are Inefficiently Measured by Enzyme-Linked Immunosorbent Assay," Ann. Neurol. 58: 147-150, 2005.249Stenh et al., "The Arctic Mutation Interferes with Processing of the Amyloid Precursor Protein," NeuroReport 13: 1857-1860, 2002.250Stine et al., "Supramolecular Structures of Aβ Aggregates and Cellular Responses," Biophysical Journal Program and Abstracts:40th Annual Meeting Feb. 17-21, 1996, Biophys J. 70: Abstract 239.251Stine et al., "The Nanometer-Scale Structure of Amyloid-β Visualized by Atomic Force Microscopy," J. Prot. Chem. 15(2):193-203, 1996.252Stine, et al., "The Nanometer-Scale Structure of Amyloid-β Visualized by Atomic Force Microscopy", Journal of Protein Chemistry, 15:2:193-203 (1996).253Summons to Attend Oral Proceedings (European Patent Application No. 01945896.7-2402), dated Jul. 25, 2008.254Supplemental Reply to Restriction Requirement (U.S. Appl. No. 09/899,815; U.S. Patent No. 7,179,463), filed Sep. 21, 2004.255Suzuki et al., "An Increased Percentage of Long Amyloid β Protein Secreted by Familial Amyloid β Protein Precursor (βAPP717) Mutants," Science 264:1336-1340, 1994.256Tagliavani et al., "A New betaAPP Mutation Related to Hereditary Cerebral Haemorrhage," Alz. Report 2(Suppl. 1):S28, Abstract 23, 1999.257Tagliavani et al., "A New βAPP Mutation Related to Hereditary Cerebral Haemorrhage," Alz. Report 2(Suppl. 1):S28, Abstract 23, 1999.258U.S. Appl. No. 09/369,236, filed Aug. 4, 1999, Krafft et al.259U.S. Appl. No. 09/745,057, filed Dec. 20, 2000, Krafft et al.260U.S. Appl. No. 10/166,856, filed Jun. 11, 2002, Klein et al.261U.S. Appl. No. 11/570,995, filed Dec. 20, 2006, Gellerfors et al.262U.S. Appl. No. 12/294,207, filed Sep. 23, 2008, Gellerfors et al.263U.S. Appl. No. 60/217,098, filed Jul. 10, 2000, Lannfelt et al.264U.S. Appl. No. 60/621,776, filed Oct. 25, 2004, Lambert et al.265U.S. Appl. No. 60/652,538, filed Feb. 14, 2005, Shughrue et al.266Vickers, "A Vaccine Against Alzheimer's Disease: Developments to Date," Drugs Aging 19(7):487-494, 2002.267Walsh et al., "Amyloid β-Protein Fibrillogenesis. Detection of a Protofibrillar Intermediate," J. Biol. Chem. 272(35):22364-22372, 1997.268Walsh et al., "Amyloid β-Protein Fibrillogenesis. Detection of a Protofibrillar Intermediate," J. Biol. Chem. 272:22364-22372, 1997.269Walsh et al., "Amyloid β-Protein Fibrillogenesis. Structure and Biological Activity of Protofibrillar Intermediates," J. Biol. Chem. 274(35):25945-25952, 1999.270Walsh et al., "Amyloid β-Protein Fibrillogenesis. Structure and Biological Activity of Protofibrillar Intermediates," J. Biol. Chem. 274(36):25945-25952, 1999.271Walsh et al., "Amyloid-β Oligomers: Their Production, Toxicity and Therapeutic Inhibition," Biochem. Soc. Trans. 30:552-7, 2002.272Walsh et al., "Naturally Secreted Oligomers of Amyloid β Protein Potently Inhibit Hippocampal Long-Term Potentiation In Vivo," Nature 416:535-539, 2002.273Walsh et al., "Oligomers on the Brain: The Emerging Role of Soluble Protein Aggregates in Neurodegeneration," Protein Pept. Lett., 11: 213-28, 2004.274Walsh, et al., "Amyloid β-Protein Fibrillogenesis", The Journal of Biological Chemistry, 274:36:25945-25952 (Sep. 3, 1999).275Walsh, et al., "Naturally secreted oligomers of amyloid β protein potently inhibit hippocampal in vivo", Nature, 416:535-539 (Apr. 4, 2002).276Ward et al., "Fractionation and Characterization of Oligomeric, Protofibrillar Forms of β-Amyloid Peptide," Biochem. J. 348:137-144, 2000.277Ward, et al., "Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of β-amyloid peptide", Biochem. J., 348:137-144 (2000).278Weidemann et al., "Identification, Biogenesis, and Localization of Precursors of Alzheimer's Disease A4 Amyloid Protein," Cell 57:115-126, 1989.279Weiner et al., "Nasal Administration of Amyloid-β Peptide Decreases Cerebral Amyloid Burden in a Mouse Model of Alzheimer's Disease," Annals Neurol. 48(4):567-579, 2000.280Weiner, et al., "Nasal Administration of Amyloid-β Peptide Decreases Cerebral Amyloid Burden in a Mouse Model of Alzheimer's Disease", Annals. of Neurology, 48:4:567-579 (2000).281Westlind-Danielsson and Arnerup, "Spontaneous In Vitro Formation of Supramolecular β-Amyloid Structures, "βamy Balls", by β-Amyloid 1-40 peptide," Biochemistry 40:14736-43, 2001.282Williams et al., "Structural Properties of Aβ Protofibrils Stabilized by a Small Molecule," Proc. Natl. Acad. Sci. U.S.A. 102(20):7115-20, 2005.283Wirak et al., "Deposits of Amyloid β Protein in the Central Nervous System of Transgenic Mice," Science 253:323-325, 1991.284WO 05/123775 A1, pp. 7-8, published Dec. 29, 2005.285Written Opinion of the International Searching Authority (PCT/SE05/000993), mailed Oct. 4, 2005.286Written Opinion of the International Searching Authority (PCT/SE07/000292), mailed Jul. 20, 2007.287Ye et al., "Protofibrils of Amyloid β-Protein Inhibit Specific K+ Currents in Neocortical Cultures," Neurobiol. Dis. 13:177-190, 2003.288Ye, et al., "Protofibrils of amyloid β-protein inhibit specific K+ currents in neocortical cultures", Neurobiology of Disease, 13:177-190 (2003).289Yoritaka et al., "Immunohistochemical Detection of 4-Hydroxynonenal Protein Adducts in Parkinson Disease," Proc. Natl. Acad. Sci. U.S.A. 93:2696-2701, 1996.290Yoshikai et al., "Genomic Organization of the Human Amyloid Beta-protein Precursor Gene," Gene 87:257-263, 1990.Referenced byCiting PatentFiling datePublication dateApplicantTitleUS8409575Aug 26, 2011Apr 2, 2013Bioarctic Neuroscience AbAntibodies specific for amyloid beta protofibrilUS8613923Jun 12, 2008Dec 24, 2013Ac Immune S.A.Monoclonal antibodyUS8796439Aug 7, 2012Aug 5, 2014Ac Immune S.A.Nucleic acid molecules encoding a humanized antibodyUS8906367 *Jan 7, 2008Dec 9, 2014University Of ZurichMethod of providing disease-specific binding molecules and targetsUS8999936 *Feb 28, 2013Apr 7, 2015Bioarctic Neuroscience AbAntibodies specific for soluble amyloid beta peptide protofibrils and uses thereofUS9034334Aug 26, 2011May 19, 2015Bioarctic Neuroscience AbProtofibril selective antibodies and the use thereofUS9146244Oct 13, 2011Sep 29, 2015Ac Immune S.A.Polynucleotides encoding an anti-beta-amyloid monoclonal antibodyUS9175094Nov 12, 2013Nov 3, 2015Ac Immune S.A.Monoclonal antibodyUS9221900Jul 29, 2011Dec 29, 2015Ac Immune S.A.Methods for identifying safe and functional humanized antibodiesUS9403902Oct 3, 2008Aug 2, 2016Ac Immune S.A.Methods of treating ocular disease associated with amyloid-beta-related pathology using an anti-amyloid-beta antibodyUS9573994Jul 8, 2015Feb 21, 2017Bioarctic Neuroscience AbAβ protofibril binding antibodiesUS9580493Jun 22, 2012Feb 28, 2017Biogen International Neuroscience GmbhAnti-α synuclein binding moleculesUS9585956Sep 22, 2015Mar 7, 2017Ac Immune S.A.Polynucleotides encoding anti-amyloid beta monoclonal antibodiesUS20090155249 *Jun 12, 2008Jun 18, 2009Ac Immune S.A.Humanized antibody igg1US20100202968 *Jan 7, 2008Aug 12, 2010University Of ZurichMethod of Providing Disease-Specific Binding Molecules and TargetsUS20100291097 *Oct 3, 2008Nov 18, 2010Andrea PfeiferMonoclonal antibodyUS20100297013 *Oct 3, 2008Nov 25, 2010Andrea PfeiferHumanized antibodyUS20120100129 *Jun 29, 2010Apr 26, 2012Gellerfors PaerN-Terminal Truncated Protofibrils/Oligomers for Use in Therapeutic and Diagnostic Methods for Alzheimer's Disease and Related DisordersUS20130236452 *Feb 28, 2013Sep 12, 2013Bioarctic Neuroscience AbAntibodies specific for soluble amyloid beta peptide protofibrils and uses thereof* Cited by examinerClassifications U.S. Classification530/388.24, 530/387.3, 530/387.1, 530/388.1, 530/387.9, 424/141.1, 424/158.1International ClassificationC07K16/00, C07K14/47, C07K, A61K39/395, A61K39/35, C07K16/18, A61P25/28Cooperative ClassificationC07K2317/24, G01N2800/387, G01N2800/2821, C07K14/4711, C07K16/18European ClassificationC07K16/18, C07K14/47A3Legal EventsDateCodeEventDescriptionDec 20, 2006ASAssignmentOwner name: BIOARTIC NEUROSCIENCE AB, SWEDENFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GELLERFORS, PAR;LANNFELT, LARS;REEL/FRAME:018660/0217Effective date: 20061219Sep 24, 2008ASAssignmentOwner name: BIOARCTIC NEUROSCIENCE AB, SWEDENFree format text: CORRECTIVE ASSIGNMENT TO CORRECT THE MISSPELLING OF ASSIGNEE S NAME (WHICH IS CORRECTLY SPELLED ON THE ATTACHED ASSIGNMENT DATED JANUARY 2007) AND ADDRESS PREVIOUSLY RECORDED ON REEL 018660 FRAME 0217;ASSIGNORS:GELLERFORS, PAR;LANNFELT, LARS;REEL/FRAME:021577/0805Effective date: 20061219Owner name: BIOARCTIC NEUROSCIENCE AB, SWEDENFree format text: CORRECTIVE ASSIGNMENT TO CORRECT THE MISSPELLING OF ASSIGNEE S NAME (WHICH IS CORRECTLY SPELLED ON THE ATTACHED ASSIGNMENT DATED JANUARY 2007) AND ADDRESS PREVIOUSLY RECORDED ON REEL 018660 FRAME 0217. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT;ASSIGNORS:GELLERFORS, PAR;LANNFELT, LARS;REEL/FRAME:021577/0805Effective date: 20061219Feb 12, 2013CCCertificate of correctionJul 9, 2015FPAYFee paymentYear of fee payment: 4RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services