No assignee for this patent application has been made.
Reporters obtained the following quote from the background information supplied by the inventors: "Aggregation and deposition of amyloid .beta.-protein (A.beta. or beta amyloid) is considered to be a primary pathological event in Alzheimer's disease (AD) (1). While the longer 42-43 amino acid A.beta. forms have been implicated in the formation of amyloid plaques (2-4), the aggregation state of the peptide is critical in determining its neurotoxicity. Many different forms of A.beta. have been identified and characterized including fibrils, proto-fibrils, annular structures, globular structures, amorphous aggregates and various soluble oligomers (5-9). Numerous studies indicate that small oligomeric morphologies of A.beta. are the primary toxic species in AD (10). These small oligomers are also called 'low-n oligomers' (i.e., dimers, trimers, or tetramers).
"One type of naturally occurring oligomeric A.beta. species is a low-n oligomer that is SDS-stable, and inhibits long term potentiation in mammalian hippocampus (13). The naturally occurring low-n SDS-stable A.beta. oligomers cause short term memory loss in rats, one of the earliest symptom associated with AD (19), and also affect dendritic morphology in neuronal cells resulting in synaptic losses (20). Concentration levels of this SDS-stable oligomeric form correlate strongly with dementia in AD patients (14). Unlike in vitro generated A.beta., naturally occurring low-n oligomeric aggregate does not dissociate in SDS (e.g., 1-10%) or chaotropic salts such as guanidine hydrochloride (e.g., 1-10%), and cannot be pelleted from physiological fluids by ultra-centrifugation (15). The naturally occurring low-n oligomeric aggregates can be detected in brain tissue by Western blot analysis (16-17). They are also resistant to the A.beta. degrading protease insulin degrading enzyme (IDE) (18).
"Synthetically generated (i.e., in vitro generated) A.beta. aggregates function similarly to naturally occurring A.beta. species (11). Antibody fragments generated against synthetic oligomeric A.beta. recognize naturally occurring oligomeric A.beta. in human brain tissue (12). While intracerebral injections of synthetic A.beta. oligomers exerted a deleterious effect on learned behavior in rats (21-22), these responses were delayed and the concentrations and amounts of synthetic A.beta. were much higher than concentrations of naturally derived SDS-stable low-n A.beta. oligomers required to interfere with the memory of a complex learned behavior (19). Together these studies clearly suggest that the naturally derived SDS-stable A.beta. oligomers may be important mediators in synaptic dysfunction in early AD and that these naturally derived oligomers behave differently than in vitro derived oligomers.
"Antibodies and small molecule inhibitors of A.beta. aggregation have been shown to prevent spine loss induced by the SDS-stable A.beta. oligomers (23-24). Since they have not been raised against a particular oligomeric form, however, anti-A.beta. antibodies have a wide range of specificities, targeting different regions or multiple morphologies of A.beta.. Therefore, reagents that can specifically target these naturally derived SDS-stable oligomers would be valuable tools for diagnostic and therapeutic applications for AD.
"Accordingly, there exists the need for new therapies and reagents for the treatment of Alzheimer's disease, in particular, therapies and reagents capable of effecting a therapeutic and diagnostic benefit at physiologic (e.g., non-toxic) doses."
In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "The present invention discloses an antibody or antibody fragment that specifically recognizes oligomeric A.beta. that is at least partially resistant to denaturation by SDS (i.e., is SDS-stable) but does not bind monomeric or fibrillar A.beta. or in vitro-generated oligomeric A.beta.. As used herein, the term 'antibody' includes scFv (also called a 'nanobody'), humanized, fully human or chimeric antibodies, single-chain antibodies, diabodies, and antigen-binding fragments of antibodies (e.g., Fab fragments). As used herein, the term 'oligomer' refers to a dimer, trimer, or tetramer. As used herein, 'SDS-stable' means that the oligomeric aggregate does not dissociate into the monomer units in SDS (such as in 1-10% SDS. In exemplary embodiments, the antibody fragment does not contain the constant domain region of an antibody.
"The antibody fragment described herein may be isolated according to a method comprising the steps of: (a) negative panning of a scFV phage library comprising serially contacting phage with: (i) brain derived control samples that do not contain oligomeric A.beta., (ii) brain-derived mononeric forms of A.beta., and (iii) synthetic monomeric forms of A.beta., and monitoring the binding of said phage to said synthetic synthetic monomeric forms of A.beta. and repeating the negative panning until no phage was observed binding to antigen to produce an aliquot of phage that does not bind to monomeric, fibrillar or other oligomeric forms of A.beta.; (b) positive panning of the aliquot from step (a) comprising contacting the aliquot of phage from step (a) with natural brain derived, SDS-stable oligomers of A.beta., and incubating for time sufficient to allow binding of phage to said SDS-stable oligomers of A.beta.; and © eluting the bound phage particles from step (b).
"In certain embodiments the observing of the binding of the phage to the synthetic monomeric forms of A.beta. is by using Atomic Force Microscope (AFM) Imaging. In certain embodiments, the negative panning is repeated until less than 0-10% phage was observed by AFM imaging as binding to antigen in step (a).
"In certain embodiments, the antibody fragment is less than 500 amino acids in length, such as between 200-450 amino acids in length, or less than 300 amino acids in length. In certain embodiments, the antibody fragment comprises (consists essentially of, or consists of) amino acid residues 16-292 of SEQ ID NO:1. In specific embodiments, the antibody fragment has an amino acid sequence of SEQ ID NO:1. The antibody fragment is specific for a 12-16 kDa oligomeric species of A.beta.
"Also contemplated herein is a binding molecule that binds to oligomeric A.beta. that is at least partially resistant to denaturation by SDS but does not bind monomeric A.beta., fibrillar A.beta. or oligomeric forms of A.beta. that are generated in vitro, wherein the binding molecule comprises the sequence of SEQ ID NO:1.
"Another embodiment contemplates a method of inhibiting the aggregation of A.beta. comprising contacting a composition that comprises A.beta. monomers with an antibody, antibody fragment or binding molecule of the invention. In certain embodiments, the aggregation of A.beta. is in a cell. In more specific embodiments, the aggregation of A.beta. is in brain tissue. In the methods of the invention, the contacting with an antibody fragment or binding molecule decreases the rate of formation of A.beta. aggregates as compared to the rate in the absence of composition or binding molecule.
"Another embodiment is directed to a method of detecting the presence of A.beta. in a physiological sample comprising contacting a sample with an antibody composition of the invention and determining the binding of the composition with said tissue sample wherein binding of the composition to the tissue sample is indicative of the presence of SDS-stable A.beta. oligomers in the tissue sample wherein said presence of said SDS-stable A.beta. oligomers is indicative of early stage AD. In certain embodiments, the physiological sample is brain tissue, serum, cerebrospinal fluid (CSF), urine or saliva.
"In a further embodiment, there is a method of preventing or inhibiting the accumulation of A.beta. in the brain of a mammal comprising administering to said mammal a composition comprising an antibody, antibody fragment or a binding molecule of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
"FIG. 1. Bio-panning against natural A.beta. oligomers--Schematic. Panning protocol showing different steps involved in isolating scFv against low concentrations of natural brain derived A.beta. oligomers.
"FIGS. 2A-2F. Bio-panning process visualized by AFM. Serial negative panning against A) 1 ng brain derived proteins from which A.beta. has been depleted; B) 1 ng brain derived A.beta. monomer; and multiple pieces containing 1 .mu.g synthetic A.beta. monomers. Panning process was visualized by AFM until no phage binding to synthetic monomers was observed. C) 1st synthetic monomer mica; D) 3rd synthetic monomer mica; E) 5th synthetic monomer mica. F) Recovered phage from last monomer mica was added to mica containing 1 ng dimer sample. Scale bar represents 1 .mu.m.
"FIGS. 3A-3D. Phage from 400 clones binds specifically to natural A.beta. oligomers. Phage was produced from the 400 clones obtained after the bio-panning process, and added to A) 1 ng A.beta. depleted brain sample; B) 1 ng brain derived A.beta. monomer; C) 1 .mu.g synthetic A.beta. monomer; D) 1 ng brain derived A.beta. oligomers. Scale bar represents 1 .mu.m.
"FIG. 4. Dot-blot to determine expression levels of isolated clones. Phage binding to the 10 pg brain derived dimers were eluted and transformed into Hb2151 competent cells. Single clones were picked and tested for levels of soluble scFv expression by dot blot analysis. The clone with the strongest expression (.star-solid.) C6, was picked for further study.
"FIGS. 5A-5D. C6 specifically recognizes brain derived oligomers. Binding of C6 phage to A) natural brain derived oligomers and B) 3D synthetic A.beta. aggregates was compared to binding of A4 to the same samples (C, D). Scale bar represents 1 .mu.m.
"FIG. 6. C6 nanobody detects A.beta. oligomers in 7PA2 cell medium. When 25 .mu.g of the cell media was probed with C6 nanobody, a band of around 12-16 kDa could be detected, corresponding to trimeric or tetrameric morphology of A.beta.
"FIG. 7. ThT fluorescence assay. Aggregation of 50 .mu.M A.beta. incubated with and without 5 .mu.M C6 was monitored by ThT fluorescence. Each experiment was performed in triplicate.
"FIGS. 8A-8H. Morphology of A.beta. incubated with or without C6. AFM images of 50 .mu.M A.beta. incubated without C6 for A) 0 hour, B) 3 day, C) 7 days, D) 10 days; or with C6 for E) 0 hour, F) 3 day,
"FIGS. 9A-9B: C6 stabilizes SDS-stable oligomers. 50 .mu.M A.beta. was incubated alone or in the presence of 5 .mu.M C6 nanobody. Aliquots corresponding to monomers (Lane 1), early oligomers corresponding to 3 h-1 D of aggregation (Lane 2) and late stage oligomers corresponding to 2-4 D of aggregation (Lane 3) were separated on a 10% Tris-Tricine gel followed by transfer onto a nitrocellulose membrane. The membrane was probed with 1/1000 dilution of 6E10 monoclonal antibody followed by 1/1000 dilution of goat anti-mouse IgG-HRP secondary antibody. A) Comparison of A.beta. monomer levels when incubated with or without C6. B) Depletion of monomers and appearance of SDS stable oligomers when A.beta. is incubated with C6.
"FIG. 10. C6 stabilizes toxic oligomeric form of A.beta.. Co-incubation of 50 .mu.M A.beta. with 5 .mu.M C6 maintains A.beta.-induced toxicity towards SH-SY5Y human neuroblastoma cells. The final concentrations of A.beta. and C6 nanobody added to the cells were 1 .mu.M and 0.1 .mu.M respectively. The error bars indicate SEM. Line at 100% indicates cells incubated with buffer alone."
For more information, see this patent application: Sierks, Michael; Kasturirangan, Srinath. Antibody Based Reagent That Specifically Recognizes Toxic Oligomeric Form of Beta-Amyloid. Filed
Keywords for this news article include: Antibodies, Amyloid, Patents, Therapy, Peptides, Immunology, Nanobodies, Amino Acids, Blood Proteins, Nanotechnology, Immunoglobulins, Emerging Technologies.
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