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WO2010012004A2 - Anticorps monoclonaux spécifiques d'agrégats amyloïdes pathologiques communs aux amyloïdes formés à partir de protéines ayant des séquences différentes - Google Patents

Anticorps monoclonaux spécifiques d'agrégats amyloïdes pathologiques communs aux amyloïdes formés à partir de protéines ayant des séquences différentes Download PDF

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WO2010012004A2
WO2010012004A2 PCT/US2009/051880 US2009051880W WO2010012004A2 WO 2010012004 A2 WO2010012004 A2 WO 2010012004A2 US 2009051880 W US2009051880 W US 2009051880W WO 2010012004 A2 WO2010012004 A2 WO 2010012004A2
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disease
seq
amyloid
antibody
monoclonal antibody
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PCT/US2009/051880
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WO2010012004A3 (fr
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Charles G. Glabe
Rakez Kayed
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The Regents Of The University Of California
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Priority to US13/055,908 priority Critical patent/US20110200609A1/en
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Publication of WO2010012004A3 publication Critical patent/WO2010012004A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • This invention relates generally to the fields of medicine, immunology and protein biochemistry and more particularly to a) methods for the production of monoclonal antibodies specific to conformational epitope(s) of a prefibrillar aggregate(s) which contribute to amyloid fibril formation in human or animal subjects, b) the hybridomas and monoclonal antibodies produced therefrom, c) the use of such monoclonal antibodies in the immunization of human or animal subjects against Alzheimer's Disease or other amyloid diseases and d) the use of such monoclonal antibodies in the diagnosis or detection of Alzheimer's Disease or other amyloid diseases in human or animal subjects.
  • amyloid fibrils are thought to be involved in the pathogenesis of various amyloid diseases of genetic, infectious and/or spontaneous origin, including spongiform encephalopathies, Alzheimer's disease, Parkinson's disease, type I! diabetes, Creutzfeldt-Jakob disease, Huntington's disease, possibly macular degeneration, various prion diseases and numerous others. In at least some of these amyloid diseases, amyloid fibrils lead to the development of amyloid plaques.
  • Amyloid peptides are the principal constituent of amyloid plaques. In the case of Alzheimer's disease, the peptides are termed A ⁇ or ⁇ -amyloid peptide.
  • a ⁇ peptide is an internal fragment of 39 to 43 amino acids of amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • Several mutations within the APP protein have been correlated with the presence of AD. See, for example, Goate et al., Nature, (1991) 349, 704 (valine to isoleucine); Chartier Marian et al., Nature (1991)353,844 (valine to glycine); Murrell et al. Science (1991) 21 ,97 (valine to phenylalanine); Mullan et al., Nature Genet.
  • cytotoxic amyloid-beta peptide aggregates disrupt the integrity of cell membranes and elaborate reactive oxygen intermediates, thereby giving rise to elevations in cytosolic calcium and eventual cell death.
  • Cell surface receptors for amyloid-beta peptide may also activate signal transduction mechanisms.
  • Soluble A ⁇ includes A ⁇ monomers as well as aggregations of such monomers referred to as prefibrillar aggregates. These prefibrillar aggregates lead to the development of amyloid fibrils.
  • Soluble A ⁇ content of the human brain is better correlated with the severity of AD than is the accumulation of amyloid plaques. See, for example, Y. M. Kuo et al. (1996) J. Biol. Chem.
  • PCT International Patent Application PCT/US2003/028829 entitled MONOCLONAL ANTIBODYS AND CORRESPONDING ANTIBODIES SPECIFIC FOR HIGH MOLECULAR WEIGHT AGGREGATION INTERMEDIATES COMMON TO AMYLOIDS FORMED FROM PROTEINS OFDIFFERING SEQUENCE (Kayed and Glabe) describes compositions of matter comprising one or more conformational epitopes found on amyloid peptide aggregates, antibodies to such epitopes and methods for making and using the compositions, epitopes and/or antibodies.
  • compositions described in PCT/US2003/028829 include synthetic or isolated compositions that contain or consist of certain conformational epitopes found on peptide aggregates (e.g., toxic peptide aggregates) present in human or veterinary patients who suffer from, or who are likely to develop, amyloid diseases (e.g., Alzheimer's Disease).
  • the invention described in PCT/US2003/028829 also includes methods for using such compositions in the detection, treatment and prevention of diseases in humans or animals and/or in the testing and identification of potential therapies (e.g., drug screening) using such antibodies.
  • the entirety of PCT International Patent Application PCT/US2003/028829 is expressly incorporated herein by reference.
  • Monoclonal antibodies are homogeneous preparations of immunoglobulin proteins that specifically recognize and bind to regions, or epitopes, of their corresponding antigens. In some cases, monoclonal antibodies can bind to and inhibit the activity of endogenous chemical entities that are toxic or deleterious. In view of this, there is a need for the development of new monoclonal antibodies that bind to and inhibit toxic forms of amyloid (e.g., cytotoxic amyloid-beta peptide aggregates or protofibrils) with high specificity, thereby providing for diagnosis and treatment of amyloid diseases.
  • amyloid e.g., cytotoxic amyloid-beta peptide aggregates or protofibrils
  • United States Patent Application Publication 007/0218499 entitled MONOCLONAL ANTIBODIES THAT TARGET PATHOLOGICAL ASSEMBLIES OF AMYLOID ⁇
  • a ⁇ describes monoclonal antibodies that purportedly bind with high specificity to soluble oligomers of amyloid ⁇ peptide. (A ⁇ ) and methods of employing those antibodies. The antibodies are described to be able to distinguish between Alzheimer's Disease (AD) and control human brain extracts. The antibodies identify endogenous A ⁇ oligomers in AD brain slices and also bind to A ⁇ oligomers on cultured hippocampal cells. The antibodies neutralize endogenous A ⁇ oligomers and A ⁇ oligomers produced in solution.
  • AD Alzheimer's Disease
  • Amyloid formation begins with amylodogenic or partially folded monomer (dark blue) which has the ability to aggregate. It can form fibrils by forming soluble fibril nuclei (light blue) which can grow by addition of monomers to form insoluble fibrils (light blue). Fibrillar oligomers and fibrils are recognized by a fibril specific polyclonal serum, "OC". Alternatively, monomers can aggregate to form prefibrillar oligomers that are immunologically distinct from fibrils and monomer. The oligomers co-aggregate to form curvilinear protofibrils and may ultimately undergo a conformation change to form fibrils.
  • Prefibrillar oligomers and protofibrils are specifically recognized by A11.
  • Prefibrllar oligomers also appear to be building blocks for annular protofibrils that appear to be pore like structures.
  • Annular protofibrils are preferentially recognized by "officer" polyclonal serum.
  • the hallmark lesions of AD include amyloid deposits, neurofibrillary tangles, and dystrophic neurites.
  • Several types of amyloid deposits are found including diffuse amyloid deposits, "cored”, “neuritic” and “compact or burned out” senile plaques and cerebrovascular amyloid deposits.
  • Neurofibrillary tangles comprised of the misfolded microtubule-associated protein tau, are frequently found frequently in association with dystrophic neurites.
  • curvilinear fibers form that have a beaded appearance form.
  • These structures have also been called “protofibrils” because they appear to be formed by the coalescence of the spherical subunits.
  • Pore-like structures known as “annular protofibrils” have been observed in solutions of oligomers. This same spectrum of aggregation intermediates and morphologies have been observed for many types of amyloids, such as alpha synuclein, islet amyloid and non-disease associated "neoamyloids”.
  • these protofibrillar structures appear to either anneal or undergo a conformational change to form mature 6-10 nm cross beta fibrils that have either a smooth or twisted morphology.
  • amyloid fibril lattice Once the amyloid fibril lattice has been established, it can grow by the addition of monomer onto the ends of the fibrils.
  • a ⁇ assembly states One of the problems intrinsic to studies of in vitro toxicity of A ⁇ assembly states is that while you may have a good idea of the size and structure of an aggregate when you add it to cells, it is difficult to be sure that it has not aggregated further or changed conformation during the time required to read out measurements of toxicity.
  • the anti-oligomer antibody blocks the toxicity soluble prefibrillar oligomers in vitro, so we will use this blocking ability to confirm the conformation of the active aggregation state as it is presented to cells.
  • the availability of pure and homogeneous populations of different assembly states of AB and novel monoclonal antibodies that specifically recognize these assembly states affords us a unique opportunity to identify distinct aggregation states and clarify their roles in A ⁇ fibril assembly and cellular toxicity.
  • compositions comprising isolated monoclonal antibodies which bind to one or more conformational epitope(s) of prefibrillar aggregate(s) that contribute to amyloid fibril formation in the brains of humans or animals (e.g., toxic species of prefibrillar aggregate(s)).
  • the monoclonal antibodies may be administered, in therapeutic amounts, to human or animal subjects to reduce the toxicity of the prefibrillar aggregate, thereby preventing or limiting the formation of amyloid deposits and the associated occurrence or progression of a disease or disorder in which amyloid deposits form within the brain or nervous tissue.
  • amyloid diseases include, but are not necessarily limited to, Alzheimer's Disease, early onset Alzheimer's Disease associated with Down's syndrome, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathies, including mad cow disease, sheep scrapie, and mink spongiform encephalopathy, Parkinson's disease, Huntington's disease, amyotropic lateral sclerosis, Creutzfeld Jakob disease, Gerstmann-Straussler-Scheinker syndrome, kuru, fatal familial insomnia, chronic wasting syndrome, familial amyloid polyneuropathy, frontotemporal dementia, type Il diabetes, systemic amyloidosis, serum amyloidosis, British familial dementia, Danish familial dementia, macular degeneration and cerebrovascular amyloidosis.
  • Alzheimer's Disease early onset Alzheimer's Disease associated with Down's syndrome
  • SAA amyloidosis hereditary Icelandic syndrome
  • the monoclonal antibodies of the present invention are identified as follows: M118, M121 , M201 , M204, M205, M206 These clones were prepared by immunizing rabbits with a conformationally- constrained antigen consisting of amyloid A ⁇ covalently coupled to colloidal gold via a thioester linkage.
  • the prefibrillar aggregate may have a molecular weight in a range of about 9 kDa to about 100,000,000 kDa.
  • the prefibrillar aggregate may comprise any suitable number of monomers.
  • the prefibrillar aggregate may comprise five monomers and in other embodiments, the prefibrillar aggregate may comprise eight monomers.
  • amyloid peptide monomers and/or amyloid fibrils may be substantially free of the conformational epitope to which the monoclonal antibodies M 118, M201 , M204, M205 and M206 binds.
  • amyloid peptide monomers and prefibrillar oligomers may be substantially free of the conformational epitope to which monoclonal antibody M121 binds because this antibody is specific for fibrils and fibrillar oligomers or fibril nuclei.
  • Figure 1 shows dot blot analysis of specificity for a number of rabbit monoclonal antibodies of the present invention.
  • Figure 2A shows dot blot analysis of A ⁇ 40 and A ⁇ 42 incubated under three different conditions.
  • Figure 2B shows dot blot analysis of four different samples of prefibrillar oligomers prepared at pH 2.5 and stained with A11 , M204 and M205.
  • Figure 2C shows a Western blot of A ⁇ 42 prefibrillar oligomers stained with A11 and M204.
  • Figure 3A shows a dot blot analysis of A ⁇ 40 prefibrillar oligomers (1), soluble monomer (2) and fibrils (3) probed with anti-oligomer (top) or stripped and reprobed with 6E10 (bottom).
  • Figure 3B is a graph showing results of an ELISA assay wherein increasing amounts of A ⁇ 40 prefibrillar oligomers (open circles) monomer (filled triangles) and fibrils (filled squares) were applied to the wells and developed with anti-oligomer antibody.
  • Figure 4A shows an ELISA assay of different types of fibrils and soluble A ⁇ monomer and prefibrillar oligomers.
  • Figure 4B shows a dot blot of A ⁇ 42 fibrils and prefibrillar oligomers stained with anti-fibril (OC) or anti-oligomer (A11).
  • Figure 4C shows a Western blot of fibrillar (F) and prefibrillar oligomer (O) A ⁇ 42 samples.
  • Figure 5A shows negative stained electron micrograph of relatively homogeneous populations of A ⁇ 42 annular protofibrils prepared from solutions of prefibrillar oligomers by treatment with 5% hexane in water.
  • Figure 5B shows the specificity of anti-annular protofibrils antisera. Homogeneous samples of A ⁇ 42 annular protofibrils, spherical oligomers, fibrils and monomer (soluble) were plated in the wells of an ELISA plate and reacted with anti- annular protofibrils antibody.
  • FIG 5C shows that the anti-annular protofibrils antibody reacts efficiently with annular protofibrils from alpha synuclein and IAPP (amylin).
  • Figure 5D shows immunoprecipitation of soluble AD brain extract with anti- oligomer antibody.
  • Figure 5E shows immunoprecipitation of non-demented control brain extract with anti-annular protofibril antibody.
  • Figure 6A shows the structure of monoclonal M204 Fab as determined by x- ray crystallography in a view (View 1) wherein the antibody fragment with the antigen combining site faces forward toward the viewer.
  • Figure 6B shows the structure of monoclonal M204 Fab as determined by x- ray crystallography in a view (View 2) wherein the antibody fragment with the antigen combining site faces toward the viewer's right.
  • Figure 7A is a bar graph comparing the initial latency to cross the platform location in a Morris Wayer Maize test of animals treated with Control, anti-oligomer A11 , 204 antibody and 205 antibody.
  • Figure 7B is a bar graph comparing the time spent during training in the quadrant opposite to the one containing the platform in a Morris Wayer Maize test of mice treated with Control, A11 , 204 and 205.
  • Figure 7C is a bar graph comparing the numbers of platform location crosses in a Morris Wayer Maize test of mice treated with Control, A11 , 204 and 205.
  • Figure 8 is a bar graph showing Recognition Index (Rl) determined by the Object Recognition test of mice treated with Control, A11 , 204 and 205.
  • Figure 9A is a bar graph of comparing the mean latency (seconds) in an electroshock passive avoidance test of mice treated with Control, A11 , 204 and 205.
  • Figure 10 is a bar graph showing A ⁇ plaque load determined by 6E10 antibody uptake in brain tissue of mice treated with Control, A11 , 204 and 205.
  • adjuvant refers to a compound that when administered in conjunction with an antigen augments the immune response to the antigen, but when administered alone does not generate an immune response to the antigen.
  • adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages.
  • a ⁇ or "A ⁇ peptide” refers to peptides which comprise low molecular weight soluble oligomers, prefibrillar aggregates, fibrils and amyloid deposits each associated with AD.
  • Amyloid A ⁇ peptides include, without limitation, A ⁇ 39, A ⁇ 40, A ⁇ 41 A ⁇ 42 and A ⁇ 43 which are 39, 40, 41 , 42 and 43 amino acid amino acids in length, respectively.
  • amyloid peptide is a peptide that is present in amyloid forms including amyloid peptide intermediates, low molecular weight soluble oligomers, amyloid fibrils and amyloid plaques.
  • antibody is used to include intact antibodies and binding fragments thereof, including but not limited to, for example, full-length antibodies (e.g., an IgG antibody) or only an antigen binding portion (e.g., a Fab, F(ab ! ) 2 or scFv fragment). Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen.
  • antibodies or binding fragments thereof can be chemically conjugated to, or expressed as, fusion proteins with other proteins.
  • Anti-oligomer antibody or “Anti-oligomer” refer to an antibody that binds to amyloid peptide aggregate intermediates but does not bind to or does not specifically bind to amyloid peptide monomers, dimers, trimers or tetramers.
  • compositions or methods "comprising" one or more recited elements may include other elements not specifically recited.
  • a composition that comprises an amyloid A ⁇ peptide may encompass both an isolated amyloid A ⁇ peptide as a component of a larger polypeptide sequence or as part of a composition which includes multiple elements.
  • epitope refers to a site on an antigen to which B and/or T cells respond or a site on a molecule against which an antibody will be produced and/or to which an antibody will bind.
  • an epitope can be recognized by an antibody defining the epitope.
  • a “linear epitope” is an epitope wherein an amino acid primary sequence comprises the epitope recognized.
  • a linear epitope typically includes at least 3, and more usually, at least 5, for example, about 8 to about 10 amino acids in a unique sequence.
  • a “conformational epitope”, in contrast to a linear epitope, is an epitope wherein the primary sequence of the amino acids comprising the epitope is not the sole defining component of the epitope recognized (e.g., an epitope wherein the primary sequence of amino acids is not necessarily recognized by the antibody defining the epitope).
  • a conformational epitope comprises an increased number of amino acids relative to a linear epitope.
  • the antibody recognizes a 3-dimensional structure of the peptide or protein.
  • a protein molecule folds to form a three dimensional structure, certain amino acids and/or the polypeptide backbone forming the conformational epitope become juxtaposed enabling the antibody to recognize the epitope.
  • Methods of determining conformation of epitopes include but are not limited to, for example, x-ray crystallography 2-dimensional nuclear magnetic resonance spectroscopy and site-directed spin labeling and electron paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996), the disclosure of which is incorporated in its entirety herein by reference.
  • immunological response relates to the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against an amyloid peptide in a recipient patient.
  • Such a response can be an active response induced by administration of monoclonal antibody or a passive response induced by administration of antibody or primed T-cells.
  • a cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class Il MHC molecules to activate antigen-specific CD4 + T helper cells and/or CD8 + cytotoxic T cells.
  • the response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells,
  • a "monoclonal antibodyic agent” or “monoclonal antibody” or “antigen” is capable of inducing an immunological response against itself upon administration to a subject, optionally in conjunction with an adjuvant.
  • Isolated means purified, substantially purified or partially purified. Isolated can also mean present in an environment other than a naturally occurring environment. For example, an antibody that is not present in the whole blood serum in which the antibody would ordinarily be found when naturally occurring is an isolated antibody.
  • Low molecular weight aggregate refers to amyloid peptides present in aggregates of less than four or five peptides.
  • low molecular weight A ⁇ refers to the low molecular weight soluble oligomers found associated with AD.
  • patient includes human and other animal subjects that receive therapeutic, preventative or diagnostic treatment or a human or animal having a disease or being predisposed to a disease.
  • prefibrillar aggregates "micellar aggregates”, “high molecular weight aggregation intermediates,” “high molecular weight amyloid peptide aggregates”, “high molecular weight soluble amyloid peptide aggregates” "amyloid peptide aggregates”, “soluble aggregate intermediates”, “amyloid oligomeric intermediates”, “oligomeric intermediates” and “oligomeric aggregates” or simply, “intermediates” refer to aggregations which include more than three individual peptide or protein monomers, for example, more than four peptide or protein monomers.
  • the upper size of prefibrillar aggregates includes aggregations of oligomers which form spherical structures or micelles and stings of micelles which lead to fibril formation.
  • Annular protofibrils are a particular subset of prefibrillar aggregates in which 3 to 10 spherical oligomer subunits are arranged in an annular or circular fashion with a hollow center that appears as a pore in electron or atomic force micrographs.
  • the molecular weight of a prefibrillar aggregate may be in a range of about 10 kDa to about 100,000,000 KDa, for example, about 10 kDa to about 10,000,000 or 1 ,000,000 KDa. However, this size range is not intended to be limiting and prefibrillar aggregates are not defined by a molecular weight range.
  • Protofibrils are prefibrillar aggregates which include spherical structures comprising amyloid A ⁇ peptides that appear to represent strings of the spherical structures forming curvilinear structures.
  • Specific binding between two entities means an affinity of at least 10 6 ,10 7 , 1O 8 10 9 M “1 , or 10 10 M "1 . Affinities greater than 10 8 M "1 are preferred for specific binding.
  • substantially identical means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 65 percent sequence identity, for example, at least 80 percent or 90 percent sequence identity, or at least 95 percent sequence identity or more, for example, 99 percent sequence identity or higher.
  • residue positions in an alignment which are not identical differ by conservative amino acid substitutions, i.e., substitution of an amino acid for another amino acid of the same class or group.
  • Some amino acids may be grouped as follows: Group I (hydrophobic side chains): leu, met, ala, val, leu, ile; Group Il (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group Vl (aromatic side chains): trp, tyr, phe.
  • Non- conservative substitutions may include exchanging a member of one of these classes for a member of another class.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm may then be used to calculate the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol.
  • BLAST Altschul et al., J. MoI. Biol. 215: 403-410 (1990).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • default program parameters can be used to perform the sequence comparison, although customized parameters can also be used.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix, see for example, Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89,10915 (1989).
  • Conservative substitutions involve substitutions between amino acids in the same class.
  • a “therapeutic agent” or “therapeutic” is a substance useful for the treatment or prevention of a disease in a patient.
  • Therapeutic agents of the invention are typically substantially pure. This means that an agent is typically at least about 50% w/w (weight/weight) pure, as well as being substantially free from proteins and contaminants which interfere with the efficacy of the therapeutic.
  • the agents may be at least about 80% w/w and, more preferably at least 90 % w/w or about 95% w/w in purity.
  • homogeneous peptides of 99% w/w or more can be produced.
  • Amyloid diseases are characterized by the accumulation of amyloid plaques or precursors to amyloid plaques in patients or the predisposition to the accumulation of amyloid plaques or precursors to amyloid plaques in patients.
  • One of the primary constituents of amyloid plaques are amyloid peptides.
  • the general conformation of amyloid peptides may vary from disease to disease, but often the peptide has a characteristic . -pleated sheet structure.
  • Amyloid peptides include peptides and proteins of about 10 or about 20 amino acids to about 200 amino acids in length. Though this size range is not intended as a limitation and amyloid peptides or proteins having fewer or more amino acids are contemplated in the present invention.
  • Prefibrillar aggregates are intermediates in the production of insoluble fibrils that accumulate in amyloid plaques of humans or animals having a disease characterized by amyloid deposits, for example, Alzheimer's disease.
  • Prefibrillar aggregates include aggregates which may be as small as four amyloid peptides, as small as five amyloid peptides, as small as six amyloid peptides, as small as seven amyloid peptides or as small as eight amyloid peptides.
  • prefibrillar aggregates are micellar aggregates or micelles or strings of micelles. Prefibrillar aggregates are effective to form a conformational epitope which is recognized by an antibody of the present invention.
  • the conformational epitopes found on prefibrillar aggregates are substantially not found in the native precursor proteins for amyloid peptides, for example, amyloid peptide monomers, the amyloid precursor protein, nor in the amyloid fibrils that are defined by their characteristic cross ⁇ x-ray fiber diffraction pattern or in amyloid plaques.
  • the prefibrillar aggregates that contain the specific polypeptide structure which results in conformational epitopes that are recognized by antibodies of the present invention have a size range of approximately a dimer, trimer, tetramer, pentamer, a hexamer, a heptamer or an octamer, dodecamer to micellar forms or protofibrils which have a molecular weight in excess of 1 ,000,000 Daltons.
  • Antibodies of the invention are effective to bind to these epitopes.
  • Monoclonal antibodies of the present invention are specific for a conformation-dependent epitope associated with amyloid oligomers or protofibrils or in the case of M 121 , amyloid fibrils.
  • the monoclonal antibodies may be prepared by immunizing mice with a conformationally-constrained antigen consisting of amyloid A ⁇ covalently coupled to colloidal gold via a thioester linkage. Such monoclonal antibodies will provide for diagnostic and therapeutic uses. The antibody is also useful for determining the three dimensional structure of amyloid oligomers bound to the antibody by co- crystallization of the antibody Fab with the antigen and X-ray crystallography.
  • Tissue culture supernatant from the hybridomas was added to the wells at 1 :200, 1 :500, 1 :1000, 1 :2000 and 1 :5000 and incubated at 37 degrees for 1 hr.
  • the plates were washed 3x with phosphate buffered saline (PBS) and 100 ul of goat anti rabbit-horseradish peroxidase conjugate 1 :10,000 dilution was added to each well and incubated for 1 hr.
  • the plates were washed 3 times with PBS and then assayed for HRP activity by adding 100 ul of color diction substrate, TMB.
  • the plates were read at 450 nm. Clones that show high reactivity against oligomers and low reactivity against monomer and fibrils were selected.
  • Appendix A which forms a part of this specification, describes in detail the production of certain monoclonal antibodies of the present invention.
  • a typical dot blot is shown in Figure 1 for clones M118, M121 , M201 , M204, M205 and M206.
  • Lane 1 is A ⁇ 42 monomer.
  • Lane 2 is A ⁇ 42 prefibrillar oligomers.
  • Lane 3 is A ⁇ 42 fibrils.
  • Lane 4 is alpha synuclein prefibrillar oligomers,
  • Lane 5 is immunoglobulin light chain prefibrillar oligomers,
  • Lane 6 is prion 106-126 prefibrillar oligomers,
  • Lane 7 is KK(Q40)KK prefibrillar oligomers,
  • Lane 8 is calcitonin prefibrillar oligomers.
  • amyloid peptides have been shown to form amyloid peptide aggregates which produce a conformational epitope recognized by the antibodies of the present invention, for example, antibodies produced against A ⁇ peptide oligomeric intermediates. Some of these peptides are present in amyloid deposits of humans or animals having a disease characterized by the amyloid deposits.
  • the present invention is not limited to the listed peptide or protein sequences or the specific diseases associated with some of the sequences.
  • the present invention contemplates antibodies as described herein binding to other amyloid peptide aggregates or all other amyloid peptide aggregates.
  • the present invention contemplates and includes the application of methods and compositions of the present invention to other peptide or protein sequences which form amyloid precursor aggregates associated with other diseases.
  • oligomeric intermediates formed from variants and fragments of wild type A ⁇ 42, AE40 including, without limitation A ⁇ 42 (A21G) Flemish mutation), AS42 (E22Q) Dutch mutation, A ⁇ 42 (E22G) Arctic mutation, AS42 (D23N) Iowa mutation, AB40 (A21G) Flemish mutation), A ⁇ 40 (E22Q) Dutch mutation, A ⁇ 40 (E22G) Arctic mutation, A ⁇ 40 (D23N) Iowa mutation, A ⁇ 40 (E22Q &D23N) Dutch & Iowa mutations, A ⁇ 3-42 (pGlu 3), A ⁇ 3-40 (pGlu 3), A ⁇ 8-42, A ⁇ 17-42, AB1-16, AB3-11 , A ⁇ 25-35, A ⁇ 4-16 (3 analogues, Cys 16 AU4-16, Ala 4 A ⁇ 4-16,and Ala 10 A ⁇ 4- 16 ), His6 A ⁇ 40C40 (6 histidines appended to the amino terminus of A ⁇ C40) are recognized
  • oligomeric intermediates recognized by antibodies of the invention include, without limitation, oligomeric intermediates formed from IAPP(C2AandC7A) where alanine is substituted for the naturally occurring cysteine in IAPP, Polyglutamine KKQ40KK or poly glutamine where the number of Q residues is greater than 32, Calcitonin, TTR and its mutants TTR Pro 55 , TTR Phe 78 , vitronectin, poly Lysine, poly arginine, serum amyloid A, cystatin C, IgG kappa light chain, oligomeric intermediates produced from other amyloid peptides disclosed herein and amyloid intermediates associated with amyloid diseases disclosed herein.
  • Examples of Rabbit Monoclonal Antibodies include, without limitation, oligomeric intermediates formed from IAPP(C2AandC7A) where alanine is substituted for the naturally occurring cysteine in IAPP, Polyglutamine KKQ40KK or poly glutamine where the number of Q residues
  • Figure 1 shows dot blot analysis of specificity for a number of rabbit monoclonal antibodies of the present invention.
  • Nitrocellulose strips containing 1 ug dots of A ⁇ 42 monomer, prefibrillar oligomers or fibrils and 5 additional types of prefibrillar oligomers were stained with polyclonal A11 and 6 distinct types of rabbit monoclonal antibodes.
  • A11 stains all types of prefibrillar oligomers as previously reported, however, the rabbit monoclonal antibodies display a more restricted specificity than A11 and have distinct preferences for different types of oligomers. For example, M204 recognizes all types of oligomers, but it reacts only weakly with light chain oligomers.
  • M201 only recognizes A ⁇ oligomers.
  • M 118, M205 and M206 have intermediate degrees of specificity and display distinct preferences.
  • M 121 is specific for A ⁇ fibrils and does not recognize A ⁇ monomer or prefibrillar oligomers.
  • M201 and M121 are IgMs, while the other monoclonals are IgG.
  • antibodies of the present invention distinguish different subclasses of A ⁇ prefibrillar oligomers.
  • a ⁇ 40 and AB42 were incubated under three different conditions (HBS, hepes buffered saline pH7.4; water pH 2.5; or PBS pH 7.4) .
  • Monomer samples were spotted at time zero, while prefibrillar oligomers and fibrils were spotted at 72 and 240 hours of incubation respectively. All of the oligomer samples stain with A11 , but M118 only reacts with samples prepared at pH 2.5.
  • FIG 2B four different samples of prefibrillar oligomers were prepared in water pH 2.5 and stained with A11 , M204 and M205. All samples stain with A11. Sample 1 stains with both 204 and 205 antibodies while sample 2 stains with only M204, sample 3 stains with M205 and sample 4 stains with neither monoclonal.
  • Figure 2C shows a Western blot of A ⁇ 42 prefibrillar oligomers stained with A11 and M204. Although the sample stains with both antibodies, the oligomer bands stained by M204 are larger than those stained by A11 , indicating that the distinct sizes are also immunologically distinct.
  • FIG. 3A The specificity of anti-prefibrillar oligomer (A11) antibody is demonstrated by Figures 3A and 3B.
  • Figure 3A 1 ug each of A ⁇ 40 prefibrillar oligomers (1), soluble monomer (2) and fibrils (3) was spotted on a nitrocellulose membrane and probed with anti-oligomer (top panel) or stripped and reprobed with 6E10 (bottom panel).
  • Figure 3B is a graph showing results of an ELISA assay wherein increasing amounts of A ⁇ 40 prefibrillar oligomers (open circles) monomer (filled triangles) and fibrils (filled squares) were applied to the wells and developed with anti-oligomer antibody.
  • prefibrillar oligomer specificity was obtained for amylin (IAPP) 1 alpha synuclein, insulin, lysozyme, prion 106-128 peptide and polyglutamine (KKQ40KK) and many other types of prefibrillar oligomers.
  • the conformation-dependent monoclonal antibodies of the present invention recognize distinct epitopes that are associated with different types of amyloids. Many of the antibodies and antisera we have produced so far recognize generic epitopes that may arise from the conformation, shape or pattern of the polypeptide aggregates and are independent of the specific amino acid sequence of the protein. These polyclonal antibodies include anti-oligomer, and two new antisera: one that recognizes an epitope found in fibrils and soluble fibril oligomers or nuclei and one associated with pore-like annular protofibrils. Amyloids of different types, including A ⁇ seem to share common pathways of aggregation that result in several distinct assembly states.
  • the protein adopts an amyloidogenic conformation that facilitates its assembly along two distinct pathways as described in the Background of the Invention Section set forth above. This results in the formation of prefibrillar oligomers or the formation of an amyloid fibril lattice. We believe that these are alternative pathways, rather than prefibrillar oligomers representing an obligate intermediate in the fibril formation pathway because the formation of prefibrillar oligomers and fibrils is differentially sensitive to disruption by protein denaturants like urea and aggregation inhibitors.
  • Prefibrillar spherical oligomers appear to be building blocks for higher order assemblies, including protofibrils and annular protofibrils because annular protofibrils can be prepared in high yield from spherical oligomers (see below).
  • Prefibrillar oligomers and protofibrils (which appear morphologically as curvilinear strings of spherical oligomers) are unstable kinetic intermediates because they disappear as mature fibrils form (20). It is not clear whether protofibrils undergo a conformation change to become mature fibrils or whether they dissociate into monomer, which then adds on to fibril nuclei during fibril growth.
  • Prefibrillar oligomers and protofibrils are recognized by anti-prefibrillar oligomer polyclonal antibodies (A11). Fibrils (both insoluble and 100,000 x G soluble oligomeric species) are recognized by anti-fibril polyclonal antibodies (OC). Rough and smooth annular protofibrils are recognized by anti-annular protofibril polyclonal antibodies (officer). There is minimal cross- reactivity between these antibodies and the epitopes appear to be quite mutually exclusive at least at the limit of the purity of the samples used to characterize them (see Figure 4 below).
  • Figures 4A through 4C show the specificity of anti-Fibril (OC) antibody.
  • Figure 4A shows an ELISA assay of different types of fibrils and soluble A ⁇ monomer and prefibrillar oligomers. Wells were plated with the different samples indicated in the inset and reacted with dilutions of OC antiserum. All fibrillar samples react with OC.
  • Figure 4B shows a dot blot of A ⁇ 42 fibrils and prefibrillar oligomers stained with anti-fibril (OC) or anti-oligomer (A11). OC only stains the fibrillar sample while A11 only stains the oligomeric sample.
  • Figure 4C shows a Western blot of fibrillar (F) and prefibrillar oligomer (O) A ⁇ 42 samples.
  • Prefibrillar oligomers were prepared by dilution of a stock solution of A ⁇ 40 in urea to 45 mM in 10 mM phosphate buffer, pH 7.4 and incubation for 4 days.
  • Both fibrillar and prefibrillar oligomer samples contain bands that react with 4G8 ranging from monomer up to the size of material that accumulates at the top of the gel.
  • OC only stains the bands from fibrillar samples of approximately dimer and above.
  • A11 only stains the prefibrillar oligomer samples.
  • the sizes of the OC and A11 positive oligomers broadly overlap, although they are immunologically distinct. 6E10 does not stain prefibrillar A ⁇ oligomer samples formed at pH 7.4 as previously reported.
  • A11 conformation-dependent rabbit polyclonal antibody
  • This anti-oligomer antibody specifically recognizes soluble oligomers in the same conformation-dependent fashion from many different amyloids, including lysozyme, IAPP, synuclein, prion 106-126, polyQ and insulin. This indicates that the oligomeric aggregates display a common epitope that is distinct from that displayed by mature fibrils.
  • the epitope is likely to be a common peptide backbone structure, such as the edge of a ⁇ sheet or a turn motif that is either absent or structurally distinct in the fibril structure.
  • the availability of antibodies that specifically recognize the generic oligomeric or prefibrillar assembly states of amyloids provides the opportunity to examine early aggregation events and for distinguishing the specific misfolded conformation from a vast excess of native and other folded structures in complex mixtures or tissues.
  • Anti-oligomer antibody stains deposits of oligomers in AD brain sections that are reduced or absent from age-matched control brain and in brain tissue from transgenic mouse models of AD.
  • the oligomeric deposits are distinct from the thioflavin-S positive fibrillar amyloid deposits and where they are found in the vicinity of diffuse amyloid deposits, their localizations remain distinct and non overlapping. Even when A11 immunoreactivity is found in the vicinity of a plaque, their distributions are distinct, with A11 being found in the periphery as if oligomers are either being recruited to a growing plaque or they are dissociating from it. At the ultrastructural level, A11 immunoreactivity has been localized in cell processes and axon terminals in human AD and Tg2576 mouse brain, suggesting that A11 positive prefibrillar oligomers may be related to neuronal dysfunction.
  • the anti-oligomer antibody provides a facile means of assessing the significance of oligomers in pathogenesis, identifying other diseases in which oligomers are implicated, the role of oligomers in pathogenesis and potentially targeting oligomers in the development of therapeutics for amyloid-related degenerative diseases.
  • Antibodies that are specific for a generic epitope that is associated with amyloid fibrils have also been reported. We have also produced such a conformation-dependent antibody that specifically recognizes many different types of fibrils, by immunizing rabbits with homogeneous fibril preparations as described herein. This polyclonal antibody recognizes fibrils, but not prefibrillar oligomers, monomer or natively folded precursor proteins such as APP.
  • the epitopes displayed by mature fibrils and soluble oligomers appears to be distinct and mutually exclusive as ELISA assays, dot blots and western blots of fibrillar and prefibrillar oligomers stain only with the fibril and prefibrillar oligomer antibody respectively (Fig. 2A, B, C).
  • the size distributions of soluble A ⁇ species staining with these antibodies broadly overlap, indicating that size is not a good indicator of their immunologically defined conformation.
  • soluble oligomers that stain with anti-oligomer (A11) and “fibrillar” oligomers or fibril nuclei that stain with anti-fibril (OC) and may represent small pieces of fibrils or fibril nuclei.
  • Staining of AD brain tissue with this antibody reveals that all types of amyloid deposits stain intensely, including thioflavin negative diffuse plaques that are commonly referred to as “non-fibrillar” because of their tinctorial properties. The staining is completely blocked by preincubation of the antiserum with excess A ⁇ fibrils.
  • amylin amyloid deposits in transgenic mouse models of type Ii diabetes and light chain amyloid in systemic amyloidosis.
  • Annular protofibrils are ring-like structures that resemble pores and have been described as occurring occasionally or individually in solutions containing predominantly spherical prefibrillar amyloid oligomers. Based on their morphological resemblance, it has been proposed that they represent membrane pores that can account for the membrane permeabilizing activity associated with amyloid aggregates.
  • Figures 5A through 5E relate to these annular protofibrils and anti annular protofibrils antibody.
  • Figure 5A shows negative stained electron micrograph of relatively homogeneous populations of A ⁇ 42 annular protofibrils prepared from solutions of prefibrillar oligomers by treatment with 5% hexane in water. Inset: Incubation of the sample for 2 weeks results in a loss of the beaded or rough morphology and the appearance of a smooth morphology. The same method is used for preparing annular protofibrils from alpha synuclein and IAPP.
  • Figure 5B shows the specificity of anti-annular protofibrils antisera.
  • FIG. 5C shows that the anti-annular protofibrils antibody reacts efficiently with annular protofibrils from alpha synuclein and IAPP (amylin).
  • Figure 5D shows immunoprecipitation of soluble AD brain extract with anti-oligomer antibody.
  • Figure 5E shows immunoprecipitation of non-demented control brain extract with anti-annular protofibril antibody.
  • annular protofibrils in high yield and purity ( Figure 5A above). Homogeneous samples of annular protofibrils were used as an immunogen to raise polyclonal antisera in rabbits. The resulting antibody is remarkably selective for annular protofibrils (Fig. 5B). The shorthand laboratory jargon for this anti annular protofibril antibody is "officer" (officers like donuts). In ELISA assays using pure preparations of A ⁇ monomer, fibrils, prefibrillar oligomers and annular protofibrils, officer antibody is selective for annular protofibrils.
  • Electron microscopy of the eluted fraction reveals numerous naturally occurring ring-like annular structures that have the exact same morphology and size as the aged samples prepared in vitro (compare Fig. 5D and 5A inset). Very few if any ring-like structures were immunoprecipitated from age matched control brains (Fig. 5E).
  • M201 only recognizes A ⁇ oligomers.
  • M118, M205 and M206 have intermediate degrees of specificity and display distinct preferences.
  • M 121 is specific for A ⁇ fibrils and does not recognize A ⁇ monomer or prefibrillar oligomers.
  • M201 , M206 and M 121 are IgMs, while the other monoclonals are IgG
  • the specificity of the monoclonal antibodies is of interest for several reasons. Firstly, all of the monoclonals we obtained in response to vaccination with the A11 A ⁇ C-terminal thioester colloidal gold antigen are conformation specific even though we selected all clones that reacted with A ⁇ monomer, oligomers and fibrils. None of the clones recognize monomer like 6E10. This indicates that the immune response to the solid phase antigen is highly conformation specific. None of the antibodies recognize both pure fibril and pure oligomer samples, indicating that the distribution of these epitopes is mutually exclusive.
  • M205 reacts strongly with alpha synuclein and light chain oligomers, but does not react well with prion 106-126, polyQ and calcitonin prefibrillar oligomers. M118 prefers light chain and polyQ oligomers, but not synuclein, prion or calcitonin oligomers. These results indicate that there are multiple distinct epitopes associated with prefibrillar oligomers that are widely distributed within this class and that monoclonal antibodies can recognize these unique epitopes. Thirdly, some monoclonals are both conformation dependent and sequence specific. M201 recognizes only A ⁇ oligomers, while M121 only recognizes A ⁇ fibrils. M118 , M204 and M205 are IgG, while the other antibodies are IgM.
  • Monoclonal antibodies distinguish different subtypes of A11 positive prefihrillar A ⁇ oligomers.
  • Electrophoretic variability of prefibrillar oligomers Electrophoretic variability of prefibrillar oligomers.
  • Prefibrillar oligomers display a range of apparent molecular weights and electrophoretic patterns, depending on the sample preparation and on the type of gel system used.
  • Samples prepared by dilution from a stock solution of urea into 10 mM phosphate to a final concentration 45 mM A ⁇ 40 run as broad band centered on an apparent Mw of approximately 56 kDa, with a number of distinct lower MW bands extending as a "ladder" to approximately 16 kDa (Fig. 2C).
  • Samples prepared by dilution of HFIP stock solution in distilled water at pH 2.5 also run as a broad band centered at approximately 56 kDa, but lack the distinct ladder of lower MW bands (Fig. 2C).
  • Prefibrillar oligomers prepared by dilution of NaOH stock solutions of A ⁇ into 10 mM phosphate buffer at pH 7.4 run as a distinct ladder with a major band at an apparent MW of 14 kDa (trimer) all the way up to approximately 100 kDa.
  • the interpretation of the exact molecular weights should be viewed with caution as there is some evidence that the electrophoretic mobility of the oligomers may not reflect their true mass. You would expect that the ladder pattern may represent oligomers that differ by a single polypeptide, but if this is true, then their electrophoretic mobility does not accurately reflect their molecular weight.
  • M118 exhibits intracellular staining of CA1 neurons in PSAPP Tg mouse brain.
  • M204 staining is finely punctuate and enriched in synaptic regions, such as the outer molecular layer.
  • the plaques stain negatively with M204, as the staining intensity of the plaques is lower than the surrounding tissue. Similar staining is observed for M205.
  • FIG. 6A View 1
  • Figure 6B View 2
  • the complimentary determining regions are rendered in atomic level resolution including the side chains.
  • the other regions of the antibody fragment outside the CDRs is rendered in ribbon format.
  • the present invention provides for amyloid disease therapeutics which induce a specific immune response against amyloid oligomeric intermediates.
  • Therapeutics of the invention include antibodies that specifically bind to oligomeric intermediates. Such antibodies can be monoclonal antibodies as described in this provisional patent application and/or in the above-incorporated copending United States Patent Application Serial No. Serial No. 10/572,001 or polyclonal antibodies as described in PCT International Application No. PCT/US2003/028829, which is also incorporated herein by reference. In one useful embodiment, the antibodies bind to a conformational epitope.
  • non-human monoclonal antibodies of the present invention can be accomplished by, for example, immunizing the animal with an oligomeric intermediate mimic of the invention. Also contemplated is immunizing the animal with a purified amyloid intermediate.
  • Humanized forms of mouse antibodies of the invention can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86,10029-10033 (1989) and WO 90/07861 (incorporated by reference for all purposes).
  • Human antibodies may be obtained using phage-display methods. See, for example, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Phage displaying antibodies with a desired specificity are selected by affinity enrichment. Human antibodies against oligomeric intermediates may also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus.
  • Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies.
  • Human or humanized antibodies can be designed to have IgG, IgD, IgA and IgE constant region, and any isotype, including IgGI, lgG2, lgG3 and lgG4.
  • Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab' F(ab')2 and Fv, or as single chain antibodies in which heavy and light chain variable domains are linked through a spacer.
  • Suitable carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria, E. coli, cholera, or H. pylori, or an attenuated toxin derivative.
  • Other carriers which may act as adjuvants for stimulating or enhancing an immune response include cytokines such as IL-1 , IL-1 ⁇ and.
  • Human or animal subjects or patients amenable to treatment with monoclonal antibodies of the present invention include individuals at risk of amyloid disease but not showing symptoms, as well as those who already show symptoms or other evidence of amyloid disease. In the case of certain amyloid diseases including AD, virtually anyone is at risk of suffering from the disease.
  • monoclonal antibodies of the present invention could be administered prophylactically, for example, as a vaccine, to the general population without any assessment of the risk of the subject patient.
  • the present methods are especially useful for individuals who do have a known genetic risk of an amyloid disease, for example, AD.
  • Such individuals may include those having relatives who have experienced an amyloid disease, and those whose risk is determined by analysis of genetic or biochemical markers or who exhibit symptoms or prodromes indicative of the potential for development of, or the actual presence of, such diseases .
  • genetic markers of risk toward AD include mutations in the APP gene, particularly mutations at position 717 and positions 670 and 671 referred to as the Hardy and Swedish mutations respectively (see Hardy, TINS, supra).
  • Other markers of risk for AD are mutations in the presenilin genes, PS1 and PS2, and ApoE4, family history of AD, hypercholesterolemia or atherosclerosis.
  • Symptoms of amyloid disease are apparent to a physician of ordinary skill. For example, individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above.
  • a number of diagnostic tests are available for identifying individuals who have amyloid diseases. For example, in the case of AD these include measurement of CSF tau and A ⁇ 42 levels. Elevated tau and decreased A ⁇ 42 levels signify the presence of AD.
  • treatment can begin at any age, for example, at the age of 10, 20, 30, 40, 50, 60 or 70.
  • Treatment may entail one or more doses, for example, multiple dosages over a period of time.
  • Treatment can be monitored by assaying antibody, or activated T-cell or B-cell responses to the therapeutic (for example, oligomeric intermediate mimic) or assaying the levels of prefibrillar aggregate present, each over time.
  • treatment by administering a single therapeutic of the invention such as a preparation containing a single monoclonal antibody of the invention, may serve as a treatment for or preventive measure against more than one amyloid disease, for example all amyloid diseases.
  • compositions of the invention or medians are administered to a patient susceptible to, or otherwise at risk of, a particular disease in an amount sufficient to eliminate or reduce the risk or delay the outset of the disease.
  • compositions or medians are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a therapeutically-or pharmaceutically-effective dose.
  • therapeutics are usually administered in several dosages until a sufficient immune response has been achieved. Typically, the immune response is monitored and repeated dosages are given if the immune response starts to fade.
  • Effective doses of the compositions of the present invention, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human, but in some diseases, such as mad cow disease, the patient can be a nonhuman mammal, such as a bovine or in the case of Alzheimer's disease, the patient may be a dog.
  • Treatment dosages need to be titrated to optimize safety and efficacy.
  • the dosage ranges from about 0.0001 mg/kg of body weight to about 100 mg/kg of body weight, and more usually about 0.01 mg/kg of body weight to about 5 mg/kg of body weight of the host.
  • the amount of monoclonal antibody to be administered may depend on whether any adjuvant is also administered, with higher dosages being required in the absence of adjuvant.
  • 0.1 to 100cc of a solution containing approximately 1% by weight of the desired monoclonal antibody(ies) my be injected subcutaneously, thereby delivering a dose of 1mg to 1g of the monoclonal antibody(ies) per injection.
  • the timing of injections can vary significantly from once a day, to once a year, to once a decade.
  • One typical regimen consists of an immunization followed by booster injections at 6 weekly intervals.
  • Another regimen consists of an immunization followed by booster injections 1 , 2 and 12 months later.
  • Another regimen entails an injection every two months for life.
  • booster injections can be on an irregular basis as indicated by monitoring of immune response.
  • Therapeutics for inducing an immune response can be administered by any suitable route of administration, for example, parenteral, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal or intramuscular.
  • the most typical route of administration is subcutaneous although others can be equally effective.
  • the next most common is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles.
  • Intravenous injections as well as intraperitoneal injections, intraarterial, intracranial, or intradermal injections may also be effective in generating an immune response.
  • therapeutics are injected directly into a particular tissue where deposits have accumulated or may accumulate.
  • Monoclonal antibodies of the invention can optionally be administered in combination with other agents that are at least partly effective in treatment of amyloidogenic disease.
  • therapeutics of the invention can also be administered in conjunction with other agents that increase passage of the compositions of the invention across the blood-brain barrier.
  • anti-inflammatory dosages of colloidal gold or gold salts may be administered concomitantly (e.g., before, concurrently with or after) the monoclonal antibody to deter the brain inflammation associated with AD and other amyloid diseases.
  • Monoclonal antibodies of the invention may sometimes be administered in combination with an adjuvant.
  • adjuvants can be used in combination with an monoclonal antibody of the invention to elicit an immune response.
  • Preferred adjuvants augment the intrinsic response to an monoclonal antibody without causing conformational changes in the monoclonal antibody that affect the qualitative form of the response.
  • Preferred adjuvants include alum, 3 de-O-acylated monophosphoryl lipid A (MPL) (see Great Britain Patent No. 2220211).
  • QS21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al., in Vaccine Design: The subunit and Ajuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); and US Patent No. 5,057,540).
  • Other adjuvants are oil in water emulsions, such as squalene or peanut oil, optionally in combination with immune stimulants, such as monophosphoryl lipid A. See, for example, Stoute et al., N. Engl. J. Med. (1997) 336,86-91.
  • Another useful adjuvant is CpG described in Bioworld Today, Nov. 15,1998.
  • a monoclonal antibody can be coupled to an adjuvant.
  • Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic.
  • a preferred class of adjuvants is aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate.
  • alum aluminum hydroxide, aluminum phosphate, aluminum sulfate.
  • Such adjuvants can be used with or without other specific immunostimulating agents such as MPL or 3-DMP, QS21 , polymeric or monomeric amino acids such as polyglutamic acid or polylysine.
  • adjuvants is oil-in-water emulsion formulations.
  • Such adjuvants can be used with or without other specific immunostimulating agents such as muramyl peptides (for example, N-acetylmuramyl-L-threonyl-D- isoglutamine (thr- MDP), -acetyl-normuramyl-L-alanyl-D- isoglutamine (nor-MDP), N-acetylmuramyl-L- alanyl-D-isoglutamyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero- 3- hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N- acetylmuramyl-L-AI-D-isoglu-L-Ala- dipalmitoxy propylamide (DTP-DPP) theramideTM
  • Oil-in-water emulsions include (a) MF59 (WO 90/14837), containing 5% Squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80,5% pluroinic-blocked polymer L121 , and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall
  • Another class of preferred adjuvants is saponin adjuvants, such as Stimulons (QS21 , Aquila, Worcester, MA) or particles generated therefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX.
  • Other adjuvants include Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA).
  • Other adjuvants include cytokines, such as interleukins, for example, IL-1 , IL-2, and IL-12, macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF) and/or chemokines such as CXCL10 and CCL5.
  • An adjuvant can be administered with an monoclonal antibody as a single composition, or can be administered before, concurrent with or after administration of the monoclonal antibody.
  • Monoclonal antibody and adjuvant can be packaged and supplied in the same vial or can be packaged in separate vials and mixed before use.
  • Monoclonal antibody and adjuvant are typically packaged with a label indicating the intended therapeutic application. If monoclonal antibody and adjuvant are packaged separately, the packaging typically includes instructions for mixing before use.
  • an adjuvant and/or carrier depends on the stability of the vaccine containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies.
  • Complete Freund's adjuvant is not suitable for human administration.
  • two or more different adjuvants can be used simultaneously. Preferred combinations include alum with MPL, alum with QS21 , MPL with QS21 , and alum, QS21 and MPL together.
  • Incomplete Freund's adjuvant can be used (Chang et al., Advanced Drug Delivery Reviews 32,173-186 (1998)), optionally in combination with any of alum, QS21 , and MPL and all combinations thereof.
  • compositions of the invention are often administered as pharmaceutical compositions comprising a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980). The preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animai or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • compositions or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonmonoclonal antibodyic stabilizers and the like.
  • adjuvants or nontoxic, nontherapeutic, nonmonoclonal antibodyic stabilizers and the like.
  • some reagents suitable for administration to animals, such as complete Freund's adjuvant are not typically included in compositions for human use.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (e.g., adjuvants).
  • immunostimulating agents e.g., adjuvants.
  • compositions of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • a pharmaceutical carrier which can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • Auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions.
  • Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. See Langer, Science (1990) 249, 1527and Hanes, Advanced Drug Delivery Reviews (1997) 28,97- 119.
  • the compositions of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.
  • binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to about 10%, for example, about 1 % to about 2%.
  • Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and may contain about 10% about 95% of active ingredient, for example, about 25% to about 70%.
  • Topical application can result in transdermal or intradermal delivery.
  • Topical administration can be facilitated by co-administration of the composition with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. See Glenn et al., Nature (1998) 391 ,851.
  • Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
  • transdermal delivery can be achieved using a skin path or using transferosomes. See for example, Paul et al., Eur. J. Immunol. (1995) 25,3521-24; Cevc et al., Biochem. Biophys. Acta (1998) 1368,201-15.
  • injectable colloidal gold preparations (MyochrysineTM or SolganalTM) are commercially available for the treatment of rheumatoid arthritis.
  • a gold preparation for oral administration (Auranofin TM) is also available.
  • Inflammation of in the brain is thought to be a cause or contributing factor Alzheimer's Disease, primarily because amyloid-beta (protein) which is found in the brains of Alzheimer's patients is known to be an inflammatory protein.
  • nonsteroidal anti-inflammatory drugs such as rofecoxib (Vioxx) and naproxen (Aleve) to slow the progression of Alzheimer's Disease.
  • Applicants have determined, on the basis of histopathological observations, that the subcutaneous administration of colloidal gold can reduce microglial activation in the brains of mice modeling for amyloid disease.
  • the present invention includes the administration of colloidal gold, gold salts or other antiinflammatory agents to the subject in an amount that is therapeutically effective to decrease neural inflammation.
  • the gold or anti-inflammatory agent may be combined with the monoclonal antibody.
  • the gold or antiinflammatory agent may be administered separately from the monoclonal antibody. Any suitable dose, dosing schedule or route of administration may be used.
  • commercially available gold preparations for treatment of rheumatoid arthritis may be administered by the same routes of administration (subcutaneous injection of MyochrysineTM or SolganalTM or oral administration of AuranofinTM and dosages/dosing schedules recommended for treatment of rheumatoid arthritis.
  • CAA cerebral amyloid angiopathy
  • mice were passively immunized with one of the following antibodies: A11 , 204, 205 (described in Appendix A) or rabbit IgG control Ab. Immunization was for 5 weeks. The immunization was done intraperitonialy (300 ⁇ g/immunization).
  • mice were pre-tested longitudinally at hidden and cued platform Morris water maze (MWM) for 7 days.
  • the mice were divided into four groups receiving A11 , 204, 205 Abs and the other group receiving vehicle Ab (rabbit IgG).
  • Abs the other group receiving vehicle Ab
  • mice were evaluated for context object recognition after every 2 weeks. Before sacrificing, the mice were again re-evaluated for MWM test. Inhibitory avoidance test (which is related to amygdala) was also done.
  • the Morris water maze tests a special memory task related to hippocampal function.
  • the apparatus used for this test was a circular aluminum tank (1.5 m diameter) painted white and filled with water maintained at 26°C-29°C.
  • the maze WAS located in a room containing simple visual, extramaze cues.
  • mice were placed on the platform in both the hidden and cued versions of the task for 15 sec prior to the first training trial
  • Mice were trained to swim to a circular clear Plexiglas platform (14 cm diameter) submerged 1.5 cm beneath the surface of the water and invisible to the mice while swimming.
  • the platform location was selected randomly for the 6 and 10 months test, but was kept constant for each individual mouse throughout training.
  • mice On each trial, the mouse was placed into the tank at one of four designated start points in a pseudorandom order. Mice were allowed 60 seconds to find the submerged platform. If a mouse failed to find the platform within 60 s, it was manually guided to the platform and allowed to remain there for 15 s. After this, each mouse was placed into a holding cage under a warming lamp for 30 s before beginning the next trial. To ensure that memory differences were not due to lack of task learning, mice were given four trials a day for as many days as were required to train the 3xTg-AD-h mice to reach the criterion ( ⁇ 20 s). To control for overtraining, probe trials were run for each group, both as soon as they reached group criterion and after all groups had reached criterion.
  • mice were deeply anesthetized with an overdose of pentobarbital (150 mg/kg, IP), blood collected by cardiac puncture, and then animals perfused transcardially with cold phosphate-buffered saline (PBS). After dissection, brain tissue was fixed overnight with 4% paraformaldehyde in PBS, pH 7.4 at 4°C. Thereafter, fixed tissue was stored in PBS/0.02% sodium azide (NaN3) at 4°C until use.
  • PBS cold phosphate-buffered saline
  • Sections were then incubated with primary antibodies (6E10) overnight at 4°C, rinsed 3 times with PBS with 0.1% TX and incubated with biotinylated secondary antibody followed by ABC kit reagent (Vector, Burlingame, CA) for 1 hour each at room temperature. Finally, after washing three times, the sections were incubated for approximately 2 ⁇ 5 min with diamino-benzidine (DAB), (Vector). Sections were mounted on slides, dehydrated in a series of graded ethanol, cleared with Histoclear, and then coverslipped with DePeX (Biomedical Specialities, CA).
  • DAB diamino-benzidine
  • the NIH image processing and analysis program (available from the National Institute of Mental Health, Bethesda, Maryland) was used to analyze the area occupied by ⁇ -amyloid, lmmunostaining was captured using a Sony high-resolution CCD video camera (XC-77) and NIH image 1.59b5 software. For every animal, 7-8 images from the superficial layer of the frontal parietal region in the cortex were captured with a 206objective. For hippocampal areas, similar imaging was performed for CA1 , subiculum and dentate gyrus.
  • FIGS Ik-IO are bar graphs showing the results of the Morris Water Maize test conducted 5 weeks following vaccination of mice with control (saline), A11 , 204 and 205 antibodies.
  • the mice vaccinated with A11 , 204 and 205 demonstrated significantly shorter initial latency to cross the platform location (Figure 7A), significantly less time spent during training in the quadrant opposite to the one containing the platform ( Figure 7B) and significantly greater numbers of platform location crosses (Figure 7C).
  • Figure 7A shows that in this test (at the 5 week time point), A11 , 204 and 205 anitbodies had significantly better retention memory than control animals.
  • Figure 8 is a bar graph showing Recognition Index (Rl) determined by the Object Recognition test conducted 4 weeks following vaccination of mice with control (saline), A11 , 204 and 205.
  • the Rl is the percentage of time spent exploring the novel object as opposed to the familiar object. Mice vaccinated with A11 and 205 antibodies had significantly higher RIs in some trials, thereby indicating potentially greater retention memory than controls.
  • FIGS 9A and 9B show results of the passive avoidance test.
  • Passive avoidance memory retention (mean latency ⁇ S. E. M.) was measured as a function of each animal's ability to remember an electrical shock at 90 minutes ( Figure 9A) and 24 hours (Figure 9B) following shock administration.
  • Figure 9A at 90 minutes post shock animals vaccinated with the 204 and 205 antibodies exhibited greater latency (i.e., better passive avoidance memory) than controls.
  • FIG 9B at 24 hours post shock, animals vaccinated with the A11 , 204 and 205 antibodies exhibited greater latency than control animals.
  • Figure 10 is a bar graph showing A ⁇ plaque load determined by 6E10 antibody uptake in brain tissue of mice following vaccination with control (saline), A11 , 204 and 205.
  • animals vaccinated with A11 , 204 and 205 had significantly lower A ⁇ plaque loads in the hippocampus than control animals.
  • A11 , 204 and 205 antibodies inhibited the formation of and/or caused regression of A ⁇ plaque in this study.
  • the supernatants from multiclone wells were screened for the presence of conformation dependent prefibrillar oligomer specific antibodies using A ⁇ prefibrillar oligomers as a primary screening agent.
  • a ⁇ monomer, prefibrillar oligomers and fibrils were used as a secondary means of excluding antibodies that are not conformation dependent and interact with all A ⁇ conformations.
  • Approximately 118 multiclone wells were selected as having immunoreactivity above a criterion of 0.5 AU (Table 1). These multi clones were sub-cloned and the resulting monoclones were subjected to additional screening and characterization using A ⁇ 40 monomer, prefibrillar oligomers and fibrils.
  • clones having a distinct preference for either prefibrillar oligomers or fibrils were selected. Representative clones selected are shown in Figure 1 in comparison to the A11 polyclonal antibody and 6E10, a sequence dependent mouse monoclonal antibody. The conformational and sequence specificity of the clones was analyzed by dot blot. Dot blot analysis was conducted by spotting 1 ug of A ⁇ 40 monomer, prefibrillar oligomers and fibrils and 1 ug of prefibrillar oligomers of alpha synuclein, immunoglobulin light chain, prion 106-126 peptide, KK(Q40)KK and calcitonin.
  • A11 polyclonal antibody reacts with all types of prefibrillar oligomers, but not A ⁇ monomer or fibrils. 6E10 recognizes only samples containing A ⁇ .
  • Clones 118, 201 , 204, 205 and 206 are specific for prefibrillar oligomers and do not recognize monomer or fibrils. This group of clones displays distinct specificities in terms of the other types of prefibrillar oligomers recognized.
  • Clone 121 is specific for A ⁇ fibrils and does not recognize prefibrillar oligomers of any type or A ⁇ monomer.
  • a number of clones appear to secrete antibodies of identical specificity. The most abundant class is similar to clone 201 , which only recognize A ⁇ prefibrillar oligomers and not oligomers of other types Fig. 4. All of these clones are also IgMs. Clone 118 is also indistinguishable from clones 48 and 55 (data not shown).
  • monoclonal antibodies offer unique advantages in terms of defining fine structural variation in amyloid aggregates and for determining the structures of these aggregates and their pathological significance.
  • the monoclonals obtained in response to vaccination with the A11 A ⁇ C-terminal thioester colloidal gold antigen are conformation specific even though we selected all clones that reacted with A ⁇ monomer, oligomers and fibrils. None of the clones recognize monomer like 6E10. This indicates that the immune response to the solid phase antigen is highly conformation specific. None of the antibodies recognize both pure fibril and pure oligomer samples, indicating that the distribution of these epitopes is mutually exclusive.
  • M205 reacts strongly with alpha synuclein and light chain oligomers, but does not react will with prion 106-126, polyQ and calcitonin oligomers. M118 prefers light chain and polyQ oligomers, but not synuclein, prion or calcitonin oligomers. These results indicate that there are multiple distinct epitopes associated with prefibrillar oligomers that are widely distributed within this class and that monoclonal antibodies can recognize these unique epitopes. Some monoclonals are both conformation dependent and sequence specific. M201 recognizes only A ⁇ oligomers, while M121 only recognizes A ⁇ fibrils. M118, M204 and M205 are IgG, while the other antibodies are IgM.
  • Monoclonal antibodies distinguish different types of A11 positive prefibrillar A ⁇ oligomers.
  • Anti-prefibrillar oligomer monoclonals do not stain plaques, but display a punctuate distribution in synapse rich areas.

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Abstract

Cette invention concerne des compositions d'anticorps monoclonaux, des procédés pour les produire et les utiliser. Les anticorps monoclonaux selon l'invention sont spécifiques d'épitope(s) conformationnel(s) d'agrégat(s) préfibrillaire(s) qui contribuent à la formation de fibrilles amyloïdes chez les sujets humains ou les animaux qui souffrent de maladies amyloïdes (par exemple, maladie d'Alzheimer). Cette invention concerne également les hybridomes et anticorps monoclonaux produits à partir de ceux-ci. Ces anticorps monoclonaux peuvent être utilisés pour immuniser des sujets humains ou des animaux contre la maladie d'Alzheimer ou d'autres maladies amyloïdes et/ou pour diagnostiquer ou détecter la maladie d'Alzheimer ou d'autres maladies amyloïdes. Ils peuvent être administrés concomitamment ou en combinaison avec des agents anti-inflammatoires, tels que des composés d'or ou contenant de l'or, pour réduire l'inflammation neurale associée aux maladies amyloïdes (par exemple, maladie d'Alzheimer).
PCT/US2009/051880 2002-09-12 2009-07-27 Anticorps monoclonaux spécifiques d'agrégats amyloïdes pathologiques communs aux amyloïdes formés à partir de protéines ayant des séquences différentes WO2010012004A2 (fr)

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WO2010119704A1 (fr) * 2009-04-17 2010-10-21 Immunas Pharma, Inc. Anticorps qui se lient spécifiquement aux oligomères a bêta et leur utilisation
WO2011029920A1 (fr) 2009-09-11 2011-03-17 Probiodrug Ag Dérivés hétérocycliques en tant qu'inhibiteurs de glutaminyle cyclase
US8378081B2 (en) 2008-02-08 2013-02-19 Immunas Pharma, Inc. Antibodies that specifically bind to Aβ oligomers and uses thereof
US8546532B2 (en) 2008-04-17 2013-10-01 Declion Pharmaceuticals, Inc. Synthesis of directed sequence polymer compositions and antibodies thereof for the treatment of protein conformational disorders
US8613924B2 (en) 2009-08-06 2013-12-24 Immunas Pharma, Inc. Antibodies that specifically bind to A beta oligomers and use thereof
US8858949B2 (en) 2009-08-06 2014-10-14 Immunas Pharma, Inc. Antibodies that specifically bind to a beta oligomers and use thereof
US9085614B2 (en) 2008-08-01 2015-07-21 Immunas Pharma, Inc. Antibodies that specifically bind to Aβ oligomers and uses thereof
EP3461819A1 (fr) 2017-09-29 2019-04-03 Probiodrug AG Inhibiteurs de la glutaminyl-cyclase
WO2021035210A1 (fr) * 2019-08-22 2021-02-25 The Regents Of The University Of California Anticorps à chaîne unique se liant à des oligomères de protéines tau et inhibant l'ensemencement par des extraits pathologiques de la maladie d'alzheimer
CN112540180A (zh) * 2020-11-06 2021-03-23 华中科技大学 用于检测人淀粉样蛋白-β双抗夹心ELISA检测试剂盒
CN112881708A (zh) * 2021-01-19 2021-06-01 华中科技大学 用于检测人淀粉样蛋白-β的胶体金免疫层析试纸及其制备
WO2021216667A3 (fr) * 2020-04-21 2021-12-02 The Board Of Regents Of The University Of Texas System Anticorps dirigés contre la chitinase 3-like -1 et leurs méthodes d'utilisation
US11267877B2 (en) 2017-10-06 2022-03-08 Prothena Biosciences Limited Anti-transthyretin antibodies
US11267878B2 (en) 2015-01-28 2022-03-08 Neotope Neuroscience Limited Anti-transthyretin antibodies
US11629185B2 (en) 2015-01-28 2023-04-18 Novo Nordisk A/S Anti-transthyretin antibodies
EP3998280A4 (fr) * 2019-07-08 2023-06-21 TERUMO Kabushiki Kaisha Hybridome et son procédé de production, anticorps monoclonal et son procédé de production
US11873332B2 (en) 2017-11-29 2024-01-16 Novo Nordisk A/S Lyophilized formulation of a monoclonal antibody against transthyretin
US11912759B2 (en) 2015-01-28 2024-02-27 Novo Nordisk A/S Anti-transthyretin antibodies
US12115141B2 (en) 2018-11-14 2024-10-15 University Of Kentucky Research Foundation Diagnosis of diabetes by detecting aggregated amylin in erythrocytes
US12247985B2 (en) 2017-10-06 2025-03-11 Novo Nordisk A/S Methods of detecting transthyretin

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US8378081B2 (en) 2008-02-08 2013-02-19 Immunas Pharma, Inc. Antibodies that specifically bind to Aβ oligomers and uses thereof
US9090680B2 (en) 2008-02-08 2015-07-28 Immunas Pharma, Inc. Antibodies that specifically bind to abeta oligomers and uses thereof
US8546532B2 (en) 2008-04-17 2013-10-01 Declion Pharmaceuticals, Inc. Synthesis of directed sequence polymer compositions and antibodies thereof for the treatment of protein conformational disorders
US9085614B2 (en) 2008-08-01 2015-07-21 Immunas Pharma, Inc. Antibodies that specifically bind to Aβ oligomers and uses thereof
WO2010119704A1 (fr) * 2009-04-17 2010-10-21 Immunas Pharma, Inc. Anticorps qui se lient spécifiquement aux oligomères a bêta et leur utilisation
US9090679B2 (en) 2009-04-17 2015-07-28 Immunas Pharma, Inc. Antibodies that specifically bind to A beta oligomers and use thereof
US8613924B2 (en) 2009-08-06 2013-12-24 Immunas Pharma, Inc. Antibodies that specifically bind to A beta oligomers and use thereof
US8858949B2 (en) 2009-08-06 2014-10-14 Immunas Pharma, Inc. Antibodies that specifically bind to a beta oligomers and use thereof
WO2011029920A1 (fr) 2009-09-11 2011-03-17 Probiodrug Ag Dérivés hétérocycliques en tant qu'inhibiteurs de glutaminyle cyclase
US11629185B2 (en) 2015-01-28 2023-04-18 Novo Nordisk A/S Anti-transthyretin antibodies
US11267878B2 (en) 2015-01-28 2022-03-08 Neotope Neuroscience Limited Anti-transthyretin antibodies
US11912759B2 (en) 2015-01-28 2024-02-27 Novo Nordisk A/S Anti-transthyretin antibodies
EP3461819A1 (fr) 2017-09-29 2019-04-03 Probiodrug AG Inhibiteurs de la glutaminyl-cyclase
US12247985B2 (en) 2017-10-06 2025-03-11 Novo Nordisk A/S Methods of detecting transthyretin
US11267877B2 (en) 2017-10-06 2022-03-08 Prothena Biosciences Limited Anti-transthyretin antibodies
US11873332B2 (en) 2017-11-29 2024-01-16 Novo Nordisk A/S Lyophilized formulation of a monoclonal antibody against transthyretin
US12115141B2 (en) 2018-11-14 2024-10-15 University Of Kentucky Research Foundation Diagnosis of diabetes by detecting aggregated amylin in erythrocytes
EP3998280A4 (fr) * 2019-07-08 2023-06-21 TERUMO Kabushiki Kaisha Hybridome et son procédé de production, anticorps monoclonal et son procédé de production
WO2021035210A1 (fr) * 2019-08-22 2021-02-25 The Regents Of The University Of California Anticorps à chaîne unique se liant à des oligomères de protéines tau et inhibant l'ensemencement par des extraits pathologiques de la maladie d'alzheimer
WO2021216667A3 (fr) * 2020-04-21 2021-12-02 The Board Of Regents Of The University Of Texas System Anticorps dirigés contre la chitinase 3-like -1 et leurs méthodes d'utilisation
CN112540180B (zh) * 2020-11-06 2022-03-29 华中科技大学 用于检测人淀粉样蛋白-β双抗夹心ELISA检测试剂盒
CN112540180A (zh) * 2020-11-06 2021-03-23 华中科技大学 用于检测人淀粉样蛋白-β双抗夹心ELISA检测试剂盒
CN112881708B (zh) * 2021-01-19 2022-03-25 华中科技大学 用于检测人淀粉样蛋白-β的胶体金免疫层析试纸及其制备
CN112881708A (zh) * 2021-01-19 2021-06-01 华中科技大学 用于检测人淀粉样蛋白-β的胶体金免疫层析试纸及其制备

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