TWI508976B - Antibodies specific for the protofibril form of beta-amyloid protein - Google Patents

Description

Specific antibody to the fibril form of β-amyloid

The present invention relates to isolated antibodies that specifically interact with the conformational epitope of the fibrillar form of the human beta-beta peptide. These antibodies display minimal or no detectable affinity for the lower molecular weight form of the beta-beta peptide. The antibodies disclosed herein will be useful for the diagnosis, treatment, and/or prevention of beta-type amyloid plaque deposits associated with the onset and progression of Alzheimer's disease.

The beta-based starch (Aβ) peptide is considered to be a pathogen that causes Alzheimer's disease ("AD") by forming insoluble Aβ peptide fibrils and depositing such fibrils to form amyloid plaques. It is believed that the formation of such plaques in brain regions critical to memory and other cognitive functions causes dementia associated with the disease (see Selkoe, 1994, J. N europathol . Exp . Neurol . 53: 438-447). The β-amyloid peptide comprises a group of peptides of 39-43 amino acids, which are processed by β-secretase and γ-secretase at the amino and carboxy terminal proteolysis from starch-like precursor protein (APP), respectively. . APP has at least five distinct isoforms: 563, 695, 714, 751, and 770 amino acids in length (see Wirak et al., 1991 Science 253:323). This equivalent work isoform of APP is produced by alternatively splicing the primary transcript of the APP gene. Numerous misidentification mutations have been identified in APP in families with somatically dominant early onset Alzheimer's disease. Some mutations cluster near the secretase cleavage site and affect APP metabolism by increasing the yield or ratio of A[beta] forms (eg, A[beta] ₄₂ ), which tend to be more fibrogenic and aggregate faster than other forms. Neuronal toxicity may be present in large molecular weight fibrils formed by aggregating soluble A[beta] peptides into insoluble fibrils and subsequent incorporation of fibrils into the amyloid plaques. The intermediate fibril form is in the form of a fibril (PF), a large molecular weight oligomeric form of the A[beta] peptide that is soluble in vitro and can be isolated in a solid form of about 670 kDa. Thus, the formation of insoluble A[beta] peptide fibrils in vitro is the end result of the initial oligomerization of the A[beta] peptide to form a structurally unique soluble higher molecular weight fibril form. These temporary fibrillar structures are precursors of starch fibers that cause cell dysfunction and neuronal loss in Alzheimer's disease (AD) and other protein aggregation diseases.

Various treatments have been developed to try to prevent the formation of A[beta] peptides, such as inhibitors that prevent proteolytic processing of APP. Similarly, immunotherapeutic strategies such as administration of anti-A[beta] antibodies (to induce clearance of starch-like deposits) or immunization with A[beta] peptide antigens (to promote humoral responses) have been sought to reduce plaque size and density.

US Patent No. 7,179,463 to Lannfelt et al. discloses a method of treating Alzheimer's disease by administering an antibody produced against a fibril composed of an Arctic mutation in the Aβ peptide coding region. An illustration of the antibodies produced is not provided in the specification and no comparison is made regarding the affinity for the low molecular weight form of the A[beta] peptide.

A series of antibodies recognizing various epitopes along the Aβ ₄₂ amino acid sequence are disclosed in U.S. Patent Nos. 6,761,888 and 6,750,324, both to Schenk et al. The specific antibody to the N-terminal and central regions of Aβ ₄₂ demonstrates the efficacy of reducing plaques both in vitro and in vivo.

Despite the current knowledge in the field of treating and preventing Alzheimer's disease, there remains a need for improved compositions and methods for treating and/or preventing such diseases. The compositions and methods of the present invention address and meet such needs by revealing antibodies that are specific for the fibril form of the A[beta] peptide while exhibiting minimal detectable affinity for the low molecular weight form of the A[beta] peptide. A pharmaceutically effective composition comprising such an antibody would be suitable for the treatment and/or prevention of beta-like amyloid deposits known to be associated with Alzheimer's onset and progression.

The present invention relates to an isolated antibody which exhibits specific binding to a conformational epitope of a fibrillar form of a beta-type amyloid peptide. Monomers of wild-type beta-type starch (A[beta]) peptides are known in the art and are shown herein as SEQ ID NO: 1. The isolated antibody of the present invention has an affinity for this repetitive conformation epitope of the larger molecular weight fibril form of the A[beta] peptide, while exhibiting other forms of the A[beta] peptide (such as the monomeric or dimeric form of the A[beta] peptide) Minimum or no affinity.

The present invention also relates to an isolated antibody which specifically interacts with a conformational epitope of the fibril form of the Aβ peptide and which exhibits a measurable affinity for a conformational epitope of the fibril form of the Aβ peptide, Wherein the fibril epitope is represented by an exposed region in the form of an Aβ-based fibril comprising an amine terminal portion of the exposed portion of the Aβ peptide.

The present invention further relates to an isolated antibody which specifically interacts with a conformational epitope of the A fibril form of the Aβ peptide and which exhibits a measurable affinity for a conformational epitope of the fibrillar form of the Aβ peptide, Wherein the fibril epitope is represented by the exposed region of the A[beta]-based fibril comprising amino acid 1-20 (SEQ ID NO: 2) of the exposed portion of the A[beta] peptide.

The present invention also relates to an isolated antibody which specifically interacts with a conformational epitope of the A fibril form of the Aβ peptide and which exhibits a measurable affinity for a conformational epitope of the fibrillar form of the Aβ peptide, wherein The fibril epitope is represented by an exposed region in the form of Aβ-based fibrils comprising amino acids 4-12 and 9-20 (SEQ ID NOS: 3 and 4, respectively) of the exposed portion of the Aβ peptide.

The present invention is directed, in part, to any affinity matured form of monoclonal antibodies 13C3, 1D1 and 19A6, and 13C3, 1D1 and 19A6. The invention further relates to antibodies which mimic the functional specificity of 13C3, 1D1 and 19A6 as described herein. For this purpose, the invention also relates to biologically active fragments and/or mutants of 13C3, 1D1, 19A6 or 13C3-like, 1D1-like or 19A6-like antibodies, including, but not necessarily limited to, amino acid substitutions (eg, such as VH ₎ Or a fixed-point format of affinity matured in the V _L region), deletions, additions, amino-terminal truncation, and carboxy-terminal truncation, such that the mutations are antibodies that produce similar or improved versions of the 13C3, 1D1, 19A6, or 13C3-like antibody binding proteins. Or the antibody binding moiety provides the basis. In one embodiment of this embodiment of the present invention in section, V _H and V _L region comprising the 13C3 respectively of _{SEQ ID NO: 7 (V H} ) and / or SEQ ID NO: 5 clarify the (V _L) in the group Acid sequence.

The invention furthermore relates to an isolated nucleic acid molecule comprising or V _H 19A6 antibody and / or nucleotide sequences encoding the V _L region of 13C3, IDl; and in particular an isolated nucleic acid molecule encoding a biologically relevant portion of 13C3 (poly Affinity maturation pattern or other mutant version of the 13C3, 1D1 or 19A6 antibody. To this end, an embodiment of the invention relates to a nucleic acid molecule comprising the coding as set forth in SEQ ID NO: 8 (13C3: _VH region) and SEQ ID NO: 6 (13C3: V _L region, respectively) the nucleotide sequence of V _H and V _L regions of the 13C.

The invention also relates to the isolated antibody 13C3, 1D1 or 19A6 as disclosed herein, i.e., specifically interacts with the conformational epitope of the A fibril form of the Aβ peptide and exhibits a fibril form for the Aβ peptide group. Configurable epitopes are antibodies with measurable affinity.

The present invention also relates to a fusion tumor capable of producing a monoclonal antibody of the present invention. Specific fusion tumors of the invention include fusion tumors producing exemplary monoclonal antibodies 13C3, 19A6 and 1D1, respectively.

The present invention relates to a pharmaceutically effective composition comprising an isolated antibody as disclosed and further defined herein: a specific interaction with a repeating conformation epitope of the A fibril form of A[beta] and exhibiting for A[beta] The repeating conformation epitope of the fibrillar form has the ability to measure affinity and specifically bind to the repeating conformational epitope of the A[beta]-based fibril form, while exhibiting minimal or no detectability for the low molecular weight form of A[beta] Affinity of isolated antibodies. Such compositions may optionally comprise one or more carriers, one or more excipients, and/or one or more chemical derivatives.

The invention also relates to a method of treating an individual having Alzheimer's disease comprising administering to the individual a pharmaceutically effective composition comprising the isolated antibody disclosed herein, the isolated antibody is also Repeated conformation epitope-based epitopes of the Aβ peptide form a specific interaction and exhibit a measured affinity for the repetitive conformation epitope of the fibrillar form of the Aβ peptide, while exhibiting a lower molecular weight form for the Aβ peptide Minimal or no antibodies that detect affinity. These methods will provide therapeutic intervention to reduce the amount of starch-like deposits in the brain of individuals with Alzheimer's disease. A specific embodiment of this part of the invention relates to a method of treating an individual suffering from Alzheimer's disease comprising administering a pharmaceutically effective composition formulated with an antibody which exhibits a base for the A[beta] peptide The conformational epitope of the fibril form has a specific affinity (e.g., at least compared to the low molecular weight form of the A[beta] peptide), particularly where the fibril epitope is comprised of the amino acid 1-20 comprising the exposed portion of the A[beta] peptide ( The exposed region of the Aβ-based fibril form of SEQ ID NO: 2) is represented. Specific embodiments disclosed herein for such therapeutic and prophylactic methods may utilize exemplary mouse monoclonal antibodies 13C3, 19A6, 1D1, as well as affinity maturation patterns, chimeric antibodies, humanized antibodies, Human monoclonal antibodies, and/or any other such antibody forms known in the art, including, but not limited to, antibodies or specific binding members reviewed herein. Any such antibody or specific binding member may be referred to as a "13C3-like antibody" in this specification. Thus, "13C3-like antibody" means also encompasses the 13C3 monoclonal antibody disclosed herein.

The invention is also directed to methods of screening and selecting compounds that act as inhibitors of fibrils and/or senile plaque formation associated with Alzheimer's disease. This method involves the use of antibodies having 13C3-like characteristics (eg, specific affinity for PF compared to the LMW form of Aβ peptide) to selectively modulate fibrils and/or plaque formation in various antibody/peptide/test compound interaction assays. The compound of the process.

The invention further relates to a diagnostic assay for the specific determination of the fibril content in an individual or patient. Such assays may be performed by any technique known and available to the skilled artisan, including but not limited to Western blot, ELISA, radioimmunoassay, immunohistochemical assay, immunoprecipitation or Other immunochemical assays are known in the art. Accordingly, one embodiment of this aspect of the invention relates to obtaining a tissue sample from an individual or patient and using a diagnostic kit and associated assay to determine the amount of PF Aβ in the sample; wherein the kit comprises a 13C3-like antibody, thus allowing for specificity The PF Aβ content in the tissue sample was determined. Tissue samples for analysis are typically blood, plasma, serum, mucus or cerebrospinal fluid from an individual or patient.

For this purpose, the antibodies of the invention may be used for at least the following purposes: (1) alone or in combination with any available combination therapy as a prophylactic or therapeutic agent for preventing or reducing plaque deposits associated with Alzheimer's disease; 2) designing peptide immunogens that can be used to elicit an effective antibody response in a prophylactic or therapeutic vaccination strategy associated with the treatment of Alzheimer's disease; (3) producing a secret epitope that mimics the binding of an antibody of the invention a prophylactic or therapeutic anti-genetic antibody (Ab2); and, (4) designing a peptide derived from a complementarity determining region (CDR) of an antibody of the present invention for screening for prophylactic and/or therapeutic A fibrillation inhibitor of the formula, and (5) a diagnostic reagent for determining the amount of fibril Aβ in serum or CSF of a patient at risk of developing AD.

It is an object of the present invention to provide an antibody which specifically interacts with an exposed conformation epitope in the form of a fibrillar form of the A[beta] peptide and which exhibits affinity for an exposed conformation epitope of the A[beta] peptide based fibril form, the exposure The conformational epitope comprises an amino acid 1-20 (SEQ ID NO: 2) of the exposed portion of the A[beta] peptide.

Another object of the present invention is to provide an antibody which interacts with an exposed conformation epitope in the form of a fibrillar form of the Aβ peptide and which exhibits affinity for an exposed conformation epitope of the A fibril form of the Aβ peptide. The epitopes comprise the amino acids 4-12 and 9-20 of the exposed portion of the A[beta] peptide (SEQ ID NO: 3, 4).

Another objective of this is to provide a 13C3-like antibody that prevents or reduces A[beta] peptidyl fibril formation associated with plaque deposition associated with Alzheimer's disease.

Another object of the present invention is to provide assays for the use of 13C3-like antibodies to select compounds suitable for the treatment of plaque deposits associated with Alzheimer's disease in an antibody/peptide/test compound interaction assay.

As used herein, "Ka" is intended to mean the association constant for a particular antibody antigen interaction, and "Kd" is intended to mean the dissociation constant for a particular antibody-antigen interaction.

As used herein, the term "antigenic determinant" or "antigenic determinant" refers to a site at which a B cell and/or T cell on an antigen reacts or a site on which an antibody is raised and/or the antibody will bind. For example, an epitope can be identified by an antibody that defines an epitope. An epitope may be a "linear epitope" (wherein the primary amino acid primary sequence comprises an epitope; typically comprises at least 3 contiguous amino acid residues in the unique sequence and more typically at least 5 and up to about 8 Up to about 10 amino acids) or "configurational epitopes" (the first-order contiguous amino acid sequence is not the epitope of the only defined component of the epitope). The conformational epitope may comprise an increased number of amino acids relative to a linear epitope, as this conformational epitope recognizes the three dimensional structure of the peptide or protein. For example, when a protein molecule is folded to form a three-dimensional structure, certain amino acids and/or polypeptide backbones that form a conformational epitope are contiguous, enabling the antibody to recognize the epitope. Methods for determining the configuration of an epitope include, but are not limited to, for example, X-ray crystallography, two-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, edited by Glenn E. Morris (1996), the disclosure of which is incorporated herein by reference in its entirety.

As used herein, "specifically combined" between two entities means at least 10 ⁶ M ^-1 , 10 ⁷ M ^-1 , 10 ⁸ M ^-1 , 10 ⁹ M ^-1 or 10 ¹⁰ M ^-1 Affinity.

As used herein, "base fibril" is a group of fibril aggregates that appear to represent a sequence of spherical structures forming a curved structure, including a spherical structure comprising an A[beta] peptide.

As used herein, the term "isolated" is used herein as it is used in the art. That is, the state in which the antibody/specificity binds to members, nucleic acid molecules, and the like. An antibody/specific binding member and a nucleic acid molecule will be free or substantially free of the substance to which it is naturally associated, such as with it in its natural environment or its preparation environment (eg, cell culture) (when this preparation is made by Other polypeptides or nucleic acids in recombinant DNA technology (when performed in vitro or in vivo). "Separated" encompasses any form containing the identified and characterized components of the invention after removal from the initial environment. Examples (but of course not limiting) include pharmaceutical formulations with diluents, antibody/specific binding members, nucleic acid molecules and their in vitro or in vivo modifications (eg, antibody glycosylation) and removal from the environment Part of the formulation.

As used herein, the term "recombinant human antibody" refers to a "antibody" produced by various recombinant DNA techniques and a viable subset of non-human transgenic genes well known in the art. The method is for producing antibodies from one or a source: (i) an isolated scFv or surrogate antibody from a human antibody library; (ii) self-stabilized or transiently transfected into a host cell, preferably a mammalian host cell. partial or complete antibody generated of the respective expression vector (e.g., in combination with the respective _L and C _H C nucleotide sequences encoding the V _H nucleotide chains and V _L sequences cloned in expression vector times in order to Promoting the display of a predetermined form of an antibody specific for the PF form of Aβ; and/or (iii) from a non-human transgenic animal containing a human immunoglobulin gene or by any other known human immunoreactivity An antibody that is "mixed and matched" to the recombination of a globin gene sequence with other DNA sequences to produce a recombinant human antibody of interest.

The term "individual" or "patient" is intended to include any member of the chordate, including but not limited to humans and other primates, including non-human primates, such as chimpanzees and other baboons and monkey species; Livestock, such as cattle, sheep, pigs, goats and horses; domesticated mammals such as dogs and cats; experimental animals, including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds Such as chicken, turkey and other pheasant birds, ducks, geese and the like.

The term "treating" a disease means performing a regimen that can include administering one or more drugs to an individual (human or other) to alleviate the signs or symptoms of the disease. Relief can occur before, and after, the signs or symptoms of the disease. Therefore, "treatment" includes "preventing" the disease. In the case of Alzheimer's disease, "prevention" can also occur in the course of advancing the treatment to prevent or stop the onset of symptoms associated with Alzheimer's disease. In addition, "treatment" does not require complete relief of signs or symptoms, does not require a cure, and includes, inter alia, a regime that has only a marginal positive effect on the individual.

As used herein, the term "active ingredient" refers to a 13C3-like antibody that exhibits affinity and specificity (eg, specific binding) to the amino terminal portion of the fibrillar structure of the beta-based starch.

As used herein, the term "effective amount" or "pharmaceutically effective amount" of an antibody as used herein refers to an amount of the active ingredient that is non-toxic but sufficient to provide the desired biological result. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

As used herein, the term "pharmaceutically acceptable" or "pharmacologically acceptable" means that the substance can be administered to an individual in a drug delivery device along with the formulated biological agent without causing any undesirable biological effects. Or any component of the composition (eg, "pharmaceutically acceptable composition") contained therein interacts in a detrimental manner.

As used herein, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably to refer to an organism that does not cause significant irritation to the organism and does not abolish the administered compound. Carriers, diluents and excipients of activity and characteristics. Adjuvants are included under these phrases.

As used herein, the term "excipient" refers to an inert substance that is added to a pharmaceutical composition to further facilitate administration of the active ingredient.

The term "minimum affinity" is used when comparing the affinity of an antibody for the fibril form of the A[beta] peptide to the affinity of the antibody for other forms of the A[beta] peptide, such as fibrils, flaky structures and low molecular weight oligomers and monomers. The ratio of the affinity for the fibril A[beta] form to the affinity for other A[beta] forms is greater than about 2. The ratio is preferably greater than about 3, or about 4, or about 5.

The starch-like precursor protein (APP) plays an important role in the pathogenesis of Alzheimer's disease (AD). The proteolytic processing of APP by β-secretase and γ-secretase produces Aβ peptide (Aβ) which is usually in the range of 39 to 43 amino acids in length. The onset of Alzheimer's disease is characterized by the accumulation of oligomeric or aggregated forms of Aβ in the brain. The immunological compositions of the present invention are useful in the treatment or prevention of Alzheimer's disease, as reagents for use in diagnostic assays, and as small molecule inhibitors for the design of starch-like deposits. The 13C3-like antibodies of the invention can be administered prophylactically to a general population of mammals, particularly humans, in a formulation that is expected to be pharmaceutically acceptable, in an amount and/or dosage regimen sufficient to eliminate, reduce or delay the onset of the disease. For individuals known to be at the genetic or family risk of Alzheimer's disease, a method of prophylactic treatment is particularly permitted. Numerous genetic markers for the risk of Alzheimer's disease have been identified, including but not limited to APP mutations (eg, Indian mutation (Va1717Phe), Swedish mutation (Lys670Asn, Met671Leu), Hendrick Hendricks mutation (Ala692Gly), Dutch mutation (Glu693Gln), Iranian mutation (Thr714Ala), German mutation (Va1715Ala), and Florida mutation (Ile716Val), etc. ). Other mutations that may indicate an increased risk of Alzheimer's disease include mutations in the presenilin genes (PS1 and PS2) and ApoE4. The present invention is also directed to a pharmaceutically acceptable composition comprising an 13C3-like antibody to an individual currently suffering from Alzheimer's disease (identified by characteristic dementia), particularly in the presence or absence of such risk factors, Therapeutic intervention is performed in an amount sufficient to cure or at least partially arrest the symptoms and complications of Alzheimer's disease. Methods based on prophylactic or therapeutic treatments encompassed herein can be used to address early-onset or late-onset Alzheimer's disease. In view of the importance of the oligomeric form of A[beta] in the onset of Alzheimer's disease, the present invention relates to an isolated antibody that specifically interacts with a repeating conformation epitope of the A fibril form of the A[beta] peptide and exhibits Repeated conformation epitopes in the form of fibrils of the A[beta] peptide have measured affinities. The monomer of the wild-type A[beta] peptide (A[beta] ₄₂ ; the form of 42 amino acids) is known in the art and is shown herein as SEQ ID NO:1:

The isolated antibodies of the invention will exhibit affinity for the repetitive conformation epitope of the larger molecular weight oligomeric fibril form of the A[beta] peptide, while exhibiting other forms of the A[beta] peptide (such as low molecular weight monomers and dimers) ) with minimal affinity.

The invention also relates to an isolated antibody that interacts with a conformational epitope of the fibrillar form of the A[beta] peptide and exhibits a measurable affinity for a conformational epitope of the fibrillar form of the A[beta] peptide, wherein The fibril epitope is represented by an exposed region in the form of an Aβ-based fibril comprising an amine-terminal portion of the exposed portion of the Aβ peptide.

The invention further relates to an isolated antibody that specifically interacts with a conformational epitope of the fibrillar form of the A[beta] peptide and exhibits a measurable affinity for a conformational epitope of the fibrillar form of the A[beta] peptide Wherein the base fibril epitope is represented by an exposed region of the Aβ-based fibril in the form of the amino acid 1-20 (SEQ ID NO: 2) comprising the exposed portion of the Aβ peptide:

As exemplified herein, mouse monoclonal antibodies have been identified that specifically exhibit a specific affinity for the fibril (PF) form of the A[beta] peptide while exhibiting minimal affinity for the low molecular weight species of the A[beta] peptide. The dimeric form of A[beta] (about 15 kDa) polymerizes over time to form a soluble PF form having a molecular weight of A[beta] of about 670 kDa. Mice were immunized with this higher molecular weight PF Aβ. Individual antibodies were screened for their specificity for the high molecular weight PF form of the A[beta] peptide while exhibiting minimal or no ability to bind to the lower molecular weight form of A[beta]. This part of the invention consists in screening, isolating and characterizing the 13C3 series of monoclonal antibodies (i.e., 13C3, 1D1 and 19A6) produced against the approximately 670 kDa high molecular weight fibrils of the A[beta] peptide. This series of monoclonal antibodies demonstrates predetermined in vitro specificity in the transgenic mouse Alzheimer's model and also reduces Alzheimer's-related plaque formation. Thus, in a particular embodiment of the invention, the isolated antibody specifically interacts with the conformational epitope of the A fibril form of the A[beta] peptide and exhibits a conformational epitope of the fibrillar form of the A[beta] peptide. Affinity can be measured, wherein the fibril epitope is composed of an Aβ-primor comprising amino acids 4-12 (SEQ ID NO: 3) and 9-20 (SEQ ID NO: 4) of the exposed portion of the Aβ peptide. The exposed areas of the fiber form indicate:

Example embodiments of the present invention based on an antibody, comprising as disclosed on the _{13C3 V H (SEQ ID NO:} 7) and / or the _{V L (SEQ ID NO: 5} ) compared to the region in order to impart the LMW Aβ peptide The form is specific to the 13C3 type of PF. Another embodiment is a 13C3-like antibody or a biologically relevant fragment thereof that exhibits specificity for the PF form over the LMW form of the A[beta] peptide. Accordingly, the present invention is also based on 13C3,1D1,19A6 biologically active fragment or 13C3-like antibody and / or mutants thereof, including (but not necessarily limited) substituted amino acid (e.g., as affinity V _H or V _L regions of the mature The site-specific forms, deletions, additions, amino terminal truncations, and carboxy-terminal truncations provide a basis for such mutations to produce similar or modified versions of antibodies or antibody binding portions of 13C3, 1D1, 19A6 or 13C3-like antibody binding proteins. As described herein, one embodiment of this aspect of the invention pertains to _VH and/or such antibodies comprising an amino acid sequence as set forth in SEQ ID NO: 7 and/or SEQ ID NO: 5, respectively. Or V _L area. Noting the existence of codon redundancy of the present invention, which can produce the same performance antibody or portion thereof (e.g., the same encoding scFv or IgG of V _H and / or alternative portions of a nucleic acid molecule _L V) of different DNA molecules. For the purposes of this specification, a sequence with one or more replaced codons will be defined as a degenerate variation. Another source of sequence variation can occur via RNA editing. This RNA editing can create another form of codon redundancy in which changes in the open reading frame do not cause changes in the amino acid residues in the expressed protein. Also included within the scope of the invention are mutations in the DNA sequence or in the translated antibody that improve the final physical properties of the expressed antibody. For this purpose, the invention relates to affinity maturation of (i) 13C3, 1D1, 19A6 or any other such 13C3-like antibody, and/or (ii) mutation of 13C3, 1D1, 19A6 or any other such 13C3-like antibody Forms include, but are not limited to, one or more mutations in the CDR1, CDR2 and/or CDR3 regions as produced by known affinity maturation methods and recombinant DNA techniques known to introduce site-specific mutations. Thus, the isolated antibody of the present invention is an antibody that specifically interacts with a conformational epitope of the A fibril form of the Aβ peptide. The isolated antibody of the present invention will exhibit affinity for this conformational epitope of the larger molecular weight fibril form of the A[beta] peptide, while exhibiting other forms of the A[beta] peptide (such as fibrils, lamella structures, and low molecular weight oligos). Polymer and monomer) have minimal affinity.

The invention also relates to isolated monoclonal antibody 13C3. This part of the invention is also directed to the production of a fusion tumor of monoclonal antibody 13C3. A fusion tumor producing monoclonal antibody 13C3 can be obtained by ATCC accession number PTA-8830.

The invention also relates to isolated monoclonal antibody 1D1. This part of the invention is also directed to the production of a fusion tumor of monoclonal antibody 1D1.

The invention also relates to isolated monoclonal antibody 19A6. This part of the invention is also directed to the production of a fusion tumor of monoclonal antibody 19A6.

The invention is also directed to methods of screening and selecting compounds that act as inhibitors of fibrils and/or senile plaque formation associated with Alzheimer's disease. This method involves the use of antibodies having a 13C3-like affinity for the PF form of the A[beta] peptide in various antibody/peptide/test compound interaction assays to select for compounds that modulate fibril and/or plaque formation processes. The compound can be a non-proteinaceous organic or inorganic molecule, a peptide (eg, in the form of a potential prophylactic or therapeutic peptide vaccine), a protein, DNA (single or double stranded), or RNA (such as siRNA or shRNA). After reviewing the disclosure and teachings of the present specification, any such peptide or small molecule that effectively competes with the 13C3-like antibody for binding to the PF form of the A[beta] peptide represents a possible dominant compound associated with prophylactic or therapeutic treatment of Alzheimer's disease. Will become obvious. For this purpose, interaction assays can be used for high yield screening purposes to identify compounds that occupy the 13C3 epitope of the PF form of the A[beta] peptide or interact with the 13C3 epitope of the PF form of the A[beta] peptide and replace the antibody.

Various antibody/antigen-based assays known in the art can be used, which are also dependent on the 13C3-like antibody of the present invention as an essential reagent for screening compounds suitable for prophylactic or therapeutic treatment of Alzheimer's disease. (eg, inorganic small molecule or candidate peptide vaccines), including, but not limited to, ELISA assays, radioimmunoassays, Western blot analysis, any homogenization assay that relies on detectable biological interactions without separation or washing steps (See, for example, PerkinElmer's AlphaScreen) and/or SPR-based technology (see, for example, BIACore). Compounds and/or peptide vaccine candidates identified using 13C3-like antibodies can be detected by a variety of assays. The assay can be a simple "yes/no" assay to determine if the ability to form a known antibody/antigen complex is altered, or can be quantified using, for example, an ELISA-based assay, a homogenization assay, or an SPR-based assay. To this end, the present invention relates to any such assay, regardless of which known method is employed, which measures the appropriate peptide or protein of the amine-terminal portion of the 13C3 epitope of the PF form of the Aβ peptide that competes with the 13C3-like antibody. The ability to simulate objects.

The antibodies described herein can be used as a primary reagent for determining the presence of A[beta]-based fibril forms in tissue samples in a number of different immunoassays. In general, such antibodies can be used in any type of immunoassay, whether qualitative or quantitative. This includes non-competitive types as well as two-point sandwich assays and single-site immunoassays in traditional competitive binding assays. For ease of detection and quantification, one embodiment of interest is a sandwich or double antibody assay in which there are many variations, all of which are intended to be encompassed in this part of the invention. For example, in a typical forward sandwich assay, an unlabeled antibody is immobilized on a solid substrate (eg, a microtiter well) and the sample to be tested is contacted with the bound molecule. After a suitable incubation period for a period of time sufficient to allow formation of the antibody-antigen binary complex, followed by addition of a second antibody labeled with a reporter molecule capable of inducing a detectable signal, and continued incubation, allowing for sufficient sites at different sites The time at which the antigen binds and forms a ternary complex of the antibody-antigen-labeled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observing the signal, which can be quantified by comparison to a control sample containing a known amount of antigen. Variations of the forward sandwich assay include simultaneous assays in which the sample is added to the bound antibody simultaneously with the antibody, or a reverse sandwich assay in which the labeled antibody is first combined with the sample to be tested, incubated and added to the unlabeled The surface binds to the antibody. These techniques are well known to those skilled in the art and the possibilities for minor variations will be apparent. As used herein, a "sandwich check" is intended to encompass all variations of the basic two-point technique.

For the sandwich assay of the present invention, the only limiting factor is that both antibodies have different binding specificities for the A[beta]-based fibril form. Therefore, many possible combinations are possible. As a more specific example, in a typical forward sandwich assay, the primary antibody is covalently or passively bound to the solid support. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polypropylene decylamine, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid carrier can be in the form of a tube, bead, disc or microplate, or any other surface suitable for performing immunoassays. Binding methods are well known in the art. After binding, the solid phase-antibody complex is washed to prepare for the test sample. An aliquot containing the body fluid in the form of the Aβ-based fibril to be tested is then added to the solid phase complex and incubated at 25 ° C to allow for the presence of any Aβ-based fibril-form protein and the Aβ-based fibril form. A specific antibody is bound for a period of time. The second antibody is then added to the solid phase complex and incubated at 25 ° C for a further period of time sufficient to allow binding of the second antibody to the primary antibody-antigen solid phase complex. The second antibody is linked to a reporter molecule, and the visible signal of the reporter molecule is used to indicate binding of the second antibody to any antigen in the sample. As used herein, a "reporter molecule" means a signal that allows for the detection of an antigen-binding antibody that can be detected analytically due to its chemical nature. Detection must be at least relatively quantitative to allow determination of the amount of antigen in the sample, which can be calculated as absolute terms, or can be compared to a standard (or series of standards) containing known normal levels of antigen.

The most commonly used reporters in this type of assay are enzymes or fluorophores. In the case of an enzyme immunoassay, the enzyme is often conjugated to a second antibody by means of glutaraldehyde or periodate. However, it will be readily appreciated that there are a variety of different conjugate techniques that are well known to those skilled in the art. Commonly used enzymes include, in particular, horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase. The receptor to be used with the specific enzyme is generally selected to produce a detectable color change upon hydrolysis by the corresponding enzyme. For example, p-nitrophenyl phosphate is suitable for alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidine is typically used. It is also possible to use a fluorescent receptor that produces a fluorescent product rather than the chromogenic substrate described above. In all cases, the enzyme-labeled antibody is added to the first antibody-A[beta]-based fibrillin complex and allowed to bind to the complex, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the ternary complex of the antibody-antigen-labeled antibody. The substrate is reacted with an enzyme linked to a second antibody to obtain a qualitatively visible signal which can be further quantified (typically in spectrophotometric manner) to obtain an estimate of the amount of antigen present in the serum sample.

In addition, a fluorescent compound such as luciferin or rhodamine can be chemically coupled to an antibody without changing the binding ability of the antibody. When activated by irradiation with light of a specific wavelength, the fluorescent dye-labeled antibody absorbs light energy, induces an excited state of the molecule, and then emits light of a characteristic longer wavelength. The emission appears as a characteristic color that can be visually detected by an optical microscope. The fluorescently labeled antibody is conjugated to the first antibody-A[beta] based fibril form protein complex as in an Enzyme Immunoassay (EIA). After washing the unbound reagent, the remaining ternary complex is then exposed to light of the appropriate wavelength and the observed fluorescence indicates the presence of the antigen. Both immunofluorescence and EIA techniques are well established in the art and are particularly preferred for the methods of the present invention. However, other reporter molecules such as radioisotopes, chemiluminescent or bioluminescent molecules can also be employed. How to change the program to suit the intended use will be apparent to those skilled in the art.

In another embodiment, the sample to be tested (eg, human blood or spinal fluid in the form of A[beta]-based fibrils) can be used in a single site immunoassay in which it is covalently or non-covalently attached to a solid substrate. The unlabeled anti-Aβ-based fibrillin protein antibody is contacted with a sample bound to a solid substrate. After a suitable incubation period that is sufficient for a period of time sufficient to allow formation of the antibody-antigen binary complex, a second antibody that is labeled with a reporter molecule capable of inducing a detectable signal is then added, and incubation is continued, allowing for sufficient antigen-antibody formation. The time of the ternary complex of the labeled antibody. For a single site immunoassay, the second antibody can be a universal antibody that binds to an antibody specific for the A[beta]-based fibril protein of interest (ie, an immunoglobulin heterologous antibody, particularly linked to a reporter molecule) Anti-(IgM and IgG)).

The 13C3-like antibody can be one of many forms known in the art. The antibody may employ any type of related antibody fragment, antibody binding portion, specific binding member, non-protein synthesis mimetic, or any entity known in the art to refer to an entity that at least substantially retains binding specificity/neutralizing activity. Other related naming forms. Accordingly, the term this description any case used in the "antibody" is meant to include (without limitation) any specific binding member, immunoglobulin class and / or isotype (e.g., _{IgG 1, IgG 2, IgG 3} , IgG 4 , IgM, IgA, IgD, IgE and IgM); and biologically relevant fragments or specific binding members thereof, including but not limited to Fab, F(ab') ₂ , Fv and scFv (single-chain or related entities). Thus, it is well known in the art and includes only in retrospective form that "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or Its antigen binding moiety. The heavy chain comprises a heavy chain variable region ( _VH ) and a heavy chain constant region (CH1, CH2 and CH3). Light chain comprises a light chain variable region (V _L) and a light chain constant region (C _L). The variable regions of the heavy and light chains each comprise a framework region (FWR) and a complementarity determining region (CDR). FWR four inverted relative area, while CDR regions (CDR1, CDR2 and CDR3) as representative of the hypervariable regions and are arranged from the NH ₂ terminus to the COOH terminus: FWR1, CDR1, FWR2, CDR2 , FWR3, CDR3, FWR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen, while the constant region, depending on the isotype, mediates binding of the immunoglobulin to the host tissue or factor. That is, chimeric antibodies, humanized antibodies, recombinant antibodies, such as human antibodies produced by non-human animals derived from transgenic genes, and antibodies selected from libraries using skilled enrichment techniques are also included in the working definition of "antibody". . As reviewed below, antibody fragments are obtained using techniques that are readily known and available to those of ordinary skill in the art. Thus, an "antibody" is any such entity or specific binding member that specifically binds to a conformational epitope of a fibrillar form of A[beta] as described herein. Thus, the term "antibody" describes an immunoglobulin produced naturally or partially or completely synthetically; any polypeptide or protein having a binding domain that is homologous or substantially homologous to the antibody binding domain. Such antibodies may be derived from natural sources, or they may be produced in part or in whole synthetically. As hereinafter, non-limiting discussion, examples of antibodies are immunoglobulin isotypes and isoforms thereof; fragments comprising an antigen binding domain such as Fab, scFv, Fv, dAb, Fd and bifunctional antibodies. It is known in the art to manipulate monoclonal antibodies and other antibodies and to employ techniques of recombinant DNA techniques to generate additional antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can include introducing DNA encoding an immunoglobulin variable region or complementarity determining region (CDR) of an antibody into a constant region or constant region of a different immunoglobulin plus a framework region. The fusion-producing tumor or other cell that produces the antibody can be subjected to genetic mutations or other changes that may or may not alter the binding specificity of the antibody produced. The antibody can be modified in a number of ways, and the term "antibody" should be taken to encompass any specific binding member or substance that has a binding domain with the desired specificity. Thus, such terms encompass antibody fragments, derivatives, functional equivalents and homologs of "antibodies" including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. The entity may be a binding fragment encompassed within the term "antigen-binding portion" or "specific binding member" of an antibody, including but not limited to (i) a Fab fragment, from V _L , V _H , C _L and C a monovalent fragment consisting of _H domains; (ii) F (ab ' ) 2 fragment comprising two Fab fragments linked of a double hinge zone a sulfur bridge bivalent fragment; (iii) Fd by the V _H and C _H domains of a fragment; (iv) an Fv fragment consisting of the _VL and _VH domains of a single arm of the antibody; (v) a dAb fragment comprising the _VH domain; (vi) an isolated complementarity determining region (CDR); (vii) 'scAb ', containing V _H and V _L and antibody fragments C _L or C _H's; and (viii) artificial antibody protein scaffold of the basis, including (but not limited to) the type III fibronectin polypeptide antibodies (see e.g. March 2004 U.S. Patent No. 6,703,199 to Koide, and PCT International Application Publication No. WO 02/32925. Furthermore, although the two domains of the Fv fragment (V _L and V _H) gene encoded by a separate line, but recombinant methods may be used such that it can be prepared by the pairing of V _L and V _H regions pair to form monovalent molecules of a single protein chain A synthetic linker (called a single-chain Fv (scFv)) is joined.

In one embodiment, the present invention is the separation of the light chain variable 13C3 or 13C3-like antibody (V _L) region may comprise a peptide sequence of 113 amino acids (SEQ ID NO: 5), which is a 339-based base The nucleotide sequence of the base pair (SEQ ID NO: 6) encodes:

In another embodiment, the heavy chain variable ( _VH ) region of an isolated 13C3 or 13C3-like antibody of the invention may comprise a peptide sequence of 115 amino acids (SEQ ID NO: 7) consisting of 345 The nucleotide sequence of the base pair (SEQ ID NO: 8) encodes:

In another embodiment, the framework regions of the _VL chain, FWR1, FWR2, FWR3, and FWR4 can comprise the following in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. Elucidated amino acid:

In another embodiment, the complementarity determining region, CDR1, CDR2, and CDR3 of the _VL chain can comprise the amino acid as set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, respectively:

In another embodiment, the framework regions of the _VH chain, FWR1, FWR2, FWR3, and FWR4 can comprise the following in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively. Elucidated amino acid:

In another embodiment, the complementarity determining region, CDR1, CDR2, and CDR3 of the _VH chain can comprise the amino acid as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively:

Multiple strains or monoclonal antibodies used in the disclosed therapeutic methods can be produced by known techniques. Monoclonal murine (mouse) antibodies displaying specificity for the selected epitope of the selected target may be purified from mammalian antisera containing antibodies reactive in this region, or may use Kohler and Milstein The technique (1975, Nature 256:495-497) was prepared as a monoclonal antibody. As used herein, a single specific anti-system is defined as a single antibody substance or a plurality of antibody substances having a uniform binding characteristic, such as a mouse monoclonal antibody having a 13C3 series of monoclonal antibodies exemplified herein. The fusion tumor cell line is produced by mixing spleen lymphocytes with a suitable fusion partner (preferably myeloma cells) under conditions that will allow for the formation of a stable fusion tumor. The spleen antibody-producing cells are fused with myeloma cells, selected and screened for antibody production. Fusionoma cells from antibody positive wells were cloned by techniques such as MacPherson's Soft Agar Technique (1973, Soft Agar Techniques, edited by Tissue Culture Methods and Applications, Kruse and Paterson, Academic Press). The monoclonal antibody system is produced in vivo by injecting individual fusion tumor cells into primary pre-sensitized mice (pristine primed mice) after a certain time interval, and is well known in the art. Technology is prepared.

In addition to the above-described species-specific monoclonal antibodies, the antibodies of the invention may also be in the form of "chimeric antibodies" which are derived from, for example, murine-derived variable regions and from a predetermined host source (eg, human; For review, see Monoclonal Antibodies Constructed in the Constant Region of Morrison and Oi, 1989, Advances in Immunology , 44: 65-92). For example, light and heavy chain variable DNA sequences from rodent (e.g., mouse) antibodies (e.g., SEQ ID NOS: 6 and 8, respectively) can be cloned in a mammalian expression vector. These light and heavy "chimeric" expression vectors are co-transfected into recipient cell lines by known techniques and selected and amplified. This cell line can then be subjected to known cell culture techniques such that the light and heavy chains of the chimeric antibody are produced. These chimeric antibodies have historically been shown to possess the antigen binding ability of the original rodent monoclonal antibody while significantly reducing the immunogenicity of the host after administration.

A reasonable improvement to a chimeric antibody is a "humanized antibody" which appears to reduce the likelihood of a patient developing an immune response to a therapeutic antibody when compared to the use of a chimeric or intact murine monoclonal antibody. The strategy for humanizing "mouse Mab" is based on the induction of site-directed mutagenesis by individual residues or by transplantation of the entire complementarity determining region (Jones et al., 1986, Nature 321:522-526). An amino acid residue of an amino acid residue. This technique is now again known in the art and is represented by numerous strategies for improving this technology; that is, by implementing strategies including, but not limited to, the following strategies: "reforming" (see Verhoeyen et al., 1988). , Science 239: 1534-1536), "hyperchimerization" (see Queen et al., 1991, Proc. Natl. Acad. Sci. 88: 2869-2873) or "veneering" (Mark et al) Human, 1994, Derivation of Therapeutically Active Humanized and Veneered anti-CD18 Antibodies Metcalf end Dalton, Cellular Adhesion: Molecular Definition to Therapeutic Potential. New York: Plenum Press, 291-312). These strategies include, to some extent, sequence comparisons between rodent and human sequences to determine if a particular amino acid substitution from a rodent to human consensus sequence is appropriate. Regardless of the variation, the central subject involved in the production of humanized antibodies depends on CDR grafting, in which all three antigen binding sites from the light and heavy chains are efficiently removed from rodents exhibiting antibody-only lines and Secondary selection (or "transplantation") in expression vectors encoding the framework regions of human antibodies. For example, humanized antibodies can be expressed using the above techniques, wherein the CDR1, CDR2 and CDR3 regions of the variable light chain are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the CDR1, CDR2 and CDR3 of the variable heavy chain The regions are set forth in SEQ ID NOS: 20, 21 and 22, respectively. Thus, a "humanized antibody" is effectively an antibody constructed solely of murine CDRs (minus any other modifications resulting from the incorporation of one or more of the above strategies), wherein the remainder of the variable region and all of the constant regions It is derived from human sources.

The present invention is also based on or related to the affinity of the antibody V _H 13C3 and / or V _L region of the isolated nucleic acid molecules and associated amino acid sequences, and more specific encoding other words 13C3 or 13C3-like antibody 13C3,1D1,19A6 An isolated nucleic acid molecule (polynucleotide) of a biologically relevant portion of a mature form or other mutant version. These nucleic acids are substantially free of other nucleic acids. DNA is the preferred nucleic acid for most purposes of selection. These DNA molecules can be sub-selected in a performance vector and subsequently transfected into a host cell of choice, wherein the recombinant host cell is a 13C3, 1D1, 19A6 or 13C3-like, 1D1-like or 19A6-like antibody or an affinity matured version thereof. The source of the relevant content provides the source. These procedures can be used to provide multiple benefits, such as generating scFvs or the like of this V _H and V _L chain were coded in the performance (e.g.) a mammalian C _H and C _L regions of the human IgG antibody expression vector system. For all amino acids except for the two amino acids, the degeneracy of the genetic code allows more than one codon to encode a particular amino acid. This allows for the construction of synthetic DNA encoding an antibody of the invention wherein the nucleotide sequence of the synthetic DNA is significantly different from the nucleotide sequence disclosed herein, but still encodes the antibody. Such synthetic DNA is intended to be within the scope of the invention. If such synthetic DNA is to be expressed in a particular host cell or organism, the codon usage of such synthetic DNA can be adjusted to reflect the codon usage of the particular host, thus producing a higher expression of the antibody of the invention. . In other words, this redundancy of the various codons encoding a particular amino acid is within the scope of the invention. Thus, the invention also targets such DNA sequences encoding RNAs comprising alternative codons encoding the final translation of the same amino acid as follows: A = Ala = alanine: codon GCA, GCC, GCG, GCU; C=Cys=cysteine: codon UGC, UGU; D=Asp=aspartic acid: codon GAC, GAU; E=Glu=glutamic acid: codon GAA, GAG; F= Phe = phenylalanine: codon UUC, UUU; G = Gly = glycine: codon GGA, GGC, GGG, GGU; H = His = histidine: codon CAC, CAU; I = Ile = isoleamine Acid: codon AUA, AUC; AUU; K = Lys = lysine: codon AAA, AAG; L = Leu = leucine: codon UUA, UUG, CUA, CUC, CUG, CUU; M = Met = Methionine: codon AUG; N=Asp=aspartate: codon GAU, GAC; P=Pro=proline: codon CCA, CCC, CCG, CCU; Q=Gln=glutamine : codon CAA, CAG; R = Arg = arginine: codon AGA, AGG, CGA, CGC, CGG, CGU; S = Ser = serine: codon AGC, AGU, UCA, UCC, UCG, UCU ;T=Thr=threonine: codon ACA, ACC, ACG, ACU; V=Val=proline: codon GUA, GUC, G UG, GUU; W=Trp=tryptophan: codon UGG; Y=Tyr=tyrosine: codon UAC, UAU. These recombinant expression vectors can then be stably or transiently transfected into a suitable cell line for production of a surrogate antibody form.

The present invention notes the existence of codon redundancy which may produce antibodies exhibit the same or different portions of DNA molecules (e.g., scFv or IgG same encoding the V _H and / or alternative portions of a nucleic acid molecule _L V). For the purposes of this specification, a sequence with one or more substituted codons will be defined as a degenerate variant. Another source of sequence variation can occur via RNA editing. This RNA editing can create another form of codon redundancy in which changes in the open reading frame do not cause changes in the amino acid residues in the expressed protein. Also included within the scope of the invention are mutations in the DNA sequence or in the translated antibody that improve the final physical properties of the expressed antibody. To this end, the invention relates to (i) a 13C3-like antibody affinity maturation pattern comprising, but not limited to, 13C3, 19A6 and 1D1, and/or (ii) a mutant form of a 13C3-like antibody, including but not limited to 13C3, 19A6 and/or 1D1, including but not limited to one of the CDR1, CDR2 and/or CDR3 regions, as produced by known affinity maturation methods and recombinant DNA techniques known to introduce site-specific mutations Or multiple mutations. Such isolated or purified nucleic acid molecules representative of 13C3-like antibody V _H and / or V _L portion. These nucleic acids are substantially free of other nucleic acids. DNA is the preferred nucleic acid for most purposes of selection. These DNA molecules can be sub-selected in a performance vector and subsequently transfected into a host cell of choice, wherein the recombinant host cell provides a source of substantial content of the 13C3-like antibody or a relevant portion of its affinity matured version. These procedures can be used to provide multiple benefits, such as generating scFvs or the like of this V _H and V _L chain were coded in the performance (e.g.) a mammalian C _H and C _L regions of the human IgG antibody expression vector system.

The invention also relates to recombinant vectors and recombinant hosts (prokaryotic and eukaryotic) comprising a nucleic acid molecule encoding a respective heavy chain region and/or light chain region of a 13C3-like antibody. These nucleic acid molecules may be ligated in whole or in part with other DNA molecules not naturally linked (ie, DNA molecules encoding recombinant immunoglobulin genes for the production of recombinant human antibodies) to form a "recombinant" encoding recombinant antibodies of various other classes. DNA molecule." Such vectors may comprise DNA or RNA. DNA vectors are preferred for most purposes of selection. Typical vectors include plastids, modified viruses, phage, plastids, yeast artificial chromosomes, and other forms of free or integrated DNA. It is within the skill of the art to identify appropriate vectors for specific gene transfer, production of recombinant human antibodies, or other uses. Well-known methods of subcloning a nucleic acid molecule of interest into a performance vector, transforming or transfecting a host cell containing the vector, and methods of preparing a substantially pure protein comprising introducing the respective expression vector into a host cell and under appropriate conditions The step of culturing the host cells. The antibodies thus produced, such as IgG recombinant human antibodies, can be collected from host cells in a conventional manner. This portion of the invention can be practiced using any known expression vector, including any vector containing a suitable promoter and other appropriate transcriptional regulatory elements. The resulting expression construct is transferred to a prokaryotic or eukaryotic host cell to produce a recombinant protein. A performance vector is defined herein as the DNA sequence required to transcribe a DNA and translate its mRNA in a suitable host. Such vectors can be used to express eukaryotic DNA in a variety of hosts, such as bacteria, blue-green algae, plant cells, insect cells, and animal cells. A specially designed vector allows DNA to shuttle between hosts, such as bacterial-yeast or bacterial-animal cells. A suitably constructed expression vector shall contain: an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of suitable restriction enzyme sites, the potential for a high number of copies, and an active promoter. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is a promoter that initiates mRNA at a high frequency. Techniques for such operations are described in Sambrook et al. (1989, Molecular Cloning. A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), which are well known and available to those skilled in the art. Expression vectors can include, but are not limited to, selection vectors, modified selection vectors, specially designed plastids or viruses. Commercially available mammalian expression vectors which may be suitable include, but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38, and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIanp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV -MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565). Likewise, a variety of bacterial expression vectors are available, including but not limited to, pCR2.1 (Invitrogen), pET1 1a (NoVagen), λgt1 1 (Invitrogen), and pKK223-3 (Pharmacia). In addition, a variety of fungal cell expression vectors can be used including, but not limited to, pYES2 (Invitrogen) and Pichia expression vector (Invitrogen). Likewise, a variety of insect cell expression vectors can be used including, but not limited to, pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).

The recombinant host cell may be a prokaryotic or eukaryotic cell, including but not limited to bacteria such as E. coli ; fungal cells such as yeast; mammalian cells including, but not limited to, cattle, pigs, monkeys and a cell strain derived from a rodent; and an insect cell. Suitable mammalian species (-26) include, but are not limited to, L-cell LM (TK-) (ATCCCCL 1.3), L-cell LM (ATCC CCL 1.2), Saos-2 (ATCCHTB-85), 293 ( ATCCCRL1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92 ), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL26), MRC-5 (ATCCCCL171), and CPAE (ATCC CCL 209).

Yet another improvement to the reengineered antibodies reviewed above is the production of fully human monoclonal antibodies. The first involves the use of a genetically engineered mouse strain with an immune system in which the mouse antibody gene has been deactivated and in turn replaced with a full set of functional human antibody genes while leaving the other components of the mouse immune system unchanged. These genetically engineered mice allow for a natural in vivo immune response and affinity maturation process that produces high affinity, fully human monoclonal antibodies. This technique is now again well known and fully described in the various publications in the art, including but not limited to U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and related family members ( U.S. Patent No. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299 And 5,770,429 (transferred to GenPharm International and available through Medarex under the protection of the UltraMab Human Antibody Development System). See also review from Kellerman and Green (2002, Curr. Opinion in Biotechnology 13: 593-597).

Finally, techniques for selecting antibody fragments from libraries using enrichment techniques can be obtained by the skilled artisan including, but not limited to, phage display, ribosome presentation (Hanes and Pluckthun, 1997, Proc. Nat. Acad. Sci . 94: 4937-4942) ), bacterial presentation (Georgiou et al, 1997, Nature Biotechnology 15: 29-34) and/or yeast presentation (Kieke et al, 1997, Protein Engineering 10: 1303-1310), which can be used as an alternative to the previously discussed techniques A single-chain antibody that binds to a target cell hormone specificity is selected. The single-stranded anti-system is selected from a single-chain antibody library that is directly produced using filamentous phage technology. Phage presentation techniques are known in the art (see, for example, the technology from Cambridge Antibody Technology, CAT), as disclosed in U.S. Patent Nos. 5,565,332; 5,733,743; 5,871,907; 5, 872, 215; 5, 962, 255; 5, 962, 255; 6,140, 471; 6,225,447; 6,291,650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081 and others Member of the American family, or dependent on the application of priority document GB 9206318, filed on May 24, 1992; see also Vaughn et al., 1996, Nature Biotechnology 14: 309-314. Single-chain antibodies can also be designed and constructed using available recombinant DNA techniques (such as DNA amplification methods (e.g., PCR)) or by using individual fusion tumor cDNAs as templates. Single-chain antibodies can be either monospecific or bispecific; bivalent or tetravalent antibodies. As described below, nucleotide sequences encoding single-chain antibodies can be constructed using manual or automated nucleotide synthesis, cloned into expression constructs using standard recombinant DNA methods, and introduced into cells to express coding sequences.

The invention further relates to an antibody-based pharmaceutical composition comprising an effective amount of a 13C3-like antibody or an affinity matured form which provides prophylactic or therapeutic inhibition of fibrils and/or senile plaque formation associated with Alzheimer's disease Treatment options. The antibody-based pharmaceutical compositions of the present invention can be manipulated by any number of strategies known in the art (see, for example, McGoff and Scher, 2000, Solution Formulation of Proteins/Peptides : In McNally, EJ. Protein Formulation and Delivery . New York, NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as Parenteral Solutions . in: Pharmaceutical Formulation Development of Peptides and Proteins . Philadelphia, PA: Talyor and Francis; pp. 145-177 In; Akers et al., 2002, Pharm. Biotechnol . 14:47-127). A pharmaceutically acceptable composition suitable for administration to a patient will contain an effective amount of the antibody in the formulation which retains the biological activity while also promoting maximum stability over an acceptable temperature range during storage. Depending on the desired formulation, the pharmaceutical composition may also include pharmaceutically acceptable diluents, pharmaceutically acceptable or agents and/or pharmaceutically acceptable excipients, or any conventionally formulated formulation. Such vehicles for pharmaceutical compositions for administration to animals or humans. 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 solution, dextrose solution, and Hank's solution. The amount of excipient commonly used in the pharmaceutical compositions or formulations of the present invention is such that the antibody is uniformly distributed throughout the composition such that it can be uniformly dispersed when delivered to an individual in need thereof. It can be used to dilute the antibody to a concentration that provides the desired beneficial relief or cure while at the same time minimizing any adverse side effects that can occur due to excessive concentrations. It can also have a preservation effect. Therefore, for antibodies with high physiological activity, more excipients will be employed. On the other hand, for any active ingredient which exhibits lower physiological activity, a smaller amount of excipient will be employed. Generally, the amount of excipient in the composition will be between about 50% by weight (w) and 99.9% by weight of the total composition. If the antibody shows a particularly low physiological activity, the amount of excipient can be as little as 1% by weight. On the other hand, for antibodies having particularly high physiological activity, the amount of excipient may be between about 98.0% by weight and about 99.9% by weight. Alternatively, the antibody can be administered as a "chemical derivative" (a molecule containing an additional chemical moiety that is not normally part of the basic molecule). These parts can improve the solubility, half-life, absorption, etc. of the biological agent. Alternatively, such moieties may impair the undesirable side effects of the antibody. Pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, and copolymers (such as latex functionalized agarose gel, agarose, cellulose, and Its analogs), polymeric amino acids, amino acid copolymers and lipid aggregates (such as small oil droplets or liposomes). Additionally, such carriers can be used as immunostimulating agents (i.e., adjuvants). For parenteral administration, the agent of the present invention can be administered in the form of an injectable amount of the substance in a physiologically acceptable diluent and a solution or suspension in a pharmaceutical carrier, which can be a sterile liquid. Such as water oil, salt water, glycerin or ethanol. Additionally, adjuvant materials such as wetting or emulsifying agents, surfactants, pH buffering materials, and the like can be present in the compositions. The other components of the pharmaceutical composition are components of petroleum, animal, vegetable or synthetic sources, such as peanut oil, soybean oil and mineral oil. In general, especially for injectable solutions, diols such as propylene glycol or polyethylene glycol are preferred liquid carriers.

The antibody formulation can be in liquid form or in solid form. The solid formulation is typically lyophilized and the solution is prepared for single or multiple administration prior to administration. The formulation should not be exposed to extreme temperatures or pH to avoid thermal denaturation. Therefore, the antibody composition of the present invention must be formulated in a biologically relevant pH range. Particularly for liquid formulations that are stored for a longer period of time between formulation and administration, a solution that requires buffering to maintain an appropriate pH range during storage is indicated. To date, both liquid and solid formulations need to be stored at lower temperatures (typically 2-8 ° C) to maintain stability over a longer period of time. The formulated antibody composition, especially the liquid formulation, may contain bacteriostatic agents that prevent or minimize proteolysis during storage, including but not limited to effective concentrations (typically <1% w/v) of benzyl alcohol, phenol, M-cresol, chlorobutanol, methyl p-hydroxybenzoate and/or propyl p-hydroxybenzoate. Bacteriostats may be banned for some patients. Thus, the lyophilized formulation can be reconstituted in a solution with or without this component. Additional components may be added to the buffered liquid or solid antibody formulation including, but not limited to, sugars as cryoprotectants (including but not necessarily limited to) polyhydroxy hydrocarbons such as sorbitol, mannitol Sugar alcohols, glycerol and galactitol, and/or disaccharides such as sucrose, lactose, maltose or trehalose) and, in some cases, related salts including, but not limited to, NaCl, KCl or LiCl. Such antibody formulations (especially liquid formulations) intended for long-term storage will depend on the range of total volume osmotic concentrations to promote long-term stability at temperatures above 2-8 ° C or above, while also allowing for formulation Suitable for parenteral injection. The effective range of total volume osmotic concentration (total number of molecules in solution) is from about 200 mOs/L to about 800 mOs/L. It will be apparent that the amount of cryoprotectant (such as sucrose or sorbitol) will depend on the amount of salt in the formulation to maintain the volumetric osmolality of the solution within an appropriate range. Thus, the salt-free formulation may contain from about 5% to about 25% sucrose, wherein the preferred range of sucrose is from about 7% to about 15%, with particularly preferred sucrose concentrations of from 10% to 12% in the salt-free formulation. . Alternatively, the sorbitol-based salt-free formulation may contain sorbitol in the range of from about 3% to about 12%, with a preferred range of from about 4% to 7%, and particularly preferably in the range of no salt. About 5% to about 6% sorbitol in the product. Salt-free formulations will of course permit an increase in the range of individual cryoprotectants to maintain an effective volumetric osmotic concentration level. These formulations may also contain second order cations (including, but not necessarily limited to, MgCl ₂ , CaCl _{2 ,} and MnCl ₂ ); and non-32 ionic surfactants (including but not necessarily limited to) polysorbate-80 (Tween) ), polysorbate-60 (Tween) ), Polysorbate-40 (Tween) ) and polysorbate-20 (Tween) ), polyoxyethylene alkyl ethers, including but not limited to Brij Brij And others, such as Triton Triton , , Span 85 and Pluronic series of nonionic surfactants (eg, polonic 121)). Any combination of such components, including possibly bacteriostatic agents, may be suitable for filling the antibody-containing formulations of the invention. The antibody composition of the invention may also be a "chemical derivative" which describes an antibody comprising an additional chemical moiety (e.g., PEGylated) that is not normally part of the immunoglobulin molecule. These moieties improve the solubility, half-life, absorption, etc. of the basic molecule. Alternatively, such moieties may impair the undesirable side effects of the base molecule or reduce the toxicity of the base molecule.

Numerous examples of various carriers, diluents, excipients, and the like are known in the art and are disclosed in the references cited herein, as well as Remington's Pharmaceutical Sciences (18th Edition; Mack Publishing Company, Easton) The contents of these documents are incorporated herein by reference. Briefly, it will be appreciated that suitable carriers, excipients, and other agents can be incorporated to formulate pharmaceutical compositions to provide improved transfer, delivery, tolerance, and the like. Methods of incorporating a biological agent and/or other active ingredient into a carrier are known to those of ordinary skill in the art and depend on the nature of the biological agent and the nature of the carrier selected by the inventors. Ion binding, gel encapsulation or physical entrapment in the carrier, iontophoresis, and soaking of the carrier in the biocide solution are suitable examples encompassing the formulation of pharmaceutical compositions for use in practicing the disclosed therapeutic methods. Alternatively, the carrier can be equivalent to a diluent for the biological agent. Such formulations may include, for example, powders, pastes, ointments, gelling agents, waxes, oils, lipids, anhydrous absorbent bases, oil-in-water or water-in-oil emulsions, emulsions of carbowax (multiple molecular weights) a polyethylene glycol), a semi-solid gel, and a semi-solid mixture containing a Kappa wax. The dosage regimen utilizing the compounds of the invention is selected based on a variety of factors, including the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the kidney of the patient, Liver and cardiovascular function; and the specific biological agent used. A physician or veterinarian of ordinary skill can readily determine and specify an effective amount of the drug required to prevent, combat or arrest the progression of the condition. Optimal precision in achieving a concentration of drug within a range that produces efficacy without toxicity requires a regimen based on the kinetics of the availability of the drug to the target site. This involves considering the distribution, balance and elimination of the drug. In the treatment and therapy according to the invention, any of the foregoing formulations may be suitable provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible.

The pharmaceutical compositions of the present invention can be administered to a host in any manner, strategy, and/or combination that can be used in the art in an amount sufficient to provide a therapeutic treatment against Alzheimer's disease. Such compositions may be provided to an individual by a variety of routes known in the art, particularly parenteral routes including, but in no way limited to, parenteral routes such as intravenous (IV), muscle Internal (IM); or subcutaneous (SC) administration, wherein IV administration is a typical mode of therapeutic antibody administration. Such compositions can be administered in the form of separate or multiple doses (i.e., administration of the antibody at staggered times by maintaining the sterile conditions of the formulation via a therapeutic regimen). Dosage regimens utilizing the compounds of the invention are selected based on a variety of factors, including the type, species, age, weight, sex, and medical condition of the patient (such as a human patient); the severity of the condition to be treated; the route of administration; The kidney, liver and cardiovascular function of the patient; and the specific antibodies used. A physician or veterinarian of ordinary skill can readily determine and specify an effective therapeutic amount of the antibody. The best accuracy in achieving antibody concentrations in the range that produces efficacy without toxicity requires a regimen based on the kinetics of the availability of the drug to the target site. This involves considering the distribution, balance and elimination of the drug. The antibodies described herein can be used alone in appropriate dosages. Alternatively, it may be necessary to co-administer or continuously administer other agents. Therapeutic dosing regimen of the antibodies of the invention can be provided in conjunction with administration of an alternative prophylactic or therapeutic regimen. The effective dosage regimen will vary depending on a number of different factors, including the mode of administration, the site of the subject, the physiological state of the patient, whether the patient is a human or animal, other drugs administered, and the treatment being a prophylactic or therapeutic treatment. For administration of a 13C3-like antibody, the dosage is from about 0.0001 mg to 100 mg per kg of host body weight, and more typically from 0.01 mg to 5 mg per kg of host body weight. In the case of Alzheimer's disease, a starch-like deposit is present in the brain, and the agent of the present invention can also be administered in combination with other agents that increase the throughput of the agent of the present invention across the blood-brain barrier. Another aspect of the delivery and dosage regimen of the 13C3-like antibody compositions of the present invention relates to drug delivery via the parenteral route, which may include non-injectable and injectable devices. The injectable compositions are usually prepared as a liquid solution or suspension; solid forms suitable for dissolving or suspending in a liquid vehicle prior to injection may also be prepared. As discussed above, the formulation may also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymers for enhancing adjuvant action (see Langer, 1990, Science 249: 1527-1523; and Hanes, 1997, Advanced Drug Delivery Reviews 28:97-119). The agent of the present invention can be administered in the form of a reservoir injection or an implant preparation which is formulated in such a manner as to allow continuous or pulsatile release of the active ingredient.

Particular embodiments include PLGA microspheres (as discussed herein and otherwise known in the art) and polymer-based non-degrading vehicles comprising poly(ethylene-co-vinyl acetate; PEVAc). In addition, the controlled release and localization of antibody-based therapeutic products are reviewed in Grainger et al, 2004, Expert Opin. Biol. Ther. 4(7): 1029-1044, which is incorporated herein by reference in its entirety. transfer. Suitable microcapsules capable of encapsulating the antibody may also include hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacrylate microcapsules prepared by coacervation techniques or by interfacial polymerization. See PCT Publication No. WO 99/24061, entitled "Method for Producing IGF-1 Sustained-Release Formulations", in which proteins are encapsulated in PLGA microspheres, which is incorporated herein by reference in its entirety. In this article. In addition, microemulsion or colloidal drug delivery systems such as liposomes and albumin microspheres can also be used. Other preferred sustained release compositions employ a bioadhesive to retain the antibody at the site of administration. As noted above, the sustained release formulation can comprise a biodegradable polymer with an antibody disposed therein that can provide non-immediate release. Non-injectable devices can be described herein as "implants", "medical reservoir implants", "sump implants", "non-injected reservoirs" or some similar terms. Common reservoir implants may include, but are not limited to, solid biodegradable and non-biodegradable polymer devices (such as elongated polymer or coaxial rod devices), as well as numerous pumps also known in the art. system. Injectable devices are divided into rapid injections (drugs are released and dissipated after injection), and storage or reservoir injections, which provide a reservoir at the injection site, allowing for sustained release of the biological agent over time. The reservoir implant can be surgically limited to the delivery point to provide a suitable reservoir for long-term release of antibodies over time. The device will be capable of carrying a therapeutic formulation in an amount that is therapeutically or prophylactically required for a pre-selected period of time. The reservoir implant can also provide protection from degradation of bodily processes such as proteases for the duration of treatment. As is known in the art, the term "sustained release" refers to the gradual (continuous or intermittent) release of the agent from the block copolymer matrix over an extended period of time. The sustained release of the 13C3-like antibody composition will produce a local biologically effective concentration of the antibody, regardless of the particular device. Depending on the formulation, sustained release of the biological agent will last for a period of one day, several days, one week or more; but most likely to last for one month or more, or up to about six months. Natural or synthetic polymers known in the art will be suitable for use as reservoir implants due to features such as general degradation kinetics, safety and biocompatibility. These copolymers can be manipulated to improve the pharmacokinetics of the active ingredient, to protect the agent from enzymatic attack, and to degrade over time at the attachment or injection site. The skilled artisan will appreciate that there are a wealth of teachings in the art for operating the properties of such copolymers, including the individual production process, the catalyst used, and the final molecular weight of the sustained release reservoir implant or reservoir injection. Natural polymers include, but are not limited to, proteins (eg, collagen, albumin or gelatin); polysaccharides (cellulose, starch, alginate, chitin, polyglucosamine, cyclodextrin, dextran, glass) Uronic acid) and lipids. Biodegradable synthetic polymers can include, but are not limited to, various polyesters, copolymers of L-glutamic acid and γL-glutamate (Sidman et al., 1983, Biopolymers 22: 547-556), polypropylene Esters ([PLA]; U.S. Patent Nos. 3,773,919 and EP 058,481), polylactide polyglycolate (PLGA), such as polylactide-co-glycolide (see, for example, U.S. Patent Nos. 4,767,628 and 5,654,008) No.), polyglycolide (PG), poly(α-hydroxy acid) polyethylene glycol (PEG) conjugate, polyorthoester, polyaspirin, polyphosphoric acid, vinyl pyrrolidone , polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBT copolymer (multi-activity), methacrylate, poly(N-isopropyl acrylamide), PEO-PPO-PEO (Polonik , PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, polyorthoester (POE), or any combination thereof (as described above, for example, see U.S. Patent No. 6,991,654 and U.S. Patent Application Serial No. 20050187631, each of which One is incorporated herein by reference in its entirety, for example, hydrogel (see, for example, Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277; Langer, 1982, Chem. Tech. 12 :98-105), Degradable ethylene - vinyl acetate (e.g. ethylene vinyl acetate disks and poly (ethylene - co - vinyl acetate)), degradable lactic acid - glycolic acid copolymers such as the Lupron Depot ^TM, poly -D - (-) - 3 - hydroxybutyric acid (EP 133,988), a uronic acid gel (see, for example, U.S. Patent No. 4,636,524), a suspension of alginic acid, a polyorthoester (POE), and the like. Since commercial Lupron Depot ^TM (as approved by the first PLA polymers of parenteral sustained release formulations in 1989) since, polylactide (PLA) and copolymers thereof with glycolide (PLGA) of this was It is well known in the art. Other examples of products that utilize PLA and PLGA as excipients to achieve sustained release of the active ingredient include Atridox (PLA; periodontal disease), Nutropin Depot (PLGA; with hGH), and Trelstar Depot (PLGA; prostate cancer). Other synthetic polymers include, but are not limited to, poly(c-caprolactone), poly-3-hydroxybutyrate, poly(beta-malic acid), and poly(dioxanone); polyanhydrides, polyamines Carbamate (see WO 2005/013936), polydecylamine, cyclodextrin, polyorthoester, n-vinyl alcohol, polyethylene oxide/polyethylene terephthalate, polyphosphazene, polyphosphate , polyphosphonates, polyorthoesters, polycyanoacrylates, polyethylene glycols, polydihydropyrans and polyacetals. Non-biodegradable devices include, but are not limited to, various cellulose derivatives (carboxymethyl cellulose, cellulose acetate, cellulose acetate propionate, ethyl cellulose, hydroxypropyl methyl cellulose), sulfhydryl implants (polydimethyl siloxane), acrylic polymer, (polymethacrylate, polymethyl methacrylate, polyhydroxy (ethyl methacrylate), and polyethylene-co-(vinyl acetate) ), poloxamer, polyvinylpyrrolidone, decanoic acid, polypropylene, polyamine, polyacetal, polyester, polyethylene-chlorotrifluoroethylene, polytetrafluoroethylene (PTFE or " Teflon ^TM "), styrene butadiene rubber, polyethylene, polypropylene, polyphenylene ether-polystyrene, poly-a-chloro-p-xylene, polymethylpentene, polyfluorene and other related biostable polymers. Carriers for sustained release of the reservoir formulation include, but are not limited to, microspheres, membranes, capsules, particles, gels, coatings, matrices, wafers, pills, or other pharmaceutical delivery compositions. Examples of such sustained release formulations. See also U.S. Patent No. 6,953,593; No. 6,946,146; , No. 6, 541, 033; and No. 6, 451, 346, each of which is incorporated herein by reference. The dosage form must be capable of carrying the amount and concentration of the drug in the treatment required for the pre-selected stage of treatment. And must provide protection against degradation by bodily processes for a duration of treatment. For example, the dosage form can be protected from degradation by metabolic processes and, for example, leakage, rupture, The outer circumference of the material of the nature of the risk of breakage or distortion. This prevents the dosage form contents from being discharged in an uncontrolled manner under the stresses that it will experience during use, such as due to the normal joints of the individual The physical force exerted on the drug delivery device by the coupling and other movements or the physical force associated with the pressure generated in the reservoir, for example in a convective drug delivery device. The drug reservoir or other means for holding or containing the drug must be This material is also present to avoid unintended reactions with the active agent formulation, and is preferably biocompatible (eg, in the case of implanted dosage forms) , which does not substantially react with respect to the body or body fluids of the individual. In general, it lasts at least 12 hours to at least one week, and most likely via design to take at least 10, 20, 30, 100 days or at least 4 as needed. The implant of the drug is delivered for a month or at least 6 months or more, and the individual is administered to the individual. At such relatively low volume rates (eg, about 0.001 ml/day to 1 ml) / day) delivery of 13C3-like antibodies to minimize tissue interference or trauma near the site of release of the formulation. Depending on the particular biological agent, the formulation may, for example, be about 0.01 μg/hr or 0.1 μg/hr, 0.25 μg/hr, 1 μg/hr, typically at a rate of up to about 200 μg/hr, delivered at a low dose, or at a low volume rate, for example at a rate of from about 0.001 ml/day to about 1 ml/day, for example from 0.01 μg/day to about 20 mg/day. The formulation is delivered. The dosage will depend on a number of factors, such as the efficacy of the active ingredient (e.g., IgG antibody) used, bioavailability and toxicity, and the individual's needs.

These and other objects, advantages and features of the present invention will become apparent from the <RTIgt;

Instance Example 1: Preparation of a fibrillar form of a starch-like β (Aβ ₄₂ )

Aβ ₄₂ synthetic peptide (American Peptide Company, Inc., CA) was prepared according to the method described by Fezoui et al. (Fezoui et al. Amyloid 7(3): 166-178. (2000)). Briefly, lyophilized A[beta] ₄₂ was dissolved in 2 mM NaOH at a concentration of 1 mg/ml (pH about 10.5), followed by ultrasonication and lyophilization. The NaOH-treated Aβ was dissolved in water at a concentration of 1 mg/ml and filtered with a 0.22 μm ULTRAFREE-MC filter (Millipore, MA). The 0.5 mg/ml peptide solution was buffered at a final concentration of 50 mM phosphate, 100 mM sodium chloride and incubated for 4 hr at room temperature. To separate the fibrillar form from the low molecular weight protein, size exclusion chromatography was used to fractionate the supernatant. The purified SEC fractions were then stored at 4 °C.

Various forms of the A[beta] ₄₂ protein have been shown to demonstrate their ability to associate together as monomers or dimers to form high molecular weight oligomers (based fibrils) (Fig. 1). Further aggregation of the soluble fibrils produces an insoluble form of the protein, while the base fibrils can be re-dissociated into a lower molecular weight form.

To purify the base fibril form of A[beta] from low molecular weight proteins, the samples were fractionated using an AKTA chromatography system using a Superdex 75 size exclusion column. 2A shows that in the absence of incubation of the A[beta] ₄₂ synthetic peptide at room temperature, the oligomerization of the formation of fibrils is not present. Figure 2B illustrates that after incubation of the A[beta] ₄₂ synthetic peptide for 4 hr, subsequent SEC purification displays to determine the fibrils of the fibrils.

Example 2: Production of a monoclonal antibody specific for the fibril Aβ

13C3, 19A6 and 1D1 antibodies were generated by immunizing Balb/c mice with fibrillar A[beta] protein using protocols known in the art (Harlow et al., Cold Spring Harbor Laboratory (1988)). The spleen was removed and fused to SP2 myeloma cells in several 96-well plates. The growth of the fused culture was monitored and the supernatant was screened for the ability of the supernatant to bind to the fibrils of the fibrils by antibody capture immunoassay.

Example 3: Characterization of monoclonal antibodies specific for fibril A[beta]

Antibody capture assays were used to further characterize monoclonal antibodies (13C3, 19A6 and 1D1) produced from fusion tumors. 50 μl of a 2 μg/ml base fibril Aβ ₄₂ protein solution was added to each well of a microtiter plate and the plates were incubated overnight at 4 °C. After incubation, the residual antigen solution was removed and washed with PBS solution. Serial dilutions of the fused tumor supernatant were added to plates containing the bound antigen and incubated for 1 hour at room temperature. This primary antibody solution was removed and the wells were washed again with PBS solution. Enzyme-labeled secondary antibodies were subsequently added and incubated for 1 hour at room temperature. After removal of the secondary antibody solution, a conjugated chromogenic receptor with specificity is added to the reaction and detection of the captured antibody produces a quantitative result.

In addition, the secondary reagent was changed to a homologous specific anti-immunoglobulin antibody to identify the specific immunoglobulin isotype of each individual plant. In these experiments, commercially available anti-Aβ ₄₂ antibodies were used to compare the binding specificity of 13C3, 19A6 and 1D1 monoclonal antibodies.

Figures 3A and B illustrate the fibril (PF) and low molecular weight (LMW) forms of the A[beta] ₄₂ peptide used to test the specificity of the 13C3 antibody in an antibody capture immunoassay. In particular, Figure 3A illustrates a graph generated from an ELISA showing that the 13C3 antibody is specific for the fibrillar form (PF) of A[beta] ₄₂ and does not recognize the low molecular weight (LMW) form of the protein. Figure 3B illustrates ELISA data obtained using commercially available 4G8 antibodies demonstrating their recognition of the low molecular weight and fibril forms of A[beta] ₄₂ protein.

Example 4: Specificity of a single antibody to the base fibril form of Aβ ₄₂ as measured by surface plasma resonance (bIACORE)

Purified monoclonal antibodies listed in Table 1 (below) were immobilized on BIAcore sensing wafers according to the published protocol (Nice et al, BioEssays 21:339-352 (1999)). The high sensitivity of the BIAcore optical reaction quantifies the change in reflectance and produces a baseline response to the individual ligand. Interaction analysis was performed when an analyte (LMW form or PF form of A[beta] ₄₂ ) was injected over the induction wafer in solution, and changes in surface plasma resonance produced specificity to identify the ability of each antibody to bind LMW and PF A[beta] ₄₂ . The reaction. All 13C3 and 19A6 antibodies bind to the PF form of A[beta] ₄₂ with specificity above the LMW form. In all of the antibodies used in this experiment, as indicated by the ratio of PF binding/LMW binding, the commercially available antibodies exhibited specificity for LMW A[beta] ₄₂ that was higher than the PF form of A[beta] ₄₂ .

Surface plasmon resonance analysis showed that the 13L3 (Fig. 4B) does not bind to the LMW form of the A[beta] ₄₂ protein by means of an induction map. However, 4G8 (Fig. 4A) shows a standard association/dissociation curve for LMW A[beta] ₄₂ protein. The antibody isotype control IgG1 (panel C) also did not bind LMW Aβ. An automated BIAcore system utilizing the principle of surface plasmon resonance detection was used in these experiments. The binding specificity data for the 19A6 antibody shows that 19A6 has a binding ratio of 5.8, which is similar to 13C3 with a ratio of 5.3.

Example 5: epitope mapping of 13C3 antibody

The epitope mapping of 13C3, 1D1 and 19A6 was carried out according to the published protocol (Korth et al., 390: 74 (1997)) using a RepliTope microarray system (JPT Peptide Technologies GmbH). Each point on the microarray contains a peptide of 13 amino acids of A[beta] ₄₂ , wherein each position shift on the microarray represents an amino acid shift (from N-terminus to C-terminus), ie SEQ ID NO:23, SEQ ID NO:24:SEQ ID NO:51; and SEQ ID NO:52. The peptides and their precise amino acid sequences are listed below, corresponding to positions on their slide arrays. After immobilizing the peptide to the RepliTope microarray, the sample is incubated with the 13C3 antibody and then subsequently labeled with a secondary antibody conjugated to the selected chemiluminescent tag. The point at which the signal is generated represents the epitope binding site on the protein bound by the antibody.

Figure 5A illustrates spot blotting from RepliTope microarray experiments to identify epitopes for antibodies 13C3, 1D1 and 4G8 on the Aβ _1-42 peptide. The bound antibody is presented by a chemiluminescent signal. Figure 5B illustrates the A[beta] _1-42 amino acid sequence of the polypeptide segment displaying the 13C3 epitope (when it is present in the sequence). The 1D1 antibody displays the same epitope as 13C3, while the commercially available 4G8 antibody identifies different epitopes.

Example 6: Characterization of 13C3 specificity

Figure 6 illustrates the fractions from size exclusion chromatography performed on the supernatant of the 7PA2 cell line (cell line secreting A? oligomer). Antibody capture assays were used to further bind the 13C3 antibody to the fibrils and the low molecular weight dissolving fraction from the SEC purified 7PA2. 100 μl of each 1:120 dilution of each fraction was added to each well of the microtiter plate and the plates were incubated overnight at 4 °C. After incubation, the residual antigen solution was removed and washed with PBS solution. Serial dilutions of the 13C3 supernatant were added to plates containing antigen-binding and incubated for 1 hour at room temperature. This primary antibody solution was removed and the wells were washed again with PBS solution. Enzyme-labeled secondary antibodies were subsequently added and incubated for 1 hour at room temperature. After removal of the secondary antibody solution, a conjugated chromogenic receptor with specificity is added to the reaction and detection of the captured antibody produces a quantitative result. This assay identified that the 13C3 antibody only specifically recognized the fibril dissolving fraction, while the 4G8 antibody recognized all dissolving fractions. The 7PA2 cell line was supplied by Dennis J. Selkoe, M.D., of Harvard Medical School.

Example 7: 13C3 reactivity was characterized by EM.

The staining method was carried out using a standard protocol (Brenner et al. Biochim. Biophys. Ada 34, 103-110 (1959)). A small volume (10 microliters) of a 0.2 mg/ml base fibril solution was applied to a carbon coated polyacetate (formvar) grid (400 mesh) for 2 min. The grid was then blocked in 1% BSA and incubated with the 13C3 antibody followed by subsequent incubation with secondary antibody (conjugated to colloidal gold). Samples were negatively stained by placing on 2 consecutive drops of 2% phosphotungstic acid for 30 seconds each. Excess stain solution was blotted with filter paper, the grid was air dried, and observed on a JEOL 100 CX transmission electron microscope at 80 kV. Images were recorded on a large Kodak 4489 negative and digitized on a flatbed scanner.

IEM (Immunoelectron Microscopy) images showed binding specificity of anti-A[beta] antibody-line 13C3 to A[beta] ₄₂ fibers (Figures 7B and 7C), while isotype control antibody IgGl did not display binding (Figure 7A). The secondary resistance system is conjugated to the colloidal gold particles.

Example 8: 13C3 treatment of a mouse model of human AD

13C3 monoclonal antibody was used to treat Aβ plaques in the Alzheimer's mouse model (TgCRND8). The mouse contains the human APP695 cDNA transgene, which accelerates the deposition of A[beta]-like amyloid plaques in the brain of mice (appearing within 1 month of age). A sample group of five five-week-old TgCRND8 mice was administered once a week for a duration of seven weeks of immunization of 13 C3 monoclonal antibodies at a concentration of 10 mg per kg of mice. A second group of 5 TgCRND8 mice was given a course of treatment, however, an isotype control IgG1 antibody was administered. The experiment was repeated with a weekly treatment instead of once a week.

Control and experimental animals were sacrificed at 12 weeks of age. The histological preparation of the brain revealed a reduction in A[beta] plaques in 13C3-treated mice.

Serial sections of cryopreserved brain from TgCRND8 mice were treated with 13C3 or IgG1 monoclonal antibodies. Figures 8A and 8B illustrate the difference in the number of A[beta]-like starch plaques between each individual antibody.

The statistical T test showed that once-a-week 13C3 antibody treatment reduced A[beta] amyloid plaques in the Alzheimer's disease model (Fig. 9A). However, treatment twice a week (Figure 9B) showed the same degree of plaque reduction.

All of the above TgCRND8 mice were obtained from Dr. David Westaway of the University of Toronto.

Example 9: Molecular Characterization of the Variable Region of MAB 13C3

The IgG heavy chain variable region and the IgG κ light chain region were selected from the 13C3 fusion tumor. Analysis of heavy and light chain sequences using the database of germline variable genes from human and mouse immunoglobulin loci extracted from the EMBL-Bank and Ensembl databases VBASE2 (http://www.vbase2.org) (Figure 10). (Retter et al. Nucleic Acids Res. 33: D671-4 (2005)). The results of the analysis identified both the heavy and light chain variable regions from newly identified immunoglobulins, but were 73% and 81% identical to the other immunoglobulin variable regions in the database, respectively. For these databases, framework regions (FWR) and complementarity determining regions (CDRs) are also identified in these sequences. When analyzing sequences for the VBASE, KABAT, and IMGT/LIGM databases, the results were only slightly different.

Example 10: Unlike the reference antibody 3D6, short-term peripheral administration of 13C3 in APP transgenic mice did not cause an increase in plasma Aβ.

At a dose of 10 mg/kg (ie, 300 μg/mouse), antibody 13C3, control IgG1 (DM4, no recognition of Aβ), and reference anti-Aβ antibody 3D6 (recognition of all conformations of Aβ) were small for APP transgenes. Rats (Thy APPSL, 10-14 weeks old) were injected intraperitoneally. Plasma Aβ was quantified at the time of pre-injection at zero time in the same mouse, at 6h, 24h and 7 days after injection. Plasma Aβ quantification was performed using an immunoassay performed with an anti-Aβ antibody pair that does not interfere with the binding of 13C3 or 3D6 to Aβ.

Administration of 3D6 (an antibody against all conformations of A[beta]) may result in a substantial increase in plasma A[beta] by protecting the A[beta] molecule from degradation. This effect was used to propose a potential "peripheral sink" hypothesis as a mechanism of action against Aβ immunotherapy (Demattos et al., 2001, PNAS 17: 8850). Unlike 3D6, administration of 13C3 did not cause any increase in plasma A[beta] levels. This is consistent with the properties of the soluble monomer or oligomeric form of antibody 13C3 which is specific for the fibril form of A[beta] and does not recognize the A[beta] peptide. These forms are possible forms that are present in plasma.

Example 11: Unlike the reference 3D6 anti-Aβ antibody, 13C3 recognizes human amylofid neuritis plaques (aggregation) in AD brain rather than diffuse Aβ deposits

Immunohistochemical studies of human brain slices diagnosed with Alzheimer's disease were performed using 13C3 and 3D6 antibodies using standard techniques. Immunostaining with DAB chromogen detection antibody (Fig. 12). 13C3 marks a starch-like deposit with a typical morphology of mature amyloin neuritis plaques (also known as dense plaques), which have a very dense core surrounded by a shallow halo or Very strong staining for large plaques. In adjacent brain sections, as seen at lower magnification, 3D6 stained more targets than 13C3 (Figure 12, left panel). Further characterization at higher magnification (Figure 12, right panel) indicates that 3D6 and 13C3 label the same mature amylin plaques, and additionally labeled a number of diffuse starch-like deposits traditionally described using anti-Aβ immunolabels . Diffuse plaque does not have fibril properties as described in the literature because it cannot be detected by thioflavin S and other tissue markers of fibrils (Mann, 1989, Ann. Med). 21:133). To rule out the difference in sensitivity between the two antibodies, a similar experiment was performed at a higher concentration (20 μg/ml) of 13C3 and diffuse deposits could no longer be detected. This data is consistent with the specificity of the fibril form of A[beta] and differs from the characteristic of 13C3, a soluble monomer or oligomeric form of 3D6 that does not recognize the A[beta] peptide.

Industrial applicability

The invention has utility in the treatment and diagnosis of Alzheimer's disease.

All publications (patent publications and non-patent publications) cited in the specification are intended to be within the technical scope of those skilled in the art. All such publications are hereby incorporated by reference in their entirety to the extent of the extent of the disclosure of the disclosure of the disclosures of

Although the present invention has been described herein with reference to the specific embodiments thereof, it is understood that these embodiments illustrate only the principles and applications of the invention. It is understood that numerous modifications may be made to the illustrative embodiments and other configurations may be devised without departing from the spirit and scope of the invention as defined by the appended claims.

Figure 1 shows a process for the formation of Aβ fibrils comprising forming a base fibril oligomer;

2A-B show the process of Aβ fibril formation and the purification of the Aβ form at time 0 (A) and 4 hours (B), as indicated by the absorbance at a mAU ₂₁₅ (absorbance at 215 nm) for the dissolved volume. . At 4 hours (B), the low molecular weight (LMW) form is dissolved to about 15 kDa dimer, while the base fibril form size is dissolved at about 670 kDa;

Figure 3A-B shows the monoclonal antibodies 13C3 (A) and 4G8 (B) to Aβ based fibrils (PF: -◆-) and low molecular weight (LMW: The specificity of the form, as indicated by the optical density (OD) read at 450/650 nm for PF and LMW forms of increasing concentrations of Aβ;

Figure 4A-C shows from Combined with the assay data, it displays the low molecular weight (LMW) forms of individual antibodies 4G8 (A), 13C3 (B) and control IgG ₁ (C) for different concentrations of Aβ (0.25 μg/mL LMW Aβ to 4.0 μg/ Affinity of mL LMW Aβ);

Figures 5A-B show data identifying the epitopes recognized by the anti-Aβ antibodies described above. Figure 5A illustrates Western blotting dot analysis using a single antibody 13C3 (top plate), ID1 (middle plate), and 4G8 (bottom plate) for a series of overlapping 13 amino acid peptides as described in Example 5. Figure 5B illustrates the amino acid sequence of Aβ _1-42 (SEQ ID NO: 1), and the predicted epitope of monoclonal antibodies 13C3 and 1D1;

Figure 6 shows monoclonal antibody 13C3 ( And the reactivity of 4G8 (-◆-) with the SEC dissolving fraction from the 7PA2 supernatant secreting Aβ oligomer. The fibril (PF) and low molecular weight (LMW) dissolving fractions are indicated on the x-axis as measured by optical density (OD) read at 450/650;

Figures 7A-C show micrographs from thin section immunoelectron microscopy showing the affinity of monoclonal antibody 13C3 for repeating structures on A[beta] fibrils (B, C). Control immune EM is IgG ₁ (A);

Figures 8A-B show data from electron micrographs showing plaques in representative TgCRND8 transgenic mice after administration of control IgG ₁ (A) antibodies compared to administration of 13C3 monoclonal antibody (B) Decrease in number;

Figure 9A-B shows treatment of TgCRND8 transgenic mice with 13C3 monoclonal antibody by once-a-week (A) or twice-weekly (B) therapy to reduce senile plaque formation;

Figure 10 shows the nucleotide and amino acid sequences of the selected light and heavy chain variable regions of mAb 13C3;

Figure 11A-B shows that short-term peripheral administration of 13c3 does not cause an increase in plasma Aβ in APP transgenic mice, unlike the reference antibody 3D6;

Figure 12 shows human amyloated neuritis plaques, which are different from the reference 3D6 anti-A[beta] antibody, which are recognized (aggregated) in the AD brain, rather than diffuse A[beta] deposits.

(no component symbol description)

Claims (41)

An isolated antibody that specifically binds to a conformational epitope of a fibrillar form of an A[beta] peptide and exhibits a measurable affinity, wherein the fibril epitope is represented by an exposed region of the A[beta]-based fibril form The exposed region consists of the amino acid sequence set forth in SEQ ID NO: 2, wherein the antibody exhibits minimal or no affinity for the monomeric or dimeric form of the A[beta] peptide. The antibody of claim 1, which is a monoclonal antibody. An antibody according to claim 2 which is a humanized monoclonal antibody. An antibody according to claim 2 which is a human monoclonal antibody. An isolated antibody that specifically binds to a conformational epitope of a fibrillar form of an A[beta] peptide and exhibits a measurable affinity, wherein the fibril epitope is represented by an exposed region of the A[beta]-based fibril form The exposed region consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4, wherein the antibody exhibits minimal or no affinity for the monomeric or dimeric form of the Aβ peptide. . An antibody according to claim 5 which is a monoclonal antibody. The antibody of claim 6, which is designated 13C3 and is produced from a fusion tumor obtainable by ATCC Accession No. PTA-8830. An antibody according to claim 6 which is a humanized monoclonal antibody. An antibody according to claim 6 which is a human monoclonal antibody. The antibody of claim 5, which further comprises a variable light chain comprising the amino acid sequence set forth in SEQ ID NO: 5. The antibody of claim 5, which further comprises the SEQ ID NO: 7 The variable heavy chain of the amino acid sequence is elucidated. The antibody of claim 5, which further comprises a variable light chain comprising the CDR1 region set forth in SEQ ID NO: 13, the CDR2 region set forth in SEQ ID NO: 14, and the CDR3 set forth in SEQ ID NO: 15. . The antibody of claim 5, which further comprises a variable heavy chain comprising the CDR1 region set forth in SEQ ID NO: 20, the CDR2 region set forth in SEQ ID NO: 21, and the CDR3 set forth in SEQ ID NO: . A method of producing an antibody according to any one of claims 1 to 13, which comprises: (a) immunizing a mammal in a protofibril form of Aβ peptide; (b) collecting B cells of the mammal (c) producing a fusion tumor from the B cells collected, wherein the fusion tumor produces an antibody; (d) selecting to produce an antigenic determinant specific binding to the fibril form of the Aβ peptide while simultaneously displaying the Aβ peptide A fusion tumor of a monomer or a dimeric form of antibody having minimal affinity, wherein the epitope of the base fibril consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4; and (e) an antibody produced by isolating the fusion tumor. A method of quantifying the amount of a fibrillar form of an A[beta] peptide in a tissue or liquid sample, the method comprising: (a) taking a tissue or liquid sample of a body; (b) contacting the tissue or liquid sample as in claim 1 Any one of 13 The antibody; and (c) the amount of the fibrillar form of the Aβ peptide in the sample. The method of claim 15, wherein the antibody is a monoclonal antibody produced from a fusion tumor obtainable by ATCC Accession No. PTA-8830. A kit for detecting a fibril form of an Aβ peptide and exhibiting a minimum affinity for a low molecular weight form of the Aβ peptide, the kit comprising: (a) the antibody of any one of claims 1 to 13 or a fragment; and (b) an agent that binds directly or indirectly to the antibody or fragment thereof. The kit of claim 17, wherein the antibody is a monoclonal antibody produced from a fusion tumor obtainable by ATCC Accession No. PTA-8830. A pharmaceutical composition for treating or preventing deposition of Aβ plaque in a mammal, comprising at least a pharmaceutically effective amount of the antibody of any one of claims 1 to 13 or a variable region thereof, the antibody or The variable region fragment specifically interacts with the fibril form of the A[beta] peptide to inhibit the formation and deposition of A[beta] fiber plaques. The pharmaceutical composition of claim 19, wherein the antibody is a monoclonal antibody. The pharmaceutical composition of claim 20, wherein the monoclonal antibody is an antibody produced from a fusion tumor obtainable by ATCC Accession No. PTA-8830. The pharmaceutical composition of claim 20, wherein the monoclonal antibody is a humanized monoclonal antibody. The pharmaceutical composition of claim 20, wherein the monoclonal antibody is a human monoclonal antibody. A fusion tumor available from ATCC Accession No. PTA-8830. An isolated nucleic acid molecule encoding a variable heavy chain of monoclonal antibody 13C3 A fragment, wherein the variable heavy chain fragment comprises the amino acid sequence set forth in SEQ ID NO: 7. An isolated nucleic acid molecule encoding a variable heavy chain fragment of monoclonal antibody 13C3, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 8. A expression vector for expressing a variable heavy chain fragment of monoclonal antibody 13C3 in a recombinant host cell, wherein the expression vector comprises the nucleic acid molecule of claim 26. A host cell which expresses a variable heavy chain fragment of monoclonal antibody 13C3, wherein the host cell contains the expression vector of claim 27. An isolated nucleic acid molecule encoding a variable light chain fragment of monoclonal antibody 13C3, wherein the variable light chain fragment comprises the amino acid sequence set forth in SEQ ID NO: 5. An isolated nucleic acid molecule encoding a variable light chain fragment of monoclonal antibody 13C3, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 6. An expression vector for expressing a variable light chain fragment of monoclonal antibody 13C3 in a recombinant host cell, wherein the expression vector comprises the nucleic acid molecule of claim 30. A host cell which expresses a variable light chain fragment of monoclonal antibody 13C3, wherein the host cell contains a expression vector as claimed in claim 31. A variable heavy chain fragment of an isolated monoclonal antibody 13C3 comprising the amino acid sequence set forth in SEQ ID NO: 7. A variable light chain fragment of an isolated monoclonal antibody 13C3 comprising SEQ ID NO: The amino acid sequence set forth in 5. Use of a variable region fragment of an antibody according to any one of claims 1 to 13 for inhibiting the formation and deposition of A[beta] plaques for the treatment or prevention of A[beta] plaques in a mammal Deposition of the drug. The use of claim 35, wherein the antibody is a monoclonal antibody. The use of claim 36, wherein the monoclonal antibody is an antibody produced from a fusion tumor obtainable by ATCC Accession No. PTA-8830. The use of claim 36, wherein the monoclonal antibody is a humanized monoclonal antibody. The use of claim 36, wherein the monoclonal antibody is a human monoclonal antibody. A pharmaceutical composition for the treatment or prevention of Alzheimer's disease, comprising the antibody of any one of claims 1 to 13 or a variable region fragment thereof. The pharmaceutical composition of claim 40, wherein the antibody specifically binds to a fibril form of Aβ, wherein the specific binding is characterized by affinity of the antibody or variable region fragment thereof for the fibril form of Aβ and other The ratio of the affinity of the Aβ form is greater than 2.