HK1081647B - N-11 truncated amyloid-beta monoclonal antibodies, compositions, methods and uses - Google Patents

Description

Monoclonal antibodies to N-11 truncated beta-amyloid, compositions, methods and uses

The invention relates to antibodies, including particular moieties or variants, that are specific for at least the N-terminal 11 site of human beta-amyloid, e.g., an A β 11-x polypeptide.

Background

In general, the present invention relates to methods and compositions for controlling the development of beta-amyloid precursor protein. More particularly, the invention relates to the use of the methods and compositions for diagnosis, prognosis and monitoring associated with the treatment of Alzheimer's disease and other beta-amyloid related diseases, and the use of the disclosed antibodies in passive immunization as a method for treating Alzheimer's disease and other beta-amyloid related diseases.

Alzheimer's Disease (AD) is a degenerative brain disorder characterized clinically by a continuous and gradual loss of memory, cognition, reasoning, judgment and emotional stability, leading to profound intellectual decline and ultimately death. In the united states alone, it is estimated that the disease currently affects about two to three million people. It is known that there is no effective treatment for preventing AD or eliminating its symptoms and causes.

The brains of patients with AD exhibit characteristic lesions known as senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits on blood vessels), and neurofibrillary tangles. A large number of these lesions, particularly amyloid plaques and neurofibrillary tangles, are prevalent in several areas of the brain important for memory and cognition in patients with AD A small number of these lesions are found on the brain of the vast majority of elderly people who do not have clinical AD via more rigorous anatomical examination. Amyloid plaques and amyloid angiopathy are also characteristic of the brain of patients with trisomy 21 (Down's syndrome), diffuse Lewy body disease, hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D).

The major components of amyloid plaques are a class of beta-amyloid (a β) polypeptides produced by cleavage of beta-Amyloid Precursor Protein (APP). There has been intense scientific debate in the past that plaques and tangles are causative or merely a consequence of alzheimer's disease, but recent findings suggest that amyloid plaques are a precursor and a factor for. In particular, it was found that production of a β polypeptide results from mutation of a gene encoding amyloid precursor protein that does not produce a β polypeptide upon normal processing. The identification of the mutations in the amyloid precursor protein gene that cause familial, early-onset alzheimer's disease most strongly suggests that amyloid metabolism is an important change in the pathogenesis of these diseases. It is now believed that the normal process of processing (non-pathogenic) the APP protein is accomplished by cleavage by an alpha-secretase which cleaves between 16 and 17 amino acids of the A.beta.polypeptide region of the protein.

BACE-1 has been shown to be an important β -secretase required for cleavage of APP at the +1 position overexpression of BACE-1 results in additional cleavage of A β at the +11 position, yielding shorter A β 11-40 and A β 11-42 fragments, hereinafter also referred to as A β 11-x polypeptides. It is noteworthy that these shorter a β fragments have been shown by biochemical analysis (6) to be the major species in the AD brain and normal aged brain, as well as by immunohistochemical studies (7) in the brain of Down's syndrome with pathological features of AD. These facts prompted a reevaluation of the role of a β 11-40/42 in the pathogenesis of alzheimer's disease, especially considering the fact that the a β species starting from Glu11 appeared to be less soluble than the a β species starting from position 1.

In addition to understanding the mechanisms involved in AD and other a β -related diseases, there is a need to develop methods and compositions for diagnosing and treating these diseases. Thus, the ability to monitor cellular processing of amyloid precursor protein is of great interest for the diagnostic, prognostic, therapeutic monitoring of alzheimer's disease. In particular, it would be desirable to be able to identify a minimally invasive and reproducible procedure for screening and evaluating detectable diagnostic markers in readily available patient samples, such as serum, cerebrospinal fluid (CSF), and the like. Polyclonal antibodies, such as Said, t.c.et al, neuroscience letters 215 (1996); 173-176, but given the fact that each batch of polyclonal antibodies is different, these antibodies are not useful as a tool for performing a reproducible procedure for screening and evaluating detectable diagnostic markers in readily available patient samples. Furthermore, nonspecific binding is particularly high with polyclonal antibodies and the accuracy in Western blotting is low.

A number of possible diagnostic markers for alzheimer's disease have been envisaged. Of particular interest to the present invention are the shorter carboxy-terminal fragments of the A β precursor protein obtained after cleavage of the APP protein by β -secretase. The diagnostic marker is preferably detectable in body fluids and tissues such as CSF, blood, plasma, serum, urine, and the like, to facilitate the use of minimally invasive diagnostic methods.

A particular detection method for A.beta.11-x detection should be able to detect A.beta.11-x in a reproducible and stepwise manner in very low concentration liquid samples, yet be able to distinguish A.beta.11-x polypeptides from other APP fragments that may be present in the sample.

Various aspects of the invention are described in greater detail herein.

Summary of The Invention

The present invention provides monoclonal antibodies that specifically recognize shorter A.beta.polypeptides resulting from cleavage of the APP protein at Glu-11 by BACE-1, such as the A.beta.polypeptide fragments A.beta.11-40 and A.beta.11-42, hereinafter referred to as A.beta.11-x polypeptides. Also provided are hybridoma cells that produce monoclonal antibodies and methods of making the antibodies and hybridoma cells. And an immunoassay for a β polypeptide by a competition method or a sandwich method using an antibody.

The invention specifically provides monoclonal antibodies that are the first 5-7 human amino acids using a beta-secretase 11 cleavage site, namely EVHHQ-C (human A. beta. -11 (6AA) -Seq Id No.: 1) and EVHHQKI-C (human A. beta. -11 (8AA) -Seq Id No.: 2) or the first 5-7 mouse amino acids using the cleavage site for beta-secretase 11, namely EVRHQ-C (mouse A.beta.11 (6AA) -Seq Id No.: 3) and EVRHQKL-C (mouse A.beta.11 (8AA) -Seq Id No.: 4) as immunogens the antibodies react specifically with A.beta.11-x polypeptides without cross-reactivity with other APP fragments, it is accordingly used in immunoassays to assess the role of a β 11-x in the pathogenesis of alzheimer's disease.

In a more specific embodiment, the monoclonal antibody reacts with human A β _11(6AA) immunogen and is expressed by hybridoma cells J & JPRD/hA β 11/1 and J & JPRD/hA β 11/2, which were deposited at the Belgian collections of microorganisms Association on 8/19 2002 under accession numbers LMBP5896CB and LMBP5897CB, respectively. In a further embodiment, the invention provides the aforementioned hybridoma cells expressing a monoclonal antibody of the invention.

In another aspect of the invention, the antibodies of the invention are used in conventional immunological techniques for detecting A β 11-x polypeptides, including in any case biological samples for monitoring β -amyloid associated diseases and conditioned media from cell cultures for monitoring intracellular processing of APP. Furthermore, it is well known that they all rely on the formation of antigen-antibody immune complexes, the detection of which is aimed at the antibodies being labelled, for example, with radioactive, enzymatic or fluorescent labels, or being immobilized on insoluble supports and being able to be detected.

The invention also includes the use of a humanized antibody of the invention in the preparation of a medicament for the treatment, prevention or reversal of alzheimer's disease, Down's syndrome, HCHWA-D, cerebral amyloid angiopathy or other beta-amyloid related diseases; for treating, preventing or reversing cognitive decline in clinical and latent Alzheimer's disease, Down's syndrome, HCHWA-D, cerebral amyloid angiopathy; or inhibiting the formation of amyloid plaques or the action of soluble toxic A β groups in humans.

Brief Description of Drawings

FIG. 1A: serum titers of mice injected with the first 5-7 human amino acids of the beta-secretase 11 cleavage site as immunogens, i.e., EVHHQ-C (human A. beta. -11 (5AA) -Seq Id No.: 1) and EVHHQKI-C (human A. beta. -11 (7AA) -Seq Id No.: 2) or the first 5-7 mouse amino acids of the beta-secretase 11 cleavage site, i.e., EVRHQ-C (mouse A. beta. -11 (5AA) -Seq Id No.: 3) and EVRHQKL-C (mouse A. beta. -11 (7AA) -Seq Id No.: 4). The coating antigen used was 2.0. mu.g/ml hA β (11-40) (American peptide Company).

TABLE 1 immunization course and time profile of spleen collections and fusions from mice injected with EVHHQ-C (human A β 11(5AA) -Seq Id No.: 1).

Table 3 shows the Western blot results for the specific detection of A.beta.11-x polypeptide in brain sections from AD patients.

FIG. 2 Sandwich ELISA using purified monoclonal antibodies JRF/A β N/25, J & JPRD/hA β 11/1 and J & JPRD/hA β 11/2 as capture antibodies and JRF/cA β 40/10-HRPO as detection antibodies. Antibody combinations were evaluated for reactivity with human A β 1-40 and human A β 11-40(American peptide company).

A: JRF/Abeta N/25 and JRF/cA beta 40/10-HRPO reacted specifically with human Abeta 1-40, but not cross-reacted with hA beta 11-40 (positive control for A beta 1-40 detection).

B: j & JPRD/hA β 11/1 and JRF/cA β 40/10-HRPO reacted specifically with hA β 11-40, but not cross-reacted with human A β 1-40.

C: j & JPRD/hA β 11/2 and JRF/cA β 40/10-HRPO reacted specifically with hA β 11-40, but not cross-reacted with human A β 1-40.

FIG. 3Western blot shows specific reaction of J & JPRD/hA β 11/1 with β 11 cleaved CTF fragment of APP in membrane extracts of HEK cells stably transfected with human APPswe and human BACE 1. C6/6.1 is directed to the C-terminus of APP and reacts with CTF fragments that cleave at β 1 and β 11 of APP.

Detailed Description

The present invention provides monoclonal antibodies that specifically recognize shorter A β polypeptides that are obtained by cleaving the APP protein at Glu-11 with BACE-1. The antibodies of the invention have specificity for one or more epitopes present on the first 5 to 7 amino acids of the beta-secretase _11 cleavage site of human a β or the first 5 to 7 amino acids of the beta-secretase _11 cleavage site of mouse a β.

The invention specifically provides monoclonal antibodies made using as immunogens the first 5-7 human amino acids that include the beta-secretase _11 cleavage site, i.e., EVHHQ-C (human A. beta. -11 (6AA) -Seq Idno.: 1) and EVHHQKI-C (human A. beta. -11 (8AA) -Seq Id No.: 2) or the first 5-7 mouse amino acids of the beta-secretase _11 cleavage site, e.g., EVRHQ-C (mouse A. beta. -11 (6AA) -Seq Id No.: 3) and EVRHQKL-C (mouse A. beta. -11 (8AA) -Seq Id No.: 4).

The aforementioned polypeptides may be prepared by methods known in the art, such as the well-known Merrifield solid phase synthesis technique, in which amino acids are sequentially added to form long chains (Merrifield (1963) J.Am.chem.Soc.85: 2149-. When used as immunogens, the polypeptides thus obtained can be used alone or can be linked to suitable immunoactive natural or synthetic carriers, such as maleimide-activated serum albumin of mammals such as bovine, rabbit, and human, thyroglobulin of mammals such as bovine, rabbit, human, and ovine, and Keyhole Limpet Hemocyanin (KLH) or other suitable protein carriers such as synthetic polymers including styrene, acrylic, vinyl, and propylene polymers.

Once an effective amount of immunogen is obtained, polyclonal antibodies specific for a β 11-x polypeptides can be prepared by using various methods, including in vitro or in vivo techniques. In vitro techniques involve exposure of lymphocytes to an immunogen, while in vivo techniques require injection of the immunogen into a suitable vertebrate host. Suitable vertebrate hosts are non-human and include mice, rats, rabbits, sheep, goats, and the like. According to a predetermined protocol, an immunogen is injected into the animal and blood is drawn from the animal periodically to obtain a continuous blood stream with elevated titer and specificity. The injection can be intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. adjuvants, such as Freund's complete adjuvant or Freund's incomplete adjuvant, can be used to enhance the antibody-producing ability. For example, in an ELISA screening procedure, serum is added to a solid phase (e.g., the bottom of a microwell plate) coated with an A β 11-x polypeptide or an A β 11-x polypeptide coupled to a carrier (e.g., BSA), followed by addition of an anti-immunoglobulin antibody (e.g., when immunization occurs in mice, anti-mouse immunoglobulin antibodies such as sheep anti-mouse immunoglobulin (Ig)) coupled to a detectable label such as an enzyme, preferably horseradish peroxidase or an antibody such as I¹²⁵The radioisotope of (1).

If desired, monoclonal antibodies can be prepared therefrom using techniques well known to those of ordinary skill in the art and by hyperimmunizing a vertebrate host, such as a mouse, with the immunogen of interest by the methods described above. Vertebrate hosts that exhibit high titers of antibodies are conveniently selected from animals immunized with the target immunogen. Typically, 2 to 5 days, preferably 4 days, after the last immunization, the spleen and lymph nodes thereof are collected and the antibody-producing cells contained therein are immortalized. The manner in which it is immortalized is not critical. Currently, the most commonly used technique is fusion with a myeloma cell fusion partner. The fusion process is carried out according to methods known in the art, for example the method of Kohler and Milstein (Nature, 256, 495-497 (1975)). Other techniques include EBV transformation, transformation with naked DNA, e.g., oncogenes, retroviruses, etc., or any other method that provides for the maintenance of cell line stability and the production of monoclonal antibodies. Fusion enhancers including polyethylene glycol (PEG) and Sendai virus may be used. Particularly preferably, PEG is used, and examples of myeloma cells include NS-11, P3U1, SP2/0 and AP-1, and SP2/0 cells are preferably used.

The most efficient method of producing monoclonal antibodies having specificity for an epitope present on the first 5 to 7 amino acids of the beta-secretase _11 cleavage site of human a β or the first 5 to 7 amino acids of the beta-secretase _11 cleavage site of mouse a β: hybridoma-producing animals, such as Balb/c mice, are first immunized by intraperitoneal injection of the immunogen of interest in Freund's adjuvant, followed by booster injections every two weeks. The isolated spleens are then fused using any technique well known to those of ordinary skill in the art, preferably using SP2/0 cells according to the modified method of Kohler and Milstein (Eur. J. Immunol., 6, 292-295(1976)) to determine the selection of hybridoma cells for production of antibodies specific for the A β 11-x polypeptide using the standard ELISA or RIA methods described previously, selection and culturing of hybridoma cells producing the monoclonal antibodies of interest is typically performed in animal media (e.g., Dulbecco's Modified Eagle's Medium (DMEM) or Eagle's Minimal Essential Medium (MEM)) supplemented with 10-20% fetal bovine serum and other components such as HAT (hypoxanthine, aminopterin, and thymidine) or hybridoma additives, accordingly, in one embodiment of the invention, the invention provides hybridoma cells J & JhA/PRJhA 11/1 and PRJhA & 11/2, they were deposited at the belgian collection of microorganisms on year 2002, 8/19, under the respective accession numbers LMBP5896CB and LMBP5897CB.

The isolation and purification of anti-A.beta.11-x monoclonal antibodies is similar to conventional isolation and purification procedures for polyclonal antibodies, such as salt precipitation, ethanol precipitation, isoelectric precipitation, electrophoresis, adsorption and desorption of ion exchange materials (e.g., DEAE), ultracentrifugation, gel filtration, and specific immunoaffinity separation techniques including antigen-binding solid phases and affinity chromatography for protein A or protein G suitable protein purification techniques are described in, for example, Methods in Enzymology, Vol.182, Deutcher, ed., Academic Press, Inc., San Diego, 1990, the disclosure of which is incorporated herein by reference.

It is therefore an object of the present invention to provide an isolated monoclonal antibody expressed by the aforementioned hybridoma cells, said antibody being capable of specifically recognizing an a β 11-x polypeptide. These isolated monoclonal antibodies are preferably expressed by hybridoma cells J & JPRD/hA β 11/1 and J & JPRD/hA β 11/2, which were deposited at the Belgian collections of microorganisms Association on 8/19 2002, under accession numbers LMBP5896CB and LMBP5897CB, respectively.

Wherever antibodies of the invention are used, detection of a β 11-x polypeptides by conventional immunological techniques involves biological samples for monitoring β -amyloid associated disease and conditioned media from cell cultures for monitoring intracellular processing of APP. Suitable immunological techniques are well known to those skilled in the art and include, for example, ELISA, Western blot analysis, competitive or sandwich immunoassays and the like. It is also well known that they all rely on the formation of antigen-antibody immune complexes, the detection of which is aimed at the antibodies being detectably labelled, e.g. by radioactive, enzymatic, luminescent or fluorescent labels, or immobilized on insoluble supports. It is therefore an object of the present invention to provide immunoassay methods for determining and detecting an A β 11-x polypeptide in a sample, comprising contacting an antibody to an A β 11-x polypeptide of the present invention with the sample, and determining whether an immunocomplex forms between the antibody and the A β 11-x polypeptide, these methods being performed either on a tissue sample or on a sample of bodily fluid, generally comprising obtaining a sample from the body of a subject, contacting said sample with an imaging effective amount of a detectably labeled antibody of the present invention; detecting the marker to determine the presence of the A β 11-x polypeptide in the sample.

Any method may be used as long as the amount of antibody, antigen or antigen-antibody complex corresponding to the amount of antigen, particularly the amount of A.beta.11-x polypeptide in a test solution can be detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. The following sandwich method is particularly preferably used in view of sensitivity and specificity.

In the measurement method using a labeling substance, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used as a labeling reagent¹²⁵I、¹³¹I、³H and¹⁴C. enzymes are typically detected by attachment to a suitable substrate and then catalyzing a detectable reaction. Examples include, for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase, preferably horseradish peroxidase.

When the immunogen or the antibody is insolubilized, physical adsorption or chemical binding which is generally used for protein or enzyme insolubilization or immobilization may be employed. Examples of the carrier include insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicone polymer, and glass.

In the sandwich method, a test solution is reacted with an insoluble anti-A β 11-x polypeptide antibody (first reaction), a labeled A β 11-x polypeptide antibody is further reacted (second reaction), and then the activity of a labeled reagent on an insoluble carrier is tested, whereby the amount of A β 11-x polypeptide in the test solution can be determined. The first reaction and the second reaction may be carried out simultaneously or sequentially.

In a further aspect, for the diagnosis of a β -amyloid associated disease, the resulting biological sample, including tissues, body fluids, such as CSF, blood, plasma, serum, urine, and the like, is contacted with an amount of the first antibody to form an immune complex. Typical contacting involves adding the sample to a solid matrix coated with a first antibody. The complex formed by contacting the sample with the first antibody is separated from the sample by elution. The recovered complex is contacted with at least one second antibody corresponding to an antigenic determinant on the antigen to bind the antigen in the complex. A detectable antibody bound to a complex consisting of the binding of the antigen to the first and second antibodies can be readily detected using techniques known in the art, by comparing the results obtained in a biological sample with those obtained in a control sample, it can be determined that there is a change in the level of A β 11-x polypeptide.

Accordingly, it is an object of the present invention to provide a sandwich assay in which a first antibody, hereinafter referred to as a coated antibody, which consists of an antibody that recognizes an A β 11-x polypeptide and either full-length A β 40 or A β 42, and a second antibody that specifically recognizes the A β 11-x polypeptide, which is detectable. Preferably, the coating antibody recognizes a human a β 11-x polypeptide and a full-length a β 40 or a β 42. In a more preferred embodiment, the coating antibody consists of the monoclonal antibody JRF/cA β 40/10 specifically recognizing A β 11-40 and full-length A β 40, said monoclonal antibody being characterized in that it comprises at least one heavy chain variable region having the amino acid sequence SEQ ID No.5 and/or at least one light chain variable region having the amino acid sequence SEQ ID No.6 (hereinafter monoclonal antibody JRF/cA β 40/10), or alternatively, the coating antibody consists of the monoclonal antibody JRF/cA β 42/12 specifically recognizing A β 11-42 and full-length A β 42, said monoclonal antibody being characterized in that it comprises at least one heavy chain variable region having the amino acid sequence SEQ ID No.7 and/or at least one light chain variable region having the amino acid sequence SEQ ID No.8 (hereinafter monoclonal antibody JRF/cA β 42/12), respectively in one preferred embodiment In one embodiment of (a), the second antibody is one of the monoclonal antibodies expressed by hybridoma cells J & JPRD/hA β 11/1 or J & JPRD/hA β 11/2, which cells were deposited at the Belgian collections of microorganisms on 8/19 2002 under the respective accession numbers LMBP5896CB and LMBP5897CB. In this embodiment, another second antibody that recognizes full-length A β 40 and A β 42, but is not cross-reactive with the A β 11-x polypeptide, is also used. Preferably, such second antibody, consisting of JRF/A β N25, characterized in that it comprises at least one heavy chain variable region having the amino acid sequence SEQ ID No.9 and/or at least one light chain variable region having the amino acid sequence SEQ ID No. 10. Accordingly, it is an object of the present invention to provide a sandwich assay in which the coating antibody consists of an antibody which specifically recognizes the A β 11-x polypeptide but which does not have cross-reactivity with the full-length A β 40 and A β 42 polypeptides, for example a monoclonal antibody expressed by hybridoma cells J & JPRD/hA β 11/1 or J & JPRD/hA β 11/2 which were deposited at the Belgian Collection of microorganisms on 8/19 of 2002 under the respective accession numbers LMBP5896CB and LMBP5897CB, and a second antibody which specifically recognizes A β 11-40 or A β 11-42, for example JRF/cA β 42/12 or JRF/cA β 40/10 having the aforementioned characteristics. In a particular embodiment, the coating antibody consists of J & JPRD/hA β 11/1 and the second antibody consists of JRF/cA β 42/26 that specifically recognizes A β 11-42 and full-length A β 42. the monoclonal antibody is characterized by comprising at least one heavy chain variable region having the amino acid sequence SEQ ID No.11 and/or at least one light chain variable region having the amino acid sequence SEQ ID No.12 (hereinafter referred to as monoclonal antibody JRF/cA β 42/26).

In an alternative sandwich assay for determining the ratio of A β 11-x polypeptide to full-length A β 40 or A β 42, the coating antibody consists of an antibody that specifically recognizes the A β 11-x polypeptide, preferably a human A β 11-x polypeptide, and a detectable second antibody specifically recognizes the polypeptide A β 11-40 or A β 11-42, preferably human A β 11-40 or human A β 11-42. in this alternative sandwich assay, the coating antibody consists of one of the monoclonal antibodies expressed by hybridoma cells J & JPRD/hA β 11/1 and J & JPRD/hA β 11/2, which were deposited at the Belgian micro-organism depositary Association on 8/19 of 2002 with respective accession numbers LMBP58 5896CB and LMBP58 5897CB. The second detectably labeled antibody consists of monoclonal antibody JRF/cA β 42/10 or monoclonal antibody JRF/cA β 40/12 having the foregoing characteristics.

The monoclonal antibody of the present invention can also be used in other detection systems than the sandwich method, for example, competitive methods and turbidimetric methods. In the competitive method, the antigen and the labeled immunogen in the test solution competitively react with the antibody. Subsequently, the labeled immunogen (B) bound to the antibody is separated from the unreacted labeled immunogen (F) (B/F separation). The amount of label in B or F is then determined to determine the amount of immunogen in the test solution. These reaction methods include a liquid phase method in which a soluble antibody is used as an antibody, and polyethylene glycol and a second antibody of the above antibody are used for B/F separation, and a solid phase method in which a solid phase antibody is used as a first antibody, or a soluble antibody is used as a first antibody and a solid phase antibody is used as a second antibody.

In the turbidimetric method, the amount of insoluble precipitated product resulting from the reaction of antigen-antibody in a gel or solution is measured. Even when the amount of antigen is small, only a small amount of precipitate is obtained, and is suitable for turbidimetry using laser light scattering.

In a further aspect, the invention relates to a method of treating or preventing a condition characterized by the formation of amyloid-beta containing plaques in a human, which comprises administering, preferably peripherally, to a human in need of such treatment a therapeutically or prophylactically effective amount of a humanized monoclonal antibody, or immunologically reactive fragment thereof, of the invention, wherein the antibody specifically binds to one or more epitopes present on the first 5 to 7 amino acids of the beta secretase _11 cleavage site of a human or mouse A β polypeptide. The invention relates to such humanized antibodies, including immunologically effective portions thereof, and methods for their preparation.

"humanized antibody" refers to an antibody that includes amino acid sequences partially or fully derived from the germline of a human antibody by altering the sequence of an antibody having non-human Complementarity Determining Regions (CDRs). "CDRs" are defined as the amino acid sequences of the complementarity determining regions of antibodies that are the highly variable regions of immunoglobulin heavy and light chains, see, e.g., Kabat et al, sequence of proteins of immunological interest, 4^thEd, U.S. department of Health and Human Services, National Institute of Health (1987). There are three heavy and three light chain CDRs (or CDRs regions) in the variable portion of an immunoglobulin. As used herein, "CDRs" refer to all three heavy chain CDRs or all three light chain CDRs (or both heavy and light chain CDRs, if desired).

The simplest such changes include replacement of the constant regions of a human antibody with murine constant regions, thereby forming a human/mouse chimera with low immunogenicity that is effectively suitable for medical use. The framework regions of the variable regions were replaced with the corresponding human framework regions, and the non-human CDRs were not processed or even replaced with sequences derived from the human genome. Fully human antibodies are prepared from genetically engineered mice whose immune systems have been altered to correspond to the human immune systems. As described above, the methods of the invention are useful for preparing immunospecific fragments of antibodies, including fragments in single chain form.

Humanized antibodies also refer to antibodies that comprise a human framework and at least one CDR from a non-human antibody, in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least about 85-90%, preferably at least 95% identical.

Humanized antibodies have at least three potential advantages when used in human therapy compared to non-human and chimeric antibodies:

1) since the effector moiety is human, it is able to interact better with other parts of the human immune system (e.g., more effectively destroying the target cell by complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ACDC)).

2) The human immune system does not recognize the framework or C region of the humanized antibody as exogenous, and therefore the antibody response against this injected antibody is less than against a fully exogenous non-human antibody or a partially exogenous chimeric antibody.

3) Injected non-human antibodies have been reported to have a much shorter half-life in the human circulation than human antibodies. The injected humanized antibody has substantially the same half-life as a naturally occurring human antibody and can be administered in smaller and less frequent doses.

In methods of treating and preventing conditions characterized by the formation of plaques containing β -amyloid, antibodies (including immunologically reactive fragments) are administered to subjects that are potentially or exhibit a β -related symptoms or pathological characteristics, such as clinical or potential alzheimer's disease, Down's syndrome, or clinical or potential amyloid angiopathy, using conventional administration techniques, preferably peripheral administration (i.e., not to the central nervous system) by intravenous, peritoneal, subcutaneous, pulmonary, transdermal, intramuscular, nasal, buccal, sublingual, or suppository administration, although the antibodies may be administered directly to the ventricles, spinal fluid, or brain parenchyma, techniques for localization to these sites are also well known in the art, but do not necessarily employ these more complex methods, when administered by simpler techniques relying on the peripheral circulatory system, the antibodies of the present invention are effective. In essence, it has been demonstrated herein that the amount of antibody that crosses the blood-brain barrier is at a plasma level of < 0.1%, the antibodies of the present invention have the ability to clear A β in peripheral circulation and are capable of altering the clearance of CNS and plasma soluble A β.

Pharmaceutical compositions are designed according to the mode of administration selected, using pharmaceutically acceptable excipients as required, such as dispersants, buffers, surfactants, preservatives, solvents, isotonicity agents, stabilizers and the like.

It is particularly useful to modify the solubility characteristics of the antibodies of the invention to render them more lipophilic, for example by encapsulating them in liposomes or by blocking polar groups.

Peripheral systems, such as intravenous, peritoneal or subcutaneous injections, are preferred. Formulations suitable for such administration are well known and typically include surfactants to facilitate transport across membranes. These surfactants are generally derived from steroids or are cationic lipids such as N- [1- (2, 3-dioleoyl) propyl ] -N, N-trimethylammonium chloride (DOTMA) or different compounds such as cholesterol hemisuccinate, phosphatidylglycerol, etc.

The concentration of the humanized antibody in the formulation is from as low as about 0.1% to as high as 15 or 20% by weight, and is selected primarily based on the volume of the liquid, viscosity, etc., particularly with the particular mode of administration selected. Thus, a typical injectable pharmaceutical composition will consist of 1mL of phosphate buffered saline in sterile buffered water and 1-100mg of a humanized antibody of the invention. A typical intravenous infusion composition has a fluid volume of 250mL, such as sterile Ringer's solution, and an antibody concentration of 1-100mg per mL or higher.

The therapeutic agents of the present invention can be stored frozen or lyophilized and formulated with a suitable sterile carrier prior to use. Lyophilization and formulation can result in varying degrees of loss of antibody activity (e.g., in conventional immunoglobulins, IgM antibodies will lose more activity than IgG antibodies). The pH of the formulation is selected to balance the stability (chemical and physical) of the antibody, making it comfortable for the patient to administer.

Typically a tolerable pH is between 4 and 8.

Although the foregoing method is most convenient and optimal for administration of proteins such as humanized antibodies, other techniques of administration, such as transdermal and oral administration, may be used with appropriate modification, so long as the formulation is appropriately designed.

Furthermore, biodegradable membranes and matrices, or micro osmotic pumps, or controlled release formulations based on dextran beads, alginate or collagen delivery systems may also be used to effect.

In summary, formulations useful for administration of the antibodies of the invention are well known in the art and may be selected in a variety of ways. Typical dosage levels can be optimized using routine clinical techniques and are also dependent upon the mode of administration and the physical condition of the patient.

The invention also provides a kit for use in the above method, which in one embodiment comprises in one or more containers an antibody of the invention, preferably a purified antibody, more preferably a monoclonal antibody, even more preferably an isolated monoclonal antibody expressed by hybridoma cells J & LPRD/hA β 11/1 and J & LPRD/hA β 11/2, which cells were deposited at the Belgian collections of microorganisms Association on 8/19 2002 under accession numbers LMBP5896CB and LMBP5897CB, respectively. In a particular embodiment, the kit of the invention comprises a substantially isolated polypeptide comprising an antigenic determinant specifically reactive with an antibody comprised in the kit. In further embodiments, the epitope is selected from the first 5-7 human amino acids of the beta-secretase 11 cleavage site as immunogens, i.e., EVHHQ-C (human A. beta. -11 (6AA) -Seq Id No.: 1) and EVHHQKI-C (human A. beta. -11 (8AA) -Seq Id No.: 2) or the first 5-7 mouse amino acids of the beta-secretase 11 cleavage site, i.e., EVRHQ-C (mouse A. beta. -11 (6AA) -Seq Id No.: 3) and EVRHQKL-C (mouse A. beta. -11 (8AA) -Seq Id No.: 4). Preferably the kit of the invention is used in a sandwich assay further comprising a coating antibody which does not specifically react with the polypeptide of interest. In a particular embodiment, the coating antibody recognizes an a β 11-x polypeptide and full-length a β 40 or a β 42, preferably the coating antibody recognizes a human a β 11-x polypeptide and full-length human a β 40 or a β 42, more preferably the coating antibody consists of a monoclonal antibody JRF/cA β 42/10 (previously characterized) that specifically recognizes a β 11-40 and full-length a β 42 or a monoclonal antibody JRF/cA β 42/12 (previously characterized) that specifically recognizes a β 11-42 and full-length a β 42. In an alternative sandwich assay of the invention, the kit comprises a coating antibody specifically recognizing an A β 11-x polypeptide, preferably a human A β 11-x polypeptide, and further comprises an antibody specifically recognizing the C-terminus of A β 40 or A β 42, preferably the C-terminus of human A β 40 or A β 42. in a more preferred case, the kit comprises as a coating antibody isolated monoclonal antibodies expressed by hybridoma cells J & LPRD/hA β 11/1 and J & LPRD/hA β 11/2, which cells were deposited at Belgian collections of microorganisms on 8/19 of 2002 under accession numbers LMBP5896CB and LMBP58 5897CB, respectively. Monoclonal antibody JRF/cA β 42/10 (defined herein before) and monoclonal antibody JRF/cA β 42/12 (defined herein before) were used as secondary antibodies, the latter being linked to a detectable label, a substrate.

In another particular embodiment, the kits of the invention include means for detecting binding of an antibody to a polypeptide of interest (e.g., the antibody may bind to a detectable substrate such as a fluorescent, enzymatic, radioactive, or luminescent compound and the second antibody that recognizes the first antibody may bind to a detectable substrate.) in particular, the kits include means for detecting binding of an antibody to an A β 11-x polypeptide, which means preferably detects binding to the first 5-7 human amino acids selected from the beta-secretase 11 cleavage site, i.e., EVHHQ-C (human A β 11(6AA) -Seq Idno.: 1) and EVHHQKI-C (human A β 11(8AA) -Seq Id No.: 2) or the first 5-7 mouse amino acids of the beta-secretase 11 cleavage site, i.e., EVRHQ-C (mouse A β 11(6AA) -Seq Id No.: 3) and RHQKL-C (mouse A β 11(6AA) -Seq Id No.: 3) L 11(8AA) -Seq IdNo.: 4) binds to an epitope of (a). In the aforementioned sandwich method, the antibody conjugated to the detectable substrate is not a coated antibody.

In further instances, the invention includes diagnostic kits for screening biological samples including tissues, body fluids such as CSF, blood, plasma, serum, urine, and the like. The biological sample comprises an A beta 11-x polypeptide, the diagnostic kit comprises a substantially isolated antibody specifically immunoreactive with the A beta 11-x polypeptide, particularly with the first 5-7 human amino acids having cleavage sites selected from the group consisting of beta-secretase 11, namely EVHHQ-C (human A. beta. -11 (6AA) -Seq Id No.: 1) and EVHHQKI-C (human A. beta. -11 (8AA) -Seq Id No.: 2) or the first 5-7 mouse amino acids of the cleavage site of beta-secretase 11, namely an antigenic determinant immune response in the group consisting of EVRHQ-C (mouse A.beta.11 (6AA) -Seq Id No.: 3) and EVRHQKL-C (mouse A.beta.11 (8AA) -Seq Id No.: 4), and means for detecting the binding of the antibody to the immunogen. In particular embodiments, the antibody may be a monoclonal antibody, particularly expressed by the hybridoma cells J & LPRD/hA β 11/1 and J & LPRD/hA β 11/2, deposited at the Belgian society for Collection of microorganisms at 19.8.2002 under the accession numbers LMBP5896CB and LMBP5897CB, respectively.

The detection means of the kit may comprise a labelled second monoclonal antibody, preferably the labelled second antibody comprises JRF/cA β 42/10 or JRF/cA β 42/12, wherein the aforementioned immobilised monoclonal antibody binds to JRF/cA β 40/10 which specifically recognizes A β 11-40 without cross-reacting with A β 1-40, and the aforementioned immobilised monoclonal antibody binds to JRF/cA β 42/12 which specifically recognizes A β 11-42 without cross-reacting with A β 1-42.

The solid surface agents used to adhere the proteinaceous material to the solid support in the above described detection methods are prepared according to well known techniques, such as polymer beads, dipsticks (dip sticks), 96-well plates, or filter materials.

The kit generally includes a support having bound to its surface an antibody of the invention, and a reporter-labeled antibody for detecting binding of the antibody to the immunogen.

The present invention will be better understood by reference to the details of the embodiments described below, but it is readily understood by those skilled in the art that these embodiments are illustrative of the invention which is more fully set forth in the claims which follow. The disclosures of these publications are hereby incorporated by reference into this application in order to fully describe the state of the art to which this invention pertains.

Examples

Feedstock and method

Preparation of monoclonal antibodies

Balb/c mice were immunized with four different polypeptides present in complete Freund's adjuvant the first two synthetic polypeptides included the first 5-7 human Amino Acids (AA) of the beta-secretase 11 cleavage site: EVHHQ (KI) -C (human A β _11(6 or 8 AA)). The other two polypeptides used for immunization comprise the mouse a β _11 amino acid sequence evrhq (kl) -C. All polypeptides were prepared using, for example, Pierce's Imject Maleimide mcKLH/BSA kit, following the manufacturer's instructions (Pierce, Rockford, IL) by coupling the polypeptide with Maleimide-activated mc (Megathurata) KLH, or Maleimide-activated bovine serum albumin using the COOH-terminal cysteine, two week weekly booster injections of 100. mu.g of KLH-coupled polypeptide were administered, the first present in complete Freund's adjuvant, and the latter in incomplete Freund's adjuvant.

The spleens of all mice were isolated, frozen in liquid nitrogen except for those immunized with the human A β _11(6AA) polypeptide. On the fourth day prior to fusion or spleen extraction, all mice were boosted intraperitoneally with 100 μ g of a β _11 polypeptide conjugated to mcKLH in saline. Mouse splenocytes were fused with SP2/0 cells according to the modified Kohler and Milstein (8) method. Hybridoma cells were cultured in 30X 96 well plates and screened 10 days later by direct ELISA using BSA-conjugated 6AA hA β _11 polypeptide and verified by unconjugated A β 11-40 polypeptide. Positive cells for the free hA β _11 polypeptide were immediately subcloned and positive clones were stored in liquid nitrogen.

All hybridoma cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, Europe), 2.5% ESG hybridoma cell supplement (Elscolab, Kruibeke, Belgium), 2% HT (Sigma, USA), 1mM sodium pyruvate, 2mM L-glutamic acid, penicillin (100U/ml) and streptomycin (50 mg/ml). All components were commercially available, purchased from Life-Technologies (Payseley, U.K.) cells were cultured in a humidified 8% CO2 air incubator.

Selection of ELISA antibodies

Screening ELISA for detection of anti-Abeta _11 antibodiesIs a direct ELISA in which 1. mu.g/ml of free human/mouse Abeta 11-40 or BSA-coupled human/mouse Abeta _11 is coated with 50. mu.l/well of coating buffer (10mM Tris, 10mM NaCl and 10mM NaN)₃Ph8.5) in U-shaped bottom high binding 96-well microtiter plates with nunc (life technologies) overnight at 4 ℃. The following day, the well plates were coated with 85 μ Ι/well of 0.1% casein PBS solution at 37 ℃ for 60 minutes to reduce non-specific binding. Then, 50. mu.l of hybridoma cell supernatant was added and cultured at 37 ℃ for 1 hour. After washing, bound monoclonal antibodies were detected with 50. mu.l/well of sheep anti-mouse Ig (Amersham-Pharmacia Biotech) coupled to horseradish peroxidase incubated for 1 hour at 37 ℃. All reagents were diluted with 0.1% casein/PBS. The well plate was washed and 50. mu.l of a solution containing 0.42mM of 3, 5, 3 ', 5' -tetramethylbenzidine and 0.003% (v/v) of H as a substrate was added₂O₂100mM citric acid solution, 100mM disodium hydrogen phosphate solution (pH 4.3). The reaction was carried out at room temperature on a well plate shaker for a maximum of 15 minutes. Then 50. mu.l/well of 2N H₂SO₄The color change was terminated and the plate was placed on a microdroplet plate reader (Thermomax, Molecular Device) and read at 450 nm. The selected monoclonal antibodies were tested for cross-reactivity with full-length human free A β 1-40 polypeptide by direct ELISA, which was identical to the screening assay except that full-length human free A β 1-40 polypeptide was used in place of the BSA-conjugated hA β _11(6AA) polypeptide. In a second validation ELISA, selected positive cultures were retested with free A.beta.11-40 polypeptide.

Sandwich ELISA for detection of amyloid beta

ELISA for detection of hA β (1-40) or hA β (11-40) in a standard dilution (American PeptideCooperation) was carried out as follows: briefly, monoclonal antibodies JRF/Abeta N/25, J at 5. mu.g/ml&JPRD/hA β 11/1 and J&JPRD/hA β 11/2 was coated overnight at 4 ℃ in NUNC flat-bottomed 96-well microtiter plates with 100. mu.l/well coating buffer. The following day, the plates were coated overnight at 37 ℃ with 125. mu.l/well of 0.1% casein PBS solutionWas incubated for 30 minutes to reduce non-specific binding and the sample was diluted with 100. mu.l/well of hA β (1-40) or hA β (11-40) polypeptide and incubated at 37 ℃ for 90 minutes. The well plates were washed and then incubated with 100. mu.l/well of HRP-labeled JRF/cA β 40/10-HRPO. The well plate was washed and 100. mu.l of a solution containing 0.42mM of 3, 5, 3 ', 5' -tetramethylbenzidine and 0.003% (v/v) of H as a substrate was added₂O₂100mM citric acid solution, 100mM disodium hydrogen phosphate solution (pH 4.3). The reaction was carried out on a well plate shaker at room temperature for a maximum of 15 minutes, followed by 50. mu.l/well of 2N H₂SO₄The color change was terminated and the plate was placed on a microtiter plate reader (Thermomax, Molecular Device) and read at 450 nm.

Immunoassay for APP CTF

For immunodetection of CTF (STUBS) fragments, HEK cells were stably transfected with human APPswe and human BACE1 at 75cm²Culture in culture flasks (Life, techniques, Paisly, u.k.) until confluent, then cells were digested and incubated in 50mM Tris: the crude digest was centrifuged at 10000g for 10min at 4 ℃ to remove nuclei and debris, pH 7.0, 0.15MNaCl, 1% Triton X-100 and the commercial protease inhibitor Cocktail (Roche, Boehringer Mannheim, Germany). The purified cell digests were normalized for protein content, samples were denatured in 2X tricine Laemmli buffer at 95 ℃ for 5 min, transferred to a previously prepared 10-20% Tristricine SDS gradient gel (NOVEX, Introvigen, Groningen, the Netherlands) at 1.5mA/cm²And semi-dried to 0.22 μm Hybond-ECL nylon membrane (APB) over 45 minutes.Small molecular weight protein gradients were used as molecular weight standards (Magicmark Western standard, Introvigen). The membranes were blocked with 10% (w/v) skim milk powder (BioRad) in PBS for 1 hour. This was then incubated with 5 μ g/ml of the appropriate monoclonal antibody at 4 ℃ overnight (monoclonal antibody C1/6.1 directed against the C-terminal epitope of APP, generously given from dr. mathews, Nathan, s. klinienstitute, Orangeburg). The membranes were then washed in 0.1% Tween20 in PBS for 5 minutes, with 5 changesAnd (4) a buffer solution. Goat anti-mouse coupled to HRP (Sigma) 1: 2000 dilutions were incubated at Room Temperature (RT) for 1 h after washing, the bands of interest were visualized by chemiluminescence according to the manufacturer's instructions (Roche, Boehringer Mannheim, Germany). The scans were performed using Lumi-Imager (Boehringer Mannheim, Germany).

Immunoassay for APP in brain sections of AD patients

Brain sections were blocked with 10% (w/v) skim milk powder (BioRad) in PBS for 1 hour. This was then incubated overnight at 4 ℃ with 5. mu.g/ml of the appropriate monoclonal antibody, then the membrane was washed in 0.1% Tween20 in PBS for 5 minutes, with 5 buffer changes, incubated with a dilution of HRP-conjugated goat anti-mouse (Sigma) 1: 2000 at Room Temperature (RT) for 1 hour after washing, the band of interest was visualized by chemiluminescence according to the manufacturer's instructions (Roche, Boehringer Mannheim, Germany).

Results and discussion

Selection of "fusion mice

Mice were injected with a panel of 4 different mcKLH-conjugated polypeptides. After the first booster, each mouse was bled, sera isolated and tested by ELISA using BSA directly coated human hA β (6 AA). The method of immunizing mice with hA β _11(6AA) was the same as for all injected mice, as shown in Table 1. In FIG. 1a, it is clearly demonstrated that mouse 1 immunized with KLH _ hA β _11(6AA) (SEQ ID No.1) shows very high serum titers for the free human A β 11-40 polypeptide, therefore, mouse 1 immunized with hA β _11(6AA) was selected for fusion.

Fusion of hA _ beta 11(6aa), spleen 1

The number of splenocytes in the hyperimmunized mice was large (6.5X 10 in total)⁸Splenocytes), two fusion processes were performed using half of the splenocytesThe culture medium containing the ESG was proliferated and ten days later, the cells were screened using a 30X 96 hybridoma well plate.

In these hybridomas, the first 65 culture wells showed a clear positive signal in the screening ELISA assay using BSA-conjugated polypeptides. Only 5 cultures were confirmed positive, or less than 10% of wells that initially showed positive, all of these cultures were negative for full-length human A β 1-40 and showed reactivity with AA present at the end of hA β 11-40/42.

The cultures were immediately cloned and the mother cultures were frozen. Of these 5, 2 hybridoma cells designated 29B5(J & JPRD/hA β 11/1) and 5C4(J & JPRD/hA β 11/2) were successfully cloned, frozen in liquid nitrogen.

TABLE 2

J&JPRD/hAβ11/1(29B5cl1F3)	J&JPRD/hAβ11/2(5C4cl3D6)
		J&JPRD/hAβ11/1(29B5cl2F5)	J&JPRD/hAβ11/2(5C4cl3F5)
J&JPRD/hA811/1(29B5cl4C1)	J&JPRD/hAβ11/2(5C4cl5B4)
		J&JPRD/hAβ11/1(29B5cl4D11)

Ass 1-40/42 and truncations in CSF samples from non-AD human controls, beagles and guinea pigs Short Abeta 11-40 determination

The ELISA for the determination of A.beta.1-40/42 and truncated A.beta.11-40 in CSF samples was performed as follows: briefly, monoclonal antibody J was added at 5. mu.g/ml&JPRD/hA β 11/1 or specific A β x-40 and A β x-42 monoclonal antibodies (Vandermeeren M. et al 2001; Pype S., et al 2003) JRF/cA β 40/10 and JRF/cA β 43/26 were coated overnight in NUNC flat bottom high binding microtitre 96-well plates with 100 μ l/well coating buffer. The following day, the plates were coated with 150. mu.l/well of 0.1% casein PBS overnight at 37 ℃ for 30 minutes to reduce non-specific binding, and incubated with 100. mu.l/well of PBS buffer diluted CSF sample at 37 ℃ for 90 minutes. Wash the well plate and then incubate with 100. mu.l/well HRP-labeled JRF/cA. beta.N/25-HRPO or JRF/cA. beta. 40/28-HRPO. the well plate is washed and 100. mu.l of a solution containing 0.42mM of 3, 5, 3 ', 5' -tetramethylbenzidine, 0.003% (v/v) H as the substrate is added₂O₂100mM citric acid solution, 100mM disodium hydrogen phosphate solution (pH 4.3). The reaction was carried out on a well plate shaker at room temperature for a maximum of 15 minutes, followed by 50. mu.l/well of 2N H₂SO4 stopped the color change and the plates were placed in a microtiter plate reader (Thermomax, molecular dynamics) and read at 450 nm.

Using the monoclonal antibodies of the invention, truncated 11-40 β amyloid isoforms can be quantitatively detected (ng/ml. + -. stdev) in CSF samples (n ═ 6) from non-AD human controls, beagle dogs and guinea pigs.

	Human ng/ml	Dog ng/ml	Guinea pig ng/ml
				Aβ1-40	5.70±0.63	5.61±0.35	5.94±0.42
Aβ11-40	0.20±0.04	0.30±0.34	0.36±0.05
				Aβ1-42	0.92±0.31	1.25±0.05	1.17±0.16

Conclusion

In a total number of hybridomas exceeding 30,000, we selected two different hybridoma cell clones that specifically recognized the free N-terminus of the human A β 11-40 polypeptide. These monoclonal antibodies are negative for full-length human A β 1-40. To evaluate the specificity of the antibodies, they were purified by protein G affinity chromatography for use in a sandwich ELISA with specificity against human cA β 40 and cA β 42 mAbs. JRF/Abeta N/25 as a monoclonal antibody specific for Abeta 1-40 and JRF/cA beta 40/10-HRPO were used together as a detection antibody, the latter specifically recognizing the C-terminal part of Abeta, and the corresponding antibodies JRF/cA beta N/25, J & JPRD/hA beta 11/1 and J & JPRD/hA beta 11/2, which specifically recognize the Abeta 11-x polypeptide, were used together as a detection antibody. FIG. 2A demonstrates that JRF/cA β N/25 specifically reacts with A β 1-40, and does not cross-react with A β 11-40 from FIGS. 2B and 2C, it can be seen that antibodies J & JPRD/hA β 11/1 and J & JPRD/hA β 11/2 specifically recognize hA β 11-40, and do not cross-react with human A β 1-40.

The ability of the antibodies of the invention to specifically label A β 11-x polypeptides in biological samples was verified by using membrane extracts of human APP and human BACE-1 stably transfected HEK cells (FIG. 3) and Western blots of brain sections of amyloid plaques in AD patients (Table 3). accordingly, the use of these antibodies and monoclonal antibodies specific for human cA β 40 and human cA β 42 in a sandwich ELISA resulted in a sensitive assay for the specific detection of human A β 11-x polypeptides in different biological samples including biological fluids and brain homogenates.

Reference to the literature

1.Jarrett，J.T.，Berger，E.P.，Lansbury，P.T.，The carboxy terminus of the beta amyloidprotein is critical for the seeding of amyloid formation：implications for thepathogenesia of Alzheimer′s disease.Biochem.32(1993)4693-4697.

2.Selkoe，DJ.，Alzheimer′s disease：genes，proteins，and therapy.Physiol.Rev.81(2001)：741-766

3.Gouras，G.K.，Xu，H.，Jovanovic，J.N.，Buxbaum，J.D.，Wang，R.，Relkin，N.R.，Gandy，S.，Generation and regulation of beta-amyloid peptide variants by neurons，J.Neurochem.，71(1998)1920-1925.

4.Wang，R.，Sweeney，D.，Gandy，S.E.，Sisodia，S.S.，The profile of soluble amyloidbeta protein in cultured cell media.Detection and quantification of amyloid beta proteinand variants by immunoprecipitation-mass spectrometry，J.Biol.Chem.，271(1996)31894-31902.

5.Vandermeeren，M.，Geraerts，M.，Pype，S.，Dillen，L.，Van Hove，C.，Mercken，M，The functional inhibitor DAPT prevents production of amyloidβ1-34in human andmurine cell lines.Neurosci.Lett.315(2001)145-148.

6.Naslund，J.，Schierhom，A.，Hellman，U.，Lannfelt，L.，Roses AD，Tjernberg，L.O.，Silberring，J.，Gandy，S.E.，Winblad，B.，Greengard，P.，Nordstedt，C.，Terenius，L.，Relative abundance of Alzheimer A beta amyloid peptide variants in Alzheimer diseaseand normal aging，Proc.Natl.Acad.Sci.U.S.A.，91(1994)8378-8382.

7.Iwatsubo，T.，Saido，T.C.，Mann D.M.，Lee，V.M.-Y.，Trojanowski，J.Q.Full-lengthamyloid-beta(1-42(43))and amino-terminally modified and truncated amyloid-beta42(43)deposit in diffuse plaques.Am.J.Pathol.149(1996)1823-1830.

8.Kohler，G.，Howe，S.C.，Milstein，C，.Fusion between immunoglobulin-secreting andnonsecreting myeloma cell lines.Eur J Immunol 6(1976)292-295.

9.Pype，S.，Moechars，D.，Dillen，L.，Mercken，M.，Characterization of amyloid betapeptides from brain extracts of transgenic mice overexpressing the London mutant ofhuman amyloid precursor protein，J.Neurochem.84(3)602-609.

TABLE 1

TABLE 3

Sequence listing

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<120> beta-amyloid monoclonal antibodies, compositions, methods and uses

<130>PRD 32

<150>PCT/EP02/11062

<151>2002-09-27

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Ser Thr Ser Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu

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Ala Asn Phe Lys Gly Arg Ala Thr Phe Thr Ala Asp Thr Ala Ser Asn

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<221>CDR2

<222>(60)..(67)

<223>

<220>

<221>CDR3

<222>(110)..(117)

<223>

<400>10

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Ile Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

35 40 45

Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser

50 55 60

Pro Lys Arg Trp Ile Tyr Asp Ser Ser Arg Leu Ala Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Gly Gly Ser Gly Thr Ser Tyr Ser Pro Thr Ile

85 90 95

Ser Asn Met Glu Ala Glu Asp Ala Ala Thr Tyr Phe Cys Gln Asn Trp

100 105 110

Arg Ser Ser Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

115 120 125

<210>11

<211>133

<212>PRT

<213> mice

<220>

<221>CDR1

<222>(50)..(54)

<223>

<220>

<221>CDR2

<222>(69)..(85)

<223>

<220>

<221>CDR3

<222>(118)..(122)

<223>

<400>11

Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly

1 5 10 15

Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Thr Ser Cys Lys Thr Ser Gly Tyr Ser Phe

35 40 45

Thr Glu Tyr Ile Met Ser Trp Val Arg Gln Ser His Gly Lys Ser Leu

50 55 60

Glu Trp Ile Gly Ser Ile Asn Pro Asn Thr Gly Gly Ser Arg Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr

115 120 125

Leu Thr Val Ser Ser

130

<210>12

<211>133

<212>PRT

<213> mice

<220>

<221>CDR1

<222>(44)..(59)

<223>

<220>

<221>CDR2

<222>(75)..(81)

<223>

<220>

<221>CDR3

<222>(114)..(122)

<223>

<400>12

Met Arg Phe Ser Ala Gln Leu Leu Gly Leu Leu Val Leu Trp Ile Pro

1 5 10 15

Gly Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro

20 25 30

Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Asn

35 40 45

Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Arg

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Ser Arg Val Ser Asn Leu Ala

65 70 75 80

Ser Gly Val Pro Asn Arg Phe Ser Gly Ser Glu Ser Gly Thr Asp Phe

85 90 95

Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Ala Gln Leu Leu Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys

115 120 125

Leu Glu Ile Lys Arg

130

Claims (10)

1. A monoclonal antibody expressed by hybridoma cells J & JPRD/hA β 11/1 or J & JPRD/hA β 11/2, which cells were deposited at the Belgian collections of microorganisms on 8/19 2002 with accession numbers LMBP5896CB or LMBP5897CB, respectively.

2. Hybridoma cells J & JPRD/hA β 11/1 or J & JPRD/hA β 11/2, deposited at the Belgian collections of microorganisms Association on 8/19 2002 under accession numbers LMBP5896CB or LMBP5897CB, respectively.

3. An immunoassay method for determining or detecting an a β 11-x polypeptide in a sample, the method comprising contacting the sample with an antibody according to claim 1, and determining whether an immune complex is formed between the antibody and the a β 11-x polypeptide.

4. A method for detecting the presence of a β 11-x polypeptide in a tissue sample, the method comprising:

contacting a tissue sample with an imaging effective amount of the antibody of claim 1; and

detecting the marker to determine the presence of the A β 11-x polypeptide in the tissue sample.

5. The method of claim 4, wherein the antibody is detectably labeled and expressed by at least one hybridoma cell of claim 2.

6. A method for detecting the presence of a β 11-x polypeptide in a body fluid sample, the method comprising:

contacting a sample of bodily fluid with an imaging effective amount of the antibody of claim 1; and

detecting the marker to determine the presence of the A β 11-x polypeptide in the body fluid sample.

7. The method of claim 6, wherein the antibody is detectably labeled and expressed by at least one hybridoma cell of claim 2.

8. Use of the antibody of claim 1 for the preparation of a composition for the diagnosis of a β -amyloid associated disease.

9. A diagnostic composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.

10. An immunoassay kit for diagnosing a beta amyloid-related disease comprising the antibody of claim 1 and a carrier means for the antibody.