HK1158946B - Diagnostic antibody assay - Google Patents

Description

Diagnostic antibody assay

The present invention relates to novel diagnostic assays for diagnosing amyloidosis, a group of disorders and abnormalities associated with amyloid protein, such as alzheimer's disease and related aspects. The invention provides, inter alia, antibody assays.

Amyloidosis is not a single disease entity, but a diverse group of progressive diseases characterized by extracellular tissue deposition of waxy amyloid protein called amyloid, which accumulates in one or more organs or body systems. As amyloid deposits accumulate, they begin to interfere with the normal function of the organ or body system. There are at least 15 different types of amyloidosis. The main forms are primary amyloidosis, without a known precedent, secondary amyloidosis following some other disorders, and hereditary amyloidosis.

Secondary amyloidosis occurs during chronic infections or inflammatory diseases such as tuberculosis, bacterial infections known as familial mediterranean fever, bone infections (osteomyelitis), rheumatoid arthritis, small bowel inflammation (granulomatous ileitis), hodgkin's disease and leprosy.

Amyloid deposits include the amyloid P (pentagon) component (AP), a glycoprotein associated with normal serum amyloid P (sap); and sulfated glycosaminoglycans (GAGs), which are complex carbohydrates of connective tissues. Amyloid fibrils comprise approximately 90% of amyloid material, and comprise one of several different types of proteins. These proteins are capable of folding into so-called "β -sheet" fibrils, a unique protein configuration that presents a congo red binding site that leads to the unique staining properties of amyloid proteins.

Many aging diseases are based on or associated with amyloid proteins and are characterized in part by the formation of extracellular accumulations of amyloid proteins or amyloid-like materials that contribute to pathogenesis and the progression of the disease. These include, but are not limited to, neurological disorders such as Mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD) such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome, lewy body dementia, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinson-dementia syndrome (guam parkinson-dementia complex). Other diseases based on or associated with amyloid are progressive supranuclear palsy, multiple sclerosis; creutzfeld jacobdisease, parkinson's disease, HIV-related dementia, ALS (amyotrophic lateral sclerosis), adult onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases, including macular degeneration.

Although the pathogenesis of these diseases may vary, their characteristic deposits often contain many common molecular components. This can be attributed in large part to the local activation of pro-inflammatory pathways, resulting in the simultaneous deposition of activated complement components, acute phase reactants, immune modulators and other inflammatory mediators (McGeeretal, TohokuJExpmed.174 (3): 269-277 (1994)).

Recently, there is increasing evidence that alzheimer's disease involves N-terminally modified a β peptide variants. Targeted biopsy revealed that a β 1-40 and a β 1-42 are present not only in the brain of alzheimer's patients, but also in senile plaques in unaffected individuals. However, N-terminally truncated and pyroglutamate modified a β N3pE-40/a β N3pE-42 are present almost exclusively in plaques in alzheimer's patients, making this a β variant a suitable diagnostic marker and a potential drug development target.

Currently, several commercial manufacturers provide ELISA kits that allow detection of the low picogram (pg) range of A β 1-40/1-42 and A β N3pE-40/A β N3 pE-42.

The morphological features of the brain of Alzheimer's Disease (AD) patients are the presence of neurofibrillary tangles and the deposition of A β peptides in the neocortical brain structure (Selkoe, D.J. & Schenk, D. Alzheimer's disease: molecular breakdown expressing prediction of cardiac-based hereophilics, Annu. Rev. Pharmacol. Toxicol.43, 545-584 (2003)). After successive cleavage by β -and γ -secretases, the a β peptide is released from Amyloid Precursor Protein (APP). Gamma-secretase cleavage results in the production of A beta 1-40 and A beta1-42 peptides which differ in their C-terminal and exhibit different aggregation, fibril formation and neurotoxicity efficacy (Shin, R.W. ethyl. amyloidodbeta-protein (Abeta)1-40 butnotbetta 1-42 conjugate of Alzheimer's disease and fibrilar breakdown, J.Neurosci.17, 8187 and msn 8193 (1997); Iwatsubo, T.ethyl. hyaluronic acid of Abeta42(43) and D.40 engineering and specific amyloid, and S.J.Neurosci.17, 8187 and msn.32. I.J.E.S. and S.E.S. Pat. No. 35 and S.E.S. No. 7 and S.S. 7. J.S.35 and S.S.7. J.S. 2. and S.S.7. J.S.J.S.A.No. 4. 7 and S.S.32. J.S.S.S.S.35, J.S.S.S.S.A.A.A.A.A.No. 7. No. 7. aberration and S.23. 7. No. 7. No. 7. aberration and S.S.23. No. 7. No. 7. 9. No. 7. No. 7. aberration et 35, S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.A.S.S.A.A.A.A.A.No. 4, et 35, S.A.A.A.No. 4, S.No. 4, et 35, et No. 7, et 35, No. 7, S.7, No. 7, No. 4, et 35, S.S.S.A.S.S.A.A.A.A.A.A.A.A.A.S.A.A.A.No. 4, No. 7, et 35, No. 7, et 35, et. In addition to C-terminal variability, N-terminally modified A.beta.peptides are abundant (Saido, T.C. et al. minor. introduction of peptides of beta-amyloid peptides, AbetaN3(pE), introduction of peptides of Neuron14, 457-. It appears that most of the A β peptides undergo N-terminal truncation by two amino acids, thereby exposing a glutamic acid residue, which is subsequently cyclized to pyroglutamic acid (pE), which results in the A β 3(pE) -42 peptide (Saido, T.C. et al. dominant and mutagenesis of cleavage-amyloidodepsipetides, AbetaN3(pE), endonexique. Neuron14, 457-466 (1995); Saido, T.C. Yamao, H.Iwatsubo, T.Ka& washima, S.Amino-and carboxy-terminatologenefbeta-amylodepetidepsipersidin hubrain. Neurosci. Lett.215, 173- (1996)). Alternatively, pE may be formed following β '-cleavage of BACE1, thereby producing A β N11(pE) -42(Naslund, J.et.Relativeaborland of Alzheimer's Absetaamyloylated mutated amino acids and amplified peptide derived peptide. In particular, A β N3(pE) -42 has been shown to be the major component of A β deposits in sporadic and Familial Alzheimer's Disease (FAD) (Saido, T.C. et al., dominant and discovery and diagnosis of beta-amyloid peptides, AbetaN3(pE), insidileplates. Neuron14, 457-466 (1995); Miravale, L.et al., amino-tert.

The A β N3pE-42 peptide coexists with the A β 1-40/1-42 peptide (Saido, T.C. et. dominant and transformed of diagnosis beta-amyloid peptides, AbetaN3pE, endogenephalas Neuron14, 457-466 (1995); Saido, T.C., Yamao, H., Iwatsubo, T. & Kawashima, S.Amino-and carboxy-termialterogeneogyfbeta-amyloid peptides, Neurosci. Lett.215, 173-176(1996)), and may play an important role in the pathogenesis of AD based on a number of observations. For example, specific neurotoxicity of A.beta.N 3pE-42 peptide (Russo, C.ethyl. heterologous-modifying gene associated beta-peptides- -AbetaN3(pE) -STRENGGLYCUTERDENDETHYLCYTESVIVAL.J.Neurochem.82, 1480-1489 (2002)) has been outlined, and pE-modification of N-truncated A.beta.peptide confers resistance to most aminopeptidases and A.beta.degrading endopeptidases (Russo, C.ethyl. heterologous-modifying gene associated beta-peptides- -AbetaN3(pE) -STRENGTHURETURETURETURETDENDE.J.Neurochem.82, the modification of Sainta.N 3-pE-42 peptide (Abetan-beta.10819, Abetamap-12, Abetan-beta.37) causes accelerated aggregation of the polypeptide, N-beta.37, Abetapeptide, Abetan-beta.10819, Abetatoxin, Abetane-encoding protein, Abetan-beta.12, Abetan-beta.10819, Abetatoxin, beta.7, Abetatoxin, beta.12, Abetatoxin, beta.12, Abetatoxin, beta.12, Abetatoxin, beta.12, Abetatoxin, beta.12, Abetatoxin, beta.12, the reduction in a β N3pE-42 formation should destabilize the peptides by making them more degradable and subsequently preventing the formation of higher molecular weight a β aggregates and enhancing neuronal survival.

However, it has not been known for a long time how pE-modification of a β peptides occurs. In recent years, glutaminyl cyclase (QC) has been shown to catalyze the formation of A.beta.N 3pE-42 under slightly acidic conditions, and specific QC inhibitors prevent A.beta.N 3pE-42 from being produced in vitro (Schilling, S., Hoffmann, T., Manhart, S., Hoffmann, M. & Demuth, H. -U.S.Glutamylcyclesns underlying glutamylcycleactional activity and minor comparisons, FEBSLett.191-196 (2004); Cynis, H.et. inhibiting of glutamylcyclassanlaceltylpropylamino chemotherapeutics amino acids, 2006. Biophys.Acta1624, 1618-1625 (2004)).

Lewy Body Dementia (LBD) is a neurodegenerative disorder that can occur in people over the age of 65 and often results in symptoms of cognitive (thought) disorders and abnormal behavioral changes. Symptoms may include cognitive disorders, neurological signs, sleep disorders, and autonomic failure. Cognitive disorders are in most cases a representative feature of LBD. Patients often develop a progressive and worsening mental disorder. Fluctuations in cognitive abilities are often associated with varying degrees of attention and alertness. Cognitive disorders and thought fluctuations may vary within minutes, hours, or days. Lewy forms autophosphorylated and unphosphorylated neurofilament proteins; they contain synaptoprotein alpha-synuclein as well as ubiquitin, which is involved in the clearance of damaged or abnormal proteins. In addition to lewy bodies, lewy neurites, which are inclusion bodies in the cellular process of nerve cells, may also be present. Amyloid plaques may form in the brain of DLB patients, however their numbers are lower than those observed in alzheimer patients. Another micropathological feature of AD neurofibrillary tangles are not a major feature of LBD, but are frequently present in addition to amyloid plaques.

Amyotrophic Lateral Sclerosis (ALS) is characterized by degeneration of both upper and lower motor neurons. In some ALS patients, dementia or aphasia (ALS-D) may be present. Dementia is the most common frontotemporal dementia (FTD), and many of these cases have ubiquitin-positive, tau-negative inclusions in the dentate gyrus and superficial neurons of the frontal and temporal lobes.

Inclusion Body Myositis (IBM) is a disabling disease, commonly found in people over the age of 50, where muscle fibers are inflamed and begin to atrophy, but the brain is unaffected and the patient retains his full intelligence. Two enzymes involved in amyloid beta production were found to be increased in the myocytes of this elderly most commonly seen in patients with advanced myopathy, where amyloid-beta was also increased.

Another disease that is based on or associated with the accumulation and deposition of amyloid is macular degeneration. Macular degeneration is a common eye disease that results in macular degeneration in the central region of the retina (the very thin tissue behind the eye in which light-sensitive cells transmit visual signals to the brain). Acute, clear, "straight" vision results from macular processing. Macular damage results in blind spots and blurred or distorted vision. Age-related macular degeneration (AMD) is a major cause of vision impairment in the united states and is the leading cause of legal blindness in caucasians over the age of 65. Americans aged about 180 million and 40 years and older suffer from advanced AMD, and an additional 730 million people who suffer from intermediate AMD are at risk for vision loss. The government estimates that 290 thousands of people will have advanced AMD by the year 2020. Little understanding and treatment of the causes of this blindness condition in AMD patients has often been surprisingly and frustratingly discovered.

There are two forms of macular degeneration: dry macular degeneration and wet macular degeneration. The dry form in which cells of the macula slowly begin to break accounts for 85% of cases of macular degeneration diagnosed. Both eyes are usually affected by dry AMD, but one eye may lose vision while the other remains unaffected. Drusen are yellow deposits under the retina, which are usually early signs of dry AMD. The risk of developing advanced dry AMD or wet AMD increases with the number or size of drusen. Dry AMD progresses and results in loss of vision without becoming the wet form of the disease; however, it is also possible that early dry AMD rapidly changes to the wet form.

Wet form AMD, although accounting for only 15% of cases, causes blindness in 90% of patients and is considered advanced AMD (no early and intermediate stage wet AMD). The dry form always precedes wet AMD. As the dry form worsens, some people begin to have abnormal blood vessel growth behind the macula. These blood vessels are very fragile and can leak fluid and blood (thus "wet" macular degeneration) leading to rapid damage of the macula.

The dry form of AMD initially usually results in slight blurring of vision. In particular, the center of vision can become blurred and this area gradually enlarges as the disease progresses. If only one eye is affected, there are no noticeable symptoms. In wet AMD, straight lines can appear wavy and central vision loss can occur rapidly.

Diagnosis of macular degeneration typically involves mydriatic fundus examination, visual acuity testing and examination of the back of the eye using a procedure known as fundus examination to aid in the diagnosis of AMD, and if wet AMD is suspected, fluorescein angiography may also be performed. If dry AMD reaches an advanced stage, there is currently no treatment to prevent vision loss. However, particular high dose antioxidant and zinc formulations may retard or prevent the progression of intermediate AMD to the advanced stage. Macugen(pegaptanib sodium) injections, laser photocoagulation and photodynamic therapy can control abnormal blood vessel growth and bleeding in the macula, which is helpful in some people with wet AMD; however, vision that has been lost cannot be recovered by these techniques. Existing low vision aids can help improve quality of life if vision has been lost.

One of the earliest signs of age-related macular degeneration (AMD) is the accumulation of extracellular deposits called drusen between the basal layer of the Retinal Pigment Epithelium (RPE) and Bruch's Membrane (BM). Recent studies by Anderson et al have demonstrated that drusen contain amyloid beta (Experimental eye research78(2004)243- & 256).

The aim of the present invention is to establish a highly sensitive and at the same time robust (robust) detection technique which allows quantitative determination of a β variants, in particular pGlu-a β peptides, in biological samples such as liquid or serum samples, preferably serum samples. This is a great challenge given the low abundance of a β peptide in blood. However, having such detection techniques is a prerequisite for studying the efficacy of small molecule inhibitors in drug screening programs.

The present invention provides novel methods and compositions comprising highly specific and highly effective antibodies, including chimeric antibodies and fragments thereof, including partially or fully humanized antibodies and fragments thereof, having the ability to specifically recognize and bind specific epitopes of a range of beta-amyloid antigens, particularly pGlu-a β peptide, which may be presented to the antibodies in monomeric, dimeric, trimeric, etc. forms or in polymeric forms, aggregates (aggregates), fibers, filaments, or concentrated forms of plaques. The antibodies taught by the present invention are particularly useful for diagnosing amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis including, but not limited to, neurological disorders such as Alzheimer's Disease (AD), lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); parkinson-dementia complex of guam; and other diseases based on or associated with amyloid proteins, such as progressive supranuclear palsy, multiple sclerosis; creutzfeldt-jakob disease, hereditary cerebral hemorrhage with amyloidosis (Dutch type), parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases, including macular degeneration and the like.

In order to meet all the above requirements, techniques based on ELISA are particularly preferred. The study was started using a β N3pEELISA, as for this a β variant, an ELISA system (humanamyl β (N3pE) AssayKit-IBL, codeno.27716) is already commercially available, which is used as reference and internal quality control. Capture of a β N3pE-40 peptide was performed using TGC hA β (x-40) elisa (hs) (the genetics company, inc., wagastrase 23, 8952Schlieren, zurich switzerland), which should facilitate and accelerate the development process.

Summary of The Invention

The invention relates in particular to antibodies or variants thereof, characterized in that they bind to the A β -peptide with high affinity_DValue of 10^-7M or better, preferably K_DValue of 10^-8M or better, even more preferably K_DValue of 10^-9M-10^-12M。

In particular, the antibody is preferably a monoclonal antibody and is selected from the group consisting of:

Aβ5-5-6

Aβ6-1-6

Aβ17-4-3

Aβ24-2-3

the antibodies of the invention are particularly useful in diagnostic methods for detecting amyloidosis, particularly Alzheimer's disease.

Description of the drawings

FIG. 1 shows a schematic view of a

A) Detection of 10ng/ml amyloid by increasing the concentration of pGlu-6166 antibody (clone 12-1)βN3pE-40。

B) Determining and detecting 10ng/ml amyloid eggβ N3pE-40 required the highest concentration of pGlu-6166 antibody (clone 12-1).

FIG. 2

Dot blot analysis of hybridoma cell culture supernatants of individual IgG-producing clones.

FIG. 3

PepSpot analysis of pGlu-6166 hybridoma cell clone and IBL-A β N3pE antibody.

FIG. 4

20 μ g of pGlu-6166 antibody and 12% SDS-PAGE of hybridoma cell culture supernatant.

FIG. 5

Biacore analysis of hybridoma cell culture supernatants. A superposition of the monitored binding processes is illustrated.

FIG. 6

Sensorgram of the interaction of anti-A.beta.N 3pE antibody clone 6-1-6 with A.beta.pE 3-40.

FIG. 7

Sensorgram of the interaction of anti-A.beta.N 3pE antibody clone 24-2-3 with A.beta.pE 3-40.

FIG. 8

Clones 6-1-6 were subjected to N3pE-ELISA, a standard curve for A β pE 3-40.

FIG. 9

Sensorgram for N3pE antibody clone 6-1-6.

FIG. 10 shows a schematic view of a

A β pE3-42 quantification using the following method: the resulting solution was diluted 1: 20 in EIA buffer, and neutralized by pH titration with 860. mu.l of 3.5 MTris.

FIG. 11

Stained brain sections from patients with Alzheimer's Disease (AD).

(A) Sporadic AD (SAD) patient brains stained with anti-A β antibody 6E10 recognizing total A β,

(B) sporadic AD (SAD) patient brains stained with N3pE antibody clone 24-2-3 recognizing A β pE3-x,

(C) brains of Familial AD (FAD) patients stained with N3pE antibody clone 24-2-3 recognizing A β pE 3-x.

Detailed Description

Definition of

The term "antibody" is used in its broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The antibody may be an IgM, an IgG (e.g. IgG1, IgG2, IgG3 or IgG4), an IgD, an IgA or an IgE. Preferably, however, the antibody is not an IgM antibody.

An "antibody fragment" comprises a portion of an intact antibody, typically the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments: diabodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the population comprises individual antibodies that are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to "polyclonal antibody" preparations of different antibodies, which typically contain different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are often advantageous in that they are synthesized by hybridoma culture, uncontaminated by other immunoglobulins. "monoclonal" refers to the property of an antibody that results from a substantially homogeneous anti-bodyA population is not to be understood as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in the present invention can be prepared by first preparing a monoclonal antibody fromet al, Nature, 256: 495(1975), or can be produced by a generally known recombinant DNA method. "monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the following methods such as Clacksonet, Nature, 352: 624-: 581-597 (1991).

Monoclonal antibodies herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, so long as they exhibit the desired biological activity.

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., Fv, Fab ', F (ab') 2, or other antigen-binding subsequences of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. The majority of humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the acceptor antibody and the introduced CDR or framework sequences.

These modifications were made to further refine and optimize the performance of the antibody. Typically, the humanized antibody comprises substantially all of at least one, and typically two, variable domains thereofWherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. Most preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see joneset al, Nature, 321: 522-525(1986), Reichmann et al, Nature.332: 323-329(1988) and Presta, curr. op. struct. biel, 2: 593-596(1992). Humanized antibodies include Primatized^TMAn antibody, wherein the antigen binding region of the antibody is derived from an antibody produced by immunizing a rhesus monkey with an antigen of interest.

"Single chain Fv" or "sFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises V_HAnd V_LPolypeptide linkers (linkers) between the domains, which allow sFv formation of the desired structure for antigen binding. For an overview of sFv see Pluckthunin the Pharmacology of monoclonal antibodies, vol.113, Rosenburg and Mooreeds, Springer-Verlag, New York, pp.269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable domain (V) in the same polypeptide chain as the light chain_D) Linked heavy chain variable domains (V)_H)(V_H-V_D). By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with the complementary domains of the other strand and two antigen binding sites are created. Diabodies are described in hollingerial, proc.natl.acad.sol.usa, 90: 6444-.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are materials that interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is purified as: (1) greater than 95% by weight of antibody, most preferably greater than 99% by weight, as determined by the Lowry method; (2) to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spin cup sequencer; or (3) homogeneity of purification by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Isolated antibodies include recombinant intracellular in situ antibodies because at least one component of the antibody's natural environment is not present. Typically, however, the isolated antibody is prepared by at least one purification step.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all of these designations include progeny. Thus, the words "transformant" and "transformed cell" include the original subject cell and cultures derived therefrom regardless of the number transferred. It is also understood that the DNA content of all progeny may not be identical due to deliberate or inadvertent mutations. The invention includes mutant progeny that are screened for the same function or biological activity as the originally transformed cell. In the case of using different names, it is clear from the context.

As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a biomolecule composed of amino acids linked by peptide bonds.

As used herein, the terms "a", "an" and "the" mean "one or more" and include the plural unless the context does not apply.

The expression "diseases and disorders caused by or associated with amyloid or amyloid-like proteins" includes, but is not limited to, diseases and disorders caused by the presence or activity of amyloid in monomeric, fibrillar or multimeric states or any combination of these. Such diseases and disorders include, but are not limited to, amyloidosis, endocrine tumors, and macular degeneration.

The term "amyloidosis" refers to a group of diseases and disorders associated with amyloid plaque formation including, but not limited to, secondary amyloidosis and age-related amyloidosis, e.g., diseases including, but not limited to, neurological disorders such as Alzheimer's Disease (AD), including diseases or conditions characterized by loss of cognitive memory capacity (conditions), such as Mild Cognitive Impairment (MCI), sporadic alzheimer's disease, lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); guam parkinsonism-dementia syndrome, familial forms of alzheimer's disease such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD); and other diseases based on or associated with amyloid proteins, such as progressive supranuclear palsy, multiple sclerosis; creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), Inclusion Body Myositis (IBM), adult onset diabetes and senile cardiac amyloidosis; and various ocular diseases including macular degeneration, optic neuropathy associated with drusen, and cataracts due to beta-amyloid deposition.

"amyloid β, A β, or/β -amyloid" is a term recognized in the art and refers to amyloid β protein and peptide, amyloid β precursor protein (APP) and modifications, fragments, and any functional equivalents thereof specifically, as used herein, amyloid β refers to any fragment produced by the proteolytic cleavage of APP, but specifically those fragments involved in or associated with amyloid pathology, including but not limited to A β_1-38、Aβ_1-40、Aβ_1-42The amino acid sequence of these a β peptides is shown below:

Aβ1-42(SEQIDNO.1)：

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala

Aβ1-40(SEQIDNO.2)：

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val

Aβ1-38(SEQIDNO.3)：

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly

"pGlu-A β" or "A β N3 pE" refers to an N-terminally truncated form of A β starting at the glutamic acid residue at position 3 of the amino acid sequence of A β, wherein said glutamic acid residue is cyclized to form a pyroglutamic acid residue_3-38、pGlu-Aβ_3-40、p-Glu-Aβ_3-42。

N-terminally truncated forms of A β, A β_3-38、Aβ_3-40、Aβ_3-42The sequence of (a) is as follows:

Aβ3-42(SEQIDNO.4)：

Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala

Aβ3-40(SEQIDNO.5)：

Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-LeuMet-Val-Gly-Gly-Val-Val

Aβ3-38(SEQIDNO.6)：

Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly

in particular, the invention relates to the following:

1. antibody, characterized in that it binds, preferably with high affinity, to the a β peptide or variant thereof.

2. The antibody of item 1, wherein the high affinity refers to dissociation constant (K)_D) Is 10^-7M or better.

3. The antibody of item 1 or 2, wherein the antibody is a monoclonal antibody.

4. The antibody of any one of the preceding, wherein the variable portion of the light chain of said antibody has an amino acid sequence selected from the group consisting of seq id nos: 49. 53, 57 and 61 or a nucleotide sequence selected from seq id no: 50. 54, 58 and 62.

5. The antibody of any one of the preceding, wherein the variable portion of the heavy chain of said antibody has an amino acid sequence selected from the group consisting of seq id nos: 51. 55, 59 and 63 or a nucleotide sequence selected from seq id no: 52. 56, 60 and 64.

6. The antibody of any one of the preceding, wherein the variable portion of the light chain of the antibody has the amino acid sequence of seq id no: 49 or the nucleotide sequence of seq id no: 50, and wherein the variable portion of the heavy chain of said antibody has the amino acid sequence of seq id no: 51 or the nucleotide sequence of seq id no: 52.

7. The antibody of any one of the preceding, wherein the variable portion of the light chain of the antibody has the amino acid sequence of seq id no:53 or the nucleotide sequence of seq id no:54, and wherein the variable portion of the heavy chain of said antibody has the amino acid sequence of seq id no:55 or the nucleotide sequence of seq id no: 56.

8. The antibody of any one of the preceding, wherein the variable portion of the light chain of the antibody has the amino acid sequence of seq id no: 57 or the nucleotide sequence of seq id no: 58, and wherein the variable portion of the heavy chain of said antibody has the amino acid sequence of seq id no: 59 or the nucleotide sequence of SEQ ID NO: 60.

9. The antibody of any one of the preceding, wherein the variable portion of the light chain of the antibody has the amino acid sequence of seq id no: 61 or the nucleotide sequence of seq id no: 62, and wherein the variable portion of the heavy chain of said antibody has the amino acid sequence of seq id no: 63 or the nucleotide sequence of seq id no: 64.

10. The antibody of any one of the preceding claims, wherein the antibody is selected from the group consisting of:

abeta 5-5-6 (accession number DSMACC2923)

Abeta 6-1-6 (accession number DSMACC2924)

Abeta 17-4-3 (accession number DSMACC2925)

Abeta 24-2-3 (accession number DSMACC 2926).

11. The antibody of any one of the preceding, wherein the antibody is a β 6-1-6 (accession number DSMACC 2924).

12. The antibody of any one of the preceding, wherein the antibody is a β 24-2-3 (accession number DSMACC 2926).

13. The antibody of any one of the preceding, wherein the antibody is a humanized or chimeric antibody or antibody fragment that retains high affinity.

14. An antibody of any one of the preceding claims for use in the detection of a β peptide or a variant thereof.

15. The antibody of item 14, wherein the variant is selected from the group consisting of:

pGlu-Aβ_3-38

pGlu-Aβ_3-40

pGlu-Aβ_3-42and are and

pGlu-Aβ_3-xin a variant thereof,

wherein x is an integer from 10 to 42, preferably from 18 to 42, more preferably from 30 to 42.

16. The antibody of any one of the preceding claims, which is human.

17. The antibody of any one of the preceding, which is a double or single chain antibody retaining high affinity.

18. An antibody of any one of the preceding claims which binds to an epitope bound by an antibody as defined in claim 15.

19. An antibody according to any one of the preceding claims, which has the complementarity determining regions of an antibody as defined in claim 15.

20. The antibody of any one of the preceding claims, which is labeled.

21. The antibody of any one of the preceding claims, which is immobilized on a solid phase.

22. An antibody obtainable from any one of the hybridoma cell lines DSMACC2923, DSMACC2924, DSMACC2925, DSMACC 2926.

23. A composition comprising the antibody of any one of the preceding.

24. The composition of item 23 for use in treating, preventing or delaying amyloidosis.

25. The composition of claim 23 or 24, wherein the amyloidosis is a neurodegenerative disease selected from the group consisting of mild cognitive impairment, alzheimer's disease and neurodegeneration in down syndrome.

26. The composition of claim 23 or 24, wherein said amyloidosis is sporadic alzheimer's disease or familial alzheimer's dementia.

27. The composition of item 26, wherein said familial alzheimer's dementia is familial british dementia or familial danish dementia.

28. Hybridoma cell line DSMACC 2923.

29. Hybridoma cell line DSMACC 2924.

30. Hybridoma cell line DSMACC 2925.

31. Hybridoma cell line DSMACC 2926.

32. Use of an antibody as defined in any one of claims 1 to 22 or a composition as defined in any one of claims 23 to 27 in a method of diagnosis or treatment.

33. The use of item 32 for diagnosing an amyloidosis-associated disease or condition.

34. The use of item 33, wherein the amyloidosis is a neurodegenerative disease selected from the group consisting of mild cognitive impairment, alzheimer's disease, and down's syndrome.

35. The use of item 33, wherein said amyloidosis is sporadic alzheimer's disease or familial alzheimer's dementia.

36. The use of item 35, wherein said familial alzheimer's dementia is familial british dementia or familial danish dementia.

37. An in vitro diagnostic method for diagnosing an amyloidosis-associated disease or condition, in particular alzheimer's disease, comprising the steps of:

contacting the antibody of any one of items 1 to 22 with a sample of an individual suspected of having the disease or disease condition, and

detecting binding of said antibody to pGlu-amyloid, preferably pGlu-A β peptide, from said sample.

38. A diagnostic kit comprising an antibody as defined in any one of claims 1 to 22 and instructions for use, and optionally other biologically active substances.

39. The diagnostic kit of item 32, wherein said other biological substance is a glutaminyl cyclase inhibitor.

40. An oligonucleotide selected from the group consisting of SEQ ID NO: 23-48.

The antibodies of the invention are useful for diagnosing amyloidosis.

The antibodies of the invention are useful as affinity purifiers. In this method, the antibody is immobilized on a solid phase such as Sephadex resin or filter paper using methods known in the art. Contacting the immobilized antibody with a sample containing the a β -peptide (or fragment thereof) to be purified, and then washing the support with a suitable solvent will remove substantially all material from the sample except the a β -peptide bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer at ph5.0, which releases the a β peptide from the antibody.

anti-A.beta.peptide antibodies may also be used in diagnostic assays for A.beta.peptide, for example to detect its presence in a particular cell, tissue or serum. Thus, the antibodies are useful in the diagnosis of amyloidosis, in particular neurodegenerative diseases selected from: mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD), such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), and neurodegeneration in down's syndrome; preferably Alzheimer's disease.

For diagnostic applications, the antibody is typically labeled with a detectable moiety. A number of markers can be used, which can be generally divided into the following groups:

(a) radioisotopes, e.g.³⁵S、¹⁴C、¹²⁵I、³H and¹³¹I. the antibodies can be labeled with a radioisotope using techniques such as those described in Current protocols immunology, Volumes1and2, G ü tigenetional, Ed., Wiley-Interscience, New York, Pubs, (1991), and radioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth metal chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine (Lissamine), phycoerythrin and texas red (TexasRed) may be used. Fluorescent labels can be coupled to antibodies using, for example, the techniques disclosed in currentprotocol immunology, supra. Fluorescence can be quantified using a fluorometer.

(c) Various enzyme-substrate labels may be utilized. Enzymes typically catalyze chemical alteration of chromogenic substrates, which can be measured using various techniques. For example, the enzyme may catalyze a substrate color change, which can be measured by a spectrophotometer. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying fluorescence changes are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and can then emit measurable light (as measured using a chemiluminometer) or deliver energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferases; U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydro-2, 3-naphthyridine dione (2, 3-dihydrophthalazinedione), malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, 0-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., urate oxidase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for coupling enzymes to antibodies are described in O' solvanet, methods for the preparation of enzymes-antibodies conjugates for use of enzymes elmunamenmoassay, inmethodsinenzyme (edLangone & h. vanvunaks), AcademicPress, new york, 73: 147, 166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) horseradish peroxidase (HRPO) with catalase as a substrate, wherein the catalase oxidizes a dye precursor (e.g., o-phenylenediamine (OPD) or 3, 3 ', 5, 5' -tetramethylbenzidine hydrochloride (TMB));

(ii) alkaline Phosphatase (AP) with p-nitrophenyl phosphate as chromogenic substrate; and

(iii) beta-D-galactosidase (. beta. -D-Gal) with either a chromogenic substrate (e.g.p-nitrophenyl-. beta. -D-galactosidase) or a fluorogenic substrate 4-methylumbelliferyl-. beta. -D-galactosidase (4-methylumbellifery 1-. beta. -D-galactosidase).

Many other enzyme-substrate combinations are available to those skilled in the art.

Sometimes, the label is indirectly coupled to the antibody. The skilled person will be aware of various techniques for achieving this. For example, an antibody may be conjugated to biotin, and any of the three broad classes of labels described above may be conjugated to avidin, or vice versa. Biotin binds selectively to avidin and thus the label can be coupled to the antibody in this indirect manner. Alternatively, to achieve indirect coupling of the label to the antibody, the antibody is coupled to a small hapten (e.g., digoxigenin) and one of the different types of labels described above is coupled to an anti-hapten antibody (e.g., anti-digoxigenin). Thus, indirect coupling of the label to the antibody can be achieved.

The a β -antibody does not require a label, and its presence can be detected using a labeled antibody that binds to the a β -antibody.

The antibodies of the invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, MonoclonaLthiodiethaneofatechniques, pp.147-158(CRCPress. Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding to a limited amount of antibody. The amount of a β -peptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibody. To facilitate determination of the amount of standard that becomes bound, the antibody is generally insoluble before or after competition, whereby standards and analytes bound to the antibody can be conveniently separated from standards and analytes that remain unbound.

Sandwich assays involve the use of two antibodies, each antibody being capable of binding to a different immunogenic portion or epitope of the protein to be detected. In a sandwich assay, a test sample analyte is bound by a first antibody immobilized on a solid support, and then a second antibody binds to the analyte, thus forming an insoluble three-part complex. The second antibody may itself be labeled with a detectable moiety (direct sandwich assay) or may be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one preferred type of sandwich assay is an ELISA assay, wherein the detectable moiety is an enzyme.

For immunohistochemistry, the tissue sample may be fresh or frozen, or may be embedded in paraffin and fixed with a preservative such as formalin.

Diagnostic kit

Conveniently, the antibodies of the invention may be provided in a kit, i.e. a combination of a predetermined amount of reagents and a package of instructions for performing a diagnostic assay. In the case of an antibody labeled with an enzyme, the kit contains the substrate and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents may be wide to provide concentrations of reagent solutions that substantially optimize assay sensitivity. In particular, the agent may be provided as a dry powder, typically a lyophilized powder, comprising excipients which, when dissolved, provide a solution of the agent with a suitable concentration.

The diagnostic kit of the present invention may contain other biologically active substances as described below. Particularly preferred for use in the diagnostic kit is a glutaminyl cyclase inhibitor.

The diagnostic kit of the invention may be particularly useful for the detection and diagnosis of diseases and conditions associated with amyloidosis, in particular neurodegenerative diseases selected from the group consisting of: mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD), such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome; preferably Alzheimer's disease.

The invention relates in particular to antibodies characterized by binding with high affinity to the A β -peptide, to antibodies characterized by binding with high affinity to the A β -peptide or a variant thereof_DValue of 10^-7M or better affinity, preferably K_DValue of 10^-8M or better, even more preferably K_DValue of 10^-9M-10^-12Therefore, the antibodies of the invention bind to the a β -peptide with higher affinity than previously known antibodies.

In particular, the antibody is preferably a monoclonal antibody and is selected from the group consisting of:

Aβ5-5-6(DSMACC2923)

Aβ6-1-6(DSMACC2924)

Aβ17-4-3(DSMACC2925)

Aβ24-2-3(DSMACC2926)

the antibodies of the invention are particularly useful in diagnostic methods to detect amyloidosis, particularly a neurodegenerative disease selected from the group consisting of: mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD), such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome; preferably Alzheimer's disease.

According to a preferred embodiment, the antibody may be a humanized or chimeric antibody or a human antibody.

Furthermore, an antibody selected from the above group may also be a functional variant of said group.

In the present invention, the variant of the p-Glu-A β -peptide is in particular:

pGlu-Aβ_3-38，

pGlu-Aβ_3-40，

pGlu-Aβ_3-42

other variants of the A β -peptide are all pGlu-A β_3-xVariants which have been shown to accumulate in the brain as a result of or prior to Alzheimer's disease x is defined as an integer from 10 to 42, for example in pGlu-A β above_3-42In "42" is an integer represented by x.

In the context of the present invention, a "functional variant" of an antibody of the invention is an antibody or a functional variant thereof which retains the binding capacity, in particular with pGlu-A β_3-xThe ability of peptides to bind with high affinity. In the field ofDomains are known for the conditions of such functional variants and include the above possibilities, which are expressed under the definition of antibodies and fragments thereof.

In a preferred embodiment, the antibody is an antibody fragment as defined above.

In another preferred embodiment, the antibody of the invention is an antibody that binds to an epitope bound by an antibody as defined above, in particular antibody 5-5-6, antibody 6-1-6, antibody 17-4-3 and antibody 24-2-3.

In another preferred embodiment, the antibody of the invention is an antibody having the Complementarity Determining Regions (CDRs) of the antibody as defined above. Preferably, the antibody may be labeled; possible markers are, in particular, those mentioned above and all markers known to the person skilled in the art for diagnostic use of antibodies.

Preferably, the antibody is immobilized on a solid phase.

The invention also relates to antibodies obtainable from the hybridoma cell line 6-1-6(DSMAC 2924).

The invention also relates to compositions comprising the antibodies defined above. In particular, the composition is a composition for use in diagnosis, in particular for use in diagnosis of a neurodegenerative disease selected from Mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD), such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome; preferably alzheimer's disease; in particular by detecting A.beta.peptide or a variant thereof in a biological sample.

In another embodiment, the antibody or fragment thereof of the invention and described herein before exhibits a binding affinity for a β oligomers, fibers, fibrils or filaments which is at least 2 times, in particular at least 4 times, in particular at least 10 times, in particular at least 15 times, more in particular at least 20 times, but especially at least 25 times higher than the binding affinity of a β monomers.

In still another embodiment, there is provided an antibody or fragment thereof as described herein before, or a chimeric antibody or fragment, or a humanized antibody or fragment thereof, which antibody binds well to aggregated a β, including a β plaques, in mammals, particularly in the human brain, but preferably does not exhibit any significant cross-reactivity with Amyloid Precursor Protein (APP).

In another aspect of the invention there is provided an antibody or fragment thereof as hereinbefore described, or a chimeric antibody or fragment thereof, or a humanized antibody or fragment thereof, which antibody substantially binds soluble polymeric amyloid protein, particularly amyloid beta (a β), including a β monomers, in mammals, particularly in the human brain, but preferably does not exhibit any significant cross-reactivity with Amyloid Precursor Protein (APP).

The invention also relates to humanized forms of an antibody as defined above, compositions comprising said humanized antibody and the use of said compositions in the treatment of amyloidosis, in particular in the treatment of neurodegenerative diseases in mammals, especially in humans. The neurodegenerative disease is in particular selected from Mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD), such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome. Preferably, the neurodegenerative disease is alzheimer's disease.

The invention also relates to hybridoma cell lines 5-5-6, 6-1-6, 17-4-3, and 24-2-3.

The invention also relates to the use of an antibody as defined above or a composition comprising said antibody in an in vitro diagnostic method. In particular, this diagnostic method is useful for diagnosing a neurodegenerative disease selected from the group consisting of: mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD), such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome; preferably alzheimer's disease; in particular by detecting A.beta.peptide or a variant thereof in a biological sample.

Preferably, the sample is a serum sample.

According to another preferred embodiment, the sample is a fluid or cerebrospinal fluid (CSF) sample.

In a particularly preferred embodiment, the present invention relates to an in vitro or in situ diagnostic method for diagnosing an amyloidosis-associated disease or condition, preferably alzheimer's disease, comprising the steps of:

contacting an antibody of the invention with a sample of an individual suspected to be suffering from said disease condition or disease, preferably selected from a serum, liquid or CSF sample, most preferably a serum sample; or contact with a specific body part or body area, an

Detecting binding of said antibody to pGlu-amyloid, preferably pGlu-A β peptide of the sample.

More particularly, the present invention relates to a method for the diagnosis of an amyloidosis-associated disease or condition, preferably alzheimer's disease, comprising detecting immunospecific binding of an antibody or an active fragment thereof to pGlu-amyloid protein, preferably pGlu-a β peptide, in a sample or in situ, comprising the steps of:

(a) contacting a sample or a specific body part or body area suspected to contain amyloid with an antibody, in particular a monoclonal antibody, of the invention, or a chimeric antibody or fragment thereof of the invention and as described herein before, or a humanized antibody or fragment thereof and/or a functional part thereof, which antibody binds to pGlu-a β peptide;

(b) binding the antibody and/or functional portion thereof to pGlu-a β peptide to form an immune complex;

(c) detecting the formation of the immune complex; and

(d) correlating the presence or absence of said immune complex with the presence or absence of pGlu-A β peptide in said sample or specific body part or region.

The invention also includes a method of determining the degree of amyloidogenic plaque burden (amyloidogenic plaque burden) in a tissue and/or a body fluid, the method comprising:

(a) obtaining a representative sample of the tissue and/or body fluid under investigation;

(b) detecting said sample with an antibody, in particular a monoclonal antibody, of the invention, or a chimeric antibody or fragment thereof of the invention and as described herein before, or a humanized antibody or fragment thereof and/or a functional part thereof;

(c) determining the amount of antibody bound to the protein; and

(d) calculating plaque burden in the tissue and/or fluid.

In particular, the present invention relates to a method for determining the extent of the amyloidogenic plaque burden in a tissue and/or a body fluid, wherein the formation of an immune complex in step c) is determined, whereby the presence or absence of said immune complex is correlated with the presence or absence of amyloid proteins, in particular pGlu-a β peptide.

In yet another embodiment, the invention relates to a composition comprising a therapeutically effective amount of an antibody of the invention, or a chimeric antibody or fragment thereof of the invention and described herein before, or a humanized antibody or fragment thereof, including any functionally equivalent antibody or any derivative or functional part thereof; particularly a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable carrier.

In another embodiment of the invention, the composition comprises a therapeutically effective amount of an antibody.

The invention also includes mixtures comprising a therapeutically effective amount of an antibody of the invention, particularly a monoclonal antibody, or a chimeric antibody or fragment thereof of the invention and described herein before, or a humanized antibody or fragment thereof, including any functionally equivalent antibody or any derivative or functional part thereof; and optionally other biologically active substances and/or pharmaceutically acceptable carriers and/or diluents and/or excipients.

In particular, the invention relates to such mixtures wherein the other biologically active substance is a compound for use in an amyloidosis drug, said amyloidosis being a group of diseases or conditions associated with amyloid or amyloid-like a β protein involved in neurodegenerative diseases selected from Mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD) such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome; preferably Alzheimer's disease.

In another embodiment of the invention, the other biologically active substance or compound may also be a therapeutic agent useful in the treatment of amyloidosis caused by amyloid beta, or in the treatment of other neurological disorders.

The other biologically active substance or compound may exert its biological effect by the same or similar mechanism as the antibody of the invention, or by an unrelated mechanism of action, or by multiple related and/or unrelated mechanisms of action.

In general, the additional biologically active compound may include neuronal transmission enhancers, psychotherapeutic agents, acetylcholinesterase inhibitors, calcium channel blockers, biogenic amines, benzodiazepinesSedatives, acetylcholine synthesis, storage or release enhancers, post-acetylcholine receptor agonists, monoamine oxidase-A or-B inhibitors, N-methyl-D-aspartate receptor antagonists, non-steroidal anti-inflammatory drugs, antioxidants and 5-hydroxytryptamine receptor antagonists.

More particularly, the invention relates to a mixture comprising at least one compound selected from the group consisting of: a compound effective against oxidative stress; an anti-apoptotic compound; a metal chelator; DNA repair inhibitors such as pirenzepine and metabolites; 3-amino-1-propanesulfonic acid (3-APS); 1, 3-propanedisulfonate (1, 3 PDS); an alpha-secretase activator; inhibitors of beta-and gamma-secretase; a Tau protein; a neurotransmitter; beta-sheet disruptor (/ 3-sheetbreaker); an amyloid β scavenger/depleting cellular component attractant; inhibitors of N-terminally truncated amyloid beta including pyroglutamated amyloid beta 3-42, such as glutaminyl cyclase inhibitors; an anti-inflammatory molecule; or cholinesterase inhibitors (ChEI), such as tacrine, rivastigmine, donepezil and/or galantamine; an M1 agonist; and other drugs, including any amyloid or tau modifying drugs and nutritional supplements.

The invention also relates to such mixtures wherein the compound is a cholinesterase inhibitor (ChEI), in particular such mixtures wherein the compound is selected from tacrine, rivastigmine, donepezil, galantamine, nicotinic acid and memantine.

In another embodiment, the mixture of the invention may comprise niacin or memantine with an antibody of the invention, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

In another embodiment, the mixture of the invention may comprise a glutaminyl cyclase inhibitor together with an antibody of the invention, and optionally a pharmaceutically acceptable carrier and/or diluent and/or excipient.

Preferred glutaminyl cyclase inhibitors are described in WO2005/075436, especially examples 1 to 141, pages 31 to 40. The syntheses of examples 1 to 141 are shown on pages 40 to 48 of WO 2005/075436. The disclosure of WO2005/075436 on examples 1 to 141, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/055945, especially examples 1 to 473 on pages 46 to 155. The syntheses of examples 1-473 are shown in WO2008/055945, page 156 and 192. The disclosure of WO2008/055945 regarding examples 1-473, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/055947, especially examples 1 to 345 on pages 53 to 118. The syntheses of examples 1-345 are shown in WO2008/055947, page 119-133. The disclosure of WO2008/055947 regarding examples 1-345, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/055950, especially examples 1 to 212, pages 57 to 120. The syntheses of examples 1 to 212 are shown in WO2008/055950, pages 121 and 128. The disclosure of WO2008/055950 regarding examples 1-212, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/065141, especially examples 1 to 25 on pages 56 to 59. The syntheses of examples 1 to 25 are shown on pages 60 to 67 of WO 2008/065141. The disclosure of WO2008/065141 regarding examples 1-25, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/110523, especially examples 1 to 27, pages 55 to 59. The syntheses of examples 1 to 27 are shown on pages 59 to 71 of WO 2008/110523. The disclosure of WO2008/110523 regarding examples 1-27, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/128981, especially examples 1 to 18 on pages 62 to 65. The syntheses of examples 1 to 18 are shown on pages 65 to 74 of WO 2008/128981. The disclosure of WO2008/128981 regarding examples 1-18, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/128982, especially examples 1 to 44 on pages 61 to 67. The syntheses of examples 1 to 44 are shown on pages 68 to 83 of WO 2008/128982. The disclosure of WO2008/128982 regarding examples 1-44, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/128983, especially examples 1 to 30 on pages 64 to 68. The syntheses of examples 1 to 30 are shown on pages 68 to 80 of WO 2008/128983. The disclosure of WO2008/128983 regarding examples 1-30, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/128984, especially examples 1 to 36, pages 63 to 69. The syntheses of examples 1 to 36 are shown on pages 69 to 81 of WO 2008/128984. The disclosure of WO2008/128984 regarding examples 1-36, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/128985, especially examples 1 to 71 on pages 66 to 76. The syntheses of examples 1 to 71 are shown on pages 76 to 98 of WO 2008/128985. The disclosure of WO2008/128985 regarding examples 1-71, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

Other preferred glutaminyl cyclase inhibitors are described in WO2008/128986, especially examples 1 to 7 on pages 65 to 66. The syntheses of examples 1 to 7 are shown on pages 66 to 73 of WO 2008/128986. The disclosure of WO2008/128986 regarding examples 1-7, their synthesis and their use as glutaminyl cyclase inhibitors is incorporated herein by reference.

In yet another embodiment of the present invention, there is provided a mixture comprising an "atypical antipsychotic"; and an antibody of the invention, particularly a monoclonal antibody, but particularly a chimeric antibody or fragment thereof of the invention and described herein before, or a humanized antibody or fragment thereof; and optionally pharmaceutically acceptable carriers and/or diluents and/or excipients, such as clozapine, ziprasidone, risperidone, aripiprazole or olanzapine, for the treatment of positive and negative psychotic symptoms including hallucinations, delusions, thought disorders (thorugh disorders) (manifested by marked incoherence, derailment, mispresentation) and monster or disorganized behaviour, as well as anhedonia, affective disorders (flantenedaffect), apathy and social withdrawal (social without driving).

In a specific embodiment of the invention, the compositions and mixtures of the invention and as described herein before comprise a therapeutically effective amount of the antibody and the biologically active substance, respectively.

Other compounds that may suitably be used in mixtures in combination with the antibodies of the invention are described in WO2008/065141 (see especially page 37/38), including PEP inhibitors (page 43/44); LiCl; inhibitors of dipeptidyl aminopeptidases, preferably inhibitors of DPIV or DPIV-like enzymes (see page 48/49); acetylcholinesterase (ACE) inhibitors (see page 47); a PIMT enhancer; inhibitors of beta-secretase (see page 41); inhibitors of gamma secretase (see page 41/42); inhibitors of neutral endopeptidase; inhibitors of phosphodiesterase-4 (PDE-4) (see page 42/43); a TNF α inhibitor; muscarinic M1 receptor antagonists (see page 46); NMDA receptor antagonists (see page 47/48); sigma-1 receptor inhibitors; histamine H3 antagonist (see page 43); an immunomodulator, immunosuppressant or an agent selected from the group consisting of antegren (natalizumab), neurolan (aminopyridine-SR), campath (alemtuzumab), IR208, NBI5788/MSP771 (telimotide), paclitaxel, anergix.ms (AG284), SH636, davvin (CD271, adapalene), BAY361677 (interleukin-4), matrix-metalloproteinase inhibitors (e.g. BB76163), interferon-tau (trophoblastin), and SAIK-MS; beta-amyloid antibody (see page 44); cystatins (see page 44); MCP-1 antagonists (see page 44/45); amyloid deposition inhibitors (see page 42) and beta amyloid synthesis inhibitors (see page 42), which are incorporated herein by reference.

In another embodiment, the invention relates to a mixture comprising a therapeutically effective amount of an antibody, in particular a monoclonal antibody, of the invention or a chimeric antibody or fragment thereof, or a humanized antibody or fragment thereof, of the invention and as described herein before, and/or a biologically active substance.

The invention also relates to the use of an antibody of the invention, in particular a monoclonal antibody, but in particular a chimeric antibody or fragment thereof, or a humanized antibody or fragment thereof, and/or a functional part thereof, of the invention and as described herein before, and/or a pharmaceutical composition or mixture comprising said antibody for the manufacture of a medicament for the treatment or alleviation of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, e.g. diseases including but not limited to neurological disorders such as Alzheimer's Disease (AD), lewy body dementia, down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutch type); parkinson-dementia complex of guam; and other diseases based on or associated with amyloid proteins, such as progressive supranuclear palsy, multiple sclerosis; creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases, including macular degeneration.

The invention also includes methods for preparing an antibody, particularly a monoclonal antibody, but particularly a chimeric antibody or fragment thereof, or a humanized antibody or fragment thereof, and/or functional parts thereof, of the invention, and particularly the invention and the aforementioned herein, and/or a pharmaceutical composition or mixture comprising, particularly a therapeutically effective amount of said antibody and/or functional parts thereof, for use in a method of preventing, treating or mitigating the effects of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, e.g., diseases including, but not limited to, neurological disorders such as Mild Cognitive Impairment (MCI), Alzheimer's Disease (AD), e.g., Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD), such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down syndrome; lewy body dementia, hereditary cerebral hemorrhage with amyloidosis (Dutch type); parkinson-dementia complex of guam; and other diseases based on or associated with amyloid proteins, such as progressive supranuclear palsy, multiple sclerosis; creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases, including macular degeneration, comprising formulating an antibody, particularly a monoclonal antibody, but particularly a chimeric antibody or fragment thereof, or a humanized antibody or fragment thereof, of the invention into a pharmaceutically acceptable form.

The invention also includes methods of preventing, treating, or mitigating the effects of amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis, such as, but not limited to, neurological disorders such as Mild Cognitive Impairment (MCI), Alzheimer's Disease (AD) such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD) such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome; lewy body (Lewybody) dementia, hereditary cerebral hemorrhage with amyloidosis (Dutch type); parkinson-dementia complex of guam; and other diseases based on or associated with amyloid proteins, such as progressive supranuclear palsy, multiple sclerosis; creutzfeldt-jakob disease, parkinson's disease, HIV-associated dementia, ALS (amyotrophic lateral sclerosis), adult-onset diabetes; senile cardiac amyloidosis; endocrine tumors and other diseases, including macular degeneration, by administering an antibody and/or a functional part thereof, but in particular a humanized antibody and/or a functional part thereof, or a composition or mixture comprising such an antibody and/or a functional part thereof, to an animal or human suffering from such a condition, comprising administering a therapeutically effective amount of the antibody.

It is another object of the present invention to provide methods for treating amyloidosis, a group of diseases and disorders associated with amyloid plaque formation including secondary amyloidosis and age-related amyloidosis, including but not limited to neurodegenerative diseases such as Mild Cognitive Impairment (MCI), Alzheimer's Disease (AD) such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD) such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome, by administering the antibodies, particularly the pharmaceutical compositions, of the present invention and described herein to an animal, particularly a mammal or a human; particularly diseases or conditions characterized by a loss of cognitive memory.

In a specific embodiment, the present invention provides a method of maintaining or increasing cognitive memory, but in particular restoring cognitive memory, in an animal, particularly a mammal or a human, suffering from a memory disorder by administering to the animal, particularly a mammal or a human, an antibody, in particular a pharmaceutical composition, of the invention and as hereinbefore described.

It is another object of the invention to provide therapeutic compositions and methods of producing such compositions, as well as methods of treating amyloidosis, a group of diseases and disorders associated with amyloid plaque formation, including secondary amyloidosis and age-related amyloidosis, including, but not limited to, neurodegenerative diseases such as Mild Cognitive Impairment (MCI), Alzheimer's Disease (AD) such as Sporadic Alzheimer's Disease (SAD) or Familial Alzheimer's Dementia (FAD) such as Familial British Dementia (FBD) and Familial Danish Dementia (FDD), neurodegeneration in down's syndrome, using the antibodies of the invention and of the foregoing description; particularly diseases or conditions characterized by a loss of cognitive memory.

In particular, the invention relates to the treatment of animals, in particular mammals or humans, suffering from amyloidosis-associated disease condition characterized by a loss of cognitive memory capacity, which treatment results in the retention of cognitive memory capacity.

Examples

1. Materials and methods

1.1 Generation of antibodies

Mouse

For hybridoma production, 8-week-old female BALB/C mice (Charles river) were used.

Myeloma cell line

For hybridoma generation, the myeloma cell line SP2/0-Ag14 from Deutsche Stammsamlungvon MikorganismenundZellkulturen was used.

Antigens

The peptide pGlu-6166 (sequence pGlu-FRHDSGC, SEQ ID NO: 65) was used.

Policy

As an immunogen, the peptide was coupled to bovine thyroglobulin (BTG, SIGMA) via the maleimide group of 3 different linkers. Three different length linkers were used from N- [ e-maleimidocaproyloxy ] succinimidyl Ester (EMCS), succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidocaproate) (LCDSMCC) and N- [ b-maleimidopropoxy ] succinimidyl ester (BMPS).

To detect the antibodies produced, the same peptide was coupled to bovine serum albumin (BSA, SIGMA) via the maleimido group of succinimidyl-6- [ (b-maleimido-propionylamino) hexanoate ] (SMPH).

Method of producing a composite material

Coupling of peptides for immunization

Coupling via the SH-group of the cysteine residue of the peptide is carried out in two steps.

1. Maloylation of Carrier proteins (Maleoylation)

2 to 5mg of the corresponding linker (50mg/ml in N-methylpyrrolidone (NMP)) were added to 2ml of carrier protein solution (10mg/ml in 0.1mM NaHCO3, pH 8.0). The reaction mixture was incubated at Room Temperature (RT) for 1 h. The reaction mixture was then desalted using a SephadexG-50 column (1.5X14cm) equilibrated with 50mM sodium phosphate, 250mM NaCl, pH 6.8.

2. Coupling of maleylated BTG to peptides

Mu.l of the peptide solution (10mg/ml in double distilled water (Aquabidest)) was mixed with 2ml of a solution containing 50mM sodium phosphate, 250ml NaCl, maleylated carrier protein (2.5mg/ml) in pH6.8, incubated at 4 ℃ for 2h and then at RT for 4 h. Unreacted maleimide groups were blocked by addition of 2-mercaptoethanol to a concentration of 10mM and incubation at 4 ℃ overnight. The resulting conjugate was dialyzed against 10mM sodium phosphate, 150mM NaCl, pH7.5 at 4 deg.C (3 changes of buffer, MW cut-off 10.000).

Coupling of peptides for ELISA

Coupling via the SH-group of the cysteine residue of the peptide is carried out in two steps.

1. Malylation of carrier proteins

2mg of SMPH (50mg/ml in N-methylpyrrolidone, NMP) was added to 2ml of a carrier protein solution (BSA, 10mg/ml in 0.1mM NaHCO3, pH 8.0). The reaction mixture was incubated at Room Temperature (RT) for 1 h. The reaction mixture was then desalted using a SephadexG-50 column (1.5X14cm) equilibrated with 50mM sodium phosphate, 250mM NaCl, pH 6.8.

2. Coupling of maleylated BTG to peptides

Mu.l of the peptide solution (10mg/ml in 50mM sodium phosphate, 250mM NaCl, pH 6.8) was mixed with 1ml of a solution containing 50mM sodium phosphate, 250ml NaCl, maleated carrier protein (2.5mg/ml) in pH6.8, incubated at 4 ℃ for 2h and then at RT for 4 h. Unreacted maleimide groups were blocked by addition of 2-mercaptoethanol to a concentration of 10mM and incubation at 4 ℃ overnight. The resulting conjugate was dialyzed against 10mM sodium phosphate, 150mM NaCl, pH7.5 at 4 deg.C (3 changes of buffer, MW cut-off 10.000).

Immunization

5 mice were immunized intraperitoneally for 39 days. For immunization, a water-in-oil emulsion was used, which consisted of equal parts of antigen solution (consisting of equal parts of three different peptide-BTG-conjugates) and complete or incomplete freund's adjuvant.

Fusion

3 immunized mice were sacrificed by CO2 incubation (incubation). The spleen was removed and homogenized under sterile conditions. Spleen cells and myeloma SP2/0 cells were washed several times in Dulbecco's modified eagleMedium (DMEM, SIGMA) and fused with polyethylene glycol 3350(1ml 50% (w/v)) at a ratio of 2, 3 spleen cells: 1SP2/0 cells. Further manipulation of the fusion hybridomas was performed according to standard methodologies.

ELISA

ELISA against IgG was used to screen cell culture supernatants. The tests were performed in 96-well polystyrene (polystyrol) microtiter plates (Greiner, No. 655061). Plates were coated with BSA-pGlu-6166 peptide. 100 μ l of undiluted cell culture supernatant was added to each well and incubated for 1 hour at RT. The supernatant from SP2/0 cells was used as a negative control. Supernatants from splenocytes were used as positive controls.

Positive wells were detected using goat anti-mouse IgG coupled to alkaline phosphatase. The Optical Density (OD) was measured at 405nm in a DynexOpsysMR microplate reader (DynexOpsysMRMicroplateReader).

Selection of hybridoma cells producing stable antibodies

Cells from positive wells were transferred to 24-well plates and cultured for several days. The cells were again transferred and tested for BSA-pGlu-6199 binding and cross-reactivity in ELISA. Positive wells were used for cryopreservation of hybridoma cell lines.

Cloning by limiting dilution

Two sequential cloning steps were performed to separate antibody-producing cells from non-antibody-producing cells and to ensure that the selected cells were monoclonal. Both cloning steps were performed according to the limiting dilution method.

Low temperature preservation

Selected hybridomas were cryopreserved using DMSO and standard methods.

ELISA assay

The capture of A.beta.N 3pE-40 was performed using hA.beta. (x-40) ELISA (HS) from TGC (the GENETICCSCcompany; Switzerland) essentially according to the manufacturer's instructions.

Biotinylated detection antibody for A.beta.N 3pE (pGlu-6166) was generated. When mentioned, the IBLHRP-conjugated a β N3pE antibody was used as a positive control (available only in combination with the iblelisia human amyloid β (N3pE) assay kit). The corresponding A.beta.N 3pE-40 peptide was synthesized (50. mu.g aliquots in Hexafluoroisopropanol (HFIP) and stored at-80 ℃ C.). Immediately prior to use, the HFIP was evaporated and the peptide was diluted to 1. mu.g/. mu.l with 100mM Tris/HClpH10, 4. This stock solution was further diluted with TGC antibody diluent. Subsequent capture and detection was performed according to the manufacturer's instructions.

1.3.PepSpot^TMAnalysis of

Specificity and biological integrity of A β N3pE antibody and cell culture supernatant JPTPepside technologies GmbH, Volmeerstrasse 5(UTZ), 12489Berlin, PepSpot of Germany^TMAnd (4) determining the technology.

Corresponding PepSpot^TMMembranes were prepared in JPT. The principle of the method is described and illustrated by Krameret al 1997Cell91, 799-.

For the assay, membranes were blocked with TBST-M (10mM Tris-HCl, pH7.5, 150mM NaCl, 0.005% Tween 20+ 5% skim milk) for 2 hours at room temperature with gentle shaking. The membranes were incubated overnight at 4 ℃ with each cell culture supernatant diluted in an equal volume of TBST-M on a shaking platform. Signal detection was performed according to standard procedures using an anti-mouse secondary antibody coupled to alkaline phosphatase.

1.4. Dot blot analysis

To this end, decreasing concentrations of A β N3pE-40 peptide (1000ng-8ng) were spotted onto small pieces of nitrocellulose membrane, followed by a PepSpot protocol^TMMembrane-like experimental procedure.

1.5.SDSPAGE

The 12% SDS polyacrylamide gel was shaped according to standard protocols. Mu.l of cell culture supernatant and 10. mu.g of biotinylated antibody were separated on a 12% SDS polyacrylamide gel. Electrophoresis was performed at a constant voltage of 100V for 2 hours.

BIACORE assay

A.beta.N 3pE-40 peptide (positive control) and A.beta.N 3E-40 peptide (negative control) were coupled to a BiacoreCM5 chip. Unmodified chips were used to determine blank values. Association and dissociation of biotinylated antibodies diluted from 20 μ g/ml to 1 μ g/ml in TGC diluent was monitored to allow subsequent determination of individual KD values. In this way, the binding characteristics of the individual cell culture supernatants were also determined.

1.6.1 affinity of Abeta N3 pE-specific antibody clones 6-1-6 and 24-2-3

Purified antibody clone 6-1-6 was diluted down to 100, 50, 30, 20, 15, 10, 7,4, 2, 1nM in HBS-EP buffer (Biacore). Affinity was determined using a Biacore3000 with a CM 5-chip immobilized with a β pE 3-40. The system was run at 30. mu.l/min. The measured bulk effect and non-specific binding to the chip surface were corrected by subtracting the signal of the flow cell 4 immobilized with a β pE3-40 and the signal of the blank flow cell 3. Association (10min) was obtained by injection of 300. mu.l of each concentration. Dissociation was observed for more than 10 min. The remaining antibody molecules were removed by injection of 5. mu.l of 0.1MHCL. For each antibody concentration, association and dissociation were recorded. Association and dissociation rates and dissociation constants were determined using a "bivalent analyte" model by fitting the association and dissociation phases globally simultaneously to all recorded antibody concentrations.

1.7. Sequencing antibody variable regions

Culturing of hybridoma:

hybridoma cells were cultured in D-MEM (+ L-glutamine, + sodium pyruvate, 4,5g/L glucose, Gibco) supplemented with 15% FBS, 1% MEM-NEA (nonessential amino acids, Gibco), 50. mu.g/ml gentamicin (Gibco), and 50. mu.M β -mercaptoethanol at 37 ℃ and 5% CO₂Medium growth. Subculture was performed after 3-4 days according to cell density. Cells were plated at 0.5 × 10⁶Cells/ml were inoculated in separate flasks (split) at 2-5X10⁶Cell density of cells/ml.

cDNA synthesis and reverse transcription:

from 2X10 according to the manual of the Nucleospinn RNA isolation kit (Macherey-Nagel)⁶Total RNA was isolated from individual cells. Using oligo (dT)₁₅Primers (Promega) and SuperScriptIII reverse transcriptase (Invitrogen), 100ngRNA was used for cDNA synthesis.

PCR amplification of heavy and light chain variable regions:

using Phusion^TMHigh fidelity DNA polymerase (NEWENGLANDBOLAB) using primers MHCG1 (in the case of clones 5-5-6 and 6-1-6) and MHCG2b (clones 7-4-3 and 24-2-3) in combination with primer MHV1-12The heavy chain variable region was amplified from the template cDNA. To amplify the light chain variable region, primer MKC was used in combination with primer MKV1-MKV 11. The primer sequences are shown in Table 1.

The PCR product was cloned in pjett 1.2:

PCR amplified heavy and light chain variable regions according to CloneJET^TMThe protocol of the PCR cloning kit (Fermentas) was cloned into pJET1.2/blunt (blunt) vector. Sequencing was performed using pJET1.2 sequencing primers.

Table 1: primer sequence for PCR amplification of heavy chain and light chain variable region

Name (R)	Sequence of	SEQ ID NO.
			MKV1	ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG	23
MKV2	ATGGAGWCAGACACACTCCTGYTATGGGTG	24
			MKV3	ATGAGTGTGCTCACTCAGGTCCTGGSGTTG	25
MKV4	ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG	26
			MKV5	ATGGATTTWCAGGTGCAGATTWTCAGCTTC	27
MKV6	ATGAGGTKCYYTGYTSAGYTYCTGRGG	28
			MKV7	ATGGGCWTCAAGATGGAGTCACAKWYYCWGG	29
MKV8	ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG	30
			MKV9	ATGGTRTCCWCASCTCAGTTCCTTG	31
MKV10	ATGTATATATGTTTGTTGTCTATTTCT	32
			MKV11	ATGGAAGCCCCAGCTCAGCTTCTCTTCC	33
MKC	ACTGGATGGTGGGAAGATGG	34
			MHV1	ATGAAATGCAGCTGGGGCATSTTCTTC	35
MHV2	ATGGGATGGAGCTRTATCATSYTCTT	36
			MHV3	ATGAAGWTGTGGTTAAACTGGGTTTTT	37
MHV4	ATGRACTTTGGGYTCAGCTTGRTTT	38
			MHV5	ATGGACTCCAGGCTCAATTTAGTTTTCCTT	39
MHV6	ATGGCTTGTCYTRGSGCTRCTCTTCTGC	40
			MHV7	ATGGRATGGAGCKGGRTCTTTMTCTT	41
MHV8	ATGAGAGTGCTGATTCTTTTGTG	42
			MHV9	ATGGMTTGGGTGTGGAMCTTGCTATTCCTG	43
MHV10	ATGGGCAGACTTACATTCTCATTCCTG	44
			MHV11	ATGGATTTTGGGCTGATTTTTTTTATTG	45
MHV12	ATGATGGTGTTAAGTCTTCTGTACCTG	46
			MHCG1	CAGTGGATAGACAGATGGGGG	47
MHCG2b	CAGTGGATAGACTGATGGGGG	48

1.8 antibody clone 6-1-6 for N3pEELISA

The plates were sealed with capture antibody-coated 96-well maxisorb plates (Nunc) by incubating 100. mu.l per well of 2. mu.g/ml anti-A β antibody 4G8 diluted in D-PBS overnight at 4 ℃. the plates were sealed, the coating solution was removed, the plate surfaces were blocked with 200. mu.l per well of PIERCE protein-free ELISA Blocker (PIERCEProtein-freeELISA-Blocker) (Tween-20-free) at room temperature for 2 hours. the plates were then washed 6 times with TBS + 0.05% (v/v) Tween-20. the remaining wash solution was removed by tapping the plates A β pE3-40 standard peptide in PIERCE protein-free ELISA Blocker (Tween-20-containing) diluted down to 200, 100, 50, 25, 12.5, 6.25, 3.125/ml. 100. mu.l of each concentration and 100. mu.l of blank diluent buffer (plate) were pipetted onto the plates and sealed at 4 ℃ and the plates were pipettedIncubate for 2 hours then plate with TBS + 0.05% (v/v) Tween-20 6 times, remove the remaining wash solution by tapping the plate 100. mu.l of detection antibody-enzyme conjugate solution containing 1. mu.g/ml A β N3pE specific antibody clone 6-1-6 and 2. mu.g/ml streptavidin-HRP conjugate (Sigma) in PIERCE protein-free ELISA blocker (containing Tween-20) was pipetted into each well, the plate was sealed and incubated at 4 ℃ for 1 hour, then the plate was washed 6 times with TBS + 0.05% (v/v) Tween-20. remove the remaining wash solution by tapping the plate, 100. mu.l SureBlue substrate solution (KPL) was pipetted into each well, the plate was incubated at room temperature for 30min, by adding 100. mu.l 1MH per well₂SO₄To terminate the reaction. The absorbance was measured at 450nm with tecansunerise and corrected by the absorbance at 540 nm.

1.9 Cross-reactivity Studies by ELISA and Surface Plasmon Resonance (SPR) analysis

ELISA：

Mu.l of anti-A β antibody 4G8 diluted in D-PBS per well 100. mu.l/ml, the plates were coated with capture antibody 4G8, the plates were sealed, the coating solution was removed, the plate surfaces were blocked with 200. mu.l of PIERCE protein-free ELISA blocking agent (no Tween-20) per well for 2 hours at room temperature, then the plates were washed 6 times with TBS + 0.05% (v/v) Tween-20, the remaining washing solution was removed by tapping the plates A β pE3-40 standard peptide and other A β peptides (2-40, 3-40, 4-40, 1-42, 3-42 and pE11-40) were diluted down in PIERCE protein-free blocking agent (containing Tween-20) to 800, 400, 200, 100, 50, 25, 12.5. mu.5. mu.l of each concentration and 100. mu.l of each dilution (100. mu.l) were tapped onto a Tween-free blocking agent (containing Tween-20) and the plates were then diluted with 100. mu.l of anti-A β antibody 4G buffer (0. mu.v) and the plates were pipetted in ELISA blocking agent (0. mu.5) and the remaining washing solution was removed by tapping the plates after incubation at 0. mu.4. mu.1. mu.4G/v) and the plates were pipetted in ELISA blocking agent (10. mu.4. mu.1. mu.4. mu.1. mu.4. mu.l of the plates and theTween-20 was washed 6 times 05% (v/v). The remaining wash solution was removed by tapping the plate. Mu.l of SureBlue substrate solution (KPL) was pipetted into each well and the plates were incubated for 30min at room temperature in the dark. Add 100. mu.l of 1MH per well₂SO₄The reaction was terminated. The absorbance was measured at 450nm with tecansunerise and corrected by the absorbance at 540 nm.

SPR：

In addition to the different A.beta.species, cross-reactivity to other pGlu-peptides present in humans was also determined. This is done by surface plasmon resonance. The following peptides or their N-terminal regions were immobilized on the surface of a CM 5-chip:

MCP1, MCP2, digastrin (biggastrin), gonadotropin releasing hormone, neurotensin, orexin a, fibronectin, collagen 1and TRH. As a positive control, binding to A β pE3-40 was also analyzed. N3pE antibody clones 6-1-6 and 24-2-3 were diluted down to 25. mu.g/ml in HBS-EP (Biacore). Binding was observed using a Biacore3000 with several CM 5-chips immobilized with the respective peptides (on flow cells 2, 3 and 4). The system was run at 20. mu.l/min. The measured bulk effect and non-specific binding to the chip surface was corrected by subtracting the signals of the test peptide immobilized flow cells 2, 3 and 4 and the signal of the blank flow cell 1. Association (9min) was obtained by injection of 180. mu.l each of antibody clones 6-1-6 and 24-2-3. Dissociation was observed for more than 9 min. The remaining antibody molecules were removed by injection of 5. mu.l of 0.1MHCL. Association and dissociation were recorded for each antibody interaction with a different peptide. Cross-reactivity was determined by evaluating the signals at the end and rate of the association phase. The values for all pGlu-peptides were compared with the signal for A β pE 3-40.

1.10 optimization and validation of N3pEELISA for brain analysis

The N3pEELISA we developed should be used to analyze A β pE3-42 concentrations in the brains of transgenic mice. Hemispheres and brainstems are typically analyzed for A β pE3-42 content, respectively. Mouse brains were homogenized using ceramic beads in 500 μ l of a 2% SDS solution containing protease inhibitors in a precell (peqlab) homogenizer. The suspension was aspirated from the beads and transferred to a centrifuge tube. The beads were washed again with 250 μ l of 2% SDS solution containing protease inhibitor and the solution was transferred to a centrifuge tube. 750 μ l SDS brain suspension was sonicated on crushed ice for 20 sec. The samples were centrifuged at 75000Xg for 1 hour at 4 ℃. The supernatant was then taken, aliquoted and stored at-80 ℃ until ELISA analysis. The remaining SDS insoluble pellet (pellet) was mixed with 150. mu.l 70% formic acid and sonicated on crushed ice for 20 sec. Immediately after sonication, the solution was neutralized with 2850 μ l1MTris (this is the old method); alternatively, the solution was neutralized with 2850. mu.l EIA buffer (PBS +10mg/ml BSA + 0.05% Tween-20) + 860. mu.l 3.5MTris, which represents the novel process. Samples of the formic acid fraction were stored at-80 ℃ until ELISA.

N3pEELISA was performed by the following protocol:

coating a 96-well maxisorb plate (Nunc) with a capture antibody by incubating 100. mu.l per well of anti-A β antibody 4G8 diluted in D-PBS overnight at 4 ℃ the plate is sealed, the coating solution is removed, the plate surface is blocked with 200. mu.l of the PIERCE protein-free ELISA blocker (no Tween-20) per well for 2 hours at room temperature. then the plate is washed 6 times with TBS + 0.05% (v/v) Tween-20. the remaining wash solution is removed by tapping the plate A2 pE3-42 standard peptide in PIERCE protein-free ELISA blocker (containing TBS-20) (old method) or EIA buffer (new method) down to pg64, 514.6, 257.3, 128.65, 64.32, 31.16, 16.08 Tris/ml 100. mu.l of each concentration and 100. mu.l of the blank buffer (containing TBS-20) (old method) or EIA buffer (new method) is not pipetted onto the plate and the sample is pipetted in 100. mu.1. mu.l of the new method after thawing the plate (ELISA) and the remaining wash solution is pipetted in 100. mu.1. mu.5. mu.20. mu.4. mu.5. mu.4. mu.20. mu.4. mu.1. mu.l of the ELISA buffer (old buffer) and the old buffer is pipetted into the plate is removed by the new method and the new method is pipetted with the ELISA buffer (New method after the ELISA buffer (New method) and the wash buffer (New method is addedClones 6-1-6 and 2. mu.g/ml streptavidin-HRP conjugate (Sigma). The plates were sealed and incubated at 4 ℃ for 1 hour. The plates were then washed 6 times with TBS + 0.05% (v/v) Tween-20. The remaining wash solution was removed by tapping the plate. Mu.l of SureBlue substrate solution (KPL) was pipetted into each well and the plates were incubated for 30min at room temperature in the dark. Add 100. mu.l of 1MH per well₂SO₄The reaction was terminated. The absorbance was measured at 450nm with tecansunerise and corrected by the absorbance at 540 nm.

1.11N3pE antibody cloning for immunohistochemistry

Formalin-fixed and paraffin-embedded sections from human brain (cortex) were processed as follows:

1. deparaffinizing and rehydrating the sections (mounted on slides):

a. incubate slides in Histonclear or xylene for 3 min

b. Removing cleaning solution

c. The slides were again incubated in Histonclear or xylene for 3 minutes

d. Slides were incubated for 3 minutes in 1: 1 Histonclear or xylene with 100% ethanol

e. Incubate slides in 100% ethanol for 3 minutes, remove solution

f. The slides were again incubated in 100% ethanol for 3 minutes

g. Incubate slides in 95% ethanol for 3 min

h. Incubate slides in 70% ethanol for 3 min

i. Incubate slides in 50% ethanol for 3 min

j. Incubate slides in distilled water for 3 minutes

2. Discontinuation of endogenous peroxidase activity:

slides were incubated with 99ml methanol +1ml 30% hydrogen peroxide for 10 minutes at room temperature.

3. Washing the slide with water: 2X5 min

4. The water was removed from each slide and the slides were placed on a slide rack in a humid chamber to prevent the slides from drying. Sections were covered with 88% formic acid for 10min at room temperature in a fume hood. Rinsed several times in water and shaken for 10 minutes in a staining dish filled with water.

5. Blocking in 10% horse serum for 20 min at room temperature.

6. The blocking solution was spun off (or aspirated) and the primary antibody (N3pE antibody clone 6 or 24) was added overnight at 4 ℃.

7. Slides were washed separately with TBS for 10 minutes to avoid dragging one slide over the other.

8. Biotinylated secondary antibody (goat anti-mouse from VectorLaboratories) was added: 9ml TBS, 1ml goat serum, 45. mu.l secondary antibody). Incubate at room temperature for 30 minutes.

9. Slides were washed separately with TBS for 10 minutes to avoid dragging one slide over the other.

10. ABC solution (10ml TBS, 100. mu.l horse serum, 90. mu.l fraction A, 90. mu.l fraction B) was added. Incubate at room temperature for 30 minutes.

11. Wash slides with 50mm tris: 2X10 min

12. Color reaction: sections were incubated with DAB solution (20 mg of DAB from Sigma in 100ml of 50mM Tris, filtered, and 33. mu.l of 33% hydrogen peroxide was added). The color reaction was observed with a microscope. The reaction product was brown. The reaction was stopped by placing the slide into a staining dish containing water.

13. Wash slide with water for 10 minutes

14. Counterstaining with hematoxylin, washing with water

15. Dewatering and clarifying: step 1 is performed in reverse order (e.g. water, ethanol to 100% histoclean)

16. The slides were covered with permount (FisherScientific). The slides were air dried. The slides were cleaned with a razor blade and ethanol.

2. Results

2.1 Generation of antibodies

6 clones stably producing antibodies against pGlu-6166-BSA peptide were isolated: clones 1-8-12, 5-5-6, 6-1-6, 12-1-8, 17-4-3 and 24-2-3. These clones were further characterized.

2.2. Determination of the desired antibody concentration:

the signal intensity in the ELISA assay is not only related to the analyte/Α β variant concentration, but is also strongly dependent on the concentration of antibody employed. Since a β variants are only present in low concentrations in serum samples, it is necessary to determine the antibody concentration that enables the detection of low concentrations of the corresponding a β variants. The specified limit of detection for a β is the low pg range for commercially available a β ELISA kits. In the standard curve, the highest concentration is typically 500 pg/ml. General information on the antibody concentrations employed is typically missing in the data sheet/instruction manual, however, due to further information deduced from the general literature, 1 μ g/ml antibody was used as a default.

In the first series of experiments, 500pg/ml A.beta.N 3pE-40 could not be detected with the corresponding pGlu-616612-1 biotin-conjugated antibody. In fact, a relatively high A.beta.N 3pE concentration (10ng/ml) was required to obtain a signal with 1. mu.g/ml antibody (see FIG. 1A: middle bar). The intensity of this signal was greatly enhanced by increasing the antibody concentration to 10. mu.g/ml (see FIG. 1A: left-hand bar). The signal intensity can be further increased up to 20. mu.g/ml antibody (see FIG. 1B). Beyond this concentration, no further increase in signal intensity can be achieved. Therefore, 20. mu.g/ml antibody was used to determine the limit of detection of pGlu-6166 antibody.

2.3. Dot blot analysis

The A.beta.N 3pE-x antibody pGlu-6166 was selected in the screening procedure because the primary cell clone (named 12-1-8) showed strong binding to the peptide used for immunization and very low cross-reactivity (see Table 2).

Table 2: screening results to confirm signals obtained with supernatants from several hybridoma cell clones in ELISA assays

Cloning	N3pE-BSA	isoDAE-BSA	N3E-BSA	N11E-BSA	N11pE-BSA
						1-8-12	1.787	0.012	0.142	0.011	0.005
5-5-6	1.649	0.015	0.126	0.004	0.006
						6-1-6	1.377	0.013	0.125	0.007	0.014
12-1-8	2.123	0.005	0.009	0.001	0.005
						17-4-3	1.915	0.007	0.320	0.003	0.004
24-2-3	1.768	0.014	0.218	0.003	0.002
						Positive control	1.824	1.227	1.596	1.243	1.346
Negative control	0.045	0.005	0.008	0.001	0.003

Screening procedures for the full-length native A β N3pE-40 peptide have not been included. Therefore, available pools of pGlu6166 hybridoma cell clones were screened to find clones expressing antibodies that may exhibit higher affinity for the native Α β N3pE-40 peptide.

As shown in fig. 2, cell clones can be identified that do exhibit higher sensitivity to the native full-length Α β N3pE-40 peptide. Although pGlu-6166 antibody clone 12-1 was only able to detect 1. mu.g of peptide, clones 6-1-6 and 24-2-3 also gave signals as low as 8ng of peptide. Therefore, clones 6-1-6 and 24-2-3 were 125 times more sensitive. Using these clones, the limit of detection of the 8pg/ml A.beta.N 3pE-40 peptide in the corresponding ELISA was obtained.

PepSpot assay

Specificity was then examined by PepSpot analysis to compare biotinylated a β N3pE-x antibody pGlu-6166 to hybridoma cell clones. In table 3, all peptides corresponding to spots on PepSpot membranes are listed. As shown in FIG. 3, pGlu-6166 clones 6-1-6 and 24-2-3 did not produce more cross-signal than pGlu-6166 antibody clone 12-1. All clones studied mainly recognized spot No. 6-specific Α β N3pE-x spot (pefrhd.., seq id no: 12), followed by spot No.5 (efrhd.. seq id no: 11) and spot No. 7 (frhd.. seq id no: 13). Weak signals were also obtained with spot 4 (AEFRHD.. SEQIDNo: 10).

Table 3: spotting to PepSpot^TMSequence of the A β peptide on the Membrane (JPTPeptideTechniques GmbH) and detection by pGlu-6166 hybridoma cell cloning

pE in Table 3 is pGlu, pyroglutamic acid

iD in Table 3 is isoAsp, isoaspartic acid

SDS-PAGE analysis

The biological integrity of the a β -N3pE antibody and the hybridoma cell culture supernatant was determined in general by SDS-PAGE analysis (see materials & methods above for details).

As shown in FIG. 4, all the samples loaded onto the gel revealed clear bands without spreading, indicating the integrity of the pGlu-616612-1 antibody and the supernatant of the hybridoma cell clone.

BIACORE analysis

Significant differences in the sensitivity of hybridoma clone supernatants to the a β N3pE-40 peptide compared to biotinylated pGlu-6166 antibody were diagnosed by dot blot analysis. However, only the endpoint results were monitored in this way. In another aspect, Biacore analysis allows real-time resolution of the binding process of a given antibody. To examine whether poor binding of the pGlu-616612-1 antibody was the result of low association with the A.beta.N 3pE-40 peptide, Biacore analysis was performed as described in the materials and methods above.

Binding was monitored for increasing concentrations of pGlu-6166 antibody with a calculated KD of 30 nM. Comparison of hybridoma clone supernatant 12-1 with cell clone supernatant 6-1-6 revealed a striking difference in binding characteristics. The association of clone 6-1-6 was about 5 times higher than that observed with clone 12-1. Most significant, however, is the difference in dissociation behavior. Although clone 6-1-6 was difficult to dissociate from the A.beta.N 3pE-40 peptide, 12-1 was easily washed away within minutes. Therefore, poor binding of clone 12-1 is likely the result of the observed "off-rate". This hypothesis is further supported by the finding that clone 24-3-2, which gave particularly favorable results in dot blot analysis, showed a very slow association with the a β N3pE-40 peptide, but no observable "off-rate" as opposed to clone 12-1 (see also fig. 5).

2.6.1 affinity of Abeta N3 pE-specific antibody clones 6-1-6 and 24-2-3

For N3pE antibody clone 6-1-6, association rates, dissociation rates and dissociation constants were calculated by global fitting to all sensorgrams shown in fig. 6.

The association rate was calculated to be 1.67e5M^-1s^-1The dissociation rate is 2.63e-4s^-1The dissociation constant was 1.57 nM.

For N3pE antibody clone 24-2-3, association rates, dissociation rates and dissociation constants were calculated by global fitting to all sensorgrams shown in fig. 7.

The association rate was calculated to be 3.25e3M^-1s^-1The dissociation rate is 3.29e-4s^-1The dissociation constant was 101 nM.

2.7 sequencing antibody variable regions

The following sequences were identified:

2.7.1 clones 5-5-6

Variable part light chain, nucleotide sequence (SEQ ID NO: 49)

ATGGTGTCCTCAGCTCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGAAACCAACGGT

GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCT

ATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGATGGAAAAACCTATTTGAATTGG

TTATTACAGAGGCCAGGCCAGTCTCCAATGCGCCTAATCTATCTGGTGTCTAAACTGGAC

TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATC

AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCA

TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTA

TCCATCTTCCCACCAT

Variable part light chain, protein sequence (SEQIDNO: 50)

MVSSAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNW

LLQRPGQSPMRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFP

FTFGSGTKLEIKRADAAPTVSIFPP

Variable part heavy chain, nucleotide sequence (SEQ ID NO.51)

ATGGGATGGAGCGGGGTCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAG

GTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCC

TGCAAGGCTTCTGGTTACTCATTCACTGGCTATACCATGAACTGGGTGAAGCAGAGCCAT

GGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAGTGGTGTTACTAGGTACAAC

CAGAAATTCAAGGGCAAGGCCACATTAATTGTAGACAAGTCATCCAGCACAGCCTACATG

GAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTATTGTACAAGAGAGGCTAAA

CGGGAGTGGGACGAGACTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAA

ACGACACCCCCATCTGTCTA

Variable part heavy chain, protein sequence (SEQIDNO: 52)

MGWSGVFLFLLSGTAGVHSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSH

GKNLEWIGLINPYSGVTRYNQKFKGKATLIVDKSSSTAYMELLSLTSEDSAVYYCTREAK

REWDETYWGQGTLVTVSAAKTTPPSV

2.7.2 cloning of 6-1-6

Variable part light chain nucleotide sequence (SEQ ID NO: 53)

ATGGTGTCCACAGCTCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGAAACCAACGGT

GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCT

ATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTGACGGAAAAACCTATTTGAATTGG

TTATTACAGAGGCCAGGCCAGTCTCCAATGCGCCTAATCTATCTGGTGTCTAAACTGGAC

TCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATC

AGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCA

TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTA

TCCATCTTCCCACCATCCAG

Variable part light chain, protein sequence (SEQIDNO: 54)

MVSTAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLNW

LLQRPGQSPMRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFP

FTFGSGTKLEIKRADAAPTVSIFPPS

Variable part heavy chain, nucleotide sequence (SEQ ID NO: 55)

ATGGGATGGAGCGGGGTCTTTATCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCC

AGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGC

TTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTT

GAGTGGATTGGACTTATTAATCCTTACAATGGTGTTACTAGGTACAACCAGAAGTTCAAGGGCA

AGGCCACATTAATTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATC

TGAGGACTCTGCAGTCTATTACTGTACAAGAGAGGCTAAACGGGAGTGGGACGAGACTTACTGG

GGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTG

Variable part heavy chain, protein sequence (SEQIDNO: 56)

MGWSGVFIFLLSGTAGVHSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSH

GKNLEWIGLINPYNGVTRYNQKFKGKATLIVDKSSSTAYMELLSLTSEDSAVYYCTREAK

REWDETYWGQGTLVTVSAAKTTPPSVYPL

2.7.3 clone 17-4-3

Variable part light chain, nucleotide sequence (SEQ ID NO: 57)

ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGGTGTTCTGGATTCCTGTTTCCAGCAGTGATGTTG

TGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAG

ATCTAGTCAGAGCCTTGTACACAGTGATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCA

GGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGT

TCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCT

GGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGACGTTCGGTGGAGGCACCAAGCTG

GAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT

Variable part light chain protein sequence (SEQIDNO: 58)

MKLPVRLLVLVFWIPVSSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSDGNTYLHWY

LQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPP

TFGGGTKLEIKRADAAPTVSIFPPSS

Variable part heavy chain nucleotide sequence (SEQ ID NO: 59)

ATGGACTTTGGGCTCAGCTTACTTATTTTTGTCCTTATTTTAAAAGGTGTCCAGTGTGAG

GTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCC

TGTGCAGCCTCTGGATTCACTTTCAGTGACTACGGAATGGCGTGGGTTCGACAGGCTCCA

GGGAAGGGGCCTGAGTGGGTAGCATTCATTAGTAATTTGGCATATAGTATCTACTATGCA

GACACTGTGACGGGCCGATTCACCATCTCTAGAGAGAATGCCAAGAACACCCTGTACCTG

GAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTACTACTGTGCAAGGTATGACTAC

GATAATATCTTGGACTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC

TCAGCCAAAACAACACCCCCATCAGTCTATCCACTG

Variable part heavy chain, protein sequence (SEQIDNO: 60)

MDFGLSLLIFVLILKGVQCEVKLVESGGGLVQPGGSRKLSCAASGFTFSDYGMAWVRQAP

GKGPEWVAFISNLAYSIYYADTVTGRFTISRENAKNTLYLEMSSLRSEDTAMYYCARYDY

DNILDYVMDYWGQGTSVTVSSAKTTPPSVYPL

2.7.4 clone 24-2-3

Variable part light chain nucleotide sequence (SEQ ID NO: 61)

ATGAAGTTGCCTGTTAGGCTGTTGGTGCTCTGGATTCAGGAAACCAAGGGTGATGTTGTGCTGA

CCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAG

TCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAG

TCTCCAAAGCGCCTAATCTATGTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTG

GCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGT

TTATTATTGCGTGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATA

AAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGT

Variable part light chain protein sequence (SEQIDNO: 62)

MKLPVRLLVLWIQETKGDVVLTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQ

RPGQSPKRLIYVVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTF

GSGTKLEIKRADAAPTVSIFPPSS

Variable part heavy chain nucleotide sequence (SEQ ID NO: 63)

ATGGGATGGAGCGGGGTCTTTCTCTTCCTCCTGTCAGTAACTGAAGGTGTCCACTCCCAGGTTC

AGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGC

TTCTGGCTATATATTCAATAACTACTGGATAAACTGGGTGAAGCAGAGGCCTGGTCAGGGTCTT

GAGTGGATTGGACAGATTTATCCTGGAGATGGTGATACTAACTACAATGGGAAGTTCAAGGGTA

AAGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTAACATC

TGAGGACTCTGCGGTCTATTTCTGTGCAAGAGAGGGATATATTGTTTATTGGGGCCAAGGGACT

CTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTG

Variable part heavy chain, protein sequence (SEQIDNO: 64)

MGWSGVFLFLLSVTEGVHSQVQLQQSGAELVRPGSSVKISCKASGYIFNNYWINWVKQRP

GQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAREGY

IVYWGOGTLVTVSAAKTTPPSVYPL

2.8 antibody clone 6-1-6 for N3pEELISA

The final N3pEELISA protocol was tested for quantitative limit ((LOQ)) and signal-to-noise ratio (S/N). The ELISA standard curve is shown in FIG. 8.

The standard curve shape looks very good for the low concentration range, which shows a nearly linear dependence of the absorbance. Based on this standard curve, LOQ was determined to be 3.125pg/ml, and S/N1.3.

2.9 investigation of Cross-reactivity by ELISA and SPR analysis

ELISA：

Cross-reactivity to other A.beta.variants was determined using our N3 pE-ELISA. The raw data are shown in table 4.

Table 4: raw data for N3pE-ELISA using clones 6-1-6: testing of Cross-reactivity

Concentration (pg/ml)	pE3-40(28.04.)	pE3-40(21.04.)	2-40	3-40	4-40	1-42	3-42	pE11-40
									800	1.8280	1.806	0.048	0.090	0.055	0.053	0.052	0.047
400	0.8750	0.912	0.045	0.065	0.044	0.048	0.052	0.044
									200	0.4350	0.484	0.044	0.057	0.046	0.048	0.049	0.048
100	0.2290	0.248	0.045	0.052	0.045	0.047	0.050	0.048
									50	0.1330	0.143	0.044	0.048	0.044	0.050	0.045	0.045
25	0.0820	0.086	0.044	0.046	0.044	0.048	0.047	0.048
									12.5	0.0570	0.063	0.039	0.042	0.040	0.043	0.042	0.046
0	0.0410	0.040	0.038	0.065	0.061	0.059	0.063	0.066

The concentration dependence of absorbance was observed only for A β pE 3-40. All tested a β variants showed less than 1% cross-reactivity, except a β 3-40. The signal at 800pg/ml (corrected for blank) was about 2.7% of the A β pE3-40 signal. This is a very good value, considering that the N-termini of both peptides have the same amino acid, which is cyclized in the case of a β pE3-40, except for the first amino acid. Overall, the a β N3pE antibody clone 6, commonly used in ELISA, has very high specificity for the N-terminus of the a β -peptide of pGlu starting from position 3.

SPR：

Cross-reactivity of clones 6-1-6 and 24-2-3 to other non A β pGlu peptides was analyzed by surface plasmon resonance. Unlike A β pE3-40, which exhibited a typical binding sensorgram, all other pGlu peptides tested showed little interaction with clones 6-1-6 and 24-2-3, respectively, see also FIG. 9 (data for clone 6-1-6 only is shown). The sensorgram for the pGlu peptide was compared with that of A β pE 3-40. The estimated cross-reactivity was less than 1%. A summary of all analyzed peptides is given in table 5.

Table 5: estimated Cross-reactivity of clones 6-1-6 and 24-2-3 to other pGlu peptides

pGlu peptides	% cross-reactivity
		MCP-1	＜1
MCP-2	＜1
		Dagaojuing liquid medicine	＜1
Gonadotropin releasing hormone	＜1
		Neurotensin	＜1
Orexin A	＜1
		Fibronectin	＜1
Collagen 1	＜1
		TRH	＜1

All experiments confirmed the fact that the N3pE antibody clones 6-1-6 and 24-2-3 are specific for the N-terminal epitope of A β pE 3-x. No other pGluN termini were identified, nor were other a β peptide variants not carrying the N-terminal pE residue identified.

2.10 optimization and validation of N3pEELISA for brain analysis

The A β pE3-42 concentration in mouse brainstem was analyzed according to the method used. The samples were derived from transgenic mice (tg) overexpressing human A β Q3-42 in the brain, which was cyclized to A β pE3-42 by QC. Samples from heterozygous (tghet) and homozygous (tghom) transgenic mice as well as from wild-type (wt) non-transgenic mice were compared. Mice used to generate samples were generated as described in WO 2009034158.

For all further experiments, samples and standards were diluted in EIA buffer. In the next step, the neutralization method used to analyze the formic acid fraction samples was optimized, i.e. the neutralization was N3 pEELISA. The resulting N3pEELISA, and developed herein, performed well, detecting significant levels of human a β pE3-42 in the tghom mouse brain, significantly lower levels of human a β pE3-42 in the tghet mouse brain, and no human a β pE3-42 in the wt mouse brain (see fig. 10).

The ELISA of the invention delivers a high signal and therefore a very acceptable LOQ and is therefore suitable for the analysis of formic acid samples, in particular formic acid brain samples.

2.11N3pE antibody cloning for immunohistochemistry

Using the N3pE antibody of the invention, a β pE3-x was stained in brain sections of patients at a late stage of Sporadic Alzheimer's Disease (SAD) and familial Forms of Alzheimer's Disease (FAD), i.e., patients carrying mutations in senescence gene 1(PS 1). Stained brain sections are shown in fig. 11. Figure 11 shows that the N3pE antibody of the invention is suitable for immunohistochemistry. The antibodies specifically detect pGlu-A β in SAD and FAD patient brains. The N3pE antibody had no background signal in the image, confirming the specific binding shown by ELISA and SPR analysis.

3. Preservation of

Monoclonal antibodies that specifically recognize A.beta.N 3pE-x were generated. All hybridoma cell lines 5-5-6, 6-1-6, 17-4-3 and 24-2-3 expressing the corresponding monoclonal antibodies have been deposited under the Budapest treaty at Deutsche Sammlung fur Mikroorganismen und Zellkulturen (DSMZ) in Braunschweig, DE, on 2008.6 and 17 days, each under a deposit number

(clone 5-5-6): DSMACC2923

(clone 6-1-6): DSMACC2924

(clone 17-4-3): DSMACC2925

(clone 24-2-3): DSMACC2926

The specificity of these antibodies for their respective target sequences can be confirmed. For a β N3pE-x, high affinity antibody clones can be identified that should give a strong signal in an ELISA setting and have the expected limit of detection in the low pg range.

4. Summary of the invention

The main object of the present invention is to establish a highly flexible and robust detection technique that allows quantitative determination of a β variants in biological samples.

Preferably, ELISA-based techniques should be developed. The task starts with a β N3pEELISA, since for this a β variant a suitable ELISA system (IBL) is already commercially available. The system is used as a reference and internal quality control.

The utility of the pGlu-6166 antibody in selected ELISA assay settings was investigated. To obtain a clear measurable signal, high antibody concentrations (20. mu.g/ml) need to be performed. High affinity A.beta.N 3pE-x antibody clones can be identified. Detection limits in the low pg range (3-8pg/ml) can be achieved with these clones.

Claims (25)

1. An antibody characterized in that it binds to pGlu-a β peptide or a variant thereof, wherein said pGlu-a β peptide or variant thereof is selected from the group consisting of:

pGlu-Aβ_3-xin a variant thereof,

wherein x is an integer from 10 to 42; and

wherein the variable portion of the light chain of said antibody consists of the nucleotide sequence of SEQ ID NO. 53, or consists of the amino acid sequence of SEQ ID NO. 54; and is

Wherein the variable part of the heavy chain of the antibody consists of the nucleotide sequence of SEQ ID NO. 55 or the amino acid sequence of SEQ ID NO. 56.

2. The antibody of claim 1, wherein the pGlu-a β peptide or variant thereof is selected from the group consisting of:

pGlu-Aβ_3-38

pGlu-Aβ_3-40and are and

pGlu-Aβ_3-42。

3. the antibody of claim 1, wherein the antibody binds to pGlu-a β peptide or a variant thereof with high affinity, wherein the high affinity is the dissociation constant (K)_D) Value of 10^-7M or better.

4. The antibody of claim 2, wherein the antibody binds to pGlu-a β peptide or a variant thereof with high affinity, wherein the high affinity is the dissociation constant (K)_D) Value of 10^-7M or better.

5. The antibody of any one of claims 1-4, wherein the antibody is a monoclonal antibody.

6. The antibody of any one of claims 1-4, wherein the antibody is a β 6-1-6 produced by hybridoma cell line DSMACC 2924.

7. The antibody of any one of claims 1-4, wherein the antibody is a humanized or chimeric antibody or antibody fragment retaining high affinity, wherein the variable part of the light chain of said antibody fragment consists of the nucleotide sequence of SEQ ID No. 53 or of the amino acid sequence of SEQ ID No. 54; and wherein the variable part of the heavy chain of said antibody fragment consists of the nucleotide sequence of SEQ ID NO. 55 or of the amino acid sequence of SEQ ID NO. 56.

8. The antibody of any one of claims 1 to 4 for use in the detection of pGlu-a β peptide or a variant thereof as defined in claim 1.

9. The antibody of any one of claims 1-4, which is humanized.

10. The antibody of any one of claims 1-4, which is a diabody or a single chain antibody that retains high affinity.

11. The antibody of any one of claims 1-4, which has the complementarity determining regions of the antibody as defined in claim 8.

12. The antibody of any one of claims 1-4, which is labeled.

13. The antibody of any one of claims 1-4, which is immobilized on a solid phase.

14. An antibody derived from the hybridoma cell line DSMACC 2924.

15. A composition comprising an antibody as defined in any preceding claim.

16. The composition of claim 15 for use in the treatment, prevention or delay of amyloidosis.

17. The composition of claim 16, wherein the amyloidosis is a neurodegenerative disease selected from the group consisting of mild cognitive impairment, alzheimer's disease and down's syndrome.

18. The composition of claim 16, wherein the amyloidosis is sporadic alzheimer's disease or familial alzheimer's dementia.

19. The composition of claim 18, wherein the familial alzheimer's dementia is familial british dementia or familial danish dementia.

20. Hybridoma cell line DSMACC 2924.

21. Use of an antibody as defined in any one of claims 1 to 14 or a composition as defined in any one of claims 15 to 19 for the manufacture of a medicament for the diagnosis of an amyloidosis-associated disease or condition.

22. The use of claim 21, wherein the amyloidosis is a neurodegenerative disease selected from the group consisting of mild cognitive impairment, alzheimer's disease and down's syndrome.

23. The use of claim 21, wherein the amyloidosis is sporadic alzheimer's disease or familial alzheimer's dementia.

24. The use of claim 23, wherein the familial alzheimer's dementia is familial british dementia or familial danish dementia.

25. Use of an antibody according to any one of claims 1 to 14 for the preparation of a diagnostic kit for the diagnosis of an amyloidosis-associated disease or condition.