US20100209346A1

US20100209346A1 - Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof

Info

Publication number: US20100209346A1
Application number: US12/559,255
Authority: US
Inventors: Heinz Hillen; Andreas Striebinger; Carsten Krantz; Achim Moeller; Reinhold Mueller
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-01-31
Filing date: 2009-09-14
Publication date: 2010-08-19
Also published as: ZA200506934B; MXPA05007964A; CN1768076A; EP2336160A2; CA2514582C; ES2567437T3; EP1594891A1; KR20050103483A; AU2010257323B8; JP2012250981A; KR20120042959A; JP2010168385A; US9176150B2; EP2336161B1; CN101830974B; AU2010257323A8; EP2336161A2; JP2007524574A; AU2004207075B2; EP2336159A2

Abstract

The invention relates to neuromodulatory oligomers of the amyloid β(1-42) protein, a particular production method, by means of which the oligomer can be obtained in a reproducible manner at high yield, the use of the oligomers and diagnostic and therapeutic agents, for the generation of oligomer-specific antibodies and for the discovery of substances which can interact with the oligomers and in the formation thereof.

Description

The invention relates to neuromodulating oligomers of the amyloid β(1-42) protein, to a specific method of preparation by which said oligomers are obtainable in a reproducible manner and with high yield, and to the use of said oligomers as diagnostic and therapeutic agents, for generating oligomer-specific antibodies and for finding substances capable of interacting with said oligomers and with the formation thereof.
The invention likewise also relates to derivatives of said oligomers, in particular to crosslinked oligomers and oligomers based on truncated forms of the amyloid β(1-42) protein thereof, to the preparation thereof and the use thereof.
Corresponding methods of generating the antibodies and of finding the substances are also described as are the antibodies themselves and the use of said antibodies and substances as diagnostic and therapeutic agents.
Amyloid β(1-42) protein, also referred to as Aβ(1-42) for short, is a central component of insoluble extracellular depositions (senile or neuritic plaques) composed of proteins, lipids, carbohydrates and salts in the brains of Alzheimer and Down's syndrome patients (C. L. Masters et al. PNAS 82, 4245-4249, 1985). This protein which tends to polymerize in an aqueous environment may be present in very different molecular forms.
A simple correlation of the deposition of insoluble protein with the occurrence or progression of dementia disorders such as, for example, Alzheimer's disease has proved to be unconvincing (R. D. Terry et al Ann. Neurol. 30. 572-580 (1991), D. W. Dickson et al. Neurobiol. Aging 16, 285-298 (1995)). In contrast, the loss of synapses and cognitive perception seems to correlate better with soluble forms of Aβ(1-42) (L. F. Lue et al. Am. J. Pathol. 155, 853-862,(1999), C. A. McLean et al. Ann. Neurol. 46, 860-866 (1999)).
With the soluble forms of Aβ(1-42), there are essentially two different hypotheses regarding the molecular forms supposedly to be the cause for dementia disorders such as Alzheimer's disease.
Firstly, a cytotoxic action of Aβ(1-42) protofibrils is' postulated. The latter are still soluble, fibrillar relatively highly aggregated Aβ(1-42) forms having molecular weights in the range from 150-250 kDa (Arispe et al. PNAS 90. 567 (1993), Lashuel et al., Nature 418, 291 (2002)) which, due to pore-forming properties, apparently cause an uncontrolled calcium influx through the membranes of neuronal cells.
Secondly, oligomeric Aβ(1-42) derivatives having molecular weights in the range from 15-30 kDa are described (M. P. Lambert et al. PNAS 95, 6448-6453 (1998)). These nonfibrillar oligomers also referred to as amyloid derived, diffusible and dementing ligands (ADDL's for Amyloid Derived Dementing Ligands, cf. U.S. Pat. No. 6,218,506 and WO 01/10900, or for Amyloid Derived Diffusible Ligands, cf. Lambert et al. supra) can be found in preparations showing an inhibiting influence on the rate of long-term potentiation of neurons in hippocampal sections.
However, the state of previous research on oligomers is characterized by great uncertainty over the actually relevant species. The information in the literature differs greatly. Thus, U.S. Pat. No. 6,218,506 describes ADDLs having from 3 to 12 subunits, whereas the ADDLs described in WO 01/10900 may have up to 24 subunits.
More specifically, at least two forms are discussed which have in a gel-electrophoretic analysis under nondenaturing conditions molecular weights in the range from 27 to 28 kDa and 23 to 24 kDa (U.S. Pat. No. 6,218,506) or from about 26 kDa to 28 kDa (WO 01/10900) and, respectively, 17 kDa and 27 kDa (M. P. Lambert et al. PNAS 95, 6448-6453 (1998)) and in an SDS gel-electrophoretic analysis under denaturing conditions molecular weights of 17 kDa and 22 kDa (M. P. Lambert et al. PNAS 95, 6448-6453 (1998)) and, respectively, of from about 22 kDa to about 24 kDa and from about 18 kDa to about 19 kDa (WO 01/10900). SDS-stable Aβ(1-42) oligomers having molecular weights in the range from 8 kDa and 12 kDa have also been detected previously by Western blot methods on brains of Alzheimer patients (C. A. McLean et al. Ann. Neurol. 46, 860-866 (1999)).
The preparation protocols described have the disadvantage of resulting in inhomogeneous oligomer preparations.
Thus it has not been possible yet to provide in a reproducible manner, let alone unambiguously identify, the molecular forms of Aβ(1-42) responsible for neuromodulation.
However, the importance of a homogeneous preparation can be gauged, for example, from the course of a first clinical study of active immunization on Alzheimer patients. Vaccination with a pre-aggregated form of Aβ(1-42) resulted in considerable side effects (mengioencephalitis, hemorrhages) in some of the patients, since the antibodies formed also recognized the Aβ(1-42) forms presumably required for cell lining, resulting in inflammatory reactions (D. Schenk.; Nat. Rev. Neurosci. 3, 824-828 (2002)).
A custom therapy which especially aims at neutralizing the actually damaging protein form therefore absolutely requires the identification and defined preparation of the latter.
In addition, the occurrence of N-terminally truncated forms of the Aβ(1-42) protein in connection with Alzheimer's disease has been reported. Apart from Aβ(1-42) N-terminally truncated forms were also detected in the depositions of brains of deceased Alzheimer patients as early as 1985 (C. Masters et al., PNAS 82, 4245-4249 (1985)). Thus, particular proteases present in the brain, such as neprilysin (NEP 24.11) or IDE (short for insulin degrading enzyme), are also known to be able to degrade Aβ(1-42) (D. J. Selkoe, Neuron 32, 177-180, (2001)).
However, the importance of the N-terminally truncated forms in the pathogenesis of Alzheimer's disease is unclear (E. B. Lee et al, JBS 278, 4458-4466 (2003)). Interestingly, some patients suffering from sporadic or familial Alzheimer's disease or Down's syndrome preferably accumulate these truncated forms (J. Näslund et al, PNAS 91, 8378-8382, (1994), C. Russo et al., Nature 405, 531-532, (2000), T. C. Saido et al, Neuron 14, 457-466, (1995)). A relatively recent study (N. Sergeant et al, J. of Neurochemistry 85, 1581-1591, (2003)) showed that 60% of all insoluble Aβ peptides in the brains of deceased Alzheimer patients are based on N-terminally truncated forms.
Antibodies directed against monomeric Aβ(1-42) protein and particular fragments thereof have already been described.
Thus, WO 03/016466 and WO 03/016467 relate to the monoclonal antibody 266 and analogs thereof which lack a particular glycosylation site in CDR2. Humanized versions thereof (Hu-266) are also known. These documents also mention other monoclonal antibodies which, like antibody 226, recognize epitopes in the region of amino acids 13-28 of Aβ(1-42). These include the antibodies 4G8 (also mentioned in Lue et al. (1999), supra) and 1C2. Furthermore, McLean et al. (1999), supra, mentions the monoclonal antibody 1 E8 which apparently recognizes an epitope in the region of amino acids 18-22 of Aβ(1-42).
Moreover, a number of further antibodies are known which recognize epitopes of N-terminal sequences of Aβ(1-42). These include commercially available, monoclonal antibody 6E10 (also mentioned in WO 01/10900; Näslund et al. (1994), supra; Sergeant et al. (2003), supra) and the monoclonal antibodies 306 and 10D5 (see WO 03/016467) and also Ban50 and NAB228 (Lee et al. (2003), supra).
Furthermore, mention must be made of the monoclonal antibodies WO2, 21F12 and 3D6 and of the polyclonal serum ADA42 (Sergeant et al. (2003), supra).
An overview over anti-Aβ(1-42) antibodies currently undergoing preclinical testing can be found in Schenk et al. (2002), supra.
The present invention is based on the object of providing the molecular forms which cause the neuromodulating and, in particular, neuron-damaging action of Aβ(1-42) and whose presence is increased in dementia disorders such as Alzheimer's disease and Down's syndrome and achieves said object by means of particular Aβ(1-42) oligomers which are obtainable in the form of homogeneous preparations from monomeric Aβ(1-42) protein, using a special method, and by means of particular derivatives of said oligomers, in particular crosslinked oligomers and oligomers based on truncated Aβ(1-42) forms. The method of preparation enables peptide-synthetic Aβ(1-42) protein which is poorly soluble in aqueous media to be converted to defined soluble oligomers with high yield.
The present invention therefore relates to the subject matters defined in the claims.
Thus, the present invention relates to oligomers of the amyloid β(1-42) protein, said oligomers having an apparent molecular weight of about 15 kDa, 20 kDa, 38 kDa or 48 kDa in SDS gel electrophoresis, or to derivatives of said oligomers having a molecular weight which may or may not have changed according to the derivatization.
Amyloid β(1-42) protein is a polypeptide having 42 amino acids which is derived from the amyloid precursor protein (APP) by proteolytic processing. This also includes, in addition to human variants, isoforms of the amyloid β(1-42) protein present in organisms other than humans, in particular other mammals, especially rats.
According to a particular embodiment, the present invention relates to oligomers of human amyloid β(1-42) proteins. Human amyloid β(1-42) proteins include in particular the protein having the amino acid sequence SEQ ID NO:1 and also muteins and allelic variants thereof derived from said sequence in particular by amino acid exchange. In this connection, very particular mention must be made of the following amino acid substitutions: A21G, E22K, E22Q, E22G and D23N. Thus, the muteins or allelic variants of the amyloid β(1-42) protein include according to the invention in particular proteins having an amino acid sequence SEQ ID NO:1 in which one or more amino acids selected from among alanine 21, glutamic acid 22 and aspartic acid 23 have been substituted by a different amino acid preferably selected from among glycine, lysine, glutamine and asparagine. Particularly important according to the invention are substitutions in position 22, in particular by glutamine or glycine.
According to another particular embodiment, the present invention relates to oligomers of rat amyloid β(1-42) proteins. Rat amyloid β(1-42) proteins include in particular the protein having the amino acid sequence SEQ ID NO:2 and also muteins and allelic variants thereof derived from said sequence in particular by amino acid exchange. In this connection, very particular mention must be made of those amino acid substitutions which correspond to the amino acid substitutions discussed for the human sequence.
Amyloid β(1-42) protein may be prepared by known peptide-synthetic methods or recombinantly. In addition, a number of these proteins are commercially available. The same applies also to muteins and allelic variants.
The oligomers of the invention are obtainable by oligomerization of amyloid β(1-42) protein. The oligomerization comprises a noncovalent aggregation of monomeric amyloid protein so that the oligomers of the invention can be assumed to be composed of a plurality of amyloid β(1-42) protein monomers.
Depending on the degree of oligomerization, the oligomers of the invention have different molecular weights. Thus it is possible to assign apparent molecular weights to said oligomers by means of denaturing gel electrophoresis. Said molecular weights are about 15 kDa for the oligomer A1, about 20 kDa for the oligomer A2, about 38 kDa for the oligomer B1 and about 48 kDa for the oligomer B2, when gel electrophoresis is carried out under standard denaturing conditions (Tris-glycine gel, 4-20%, cf. Lammli UK, Nature 227, 680-685 (1970)), with the following standard proteins having the following apparent molecular weights under identical conditions: myosin 250 kDa, bovine serum albumin 98 kDa, glutamine hydrogenase 64 kDa, carboanhydrase 36 kDa, myoglobin 30 kDa, lysozyme 16 kDa, aprotinin 6 kDa, insulin B chain 4 kDa (cf. Blue Pre-stained Standard). According to another aspect, the molecular weights for the oligomers B are from about 64 to 90 kDa, when gel electrophoresis is carried out under standard native conditions (Tris glycine gel, 4-20%), with the following standard proteins having the following apparent molecular weights under identical conditions: myosin 250 kDa, bovine serum albumin 98 kDa, glutamine hydrogenase 64 kDa, carboanhydrase 36 kDa, myoglobin 30 kDa, lysozyme 16 kDa, aprotinin 6 kDa, insulin B chain 4 kDa (cf. Blue Pre-stained Standard).
According to another aspect, the oligomers of the invention are characterized by an affinity for neuronal cells. It can be assumed that said oligomers bind to particular cell surface proteins, in particular receptors.
Accordingly, the present invention also relates to a method of determining the binding of an oligomer of the invention to a predefined cellular structure, which method comprises

i) allowing said oligomer of the invention to act on said cellular structure and
ii) determining whether said oligomer of the invention binds to said cellular structure.

According to another aspect, the oligomers of the invention are characterized by a neuromodulating action. This neuromodulating action can manifest itself in particular in a reduced survivability of neuronal cells, for example neuroblastoma cells (neuro-toxicity) when at least one oligomer of the invention is allowed to act on a culture of these cells. It is possible here to assess the survivability of said cells in a manner known per se, for example by determining the extent of apoptosis caused by the action of the oligomers of the invention. For this purpose, suitable assay methods, for example colorimetric methods based on 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) are available. This neuromodulating action can manifest itself in vivo in particular in a modulation of the firing rate of neurons.
Accordingly, the present invention also relates to a method of determining the activity, in particular neurotoxicity, of an oligomer of the invention, which method comprises

i) allowing said oligomer of the invention to act on cells and
ii) determining whether the state of said cells is modified.

It is possible to provide for said methods oligomer of the invention in the above-described manner, for example in the form of any of the abovementioned compositions. The cells or cellular structures are conveniently provided in vitro, in particular as cell culture or else, in cellular structures, as homogenates. Here, neuronal cells and, in particular, neuroblastoma cells serve to determine neuro-toxicity. Alternatively, it is also possible to provide the cells in vivo, in particular as part of an organism, for example of an experimental animal, or ex vivo.
The state of the cells is usually determined at least once prior to and at least once after the action of the oligomer. If comparison between the state prior to and the state after the action results in a deviation, the oligomer tested has activity.
The type of the state to be determined depends on the type of the activity to be determined. Neurotoxicity can be determined by determining the survivability, for example. For this purpose, the proportion of living cells based on the total number of cells prior to and after the action of the oligomer can be determined and compared to one another.
Based upon the methods above of determining binding or activity, it is possible, according to particular embodiments, to test substances on whether they inhibit binding of oligomers of the invention and/or modulate, i.e. in particular reduce or essentially completely inhibit, or enhance the activity thereof.
For this purpose, the method is in principle carried out at least twice, once in the presence of the test substance and once more without test substance. To this end, said test substance is added to the oligomers usually after they have been provided.
However, if it is intended to determine whether or not a substance to be tested influences formation of said oligomers, said substance is expediently added to the reactant(s) used for oligomer formation, already prior to formation of said oligomers, i.e. already before they have been provided. Thus it is possible to carry out the preparation method of the invention with addition of the test substance and then to determine whether and to what extent the oligomers are formed. For this purpose, it can be determined whether the method products obtained in this way have the properties of the oligomers of the invention, i.e. their molecular weight, binding ability and activity, for example.
For the purposes of a neuromodulating action as pronounced as possible, preference must be given to the oligomers B of the invention. In this regard, preference is also given to derivatives of these oligomers, with particular emphasis being given to the oligomers based on an N-terminally truncated Aβ(1-42) protein.
For process engineering reasons, the oligomers A and the oligomers B may be produced as a mixture in the form of compositions which, in addition to the oligomers, further comprise small proportions of other polypeptides, in particular monomeric amyloid β(1-42) protein and, where appropriate, also higher molecular weight forms of aggregated amyloid β(1-42) protein. Compositions of this kind are likewise subject matter of the present invention and are distinguished in particular by the proportion of oligomer(s) of the invention being at least 70% by weight, preferably at least 90% by weight, and in particular at least 95% by weight, based on the totality of proteins derived from amyloid β(1-42) protein. In a preparation of oligomers A, the proportion of oligomer A2 (20 kDa band in the SDS gel) is at least 50% by weight and preferably at least 70% by weight and in particular at least 85% by weight. In a preparation of oligomers B, the proportion of oligomers B1 and B2 (38 kDa and 48 kDa bands in the SDS gel) is at least 60% by weight, preferably at least 75% by weight and in particular at least 90% by weight.
The oligomers of the invention may also be derivatized. The purpose of such derivatizations may be, for example, to modulate the physicochemical properties of said oligomers, in particular with respect to bioavailability, to provide said oligomers with a detectable label or to immobilize said oligomers, for example to be able to couple them to supports. Labeling and immobilization are particularly important for diagnostic applications.
Suitable labels familiar in the protein-biochemical field are sufficiently known to the skilled worker. These include fluorescent labels, for example particular fluorescein and tetramethylrhodamine derivatives, luminescent labels, colorimetric labels, radio labels and magnetic labels and also labels with affinity for complementary binding partners, such as biotin and streptavidin derivatives.
With respect to the apparent molecular weights, it must be taken into account that the oligomer derivatives have molecular weights which may have increased correspondingly, compared to the nonderivatized oligomers, but with the aggregation number being identical. Thus, for example, a biotin derivative based on the Aβ(1-42) oligomer B1 having a molecular weight of 38 kDa in the SDS gel has a molecular weight of 42 kDa.
According to a particular embodiment of the present invention, the oligomers are crosslinked. Suitable crosslinkers are known to the skilled worker and are usually bifunctional reagents such as formaldehyde, glutardialdehyde, disuccinimidyl suberate, dithiobis(succinimidyl propionate), disuccinimidyl tartrate, disulfo-succinimidyl tartrate, dimethyl adipimidate, dimethyl pimelidate, dimethyl suberimidate, dimethyl 3,3′-dithiobispropionimidate, N-γ-maleinimidobutyloxy-succinimide ester, succinimidyl 4(N-maleinimidomethyl)cyclohexane-1-carboxylate, N-succinimidyl (4-iodacetyl)aminobenzoate and N-succinimidyl 3-(2-pyridyldithio)-propionate.
Such crosslinked oligomers have the advantage that they are stabilized and that their oligomerization is usually no longer reversible. They are therefore particularly suitable for use in diagnostic test systems or as immunogen for production of oligomer-specific antibodies.
The derivatives of oligomers of the invention also include oligomers of fragments of the amyloid β(1-42) protein. Preference is given to those fragments which are obtainable by the action of naturally occurring proteases. The fragments obtainable by proteolysis in physiological buffers under nondenaturing conditions, in particular, have increased proteolytic stability compared to the amyloid β(1-42) protein. Preference is given to fragments obtainable by the action of endopeptidases. According to a particular embodiment of the present invention, said fragments are obtainable by the action of trypsin, chymotrypsin, thermolysin, elastase, papain or endoproteinase GluC. According to another aspect, preference is given to those fragments whose oligomers of the invention are distinguished by a neuromodulating action. For further illustration of this aspect, reference is made to the corresponding comments on the neuromodulating action of inventive oligomers of the amyloid β(1-42) protein.
Structurally, preferred fragments are in particular characterized in that they are derived from the amyloid β(1-42) protein by removal of N-terminal sequences. According to one aspect, said N-terminal sequences may be fragments having up to 23, preferably having up to 21 and in particular having up to 19, amino acids of the N-terminal sequence of the amyloid β(1-42) protein. Accordingly, preference is given according to the invention to Aβ(1-42) protein fragments whose sequence comprises the contiguous amino acids 24 to 42, preferably 22 to 42 and in particular 20 to 42. According to another aspect, the N-terminal sequences to be removed may be sequences having at least 7, preferably having at least 9 and in particular having at least 11, amino acids of the N-terminal sequence of the amyloid β(1-42) protein. Accordingly, preference is given according to the invention in particular to fragments of the amyloid β(1-42) protein which are N-terminally truncated by from 7 to 23, preferably 9 to 21 and in particular 11 to 19, amino acids. These fragments correspond to the formula Aβ(x-42), where x is from 8 to 24, preferably 10 to 22 and in particular 12 to 20. According to the invention, the Aβ(x-42) fragments are therefore preferably to be selected from among the following fragments: Aβ(8-42), Aβ(9-42), Aβ(10-42), Aβ(11-42), Aβ(12-42), Aβ(13-42), Aβ(14-42), Aβ(15-42), Aβ(16-42), Aβ(17-42), Aβ(18-42), Aβ(19-42), Aβ(20-42), Aβ(21-42), Aβ(22-42), Aβ(23-42) and Aβ(24-42). Special fragments are the amyloid β(20-42) fragment obtainable by the action of thermolysin and the amyloid β(12-42) fragment obtainable by the action of GluC endoproteinase.
Derivatives of the invention are also those forms which are derived from an amyloid β(1-42) protein which has 1 or 2 further C-terminal amino acids. Examples thereof thus include oligomers of the Aβ(1-43) protein or derivatives thereof corresponding to the comments above.
The inventive method of preparing the oligomers may essentially comprise three steps, the first of which is optional but advantageous, the second step is absolutely required for preparation of the oligomers A and B and the third step serves to prepare the oligomers B of the invention.
Step 1 relates to unfolding of the protein. For this purpose, hydrogen bond-breaking agents such as, for example, hexafluoroisopropanol (HFIP) may be allowed to act on the protein. Times of action of a few minutes, for example about 10 to 60 minutes, are sufficient when the temperature of action is from about 20 to 50° C. and in particular about 35 to 40° C. Subsequent dissolution of the residue evaporated to dryness, preferably in concentrated form, in suitable organic solvents miscible with aqueous buffers, such as, for example, dimethyl sulfoxide (DMSO), results in a suspension of the at least partially unfolded protein, which can be used in step 2 of the method of the invention. If required, the stock suspension may be stored at low temperature, for example at about −20° C., for an interim period.
Alternatively to step 1 above, the protein may be taken up in slightly acidic, preferably aqueous, solution, for example an about 10 mM aqueous HCl solution. After an incubation time of usually a few minutes, insoluble components are removed by centrifugation. A few minutes at 10 000 g is expedient. These method steps are preferably carried out at room temperature, i.e. a temperature in the range from 20 to 30° C. The supernatant obtained after centrifugation contains the amyloid β(1-42) protein and may be stored at low temperature, for example at about −20° C., for an interim period.
Step 2 relates to oligomerization of the protein to give the oligomers A. For this purpose, a detergent is allowed to act on the, optionally, at least partially unfolded protein until sufficient oligomer A has been produced.
Preference is given to using ionic detergents, in particular anionic detergents.
According to a particular embodiment, a detergent of the formula (I):
R—X,
is used, in which
the radical R is unbranched or branched alkyl having from 6 to 20 and preferably 10 to 14 carbon atoms or unbranched or branched alkenyl having from 6 to 20 and preferably 10 to 14 carbon atoms,
the radical X is an acidic group or salt thereof, with X being preferably selected from among —COO⁻M⁺, —SO₃M⁺, and especially
—OSO₃ ⁻M⁺ and M⁺ is a hydrogen cation or an inorganic or organic cation preferably selected from alkali metal and alkaline earth metal cations and ammonium cations.
Advantageous are detergents of the formula (I), in which R is unbranched alkyl of which alk-1-yl radicals must be mentioned in particular. Particular preference is given to sodium dodecyl sulfate (SDS). Lauric acid and oleic acid can also be used advantageously. The sodium salt of the detergent lauroylsarcosin (also known as sarkosyl NL-30 or Gardol®) is also particularly advantageous.
The time of detergent action in particular depends on whether—and if yes, to what extent—the protein subjected to oligomerization has unfolded. If, according to step 1, the protein has been treated beforehand with a hydrogen bond-breaking agent, i.e. in particular with hexafluoroisopropanol, times of action in the range of a few hours, advantageously from about 1 to 20 and in particular from about 2 to 10 hours, are sufficient when the temperature of action is about 20 to 50° C. and in particular about 35 to 40° C. If a less unfolded or an essentially not unfolded protein is the starting point, correspondingly longer times of action are expedient. If the amyloid β(1-42) protein has been pretreated, for example, according to the procedure indicated above as an alternative to step 1 or said protein is directly introduced to step 2, times of action in the range from about 5 to 30 hours and in particular from about 10 to 20 hours are sufficient when the temperature of action is about 20 to 50° C. and in particular about 35 to 40° C. After incubation, insoluble components are advantageously removed by centrifugation. A few minutes at 10 000 g is expedient.
The detergent concentration to be chosen depends on the detergent used. If SDS is used, a concentration in the range from 0.01 to 1% by weight, preferably from 0.05 to 0.5% by weight, for example of about 0.2% by weight, proves expedient. If lauric acid or oleic acid are used, somewhat higher concentrations are expedient, for example in a range from 0.05 to 2% by weight, preferably from 0.1 to 0.5% by weight, for example of about 0.5% by weight.
The detergent action should take place at a salt concentration approximately in the physiological range. Thus, in particular NaCl concentrations in the range from 50 to 500 mM, preferably from 100 to 200 mM and particularly at about 140 mM are expedient.
The resulting solution containing the oligomers A, i.e. in particular oligomers A1 and/or A2, can be stored at low temperature, for example at about −20° C., for an interim period. Said solution may be subjected as such to the use of the invention or to further reaction to give the oligomers B, or further work-up or purification steps follow first which achieve in particular further concentration of at least one of the oligomers A of the invention. In particular, it is possible to remove the oligomers of the invention by means of chromatographic methods from other protein components present in the solution and in particular from those derived from the amyloid β(1-42) protein. Particularly suitable for this purpose are affinity chromatography methods which may employ, for example, specific antibodies, i.e. in particular also the oligomer-specific antibodies of the invention.
Step 3 relates to oligomerization to give the oligomers B. Preferably, a composition containing oligomer A is chosen as a reactant for this step. In this respect, the oligomers A are, according to the invention, important intermediates for the preparation of oligomers B. If said composition is from step 2, it regularly contains detergent and a salt concentration in the physiological range. It is then expedient to reduce detergent action and salt concentration. This may be carried out by reducing the concentration of detergent and salt, for example, by diluting, expediently with water or a buffer of lower salt concentration, for example Tris-HCl, pH 7.3. Dilution factors in the range from about 2 to 10, advantageously in the range from about 3 to 8 and in particular of about 4, have proved suitable. The reduction in detergent action may also be achieved by adding substances which can neutralize said detergent action. Examples of these include substances capable of complexing the detergents, like substances capable of stabilizing cells in the course of purification and extraction measures, for example particular EO/PO block copolymers, in particular the block copolymer under the trade name Pluronic® F 68. Alkoxylated and, in particular, ethoxylated alkyl phenols such as the ethoxylated t-octylphenols of the Triton® X series, in particular Triton® X100, 3-(3-cholamidopropyldimethylammonio)-1-propanesulfonate (CHAPS®) or alkoxylated and, in particular, ethoxylated sorbitan fatty esters such as those of the Tween® series, in particular Tween® 20, in concentration ranges around or above the particular critical micelle concentration.
Subsequently, the solution is incubated until sufficient oligomer B has been produced. Times of action in the range of several hours, preferably in the range from about 10 to 30 hours and in particular in the range from about 15 to 25 hours, are sufficient when the temperature of action is about 20 to 50° C. and in particular about 35 to 40° C. The solution may then be concentrated and possible residues may be removed by centrifugation. Here too, a few minutes at 10 000 g proves expedient. The supernatant obtained after centrifugation contains oligomers B.
The resulting solution containing the oligomers B, i.e. in particular the oligomers B1 and/or B2, may be stored at low temperature, for example at about −80° C., for an interim period. Said solution may be subjected as such to the use of the invention or further work-up or purification steps may follow first. Regarding this, reference is made to the comments above on corresponding measures in connection with the oligomers A.
Further work-up or purification steps may also be carried out for the purpose of essentially completely removing the detergent used for preparing the oligomers. For example, the oligomers B may first be precipitated from the detergent-containing solution, isolated and redissolved in detergent-free medium. Measures for precipitating proteins are sufficiently known to the skilled worker. According to the invention, the addition of aqueous-methanolic acetic acid has proved expedient. Without being bound to a particular mechanism, the detergent or variation of detergent and salt concentrations seems to put the protein in defined soluble forms which clearly differ from the protein starting form dissolved in aqueous physiological buffers, as can be detected, for example, by means of denaturing or native gel electrophoresis or gel permeation chromatography. This is astonishing, since detergents are normally capable of disaggregating, i.e. disassembling to subunits, protein aggregates, whereas, according to the invention, defined oligomers are obtained starting from a monomer prone to aggregation.
Derivatives of oligomers of the invention are expediently prepared by carrying out the above-described method of the invention on already appropriately derivatized amyloid β(1-42) protein. Alternatively, it is also possible to derivatize the oligomer but this should not alter the structure of said oligomer. Suitable protein-chemical measures are known to the skilled worker.
Crosslinking of oligomers of the invention or derivatives thereof may be carried out in a manner known per se. If, for example, glutardialdehyde is used as crosslinker, a solution resulting from method step 2 or 3 of the invention can be treated with a glutardialdehyde solution. After a few hours at room temperature, the oligomers will have reacted with the glutardialdehyde. The reaction may then be stopped in a manner known per se by reacting the excess glutardialdehyde with reagents generally known for this purpose, such as ethanolamine. Depending on whether oligomers A or B of the invention are crosslinked, a solution of crosslinked oligomers of the invention referred to as A-CL and B-CL, respectively, is obtained.
It is possible, in particular for the optionally crosslinked oligomers B or derivatives thereof, following the synthesis thereof, to increase again the salt concentration, without impairing the stability of said oligomers. This is important with regard to the use of said oligomers, for example in the case that for the use physiological conditions are expedient (cellular applications, in vivo applications).
Oligomers of fragments of the amyloid β(1-42) protein may be prepared in principle either starting from corresponding monomer fragments by oligomerization or starting from oligomers of said amyloid β(1-42) protein by proteolysis. Thus, according to the second method variant, a solution resulting from method steps 2 or 3 of the invention can be treated with protease. When the desired degree of proteolysis is reached, the protease is inactivated in a generally known manner. The resulting oligomers may then be isolated following the procedures already described herein and, if required, processed further by further work-up and purification steps.
The oligomers of the invention and the compositions comprising them are distinguished by their homogeneity and stability. They are soluble in physiological media, for example physiological saline, and differ from fibrillar forms by their rather globular appearance. They have the advantage of having a spatial structure which is distinctly different from other Aβ(1-42) forms, in particular the monomer, nontoxic oligomers, the Aβ(1-42)-comprising precursor molecule APP, protofibrils and fibrils. They are therefore particularly suitable for generating specific antibodies both in vivo and in vitro and make possible, for example, a specific active immunization. In this connection, it may be crucial to use for immunization only those oligomers which bring on the disease. Other forms of Aβ(1-42), such as the monomeric molecule or smaller oligomers, may be necessary for important signal functions in the organism. Likewise, removal of the fibrillar depositions which are presumably important for cell lining may damage the organism.
In the same way, it is possible to implement possible therapies using the homogeneous oligomers of the invention and compositions containing said oligomers, such as passive immunization, the use of destabilizers of the oligomeric forms and the use of receptor (partial) agonists or antagonists. Thus a homogeneous oligomer preparation makes also possible a specific production of polyclonal or monoclonal antibodies for passive immunization. It is also possible to find receptor molecules or signal molecules which are relevant to the disease and which are influenced by said oligomeric form by using the oligomers of the invention and compositions containing these.
Owing to their involvement in amyloid β protein-associated physiological processes, the oligomers of the invention have diagnostic and therapeutic value. Thus the present invention relates to the use of oligomers of the invention and of derivatives thereof, optionally in the form of a corresponding composition, in diagnostic in vitro and in vivo detection methods.
Amyloid β protein-associated physiological processes include those associated with depositions of amyloid proteins (amyloidoses). These include both those processes which result in structural modifications of nerve tissue and are important, for example, in Alzheimer's disease and Down's syndrome and those processes which affect other tissues, such as amyloid microangiopathies, for example congophilic amyloid angiopathy (CAA).
The present invention further relates to the use of oligomers of the invention and of derivatives thereof, optionally in the form of a corresponding composition, for generating oligomer-specific antibodies.
Here, the inventive oligomers based on truncated forms of the Aβ(1-42) protein are of particular importance: due to the missing N terminus, they can generate a distinctly more selective immune response than the Aβ(1-42) protein. While the highly immunogenic N terminus in the classical Aβ(1-42) protein predominantly produces antibodies which are specific for this region of the molecule, for example the antibodies 6E10 (F. Signet) and BAM-10 (Sigma, St. Louis) which, according to the manufacturers' information, are directed against the N terminus (6E10: Aβ(1-17) and BAM-10 Aβ(1-12)), the antibodies obtainable using the oligomers of the invention based on truncated Aβ(1-42) forms recognize oligomer-specific regions, thereby achieving a selectivity compared to other Aβ(1-42) forms. This advantage may be utilized in particular for active vaccination, since the more selective immune response resulting after vaccination (versus APP, monomeric forms, protofibrils, fibrils, plaques) also entails fewer side effects (e.g. brain hemorrhages, impairment of physiological neurotrophic activity of the in situ monomeric form). Superior production and selection of, in particular, monoclonal antibodies for passive immunization, i.e. an antibody therapy, are also possible.
One aspect of this use is the generation of oligomer-specific antibodies within the framework of a therapy.
The present invention therefore further relates to the use of an oligomer of the invention or of a derivative thereof in the therapeutic field, in particular as vaccine.
Such a vaccine is usually a pharmaceutical composition which comprises at least one oligomer of the invention and/or at least one derivative thereof of the invention. For this purpose, it is possible to use in particular any of the compositions of the invention which include two or more oligomers or a combination of different compositions. Thus it is possible to carry out a vaccination using in particular the oligomers B, i.e. B1 and B2, or derivatives thereof. The composition may further comprise a physiologically suitable carrier and, optionally, further excipients, for example immunostimulants.
While in principle any suitable carriers may be chosen, the type of carrier usually depends on the route of administration. Thus the vaccines of the invention may be formulated in particular in a form suitable for parenteral, for example intravenous, intramuscular and subcutaneous administration. In these cases, the carrier preferably includes water, saline, alcohol, a fat, a wax and/or a buffer.
It is possible to use any of a multiplicity of immunostimulants in the vaccines of the invention. For example, an adjuvant may be included. Most adjuvants contain a substance which ought to protect the antigen from rapid catabolism, such as aluminum hydroxide or a mineral oil, and also a protein derived from lipid A, Bortadella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are usually commercially available, for example complete or incomplete Freund's adjuvant; AS-2; aluminum salts such as aluminum hydroxide (as gel, where appropriate) or aluminum phosphate; calcium salts, iron salts or zinc salts; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biologically degradable microspheres; monophosphoryl lipid A. Cytokines such as GM-CSF or Interleukin-2, -7 or -12 may likewise be used as adjuvants.
The present invention furthermore relates to a method of producing antibodies, which comprises

- i) immunizing a host with at least one oligomer of the invention, derivative thereof or composition; and
- ii) obtaining an antibody-containing host serum produced as a response to said immunization.

According to a particular embodiment of the method of producing the antibodies, the immunization is carried out by administering immunization cocktails comprising mixtures of various oligomers or oligomer derivatives of the invention. In particular, it may be expedient to administer in the course of said method a plurality of immunization cocktails whose oligomer composition differs.
If the oligomers or oligomer derivatives to be used are not or only weakly immunogenic, their immunogenicity may be increased by coupling them to carriers, preferably to a carrier protein such as keyhole limpet hemocyanin (KLH), Limulus polyphenus hemocyanin (LPH), bovine serum albumin (BSA) or ovalbumin (OVA). For this purpose, there are a number of commonly known possible couplings available to the skilled worker. A possible expedient example is the reaction with glutardialdehyde, for example by incubating the oligomer or oligomer mixture with a suitable peptide or peptide mixture in water or an aqueous solvent. This reaction may conveniently be carried out at ambient temperature, i.e. usually room temperature. However, it may also be expedient to reduce or slightly increase the temperature. The reaction usually produces the desired result within a few hours, a reaction time of 2 h being within the usual range, for example. The glutardialdehyde concentration is usually in the ppm to % range, expediently from 10 ppm up to 1%, preferably from 10 ppm to 0.5%. Optimization of the reaction parameters is within the artisan's skills and should take into account that the oligomers A and B or oligomer derivatives are stable under the chosen reaction conditions.
The immunization cocktails are prepared by first combining the components to be used. It is of advantage to incubate the resulting component mixture initially. This is conveniently carried out at ambient temperature, i.e. usually at room temperature. However, it may be expedient to cool or slightly heat said mixture. The incubation period is usually from a few minutes to a few hours, with an incubation time of 1 h having proved advantageous.
Immunization cocktails contain, in addition to the antigen, usually further excipients, in particular adjuvants commonly used for immunization, for example Freund's adjuvant. More specifically, complete Freund's adjuvant is used for the first immunization, whereas any further immunizations are carried out with incomplete Freund's adjuvant. The immunization cocktail is prepared by adding the antigen (immunogen), preferably in the form of the above-described component mixture, to the excipient(s), with the antigen usually being emulsified.
Suitable hosts are in particular rodents or else rabbits. These or other suitable hosts are injected with the immunization cocktails, preferably subcutaneously. The antibody titers may be determined using an immunoassay, for example competitively using a sheep antiserum directed against host IgG and a labeled oligomer. Thus it may be decided toward the end of immunization whether a particular host is suitable for producing antibodies. If, for example, four immunizations are carried out, it is possible to determine the antibody titer after the third immunization and then to obtain antibodies from animals having a sufficient antibody titer.
The antibodies produced are preferably obtained by taking blood from the hosts over a period of several weeks or months. Finally, the host can be bled. Serum containing the desired antibodies may be obtained from the blood obtained in a manner known per se. The whole serum thus obtained may, if required, be further purified by the skilled worker in order to concentrate the antibody fraction present therein and in particular the oligomer-recognizing antibodies.
According to a particular embodiment of this method, at least one antibody of the serum is selected, which antibody specifically recognizes the oligomer used as immunogen or a derivative thereof or at least one oligomer or derivative thereof present in the composition used as immunogen. In this context, specificity means a higher binding affinity of the antibody for the immunogen than for other, in particular related proteins, especially in comparison with monomeric amyloid β(1-42) protein and also with oligomeric or multimeric amyloid β(1-42) protein aggregates having a higher molecular weight than the oligomers of the invention. It is also possible to obtain in this manner monoclonal oligomer-specific antibodies. To this end, however, preference is given to removing from the hosts spleen tissue and, starting from the thus obtained spleen lymphocytes, to establish in the usual manner hybridomas which produce the monoclonal antibodies.

Detailed Description of Antibody Production

B lymphocytes which, in totality, contain an antibody repertoire composed of hundreds of millions of different antibody specificities are part of the mammalian immune system. A normal immune response to a particular antigen means selection of one or more antibodies of said repertoire which specifically bind to said antigen, and the success of an immune response is based at least partially on the ability of said antibodies to specifically recognize (and ultimately to eliminate) the stimulating antigen and to ignore other molecules in the environment of said antibodies.
The usefulness of antibodies which specifically recognize one particular target antigen has led to the development of monoclonal antibody technology. Standardized hybridoma technology now allows the production of antibodies with a single specificity for an antigen of interest. More recently, recombinant antibody techniques such as in-vitro screening of antibody libraries have been developed. These techniques likewise allow antibodies having a single specificity for an antigen of interest to be produced.
In the method of the invention, the antigen of interest may be allowed to act on the antibody repertoire either in vivo or in vitro.
According to one embodiment, the antigen is allowed to act on the repertoire by immunizing an animal in vivo with said antigen. This in-vivo approach may furthermore comprise establishing from the lymphocytes of an animal a number of hybridomas and selecting a hybridoma which secretes an antibody specifically binding to said antigen. The animal to be immunized may be, for example, a mouse, rat, rabbit, chicken, camelid or sheep or may be a transgenic version of any of the animals mentioned above, for example a transgenic mouse with human immunoglobulin genes, which produces human antibodies after an antigenic stimulus. Other types of animals which may be immunized include mice with severe combined immunodeficiency (SCID) which have been reconstituted with human peripheral mononuclear blood cells (chimeric hu-PBMC SCID mice) or with lymphoid cells or precursors thereof, as well as mice which have been treated with a lethal total body irradiation, then protected against radiation with bone marrow cells from a mouse with severe combined immunodeficiency (SCID) and subsequently transplanted with functional human lymphocytes (the “Trimera” system). Another type of an animal to be immunized is an animal (e.g. a mouse) in whose genome an endogenous gene encoding the antigen of interest has been switched off (knocked out), for example by homologous recombination, so that, after immunization with the antigen, said animal recognizes said antigen as foreign. It is obvious to the skilled worker that the polyclonal or monoclonal antibodies produced by this method are characterized and selected by using known screening methods which include, but are not limited to, ELISA techniques.
According to another embodiment, the antigen is allowed to act on the antibody repertoire in vitro by screening a recombinant antibody library with said antigen. The recombinant antibody library may be expressed, for example, on the surface of bacteriophages or on the surface of yeast cells or on the surface of bacterial cells. In a variety of embodiments, the recombinant antibody library is an scFv library or an Fab library, for example. According to another embodiment, antibody libraries are expressed as RNA-protein fusions.
Another approach to producing antibodies of the invention comprises a combination of in vivo and in vitro approaches. For example, the antigen may be allowed to act on the antibody repertoire by immunizing an animal in vivo with said antigen and then screening in vitro with said antigen a recombinant antibody library prepared from lymphoid cells of said animal or a single domain antibody library (e.g. containing heavy and/or light chains). According to another approach, the antigen is allowed to act on the antibody repertoire by immunizing an animal in vivo with said antigen and then subjecting a recombinant antibody library or single domain library produced from lymphoid cells of said animal to affinity maturation. According to another approach, the antigen is allowed to act on the antibody repertoire by immunizing an animal in vivo with said antigen, then selecting individual antibody-producing cells secreting an antibody of interest and obtaining from said selected cells cDNAs for the variable region of the heavy and light chains (e.g. by means of PCR) and expressing said variable regions of the heavy and light chains in mammalian host cells in vitro (this being referred to as selected lymphocyte antibody method or SLAM), thereby being able to further select and manipulate the selected antibody gene sequences. Moreover, monoclonal antibodies may be selected by expression cloning by expressing the antibody genes for the heavy and light chains in mammalian cells and selecting those mammalian cells which secrete an antibody having the desired binding affinity.
Accordingly, one aspect of the present invention is to provide defined antigens for screening and counter screening. Thus it is possible, according to the invention, to select those polyclonal and monoclonal antibodies which bind an oligomer of the invention or derivative thereof but not other forms of the Aβ(1-42) protein, APP, amyloid fibrils or amyloid plaques and a number of other nonrelated antigens and tissues.
It is sufficiently known to the skilled worker that antibody selections are based on well-defined antigens. In contrast, less well-defined antigens are not selective enough when used. In short, the acting and selecting in vitro is similar to affinity chromatography, with “ligands” for the desired antigen being removed from those which bind the antigen with insufficient affinity. The degree of concentration of the desired antibodies from the huge pool of other antibodies is therefore a direct consequence of the quality of the antigen. Surprisingly, the oligomers of the invention and derivatives thereof are antigens which can be used to concentrate suitable, relevant and selective antibodies and efficiently remove them from antibodies which recognize other forms associated with the Aβ(1-42) protein and other nonrelated antigens.
The methods of the invention for producing antibodies can be used to produce various types of antibodies. These include essentially human antibodies, chimeric antibodies, humanized antibodies and CDR graft antibodies and also antigen-binding moieties thereof.
Methods of producing antibodies of the invention are described below. A distinction is made here between in-vivo approaches, in-vitro approaches or a combination of both.

In-Vivo Approaches

Starting from the in-vivo generated antibody-producing cells, monoclonal antibodies may be produced by means of standardized techniques such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also Brown et al. (1981) J. Immunol 127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et at (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75). The technology of producing monoclonal antibody hybridomas is sufficiently known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet., 3:231-36). Briefly, an immortalized cell line (typically a myeloma) is fused with lymphocytes (typically splenocytes or lymph node cells or peripheral blood lymphocytes) of a mammal immunized with the oligomer of the invention or derivative thereof, and the culture supernatants of the resulting hybridoma cells are screened in order to identify a hybridoma which produces a monoclonal antibody with specificity for the oligomer of the invention or for a derivative thereof. Any of the many well known protocols for fusing lymphocytes and immortalized cell lines can be applied for this purpose (see also G. Galfre et at (1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the skilled worker will appreciate that there are diverse variations of such methods, which are likewise useful. Typically, the immortalized cell line (e.g. a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas may be established by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the invention with an immortalized mouse cell line. Preferred immortalized cell lines are mouse myeloma cell lines which are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Any of a number of myeloma cell lines may be used standardwise as fusion partner, for example the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma cell lines are available from the American Type Culture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (PEG). Hybridoma cells resulting from the fusion are then selected using HAT medium, thereby killing unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing monoclonal antibodies which specifically recognize the oligomer of the invention or a derivative thereof are identified by screening the hybridoma culture supernatants for such antibodies, for example by using a standard ELISA assay in order to select those antibodies which can specifically bind the oligomer of the invention or a derivative thereof.
Depending on the type of the desired antibody, various host animals may be used for in-vivo immunization. A host expressing itself an endogenous version of the antigen of interest may be used. Alternatively, it is possible to use a host which has been made deficient in an endogenous version of the antigen of interest. For example, mice which had been made deficient in a particular endogenous protein via homologous recombination at the corresponding endogenous gene (i.e. knockout mice) have been shown to generate a humoral response to the protein with which they have been immunized and therefore to be able to be used for production of high-affinity monoclonal antibodies to the protein (see, for example, Roes, J. et al. (1995) J. Immunol. Methods 183:231-237; Lunn, M. P. et al. (2000) J. Neurochem. 75:404-412).
A multiplicity of nonhuman mammals are suitable hosts for antibody production in order to produce nonhuman antibodies to the oligomer of the invention or to a derivative thereof. They include mice, rats, chickens, camelids, rabbits and goats (and knockout versions thereof), although preference is given to mice for the production of hybridoma. Furthermore, a nonhuman host animal expressing a human antibody repertoire may be used for producing essentially human antibodies to a human antigen with dual specificity. Nonhuman animals of this kind include transgenic animals (e.g. mice) bearing human immunoglobulin transgenes (chimeric hu-PBMC SCID mice) and human/mouse irradiation chimeras which are described in more detail below.
According to one embodiment, the animal immunized with an oligomer of the invention or derivative thereof is a nonhuman mammal, preferably a mouse, which is transgenic due to human immunoglobulin genes so that said nonhuman mammal makes human antibodies upon antigenic stimulation. Typically, immunoglobulin transgenes for heavy and light chains with human germ line configuration are introduced into such animals which have been altered such that their endogenous heavy and light chain loci are inactive. If such animals are stimulated with antigen (e.g. with a human antigen), antibodies derived from the human immunoglobulin sequences (human antibodies) are produced. It is possible to make from the lymphocytes of such animals human monoclonal antibodies by means of standardized hybridoma technology. For a further description of transgenic mice with human immunoglobulins and their use in the production of human antibodies, see, for example, U.S. Pat. No. 5,939,598, WO 96/33735, WO 96/34096, WO 98/24893 and WO 99/53049 (Abgenix Inc.), and U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,877,397 and WO 99/45962 (Genpharm Inc.); see also MacQuitty, J. J. and Kay, R. M. (1992) Science 257:1188; Taylor, L. D. et al. (1992) Nucleic Acids Res. 20:6287-6295; Lonberg, N. et al. (1994) Nature 368:856-859; Lonberg, N. and Huszar, D. (1995) Int. Rev. Immunol. 13:65-93; Harding, F. A. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546; Fishwild, D. M. et al. (1996) Nature Biotechnology 14:845-851; Mendez, M. J. et al. (1997) Nature Genetics 15:146-156; Green, L. L. and Jakobovits, A. (1998) J. Exp. Med. 188:483-495; Green, L. L. (1999) J. lmmunol. Methods 231:11-23; Yang, X. D. et al. (1999) J. Leukoc. Biol. 66:401-410; Gallo, M. L. et al. (2000) Eur. J. Immunol. 30:534-540.
According to another embodiment, the animal which is immunized with an oligomer of the invention or a derivative thereof may be a mouse with severe combined immunodeficiency (SCID), which has been reconstituted with human peripheral mononuclear blood cells or lymphoid cells or precursors thereof. Such mice which are referred to as chimeric hu-PBMC SCID mice produce human immunoglobulin responses upon antigenic stimulation, as has been proved. For a further description of these mice and of their use for generating antibodies, see, for example, Leader, K. A. et al. (1992) Immunology 76:229-234; Bombil, F. et al. (1996) Immunobiol. 195:360-375; Murphy, W. J. at al. (1996) Semin. Immunol. 8:233-241; Herz, U. at al. (1997) Int. Arch. Allergy Immunol. 113:150-152; Albert, S. E. et al. (1997) J. Immunol. 159:1393-1403; Nguyen, H. et al. (1997) Microbiol. Immunol. 41:901-907; Arai, K. et al. (1998) J. Immunol. Methods 217:79-85; Yoshinari, K. and Arai, K. (1998) Hybridoma 17:41-45; Hutchins, W. A. et al. (1999) Hybridoma 18:121-129; Murphy, W. J. et al. (1999) Clin. Immunol. 90:22-27; Smithson, S. L. et al. (1999) Mol. Immunol. 36:113-124; Chamat, S. et al. (1999) J. Infect. Diseases 180:268-277; and Heard, C. et al. (1999) Molec. Med. 5:35-45.
According to another embodiment, the animal which is immunized with an oligomer of the invention or a derivative thereof is a mouse which has been treated with a lethal total body irradiation, then protected from radiation with bone marrow cells from mice with severe combined immunodeficiency (SCID) and subsequently transplanted with functional human lymphocytes. This type of chimera, referred to as the Trimera system, is used in order to produce human monoclonal antibodies by immunizing said mice with the antigen of interest and then producing monoclonal antibodies by using standardized hybridoma technology. For a further description of these mice and of their use for generating antibodies, see, for example, Eren, R. et al. (1998) Immunology 93:154-161; Reisner, Y and Dagan, S. (1998) Trends Biotechnol. 16:242-246; Han, E. et al. (1999) Hepatology 29:553-562; and Bocher, W. O. et al. (1999) Immunology 96:634-641.

In-Vitro Approaches

As an alternative to producing antibodies of the invention by immunization and selection, antibodies of the invention may be identified and isolated by screening recombinant combinatorial immunoglobulin libraries with an oligomer of the invention or derivative thereof to thereby isolate immunoglobulin library members which specifically bind to said oligomer or derivative thereof. Kits for generating and screening display libraries are commercially available (e.g. the Pharmacia Recombinant Phage Antibody System, catalog No. 27-9400-01; and the Stratagene SurfZAP® Phage Display Kit, catalog No. 240612). In many embodiments, the display library is an scFv library or an Fab library. The phage display technique for screening recombinant antibody libraries has been adequately described. Examples of methods and compounds which can be used particularly advantageously for generating and screening antibody display libraries can be found, for example, in McCafferty et al. WO 92/01047, U.S. Pat. No. 5,969,108 and EP 589 877 (describes in particular scFv display), Ladner et al. U.S. Pat. No. 5,223,409, U.S. Pat. No. 5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,837,500 and EP 436 597 (describes pill fusion, for example); Dower et al. WO 91/17271, U.S. Pat. No. 5,427,908, U.S. Pat. No. 5,580,717 and EP 527 839 (describes in particular Fab display); Winter et al. International Publication WO 92/20791 and EP 368,684 (describes in particular the cloning of sequences for variable immunoglobulin domains); Griffiths et al. U.S. Pat. No. 5,885,793 and EP 589 877 (describes in particular isolation of human antibodies to human antigens by using recombinant libraries); Garrard et al. WO 92/09690 (describes in particular phage expression techniques); Knappik et al. WO 97/08320 (describes the human recombinant antibody library HuCal); Salfeld et al. WO 97/29131, (describes production of a recombinant human antibody to a human antigen (human tumor necrosis factor alpha) and also in-vitro affinity maturation of the recombinant antibody) and Salfeld et al. U.S. Provisional Application No. 60/126,603 and the patent applications based hereupon (likewise describes production of recombinant human antibodies to human antigen (human interleukin-12), and also in-vitro affinity maturation of the recombinant antibody).
Further descriptions of screenings of recombinant antibody libraries can be found in scientific publications such as Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; McCafferty et al. Nature (1990) 348:552-554; and Knappik et al. (2000) J. Mol. Biol. 296:57-86.
As an alternative to using bacteriophage display systems, recombinant antibody libraries may be expressed on the surface of yeast cells or of bacterial cells. WO 99/36569 describes methods of preparing and screening libraries expressed on the surface of yeast cells. WO 98/49286 describes in more detail methods of preparing and screening libraries expressed on the surface of bacterial cells.
Once an antibody of interest of a combinatorial library has been identified, the DNAs encoding the light and heavy chains of said antibody are isolated by means of standardized molecular-biological techniques, for example by means of PCR amplification of DNA from the display package (e.g. the phage) which has been isolated during library screening. Nucleotide sequences of genes for light and heavy antibody chains, which may be used for preparing PCR primers, are known to the skilled worker. A multiplicity of such sequences are described, for example, in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and in the database of sequences of the human germ line VBASE.
An antibody or antibody moiety of the invention may be produced by recombinantly expressing the genes for light and heavy immunoglobulin chains in a host cell. In order to recombinantly express an antibody, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the light and heavy immunoglobulin chains of said antibody, thereby expressing the light and heavy chains in the host cell and secreting them preferably into the medium in which said host cells are cultured. The antibodies can be isolated from this medium.
Standardized recombinant DNA methods are used in order to obtain genes for heavy and light antibody chains, to insert said genes into recombinant expression vectors and to introduce said vectors into host cells. Methods of this kind are described, for example, in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al.
Once DNA fragments encoding VH and VL segments of the antibody of interest are obtained, said DNA fragments may be further manipulated using standardized recombinant DNA techniques, for example in order to convert the genes for variable regions to genes for full length antibody chains, to genes for Fab fragments or to an scFv gene. These manipulations comprise linking a VL- or VH-encoding DNA fragment operatively to another DNA fragment encoding another protein, for example a constant antibody region or a flexible linker. The term “operatively linked” is to be understood here as meaning that the two DNA fragments are linked to one another in such a way that the amino acid sequences encoded by said two DNA fragments remain in frame.
The isolated DNA encoding the VH region may be converted to a gene for a full length heavy chain by operatively linking the VH-region encoding DNA with another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are well known (see, for example, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments spanning said regions may be obtained by means of standardized PCR amplification. The heavy chain constant region may be a constant region from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, with preference being given to a constant region from IgG1 or IgG4. To obtain a gene for a heavy chain Fab fragment, the VH-encoding DNA may be operatively linked to another DNA molecule encoding merely the heavy chain constant region CH1.
The isolated DNA encoding the VL region may be converted to a gene for a full length light chain (and a gene for an Fab light chain) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of genes of the constant region of human light chain are well known (see Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments spanning said regions may be obtained by means of standardized PCR amplification. The light chain constant region may be a constant kappa or lambda region, a constant kappa region being preferred.
In order to generate an scFv gene, the VH- and VL-encoding DNA fragments may be operatively linked to another fragment encoding a flexible linker, for example the amino acid sequence (Gly₄-Ser)₃so that the VH and VL sequences are expressed as a continuous single-chain protein, with the VL and VH regions being linked to one another via said flexible linker (see Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554). Single domain VH and VL with specificity for an oligomer of the invention or for a derivative thereof may be isolated from single domain libraries by the above-described methods. Two VH single-domain chains (with or without CH1) or two VL chains or a pair of one VH chain and one VL chain with the desired specificity may be used for removing oligomers of the invention or derivatives thereof from the body.
In order to express the recombinant antibodies or antibody moieties of the invention, the DNAs encoding partial or full length light and heavy chains may be inserted into expression vectors so as to operatively link the genes to transcriptional and translational control sequences. In this context, the term “operatively linked” is to be understood as meaning that an antibody gene is ligated in a vector in such a way that transcriptional and translational control sequences within the vector fulfill their intended function of regulating transcription and translation of said antibody gene.
The expression vector and the expression control sequences are chosen so as to be compatible with the expression host cell used. The gene for the antibody light chain and the gene for the antibody heavy chain may be inserted into separate vectors or both genes are inserted into the same expression vector, this being the usual case. The antibody genes are inserted into the expression vector by means of standardized methods (for example ligation of complementary restriction cleavage sites on the antibody gene fragment and the vector, or ligation of blunt ends, if no restriction cleavage sites are present). The expression vector may already carry sequences for antibody constant regions prior to insertion of the sequences for the light and heavy chains. For example, one approach is to convert the VH and VL sequences to full length antibody genes by inserting them into expression vectors already encoding the heavy and, respectively, light chain constant regions, thereby operatively linking the VH segment to the CH segment(s) within the vector and also operatively linking the VL segment to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide which facilitates secretion of the antibody chain from the host cell. The gene for said antibody chain may be cloned into the vector, thereby linking the signal peptide in frame to the N terminus of the gene for the antibody chain. The signal peptide may be an immuno-globulin signal peptide or a heterologous signal peptide (i.e. a signal peptide from a non-immunoglobulin protein). In addition to the genes for the antibody chain, the expression vectors of the invention may have regulatory sequences controlling expression of the genes for the antibody chain in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers and further expression control elements (e.g. polyadenylation signals) which control transcription or translation of the genes for the antibody chain. Regulatory sequences of this kind are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). The skilled worker will appreciate that the expression vector design which includes selection of regulatory sequences may depend on factors such as the choice of host cell to be transformed, the desired strength of expression of the protein, etc. Preferred regulatory sequences for expression in mammalian host cells include viral elements resulting in a strong protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), simian virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus (e.g. the adenovirus major late promoter (AdMLP)) and polyoma. For a further description of viral regulatory elements and sequences thereof, see, for example, U.S. Pat. No. 5,168,062 to Stinski, U.S. Pat. No. 4,510,245 to Bell et al. and U.S. Pat. No. 4,968,615 to Schaffner et al.
Apart from the genes for the antibody chain and the regulatory sequences, the recombinant expression vectors of the invention may have additional sequences such as those which regulate replication of the vector in host cells (e.g. origins of replication) and selectable marker genes. The selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all to Axel et al.). For example, it is common for the selectable marker gene to render a host cell into which the vector has been inserted resistant to drugs such as G418, hygromycin or methotrexate. Preferred selectable marker genes include the gene for dihydrofolate reductase (DHFR) (for use in dhfr host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
For expression of the light and heavy chains, the expression vector(s) encoding said heavy and light chains is(are) transfected into a host cell by means of standardized techniques. The various forms of the term “transfection” are intended to comprise a multiplicity of techniques customarily used for introducing exogenous DNA into a prokaryotic or eukaryotic host cell, for example electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibodies of the invention either in prokaryotic or eukaryotic host cells, preference is given to expressing the antibodies in eukaryotic cells and, in particular, in mammalian host cells, since the probability of a correctly folded and immunologically active antibody being assembled and secreted is higher in such eukaryotic cells and in particular mammalian cells than in prokaryotic cells. Prokaryotic expression of antibody genes has been reported as being ineffective for production of high yields of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).
Preferred mammalian host cells for expressing recombinant antibodies of the invention include CHO cells (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, which are used together with a DHFR-selectable marker, as described, for example, in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. When introducing recombinant expression vectors encoding the antibody genes into mammalian host cells, the antibodies are produced by culturing the host cells until the antibody is expressed in said host cells or, preferably, the antibody is secreted into the culture medium in which the host cells grow. The antibodies may be isolated from the culture medium by using standardized protein purification methods.
It is likewise possible to use host cells in order to produce moieties of intact antibodies, such as Fab fragments or scFv molecules. Variations of the above-described procedure are of course included in the invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody of the invention. If either light or heavy chains are present which are not required for binding of the antigen of interest, then the DNA encoding either such a light or such a heavy chain or both may be removed partially or completely by means of recombinant DNA technology. Molecules expressed by such truncated DNA molecules are likewise included in the antibodies of the invention. In addition, it is possible to produce bifunctional antibodies in which a heavy chain and a light chain are an antibody of the invention and the other heavy chain and the other light chain have specificity for an antigen different from the antigen of interest, by crosslinking an antibody of the invention to a second antibody by means of standardized chemical methods.
In a preferred system for recombinant expression of an antibody of the invention or an antigen-binding moiety thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr CHO cells by means of calcium phosphate-mediated transfection. Within the recombinant expression vector, the genes for the heavy and light antibody chains are in each case operatively linked to regulatory CMV enhancer/AdMLP-promoter elements in order to effect strong transcription of said genes. The recombinant expression vector also carries a DHFR gene which can be used for selecting CHO cells transfected with the vector by using methotrexate selection/amplification. The selected transformed host cells are cultured so that the heavy and light antibody chains are expressed, and intact antibody is isolated from the culture medium. Standardized molecular-biological techniques are used in order to prepare the recombinant expression vector, to transfect the host cells, to select the transformants, to culture said host cells, and to obtain the antibody from the culture medium. Thus the invention relates to a method of synthesizing a recombinant antibody of the invention by culturing a host cell of the invention in a suitable culture medium until a recombinant antibody of the invention has been synthesized. The method may furthermore comprise isolating said recombinant antibody from said culture medium.
As an alternative to screening recombinant antibody libraries by phage display, other methods known to the skilled worker may be used for screening large combinatorial libraries to identify the antibodies of the invention. In one type of an alternative expression system, the recombinant antibody library is expressed in the form of RNA-protein fusions, as described in WO 98/31700 to Szostak and Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, in-vitro translation of synthetic mRNAs carrying on their 3′ end puromycin, a peptidyl acceptor antibiotic, generates a covalent fusion of an mRNA and the peptide or protein encoded by it. Thus a specific mRNA of a complex mixture of mRNAs (e.g. a combinatorial library) may be concentrated on the basis of the properties of the encoded peptide or protein (e.g. of the antibody or a moiety thereof), such as binding of said antibody or said moiety thereof to an oligomer of the invention or a derivative thereof. Nucleic acid sequences which encode antibodies or moieties thereof and which are obtained by screening of such libraries may be expressed by recombinant means in the above-described manner (e.g. in mammalian host cells) and may, in addition, be subjected to further affinity maturation by either screening in further rounds mRNA-peptide fusions, introducing mutations into the originally selected sequence(s), or using other methods of in-vitro affinity maturation of recombinant antibodies in the above-described manner.

Combinations of In-Vivo and In-Vitro Approaches

The antibodies of the invention may likewise be produced by using a combination of in-vivo and in-vitro approaches such as methods in which an oligomer of the invention or a derivative thereof is first allowed to act on an antibody repertoire in a host animal in vivo to stimulate production of oligomer- or derivative-binding antibodies and then further antibody selection and/or antibody maturation (i.e. optimization) are accomplished with the aid of one or more in-vitro techniques. According to one embodiment, a combined method of this kind may comprise firstly immunizing a nonhuman animal (e.g. a mouse, rat, rabbit, chicken, camelid, goat or a transgenic version thereof or a chimeric mouse) with said oligomer of the invention or derivative thereof to stimulate an antibody response to the antigen and then preparing and screening a phage display antibody library by using immunoglobulin sequences of lymphocytes which have been stimulated in vivo by the action of said oligomer or derivative. The first step of this combined procedure may be carried out in the manner described above in connection with the in-vivo approaches, while the second step of this procedure may be carried out in the manner described above in connection with the in-vitro approaches. Preferred methods of hyperimmunizing nonhuman animals with subsequent in-vitro screening of phage display libraries prepared from said stimulated lymphocytes include those described by BioSite Inc., see, for example, WO 98/47343, WO 91/17271, U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,580,717.
According to another embodiment, a combined method comprises firstly immunizing a nonhuman animal (e.g. a mouse, rat, rabbit, chicken, camelid, goat or a knockout and/or transgenic version thereof, or a chimeric mouse) with an oligomer of the invention or derivative thereof to stimulate an antibody response to said oligomer or derivative thereof and selecting the lymphocytes which produce the antibodies having the desired specificity by screening hybridomas (prepared, for example, from the immunized animals). The genes for the antibodies or single domain antibodies are isolated from the selected clones (by means of standardized cloning methods such as reverse transcriptase polymerase chain reaction) and subjected to in-vitro affinity maturation in order to improve thereby the binding properties of the selected antibody or the selected antibodies. The first step of this procedure may be conducted in the manner described above in connection with the in-vivo approaches, while the second step of this procedure may be conducted in the manner described above in connection with the in-vitro approaches, in particular by using methods of in-vitro affinity maturation, such as those described in WO 97/29131 and WO 00/56772.
In a further combined method, the recombinant antibodies are generated from individual isolated lymphocytes by using a procedure which is known to the skilled worker as selected lymphocyte antibody methods (SLAM) and which is described in U.S. Pat. No. 5,627,052, WO 92/02551 and Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method, a nonhuman animal (e.g. a mouse, rat, rabbit, chicken, camelid, goat, or a transgenic version thereof, or a chimeric mouse) is firstly immunized in vivo with an oligomer of the invention or a derivative thereof to stimulate an immune response to said oligomer or derivative, and then individual cells secreting antibodies of interest are selected by using an antigen-specific hemolytic plaque assay. To this end, the oligomer or derivative thereof or structurally related molecules of interest may be coupled to sheep erythrocytes, using a linker such as biotin, thereby making it possible to identify individual cells secreting antibodies with suitable specificity by using the hemolytic plaque assay. Following the identification of cells secreting antibodies of interest, cDNAs for the variable regions of the light and heavy chains are obtained from the cells by reverse transcriptase PCR, and said variable regions may then be expressed in association with suitable immunoglobulin constant regions (e.g. human constant regions) in mammalian host cells such as COS or CHO cells. The host cells transfected with the amplified immunoglobulin sequences derived from in vivo-selected lymphocytes may then be subjected to further in-vitro analysis and in-vitro selection by spreading out the transfected cells, for example, in order to isolate cells expressing antibodies with the desired specificity. The amplified immunoglobulin sequences may furthermore be manipulated in vitro.
Analogously to the above-described procedures, it is also possible to prepare antibodies having specificity for an oligomer of the invention or derivative thereof and for structurally related synthetic molecules. This comprises

- i) providing an antigen which comprises a structural feature shared by the oligomer of the invention or derivative thereof and the structurally related synthetic molecules;
- ii) exposing an antibody repertoire to said antigen; and
- iii) selecting from said repertoire an antibody which binds to two structurally related molecules, thereby obtaining the antibody having the desired specificity.

The present invention relates to the oligomer-specific antibodies obtainable by the above methods as well as to the use thereof for preparing a medicament for the treatment of amyloid β-associated dementing disorders or for preparing a composition for diagnosing amyloid β-associated dementing disorders.
The antibodies obtainable according to the invention include in particular antisera which can be obtained by the above methods. Said antisera may be whole sera, i.e. blood obtained from the host after removing the cellular and coagulable components, or fractions of said serum which contain in particular a concentrated immunoglobulin fraction and preferably a concentrated, oligomer-recognizing immunoglobulin fraction. Fractions of this kind may be obtained using the methods described above in connection with antibody purification.
The antisera of the invention are polyclonal, i.e. they contain antibodies of different specificity, usually of different classes and subclasses, normally all L-chain isotypes are represented and multiple protein epitopes are recognized.
When using various oligomers of the invention as immunogens, then the antisera of the invention are usually cross-reactive.
According to another aspect, the antibodies obtainable according to the invention also include monoclonal antibodies, in particular chimeric and humanized antibodies, and also oligomer-binding fragments thereof.
The present invention relates to proteins and in particular to antibodies binding to an oligomer of the invention or derivative thereof, i.e. antibodies having specificity for an oligomer of the invention or derivative thereof. The present invention also relates to moieties of said proteins or antibodies, in particular to antigen-binding moieties thereof, i.e. protein or antibody moieties binding to an oligomer of the invention or derivative thereof.
The antibody of the invention is preferably chosen so as to have particular binding kinetics (e.g. high affinity, low dissociation, low off rate, strong neutralizing activity) for the specific binding to an oligomer of the invention or a derivative thereof.
Thus preference is given to proteins and, in particular, antibodies having an affinity for the oligomer of the invention or derivative thereof in the range of K_D=10⁻⁸-10⁻¹²M. Particular preference is given to high-affinity proteins and in particular antibodies binding with an affinity greater than K_d=10⁻⁸M, with an affinity greater than K_d=10⁻⁹M, with an affinity greater than K_d=10⁻¹⁰M or with an affinity greater than K_d=10⁻¹¹M.
According to another aspect, preference is given to those proteins and, in particular, antibodies which bind other Aβ(1-42) forms, in particular monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein with comparatively lower affinity, in particular with lower affinity than K_d=10⁻⁸M.
Accordingly, preference is given according to the invention especially to those proteins and, in particular, antibodies which bind the oligomer or derivative thereof with higher affinity than monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein. Particular preference is given to affinity ratios of 10, 100 or 1000.
According to another aspect, the antibodies of the invention may be chosen so as to bind the oligomer or derivative thereof with a k_offrate constant of 0.1 s⁻¹or less. Increasing preference is given to rate constants k_offof 1×10⁻²s⁻¹or less, 1×10⁻³s⁻¹or less, 1×10⁻⁴s⁻¹or less, or 1×10⁻⁸s⁻¹or less, in the order indicated.
Furthermore, antibodies of the invention may be chosen so as to inhibit the activity, in particular the neurotoxic activity, of oligomers of the invention or derivatives thereof with an IC₅₀of 1×10⁻⁶M or less. Increasing preference is given to inhibition constants IC₅₀of 1×10⁻⁷M or less, 1×10⁻⁸M or less, 1×10⁹M or less, 1×10 −10 M or less, or 1×10⁻¹¹M or less, in the order indicated.
The antibodies are preferably isolated antibodies. According to another aspect, the antibodies are neutralizing antibodies. The antibodies of the invention include monoclonal and recombinant antibodies. According to a multiplicity of embodiments, the antibody may comprise an amino acid sequence derived entirely from a single species, such as a human antibody or a mouse antibody. According to other embodiments, the antibody may be a chimeric antibody or a CDR graft antibody or another form of a humanized antibody.
The term “antibody” is intended to refer to immunoglobulin molecules consisting of 4 polypeptide chains, two heavy (H) chains and two light (L) chains. The chains are usually linked to one another via disulfide bonds. Each heavy chain is composed of a variable region of said heavy chain (abbreviated here as HCVR or VH) and a constant region of said heavy chain. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain is composed of a variable region of said light chain (abbreviated here as LCVR or VL) and a constant region of said light chain. The light chain constant region consists of a CL domain. The VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region consists of three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The term “antigen-binding moiety” of an antibody (or simply “antibody moiety”) refers to one or more fragments of an antibody having specificity for an oligomer of the invention or derivative thereof, said fragment(s) still being capable of specifically binding said oligomer or derivative thereof. Fragments of a complete antibody have been shown to be able to carry out the antigen-binding function of an antibody. In accordance with the term “antigen-binding moiety” of an antibody examples of binding fragments include (i) an Fab fragment, i.e. a monovalent fragment composed of the VL, VH, CL and CH1 domains; (ii) an F(ab′)₂fragment, i.e. a bivalent fragment comprising two Fab fragments linked to one another in the hinge region via a disulfide bridge; (iii) an Fd fragment composed of the VH and CH1 domains; (iv) an Fv fragment composed of the FL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain or of VH, CH1, CH2, DH3, or VH, CH2, CH3; and (vi) an isolated complementarity-determining region (CDR). Although the two domains of the Fv fragment, namely VL and VH, are encoded by separate genes, they may further be linked to one another using a synthetic linker and recombinant methods, making it possible to prepare them as a single protein chain in which the VL and VH regions combine in order to form monovalent molecules (known as single chain Fv (ScFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). The term “antigen-binding moiety” of an antibody is also intended to comprise such single chain antibodies. Other forms of single chain antibodies such as “diabodies” are likewise included here. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker which is too short for the two domains being able to combine on the same chain, thereby forcing said domains to pair with complementary domains of a different chain and to form two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
Furthermore, an antibody or antigen-binding moiety thereof may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of said antibody or antibody moiety with one or more further proteins or peptides. Relevant to such immunoadhesion molecules are the use of the streptavidin core region in order to prepare a tetrameric scFv molecule (Kipriyanov; S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of a cystein residue, a marker peptide and a C-terminal polyhistidine tag in order to produce bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody moieties such as Fab and F(ab′)₂fragments may be produced from whole antibodies by using conventional techniques such as digestion with papain or pepsin. In addition, antibodies, antibody moieties and immunoadhesion molecules may be obtained by using standardized recombinant DNA techniques. An “isolated antibody having specificity for an oligomer of the invention or derivative thereof” means an antibody having specificity for an oligomer of the invention or derivative thereof, which is essentially free of other antibodies having different antigen specificities, i.e. in particular an antibody which is free of antibodies specifically binding to other forms of the Aβ(1-42) protein, as described above.
The term “neutralizing antibody” means an antibody whose binding to a particular antigen results in the inhibition of the biological activity of said antigen. Said inhibition of the biological activity of the antigen may be assessed by measuring one or more indicators for said biological activity of the antigen, using a suitable in-vitro or in-vivo assay.
The term “monoclonal antibody” means an antibody derived from a hybridoma (e.g. an antibody secreted by a hybridoma prepared by means of hybridoma technology such as the standardized hybridoma methods according to Kohler and Milstein). An antibody which is derived from a hybridoma and which has specificity for an oligomer of the invention or derivative thereof is therefore referred to as monoclonal antibody.
The term “recombinant antibody” refers to antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes (see, for example, Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295); or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric, CDR graft and humanized antibodies.
The term “human antibody” refers to antibodies whose variable and constant regions correspond to or derive from immunoglobulin sequences of the human germ line, as described, for example, by Kabat et al. (see Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, the human antibodies of the invention may contain amino acid residues not encoded by human germ line immunoglobulin sequences (for example mutations which have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular in CDR3. Recombinant human antibodies of the invention have variable regions and may also contain constant regions derived from immunoglobulin sequences of the human germ line (see Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). According to particular embodiments, however, such recombinant human antibodies are subjected to in-vitro mutagenesis (or to a somatic in-vivo mutagenesis, if an animal is used which is transgenic due to human Ig sequences) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences which although related to or derived from VH and VL sequences of the human germ line, do not naturally exist in vivo within the human antibody germ line repertoire. According to particular embodiments, recombinant antibodies of this kind are the result of selective mutagenesis or back mutation or of both.
The term “back mutation” refers to a method which comprises replacing some of or all of the somatically mutated amino acids of a human antibody with the corresponding germ line residues of a homologous germ line antibody sequence. The sequences for the heavy and light chains of a human antibody of the invention are compared separately with the germ line sequences in the VBASE database to identify the sequences having the highest homology. Imprints on the human antibodies of the invention are reverted to the germ line sequence by mutating defined nucleotide positions encoding such deviating amino acids. The direct or indirect importance of each amino acid identified in this way as a candidate for a back mutation for antigen binding should be investigated, and an amino acid impairing a desired property of said human antibody after mutation should not be incorporated in the final human antibody. In order to keep the number of amino acids for back mutation as low as possible, those amino acid positions which, although deviating from the closest germ line sequence, are identical to the corresponding amino acid sequence of a second germ line sequence may remain unchanged, provided that said second germ line sequence is identical and colinear with the sequence of the human antibody of the invention in at least 10 and preferably in 12 amino acids on both sides of the amino acid in question. Back mutations may be carried out at any stage of antibody optimization.
The term “chimeric antibody” refers to antibodies which contain sequences for the variable region of the heavy and light chains from one species but in which the sequences of one or more of the CDR regions of VH and/or VL have been replaced with CDR sequences of another species, such as antibodies having variable regions of the heavy and light chains from mouse, in which one or more of the mouse CDRs (e.g. CDR3) have been replaced with human CDR sequences.
The term “humanized antibody” refers to antibodies which contain sequences of the variable region of heavy and light chains from a nonhuman species (e.g. mouse, rat, rabbit, chicken, camelid, goat) but in which at least one part of the VH and/or VL sequence has been altered in order to be more “human-like”, i.e. to be more similar to variable sequences of the human germ line. One type of a humanized antibody is a CDR graft antibody in which human CDR sequences have been inserted into nonhuman VH and VL sequences to replace the corresponding nonhuman CDR sequences.
One way of measuring the binding kinetics of an antibody is by means of surface plasmon resonance. The term “surface plasmon resonance” refers to an optical phenomenon by which biospecific interactions can be analyzed by detecting changes in protein concentrations by means of a biosensor matrix, using, for example, the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.
The term “K_off” refers to the off rate constant for the dissociation of an antibody from the antibody/antigen complex.
The term “K_d” refers to the dissociation constant of a particular antibody-antigen interaction.
The binding affinity of the antibodies of the invention may be evaluated by using standardized in-vitro immunoassays such as ELISA or BIAcore analyses.
Apart from antibodies, the protein may be a T cell receptor-derived molecule or a T cell receptor-derived receptor domain or a fusion protein of said receptor domain with an Fc moiety of an immunoglobulin.
The present invention also relates to pharmaceutical agents (compositions) containing a protein of the invention and in particular an antibody of the invention and also, optionally, a pharmaceutically suitable carrier. Pharmaceutical compositions of the invention may furthermore contain at least one additional therapeutic agent, for example one or more additional therapeutic agents for the treatment of a disease for whose relief the antibodies of the invention are useful. If, for example, the antibody of the invention binds to an oligomer of the invention, the pharmaceutical composition may furthermore contain one or more additional therapeutic agents useful for the treatment of disorders in which the activity of said oligomer is important.
Pharmaceutically suitable carriers include any solvents, dispersing media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, and the like, as long as they are physiologically compatible. Pharmaceutically acceptable carriers include, for example, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases, preference is given to using isotonic agents, for example sugars, polyalcohols such as mannitol or sorbitol, or sodium chloride in addition. Pharmaceutically suitable carriers may furthermore contain relatively small amounts of auxiliary substances such as wetting agents or emulsifiers, preservatives or buffers, which increase the half life or efficacy of the antibodies.
The pharmaceutical compositions may be suitable for parenteral administration, for example. Here, the antibodies are prepared preferably as injectable solutions with an antibody content of 0.1-250 mg/ml. The injectable solutions may be prepared in liquid or lyophilized form, the dosage form being a flint glass or vial, an ampoule or a filled syringe. The buffer may contain L-histidine (1-50 mM, preferably 5-10 mM) and have a pH of 5.0-7.0, preferably of 6.0. Further suitable buffers include, without being limited thereto, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate buffers. Sodium chloride may be used in order to adjust the tonicity of the solution to a concentration of 0-300 mM (preferably 150 mM for a liquid dosage form). Cryoprotectants, for example sucrose (e.g. 0-10%, preferably 0.5-1.0%) may also be included for a lyophilized dosage form. Other suitable cryoprotectants are trehalose and lactose. Fillers, for example mannitol (e.g. 1-10%, preferably 2-4%) may also be included for a lyophilized dosage form. Stabilizers, for example L-methionine (e.g. 51-50 mM, preferably 5-10 mM) may be used both in liquid and lyophilized dosage forms. Further suitable fillers are glycine and arginine. Surfactants, for example polysorbate 80 (e.g. 0-0.05%, preferably 0.005-0.01%), may also be used. Further surfactants are polysorbate 20 and BRIJ surfactants.
The compositions of the invention may have a multiplicity of forms. These include liquid, semisolid and solid dosage forms, such as liquid solutions (e.g. injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended type of administration and on the therapeutic application. Typically, preference is given to compositions in the form of injectable or infusible solutions, for example compositions which are similar to other antibodies for passive immunization of humans. The preferred route of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). According to a preferred embodiment, the antibody is administered by intravenous infusion or injection. According to another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.
Therapeutic compositions must typically be sterile and stable under the preparation and storage conditions. The compositions may be formulated as solution, micro-emulsion, dispersion, liposome or another ordered structure suitable for high active substance concentrations. Sterile injectable solutions may be prepared by introducing the active compound (i.e. the antibody) in the required amount into a suitable solvent, where appropriate with one or a combination of the abovementioned ingredients, as required, and then sterile-filtering said solution. Dispersions are usually prepared by introducing the active compound into a sterile vehicle containing a basic dispersion medium and, where appropriate, other required ingredients. In the case of a sterile lyophilized powder for preparing sterile injectable solutions, vacuum drying and spray drying are preferred methods of preparation, which produces a powder of the active ingredient and, where appropriate, of further desired ingredients from a previously sterile-filtered solution. The correct flowability of a solution may be maintained by using, for example, a coating such as lecithin, by maintaining, in the case of dispersions the required particle size or by using surfactants. A prolonged absorption of injectable compositions may be achieved by additionally introducing into the composition an agent which delays absorption, for example monostearate salts and gelatin.
The antibodies of the invention may be administered by a multiplicity of methods known to the skilled worker, although the preferred type of administration for many therapeutic applications is subcutaneous injection, intravenous injection or infusion. The skilled worker will appreciate that the route and/or type of administration depend on the result desired. According to particular embodiments, the active compound may be prepared with a carrier which protects the compound against rapid release, such as, for example, a formulation with controlled release, which includes implants, transdermal plasters and microencapsulated release systems. Biologically degradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycol acid, collagen, polyorthoesters and polylactic acid may be used. The methods of preparing such formulations are well known to the skilled worker, see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
According to particular embodiments, an antibody of the invention may be administered orally, for example in an inert diluent or an assimilable edible carrier. The antibody (and further ingredients, if desired) may also be enclosed in a hard or soft gelatin capsule, compressed to tablets or added directly to food. For oral therapeutic administration, the antibodies may be mixed with excipients and used in the form of swallowable tablets, buccal tablets, capsules, elixirs, suspensions, syrups and the like. If it is intended to administer an antibody of the invention via a route other than the parenteral one, it may be necessary to choose a coating from a material which prevents its inactivation.
The antibodies of the invention are preferably capable of neutralizing, both in vitro and in vivo, the activity of oligomers of the invention or derivatives thereof to which they bind. Said antibodies may therefore be used for inhibiting the activity of oligomers of the invention or derivatives thereof, for example in a cell culture containing said oligomers or derivatives thereof or in human individuals or other mammals in which said oligomers or derivatives thereof are present. According to one embodiment, the invention relates to a method of inhibiting the activity of oligomers of the invention or derivatives thereof, which method comprises allowing an antibody of the invention to act on an oligomer or derivative thereof so as to inhibit the activity of said oligomer or derivative thereof. Said activity may be inhibited in vitro, for example. For example, the antibody of the invention may be added to a cell culture which contains or is suspected to contain the oligomer of the invention or derivative thereof, in order to inhibit the activity of said oligomer or derivative thereof in said culture. Alternatively, the activity of the oligomer or derivative thereof may be inhibited in an individual in vivo.
Thus the present invention further relates to a method of inhibiting the activity of oligomers of the invention or derivatives thereof in an individual who suffers from a disorder in which the amyloid β protein is involved and in which in particular the activity of said oligomer of the invention or derivative thereof is important. Said method comprises the administration of at least one antibody of the invention to the individual with the aim of inhibiting the activity of the oligomer or derivative thereof to which the antibody binds. Said individual is preferably a human being. An antibody of the invention may be administered for therapeutic purposes to a human individual. In addition, an antibody of the invention may be administered to a nonhuman mammal for veterinary purposes or within the framework of an animal model for a particular disorder. Such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (for example for testing dosages and time courses of administration).
Disorders in which the oligomers of the invention or derivatives thereof play a part include in particular disorders in whose development and/or course an oligomer of the invention or derivative thereof is involved. These are in particular those disorders in which oligomers of the invention or derivatives thereof are evidently or presumably responsible for the pathophysiology of said disorder or are a factor which contributes to the development and/or course of said disorder. Accordingly, those disorders are included here in which inhibition of the activity of oligomers of the invention or derivatives thereof can relieve symptoms and/or progression of the disorder. Such disorders can be verified, for example, by an increased concentration of oligomers of the invention or derivatives thereof in a biological fluid of an individual suffering from a particular disorder (e.g. increased concentration in serum, plasma, CSF, urine, etc.). This may be detected, for example, by using an antibody of the invention. The oligomers of the invention and derivatives thereof play an important part in the pathology associated with a multiplicity of disorders in which neurodegenerative elements, cognitive deficits, neurotoxic elements and inflammatory elements are involved.
The antibodies of the invention may be administered together with one or more additional therapeutic agents which are useful in the treatment of the above-described disorders.
The pharmaceutical compositions of the present invention usually contain a therapeutically active amount or a prophylactically active amount of at least one antibody of the invention. Depending on the treatment desired, for example whether a therapeutic or prophylactic treatment is desired, dosage plans can be chosen and adapted. For example, a single dose, multiple separate doses distributed over time or an increasing or decreasing dosage may be administered, depending on the requirements of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in single dosage form in order to facilitate administration and to ensure uniformity of the dosage.
A therapeutically or prophylactically active amount of an antibody of the invention may be, for example, in the range of 0.1-20 mg/kg and preferably 1-10 mg/kg, without being limited thereto: These amounts may, of course, vary, depending on the type and severity of the condition to be relieved.
Within the framework of diagnostic usage of the antibodies, qualitative or quantitative specific oligomer determination serves in particular to diagnose disease-relevant amyloid β(1-42) forms. In this context, specificity means the possibility of being able to detect a particular oligomer or oligomer mixture with sufficient sensitivity. The antibodies of the invention advantageously have sensitivities of less than 10 ng/ml of sample, preferably of less than 1 ng/ml of sample and particularly preferably of less than 100 pg/ml of sample, meaning that at least the concentration of oligomer per ml of sample, indicated in each case, advantageously also lower concentrations, can be detected by the antibodies of the invention.
The determination is carried out immunologically. This may be carried out in principle by using any analytical or diagnostic assay method in which antibodies are used, including agglutination and precipitation techniques, immunoassays, immunohistochemical methods and immunoblot techniques, for example Western blotting or dot blot methods. In vivo methods, for example imaging methods, are also included here.
The use in immunoassays is advantageous. Suitable are both competitive immunoassays, i.e. antigen and labeled antigen (tracer) compete for antibody binding, and sandwich immunoassays, i.e. binding of specific antibodies to the antigen is detected by a second, usually labeled antibody. These assays may be either homogeneous, i.e. without separation into solid and liquid phases, or heterogeneous, i.e. bound labels are separated from unbound ones, for example via solid phase-bound antibodies. Depending on labeling and method of measurement, the various heterogeneous and homogeneous immunoassay formats can be classified into particular classes, for example RIAs (radioimmunoassays), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescence immunoassay), LIA (luminescence immunoassay), TRFIA (time-resolved FIA), IMAC (immunoactivation), EMIT (enzyme-multiplied immune test), TIA (turbodimetric immunoassay), I-PCR (immuno-PCR).
For the oligomer determination of the invention, preference is given to competitive immunoassays in which labeled oligomer (tracer) competes with the oligomer to be quantified of the sample for binding to the antibody used. The amount of antigen, i.e. the amount of oligomer, in the sample can be determined from the amount of the displaced tracer with the aid of a standard curve.
Of the labels available for these purposes, enzymes have proved advantageous. Systems based on peroxidases, in particular horseradish peroxidase, alkaline phosphatase and β-D-galactosidase, may be used, for example. Specific substrates whose conversion can be monitored photometrically, for example, are available for these enzymes. Suitable substrate systems are based on p-nitrophenyl phosphate (p-NPP), 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NPT), Fast-Red/naphthol-AS-TS phosphate for alkaline phosphatase; 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPT), 3,3′,5,5′-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 3-dimethylamino-benzoic acid (DMAB) and 3-methyl-2-benzothiazolinehydrazone (MBTH) for peroxidases; o-nitrophenyl-β-D-galactoside (o-NPG), p-nitrophenyl-β-D-galactoside and 4-methylumbelliphenyl-β-D-galactoside (MUG) for β-D-galactosidase. In many cases, these substrate systems are commercially available in a ready-to-use form, for example in the form of tablets which may also contain further reagents such as appropriate buffers and the like.
The tracers used are labeled oligomers. In this sense, a particular oligomer can be determined by labeling the oligomer to be determined and using it as tracer.
The coupling of labels to oligomers for preparing tracers may be carried out in a manner known per se. The comments above on derivatization of oligomers of the invention are referred to by analogy. In addition, a number of labels appropriately modified for conjugation to proteins are available, for example biotin-, avidin-, extravidin- or streptavidin-conjugated enzymes, maleimide-activated enzymes and the like. These labels may be reacted directly with the oligomer or, if required, with the appropriately derivatized oligomer to give the tracer. If, for example, a streptavidin-peroxidase conjugate is used, then this firstly requires biotinylation of the oligomer. This applies correspondingly to the reverse order. For this purpose too, suitable methods are known to the skilled worker.
If a heterogeneous immunoassay format is chosen, the antigen-antibody complex may be separated by binding it to the support, for example via an anti-idiotypical antibody coupled to said support, e.g. an antibody directed against rabbit IgG. Supports, in particular microtiter plates coated with appropriate antibodies are known and partly commercially available.
The present invention further relates to immunoassay sets having at least one above-described antibody and further components. Said sets are, usually in the form of a packaging unit, a combination of means for carrying out an oligomer determination of the invention. For the purpose of handling which is as easy as possible, said means are preferably provided in an essentially ready-to-use form. An advantageous arrangement offers the immunoassay in the form of a kit. A kit usually comprises multiple containers for separate arrangement of components. All components may be provided in a ready-to-use dilution, as a concentrate for diluting or as a dry substance or lyophilisate for dissolving or suspending; individual or all components may be frozen or stored at room temperature until use. Sera are preferably shock-frozen, for example at −20° C. so that in these cases an immunoassay has to be kept preferably at temperatures below freezing prior to use.
Further components supplied with the immunoassay depend on the type of said immunoassay. Usually, standard protein, tracer which mayor may not be required and control serum are supplied together with the antiserum. Furthermore, microtiter plates, preferably antibody-coated, buffers, for example for testing, for washing or for conversion of the substrate, and the enzyme substrate itself may also be included.
General principles of immunoassays and generation and use of antibodies as auxiliaries in laboratory and hospital can be found, for example, in Antibodies, A Laboratory Manual (Harlow, E., and Lane, D., Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988).
Furthermore of interest are also substances which inhibit aggregation of the oligomers of the invention or accelerate disaggregation thereof. Such substances particularly represent possible therapeutic agents for the treatment of the above amyloid β-associated disorders such as, for example, Alzheimer's disease.
The present invention therefore also relates to a method of characterizing a substance or a substance mixture, which method comprises

- i) providing said substance or said substance mixture in a suitable manner;
- ii) allowing said substance or said substance mixture to act on at least one oligomer of the invention, derivative thereof or a composition; and
- iii) determining whether said substance or particular parts of said substance mixture bind to said oligomer, derivative thereof or to at least one oligomer or derivative thereof present in said composition.

Said method may also be carried out on mixtures of biological origin, for example a cell preparation and cell extracts, in order to identify natural binding partners with affinity for the oligomers of the invention, for example cell surface antigens, in particular receptors, and soluble ligands, for example particular proteins and mediators.
In addition to mere binding of the substances, further interactions with and influences on the oligomers of the invention may also be a subject of said method. Thus it is possible to determine, in particular, whether

- the substance is capable of modulating, in particular inhibiting, the aggregation of amyloid β protein to the oligomers of the invention;
- the substance is capable of modulating, in particular promoting, disaggregation of the oligomers of the invention;
- the oligomers of the invention cause functional changes in a binding partner, for example have an agonistic, partially agonistic, antagonistic or inverse agonistic effect on a receptor.

Said methods are usually in vitro-screening methods which can be used to select from a multiplicity of different substances those which appear to be most promising with respect to a future application. It is possible, for example, to establish by means of combinatorial chemistry extensive substance libraries comprising myriads of potential active substances. The screening of combinatorial substance libraries for substances having the desired activity can be automated. Screening robots serve to efficiently analyze the individual assays which are preferably arranged on microtiter plates. Thus the present invention also relates to screening methods, i.e. both primary and secondary screening methods, in which preferably at least one of the methods described below is applied. If several methods are applied, they may be applied to one and the same sample with a time shift or simultaneously or to different samples of a substance to be tested.
A particularly effective technique for carrying out such methods is the scintillation proximity assay, SPA for short, which is known in the field of drug screening. Kits and components for carrying out this assay may be obtained commercially, for example from Amersham Pharmacia Biotech. In principle, solubilized or membrane-bound receptors are immobilized on small fluoromicrospheres containing a scintillating substance. When, for example, a radioligand binds to the immobilized receptors, said scintillating substance is stimulated and emits light, due to the spatial proximity of scintillating substance and radioligand.
Another particularly effective technique for carrying out methods of this kind is the FlashPlateR technique known in the field of drug screening. Kits and components for carrying out this assay may be commercially obtained, for example from NENR Life Science Products. This principle is likewise based on microtiter plates (96- or 384-well) coated with scintillating substance.
The present invention relates to substances or parts of substance mixtures, which are identifiable according to this method as a ligand binding to the oligomer, derivative thereof or to at least one oligomer or derivative thereof present in a corresponding composition, as well as to the use thereof for preparing a medicament for the treatment of amyloid &associated, in particular dementing, disorders or for preparing a composition for diagnosing amyloid β-associated, in particular dementing, disorders.
The following examples are intended to illustrate the invention, without limiting its scope.

In the drawings:

FIG. 1 shows an SDS PAGE of an Aβ(1-42) oligomer A preparation (lane A); an Aβ(1-42) oligomer B preparation (lane B); of standard proteins (molecular marker proteins, lane C);

FIG. 2 shows an SDS PAGE of an Aβ(1-42) oligomer A preparation (lane A); an Aβ(1-42) oligomer A-CL preparation (lane A′); an Aβ(1-42) oligomer B preparation (lane B); an Aβ(1-42) oligomer B-CL preparation (lane B′); of standard proteins (molecular marker proteins, lane C);

FIG. 3 shows an SDS PAGE of a biotin Aβ(1-42) oligomer B preparation (lane A); of standard proteins (molecular marker proteins, lane B);

FIG. 4 shows an SDS PAGE of a fluorescein Aβ(1-42) oligomer B preparation (lane A); of standard proteins (molecular marker proteins, lane B);

FIG. 5 shows a gel permeation chromatography of a solution containing Aβ(1-42) lyophilisate in comparison with a preparation containing Aβ(1-42) oligomers B;

FIG. 6 shows a NATIVE PAGE of an Aβ(1-42) oligomer B preparation (lane A); of standard proteins (molecular marker proteins, lane B);

FIG. 7 shows the binding of (A) monomeric Aβ(1-42) protein and (B) of the Aβ(1-42) oligomers A and (C) Aβ(1-42) oligomers B to the surface of the human neuroblastoma cell line IMR-32;

FIG. 8 shows the neurotoxic effect in % after treatment of murine cortical neurons with Aβ(1-42) oligomers B, the error bar corresponding to the 95% confidence interval;

FIG. 9 shows an SDS PAGE of an Aβ(1-42) preparation treated with trypsin (lane 2), chymotrypsin (lane 3), thermolysin (lane 4), elastase (lane 5), papain (lane 6) or untreated (lane 7); and of standard proteins (molecular marker proteins, lane 1);

FIG. 10 shows dot blots of the reactivity of 100 pmol (row A); 10 pmol (row B); 1 pmol (row C); 0.1 pmol (row D) and 0.01 pmol (row E); of Aβ(1-42) oligomer B preparation of example 6b (column 1); of HFIP-treated Aβ(1-42) monomer of example 1a (column 2); of thermolysin-cleaved Aβ(1-42) oligomer B preparation of example 15a (column 3); of glutaraldehyde-crosslinked Aβ(1-42) oligomer B preparation of example 14a (column 4); of ADDL prepared according to M. P. Lambert et al., J. Neurochem. 79, 595-605 (2001) at 4° C. or room temperature or 37° C. (

columns

5, 6 and 7, respectively); of Aβ(1-42) dissolved in 0.1% NH₄OH (column 8); of an Aβ(1-42) fibril preparation of example 27 (column 9); and of PBS-diluted APP from Sigma (column 10) with a) the monoclonal antibody 6E10; b) the polyclonal antiserum (d1) of example 25d; c) the polyclonal antiserum (c1) of example 25c; d) the polyclonal antiserum (a1) of example 25a; and e) the polyclonal antiserum (a2) of example 25a;

FIG. 11 shows an SDS PAGE of an Aβ(20-42) oligomer B preparation of example 15a (lane B); of an Aβ(12-42) oligomer B preparation of example 15b (lane C); of an Aβ(1-42) oligomer B preparation of example 6b (lane A); and of standard proteins (molecular marker proteins, lane D);

FIG. 12 shows a gel permeation chromatography of the Aβ(1-42) oligomer B preparation of example 6b in comparison with the Aβ(1-42) oligomer B-CL preparation of example 14a;

FIG. 13 shows a diagrammatic representation of the monomeric Aβ(1-42) protein (left); of the Aβ(1-42) oligomer (12-mer, A); and of the Aβ(12-42) oligomer (12-mer, C) and Aβ(20-42) oligomer (12-mer, B) both of which are obtainable by proteolytic cleavage;

FIG. 14 shows the excitatory postsynaptic potential (fEPSP) as a function of the time of action of Aβ(1-42) oligomers B on hippocampal sections;

FIG. 15 shows an immunofluorescence image depicting the binding of Aβ(1-42) oligomers B to hippocampal rat neurons (a) in comparison with unspecifically bound fluorescence (b).

Unless stated otherwise, concentrations of oligomers of the invention are expressed in moles of monomeric Aβ(1-42) polypeptide. The term β amyloid(1-42) protein corresponds to the term amyloid β(1-42) protein. Unless stated otherwise, the proteins (polypeptides) used are of human origin.

Example 1

a) Preparation of a Stock Suspension of Human Aβ(1-42)

2 mg of human β-amyloid(1-42) protein (short name: Aβ(1-42); peptide-synthetic material, lyophilisate, from Bachem, Germany) are dissolved in 800 μl of 1,1,1,3,3,3-hexafluoro-2-propanol and incubated in an Eppendorf vessel at 37° C. for 30 min. This is followed by evaporation to dryness in a vacuum concentrator (Speed Vac). The residue is taken up with 88 μl of DMSO, resulting in a 5 mM Aβ(1-42) stock suspension which can be stored at −20° C.

b) Preparation of a Stock Suspension or Rat Aβ(1-42)

2 mg of rat β-amyloid(1-42) protein (short name: rat Aβ(1-42); peptide-synthetic material, lyophilisate, from Bachem, Germany) by dissolving 800 μl of 1,1,1,3,3,3-hexafluoro-2-propanol and incubated in an Eppendorf vessel at 37° C. for 30 min. This is followed by evaporation to dryness in a vacuum concentrator (Speed Vac). The residue is taken up with 88 μl of DMSO, resulting in a 5 mM rat Aβ(1-42) stock suspension which can be stored at −20° C.

Example 2

a) Preparation of Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [Aβ(1-42) Oligomers A]; use of SDS
690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) are added to 60 μl of the stock solution of example 1a and the mixture is adjusted to SDS content of 0.2% with 75 μl of 2% strength sodium dodecyl sulfate (SDS) solution. This is followed by 5 hours of incubation at 37° C. and centrifugation for 10 min at 10 000 g. This Aβ(1-42) oligomer A preparation (approx. 400 μM Aβ(1-42)) can be stored at −20° C.
b) Preparation of Rat Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [Rat Aβ(1-42) Oligomers A]; use of SDS
690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) are added to 60 μl of the stock solution of example 1b and the mixture is adjusted to SDS content of 0.2% with 75 μl of 2% strength sodium dodecyl sulfate (SDS) solution. This is followed by 5 hours of incubation at 37° C. and centrifugation for 10 min at 10 000 g. This rat Aβ(1-42) oligomer A preparation (approx. 400 μM rat Aβ(1-42)) can be stored at −20° C.
c) Preparation of Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [Aβ(1-42) Oligomers A]; Use of Lauric Acid
690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) are added to 60 μl of the stock solution of example 1a and the mixture is adjusted to lauric acid content of 0.5% with 75 μl of 5% strength lauric acid solution. This is followed by 5 hours of incubation at 37° C. and centrifugation for 10 min at 10 000 g. This Aβ(1-42) oligomer A preparation (approx. 400 μM Aβ(1-42)) can be stored at −20° C.
d) Preparation of Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [Aβ(1-42) Oligomers A]; Use of Oleic Acid
690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) are added to 60 μl of the stock solution of example 1a and the mixture is adjusted to oleic acid content of 0.5% with 75 μl of 5% strength oleic acid solution. This is followed by 5 hours of incubation at 37° C. and centrifugation for 10 min at 10 000 g. This Aβ(1-42) oligomer A preparation (approx. 400 μM Aβ(1-42)) can be stored at −20° C.
e) Preparation of Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [Aβ(1-42) Oligomers A]; Use of Lauroylsarcosine
690 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) are added to 60 μl of the stock solution of example 1a and the mixture is adjusted to lauroylsarcosine content of 0.5% with 75 μl of 5% strength lauroylsarcosine solution. This is followed by 5 hours of incubation at 37° C. and centrifugation for 10 min at 10 000 g. This Aβ(1-42) oligomer A preparation (approx. 400 μM Aβ(1-42)) can be stored at −20° C.

Example 3

a) Alternative Method of Preparing Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [Aβ(1-42) Oligomers A]
1 mg of human β-amyloid(1-42) protein (short name: Aβ(1-42); peptide-synthetic material, lyophilisate, from Bachem, Germany) is taken up in 220 μl of 10 mM aqueous HCl solution and incubated at room temperature for 10 min. Insoluble components are removed by centrifugation at 10 000 g for 5 min. The supernatant (1 mM Aβ(1-42)) contains the Aβ(1-42) protein and is processed further as follows:
9 μl of PBS buffer and 1 μl of 2% strength SDS soloution are added to 1 μl of the supernatant and the mixture is incubated at 37° C. for 16 h. The hAβ(1-42) oligomer A preparation (100 μM) can be stored at −20° C.
b) Alternative Method of Preparing rat Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [Rat Aβ(1-42) Oligomers A]
1 mg of rat β-amyloid(1-42) protein (short name: rat Aβ(1-42); peptide-synthetic material, lyophilisate, from Bachem, Germany) is taken up in 220 μl of 10 mM aqueous HCl solution and incubated at room temperature for 10 min. Insoluble components are removed by centrifugation at 10 000 g for 5 min. The supernatant (1 mM rat Aβ(1-42)) contains the rat amyloid-β(1-42) protein and is processed further as follows:
9 μl of PBS buffer and 1 μl of 2% strength SDS soloution are added to 1 μl of the supernatant and the mixture is incubated at 37° C. for 16 h. The rat Aβ(1-42) oligomer A preparation (100 μM) can be stored at −20° C.

Example 4

a) Alternative Method of Preparing Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [Aβ(1-42) Oligomers A]
1 mg of human β-amyloid(1-42) protein (short name: Aβ(1-42); peptide-synthetic material, lyophilisate, from Bachem, Germany) is dissolved in 44 μl of 1% SDS/H₂O (5 mM Aβ(1-42)). 5 μl of the solution are admixed with 40 μl PBS and 5 μl of 2% SDS and incubated at 37° C. for 16 h. Insoluble components are removed by centrifugation at 10 000 g for 5 min. The thus obtained Aβ(1-42) oligomer A preparation (500 μM Aβ(1-42)) can be stored at −20° C.
b) Alternative Method of Preparing Rat Aβ(1-42) Oligomers having Molecular Weights of 15 kDa and 20 kDa [rat Aβ(1-42) Oligomers A]
1 mg of rat β-amyloid(1-42) protein (short name: rat A(3(1-42); peptide-synthetic material, lyophilisate, from Bachem, Germany) is dissolved in 44 μl of 1% SDS/H₂O (5 mM rat Aβ(1-42)). 5 μl of the solution are admixed with 40 μl PBS and 5 μl of 2% SDS and incubated at 37° C. for 16 h. Insoluble components are removed by centrifugation at 10 000 g for 5 min. The thus obtained rat Aβ(1-42) oligomer A preparation (500 μM rat Aβ(1-42)) can be stored at −20° C.

Example 5

a) Preparation of Aβ(1-42) Oligomers having Molecular Weights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]
An Aβ(1-42) oligomer A solution obtained according to example 2a is diluted with 2.475 ml of water (0.05% SDS content, 0.1 mM Aβ(1-42)) and incubated at 37° C. for 20 hours. Aliquots of this Aβ(1-42) oligomer B preparation can be frozen at −80° C. and stored for further studies.
b) Preparation of Rat Aβ(1-42) Oligomers having Molecular Weights of 38 kDa and 48 kDa [Rat Aβ(1-42) Oligomers B]
A rat Aβ(1-42) oligomer A solution obtained according to example 2a is diluted with 2.475 ml of water (0.05% SDS content, 0.1 mM rat Aβ(1-42)) and incubated at 37° C. for 20 hours. Aliquots of this rat Aβ(1-42) oligomer B preparation can be frozen at −80° C. and stored for further studies.
c) Alternative Preparation of Aβ(1-42) Oligomers having Molecular Weights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]
An Aβ(1-42) oligomer A solution obtained according to example 2c is diluted with 2.475 ml of water (0.125% lauric acid content, 0.1 mM Aβ(1-42)) and incubated at 37° C. for 20 hours. Aliquots of this Aβ(1-42) oligomer B preparation can be frozen at −80° C. and stored for further studies.
d) Alternative Preparation of Aβ(1-42) Oligomers having Molecular Weights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]
An Aβ(1-42) oligomer A solution obtained according to example 2d is diluted with 2.475 ml of water (0.125% oleic acid content, 0.1 mM Aβ(1-42)) and incubated at 37° C. for 20 hours. Aliquots of this Aβ(1-42) oligomer B preparation can be frozen at −80° C. and stored for further studies.
e) Alternative preparation of Aβ(1-42) Oligomers B having Molecular Weights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]
An Aβ(1-42) oligomer A solution obtained according to example 2e is diluted with 2.475 ml of water (0.125% laurylsarcosine content, 0.1 mM Aβ(1-42)) and incubated at 37° C. for 20 hours. Aliquots of this Aβ(1-42) oligomer B preparation can be frozen at −80° C. and stored for further studies.
f) Preparation of SDS-Free Aβ(1-42) Oligomers B having Molecular Weights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]
10 μl of an Aβ(1-42)oligomer B preparation prepared according to example 6b are admixed with 250 μl of an acetic acid/methanol/water mixture in a 4%/33%/63% ratio and incubated at 0° C. on ice for 30 min. After centrifugation (10 000 g for 10 min), the supernatant is removed and the precipitated protein residue taken up to 200 μl of buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4). The preparation obtained in this way contains the dissolved Aβ(1-42) oligomers B in SDS-free from and can be stored at −20° C.

Example 6

a) Dialysis and Concentration of Aβ(1-42) Oligomers having Molecular Weights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]
An Aβ(1-42) oligomer B preparation prepared according to example 5a is admixed with 30 ml of PBS buffer containing 0.1% Pluronic® F68 (BASF) and concentrated to 3 ml in an Amicon Centriprep YM, 30 kD. Residues which may be present are removed by centrifugation (10 000 g for 5 min). The supernatant is removed. Aliquots of this Aβ(1-42) oligomer B preparation can be frozen at −80° C. and stored for further studies.
b) Preparation of a Concentrate of Aβ(1-42) Oligomers B having Molecular Weights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B]
72.6 ml of an Aβ(1-42) oligomer B preparation obtained according to example 5a are concentrated to 2 ml via a 30 kD Centriprep YM Tube (Amicon). The concentrate is removed by centrifugation at 10 000 g for 10 min. The supernatant is removed and dialyzed at 6° C. in a dialysis tube against 1 l of buffer (5 mM sodium phosphate, 35 mM sodium chloride, pH 7.4) for 16 h. The dialysate is removed by centrifugation at 10 000 g for 10 min. The supernatant is removed and can be stored at −80° C. for further studies.

Example 7

Preparation of Biotin-Aβ(1-42) Stock Suspension

0.5 mg of biotin-β-amyloid(1-42) protein (short name: biotin-Aβ(1-42); peptide-synthetic material, lyophilisate, AnaSpec) are dissolved in 200 μl of 1,1,1,3,3,3-hexafluoro-2-propanol and incubated in an Eppendorf vessel at 37° C. for 30 min. This is followed by evaporation to dryness in a vacuum concentrator (Speed Vac). The residue is taken up with 20.5 μl of DMSO, producing a 5 mM biotin-Aβ(1-42) stock suspension which can be stored at −20° C.

Example 8

Preparation of Biotin-Aβ(1-42) Oligomers having Molecular Weights of 17 kDa and 22 kDa [Biotin-Aβ(1-42) Oligomers A]
2 μl of the stock suspension of example 7 are admixed with 23 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) and adjusted to an SDS content of 0.2% with 2.4 μl of 2% strength. SDS solution. This is followed by 6 hours of incubation at 37° C. Insoluble components are removed by centrifugation at 10 000 g for 5 min. The biotin-Aβ(1-42) oligomer A preparation obtained in this way can be stored at −20° C.

Example 9

Preparation of Biotin-Aβ(1-42) Oligomers having Molecular Weights of 42 kDa and 52 kDa [Biotin-Aβ(1-42) Oligomers B]
A biotin-Aβ(1-42) oligomer A solution obtained according to example 8 is diluted with 82 μl of water (0.05% SDS content, 0.1 mM Aβ) and incubated at 37° C. for 16 hours. Insoluble components are removed by centrifugation at 10 000 g for 5 min. The biotin Aβ(1-42) oligomer B preparation can be frozen at −20° C. and stored for further studies. (FIG. 3).

Example 10

Preparation of Fluorescein-Aβ(1-42) Stock Suspension

0.5 mg of fluorescein-β-amyloid(1-42) protein (short name: fluorescein-Aβ(1-42); peptide-synthetic material, lyophilisate, AnaSpec) are dissolved in 200 μl of 1,1,1,3,3,3-hexafluoro-2-propanol and incubated in an Eppendorf vessel at 37° C. for 30 min. This is followed by evaporation to dryness in a vacuum concentrator (Speed Vac). The residue is taken up with 20.5 μl of DMSO, producing a 5 mM fluorescein-Aβ(1-42) stock suspension which can be stored at −20° C.

Example 11

Preparation of Fluorescein-Aβ(1-42) Oligomers having Molecular Weights of 17 kDa and 22 kDa [Fluorescein-Aβ(1-42) Oligomer A]
2 μl of the stock suspension of example 10 are admixed with 23 μl of PBS buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) and adjusted to an SDS content of 0.2% with 2.4 μl of 2% strength SDS solution. This is followed by 6 hours of incubation at 37° C. Insoluble components are removed by centrifugation at 10 000 g for 5 min. The fluorescein-Aβ(1-42) oligomer A preparation obtained in this way can be stored at −20° C.

Example 12

Preparation of Fluorescein-Aβ(1-42) Oligomers having Molecular Weights of 42 kDa and 52 kDa [Fluorescein-Aβ(1-42) Oligomers B]
A fluorescein-Aβ(1-42) oligomer A solution obtained according to example 11 is diluted with 82 μl of water (0.05% SDS content, 0.1 mM Aβ) and incubated at 37° C. for 16 hours. The fluorescein Aβ(1-42) oligomer B preparation can be frozen at −80° C. and stored for further studies. (FIG. 4).

Example 13

Crosslinking of Aβ(1-42) Oligomers A of Example 2a [Aβ(1-42) Oligomers A-CL]

10 μl of an Aβ(1-42) oligomer A solution prepared according to example 2a are diluted to 100 μM Aβ(1-42) content with 7.5 μl of PBS, 0.2% SDS. To this solution 1 μl of freshly prepared 10 mM glutardialdehyde solution in water is added, followed by stirring at RT for 3 h. The excess glutardialdehyde is saturated by adding 1 μl of 100 mM ethanolamine solution in water, pH 7.4, to the sample and stirring for 1 h. The preparation obtained in this way contains crosslinked Aβ(1-42) oligomers A and is referred to as Aβ(1-42) oligomer A-CL preparation.

Example 14

a) Crosslinking of Aβ(1-42) Oligomers B of Example 5a [Aβ(1-42) Oligomers B-CL]

10 μl of an Aβ(1-42) oligomer B solution prepared according to example 5a are admixed with 1 μl of freshly prepared 10 mM glutardialdehyde solution in water and stirred at RT for 3 h. The excess glutardialdehyde is saturated by adding 1 μl of 100 mM ethanolamine solution in water, pH 7.4, to the sample and stirring for 1 h. The preparation obtained in this way contains crosslinked Aβ(1-42) oligomers B and is referred to as Aβ(1-42) oligomer B-CL preparation.

b) Alternative Procedure for Crosslinking Aβ(1-42) Oligomers [Aβ(1-42) Oligomers B-CL]

72.6 ml of an Aβ(1-42) oligomer B solution prepared according to example 5a are admixed with 7.26 ml of freshly prepared 10 mM glutardialdehyde solution in water and stirred at RT for 2 h. The excess glutardialdehyde is saturated by adding 726 μl of buffer (20 mM sodium phosphate, 140 mM NaCl, 500 mM ethanolamine, pH 7.4) to the sample and stirring at RT for 30 min. The reaction mixture is concentrated to 3 ml via a 15 ml 30 kDa Centriprep tube. The concentrate is removed by centrifugation at 10 000 g for 10 min. The supernatant is removed and dialyzed at 6° C. in a dialysis tube against 1 l of 5 mM sodium phosphate, 35 mM NaCl, pH 7.4 for 16 h. The dialysate is subsequently removed by centrifugation at 10 000 g for 10 min and the supernatant is removed and can be stored at −80° C. for further studies. The preparation obtained in this way contains crosslinked Aβ(1-42) oligomers B and is referred to as Aβ(1-42) oligomer B-CL preparation.

Example 15

a) Preparation of Truncated Aβ(20-42) Oligomers, Starting from Aβ(1-42) Oligomers B, by Cleavage with Thermolysin:
1.59 ml of Aβ(1-42) oligomer B preparation prepared according to example 6b are admixed with 38 ml of buffer (50 mM MES/NaOH, pH 7.4) and 200 μl of a 1 mg/ml thermolysin solution (Roche) in water. The reaction mixture is stirred at RT for 20 h. Then 80 μl of a 100 mM EDTA solution, pH 7.4, in water are added and the mixture is furthermore adjusted to an SDS content of 0.01% with 400 μl of a 1% strength SOS solution. The reaction mixture is concentrated to approx. 1 ml via a 15 ml 30 kDa Centriprep tube. The concentrate is admixed with 9 ml of buffer (50 mM MES/NaOH, 0.02% SDS, pH 7.4) and again concentrated to 1 ml. The concentrate is dialyzed at 6° C. against 1 l of buffer (5 mM sodium phosphate, 35 mM NaCl) in a dialysis tube for 16 h. The dialysate is adjusted to an SDS content of 0.1% with a 2% strength SDS solution in water. The sample is removed by centrifugation at 10 000 g for 10 min and the supernatant is removed.
The material thus obtained is analyzed further (SDS polyacrylamide gel electrophoresis; cf. FIG. 11); the mass-spectrometric analysis of the truncated oligomers produced reveals that the oligomer is composed of truncated Aβ(20-42).
b) Preparation of Truncated Aβ(12-42) Oligomers, Starting from Aβ(1-42) Oligomers B, by Cleavage with Endoproteinase GluC:
2 ml of an Aβ(1-42) oligomer B preparation prepared according to example 6b are admixed with 38 ml buffer (5 mM sodium phosphate, 35 mM sodium chloride, pH 7.4) and 150 μl of a 1 mg/ml GluC endoproteinase (Roche) in water. The reaction mixture is stirred for 6 h at RT, and a further 150 μl of a 1 mg/ml GluC endoproteinase (Roche) in water are subsequently added. The reaction mixture is stirred at RT for another 16 h, followed by addition of 8 μl of a 5 M DIFP solution. The reaction mixture is concentrated to approx. 1 ml via a 15 ml 30 kDa Centriprep tube. The concentrate is admixed with 9 ml of buffer (5 mM sodium phosphate, 35 mM sodium chloride, pH 7.4) and again concentrated to 1 ml. The concentrate is dialyzed at 6° C. against 1 l of buffer (5 mM sodium phosphate, 35 mM NaCl) in a dialysis tube for 16 h. The dialysate is adjusted to an SDS content of 0.1% with a 1% strength SDS solution in water. The sample is removed by centrifugation at 10 000 g for 10 min and the supernatant is removed.
The material thus obtained is analyzed further (SDS polyacrylamide gel electrophoresis; cf. FIG. 11). Mass-spectrometric analysis of the truncated oligomer produced reveal that the oligomer is composed of truncated Aβ(12-42).
Characterization of Aβ(1-42) oligomers

Example 16

SDS Polyacrylamide Gel Electrophoresis (SDS PAGE)

The molecular weight under denaturing conditions is characterized by analyzing the preparations of examples 2a, 5a, 9, 12, 13 and 14a, all of which contain oligomers A and B, under denaturing conditions according to standard conditions in a 4-20% strength Tris-glycine SDS PAGE.
The evaluation of said SDS PAGE (FIG. 1) reveals that starting protein Aβ(1-42) still present in the oligomer A preparation appears as a band at about 4 kDa, while the oligomers A1 and A2 of example 2a, denoted 1 and 2, respectively, in FIG. 1, are visible at about 15 kDa (weaker band) and at about 20 kDa (main band).
In the oligomer B preparation comparatively little starting protein Aβ(1-42) can be detected (relatively weak band at about 4 kDa). In contrast, the oligomers B1 and B2 of example 5a, denoted 3 and 4, respectively, in FIG. 1 appear at about 38 kDa and about 48 kDa (see arrows). Correspondingly higher molecular weights of about 42 kDa and about 52 kDa arise for the biotin- and fluorescein-derivatized oligomers B of examples 9 and 12 (FIGS. 3, 4).
Analysis of the preparations containing the respective crosslinking products A-CL and B-CL (examples 13 and 14a; FIG. 2) reveals that the two samples essentially maintain their degree of oligomerization (cf. lane A with A′ and lane B with B′). The slightly different migration behavior compared to the oligomers A and B can be explained by a slightly altered SDS binding capacity and by modification of the amino groups of the N terminus and, respectively, the lysine residue.
Analysis of the truncated Aβ(20-42) oligomers (of Example 15a) reveals that the 38/48 kDa double band (FIG. 11, lane A) is converted to a 28/38 kDa double band (FIG. 11; lane B) by proteolytic removal of the N-terminal peptide with themolysine. Similarly, analysis of the truncated Aβ(12-42) oligomers (of Example 15b) reveals that the 38/48 kDa double band (FIG. 11; lane A) is converted to a 33/40 kDa double band (FIG. 11; ring C) by proteolytic removal of the N-terminal peptide with Glu-C endoprotease.

Example 17

Gel Permeation Chromatography

In order to study the molecular weight behavior under nondenaturing conditions in more detail, a gel permeation chromatography (GPC) is carried out by way of an FPLC process using a Superose 12 HR10/30 column. The GPC is run at 4° C.
The column is equilibrated with 5 volumes of PBS buffer (flow rate 0.5 ml/min, UV detection, 214 nm) and first calibrated using protein standards. Subsequently, the Aβ(1-42) oligomer B preparation of example 5a (FIG. 5, bottom) and, for comparison, the same concentration of freshly weighed Aβ(1-42) lyophilisate dissolved in PBS are analyzed, after removing the insoluble components by centrifugation at 10 000 g for 5 minutes (FIG. 5, top).
The evaluation reveals that the Aβ(1-42) oligomer B preparation has a protein fraction characterized by a main peak in the molecular weight range of around approximately 50 kDa. As under denaturing conditions in the SDS PAGE, this protein fraction differs significantly from the monomeric Aβ(1-42) protein characterized by a main peak in the molecular weight range of around aproximately 16 kDa.
In order to study the molecular weight behavior of Aβ(1-42) oligomers B of example 6b and of Aβ(1-42) oligomers B-CL of example 14a under nondenaturing conditions, a gel permeation chromatography (GPC) is carried out by way of an FPLC process using a Superose 12 HR10/30 column at room temperature. The column is equilibrated with 5 column volumes of PBS buffer (flow rate 0.5 ml/min, UV detection, 214 nm) and first calibrated using protein standards. Subsequently, Aβ(1-42) oligomer B of example 6b is diluted to 1 mg/ml with PBS buffer and Aβ(1-42) oligomer B-CL of example 14a is diluted to 1 mg/ml with PBS buffer, and both mixtures are analyzed (FIG. 12).
The evaluation reveals that the Aβ(1-42) oligomer B preparation with reduced SDS content has a protein fraction characterized by a main peak in the molecular weight range around approximately 100 kDa. In comparison therewith, the Aβ(1-42) oligomer B-CL preparation with reduced SDS content has a protein fraction characterized by a main peak in the molecular weight range around approximately 60 kDa.

Example 18

Native Polyacrylamide Gel Electrophoresis (NATIVE PAGE) of Aβ(1-42) Oligomers B of Example 5a

The molecular weight under native conditions is characterized by analyzing the oligomers B-containing preparations of example 5a under nondenaturing conditions in a 4-20% Tris-glycine gel.
The detergent present in the preparations is neutralized by the nonionic detergent Triton X-100. To this end, 1 μl (4% Triton X-100) is pipetted to 10 μl of the preparation of the example 5a and incubated at room temperature for 5 min. Subsequently, 10 μl are admixed with the same volume of native sample buffer (4 ml of 1M Tris, pH 6.8, 8 ml of glycerol, 1 ml of bromophenol blue in 50 ml of H₂O ) and the electrophoresis (running buffer: 7.5 g of Tris, 36 g of glycine to 2.5 l of H₂O).
The NATIVE PAGE evaluation reveals that the starting peptide Aβ(1-42) still present in the oligomer B preparation appears as a band at about 28 kDa (lane A, denoted 1), while the main bands of the preparation of example 5a are visible at an apparent molecular weight of 64-90 kDa (lane A, denoted 2) (FIG. 6).
It is important that molecular weights can be assigned to the oligomers of the invention, in particular the oligomers B, also in native molecular weight analytical methods such as gel permeation chromatography (see example 17) or native gel electrophoresis (see example 18).
After complexing of the SDS, the oligomers B to which molecular weights of about 38 and 48 kDa can be assigned in the SDS PAGE are detected in the native gel electrophoresis as a band in the molecular weight range of about 64-90 kDa, based on selected standard proteins. This method cannot be expected to provide an exact reading of the molecular weight, since the migration behavior of the oligomers is substantially determined by their intrinsic charges, in addition to their size. However, the result leads to the conclusion that a defined oligomeric species is present.

Example 19

a) Stability of Aβ(1-42) Oligomers B at Various Protein Concentrations in Physiological Buffers

The Aβ(1-42) oligomers B obtained according to example 6b are tested for stability in PBS buffer under the following conditions.
5 mg/ml are diluted with PBS in 2× dilution steps down to 0.08 mg/ml. All solutions obtained are incubated at room temperature for 24 hours. This was followed by analyzing the band pattern in an SDS PAGE in comparison with a frozen control preparation. The band pattern is identical in all samples.

b) Stability of Aβ(1-42) Oligomers B at Various Temperatures and After Different Periods of Time in Physiological Buffers

The Aβ(1-42) oligomers B obtained according to example 6b are diluted to 0.5 mg/ml with PBS buffer and incubated at room temperature or at 37° C. for 24 h or 96 h.
Subsequently, the band pattern was analyzed in an SDS PAGE. The band pattern is identical in all samples.

Example 20

Proteolysis of Aβ(1-42) Oligomers B by Means of Various Proteases (Trypsin, Chymotrypsin, Thermolysin, Elastase, Papain)

Aliquots of the Aβ(1-42) oligomer B preparation obtained according to example 6b are diluted to 0.5 mg/ml with buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) and incubated with in each case 1/50 of the amount by weight of the protease solutions indicated in FIG. 9 under the following conditions at 37° C. and pH 7.4 for 20 h. 1 μg of reactive mixture aliquots are then analyzed in an SDS-PAGE (FIG. 9).
The SDS PAGE reveals that, starting from the Aβ(1-42) oligomers B preparation, all proteases revert the double band at about 38/48 kDa to a kDa double band at about 32/28 kDa under the chosen limited proteolysis conditions.

Example 21

Stability of Aβ(1-42) Oligomers B in Rat Plasma

4 μl of the Aβ(1-42) oligomer B preparation prepared according to example 6b are incubated with 76 μl of rat plasma at room temperature for 0 h, 1 h, 2 h, 4 h and 8 h. The incubations are stopped by freezing in dry ice.
Subsequently, all samples are analyzed in an SDS PAGE, after addition of SDS sample buffer. This is followed by an evaluation of the stability via staining of the Aβ(1-42) oligomers B in a Western blot. The anti-Aβ(1-42) antibody 6E10 (Signet) was used for detection. The bands are made visible by an anti-mouse IgG antibody coupled to alkaliphosphatase and addition of the substrate NBT/BCIP. Both molecular weight and intensity of the observed band pattern remain nearly unchanged over a period of 2 h, 4 h and 8 h.
This result suggests that the Aβ(1-42) oligomers B have high plasma stability. According to this, the biological half life in the plasma is in the range of about 8 hours or longer.

Example 22

Binding of biotin-Aβ(1-42) oligomers having molecular weights of 17 kDa and 22 kDa [biotin-Aβ(1-42) oligomers A] and, respectively, of 42 kDa and 52 kDa [biotin-Aβ(1-42) oligomers B] to the surface of human neuronal cells.
Binding of human β-amyloid(1-42) protein and the two Aβ(1-42) oligomer A and Aβ(1-42) oligomer B preparations to the human neuroblastoma cell line IMR-32 (ATCC Number: CCL-127) is studied by means of FACScan (Beckton Dickinson). A suspension of IMR-32 cells (1,5×10⁶cells/0,1 ml PBS) is incubated with biotin-labeled human β-amyloid(1-42) protein (peptide-synthetic material, lyophilisate, AnaSpec) and the preparations containing the biotin-labeled oligomers A and B (examples 8 and 9, respectively) at 37° C. for 20 minutes. The cells are subsequently washed with buffer (PBS plus 1% BSA) and incubated at room temperature with fluorescein coupled to streptavidin isothiocyanate (Sigma) for 20 minutes. After a washing step with buffer, binding to the surface of IMR-32 cells was analyzed by FACScan. The dashed line indicates background fluorescence in the absence of the biotin-labeled components. The addition of the individual preparations resulted in a strong increase of cell-associated fluorescence and is represented by the thick line. Binding of the oligomers A (FIG. 7B) and B (FIG. 7C) to the cell surfaces is distinctly different from the binding of the monomeric β-amyloid(1-42) protein (FIG. 7A). The data indicate specific binding sites for said oligomers on human cell surfaces.

Example 23

Detection of Aβ(1-42) Oligomers B in Rat Brain Homogenates After Icy Administration

10 nmol of an Aβ(1-42) oligomer B preparation obtained according to example 6b are administered as icv-bolus to rats. The brains are prepared after 15 min and 120 min, respectively. Brains of untreated control animals are likewise prepared. For this purpose, in each case 1 g of rat brain is admixed with 9 ml of disruption buffer A (200 ml of 5 mM sodium phosphate, 35 mM NaCl, 300 mM sucrose, adjusted to pH 7.4, are admixed with 4 tablets of Complete® protease inhibitor cocktail from Roche) in a 50 ml Falcon tube and disrupted under ultrasound (UltraTurrax) on ice for 2 min. The solution is left standing for 20 min, then shaken briefly and divided into 8×1 ml aliquots (=homogenate).
In order to carry out quantitative detection, first a series of standards of the Aβ(1-42) oligomer B preparation in PBS in the concentration range of 1.58 ng/μl-0.005 ng/μl is prepared.
Furthermore, as a positive control, a homogenate from a control brain of an untreated rat is also processed and a series of standards of the Aβ(1-42) oligomer B preparation is prepared in this brain homogenate in the same way. The homogenates are then introduced in an ultracentrifuge at 100 000 g for 1 h and the supernatants are used for the subsequent analyses.
From the comparison of the values measured on both series of standards (PBS versus control brain-homogenate supernatant), the Aβ(1-42) oligomer B content can be determined quantitatively as follows, first in the positive control and then, in comparison therewith, also in the brain sample of treated rats.

1) Detection by Dot Blot

1 μl drops of the series of standards samples and of the sample extracts from the treated animals are applied to nitrocellulose paper, and Aβ(1-42) is detected using the antibody 6E10 (anti-Aβ(1-42); Signet). Staining is carried out using an alkaliphosphatase coupled to anti-mouse IgG and by adding the staining reagent NBT/BCIP.
While no Aβ(1-42) (0.01 nmol/g) is detectible in the brain extracts of untreated rats, about 0.4 nmol/g Aβ(1-42) can be detected in the rats sacrificed 15 min after treatment by comparing the staining intensity with the corresponding concentrations of the positive control, and approx. 0.2 nmol/g can still be detected by the same method in the rats sacrificed 120 min after treatment. This results in an average biological half life of about 105 min for the exogenously administered Aβ(1-42) oligomers B.

2) Detection by Western Blot

All dot blot-analyzed samples are likewise analyzed in a Western blot. The Western blot is likewise developed with mMAb 6E10 (anti-Aβ(1-42); Signet) and furthermore with an alkaliphosphatase coupled to anti-mouse IgG and by adding the staining reagent NBT/BCIP.
Anti-Aβ(1-42) reactive bands only occur in the 38/48 kDa region, corresponding to the apparent molecular weight of the Aβ(1-42) oligomers B, i.e. the oligomeric structure is still retained with in vivo administration, even after 2 hours.
Furthermore, as in the dot blot method, the same concentrations can be estimated in the brains of the 15 min rats (0.4 nmol/g) and the 120 min rats (0.2 nmol/g) by comparing the staining intensities with correspondingly intensively stained bands of the Aβ(1-42) oligomers B in the positive control.

Example 24

Neurotoxic Action of Aβ(1-42) Oligomers having Molecular Weights of 38 kDa and 48 kDa [Aβ(1-42) Oligomers B] on Murine Cortical Neurons
Murine cortical neurons are prepared and cultured as a mixed culture with glia cells following the literature (Choi et al. (1987) J. Neurosci. 7, 357-368). The cortices of embryos on days 14-15 of development are mechanically removed from the meninges and lower brain regions. The cells are separated from one another by incubation in a 0.05% strength trypsin solution at 37° C. for 5-7 minutes and subsequently pipetting said solution several times through a pasteur pipette with reduced opening. After determining the number of cells, 430 000 cells are seeded in 0.5 ml of maintenance medium (minimum essential medium containing 0.8 mM glutamine, 18 mM glucose, 23 mM NaHCO₃and 10% of horse serum) per 2 cm²on cell culture material coated with poly-L-ornithine and laminine. Cultivation and later incubations of the cells are carried out in a humidified cell culture incubator at 37° C., 5% CO₂. After 3-5 days in culture, propagation of the glia cells is interrupted by a 1-day incubation with a mixture of (+)-5-fluoro-2′-deoxyuridine/uridine (10 μM each). After 14 days in culture, the toxic action of Aβ(1-42) oligomers B is studied. For this purpose, the cells are incubated in brain cell buffer (120 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 15 mM glucose, 25 mM HEPES, pH 7.2) for 15 minutes. Control cells 1 are incubated with 300 μM L-glutamate in brain cell buffer for the same time. The Aβ-oligomer stock solution is diluted with serum-free medium (minimum essential medium containing 0.8 mM glutamine, 20 mM glucose, 26 mM NaHCO₃) to various final concentrations and incubated for 24 h. The L-glutamate-treated control cells 1 are incubated in parallel in serum-free medium. Another group of cells (control cells 2) is treated only with brain cell buffer and serum-free medium. The cell culture supernatants are removed after 24 h, the remaining cells are destroyed by incubation in distilled water for 20 minutes and the activity of the enzyme lactate dehydrogenase (LDH) is determined enzymatically in both solutions. For evaluation, the ratio of the LDH activity of the cell culture supernatants to the sum of the LDH activities of supernatant and remaining cells is determined and the average of the quadruple determinations is formed. 3 experiments are carried out, with each experimental condition being present in quadruplicate (n=12). The average for the glutamate-treated cells (control cells 1) is set to 100% neuronal death, the average of the cells which have been treated neither with L-glutamate nor with Aβ oligomers (control cells 2) is set to 0% neuronal death and the values of the cells treated with Aβ oligomers are converted accordingly. The neurotoxicity average of all determinations at the concentrations used in each case indicates a distinct toxic action of the Aβ(1-42) oligomers having molecular weights of 38 kDa and 48 kDa [Aβ(1-42) oligomer B], cf. FIG. 8.

Example 25

Production of Antibodies

The cocktails used for immunization contain in all cases adjuvant (Biogenes) with essentially the following components:

- 95% Paraffin oil
- 2.4% Tween 40
- 0.1% Cholesterol
- 0.1% Lipopolysaccharide

The adjuvant is mixed with a solution of the antigen in a 2:1 ratio until a stable emulsion is obtained. The emulsion is injected and forms a depot from which said antigen is released steadily.
a) Production of polyclonal antisera by immunization of rabbits with truncated Aβ(20-42) oligomers B of example 15a.
2 rabbits are immunized with unconjugated Aβ(20-42) oligomer B preparation of example 15a according to a standard protocol:


	Day 1	Primary immunization and taking of preimmune serum
	Day
7	First boost
	Day 14	Second boost
	Day
28	Third boost and bleeding
	Day 35	Taking blood

The antisera are obtained from the rabbit's blood by letting the latter stand at room temperature and subsequent centrifugation at room temperature. The serum obtained in this way is referred to as serum (a1).
2 mg of Aβ(20-42) oligomer B preparation of example 15a are coupled to LPH with glutardialdehyde under standard conditions and 2 more rabbits are immunized with this conjugated Aβ(20-42) oligomer B according to a standard protocol:

The antisera are obtained from the rabbit's blood by letting the latter stand at room temperature and subsequent centrifugation at room temperature. The serum obtained in this way is referred to as serum (a2).
b) Production of polyclonal antisera by immunization of rabbits with truncated Aβ(12-42) oligomers B of example 15b.
2 rabbits are immunized with unconjugated Aβ(12-42) oligomer B preparation of example 15b according to a standard protocol:

The antisera are obtained from the rabbit's blood by letting the latter stand at room temperature and subsequent centrifugation at room temperature. The serum obtained in this way is referred to as serum (b1).
2 mg of Aβ(12-42) oligomer B preparation of example 15b are coupled to LPH with glutardialdehyde under standard conditions and 2 more rabbits are immunized with this conjugated Aβ(12-42) oligomer B according to a standard protocol:

The antisera are obtained from the rabbit's blood by letting the latter stand at room temperature and subsequent centrifugation at room temperature. The serum obtained in this way is referred to as serum (b2).
c) Production of polyclonal antisera by immunization of rabbits with Aβ(1-42) oligomer preparation B-CL of example 14a.
2 rabbits are immunized with unconjugated Aβ(1-42) oligomers B-CL preparation of example 14a according to a standard protocol:

The antisera are obtained from the rabbit's blood by letting the latter stand at room temperature and subsequent centrifugation at room temperature. The serum obtained in this way is referred to as serum (c1).
d) Production of polyclonal antisera by immunization of rabbits with Aβ(1-42) oligomer preparation B of example 6b.
2 rabbits are immunized with unconjugated Aβ(1-42) oligomers B preparation of example 6b according to a standard protocol:

The antisera are obtained from the rabbit's blood by letting the latter stand at room temperature and subsequent centrifugation at room temperature. The serum obtained in this way is referred to as serum (d1).
2 mg of Aβ(1-42) oligomer B preparation of example 6b are coupled to LPH, with glutardialdehyde under standard conditions and 2 more rabbits are immunized with this conjugated Aβ(1-42) oligomer B preparation according to a standard protocol:

The antisera are obtained from the rabbit's blood by letting the latter stand at room temperature and subsequent centrifugation at room temperature. The serum obtained in this way is referred to as serum (d2).

Example 26

Characterization of polyclonal antisera from the immunizations of example 25 regarding their cross reaction with various Aβ(1-42) forms.
In order to characterize the polyclonal antisera, serial dilution in the range of 100 pmol/μl-0.01 pmol/μl of the various Aβ(1-42) forms were prepared in PBS. In each case 1 μl of the sample was applied to a nitrocellulose membrane: detection was carried out using the appropriate rabbit sera of example 25. A detection using mMAb 6E10 (Signet) was carried out for comparison. Alkaliphosphatase coupled to anti-rabbit IgG (for comparison: alkaliphosphatase coupled to anti-mouse IgG) was used for staining together with addition of the staining reagent NBT/BClP. FIG. 10 indicates the dot blots obtained in this manner, which can be numerically evaluated as follows:


					Serum
Antigen	6E10	Serum (a1)	Serum (a2)	Serum (d1)	(c1)

Oligomer	0.1	0.5	10	0.1	0.5
Monomer	0.1	10	100	0.1	1
Fibril	1	100	>100	0.5	1
APP	0.01	>1	1	0.1	0.1
Aβ(1-40)*	0.1	100	n.d.	0.5	10

*not shown in FIG. 10

The numbers in the table indicate the minimum amount of antigen visible [in pmol; in each case based on the monomer, except for APP] (detection limit).
Comparison of the immunological reactions with rabbit serum (a1) obtained by immunization with unconjugated Aβ(20-42) oligomer of example 15a clearly indicates that the antibodies directed against these oligomers cross-react only weakly with the other forms such as fibrils, APP and monomer. In contrast, a markedly stronger cross reaction with the Aβ(1-42) oligomer B (see row 1) and, moreover, a cross reaction with the stabilized CL antigen are observed. This data indicates a distinctly different structure of the oligomer form present here, in comparison with APP, monomer and the fibril structure. The antibodies bind to oligomer-specific structures.
The immunological reactions with rabbit serum (a2) obtained by immunization with conjugated Aβ(20-42) oligomer of example 15a likewise show that antibodies directed against these oligomers cross-react only weakly with the other forms such as fibrils and monomer. In contrast, a weak cross reaction with the Aβ(1-42) oligomer B (see row 1) and, moreover, a cross reaction with the stabilized CL antigen and with APP are observed. This data indicates a likewise distinctly-different structure of the oligomer form present here, in comparison with monomer and the fibril structure.
In comparison therewith, the immunological reactions with rabbit serum (d1) obtained by immunization with Aβ(1-42) oligomer of example 6b, and also with rabbit serum (c1) obtained by immunization with Aβ(1-42) oligomer B-CL of example 14a indicate that the antibodies directed against these oligomers cross-react with the other forms such as fibrils, APP and monomer comparably as strongly as with the Aβ(1-42) oligomers B (see row 1). These antibodies exhibit an immune reaction comparable to that of mMAb 6E10 (Signet) and do not bind to oligomer-specific structures.
Correspondingly, mice were immunized with Aβ(1-42) oligomers of example 6b and monoclonal antibodies established in a manner known per se. Here, 2 out of 10 hybridomas secreted monoclonal antibodies whose binding profiles are similar to those of the antiserum (a1), in particular with respect to the reactivites assayed above.

Example 27

Preparation of Aβ(1-42) Fibrils

100 μl of 2 mg/ml Aβ(1-42) solution in 0.1% NH₄OH are diluted with 300 μl of buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4) to 0.5 mg/ml and adjusted to pH 7.4 with 0.1 M HCl (100 μM Aβ(1-42)). The sample is incubated at 37° C. for 24 h and then removed by centrifugation at 10 000 g for 10 min. The protein residue obtained is resuspended with 400 μl of buffer (20 mM sodium phosphate, 140 mM NaCl, pH 7.4). The Aβ(1-42) fibril preparation obtained in this way can be stored at −20° C. and used for further studies.

Example 28

Effects of Aβ(1-42) Oligomers B-CL (Of Example 14b) on Hippocampal Slices

Transversal hippocampal slices of 400 μM in thickness were adapted in a submersion slice chamber at 34° C. under perfusion by gassed Ringer's solution (NaCl 124 mM, KCl 4.9 mM, MgSO₄1.3 mM, CaCl₂2.5 mM, KH₂PO₄1.2 mM, NaHCO₃25.6 mM, glucose 10 mM). Subsequently, the Schaffer collateral was stimulated with the aid of a monopolar stimulating electrode and the excitatory postsynaptic potential (fEPSP) in the stratum radiatum was recorded. Test pulses were given with 30% of the stimulus level that generates a maximum fEPSP. Long-term potentiation was induced by applying 100 pulses three times every 10 minutes, with single pulses having a width of 200 μs (strong tetanus). The resulting potentiation of the fEPSPs was recorded for at least 240 minutes. The rinsing-in of the oligomer B-CL of example 14b (500 nM) started 20 minutes before the first tetanus and was stopped 10 minutes after the last tetanus. The rise (slope) of the fEPSPs was determined and plotted as a function of time.
Figure FIG. 14 indicates that the washing-in of Aβ(1-42) oligomers suppresses long-term potentiation in hippocampus, especially in the maintenance phase. Accordingly, the Aβ(1-42) oligomers BCL influence the storage of information by nerve cells (cellular memory).

Example 29

Binding of Aβ(1-42) Oligomers B (Of Example 5b) to Rat Primary Hippocampal Neurons

The binding of rat Aβ(1-42) oligomers B of example 5b to rat primary hippocampal neurons was studied. The hippocampal neurons were cultured in neurobasal medium with B27 supplement on poly-L-lysine-coated cover slips and used on day 14 of the culture. The oligomers were bound to the cell membrane of the neurons by adding 200 nM (total monomeric Aβ concentration) oligomers to fresh culture medium and incubating at 37° C. for 15 minutes. After removing the Aβ-containing medium, two washing steps with medium were carried out and the cells were then fixed in 3.7% formaldehyde. After further washing of the cells, unspecific binding sites were blocked with 10% normal donkey serum in PBS buffer at room temperature for 90 min. 6E10 (from mouse) was applied as the first antibody in a 1:2000 dilution at room temperature for 2 h. The cells were washed again and incubated with the second antibody (from donkey) which is directed against mouse and which is coupled to the fluorescent dye Cy3, at room temperature for 2 h. After the cells had been washed again, the cover slips containing the neurons were fixed to a slide with embedding medium. The hippocampal neurons with bound Aβ oligomers were depicted in a fluorescent microscope. The control used was a mixture in which the first antibody 6E10 had been omitted. This control thus exhibits unspecific fluorescence not based on Aβ.
As FIG. 15 a shows, the oligomers bind to the cell surface of the neurons in a dotted manner. In contrast, the control without the first antibody 6E10 exhibits only low unspecific fluorescence, due to binding of the second antibody to the neurons (FIG. 15 b).

Claims

1. An oligomer of the amyloid β(1-42) protein, having an apparent molecular weight of about 15 kDa in SDS gel electrophoresis, or a derivative thereof.

2. An oligomer of the amyloid β(1-42) protein, having an apparent molecular weight of about 20 kDa in SDS gel electrophoresis, or a derivative thereof.

3. An oligomer of the amyloid β(1-42) protein, having an apparent molecular weight of about 38 kDa in SDS gel electrophoresis, or a derivative thereof.

4. An oligomer of the amyloid β(1-42) protein, having an apparent molecular weight of about 48 kDa in SDS gel electrophoresis, or a derivative thereof.

5. A composition, comprising a mixture of an oligomer as claimed in claim 1 and an oligomer as claimed in claim 2, or of derivatives thereof.

6. A composition, comprising a mixture of an oligomer as claimed in claim 3 and an oligomer as claimed in claim 4, or of derivatives thereof.

7. The oligomer or composition as claimed in any of claims 1 to 6, characterized in that the derivatives have a detectable label.

8. The oligomer or composition as claimed in claim 7, characterized in that the label is a fluorescent, luminescent, colorimetric, radioactive or magnetic label or is a label having affinity to complementary binding partners.

9. The oligomer or composition as claimed in any of claims 1 to 8, characterized in that the oligomer or the derivative is crosslinked.

10. The oligomer or composition as claimed in any of claims 1 to 8, characterized in that the derivative is obtainable by proteolytic cleavage of the oligomer.

11. The oligomer or composition as claimed in claim 10, characterized in that the derivative is an oligomer of an Aβ(x-42) fragment, where x is 8 to 24, preferably from 10 to 22, and in particular from 12 to 20.

12. The oligomer or composition as claimed in claim 11, characterized in that the derivative is an oligomer of the Aβ(12-42) fragment or of the Aβ(20-42) fragment.

13. A method for preparing oligomers of a derivatized or nonderivatized amyloid β(1-42) protein, characterized in that monomeric amyloid β(1-42) protein or a derivative thereof is exposed to a detergent and, optionally, at least one oligomer or derivative thereof is obtained in a manner known per se.

14. A method for preparing oligomers of a derivatized or nonderivatized amyloid β(1-42) protein, characterized in that monomeric amyloid β(1-42) protein or a derivative thereof is exposed to a detergent, the detergent action is then reduced and incubation is continued, and, optionally, at least one oligomer or derivative thereof is obtained in a manner known per se.

15. The method as claimed in claim 14, characterized in that the detergent is ionic.

16. The method as claimed in claim 15, characterized in that the detergent is sodium dodecyl sulfate.

17. The method as claimed in any of claims 13 to 16, characterized in that the time of exposure to the detergent is from about 1 to 20 hours.

18. The method as claimed in claim 17, characterized in that the temperature of exposure is from about 20 to 50° C.

19. The method as claimed in any of claims 14 to 16, characterized in that the time of continued incubation is from 10 to 30 hours.

20. The method as claimed in claim 19, characterized in that the temperature of exposure is from about 20 to 50° C.

21. The method as claimed in any of claims 13 to 20, characterized in that the amyloid β(1-42) protein is unfolded at least partially beforehand.

22. The method as claimed in claim 21, characterized in that for this purpose the protein is exposed to a hydrogen bond-breaking agent, preferably HFIP.

23. The method as claimed in any of claims 13 to 22, characterized in that the oligomer is exposed to a crosslinker.

24. The method as claimed in any of claims 13 to 23, characterized in that the oligomer is exposed to a protease.

25. The use of an oligomer, of a derivative thereof or of a composition as claimed in any of claims 1 to 12 in a diagnostic in vitro detection method.

26. The use of an oligomer, of a derivative thereof or of a composition as claimed in any of claims 1 to 12 in a diagnostic in vivo detection method.

27. The use of an oligomer, of a derivative thereof or of a composition as claimed in any of claims 1 to 12 for generating oligomer-specific antibodies.

28. A method for characterizing a substance or a substance mixture, which comprises

i) providing said substance or said substance mixture;

ii) exposing an oligomer, a derivative thereof or a composition as claimed in any of claims 1 to 12 to said substance or said substance mixture; and

iii) determining, whether said substance or particular portions of said substance mixture bind to said oligomer, said derivative thereof or to at least one oligomer or derivative thereof present in said composition.

29. A substance or a portion of a substance mixture, characterized in that said substance or said portion is identifiable by a method as claimed in claim 28 as a ligand binding to the oligomer, the derivative thereof or to at least one oligomer or derivative thereof present in the composition.

30. The use of a substance or of a portion of a substance mixture as claimed in claim 29 for preparing a medicament for the treatment of amyloid β-associated, in particular dementing, disorders.

31. The use of a substance or of a portion of a substance mixture as claimed in claim 29 for preparing a composition for diagnosing amyloid β-associated, in particular dementing, disorders.

32. A method for producing antibodies, which comprises

i) immunizing a host with at least one oligomer, derivative thereof or composition as claimed in any of claims 1 to 12; and

ii) obtaining an antibody-containing host serum produced as a response to said immunization.

33. The method as claimed in claim 32, characterized in that at least one antibody of the serum is selected, which recognizes the oligomer or derivative thereof used as immunogen or at least one oligomer or derivative thereof present in the composition used as immunogen.

34. A method for preparing antibodies with specificity for an oligomer or derivative thereof as claimed in any of claims 1 to 12, which comprises

i) providing an antigen comprising said oligomer or derivative thereof;

ii) exposing an antibody repertoire to said antigen; and

iii) selecting from said repertoire an antibody which specifically binds to said oligomer or derivative thereof.

35. The method as claimed in claim 34, wherein the antibody repertoire is exposed to the antigen in vivo by immunizing an animal with said antigen.

36. The method as claimed in claim 34, wherein furthermore a number of hybridomas from lymphocytes of the animal are established and a hybridoma secreting an antibody which specifically binds to the oligomer or derivative thereof is selected.

37. The method as claimed in claim 34, wherein the animal is selected from among mice, rats, chickens, camelids, cynomolus monkeys, rabbits and goats.

38. The method as claimed in claim 37, wherein the animal is a knockout mouse with APP deficiency.

39. The method as claimed in claim 37, wherein the animal is a transgenic mouse having human immunoglobulin genes, which generates human antibodies after an antigenic stimulus.

40. The method as claimed in claim 37, wherein the animal is a mouse with severe combined immunodeficiency (SCID), which has been reconstituted with human peripheral mononuclear blood cells or lymphoid cells or precursors thereof.

41. The method as claimed in claim 37, wherein the animal is a mouse which has been treated with lethal total body irradiation, then protected against radiation with bone marrow cells from a mouse with severe combined immunodeficiency (SCID) and into which subsequently functional human lymphocytes or precursors thereof have been transplanted.

42. The method as claimed in claim 34, wherein the antibody repertoire is exposed to the antigen in vitro by screening a recombinant antibody library with said antigen.

43. The method as claimed in claim 42, wherein the recombinant antibody library is expressed on the surface of a bacteriophage.

44. The method as claimed in claim 42, wherein the recombinant antibody library is expressed on the surface of yeast cells.

45. The method as claimed in claim 42, wherein the recombinant antibody library is expressed on the surface of bacterial cells.

46. The method as claimed in claim 42, wherein the recombinant antibody library is expressed as RNA-protein fusions.

47. The method as claimed in claim 42, wherein the recombinant antibody library is an scFv library or an Fab library.

48. The method as claimed in claim 42, wherein the recombinant antibody library is a single domain library.

49. The method as claimed in claim 34, wherein the antibody repertoire is exposed to the antigen by immunizing an animal in vivo with said antigen and a recombinant antibody library prepared from the lymphoid cells of said animal is then screened with said antigen.

50. The method as claimed in claim 34, wherein the antibody repertoire is exposed to the antigen by immunizing an animal in vivo with said antigen and a recombinant antibody library prepared from the lymphoid cells of said animal is then subjected to affinity maturation in vitro.

51. The method as claimed in claim 34, wherein the antibody repertoire is exposed to the antigen by immunizing an animal with said antigen and individual cells secreting the antigen-binding antibody are then selected and cDNAs for the variable regions of the heavy and light chains are obtained from the single cells.

52. An antibody, obtainable by a method as claimed in any of claims 32 to 51.

53. A protein, which binds specifically to an oligomer or derivative thereof as claimed in any of claims 1 to 12.

54. The protein as claimed in claim 53, which binds the oligomer or derivative thereof with an affinity in the range of K_D=10⁻⁶-10⁻¹²M.

55. The protein as claimed in claim 54, which binds the oligomer or derivative thereof with an affinity greater than K_d=10⁻⁸M.

56. The protein as claimed in claim 54, which binds the oligomer or derivative thereof with an affinity greater than K_d=10⁻⁹M.

57. The protein as claimed in claim 54, which binds the oligomer or derivative thereof with an affinity greater than K_d=10⁻¹⁰M.

58. The protein as claimed in claim 54, which binds the oligomer or derivative thereof with an affinity greater than K_d=10⁻¹¹M.

59. The protein as claimed in any of claims 53 to 58, which binds monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein with an affinity of less than K_d=10⁻⁸M.

60. The protein as claimed in any of claims 53 to 59, wherein the affinity of said protein to the oligomer or derivative thereof is at least 10 times greater than to monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein.

61. The protein as claimed in any of claims 53 to 59, wherein the affinity of said protein to the oligomer or derivative thereof is at least 100 times greater than to monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein.

62. The protein as claimed in any of claims 53 to 59, wherein the affinity of said protein to the oligomer or derivative thereof is at least 1000 times greater than to monomeric Aβ(1-42) protein and/or monomeric Aβ(1-40) protein.

63. The protein as claimed in any of claims 53 to 62, which binds the oligomer or derivative thereof with a rate constant k_offof 0.1 s⁻¹or lower, as determined by means of surface plasmon resonance.

64. The protein as claimed in any of claims 53 to 63, which inhibits the activity of the oligomer or derivative thereof with an inhibition constant IC₅₀of 1×10⁻⁵or lower.

65. The protein as claimed in any of claims 53 to 64, which is an antibody or an antigen-binding moiety thereof.

66. The antibody as claimed in claim 65, which is a substantially human antibody or an antigen-binding moiety thereof.

67. The antibody as claimed in claim 65, which is a chimeric antibody or an antigen-binding moiety thereof.

68. The antibody as claimed in claim 65, which is a CDR graft antibody or an antigen-binding moiety thereof.

69. The protein as claimed in any of claims 53 to 64, which is a molecule derived from the T-cell receptor or a receptor domain derived from the T-cell receptor or a fusion protein of said receptor domain with an Fc moiety of an immunoglobulin.

70. A pharmaceutical agent, comprising a protein as claimed in any of claims 53 to 69 and, optionally, a pharmaceutically suitable carrier.

71. The use of a protein as claimed in any of claims 53 to 69 for preparing a medicament for the treatment of amyloid β-associated, in particular dementing, disorders.

72. The use of a protein as claimed in any of claims 53 to 69 for preparing a composition for diagnosing amyloid β-associated, in particular dementing, disorders.

73. A vaccine, comprising at least one oligomer or at least one derivative thereof or a composition as claimed in any of claims 1 to 12.

74. The use of an oligomer or derivative thereof or of a composition as claimed in any of claims 1 to 12 for preparing a vaccine for the treatment of amyloid β-associated, in particular dementing, disorders.