MX2007004909A - Anti-addl antibodies and uses thereof - Google Patents

Abstract

The present invention relates to antibodies that differentially recognize multi-dimensional conformations of Aβ-derived diffusible ligands, also known as ADDLs. The antibodies of the invention can distinguish between Alzheimer⿿s Disease and control human brain extracts and are useful in methods of detecting ADDLs and diagnosing Alzheimer⿿s Disease. The present antibodies also block binding of ADDLs to neurons, assembly of ADDLs, and tauphosphorylation and are there useful in methods for the preventing and treating diseases associated with soluble oligomers of amyloidβ1-42.

Description

DIFFUSABLE ANTI-LIGAND ANTIBODIES DERIVED FROM BETA ALPHA AND USES THEREOF BACKGROUND OF THE INVENTION Alzheimer's disease is a progressive and degenerative dementia (Ferry, et al (1991) Ann Neurol 30: 572-580; Coyle (1987) In: Encyclopedia of Neuroscience, Adelman (ed.), Birkháuser , Boson-Basel-Stutgart, pp. 29-31,). In its early stages, Alzheimer's disease manifests itself primarily as a profound inability to form new memories (Selkoe (2002) Science 298: 789-791), apparently due to the neurotoxins derived from amyloid beta (? ß). ? ß is an amphipathic peptide whose abundance is increased by mutations and risk factors linked to Alzheimer's disease. The fibrils formed from ?ß constitute the nucleus of the amyloid plaques, which are remarkable marks of a brain with Alzheimer's disease. Analogous fibrils generated in vitro are lethal to cultured brain neurons. These findings indicate that memory loss is a concentration of neuronal death caused by? ß fibrillar. Despite strong experimental support for? ß fibrillar memory loss, there is a poor correlation between dementia and amyloid plaque burden (Katzman (1988) Ann Neurol., 23: 138-144). In addition, hAPP mice REF..181410 transgenic (Dodart, et al. (2002) Nat. Neurosci.5: 452-457; Kotilinek, et al. (2002) J. Neurosci. 22: 6331-6335), who develop amyloid plaques dependent on age and, in a more important, age-dependent memory dysfunction, show that within 24 hours of vaccination with monoclonal antibodies against? ß, memory loss can be reversed without change in plate levels. Such findings are not consistent with a mechanism for loss of memory dependent on neuronal death, placed by the amyloid fibrils. The additional neurologically active molecules formed by self-assembling j e of? ß, have been suggested. These molecules include soluble ß-ß oligomers, also referred to as diffusible ligands derived from β or ADDLs. The oligomers are metastable and are formed at low concentrations of ß1-42 (Lambert, et al (1998) Proc. Nati, Acad. Sci. USA 95: 6448-6453). The? ß oligomers rapidly inhibit long-term potentiation (LTP), a classic experimental paradigm for synaptic memory and plasticity. As such, memory loss results from the failure of the synapse, before neuronal death and synapse failure by? ß oligomers, not fibrils (Hardy and Selkoe (2002) Science 297: 353- 356). The soluble oligomers have been found in the brain tissue and are strongly elevated in the disease of Alzheimer's disease (Kayed, et al. (2003) Science 300: 486-489; Gong, et al. (2003) Proc. Nati. Acad. Sci. USA 100: 10417-10422) and in models of Alzheimer's disease in mice transgenic hAPP (Kotilinek, et al. (2002) J. Neurosci.22: 6331-6335; Chang, et al. (2003) J. Mol. Neurosci. 20: 305-313). A variety of treatment options for the Alzheimer's test have been suggested. Clinical vaccine trials have shown that people who mount a vigorous immune response to the vaccine show cognitive benefit (Hock, et al. (2003) Neuron 38: 547-554); however, the frequency of central nervous system inflammation caused early termination of part of the test (Birmingham and Frantz (2002) Nat. Med. 8: 199-200). As an alternative to the vaccine, therapeutic antibodies that target ADDLs without binding to the monomers or fibrils have been suggested (Klein (2002) Neurochem Int. 41: 345-352). The ADDLs are highly antigenic, generating selective polyclonal antibodies of the oligomer in rabbits at a concentration of ~ 50 μ? / P ?? (Lambert, et al (2001) J. Neurochem, 79: 595-605). Results from transgenic mouse models also suggest that antibodies can be successful in reversing memory decline (Dodart, et al. (2002), Nat. Neurosci.5: 452-457). Accordingly, there is a need in the art for selective ADDL therapeutic antibodies for prevention and treatment of Alzheimer's disease. The present invention fulfills this need.

BRIEF DESCRIPTION OF THE INVENTION The present invention is an isolated antibody, or fragment thereof, capable of differentially recognizing a multidimensional conformation of one or more diffusible ligands derived from? -β. In particular embodiments, the antibody of the present invention is in admixture with a pharmaceutically acceptable carrier. In other embodiments, the antibody of the present invention is in a kit. Methods for preventing the binding of diffusible ligands derived from ß to a neuron are also provided, inhibiting the assembly of the diffusible ligands derived from ß, and blocking the desfoforilación of the tau protein in Ser202 / Thr205 using an antibody or fragment. of antibody that binds to a multidimensional conformation of one or more diffusible ligands derived from? β. The present invention further encompasses a method for prophylactically or therapeutically treating a disease associated with diffusible ligands derived from? -β, using an antibody of the present invention. The administration of an antibody of the invention can prevent the binding of the diffuse ligands derived from ß to a neuron, with which prevents or treats the disease associated with the diffusible ligands derived from? ß. The present invention is also a method for identifying a therapeutic agent that prevents the binding of diffusible ligands derived from ß to a neuron. This method of the invention involves contacting a neuron with diffusible ligands derived from? -β, in the presence of an agent and using an antibody of the present invention, to determine the binding of the diffuse ligands derived from? -β to the neuron in the presence of from the people . The present invention also encompasses a method for detecting the diffusible ligands derived from? -β in a sample and a method for quenching a disease associated with the diffusible ligands derived from? -β. Such methods involve contacting a sample with an antibody of the present invention so that the diffusible ligands derived from ßβ can be detected and a disease associated with the diffusible ligands derived from ßβ can be diagnosed.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows results from an alkaline phosphatase assay, in which anti-ADDL antibodies differentially block neurons. Figure 2 shows a summary of the hADDL link when the B103 cells are pre-incubated with anti-ADDL antibodies. Figure 3 shows a summary of the binding characteristics of the antibodies capable of differentially recognizing the multidimensional conformations of the ADDLs. Figure 4 shows a summary of the inhibition of ADDL assembly of the antibodies described herein. Figure 5 shows a correlation graph of the N2A link: KADDL. Figures 6A-6X show the nucleic acid sequences for the heavy and light chain variable regions, respectively, for murine anti-ADDL, 20C2 antibodies (Figures 6A and 6B), 5F10 (Figures 6C and 6D), 2D6 (Figures 6E and 6F), 2B4 (figures 6G and 6H), 4E2 (figures 61 and 6J), 2H4 (figures 6K and 6L), 2A10 (figures 6M and 6N), 3B3 (figures 60 and 6P), 1F6 (figures 6Q and 6R), 1F4 (figures 6S and 6T), 2E12 (figures 6U and 6V) and 4C2 (figures 6 and 6X). The lowercase letters indicate the antibody guide sequences and the uppercase letters indicate the sequences of the variable region of the antibody. The nucleotides that code for regions of determination of complementarity (CDRs) are underlined. Figures 7A-7F show the comparisons of the CDR1 sequences (figure 7A), CDR2 (figure 7B), CDR3 (figure 7C) for the heavy chain variable regions, and the CDR sequences (figure 7D), CDR2 (figure 7E), CDR3 (FIG. 7F) for variable chain variable regions for anti-ADDL mouse antibodies. Figures 8A-8K show the amino acid sequences for the heavy and light chain variable regions, respectively, for the humanized anti-ADDL antibodies 20C2 (Figures 8A and 8B), 26D6 (Figures 8C and 8D), 4E2 (Figures 8E and 8F), 3B3 (figures 8G and 8H), 2H4 (figures 81 and 8J) and IF6 (figures 8K) created by the CDR graft. The sequences are presented as comparisons between the mouse sequences, the most homologous human sequence obtained from the NCBI protein database, the most homologous human genomic sequence and the humanized sequence. The amino acids in the mouse and human sequences that differ from the humanized sequences are in bold. The CDRs are underlined. The important residues for the maintenance of the conformation of the CDR loop are indicated with an *. The conserved residues found in the VL / VH interface are indicated with a Potential glycosylation sites are indicated by italics. For the heavy chain 20C2, two humanized sequences (HCVRA and HCVRB) differing by an amino acid in position 24 are generated. In HCVRA of 20C2 the amino acid was used human and in HCVRB of 20C2 the mouse amino acid was used. No light chain was designed for 1F6 because it has the same sequence as that of the light chain for 4E2. Figures 9A-9D show the amino acid sequences for the heavy and light chain variable regions, respectively, for humanized anti-ADDL antibodies 20C2 (Figures 9A and 9B) and 26D6 (Figures 9C and 9D) created by plating or coating. The sequences are presented as comparisons between the mouse sequences, the most homologous human sequence obtained from the NCBI protein database, the most homologous human genomic sequence and the most humanized sequence. The amino acids in the human, mouse and human genomic sequences that differ from the humanized sequences are in bold. The CDRs are underlined. The residues important for the maintenance of the conformation of the CDR loop are indicated with an asterisk. Residues conserved and found in the VL / VH interface are indicated by a number symbol. The potential glycosylation sites are indicated with italics. For the heavy chain 20C2, two humanized sequences were generated (HCVRVenA and HCVRVenB) that differ by an amino acid at position 81. In HCVRVenA of 20C2, the mouse amino acid and the HCVRVenB of 20C2 were used, the human amino acid was used. For heavy chain 26D6, three humanized sequences were designed based on the coating (HCVR Venl, Ven2 and Ven3) that differ in amino acids 11, 23, 15, 81, 89 and 118. In HCVR Venl, the mouse amino acid was used in all positions. In Ven2, the mouse amino acid was used for residues 81 and 118 and the human amino acid for residues 11, 13, 15 and 89. In Ven3, human amino acids were used in all positions. For the light chain of 26D6, two humanized coated sequences (LCVR Venl and Ven2) were designed, which differ in amino acids 88 and 105. In LCVR Venl, the mouse amino acid was used in both positions and in Ven2 the human amino acid was used. Figures 10A-10T show the nucleic acid sequences for the heavy and light chain variable regions (HCVRs and LCVRs, respectively) for humanized anti-ADDL antibodies. The CDR-grafted HCVRs and LCVRs for 20C2, 2D6, 4E2, 3B3, 2H4 and IF6 are respectively presented in Figure 10A to Figure 10K. The coated HCVRs (VenA and VenB) and the LCVR for 20C2 are presented in Figure 10L to Figure ION, while the coated HCVRs (Venl, Ven2, Ven3) and the LCVRs (Venl, Ven2) for 26D6 are presented in the Figure 10O to Figure IOS. Capital letters indicate the sequences of the variable region of the antibody. The CDRs are underlined. The variable region sequences were cloned into the whole heavy and light chain antibody expression vectors. Figures 11A-11Y show the amino acid sequences for the humanized heavy chains IgG1 and IgG2m4 complete and the humanized Kappa light chains for anti-ADDL antibodies; Figure 11A, 20C2 HCVRA of IgGl grafted with the CDR; Figure 11B 20C2 HCVRB of IgGl grafted with CRD; Figure 11C 20C2 HCVRA of IgG2m4 grafted with CRD; Figure 11D, 20C2 HCVRB of IgG2m4 grafted with CRD; Figure HE, 20C2 LCVR Kappa grafted with CRD, Figure 11F, 26D6 HCVR of IgGl grafted with CRD; Figure 11G, 26D6 HCVR of IgG2m4 grafted with CRD; Figure 11H 26D6 HCVR Kappa grafted with CRD; Figure 111, 4E2 HCVR of IgGl grafted with CRD, 4E2 LCVR Kappa grafted with CRD, Figure 11J, 4E23 HCVR Kappa grafted with CRD, Figure 11K, 3B3 LCVR of IgGl grafted with CRD; Figure 11L, Figure 11M, 2H4 HCVR of IgGl grafted with CRD; Figure 11N, 2H4 LCVR Kappa grafted with CRD; Figure 110, 1F6 HCVR of IgGl grafted with CRD; Figure 11P, 20C2 HCVR VenA of coated IgGl; Figure 11Q, 20C2 HCVR VenB of coated IgGl; Figure 11R, 20C2 HCVR VenB of coated IgG2m4; Figure 11S, 20C2 Coated LCVR Kappa; Figure 11T, 26D6 HCVR Venl Ig coated; Figure 11U, 26D6 HCVR Venl IgGl coated; Figure 11V, 26V6 HCVR Ven2 of coated IgGl; Figure 11W, 26D6 HCVR Ven3 coated; Figure 11X, 26D6 LCVR Venl Kappa; and Figure 11Y, 26D6 LCVR Ven2 Kappa. The underline indicates the variable region sequences and the amino acids corresponding to the CDRs are doubly underlined. The remaining amino acid sequences are sequences of the constant region. Figure 12 shows a comparison of the amino acid sequence of the human antibody constant regions and the IgG2m4 sequence. The asterisk indicates a glycosylation site in Asn297. The regions that are linked to FcRn are indicated. The sequences in which IgG2m4 is different from IgG2 are underlined. Figures 13A-13B show the annotated sequence of amino acids for the heavy (FIG. 13A), and light (13B) chains of the humanized antibody 20C2 in phage display vector pFab3d or Fab. Figure 14 describes the design and primers employed in the preparation of two LC-CRD3 libraries namely LC3-1 LC3-2, for the generation of a CDR3 light chain of 20C2 matured by affinity. The restriction endonuclease recognition sites used for cloning are indicated in italics. The uppercase letters indicate the amino acids that code for the sequences of the variable regions of the antibody. The nucleic acids encoding the CDRs are underlined.

DETAILED DESCRIPTION OF THE INVENTION Monoclonal antibodies, which differentially recognize multi-dimensional conformations of the ß-diffused ligands (e.g., ADDLs), have now been generated. Advantageously, monoclonal antibodies can distinguish Alzheimer's disease and control human brain extracts, and identify the endogenous oligomers in brain slices of Alzheimer's disease and cultured hippocampal cells. In addition, the present antibodies neutralize the endogenous and synthetic ADDLs in solution. The so-called "synthetic" ADDLs are produced in vitro by mixing the purified β1-42 amyloid under conditions that generate ADDLs. See United States Patent No. 6,218,506. The particular antibodies herein show a high degree of selectivity for 3-24mers, with minimal detection of the monomeric ß-peptides. In addition, the recognition of ADDLs by selected antibodies of the invention is not blocked by short peptides spanning the linear sequence of ß1-42 or ß1-40. However, the link is blocked by? ß1-28 indicating an epitope based on the conformationally unique structure also found in? ß1-28. The delineation of the epitopes of the current antibodies indicated that these antibodies recognize similar linear core sequences, with similar characteristics of affinity and specificity as measured by ELISA. In addition, the present antibodies differentially block the ability of ADDL-containing preparations to bind to primary cultures of rat hippocampal neurons and immortalized neuroblastoma cell lines, and also block the assembly of ADDL. This finding demonstrates that these antibodies possess a differential ability to recognize a multidimensional conformation of ADDLs despite the recognition of similar linear sequences and affinities. Since it is known that ADDLs are associated with a subset of neurons and disrupt normal neuronal function, one use of the present invention is the development and / or identification of antibodies that prevent the binding of ADDLs to neurons. Such antibodies would be useful in the treatment of diseases related to ADDLs including Alzheimer's disease. A refinement of this use would be to specifically use humanized and / or affinity-matured versions of these antibodies for the prevention of ADDL binding to neurons without assembly of the ADDLs. Accordingly, the present invention is an isolated antibody that differentially recognizes one or more multidimensional conformations of the ADDLs. It is said that an antibody of the present invention is isolated when it is present in the substantial absence of other biological macromolecules of the same type. In this way, a "Isolated antibody" refers to an antibody that is substantially free of other antibodies; however, the molecule may include some additional agents or portions that do not detrimentally affect the basic characteristics of the antibody (e.g., binding specificity, neutralizing activity, etc.). Antibodies that are capable of binding specifically to one or more multidimensional conformations of the ADDLs, bind to particular ADDLs derived from oligomerization of β1-42 but do not cross-react with other ββ peptides, namely β1- 12,? ß1-28,? ß1-40 and? ß2-28 as determined by Western blot analysis as described herein; and they are preferably linked to the ADDLs in solution (see for example, example 21). The specific binding between the two entities generally refers to an affinity of at least 106, 107, 108, 109 or 1010 M. "Affinities greater than 108? G1 are desired to achieve specific binding. which is capable of specifically binding to a multidimensional conformation of one or more ADDL is also produced against (eg, an animal is immunized with) the multidimensional conformations of ADDLs In other embodiments, an antibody that is capable of binding specifically to a multidimensional conformation of one or more ADDLs, it is produced against a peptide of low n-mer formation such as β1-42 (Nle35-Dpro37). The term "epitope" refers to a site on an antigen to which the B and / or T cells respond or a site on a molecule against which an antibody will be produced and / or to which an antibody will bind. For example, an epitope can be recognized by an antibody that defines the epitope. A linear epitope is an epitope in which a primary amino acid sequence comprises the recognized epitope. A linear epitope typically includes at least 3, and more usually, at least 5, e.g., about 8 to about 10 amino acids in the unique sequence. A conformational epitope, in contrast to a linear epitope, is an epitope where the primary sequence of the amino acids comprising the epitope is not the only recognized epitope definition component (eg, an epitope where the primary amino acid sequence) it is not necessarily recognized by the antibody that defines the epitope Typically, a conformational epitope encompasses an increased number of amino acids relative to a linear epitope With respect to the recognition of conformational epitopes, the antibody recognizes a three-dimensional structure of the peptide or of the protein, for example, when a protein molecule is folded to form a three-dimensional structure, some amino acids and / or the polypeptide backbone that forms the conformational epitope become juxtaposed, making it possible for the antibody to recognize the antibody. Methods for determining the conformation of epitopes include, but are not limited to, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy and site-directed spin marking and paramagnetic electron resonance spectroscopy. See, for example, Epitope apping Protocols in Methods in Molecular Biology (1996) Vol. 66, Morris (Ed.). Diffusible ligands derived from ββ or ADDLs refer to soluble oligomers of ββ-42 amyloid which are desirably compounds of aggregates of less than eight or nine β1-42 amyloid peptides, and are found associated with Alzheimer's disease. This is in contrast to the high molecular weight aggregation intermediates, which form rows of micelles that lead to the formation of the fibrils. As exemplified herein, the present antibody binds or recognizes at least one multidimensional conformation of an ADDL (see for example, Figure 3). In particular embodiments, the present antibody binds at least two, at least three, or at least four multidimensional conformations of an ADDL. The multidimensional conformations of the ADDLs are intended to encompass dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nomomers, decamers, etc., as defined by the SDS-PAGE analysis. Because the designations of the trimer, tetramer, etc., may vary with the test method employed (see for example, Bitan, et al. (2005) Amyloid 12: 88-95) the definition of trimer, tetramer, and the like , as used herein, is in accordance with the SDS-PAGE analysis. To illustrate the differential binding capabilities of the present antibodies it has been found that certain antibodies will recognize a multidimensional conformation, for example, ADDLs tetrameters (for example, the 2D6 or 4E2 antibody), while other antibodies recognize several multidimensional conformations, for example. example, the trimer and tetramer of the ADDLs (for example, the antibody 2A10, 2B4, 5F10 or 20C2). As such, the antibodies of the present invention have specific characteristics of oligomer. In particular embodiments, a multidimensional conformation of an ADDL is associated with a specific polypeptide structure that results in a conformational epitope that is recognized by an antibody of the present invention. In other embodiments, an antibody of the present invention binds specifically to a multidimensional conformation ADDL having a size range of about one trimer or tetramer, which have molecular weights greater than 50 kDa. In certain embodiments, in addition, of the binding to a multidimensional conformation, the present antibody binds to a linear epitope selected from β1-42 amyloid. A linear epitope of an ADDL is intended to be a peptide of four, five, six or more amino acid residues, which is healthy at the N-terminal residues of 10, 11, 12, 15 or 20 amino acids of the β1-42 amyloid . In particular embodiments, an antibody of the invention binds specifically to a linear epitope within residues 1-10, 1-8, 3-10 or 3-8 of β1-42 amyloid. Exemplary linear epitopes of β1-42 amyloid include, but are not limited to, amino acid residues EFRHDS (SEQ ID No .: 177); DAEFRHDS (SEQ ID No .: 178; and EFRHDSGY (SEQ ID No .: 179) While the antibodies of the present invention may have similar linear epitopes, such linear epitopes are not completely indicative of the binding characteristics of the present antibodies (for example, the ability to block the binding of ADDL to neurons, prevent phosphorylation of tau and inhibit the assembly of ADDL) because, as is well known to the person skilled in the art, the linear epitope can only correspond to a portion of the epitope of the antigen (see for example, Breitling and Dübel (1999) In: Recombinant Antibodies, John iley & Sons, Inc., NY, pg. 115). For example, 20C2 was found to bind to assemblies of truncated? -7-42 peptide, inverted in charge, lacking the linear epitope for 20C2 (eg, amino acid residues 3-8) and contain a correspondingly different sequence to residues 7-16 of? ß. Therefore, 20C2 binds to the conformational epitopes that depend on the elements from within residues 17-42 of the? ß but only when it is in a multidimensional conformation. The antibodies of the present invention can be distinguished from those of the prior art by being able to differentially recognize the multidirectional ADDLs and consequently differentially block the ADDL that binds to the neurons, differentially foreseeing tau phosphorylation and differentially inhibiting the assembly of ADDL. An antibody, as used in accordance with the present invention includes, but should not be limited to, polyclonal or monoclonal antibodies, and chimeric, human antibodies, for example, isolated from B cells, humanized, neutralizing, bispecific or single chain the same. In one embodiment, an antibody of the present invention is monoclonal. For the production of antibodies, various hosts can be immunized, including goats, rabbits, chickens, rats, mice, humans, and others, by injection with synthetic or natural ADDL. Methods for the production of antibodies are well known in the art. See, for example, Kohler and Milstein ((1975) Nature 256: 495-497) and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York (1988)) Depending on the host species, they can be used Various adjuvants for enhancing the immune response Adjuvants used in accordance with the present invention desirably increase the intrinsic response of ADDLs without causing conformational changes in the immunogen that affects the qualitatively form of the response Particularly suitable adjuvants include monophosphoryl- 3-de-O-acylated lipid (MPLMR; RIBI Immunochem Research Inc., Hamilton, MT; see GB 2220211) and oil-in-water emulsions, such as squalene or peanut oil, optionally in combination with immunostimulants, such as monophosphoryl- lipid A (see Stoute, et al (1997) N. Engl. J. Med. 336: 86-91), muramyl peptides (eg, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP) , Na cetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanin-2- (1 '-2' dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ) -ethylamine (E- PE), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy-propylamide (DTP-DPP), or other components of the cell wall of bacteria. Specific examples of oil-in-water emulsions include MF59 (WO 90/14837), containing 5% squalene, 0.5% TWEEN ™ 80, and 0.5% SPAN 85 (optionally containing various amounts of MTP-PE) formulated into particles submicron using a microfluidizer such as the HOY microfluidizer (Microfluidics, Newton, MA); SAF containing 10% squalene, 0.4% TWEEN ™ 80, 5% polymer L121 blocked with PLURONIC® and thr-MDP, either microfluidized in a submicron emulsion or vortexed to generate a larger emulsion particle size, and the RIBIMR adjuvant system (RAS) (Ribi InmnoChem, Hamilton, MT) containing 2% squalene, 0.2% T EENMR 80, and one or more components of the bacterial cell wall such as monophosphoryl lipid A, trehalose dimycolate. (TDM) and cell wall skeleton (CWS). Yet another adjuvant class is the saponin adjuvants, such as STIMULON ™ (QS-21, Aquila, Framingham, MA) or particles generated therefrom, such as ISCOMs (immunostimulatory complexes) and ISCOMATRIZ® (CLS Ltd., Parkville, Australia) . Other suitable adjuvants include complete Freund's adjuvants (CFA), incomplete Freund's adjuvant (IFA), and mineral gels such as aluminum hydroxide and surface active substances such as lysolecithin, polyols PLURONIC®, polyanions, peptides, CpG (WO 98/40100), keyhole limpet hemocyanin dinitrophenol, and cytokines such as interleukin (IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF), and tumor necrosis factor (TNF). Among the adjuvants used in humans, BCG (Bacillus Calmette-Guerin) and Corynej acteriuffl parvum are particularly suitable. An antibody for an ADDL of multidimensional conformation is generated by the immunization of an animal with the ADDLs. In general, ADDLs can be generated synthetically or by expression and purification of recombinant fragments. Synthetic ADDLs may be prepared as described herein, or in accordance with the methods described in U.S. Patent No. 6,218,506 or in co-pending applications USSN 60 / 621,776, 60 / 652,538, 60 / 695,526 and 60 / 695,528. In addition, ADDLs can be fused with another protein such as key hole limpet hemocyanin to generate an antibody against the chimeric molecule. ADDLs can be conformationally constrained to form a useful epitope as described herein, and furthermore can be associated with a surface for example, physically linked or chemically bound to a surface of a Such as to allow the production of a conformation that is recognized by the antibodies of the present invention. Monoclonal antibodies for the multidimensional conformations of the ADDLs can be prepared using any technique that provides for the production of the antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, et al. (1975) Nature 256: 495-497; Kozbor, et al. (1985) J. Immunol., Methods 81: 31-42; Cote, et al. (1983) Proc. Nati, Acad. Sci. 80: 2026-2030; Cole, et al. (1984) Mol. Cell Biol. 62: 109-120). Exemplary monoclonal antibodies include the murine antibodies designated 2A10, 4C2, 2D6, 4E2, 20C2, 2B4, 5F10, 2H4, 2E12, 1F6, 1F4, 3B3, 5GI2, 6B7, 6B11, 11B4, 11B5, 14A11, 15G6, 17G4, 20C2 , 3B7, 1E3, 1A9, 1G3, 1A7 and 1E5. In addition, humanized and chimeric antibodies can be produced by splicing the mouse antibody genes to human antibody genes to obtain a molecule with antigen specificity and appropriate biological activity (see Morrison, et al. (1984) Proc. Nati, Acad. Sci. 81, 6851-6855; Neuberger, et al. (1984) Nature 312: 604-608; Takeda, et al. (1985) Nature 314: 452-454; Queen, et al. (1989) Proc. Nati Acad. Sci USA 86: 10029-10033; WO 90/07861). For example, a mouse antibody is expressed as the Fv or Fab fragment in a phage selection vector. The gene for the light chain (and in a parallel experiment, the gene for the heavy chain) is exchanged for a library of human antibody genes. The phage libraries, which are still linked to the antigen, are then identified. This method, commonly known as chain intermixing, provided humanized antibodies that should bind to the same epitope as the mouse antibody from which Jespers, et al. (1994) Biotechnology NY 12: 899-903). As an alternative, chain intermixing can be performed at the level of the proteins (see Figini, et al (1994) J. Mol. Biol. 239: 68-78). Human antibodies can also be obtained using phage display methods. See, for example, WO 91/17171 and WO 92/01047. In these methods, phage libraries are produced, in which members show different antibodies on their outer surfaces. The antibodies are usually shown as fragments of Fb or Fab. Phage display antibodies with a desired specificity are selected by affinity enrichment for the ADDLs. Human antibodies against ADDLs can be also produced from non-human transgenic mammals having transgenes encoding at least one segment of the human immunoglobulin locus, and an endogenous, inactivated immunoglobulin locus. See for example, WO 93/12227 and WO 91/10741, each incorporated by reference herein. Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. It is particularly likely that such antibodies share the useful functional properties of mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using the ADDLs as an affinity reagent. Humanized antibodies can also be produced during surface coating or reshaping of murine antibodies. The coating involves the replacement of the amino acids of the fixed surface region in the heavy and light variable regions of the mouse, with those of a homologous human antibody sequence. The replacement of amino acids from the mouse surface with human residues in the same position from a homologous human sequence, has been shown to reduce the immunogenicity of the mouse antibody, while preserving its binding or ligand. The replacement of external debris in general has little or no effect on internal domains, or on interdomain contacts (see for example, U.S. Patent No. 6, 797, 92). Human or humanized antibodies can be designed to have constant regions of IgG, IgD, IgA, IgM or IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG. In particular embodiments, an antibody of the invention is IgG or IgM, or a combination thereof. A particular combination encompasses a constant combination formed by the selective incorporation of human IgG4 sequences into a constant human IgG2 constant region. An exemplary mutant IgG2 Fe is IgG2m4, described herein as SEQ ID No.: 254. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, such as heavy chains and light chains separated or as antibodies to simple chain, in which the variable domains of the heavy and light region are linked through a spacer. Techniques for the production of single chain antibodies are well known in the art.

Exemplary humanized antibodies produced by CDR grafting and coating are described herein for antibodies designated 4E2, 26D6, 20C2, 3B3, 2H4 and 1F6. The amino acid sequences for the heavy chain variable regions of IgG1 and IgG2M4 as well as the light chain variable regions for the humanized 4E2, 26D6, 20C2, 3B3, 2H4 and 1F6, generated by CDR grafting and the coating are presented in the Figures 11A to 11Y, and described herein as SEQ ID No.: 152 to 176. Diabodies are also contemplated. A "diabody" refers to a genetically engineered antibody construct, prepared by isolating the binding domains (heavy chain and light chain) of a binding antibody, and supplying a binding portion that binds or operably links the chains heavy and light on the same polypeptide chain, thus preserving the binding function (see Holliger et al. (1993) Proc. Nati, Acad Sci USA 90: 6444, Poljak (1994) Structure 2: 1121-1123). This forms, in essence, a radically abbreviated antibody, which has only one variable domain necessary for antigen binding. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain, and create two antigen binding sites. These dimeric antibody fragments, or diabodies, are bivalent and bispecific. The person skilled in the art will appreciate that any method for generating the diabodies can be used. The proper methods are described by Holliger, et al. (1993) supra, Poljak (1994) supra, Zhu, et al. (1996) Biotechnology 14: 192-196, and U.S. Patent No. 6,492,123, incorporated by reference herein. Fragments of an isolated antibody of the invention are also expressly encompassed by the present invention. The fragments are intended to include Fab fragments, F (ab ') 2 fragments, F (ab') fragments, bispecific scFv fragments, Fd fragments and fragments produced by a Fab expression library, as well as peptide aptamers. For example, F (ab ') 2 fragments are produced by digestion with pepsin of the antibody molecule of the invention, while Fab fragments are generated by reducing the disulfide bridges of the F (ab') 2- fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of the monoclonal Fab fragments with the desired specificity (Huse, et al (1989) Science 254: 1275-1281). In particular embodiments, the antibody fragments of the present invention are fragments of neutralizing antibodies that retain the binding site to the variable region thereof. Exemplary are F (ab ') 2 fragments / F (ab') / fragments and Fab fragments. See in general Immunology: Basic Processes (1985) 2nd edition, J. Bellanti (Ed.) Pp. 95-97. Peptide aptamers that differentially recognize multidimensional conformations of ADDLs can be rationally designed or selected in an aptamer library (eg, provided by Aptanomics SA, Lyon, France). In general, peptide aptamers are synthetic recognition molecules whose design is based on the structure of the antibodies. Peptide aptamers consist of a variable peptide loop bound at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer at levels comparable to that of an antibody (nanomolar range). Exemplary nucleic acid sequences encoding the heavy and light chain variable regions for use in the production of the antibody and the antibody fragments of the present invention are described herein in FIGS. 6 and 10 (e.g., SEQ. ID No.: 1-24 and SEQ ID No.: 132-151). As will be appreciated by the person skilled in the art, variable regions of heavy chain described herein, can be used in combination with any of the light chain variable regions described herein to generate antibodies with modified affinities, dissociation constants, epitopes and the like. For example, the combination of the light chain variable region of 2H4 (encoded by SEQ ID NO: 12) with the heavy chain variable region of 2A10 (encoded by SEQ ID NO: 13) can provide recognition of a larger linear epitope. Exemplary heavy and light chain CDRs for use in the production of an antibody or antibody fragment of the present invention are described in Figures 7A-7F and have the amino acid sequences described in SEQ ID No.:25, 26 and 28 (heavy chain CDR1); SEQ ID No.: 29, 30, 31, 33, 34, 35 and 36 (heavy chain CDR2); SEQ ID No.: 38, 39, 40, 41, 43, 44, 45, 46, 47 and 48 (heavy chain CDR3); SEQ ID No .: 49, 50, 51 and 53 (light chain CDR1); SEQ ID No.: 54, 55, 56 and 58 (light chain CDR2); and SEQ ID No .: 59, 60, 61, 62, 63, 64 and 66 (light chain CDR3). In particular embodiments of the heavy and light chains of the antibody or of the antibody fragments of the present invention are as follows. A heavy chain CDR1 having an amino acid sequence of Ser-Phe-Gly-Met-His (SEQ ID No.: 28) or Thr-Ser-Gly-Met-Gly- Val-Xaa (SEQ ID No.:27), wherein Xaa is an amino acid without side chain or a small side chain (eg, Ser, Gly or Ala). A heavy chain CDR2 having an amino acid sequence of His-Ile-Xaai-Trp-Asp-Asp-Asp-Lys-Xaa2-Tyr-Asn-Pro-Ser-Leu-Lys-Ser (SEQ ID No.:32) ), while Xaai is an amino acid with an aromatic side chain group (e.g., Phe, Tyr or Trp) and Xaa2 is Ser, Arg or Tyr; or a heavy chain CDR2 having an amino acid sequence of Tyr-Ile-Xaai-Xaa2-Xaa3-Ser-Xaa4-Thr-Ile-Tyr-Tyr-Ala-Asp-Thr-Val-Lys-Arg (SEQ ID No .: 37), wherein Xaai, and Xaa2 are amino acids with a polar side chain group (eg, Arg, Ser, Gly, Thr, Cys, Tyr, Asn, Gln, Lys, or His); Xaa3 is Gly or Val; and Xaa4 is an amino acid with a polar and uncharged side group (eg, Gly, Ser, Thr, Cys, Tyr, Asn, or Gln). A heavy chain CDR3 having an amino acid sequence of Arg-Ser-Ile-Xaai-Xaa2-Xaa3-Xaa4-Pro-Glu-Asp-Tyr-Phe-Xaa5-Tyr (SEQ ID NO: 42), wherein Xaai is an amino acid with an uncharged side polar group (eg Gly, Ser, Thr, Cys, Tyr, Asn, or Gln); Xaa2 is an amino acid with a hydroxyl side chain group (e.g., Ser or Thr); Xaa3 and Xaa4 are amino acids with an aliphatic side chain group (e.g., Ala, Val, Leu, lie, or Pro); and Xaa5 is Asp or Ala. A light chain CDR1 having an amino acid sequence of Arg-Ser-Ser-Gln-Ser-Xaai-Xaa2-His-Ser-Asn-Gly-Asn-Thr-Tyr-Leu-Xaa3 (SEQ ID No.: 52), wherein Xaai and Xaa2 are amino acids with an aliphatic side chain group (e.g., Ala, Val, Leu, Lie or Pro) and Xaa3 is an amino acid with a charged side-chain group (e.g., Asp, Glu , Arg, His or Lys). A light chain CDR2 having an amino acid sequence of Lys-Xaai-Ser-Asn-Arg-Phe-Xaa2 (SEQ ID No.: 57), wherein Xaai is an amino acid with an aliphatic side chain group (e.g. , Ala, Val, Leu, lie or Pro) and Xaa is Ser or Phe. A light chain CDR3 having an amino acid sequence of Xaai-Gln-Xaa2-Xaa3-Xaa4-Val-Pro-Xaa5-Thr (SEQ ID No.: 65), wherein Xaai is Ser or Phe; Xaa2 is an amino acid without side chain (eg, Gly) or a hydroxyl side chain group (e.g., Ser or Thr), Xaa3 is an amino acid with a hydroxyl side chain group (e.g., Ser or Thr); Xaa4 is His, Tyr or Leu; Xaa5 is an amino acid with an aliphatic side chain group (e.g., Ala, Val, Leu, lie, or Pro). As will be appreciated by the skilled person, one or more of the CDRs within the heavy and light chain variable regions of an antibody can be replaced with one or more CDRs from another antibody to generate a completely new antibody or antibody fragment. . For example, replacement of CDR3 of the heavy chain of 5F10 with CDR3 of the heavy chain of 4E2 (SEQ ID No.: 41) may improve the ability of 5F10 to block the binding of ADDLs to cells neuronal Antibodies with particular characteristics are contemplated. In one embodiment, an antibody that binds to the 3 to 8 amino acid epitope of? Β1-42 has a heavy chain CDR1 amino acid sequence of Thr-Ser-Gly-Met-Gly-Val-Xaa (SEQ ID No. : 27), wherein Xaa is an amino acid without side chain or a small side chain (e.g., Ser, Gly or Ala); or an amino acid sequence of the heavy chain CDR2 of His-Ile-Xa ax-Trp-Asp-Asp-Asp-Lys-Xaa2-Tyr-Asn-Pro-Ser-Leu-Lys-Ser (SEQ ID No .: 32), wherein Xaai is an amino acid with an aromatic side chain group (e.g., Phe, Tyr or Trp) and Xaa2 is Ser, Arg or Tyr. In yet another embodiment, an antibody with a moderate affinity for large ADDL aggregates (> 50 kDa) on small aggregates (<30 kDa) (e.g., peak 1 and peak 2 of SEC, respectively), has a sequence of heavy chain CDR3 amino acids of Arg-Ser-Ile-Xaai-Xaa2-Xaa3-Xaa4-Pro-Glu-Asp-Tyr-Phe-Xaa5-Tyr (SEQ ID No .: 42), wherein Xaa! is an amino acid with a polar and uncharged side group (eg, Gly, Ser, Thr, Cys, Tyr, Asn or Gln); Xaa2 is an amino acid with hydroxyl side chain group (eg, Ser or Thr), Xaa3 and Xaa4 are amino acids with an aliphatic side chain group (eg, Ala, Val, Leu, Lie or Pro) and Xaa5 is Asp or To. Antibodies or antibody fragments of the present invention may have additional portions linked to these. For example, a microsphere or microparticle can be linked to the antibody or antibody fragment, as described in U.S. Patent No. 4,493,825, the disclosure of which is incorporated by reference herein. In addition, the antibody or antibody fragments of the invention can be mutated and selected for increased antigen affinity, neutralizing activity (eg, the ability to block the binding of ADDLs to neuronal cells or the ability to block the assembly), or a modified dissociation constant. The mutant strains of E. coli (Low, et al (1996) J. Mol. Biol. 260: 359-368), the intermixing of chains (Figini, et al, (1994) supra), and PCR mutagenesis they are established methods for mutating nucleic acid molecules that code for antibodies. By way of illustration, increased affinity can be selected by contacting a large number of phage antibodies with a small amount of biotinylated antigen, so that the antibody competes for the binding. In this case, the number of antigen molecules must exceed the number of phage antibodies, for the antigen concentration it must be somewhat below the dissociation constant. In this way, phage antibodies predominantly mutated with increased affinity binds to the biotinylated linkage, while the larger part of the weaker affinity phage antibodies remains unbound. Streptavidin can then help enrich the highest affinity mutated phage antibodies from the mixture (Schier, et al (1996) J. Mol. Biol. 255: 28-43). The exemplary affinity-matured light chain CDR3 amino acid sequences are described herein (see tables 11 and 12), with the particular modalities encompassing a light chain CDR3 amino acid sequence of Xaax-Gln-Xaa2 -Thr-Arg-Val-Pro-Leu-Thr (SEQ ID No .: 316), where Xaai is Phe or Leu, and Xaai is Ala or Thr. For some therapeutic applications it may be desirable to produce the dissociation of the antibody from the antigen. To achieve this, the phage antibodies are linked to the biotinylated antigen and an excess of non-biotinylated antigen is added. After a period of time, predominantly phage antibodies with constant lower dissociation can be harvested with streptavidin (Hawkins, et al (1992) J. Mol. Biol. 226: 889-96). Various immunoassays, including those described herein, can be used for screening to identify antibodies or fragments thereof, which have the desired specificity for multidimensional conformations of the ADDLs. Numerous protocols for competitive binding (e.g., ELISA), latex agglutination assays, immunoradiometric assays, kinetic assays (e.g., BIACOREMR analysis) using either polyclonal or monoclonal antibodies, or fragments thereof, are well known in the art. The technique. Such immunoassays typically involve measuring the complex formation between a specific antibody and its cognate antigen. A two-site monoclonal antibody-based immunoassay using monoclonal antibodies reactive for two non-interfering epitopes is adequate, but a competitive binding assay may also be employed. Such assays can also be used in the detection of multidimensional conformations of the ADDLs in a sample. An antibody or antibody fragment can also be subjected to other assays of biological activity, for example, the displacement of the ADDL binding to the neurons or cultured hippocampal cells of the ADDL assembly block, in order to evaluate the neutralizing activity or pharmacological and potential efficacy as a prophylactic or therapeutic agent. Such assays are described herein and are well known in the art. Antibodies and antibody fragments can be produced and maintained as hybridomas or alternatively may be recombinantly produced in any well-established expression system, including but not limited to, E. coli, yeast (eg, Saccharomyces spp., and Pichia app.), baculovirus, mammalian cells ( for example, myeloma, CHO, COS), transgenic plants or animals (Breitling and Dübel (1999) In: Recombinant Antibodies, John Wiley &Sons, Inc., NY, pp. 119-132). The exemplary amino acid sequences of the heavy chain variable regions of IgG1 and IgG2m4, as well as the variable regions of the Kappa light chain for the humanized antibodies 4E2, 26D6, 20C2, 3B3, 2H4 and 1F6 generated by the CDR graft and the coating, are presented in Figures 10A to IOS and described herein as SEQ ID No.: 132 to 151. So that antibodies and antibody fragments can be isolated using any suitable methods including, but not limited to, affinity, immunoglobulin binding molecules (eg, proteins A, L, G or H), markers operably linked to the antibody or antibody fragment (eg, His-tag, FLAG®-tag, Strep tag, c- myc tag) and similar. See, Breitling and Dübel (1999) supra. The antibodies and antibody fragments of the present invention have a variety of uses including, diagnosis of diseases associated with the accumulation of ADDLs, the blocking or inhibition of the binding of ADDLs to neuronal cells, the blocking of ADDL assembly, the prophylactic or therapeutic treatment of a disease associated with ADDLs, the identification of therapeutic agents that prevent the binding of ADDLs to neurons, and that prevent phosphorylation of the tau protein in Ser202 / Thr205. The antibody and antibody fragments of the present invention are also useful in a method for blocking or inhibiting the binding of ADDLs to neuronal cells. This method of the invention is carried out by contacting a neuron, in vitro or in vivo, with an antibody or antibody fragment of the present invention, so that the binding of the ADDLs to the neuron is blocked. In particular embodiments, an antibody or antibody fragment of the present invention achieves at least a decrease of 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 97% in the binding of the ADDLs compared to the ADDLs link in the absence of the antibody or the antibody fragment. The degree to which an antibody can block the binding of ADDLs to a neuron can be determined according to the methods described herein, for example, the immunocytochemistry or the cell-based alkaline phosphatase assay or any other assay suitable. Antibodies particularly useful for decreasing the binding of ADDLs to neuronal cells include exemplary monoclonal antibodies 20C2, 3B3, 1F4, 1F6, 4E2, 2B4, 2D6, and 2H4. The antibody and antibody fragments of the present invention are further useful in a method for blocking or inhibiting the assembly of ADDLs. This method involves contacting a sample containing the β1-42 amyloid peptides with an antibody or antibody fragment of the present invention, so that the assembly of the ADDL is inhibited. The degree to which an antibody can block the assembly of the ADDLs can be determined according to the methods described herein, for example, FRET or fluorescence polarization or any other suitable assay. Particularly useful antibodies to block the assembly of the ADDLs include exemplary antibodies 1F4, 20C2, 4C2, 1F6, 2B4, 5F10, 2A10 and 2D6. The antibodies described herein are also useful in methods for preventing phosphorylation of the tau protein in Ser202 / Thr205. This method involves contacting a sample containing the tau protein with an antibody or antibody fragment of the present invention, so that the binding of the ADDLs to the neurons is blocked, thereby preventing phosphorylation of the Tau protein The degree to which an antibody can prevent phosphorylation of the tau protein in Ser202 / Thr205 can be determined according to the methods described herein or any other suitable assay. Blocking or decreasing the binding of ADDLs to neurons, the inhibition of the assembly of ADDLs and the prevention of tau protein phosphorylation in Ser202 / Thr205 all find application in the methods to treat prophylactically or therapeutically a disease associated with the accumulation of ADDLs. Accordingly, the present invention also encompasses the use of an antibody or antibody fragment of the present invention to prevent or treat a disease associated with the accumulation of ADDLs (e.g., disorders related to Alzheimer's disease or memory or the like). ). Suitable patients for treatment include individuals at risk of disease but not showing symptoms, as well as patients who currently show symptoms. In the case of Alzheimer's disease, virtually anyone is at risk of suffering from Alzheimer's disease, if he lives long enough. Therefore, the antibody or antibody fragments of the present invention can be administered prophylactically to the general population without the need for any evaluation of the risk of the target patient. Current methods are especially useful for inhibit those who have a known genetic risk of Alzheimer's disease. Such individuals include those who have relatives who have been diagnosed with the disease, and those whose risk is determined by analysis of genetic or biochemical markers. Genetic risk markers for Alzheimer's disease include mutations in the APP gene, particularly mutations at position 717 and positions 670 and 671 referred to as the Hardy and Swedish mutations respectively. Other risk markers are mutations in the genes in presenilin, PS1 and PS2, and ApoE4, the family history of Alzheimer's disease, hypercholesterolemia or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized for characteristic dementia, as well as the presence of risk factors described above. In addition, a number of diagnostic tests are available to identify individuals who have Alzheimer's disease. These include the measurement of tau levels in CSF and ß1-42. Individuals suffering from Alzheimer's disease can also be diagnosed by ADRDA criteria or the method described herein. In asymptomatic patients, treatment can begin at any age (eg, 10, 20, 30 years of age). Usually, however, it is not necessary to start the treatment until a patient reaches 40, 50, 60 or 70 years of age. The treatment typically involves multiple doses over a period of time. The treatment can be monitored by the evaluation for the presence of ADDLs over time. In therapeutic applications, a pharmaceutical composition or medicament containing an antibody or antibody fragment of the invention, is administered to a patient suspected of or already suffering from such a disease associated with the accumulation of the ADDLs in an amount sufficient to cure, or at least partially halting the symptoms of the disease (biochemical, histological and / or behavioral), including its complications and intermediate pathological phenotypes in the development of the disease. In prophylactic applications, a pharmaceutical composition or a medicament containing an antibody or antibody fragment of the invention is administered to a patient susceptible to, or otherwise at risk of, a disease associated with the accumulation of the ADDLs in an amount sufficient to achieve passive immunity in the patient, thereby eliminating or reducing the risk, decreasing the severity, or delaying the onset of the disease, including the histological and / or behavioral biochemical symptoms of the disease, its complications and intermediate pathological phenotypes that occur during the development of the disease. In some methods, the administration of the agent decreases or eliminates the myocognitive deterioration in patients who have not yet developed the pathology characteristic of Alzheimer's. In particular embodiments, an effective amount of an antibody or antibody fragment of the invention is an amount that reaches at least a 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% decrease. , 90%, 95%, or 97% in the link of the ADDLs to the neurons in the patient, compared to the ADDL link in the absence of treatment. As such, the impairment of long-term memory enhancement / formation is diminished. The effective doses of the compositions of the present invention for the treatment of the conditions described above vary depending on many different factors, including the means of administration, the physiological state of the patient, whether the patient is human or an animal, other medications administered , and if the treatment is prophylactic or therapeutic. Usually, the patient is a human but other non-human mammals such as dogs or transgenic mammals can also be treated. Treatment doses are generally titrated to optimize safety and efficacy. For passive immunization with an antibody or fragment of antibody, dose ranges of approximately 0.0001 to 100 mg / kg and more usually 0.01 to 5 mg / kg of host body weight are adequate. For example, the doses may be 1 mg / kg body weight or within the range of 1-10 mg / kg. An exemplary treatment regimen involves administration once every two weeks or once a month or once every three to six months. In some methods, two or more antibodies of the invention with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the indicated ranges. The antibodies are usually administered on multiple occasions, where the intervals between the single doses can be weekly, monthly or annually. The intervals may also be irregular as indicated by the measurement of blood levels of antibodies to the ADDLs in the patient. In some methods, the dose is adjusted to achieve a plasma concentration of the antibody of 1-1000 μ? / P ?? and in some methods 25-300 μ9 /? t? 1. Alternatively, the antibody or antibody fragment can be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency varies depending on the average life of the antibody in the patient. In general, humanized and humanized antibodies have longer half-lives than Chimeric antibodies and non-human antibodies. As indicated above, the dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dose is administered at relatively infrequent intervals over a prolonged period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dose at relatively short intervals is sometimes required, until the progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete improvement of the symptoms of the disease. After this, the patient can be administered with a prophylactic regimen. The antibody or antibody fragments of the present invention can be administered as a component of a pharmaceutical composition or medicament. The pharmaceutical compositions or medicaments generally contain the active therapeutic agent and a variety of other pharmaceutically acceptable components. See Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Liipincott Williams and Wilkins: Philadelphia, PA, 2000. The preferred form depends on the intended mode of administration and the therapeutic application. The Pharmaceutical compositions may contain, depending on the desired formulation, non-toxic pharmaceutically acceptable carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, saline buffered with physiological phosphate, Ringer's solutions, dextrose solution and Hank's solution. The pharmaceutical compositions also contain large, slowly metabolized macromolecules, such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as SEPHAROSEMR functionalized with latex, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers and lipid aggregates (such as oil droplets or liposomes). The administration of a pharmaceutical composition or medicament of the invention can be carried out via a variety of routes including, but not limited to, oral, topical, pulmonary, rectal, subcutaneous, intradermal, intranasal, intracranial, intramuscular, infraocular or intra-articular, and the like. The most typical route of administration is intravenous, followed by subcutaneous, although they can be equally effective other routes. The intramuscular injection can also be performed on the arm or leg muscles. In some methods, agents injected directly into a particular tissue where deposits have accumulated, for example, intracranial injection. In some embodiments, an antibody or antibody fragment injected directly into the skull. In other embodiments, the antibody or antibody fragment is administered as a composition or sustained release device, such as an EDIPADMR device. For pteral administration, the antibody or antibody fragments of the invention can be administered as injectable doses of a solution or suspension of the substance in a physiologically acceptable diluent, with a pharmaceutical carrier which can be a sterile liquid such as water, oils, saline, glycerol or ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, surfactants, pH buffering substances and the like, may be present in the compositions. Other components of the pharmaceutical compositions those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol suitable liquid carriers, particularly for injectable solutions. The antibodies can be administered in the form of a depot injection or an implant preparation which can be formulated in such a manner as to allow a sustained release and active ingredient. An exemplary composition contains an antibody at 5 mg / ml, formulated in aqueous buffer composed of 50 mM L-histidine, 150 mM sodium chloride, adjusted to pH 6.0 with hydrochloric acid. Typically, the compositions prep as injectables, either as liquid solutions or suspensions, solid forms suitable for solution in or suspension in liquid carriers can also be prep prior to injection. The preparation can also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymer for improved distribution. For suppositories, the binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formulated from mixtures containing the active ingredient in the range of 0.5% to 10%, or more desirably l% -2%. Oral formulations include excipients such as the pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations or powders, and contain 10% -95% active ingredient, or more adequately 25% -70%. Topical application can result in intradermal or transdermal distribution. Topical administration should be facilitated by co-administering the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins (see Glenn, et al (1998) Nature 391: 851). Co-administration can be achieved by using the components as a mixture or as ligated molecules obtained by chemical cross-linking or expression as a fusion protein. Alternatively, the transdermal distribution can be achieved using a skin patch or using transerosomes (Paul, et al (1995) Eur. J. Immunol., 25: 3521-24; Cevc, et al. (1998) Biochem. Biophys., Acta 1368 : 201-15). An antibody or antibody fragment of the invention can optionally be administered in combination with other agents that at least partially effective in the treatment of amyloidogenic disease. The antibody or antibody fragments of the present invention also find application in the identification of therapeutic agents that prevent the binding of ADDLs to neurons (e.g., a cell. hippocampal) with which they prevent the downstream examples (3 ') attributed to the ADDLs. Such an assay is carried out by contacting a neuron with the ADDLs in the presence of an agent and using an antibody or antibody fragment of the invention to determine the binding of the ADDLs to the neuron in the presence of the agent. As will be appreciated by the person skilled in the art, an agent that blocks the binding of the ADDLs of a neuron, will decrease the amount of ADDLs linked to the neuron compared to a neuron that has not been contacted with the agent; an amount that is detectable in an immunoassay employing an antibody or antibody fragment of the present invention. Suitable immunoassays for detecting ADDLs linked to neurons are described herein. The agents that can be selected using the method provided herein encompass numerous chemical classes, although typically these are organic molecules, preferably small organic compounds having a molecular weight of not more than 100 and less than about 2,500 daltons. The agents encompass functional groups necessary for structural interaction with proteins, particularly hydrogen bonds, and typically include at least one amino, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. Agents often contain structures carbon or heterocyclic cyclics and / or aromatic or polyaromatic structures substituted with one or more of the aforementioned functional groups. Agents can also be found among biomolecules including peptides, antibodies, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. The agents are obtained from a wide variety of sources including libraries of natural or synthetic compounds. A variety of other reagents such as neutral salts and proteins can be included in the screening assays. Also, reagents that otherwise improve assay efficiency, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like, can be used. The mixture of components can be added in any order that provides the required link. The agents identified by the screening test of the present invention will be beneficial for the treatment of amyloidogenic diseases and / or taupathies. In addition, it is contemplated that the experimental systems used to exemplify these concepts represent research tools for the evaluation, identification and selection of new pharmacological targets used with the amyloid induction of phosphorylation. of tau. The present invention also provides methods for detecting ADDLs and diagnosing a disease associated with the accumulation of ADDLs using an antibody or antibody fragment of the present invention. A disease associated with the accumulation of ADDLs is intended to include any disease wherein the accumulation of ADDLs results in physiological deterioration of long-term potentiation / memory formation. Diseases of this type include, but are not limited to, Alzheimer's disease and similar disorders related to memory. According to other methods, a sample from a patient is contacted with an antibody or antibody fragment of the invention and the binding of the antibody or antibody fragment to the sample is syndicator of the presence of the ADDLs in the sample. As used in the context of the present invention, a sample is intended to mean any body fluid or tissue that is suitable for analysis using immunoassays. Suitable samples that can be analyzed according to the methods of the invention include, but are not limited to, biopsy samples and fluid samples from the brain of a patient (e.g., a mammal such as a human). For in vitro purposes (for example, in assays that monitor the formation of oligomers), a sample can be a neuronal cell line or tissue sample. For diagnostic purposes, it is contemplated that the sample may be from an individual suspected of having a disease associated with the accumulation of ADDLs or an individual at risk of having a disease associated with the accumulation of ADDLs, for example, an individual with a family structure that predisposes the individual to a disease associated with the accumulation of ADDLs. Detection of the binding of the antibody or the antibody fragment to the solids in the sample can be carried out using any standard immunoassay (eg, as described herein), or alternatively when the antibody fragment is, for example, a peptide aptamer, the linkage can be directly detected, for example, a detectable marker protein (e.g., β-galactosidase, GFP or luciferase) fused to the aptamer. Subsequently, the presence or absence of the ADDL-antibody complex is correlated with the presence or absence, respectively, of the ADDLs in the sample, and therefore the presence or absence, respectively, of a disease associated with the accumulation of the ADDLs. It is contemplated that one or more antibodies or antibody fragments of the present invention can be used in conjunction with immuno-based, non-invasive, current imaging techniques to greatly increase detection and improve early diagnosis with a disease associated with the accumulation of ADDLs. To facilitate diagnosis, the present invention also pertains to a kit for containing an antibody or antibody fragment of the present invention. The kit includes a container holding one or more antibodies or antibody fragments, which recognize the multidimensional conformation of the ADDLs, and instructions for the use of the antibody for purposes of binding to the ADDLs to form an antibody-antigen complex and detect the formation of the antibody-antigen complex, such that the presence or absence of the antibody-antigen complex correlates with the presence or absence of the ADDLs in the sample. Container agents include multiple well plates that allow simultaneous detection of ADDLs in multiple samples. The invention is described in more detail by the following non-limiting examples.

Example 1: General Materials and Methods Preparation of ADDL. The ADDLs in the F12 medium (Biosource, Camarillo, CA) were prepared from? ß? - 42 according to established methods (Lambert, et al. (2001), supra). Briefly, the β1-42 peptide (American Peptide Co., Sunnyvale, CA or California Peptide Research, Inc., Napa, CA) was weighed and placed in a glass jar capable of maintaining a sufficient amount of HFIP (1, 1, 1, 3, 3, 3-hexafluoro-2-propanol) to reach a peptide concentration of 10 mg / ml. The HFIP was added to the anhydrous peptide, the vial was capped and gently shaken to mix, and the peptide / HFIP solution was stored at room temperature for at least 1 hour. Aliquots (50 or 100 μ ?, 0.5 or 1.0 mg, respectively) of the peptide solution were dispensed into a series of conical tubes for 1.5 ml centrifuge. The tubes were placed in a speedvac overnight to eliminate the HFIP. The tubes containing the dried peptide film were capped and stored at -70 ° C in a sealed container with a drier. Before use, the β1-42 peptide film was removed from storage at -70 ° C and allowed to warm to room temperature. Fresh DMSO (40 μg / mg of the peptide film, 5 mM) was added and the peptide / DMSO mixture was incubated in whirlpool mixer at the lowest possible speed for ten minutes. The F12 medium (2 ml / mg of peptide) was filled into each DMSO / peptide tube and the tube was capped and mixed by inversion. The preparation 100 μp? It was stored at 2-8 ° C for eighteen to twenty-four hours. The Samples were centrifuged at 14,000 x g for ten minutes at 2-8 ° C. The supernatant was transferred to a fresh tube and stored 2-8 ° C until it was used. The biotinylated ADDL preparations (bADDLs) were prepared in the same manner as described above for the ADDL preparations using the 100% N-terminal biotinylated amyloid β peptide (American Peptide Company, Sunnyvale, CA). Preparation of ADDL fibrils. At room temperature the ADDL peptide film was added 2 ml of 10 mM hydrochloric acid per mg of peptide. The solution was mixed on a whirling mixer at the lowest possible speed for five to ten minutes and then the resulting preparation was stored at 37 ° C for eighteen to twenty-four hours before use. Preparation of the monomer. Dry preparations of HFIP of amyloid beta (1-40) (? Β1-40) were prepared as described for the ββ peptide (1-42). The peptide film was dissolved in 2 ml of 25 mM borate buffer (pH 8.5) per mg of peptide, aliquoted, and frozen at -70 ° C until use. Preparation of human fibrils. Samples obtained from the human cerebral cortex were homogenized in 20X cold F12 medium with protease inhibitors (COMPLETE®, Roche Diagnostics Corporation, Indianapolis, IN) for 1 minute. The sample was then centrifuged at 10,000 x g for 1 hour at 4 ° C. After washing twice with F12, the button was resuspended in 2% SDS / F12 and incubated on ice for 30 minutes. The sample was subsequently centrifuged at 220,000 x g for 1 hour at 4 ° C. The button was resuspended in F12, cold and sonicated for 1 minute in 15 second bursts. The protein was determined using the COOMASSIE PLUSTM equipment (Pierce Biotechnology, Rockford, IL). Immunization. The resulting soluble? -somal oligomers, referred to herein as "synthetic" ADDLs were mixed 1: 1 with Freund's complete adjuvant (first and second vaccination) or incomplete Freund's adjuvant (all subsequent vaccinations) and injected subcutaneously (first two vaccinations) or intraperitoneally in three mice in a total volume of ~ 1 ml / mouse. Each injection consisted of purified ADDLs, equivalent to 194 ± 25 μg of total protein. The mice were injected approximately every three weeks. After six injections, one mouse died and his spleen was frozen. The spleen of the mouse with the highest serum titre was then fused with SP2 / 0 myeloma cells in the presence of polyethylene glycol or plated in six 96-well plates. The cells were cultured at 37 ° C with 5% C02 for ten days in 200 μ? of HAT selection medium, which is composed of ISCOV medium supplemented with 10% of fetal bovine serum (FBS), 1 μ? / ml of HYBRI AX® (azaserin-hypoxanthine); Sigma-Aldrich, St. Louis, MO), and 30% conditioned medium collected from SP2 / 0 culture medium. The cultures were fed once with the ISCOV medium supplemented with 10% FBS on day 10, and the culture supernatants were removed on day 14 to select the positive wells in ELISA. Positive cultures were also cloned by limiting dilutions with the probability of 0.3 cells per well. Positive clones were confirmed in ELISA and expanded further. The selection of supernatants involved five assays: a dot blot and western blot (Lambert, et al (2001) supra), a native immunoblot using synthetic ADDLs, and a dot blot and Western blot using endogenous fibrils obtained from human tissue. These assays tested the binding of antibodies to ADDLs (dot blotting) and identified the oligomer (s) that had the highest affinity (western). All antibodies are tested in dot blots using 5 pmol of ADDLs (576 supernatants in the first fusion and 1920 supernatants in the second). Those supernatants that proved to be positive were then screened using Western blot to 10-20 pmol of ADDLs. The selection was repeated to identify low positive or false positive. The supernatants from ten wells were expanded for the first mouse and the forty-five wells were expanded for the second mouse. The expanded cells were then frozen and subcloned. The ascites fluids containing the monoclonal antibody were produced in female balb / c mice using standard protocols (molecular biology protocol). Briefly, the mice were primed by intraperitoneal injection of 0.5 ml of pristane. One week after the priming, the mice were injected intraperitoneally with approximately 5 x 106 hybridoma cells in 1 ml of phosphate buffered saline (PBS). The ascites were collected ten to fourteen days later. The purification of IgG was carried out by using the BIO-RAD® AFFI-GEL® protein A MAPS® II equipment, according to the manufacturer's protocol. For each run, 3 ml of ascites were desalted by passage through a desalting and elution column in 4 ml of binding buffer. The sample was then applied to the protein A column. After washing with 40 ml of binding buffer, the column was eluted with the elution buffer and the 5 ml fractions were collected. The samples were neutralized by the addition of 60 μ? of sodium hydroxide 10 N. To exchange the buffer to the PBS, the Samples were applied to a second desalting column and eluted with PBS. Control antibodies. Polyclonal antibodies M71 / 2 and M90 / 1 were obtained from Bethyl Laboratories, Inc. (Montgomery, TX). The monoclonal antibodies of 6E10 anti-β (produced against residues 1-17) and 4G8 (produced against residues 17-24) were obtained from Signet Labs (Dedham, MA). The monoclonal antibody WO-2 is known in the art for its ability to recognize 1-40 and 1-42 by Western blot analysis (Ida, et al (1996) J. Biol. Chem. 271: 22908- 22914). The monoclonal antibody BAM-10 (produced against? Β-40) was obtained from ABCAM® (Cambridge, MA). The monoclonal antibody 26D6 is well known in the art for its ability to recognize amino acids 1-12 of the ß-sequence (Lu, et al. (2000), Nat. Med. 6: 397-404). Immunoblot analysis. Sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using established methods (Lambert, et al (2001) supra), except that 10-20% Tris-tricine gels were used (BIO-RAD ®, Hercules, CA) and the separation was performed at 120 V. The gels were transferred according to standard methods and the secondary antibody was routinely used at a dilution of 1: 40,000. For the initial selection, 2.7 μg of ADDLs, equivalent to -10-20 pmol / band, were separated on bidiraensional (2D) gels at 4-20%. Electrophoresis and transfer were as described above. Using the tracking dye as a guide, the nitrocellulose was placed inside a Surf-blot device (Idea Scientific, Minneapolis, MN) and 200 μ? of the hybridoma supernatant were mixed with the blocking buffer, composed of 5% fat-free dehydrated milk in buffered saline with TWEENMR 20 (TBS-T; Lambert et al. (2001) supra), and added to each well. 20-21 wells. After incubation at room temperature for 1.5 hours, with shaking, the supernatants were removed and the wells were washed with 200 μ? of blocking damper. The membrane was then removed from the Surf-blot apparatus and washed for 3 x 15 minutes in TBS-T. The secondary antibody (anti-mouse IgG conjugated to HRP, 1: 40,000; Molecular Probes, Eugene, OR) was then incubated with the membrane for 1 hour at room temperature. After washing (3 x 15 minutes) the oligomers were visualized with SUPERS IGNAL® at medium strength (Pierce, Rockland, IL). Western blotting using human fibrils was performed in the same manner using approximately 64 μg of human fibrillated tissue in each two-dimensional immunoblot of SDS-PAGE. Natural polyacrylamide gel electrophoresis was performed according to the established methods (Chromy, et al. (2003) Biochemistry 42: 12749-12760) except that the separation was performed at 120 V. Western Blot. The separated proteins were transferred to nitrocellulose. Stains or blots were blocked with 5% nonfat dry milk or 1% bovine serum albumin (BSA) in TBS-T (TBS with 0.1% TWEENMR 20) overnight, incubated with the primary antibody (s) by 1.5 hours, washed and incubated with the horseradish peroxidase conjugated secondary antibody (HRP) (Amersham Biosciences Corp., Piscataway, NJ) for 1 hour. After the final wash, the proteins were visualized with a West Femto luminescence kit (Pierce Biotechnology, Rockford, IL) and a KODAK® 440 CF or film image station (YPERFILMMR, Amersham Biosciences Corp., Piscataway, NJ). Hippocampal crops. The cultures were prepared from E18 embryos according to standard methods (1997) J. Neurosci. Methods 71: 143-155; Stevens, et al. (1996) J. Neurosci. Res. 46: 445-455). Viable cells were counted and plated on coverslips with polylysine (200 μg / ml) at densities of 1.5 x 10 -106 cells / cm2. The medium was changed by removing half of the medium and replacing it with the supplemented NEUROBASALMR medium.

Primary neurons Primary hippocampal cultures were prepared from neonatal, dissociated, frozen rat hippocampal cells (Cambrex, Corp., East Rutherford, NJ) that were thawed in plated in 96-well COSTAR® plates at a concentration of 20,000 cells per well. The cells were maintained in the NEUROBASALMR medium without L-glutamine (GIBCO-BRLMR, Gaithersburg, MD) and supplemented with B27 (GIBCOBRLMR, Gaithersburg, MD) for a period of two weeks and then used for the binding studies. B103 cells. The B103 neuroblastoma cell line (Schubert and Behl (1993) Brain Res. 629: 275-82) was developed in the DMEM medium without phenol red (GIBCO-BRLMR, Gaithersburg, MD), in the presence of 10% FBS ( Hyclone, Logan, UT) and 1% Pen-Strep (GIBCO-BRLMR, Gaithersburg, MD). B103 cells that develop exponentially were dissociated and seeded in 96-well CORNING® plates at a concentration of 5,000 cells / well. Twenty-four hours after plating, the cells were used to evaluate ADDL and the bADDL binding as well as commercial and novel anti-ADDL monoclonal antibodies. Dot Blot Analysis. These analyzes were performed according to Lambert, et al. ((2001) supra) applying either the ADDLs (5 pmol / point) or the fibrils to the nitrocellulose.

For the last Dot Blots, the ADDLs were applied to anhydrous nitrocellulose in duplicate at various pmolar concentrations in 0.5 μm. using a template derived from the Surf-blot device. The samples were then dried for 15 minutes, blocked with the blocking buffer for 1 hour, and incubated for 1.5 hours with the antibody plus or minus the peptide, which had been pre-incubated for at least 1 hour at room temperature. The solution was removed from the Surf-blot apparatus, the wells were washed with the blocking buffer, and the membrane was removed from the apparatus. The nitrocellulose was washed, treated with the secondary antibody, and visualized as indicated above. Immunocytochemistry Immunocytochemistry was performed according to established methods (Lambert, et al (2001) supra), except that secondary antibodies were conjugated to ALEXAFLUOR® 588 (Molecular Probes, Eugene, OR). Antibodies and ADDLs were pre-incubated for 1 hour at room temperature, at a molar ratio of 1: 4 of the antibody: ADDL before application to the 21-day hippocampal cell culture. For endogenous ADDLs, the human brain protein (prepared as in Lambert, et al (2001) supra) was incubated with cells for 1 hour before the cells were washed, fixed, and visualized as described above.

Slightly fixed frozen sections (4% paraformaldehyde at 4 ° C for 30 hours and cryoprotected in 40 μt sucrose) from hippocampi with Alzheimer's disease and control were incubated with the antibody (1: 1000 in phosphate buffered saline (PBS) )) overnight at 4 ° C. After removal of the antibody, the sections were washed 3 times with PBS and incubated with the secondary antibody at room temperature. The link was then visualized with DAB (SIGMAMR, St. Louis, MO). The sections were then counterstained with hematoxylin, mounted and imagined on a NIKON® ECLISPE® E600 light microscope with a SPOTMR INSIGHTMR digital video camera (see 3.2). Quantitative immunocytochemistry. Cultured hippocampal cells were incubated with 500 nM ADDLs for 1 hour at 37 ° C. The ADDLs were removed by washing and the cells were incubated with 3.7% formaldehyde. Cells were incubated with 0.1% TRITONMR X-100 in PBS-NGS (PBS with 10% normal goat serum) for 30 minutes, washed once, and incubated with the desired primary antibody (s) (diluted in PBS-NGS ) overnight at 4 ° C. Samples were washed and incubated with the appropriate secondary antibody (s), for example, or the anti-mouse and anti-rabbit IgGs ALEXAFLUOR® 488 or 594 (Molecular Probes, Inc., Eugene, OR), for 2 hours at 37 ° C. The coverslips were washed and mounted on the mounting medium PROLONG® anti-fading (Molecular Probes, Inc., Eugene, OR) and imaged using a LEICA® TCS SP2 DMRXE7 focal microscope. ELISA Polyclonal anti-ADDLs IgG (M90 / 1; Bethyl Laboratories, Inc., Montgomery, TX) was plated at 0.25 mg / well on the IMMULONMR 3 REMOVAWELL ™ strands (Dynatech Labs, Chantilly, VA) for 2 hours at room temperature , and then the wells were blocked with 2% BSA in TBS. The samples diluted with 1% BSA in F12 were added to the wells, allowed to bind for 2 hours at 4 ° C, and washed 3 times with BSA / TBS at room temperature. The monoclonal antibodies diluted in BSA / TBS were incubated for 90 minutes at room temperature and detected with a VECTASTAIN® ABC kit for mouse IgG. The HRP marker was visualized with the BIO-RAD® peroxidase substrate and read at 405 nm on a Dynex MRX-TC microplate reader.

Example 2: Development and Characterization of the Anti-ADDL antibodies Three mice were inoculated with the ADDLs (194 ± μg protein / injection) every three weeks for a total of six inoculations. Hybridomas made from the fusion of these mouse spleens with SP2 cells were developed in 96-well plates. The supernatants from these wells were selected in transfers by points with the synthetic ADDLs to identify the positive clones, which were compared with the dot transfers of the endogenous fibrils, to identify the transfers. Hybridomas that bound only to synthetic ADDLs and not to endogenous fibrils were searched. To further refine what the hybridoma products were linked to and under what conditions the linkage occurred, three Western Blots of each positive clone were made: SDS-PAGE of the ADDLs, the native ADDLs gels and SDS-PAGE with endogenous fibrils. Approximately 40 clones were selected for the additional examination. Each clone was tested for the recognition of the brain extract of soluble Alzheimer's disease, for the identification of the ADDLs linked to the cultured hypocompact cells, and for the ability to block the ADDL binding under various conditions. The selected antibodies were collected from the culture medium and subsequently purified using the G SEPHAROSEMR protein. Each time a group of hybridomas was selected by means of Dot Blot approximately -30% produced positive supernatants. Of these, only one or two hybridomas were linked to the synthetic ADDLs and not to the endogenous fibrils. Approximately 2% of the original number of clones were linked to the synthetic ADDLs and not the monomer at low concentrations of ADDL, as determined by Western blot analysis. Clone 3B7, which was linked to the synthetic ADDLs and not to the fibrils on Western blots, was maintained for further analysis. One to two clones were identified which bound to higher molecular weight material (12-24 mer) better than the trimer / tetramer oligomers. Two or three clones were identified, which could bind to the native ADDLs under native conditions, but failed to bind to the ADDLs in the presence of SDS. The results of this analysis indicated that ADDLs are good antigens in mice and monoclonal antibodies that bind to synthetic ADDLs with much higher affinity than monomers can be developed.

Example 3: Immunohistochemical analysis of ADDLs Endogenous and Synthetic Linked to Cultured Hippocampal Cells Cultured hippocampal cells were also analyzed to determine whether the monoclonal antibodies that distinguish between cerebral extracts of Alzheimer's disease and control, could or not identify the ADDLs (endogenous or synthetic) linked to the cells cultivated The hippocampal cultures were prepared according to established protocols and allowed to develop for 3-4 weeks. Synthetic ADDLs were prepared according to standard protocols (for example, U.S. Patent No. 6,218,506). The endogenous ADDLs were extracted from the brain of Alzheimer's disease according to Gong, et al. ((2003) supra). The ADDLs (100 nM in F12, or 2 mg of total protein in F12) were incubated with the cells for 1 hour and then washed and fixed according to standard methods. After washing, the cells were incubated with the monoclonal antibodies 20C2, 3B7, M94, 2A10, E2, 2D6, 4C2, 2B4, 5F10, or 5G12 and subsequently with the anti-mouse secondary antibody conjugated to ALEXAFLUOR® 488. The images were taken on a NIKON® DIAPHOTMR epifluorescent microscope with the COOLSNAPMR HQ_ camera and analyzed using METAMORPHMR software (Universal Imaging, Downingtown, PA). The endogenous and synthetic ADDLs showed the standard hot spot pattern in cultured cells when visualized by 20C2. Thus, the monoclonal antibody 20C2 identifies the synthetic and endogenous ADDLs linked to cultured hippocampal cells. Since 3B7 was not bound to the fibrils, the higher molecular weight oligomers, and the monomers, the hot spot bonding of the ADDLs by 3B7 was attributed to the oligomeric ADDLs.

The other antibodies seemed to recognize a variety of epitopes on the ADDLs bound to the cells ranging from the hot spots on the processes (M94, 2A10) to the specific coupling of the cell body (4E2) and other intermediate states (2D6, 4C2, 2B4). , 5F10, 5G12).

Example 4: Inhibition of ADDL Link to Neurons Using Anti-ADDL Murine Antibodies To determine whether the monoclonal antibodies that distinguish between cerebral extracts of Alzheimer's disease and control, they could also block the binding of ADDLs to cultured cells, cultured hippocampal cells were pre-incubated with the antibody 20C2 and the ADDL binding was determined by immunocytochemistry. Hippocampal cultures were prepared according to established methods and allowed to develop for 3-4 weeks. Synthetic ADDLs were prepared according to standard protocols (for example, see U.S. Patent No. 6,218,506 and the like). The endogenous ADDLs were extracted from the brain of Alzheimer's disease, according to Gong, et al. (2003) supra). The ADDLs (100 nM, in F12, or 2 mg of total protein in F12) were pre-incubated with the 20C2 antibody for 1 hour and subsequently added to cells for 1 hour at 37 ° C. The cells were washed, fixed and incubated with the antibody secondary anti-mouse conjugate to ALEXAFLUOR® 488. The endogenous and synthetic ADDL that bind to the cultured cells were blocked by pre-incubation with 20C2. The images of the vehicle and the non-secondary control antibody were black.

Example 5: Detection of ADDL Link to Neurons Using Biotinylated ADDLs The binding of ADDLs or bADDLs (biotinylated ADDLs) to neurons was detected using standard immunofluorescence procedures. Primary hippocampal neurons (cultured for fourteen days) or B103 cells (plated for 24 hours) were incubated with ADDLs or bADDLs at 5-25 μp? for one hour at 37 ° C and the cells were subsequently washed three to four times with the warm culture medium to remove the unbound ADDLs or bADDLs. The cells were then fixed for ten minutes at room temperature with 4% paraformaldehyde prepared from 16% formaldehyde (Electron Microscopy Sciences, Fort Washington, PA) diluted in PBS. Subsequently, the solution was removed and fresh fixative was added for an additional ten minutes at room temperature. Cells were permeabilized (4% paraformaldehyde solution with 0.1% TRIT0NMR-X 100; Sigma, St. Louis, MO) for ten minutes, washed six times times with PBS and incubated for one hour at 37 ° C with blocking buffer (PBS with 10% BSA; Sigma, St. Louis, MO). At this point, the protocols for the detection of the ADDLs and the linked bADDLs diverge. To detect the ADDL binding, the cells were incubated overnight at 37 ° C with 4G8 (diluted 1: 1,000 in PBS containing 1% BSA; Signet Labs, Dedham, MA), 6E10 (1: 1, 000; Signet Labs, Dedham, MA), or one of the anti-ADDL monoclonal antibodies described herein (diluted 1: 1,000). In addition, a polyclonal antiserum raised against tau (1: 1,000; Sigma, St. Louis, MO) was used to visualize cellular processes. The next day, the cells were washed three times with PBS, incubated for one hour at room temperature with a secondary anti-mouse antibody labeled with ALEXA® 594 (diluted 1: 500 in PBS with 1% BSA, Molecular Probes, Eugene, OR,) and a secondary anti-rabbit antibody labeled with ALEXA® 488 (diluted 1: 1100; Molecular Probes, Eugene, OR), washed three times with PBS and the observed linkage using a microscope with fluorescence capabilities. For the detection of the bADDL binding, the cells were incubated overnight with the tau antibody. Subsequently, the cells were washed three times with PBS, incubated for one hour at room temperature with a secondary anti-mouse antibody labeled with ALEXA® 488 (as described above) and labeled streptavidin.

ALEXA® 594 at a dilution of 1: 500 (Molecular Probes, Eugene, OR), washed 5-6 times in PBS and the binding is performed with a fluorescence microscope. If the staining of the cell nuclei was desired, the nuclei were labeled with DAPI (1: 1000) according to standard protocols. For the immunocytochemical analysis of the ADDLs using an ADDL-specific monoclonal antibody, the cells were washed, fixed, permeabilized and blocked after incubation with ADDLs. To detect the bADDLs linked with monoclonal antibodies, the cells were incubated overnight with 4G8, 6E10 or one of the current anti-ADDL monoclonal antibodies, and the immunoreactivity was subsequently detected with a secondary anti-mouse antibody labeled with ALEXA® 488. The linked bADDLs were visualized with a streptavidin labeled with ALEXA® 594 and the nuclei stained with DAPI. After staining, the colocalization of the bADDL binding and the ADDL immunoreactivity was detected with a fluorescence microscope. Specific immunoreactivity with primary hippocampal cells incubated with ADDLs was observed with each of the monoclonal antibodies (eg, 20C2, 2H4, 2B4 and 2A10). The linked ADDLs appeared as dotted staining throughout the neuronal processes and the cellular soma. This pattern was only observed on a subgroup of neurons, a pattern that is consistent with previous reports describing ADDL binding to primary neurons using commercial and non-commercial antibodies. The staining pattern and the results of a number of control studies demonstrated the specificity of these antibodies. The use of bADDLs offered a simplified method for detecting linked ADDLs and evaluated blocking of ADDL binding with monoclonal antibodies. When the bADDLs were added to the primary hippocampal cells and the binding assessed with a fluorescently labeled streptavidin, the specific binding was observed throughout the neuronal processes of a subset of cells in culture. If the cells were then fixed, the immunocytochemistry was performed and an anti-ADDL antibody was used to visualize the binding, a similar pattern of staining was observed. In addition, the superposition of these staining patterns revealed a perfect overlap of antibody staining and linked bADDLs, thus demonstrating that ADDLs are functionally equivalent and the use of bADDLs in binding assays.

Example 6: Displacement Detection and Measurement Differential of the Monoclonal Antibody Murine anti-ADDL of the bADDL link to neurons The ability of antibodies to block the binding of ADDLs and bADDLs to neuronal cultures (primary neurons or B103 cells) was characterized using the immunocytochemical methods described in present, with a few modifications. The monoclonal antibodies were mixed with 1-10 μ? T? of bADDLs at a molar ratio of 1: 1, 1.5 or 1:10 (antibody: bADDLs) and incubated in a siliconized centrifuge tube for one hour at 37 ° C on a slow rotor (Miltenyi Bistec, Auburn, CA). Subsequently, the antibody / bADDL mixture was added to the cells and allowed to incubate further for one hour at 37 ° C. After incubation, the cells were washed, fixed, permeabilized, blocked and incubated overnight with a polyclonal antiserum driven against tau to visualize cellular processes. The next day, the cells were washed, incubated with a secondary anti-rabbit antibody labeled with ALEXA® 488 and a re-stavidin labeled with ALEXA® 594, and the cells were stained with DAPI to allow detection of the nuclei. Once stained, the degree of bonding was visually assessed with a fluorescence microscope. To quantitatively assess the degree of link of bADDL and the ability of anti-ADDL antibodies to abate this interaction, a cell-based alkaline phosphatase assay was developed. Raonoclonal antibodies or PBS were mixed at a molar ratio of 1: 1 (B103 cells) or 1: 5 (primary neurons) with 2.5-10 μ? (final concentration) of the bADDLs and incubated for one hour at 37 ° C on a slow rotor. After preincubation, antibody / bADDL preparations were added to the B103 or primary neuron cultures and incubated for an additional hour at 37 ° C. At the end of the incubation period, the bADDLs / antibody mixture was removed and the plates washed six times with the medium. The cells were fixed in 4% paraformaldehyde for ten minutes at room temperature, the solution removed, fresh fixative was added and the cells were fixed for an additional ten minutes. The cells were permeabilized with 4% paraformaldehyde containing 0.1% TRITONMR X-100 (2 times, each time for ten minutes at room temperature), washed six times in PBS and treated with 10% BSA for one hour at 37 ° C. C. The reptavidin conjugate to the alkaline phosphatase (1: 1,500 in 1% BSA; Molecular Probes, Eugene, OR) was added to the cells for one hour at room temperature. The cells were rinsed six times with PBS, the alkaline phosphatase substrate (CDP-STAR® with SAPPHIRE-IIMR, Applied Biosystems, Foster City, CA) was added to the cells and incubated for thirty minutes before determining the luminescence on a LJL luminometer (Analyst AD, LJL BioSystems, Sunnyvale, CA). When the binding of the bADDLs to the neurons was evaluated, an antibody-dependent staining pattern was observed. Some of the investigated antibodies markedly reduced the binding of the bADDLs, while others were less effective. Unexpectedly, a third group of antibodies appeared to improve the binding of bADDLs to neurons. While the results of these studies were qualitative and not quantitative in nature, they indicated that the antibodies differentially blocked the binding of bADDL to the neurons. The quantitative evaluation showed a similar trend (figure 1). That is, some antibodies knocked down the binding of bADDLs to neurons, some were weak and had little effect, and a few improved the binding (for example, 5F10 and 4C2). In addition, a mouse Fab was unable to block the binding of the bADDLs further demonstrating the specificity of the monoclonal antibodies in this assay. Analysis of bADDL binding and blocking with monoclonal antibodies in the B103 neuroblastoma cell line demonstrated the binding of bADDL specific for B103 cells, but not to an ovarian cell line (CHO). In addition, the link was dramatically attenuated when the bADDLs were pre-incubated with an anti-ADDL monoclonal antibody before addition to B103 cells. Quantitative evaluation of the blocking of bADDL binding to B103 cells with monoclonal antibodies indicated that monoclonal antibodies were not equal in their ability to block the binding of bADDL to cells (Figure 2). As seen with primary hippocampal cells, some antibodies were very good at blocking the binding, while others were less effective. In addition, the 4C2 antibody also enhanced the ability of bADDLs to bind to B103 cells in culture. To show that bADDLs are also linked to regions of the hippocampus that are involved in learning and memory, a series of linkage studies were conducted using cultures of rat hypocompalent slices. The linkage studies showed that the neurons in CA1-3 and in the dentate gyrus regions of the hippocampus were able to bind to the bADDLs while the neurons in other regions did not. When the bADDLs were pre-incubated with an anti-ADDL monoclonal antibody, the degree of bADDL binding was attenuated in a dose-dependent manner. These results showed that monoclonal antibodies can also knock down the binding of bADDLs to a subgroup of hippocampal neurons, neurons that are critical for learning and memory.

Example 7: Linkage of Anti-ADDL Antibodies to Endogenous ADDLs from Brains with Alzheimer's and Control To further characterize the monoclonal antibodies described herein, it was determined whether monoclonal antibodies could identify ADDLs from soluble extracts of the human brain with Alzheimer's disease (endogenous ADDLs) and distinguish that from the brain control extracts. Synthetic ADDLs and human brain extracts prepared in F12 were diluted in F12 and dotted (1 pmol of ADDLs, 0.5 g of brain extract) in duplicate on anhydrous nitrocellulose HYB0NDMR ECLMR. The brain tissue, with the corresponding degrees of CERAD (Consortium to establish a registry for Alzheimer's disease (Consortium to Establish a Registry for Alzheimer's Diseasse)) and the Blaak stages, was obtained from the NU Brain Bank Core. The blot was allowed to dry for 20 minutes and then incubated in 3% H20 in TBS (20 mM Tris-HCl, pH 7.5, 0.8% NaCl) for 20 minutes at room temperature. The transfer was cut into strips and blocked with 5% milk in TBS-T (0.1% TWEEN ™ 20 in TBS) for 1 hour at room temperature. The polyclonal rabbit antibody M71 / 2 (1: 2500, 0.4 μg, Bethyl Laboratories, Inc., Montgomery, TX); the monoclonal antibody 6E10 (1: 500, 3 μg, Signet Labs, Dedham, MA), and antibodies monoclonal 20C2 (1.52 mg / ml, 5 μg), 11B5 (2.54 mg / ml, 5 μg), 2B4 (1.71 mg / ml, 5 μm), and 2A10 (1.93 mg / ml, 7.5 μm) as described in the present (figure 3) they were diluted in 1.5 ml of milk / TBS-T and incubated for 1 hour at room temperature. The transfers were washed 3 x 10 minutes with TBS-T. The blots were incubated with the horseradish peroxidase-linked secondary antibody (HRP) (1: 40,000 in milk / TBS-T; Amersham Life Science, Inc., Arlington Heights, IL) for 1 hour at room temperature. Blots were washed 3 times for 10 minutes with TBS-T, rinsed 3 times with dH20, developed with SUPERSIGNAL ™ substrate (1: 1 dilution with ddH20; Pierce, Rockland, IL) and exposed to HYPERFILM ™ ECLMR (Amersham Life Science, Inc. , Arlington Heigths, IL). All the antibodies tested identified synthetic ADDLs with robust linkage, except 2A10, which had weaker linkage, even though this was tested at a higher protein concentration. Polyclonal antibody M71 / 2 and monoclonal antibodies 20C2 and 11B5 bound strongly to samples with Alzheimer's disease, but showed only a very mild linkage, similar to the background in the control brain. In contrast, monoclonal antibodies 6E10, 2B4, and 2A10 showed weak link to the brain with Alzheimer's disease. The results of this analysis indicated that two of the monoclonal antibodies tested could distinguish between Alzheimer's disease and the control brain, where the binding to the endogenous oligomers was with a high degree of specificity. In addition, these data indicate that detection can be achieved in early stages of Alzheimer's disease.

Example 8: Nonhistochemical Analysis of Alzheimer's Disease and Brain Slices Control The immunohistochemical analysis using the monoclonal antibodies described herein was carried out to determine whether ADDLs can be visualized in brain slices using monoclonal antibodies that distinguish between Alzheimer's disease and control brain extracts; and to demonstrate the nature of ADDL labeling (eg, diffuse, perineuronal, plate-like, etc.) and its distribution in human tissue. Sections (40 of brain with Alzheimer's disease and brain control, fixed, were prepared according to standard methods. The slices were labeled with several monoclonal antibodies and a polyclonal antibody, and subsequently counteracted with hematoxylin to identify the nuclei of the cells. Images were obtained using a NIKON® ECLIPSE® E600 light microscope with a SPOTMR digital video camera INS IGHT (v. 3.2). Immunohistochemical analysis indicated that ADDL staining was manifested in the brain with Alzheimer's disease, in the hippocampus, the entorhinal cortex, and the intermediate frontal gyrus. In a severe case of Alzheimer's disease, there was abundant staining of light ADDLs in what appeared predominantly as a plaque-like distribution. Some light ADDL staining was observed as perineuronal in a case of Alzheimer's disease. In contrast, there was no staining using the antibody in any regions of control samples, nor even a rare neuron surrounded by dotted immunostaining. These data indicate that monoclonal and polyclonal antibodies can be used to identify ADDLs in fixed human tissue, where the labeling is varied, consisting of plate-like regions, vascular regions, and perineuronal marking of individual cells and some clusters . In addition, the labeling of ADDLs in the brain with Alzheimer's disease, but the control brain was observed in at least three brain regions: the hippocampus, the entorhinal cortex and the intermediate frontal gyrus.

Example 9: Immunostaining Control Similar to Monomer ? ß? -40? ß1-40 is slowly oligomerized in DMS0 / F12 compared to the ADDLs. In this way, it was determined if? ß? -40 could serve as a control similar to the monomer. The ADDLs were subjected to size exclusion chromatography (SEC) on a SUPERDEX® 75 (ADDLO 63), which resolved into two peaks. ? ß1-40 was prepared in D SO / F12 (45.5 mM), frozen and thawed. The samples were diluted with F12 and mixed ~2: 1 with the Tricine sample buffer (BIO-RAD®, Altham, MA). The SDS-PAGE was carried out on 10-20% Tris-tricine gels (BIO-RAD®, Waltham, MA) with Tris / tricine / SDS buffer (BIO-RAD®, Waltham, MA) at 120V a room temperature for 80 minutes. The gel was stained with silver (60 pmoles of β1-40 or ADDLs, 40 pmoles of peaks 1 or 2) with SILVEXPRESS ™ (INVITROGEN ™, Carlsbad, CA). Alternatively, the gels (20 pmol of β1-49 or ADDLs, 30 pmol of peaks 1 or 2) were electrotransferred onto HYBONDM® ECLMR nitrocellulose using 25 mM Tris-192 mM glycine, 20% v / v methanol, pH 8.3, 0.02 of SDS at 100V for 1 hour at 8 ° C. Transfers were blocked with 5% milk in TBS-T (0.1% TWEEN ™ 20 in 20 mM Tris-HCl, pH 7.5, 0.8% sodium chloride), overnight at 8 ° C. The monoclonal antibody 6E10 (1: 2000; Signet Labs, Dedham, MA), the monoclonal antibody 20C2 (1.52 mg / ml, 1: 2000; figure 3); or the M71 / 2 polyclonal antibody (1: 4000, Bethyl Laboratories, Inc., Montgomery, TX) was diluted in milk / TBS-T and incubated with the blots for 90 minutes at room temperature. The blots were washed 3 x 10 minutes with TBS-T and subsequently incubated with the secondary antibody conjugated to HRP (1: 40,000 in TBS-T; Amersham Life Science, Inc., Arlington Heigths, IL) for 1 hour at room temperature. . After three washes with TBS-T, 10 minutes per wash, the blots were rinsed 3 times with dH20, developed with the substrate of maximum sensitivity West Femto SU PERSIGNALO (dilution 1: 1 with ddH20; Pierce, Rockland, IL) and exposed to HYPERFILMMR ECLMR (Amersham Life Science, Inc., Arlington Heigths, IL). The silver staining analysis showed ß1-40 as a heavy monomer band. In contrast, the ADDLs and peak 1 showed the monomer, the trimer and the tetramer, although there was less tetramer. The silver staining analysis of type 2 showed the heavy monomer with a lighter trimer and a very light tetramer band. The immunostaining of β1-40 with 6E10 showed only one band of light monomer. The immunity of the ADDLs and the peak 1 with 6E10 showed the monomer, the trimer, the tetramer and 12-24 mer. Peak 2 showed the staining of the heavy monomer with 6E10 and some light trimer and the tetramer without 12-24 mer. There was no monomer staining of? ß1-40 with 20C2 or M71 / 2. While 20C2 and M71 / 2 showed minimal or no monomer staining of the ADDLs and peak 1, these samples stained the trimer, the tetramer and the 12-24 mer with 20C2 and M71 / 2. The immunottion of peak 2 with 20C2 and M71 / 2 showed the light monomer, the trimer and the tetramer without 12-24 mer observed. ? ß1-40 was immunostained lighter with 6E10 than the ADDL monomer, despite the heavier silver staining. These results indicated that, in contrast to the 6E10 antibody showing good recognition of the monomer, the gels transferred with 0.02% SDS in the transfer buffer showed minimal detection of the monomer with oligomer specific antibodies. Immunostaining of the SEC fractions showed peak 2 composed mainly of monomer with small amounts of trimer and tetramer and without 12-24 mers, while peak 1 has monomer, trimer, tetramer and 12-24 mers. To further characterize the monoclonal antibodies with respect to the binding to peak 1 and peak 2, the sandwich ELISA was developed using the M90 polyclonal antibody to the ADDLs as capture antibody. The fractions of peak 1 and of the SEC peak 2 referred to herein are the two largest peaks of the ADDLs that were fractionated on a SEPHADEX ™ 75 column to distinguish between the potentially bioactive and inactive oligomers.

Non-denaturing gel electrophoresis confirmed the separation into large (> 50 kDa) and small (<30 kDa) aggregates that were stable at 37 ° C. These peaks were used separately as the detection substance for the supernatants of the clones. The link was visualized with the VECTASTAIN® device. The differences between the recognition of two peaks were observed for all antibodies. For example, compare the ratio of peak 1 to peak 2 for antibodies 2B4 and 20C2 (figure 3). Only one antibody reflects the preference of the control antibody (6E10) for peak 2.

Example 10: Detection of ADDL Formation from? ß? -42 Polyclonal antibodies have been used in dot blots to show the time-dependent ADDL formation from ß1-42. Thus, it was shown that the monoclonal antibody 20C2, which binds preferentially to the oligomers, could also show an increased signal over time as the ADDLs are formed from ß1-42. ? ß1-42, -750 pmoles of the HFIP film, were dissolved in 1.5 ml of DMSO (0.5 mM) and aliquots of 2 μ? diluted to a final volume of 100 μ? with F12 (10 nM) and incubated on ice. Two μ? (20 fmol) of the reaction mixture were transferred by dots over dry nitrocellulose HYBOND ECL (Amersham Life Science, Inc., Arlington Heights, IL) at specified time points. Nitrocellulose was blocked with 5% non-fat dehydrated milk in TBS-T (20 mM Tris-HCl, pH 7.5, 0.8% sodium chloride, 0.1% T EENMR-20) for 1 hour at room temperature. The polyclonal antibody M90 / 1 (Bethyl Laboratories, Inc., Montgomery, TX) or the monoclonal antibody 20C2 (1.52 mg / ml) was diluted 1: 2000 in milk / TBS-T and incubated with the transfer for 90 minutes at room temperature. environment, followed by washing 3 x 10 minutes with TBS-T. The secondary antibodies conjugated to HRP (Amersham Life Science, Inc., Arlington Heights, IL) were diluted 1: 40,000 in milk / TBS-T and incubated blotting for 60 minutes at room temperature followed by washing as described above. After a brief rinse with dH20, the transfer was incubated for 60 seconds with the SUPERSIGNAL® West Femto maximum sensitivity substrate (diluted 1: 1 with ddH20; Pierce, Rockland, IL) and exposed to HYPERFILMMR ECLMR (Amersham Life Science, Inc. ., Arlington Heights, IL). The transfers by points were explored and the intensity of the points was determined with ADOBE® PHOTOSHOP®. Both antibodies detected time-dependent ADDL formation of ß1-42, where the results for 20C2 showed better signal and consistency. Nor does the antibody was able to detect β1-40 at a concentration equivalent to the ADDLs. These data further demonstrate the specificity of the oligomer of this antibody, since the monomers are present all the time and the oligomers are formed over time. In addition, M90 / 1 and 20C2 showed minimal recognition of the? 1-40 monomers even at a concentration 100 times higher than the ADDLs.

Example 11: Transfer Tests by Competition Points To determine whether the monoclonal antibodies described herein could be linked to the monomers, a dot transfer test was performed, competing with the synthetic ADDLs, 20C2 and ß1-40. The ADDLs were applied to dry nitrocellulose at 10 pmol / 0.5 μ? . While the nitrocellulose was being blocked in 5% NDM / TBS-T for 1 hour, the ADDLs and? -1-40 fresh at various concentrations were incubated with 200 μ? of each of 20C2 (final concentration of 1.5 μq / ml) in 5% of NDM / TBS-T for 1 hour. These solutions were then applied to the nitrocellulose using the SURF-BLOT apparatus and incubated at room temperature for 1.5 hours with oscillation. The transfer was subsequently visualized with anti-mouse IgG-HRP and chemiluminescence. The quantification was done using KODAK® IMAGESTATION® 440 and EXCEL®.

The results of this analysis indicated that the synthetic ADDLs in solution could effectively and specifically block the binding of 20C2 to the ADDLs and mobilized on nitrocellulose with a maximum mean inhibition observed at < 50 nM for the ADDLs. In contrast, ß? -40 in solution did not block the binding of 20C2 to the immobilized ADDLs. To determine which portions constitute the binding epitope of the β1-42 molecule, a competition transfer test was performed with the ADDLs, 20C2 and the peptides. The ADDLs were transferred by points on nitrocellulose at four concentrations (1, 0.5, 0.25, and 0.125 pmol) each in 0.5 μ? . While the nitrocellulose was being blocked in 5% NDM / TBS-T for two hours, the peptides at 50, 100 and 200 pmol were added to 200 μ? of 20C2 (final concentration of 1.52 μ9 / p? 1 = 1.9 pmol, in 5% of NDM / TBS-T) and stirred at room temperature. The solutions were subsequently incubated with the nitrocellulose using the SURF-BLOT apparatus for 1.5 hours at room temperature. The binding was visualized with anti-mouse IgG-HRP using chemiluminescence. The results of this analysis indicated that the link to the ADDLs was blocked by the ADDLs themselves and by? ß1-28, but not another combination of peptides. Thus, the binding epitope required some conformation that? ß1-28 could reach, but that was not available about? ß1-12 and? ß12-28 or their combination. Alternatively,? ß1-28 forms a dimer that blocks the binding of the ADDLs by spherical impediment. To determine if? ß1-28 is added (similar to? ß? - 42) or folded such that it blocks the binding epitope for 20C2, the SDS-PAGE gels were stained with silver and the transfer analysis was performed. Western. The ADDLs and ß1-28 (60 pmol in each of two bands used for silver staining and 20 pmol otherwise) were separated using a SDS-PAGE of 10-20% Tris-tricine. The bands at 60 pmol were excised and stained with SILVERXPRESS ™ (INVITROGEN ™, Carlsbad, CA); alternatively, the gels (20 pmoles of ADDLs and? ß1-28) were electroblotted on the HYBONDMR ECLMR nitrocellulose using 25 mM Tris-glycine 192 mM, 20% v / v methanol, pH 8.3, 0.025 SDS at 100 V for 1 hour at 8 ° C. Blots were blocked with 5% milk in TBS-T (0.1% TWEENMR-20 in 20 mM Tris-HCl, pH 7.5, 0.85 sodium chloride). The samples were incubated with 20C2 (1: 1000, 1.52 mg / ml) or 20C2 +? ß1-28 (2 nmol, preincubated for 2 hours) for 1.5 hours at room temperature in the above-mentioned blocking buffer. The binding was visualized with the anti-mouse IgG-HRP (1: 40,000 in TBS-T) and chemiluminescence.

Silver staining showed a monomer, trimer and tetramer in the ADDL band, while the? ß? -28 band had a species, which ran approximately as a dimer. The ADDLs, but not? ß1-28 were visualized by 20C2 and the link to all ADDL species by 20C2 was blocked by? ß1-28. In addition, while the binding epitope of 20C2 is blocked by β1-28, 20C2 does not recognize the β1-28 peptide in a Western blot.

Example 12: Isotype Analysis of the Anti-ADDL Antibodies To further characterize the monoclonal antibodies described herein, the isotype analysis was performed using the SIGMA IMMUNOTYPEMR kit with the Mouse Monoclonal Antibody Isotyping Reagents, following the instructions of the manufacturer (Sigma-Aldrich Co., St. Louis, MO). The results of this analysis are presented in Figure 3.

Example 13: Core Linear Epitope Mapping of Anti-ADDL Antibodies The specific interaction of the anti-ADDL monoclonal antibodies with the amyloid beta peptide was detected in standard ELISA assays. In summary, the synthetic peptides, or ADDL or the fibrila in some cases, they were used as the antigen to be coated on the NUNCMR MAXI SORBMR plate at a concentration of 4 μq / ml (approximately 800 to 1200 nM). Unless specified, the peptides were coated in 5 mM sodium bicarbonate buffer, pH 9.6, overnight at 4 ° C. After blockade, the plates with PBS containing 0.05% TWEENMR 20 and 3% (w / v) dehydrated milk without fat for one hour, the monoclonal antibody was titrated in blocking buffer at a certain concentration and the plates were incubated for one hour at room temperature with gentle shaking. After washing, goat anti-mouse IgG conjugated to HRP (H + L), diluted in the blocking buffer, was added to the plates. The colorimetric substrate, TMB, was added to the plates after intensive washings to remove the conjugate with unbound HRP. The absorbance was measured at a wavelength of 450 nm on a plate reader. To map the linear epitope of the nucleus for the anti-ADDL monoclonal antibodies, a group of ten amino acid, overlapping peptides was synthesized to cover? ß? -42 (Table 1). Three peptides of fourteen amino acids, with reverse sequence of amino acids? ß1-42, were also synthesized as non-specific control peptides.

TABLE 1 All the peptides were dissolved in DIVISO approximately 400 to 50 μ? (1 mg / ml) and stored multiple aliquots at -20 ° C. The peptides were used ^ in an ELISA assay for the determination of the core epitope of the anti-ADDL monoclonal antibodies. Each monoclonal antibody was tested at four concentrations (3, 1, 0.3 and 0.1 μq / ml) either against a group of N-terminal peptides (from residues 1 to 25) or a group of C-terminal peptides (from the residues 17 to 42), with control peptides. The linear core epitopes for the panel of monoclonal antibodies are listed in Table 2. Several commercial monoclonal antibodies (6E10, BAM-10, 4G8 and WO-2) were included in the experiment to validate the assay format, and the results confirmed their linear core epitopes as reported in the published literature.

TABLE 2 Position inside? ? -42. aIgGl, IgG2b, IgG2a, nd, not determined.

Nine of twelve ADDL-specific monoclonal antibodies evaluated were mapped to the N-terminal region of? ß? -42, and seven of these were mapped to amino acid residues 3 to 8. Two monoclonal antibodies, 2H4 and 2E12, prefer epitopes slightly bigger. Three monoclonal antibodies, 1F4, 1F6 and 3B3, failed to bind to the group of overlapping peptides, even at high concentration of 3 μg / ml, but their epitopes were estimated to be located at the N-terminus of? Β -42, since these they could bind to the β1-20 peptide, which was used as a positive control in the experiments.

Example 14: Affinity and Specificity of the Anti-ADDL Mouse Antibodies A solution-based binding assay was developed to determine the specificity and affinity of the anti-ADDL antibodies to differentiate amyloid beta peptide preparations (ADDL, fibril,? ß1-40,? ß1-20). A quantitative ELISA was established, which was able to capture the linear dose-response range of monoclonal antibodies against ADDL coated on NUNCMR plates. Based on this information, a fixed concentration of monoclonal antibody was selected that could give consistent optical density (OD) signals in ELISA just above the test noise (OD reading at 450 nm around 0.2). to 0.5). The IgG at this fixed concentration was then incubated with different substrates of amyloid beta peptide (ADDL, fibril,? -1-40,? -1- 1-20) in 20 point titrations in solution at room temperature overnight to reach equilibrium. The amount of free IgG within the mixture was determined the next day in a quantitative ELISA assay with one hour of incubation over regular ELISA plates. The bound IgG fraction was calculated and the correlations of the IgG bound to the free ligand titration (substrates) were used to derive KD, using the GraFit program (Erithacus Software, Surrey, United Kingdom). Thus, the substrate preference for each antibody to different preparations of amyloid beta peptide was presented as the intrinsic affinity (KD) values. There were several advantages to using this test format. Firstly, the interaction of the antibody and the substrate was in the solution phase, thus, there was no constraint from any solid surface such as in the regular ELISA assay or the BACOREMR experiment, where the potential influence of the solid surface from the ELISA plates or the sensor chip on the monoclonal antibody and the interaction of the substrate have to be taken into consideration for the interpretation of the data. Second, the interactions were allowed to break the balance. Therefore, the interaction of IgG and the substrate occurred in limiting concentrations of both components without problems for the precipitation of IgG or ol igomerization of the amyloid beta peptide due to the high concentration experimental. Third, the reading of the assay was independent of the antigen in the solution; thus, any heterology of beta amyloid in different peptide preparations (eg, ADDL or fibril) could not interfere with data interpretation and mathematical modeling. The sensitivity of the assay was limited to the detection limits of the ELISA assay, which allowed this assay to evaluate monoclonal antibodies with KD values in the nanomolar range. Alternative substrates such as fluorescent reagents are contemplated to improve the range of sensitivity. It is believed that the immune complex was minimally disturbed during the one hour incubation to capture the free IgG in the quantitative ELISA. The amounts of free IgG were determined by a standard curve and plotted against the titrations of different substrates. The amounts of the IgG linked to different substrates were plotted graphically and the information was used in GraFit to adjust the curve with appropriate mathematical models. The KD summary, expressed in the nM ranges, for the panel of monoclonal antibodies described herein is presented in Table 3.

TABLE 3 * All antibodies were IgG. The values listed in italics are high SE and poor adjustment. IC: inconclusive data N / T: not tested Example 15: Detection and Measurement of Phosphorylation of hyperphosphorylated Tau Tau (pTau) is a key marker of Alzheimer's disease, although little is known about the events that cause this hyperphosphorylation. Unwanted Being compromised by some theory, it is believed that ADDLs can play a role in this phosphorylation event. To investigate this, neuronal cultures (primary neurons and B103 cells) were developed as described above, adding 1 μ ?? of bADDLs or vehicle to the media, and the crops were maintained for one, six or twenty-four additional hours. At the end of each incubation, the cells were washed, fixed, permeabilized, blocked and incubated overnight with a monoclonal antiserum raised against pTau (AT8, 1: 500, Pierce, Rockland, IL). The next day, the cells were washed, incubated with a secondary anti-mouse antibody labeled with ALEXA® 488 and a streptavidin labeled with ALEXA® 594 and the cells were stained with DAPI to allow detection of the nuclei. The cells were then evaluated using a fluorescence microscope, with the degree of pTau staining and correlation with the bADDL binding that is annotated at each time point. The results of this analysis indicated that the binding of bADDL to B103 cells increased the level of pTau in cellular processes, when compared with vehicle-treated cells. A similar change was also noticed in the primary hippocampal cells. When cells were exposed to bADDLs for six hours, an increase in pTau staining was observed in a subpopulation of cells, the cells that also bound to bADDL. A study over time with B103 cells further investigated the modulation of pTau by the bADDLs. The addition of bADDLs resulted in a marginal increase in pTau at one hour. However, pTau staining was dramatically increased six hours after the addition of the bADDLs and remained elevated until 24 hours later. Thus, these data indicate that the binding of ADDL to neurons can initiate a cascade of intracellular events that result in the hyperphosphorylation of tau, the accumulation of neurofibrillary tangles and eventual cell death. For this purpose, a person skilled in the art can appreciate that blocking the binding of ADDLs to neurons could in turn prevent such events downstream, and be beneficial for the treatment of amyloidogenic diseases and / or tauopathies. In addition, a better understanding of the signaling events that are triggered by the ADDL link and result in the production of pTau, can also elucidate the additional pathways that are suitable targets for the development of new therapeutics.

Example 16: Interaction of the Peptide [beta] / ADDL-Antibody and Inhibition of Assembly The changes in the kinetics of assembly of ADDL and the oligomeric size, in the presence of antibodies Selected monoclonal antibodies described herein, were observed by the fluorescence resonance energy transfer (FRET) and fluorescence polarization (FP) using a 1: 4 mixture of the fluorescein-labeled β1-42 monomers, to the native peptide monomers . The auto-off of the fluorescein emission after the incorporation of the monomer into the ADDLs, results in a three to five-fold reduction in the fluorescence intensity over the short time scale in hours due to FRET. In addition, the increase in size when the monomers are assembled into oligomeric ADDL species results in a two-fold increase in FP. The FRET and FP kinetic progress curves of the ADDL assembly, in the presence of various anti-ADDL and anti-ββ peptide, novel and commercial, showed differences in the ability of the antibodies to inhibit the assembly of ADDL and / or the binding of the peptide oligomers (Figure 4). The tests were carried out in black opaque microtitre plates, of CORNING® Non-Bonding Surface, of 384 wells. The assay buffer was composed of 50 mM MOPS-Tris (pH 8.0) with 100 mM magnesium chloride. The assay volume, which contained ????? ß1-42 0.2 μ? A and? ß1-42 0.8 μ?, Was 50 IU and the assay temperature was 37 ° C. The ADDL assembly was monitored with a plate reader Tecan GENios Pro, exciting a wavelength of 485 nm and detecting the emission at a wavelength of 515 nm. The kinetic traces were collected by recording the fluorescence intensity and the polarization readings every five minutes over a six-hour time course. The negative control reactions, which did not appreciably resemble the ADDLs during this time, lacked magnesium chloride but contained all other components of the buffer and the peptide. The positive control reactions contained all the components of the buffer in the absence of aggregated reagents of monoclonal antibody. To test ADDL binding and inhibition of assembly, the antibodies were incubated with the peptide mixture at eight concentrations from 500 nM down to 5 nM. This assay was useful for classifying different profiles of ADDL binding behavior and inhibition of ADDL assembly. The binding and neutralization of larger ADDL species, through interaction with specific ADDL and / or conformational epitopes, serves as a viable therapeutic strategy. In addition, the inhibition of oligomerization in large ADDLs by the binding of an ADDL-specific and / or conformational epitope present in transient (non-monomeric) intermediary ADDL assembly species provides an alternative strategy for anti-ADDL therapy. The FP progress curves, which demonstrated the surprising differences between the antibodies, denote such a binding of intermediate or stable species. The correlation of FP / FRET behavior of monoclonal antibodies with other functional, cellular and in vivo effects allows the selection of desired immunotherapeutic modes of action. The results of the analyzes described herein indicate that 1F6, 2A10, 5F10, 2D6 and 2B4 show potent assembly inhibition, while 20C2, 1F4 and 4C2 show intermediate inhibition of the assembly and 2H4, 3B3 and 4E2 show weak assembly inhibition ( Figure 4). As summarized in Table 4 and illustrated in Figure 5, 20C2, 4E2, 3B3 and 5F10 show a variety of biochemical behaviors.

TABLE 4 In addition, the antibody 1A9, one of five purified antibodies (eg, 1A9, 1E3, 1G3, 1A7 and 1E5) generated against a peptide of low formation of n-merβ1-42 [Nle35-Dpro37], is secreted with 5F10 in terms of its inhibition of FP assembly and behavior.

In addition, 20C2 was found to bind to inverted mounts, to assemblies of the truncated? -7-42 peptide as determined by SE / ICC, indicating a lack of conventional linear epitope that binds to the inverted-charge peptide? -42, which has a very different sequence corresponding to residues 7-16 of? ß, for example,? ß (7-42) [Orn7Orn1iD13Di Ei6Nle35]. Therefore, 20C2 binds to the conformational epitopes that depend on the elements from within residues 17-42 of? ß, but only when they are assembled.

Example 17: Isolation of the Variable Region of Mouse Antibody The cDNAs encoding the variable domains of the mouse antibody were cloned and sequenced after a polymerase chain reaction (PCR) using specially designed primers that hybridize to the 5 'ends of the mouse constant regions and the murine guide sequences upstream (5') of the V regions. This ensured that the mouse variable region sequences obtained were complete and accurate. Briefly, the mRNA was extracted from the mouse hybrid cell lines using the Mini QIAGEN® OLIGOTEX® Direct mRNA kit and subsequently converted to cDNA using a first-strand cDNA synthesis kit. The cDNA was then used as a template in the PCR reactions to obtain the sequences of the variable regions of the antibody. To obtain the light chain variable region sequence, eleven independent PCR reactions were established using each of the eleven PCR primers 5 'light chain (MKV-1 to MKV-11) and the PCR primer MKC-1 3 '(Table 5).

TABLE 5 The underlined and italic sequences denote the restriction sites Xbal and SacI, respectively K = G or T, Y = C or T and R = A or G.

To obtain the heavy chain variable region sequences, twelve independent PCR reactions were established using each of the twelve heavy chain 5 'PCR primers (MHV-1 to MHV-12) and the appropriate isotype-specific primer 3' (HCG-1, HCG-2A, MHCG-2B, MHCG-3) (Table 6).

TABLE 6 The underlined and italic sequences denote the Xbal and SacI restriction sites, respectively. W = A or T, M = A or C, K = G or T, Y = C or T and R = A or G.

Each of the light chain PCR reactions contained 46 μ? of the Super Mix of PCR INVITROGENMR PLATINUM®, 1.0 μ? of one of the primers 5 '100 μ? (MKV-1 to MKV-11), 1.0 μ? of primer 3 '100 μ? (MKC-1), and 2.0 μ? of hybridoma cDNA. Similar PCR reactions were used to clone the variable regions of the mouse heavy chain. The reactions were placed in a DNA thermal cycler and, after an initial denaturation step at 97 ° C for 2 minutes, were subjected to 30 cycles of: 95 ° C for 30 seconds, 55 ° C for 45 seconds, and 72 ° C for 90 seconds. After the last cycle, the final extension step at 72 ° C for 10 minutes was used. To determine which PCR reactions produced the product, aliquots of 5 μ? of each reaction were separated on gels with 1.5% (w / v) agarose / ?? of TAE buffer, containing 0.5 μg / ml of ethidium bromide. The PCR products from the reactions that produced the fragments of the expected size (420 to 500 base pairs) were then gel purified, digested with Xbal and SacI and ligated into the Xbal and SacI sites in the multiclonation region of the plasmid. pNEB193 (New England Biolabs, Beverly, MA). Alternatively, the PCR products were ligated directly into plasmid pCR®2.1 using the INVITROGENMR TA CLONING® kit. The ligation products were then transformed into the XL-1 cells and the transformed E. coli aliquots were plated on LB agar plates containing 50 μg / ml ampicillin and covered with 40 μ? of the X-Gal reserve solution (50 mg / ml) and 40 μ? of IPTG (100 m) for the selection of blue / white. Plates were incubated overnight at 37 ° C and potential clones were identified as white colonies. DNA from at least 24 independent clones for each PCR product was sequenced on both strands using the forward and reverse universal primers for pNEB193 and pCR®2.1. The resulting sequences were then assembled into a contiguous to generate a consensus sequence for each variable region of light and heavy chain of antibody. Using this procedure the sequences were determined for the light and heavy variable regions of the hybridoma antibody 20C2, 5F10, 2D6, 4E2, 2H4, 2A10, 3B3, 1F6, 1F4, 2E12 and 4C2 (Figures 6A-6X). The six complementarity determination regions (CDRs), which form the structure complementary to the antigen, are underlined in Figures 6A-6X. After the analyzes of the corresponding CDRs and antigenic epitopes (Table 2), the sequence similarities were observed. Antibodies that share the 3 to 8 amino acid epitope of β1-42 (eg, 2A10, 4C2, 2D6, 4E2, 20C2, 2B4 and 5F10) shared the sequences of CDR1 (Figure 7A) and CDR2 (Figure 7B), highly homologous, heavy chain. Antibody 2H4, which was found to recognize the 1- to 8-amino acid epitope of ß1-42, appeared to have unique CDR3 sequences (Figure 7C) of single and heavy chain CDR1 (Figure 7D), CDR3 (Figure 7E) ) and CDR3 (Figure 7F) of the light chain. Similarly, the 2E12 antibody, which was found to recognize the 3 to 10 amino acid epitope of β1-42, has unique heavy chain CDR3 sequences (Figure 7C). In addition, antibodies 2A10, 2B4, 4C2 and 4E2, which have similar affinities for the Pico 1 and Pico 2 ADDLs of SEC (see Figure 3), shared highly homologous heavy chain CDR3 sequences (Figure 7C). In addition, the amino acid substitutions in CDR3 of the heavy chain of the 4E2 antibody appeared to improve blockade of the binding of ADDLs to neuronal cells, since 4E2 was more effective than antibody 2D6 in blocking the binding of ADDL to neurons, and heavy and light chain sequences of 4E2 and 2D6 were identical except for three CDR3 amino acid residues of the heavy chain; Ser versus Asn, Thr versus Ser, e lie versus Val for 2D6 and 4E2, respectively (Figure 7C).

Example 18: Humanization of the Sequences of the Variable Region of the Anti-ADDL Mouse Antibody The nucleic acids of the variable domains of the heavy and light chain of the mouse antibody, obtained from mouse hybridoma cell lines 20C2, 26D6, 4E2, 3B3 , 2H4 and 1F6 were humanized using a CDR grafting process and in the case of 20C2 and 26D6 a coating or coating strategy. It will be appreciated by those skilled in the art that the humanization of mouse antibody sequences can maximize the therapeutic potential of an antibody by improving its serum half-life and its Fe effector functions, thereby reducing the anti-globulin response . The humanization by CDR grafting was carried out by the selection of the human light and heavy chain variable regions from the database of NCBI proteins with the highest homology to the mouse variable domains. The sequences of the mouse variable region were compared to all sequences of the human variable region in the database using the Local Basic Protein-protein Alignment Search Tool (BLAST). Subsequently, the mouse CDRs were linked to the human framework regions and the preliminary amino acid sequence was analyzed. All differences between the mouse and human sequences in the structural regions were evaluated particularly if they were part of the canonical sequences for the loop structure or were residues located at the VL / VH interface (O'Brien and Jones (2001)). in: Antibody Engineering, Kontermann and Dubel (Eds.), Springer Laboratory Manuals). Structural regions were also screened for unusual or rare amino acids compared to consensus sequences for the human subgroup and for potential glycosylation sites. Where there were differences in the amino acid sequence between the sequences of the human and mouse structural regions that were not found to be involved in the canonical sequences or located at the VL / VH interface, the human residue was selected at that position. Where there was a difference in a key residue, two versions of the variable region sequence were generated for the evaluation. The CDR grafting strategy made the minimum number of changes to the human structural region so that a good binding to the antigen was achieved while maintaining human structural regions that closely resembled the sequence derived from natural human antibody. The design of the humanized amino acid sequences using the CDR graft is shown in Figure 8. The humanized sequences for 20C2 and 26D6 were also designed using a coating strategy (see, for example, US Pat. No. 6, 797, 492). Humanization was carried out by selecting the human light and heavy chain variable regions of the NCBI protein database with the highest homology to the mouse variable domains, as well as to the germ line family or families of human antibodies , closer (see, Kabat, et al. (1991) Sequences of proteins of immunological interest, 5th ed., US Dept. Health and Human Services, NIH, Washington DC). The sequences of the mouse variable region were compared to all sequences of the human variable region in the database using BLAST protein-protein. The murine variable sequences and their closest human homologs were modeled to the closest crystallized human antibody as determined by computer modeling as practice in the technique. From the model of the murine VH and VL sequences, a map of the surface area was constructed, which dictated the accessibility to the solvent of the amino acids in the variable regions of the heavy and light chains of mice. To confirm the modeling, these exposed residues were compared position by position with the known accessible surface residues (see, for example, Padlan (1994) Mol Immunol 31 (3): 169-217). A rating was assigned for each residue in the sequence designating it as exposed, mainly exposed, partially buried, mainly buried and buried according to the established methods (see, United States Patent No. 6,797,492). The mouse structural residues that qualified as exposed or mainly exposed and differed from the homologous human sequences were changed to the human residue in that position. The designed coated sequences retained the mouse CDRs, the residues that are adjacent to the CDRs, the residues known to be involved in the canonical sequences, the residues located at the VL / VH interface, and the residues in the N-terminal sequences of the heavy and light mouse chain. The N-terminal sequences are known to be contiguous with the CDR surface and are potentially involved in binding to the ligand. Similarly, care must be taken to limit changes in Pro, Gly or charged waste. Once the coated sequences were finalized they were remodeled to look for any obvious, potential structural problems. In some cases, more than one coated sequence was generated for the analysis. The design of the humanized amino acid sequences using the coating procedure is shown in Figure 9. Once the humanized amino acid sequences were selected the sequences were reverse translated to obtain the corresponding DNA sequence. The DNA sequences were optimized at the codon using methods established in the art (Lathe (1985) J. Mol. Biol. 183 (1): 1-12) and designed with flanking restriction enzyme sites for cloning within human antibody expression vectors. The synthesized DNA sequences are presented in Figures 10A-10S. For the humanized 20C2 antibodies designed by the grafting and coating of CDR, the human versions of IgGl / kappa and IgG2m4 / kappa were constructed, wherein IgG2m4 represents the selective incorporation of the human IgG4 sequences into a constant region of standard human IgG2. . The IgGl / kappa and IgG2m4 / kappa versions were also elaborated for the antibody grafted with CDR 26D6. For all other antibodies, only the IgGl / kappa versions were made. The complete sequence of amino acids of the resulting antibodies is shown in Figures 11A-11Y. The antibodies were expressed by co-transient transfection of the separated light and heavy chain expression plasmids, within 293 EBNA cells. In cases where more than one humanized heavy or light chain sequence was designed for a given antibody, all combinations of heavy and light chains were combined to generate the corresponding antibodies. The antibodies were purified from the culture supernatant 7 to 10 days after transfection using protein A columns and used in the subsequent analysis.

Example 19: Generation of IgG2m4 Antibodies IgG2m4 antibody derivatives were prepared to decrease Fe receptor coupling, Clq binding, unwanted cytotoxicity or immune complex formation, while maintaining the extended half-life of the properties pharmacokinetics of a typical human antibody. The IgG2m4 basic antibody format is that of IgG2, which has been shown to have a longer half-life in experimental models (Zuckier, et al. (1994) Cancer Suppl 73: 794-799). The IgG2 Structure was modified to eliminate the Clq link through the selective incorporation of the IgG4 sequences, while maintaining the low level typical of the FcyR binding (Canfield and Morrison (1991) J. Exp. Med. 173: 1483-1491). This was achieved by the use of crossing points where the sequences of IgG2 and IgG4 were identical, whereby an antibody containing natural Fe sequences is produced instead of any artificial mutational sequences. The IgG2m4 form of the human antibody constant region was formed by the selective incorporation of the human IgG4 sequences into a standard human IgG2 constant region, as shown in Figure 12. Conceptually, IgG2m4 resulted from a pair of exchanges of chain within the CH2 domain as shown in Figure 12. Four simple mutations corresponding to the sequences coming from IgG4 were made. The Fe residues mutated in IgG2 included His268Gln, Val309Leu, Ala330Ser, and Pro331Ser, which minimized the potential for neoepitopes. The specific IgG4 amino acid residues placed within the constant region of IgG2 are shown in Table 7, along with other alternatives from the basic structure.

TABLE 7 The positions marked with an asterisk are subject to allele variations. Hougs et al. (2001) Immunogenetics 52 (3-4): 242-8. W097 / 11971. Medgyesi, et al. (2004) Eur. J. Immunol. 34: 1127-1135. Tao et al. (1991) J. Exp. Med. 173: 1025-1028. Armor et al. (1999) Eur. J. Immunol. 29: 2613.

Xu et al. (1994) J. Biol. Chem. 269: 3469-3474. Canfield and Morrison (1991) J. Exp. Med. 173: 1483.

Example 20: Linkage Affinity of Anti-ADDL Humanized Antibodies To assess the binding affinity to ADDL of humanized antibodies, titration ELISAs were conducted as described herein. The 96-well microtiter plates, coated with streptavidin (Sigma, St. Louis, MO) were coated with 10% biotinylated ADDL antigen (1 μ?). A series of 1-fold dilutions of the purified antibody, beginning at 500 ng / ml were added to the captured ADDL plates and the plates were incubated for 2 hours at 25 ° C. After washing five times with the PBS solution using a plate washer (Bio-Tek, Winooski, VA), goat anti-human kappa light chain polyclonal antibody (Biomeda, Foster City, CA) was added to a dilution 1/2000 in a fat-free milk blocker and incubated at room temperature for 1 hour. A rabbit anti-goat IgG (H + L) antibody conjugated to HRP (Bethyl Laboratories, Inc., Montgomery, TX) was added at a dilution of 1/2000 in the blocking solution and incubated for 1 hour at room temperature. After washing with PBS, the HRP substrate, 3, 3 ', 5', 5-tetramethylbenzidine (ready for use TMB; Sigma, St. Louis, MO) was added and the reaction was stopped after 10 minutes with 0.5N sulfuric acid. The absorbance at a wavelength of 450 nm was read in a plate reader (model VICTOR V, Perkin Elmer, Boston, MA) and the data were processed using the EXCEL® spreadsheet. Test variations between plates were estimated within 20%. Different groups of humanized antibodies were compared in different experiments. A comparison of the antibodies of humanized IgG1, 20C2A, 20C2B, 3B3, 4E2, 1F6 and 2H4 by CDR grafting, indicated that all antibodies could bind to the ADDLs, where the binding to 1F6 was weaker than most and 20C2A It was the strongest. The four different humanized versions of IgGl 20C2 antibodies (two versions grafted with CDR and two coated versions) were also compared and found to show very similar ADDL binding curves, with the entire link slightly better than a chimeric 20C2 antibody. The seven different humanized versions of IgGl 26D6 (a grafted version with CDR and six coated versions) were also compared. It was found that all have ADDL binding curves similar to the chimeric form of 26D6. The IgG and IgG2m4 antibodies for the two versions of 20C2 humanized by the CDR graft were also analyzed and found to have comparable binding curves as they had the isotypes IgGl and IgG2m4 of humanized 26D6, by grafting with CDR. Example 21: Inhibition of Linkage of ADDL to Neurons Using Humanized Anti-ADDL Antibodies Humanized anti-ADDL antibodies were additionally evaluated for their ability to block the binding of ADDL to primary hippocampal neurons using the methods described herein. The relevant antibodies, or PBS as a control, were mixed at a molar ratio of 1: 1 (B103 neuroblastoma cells) or 1: 5 (primary hippocampal neurons) with 2.5 to 10 μp? (final concentration) of bADDLs and incubated for one hour at 37 ° C on a slow rotor. After preincubation, antibody / bADDL preparations were added to B103 or primary neuron cultures, and incubated for an additional 1 hour at 37 ° C. At the end of the incubation period, the bADDLs / antibody mixture was removed and the plates washed six times with the medium. The cells were then fixed in 4% paraformaldehyde for ten minutes at room temperature, the solution removed, fresh fixative was added, and the cells were fixed for an additional ten minutes. The cells were permeabilized with 4% paraformaldehyde containing 0.1% Triton ™ X-100 (2 times, each time for ten minutes at room temperature), washed six times in PBS and then treated with 10% BSA in PBS for one hour at 37 ° C. Streptavidin conjugated to alkaline phosphatase (1: 1,500 in 15 BSA; Molecular Probes, Eugene, OR) was then added to the cells for one hour at room temperature. The cells were rinsed six times with PBS, the alkaline phosphatase substrate (CDP-STAR® with SAPPHIRE-I IMR, Applied Biosystems, Foster City, CA) was added to the cells and incubated for thirty minutes before the luminescence was determined. a LJL Luminometer (Analyst AD, LJL Biosystems, Sunnyvale, CA). As with murine antibodies, the humanized versions of 26D6, 20C2, 4E2, 3B3, 2H4 and 1F6 were able to inhibit the binding of ADDL preparations to neuroblastoma B103 cells and to primary neurons.

Example 22: Maturation of Affinity of an Anti-ADDL Humanized Antibody The nucleic acid molecules encoding the humanized 20C2 version, a single variable heavy chain, the light chain only, or the heavy chain and the light chain together, were cloned within of Pfab3d vector phage display Fab. Analysis of the nucleic acid sequence confirmed the sequence and orientation in pFab3d. The 20B2 Fab sequences annotated, in pFab3d are presented in Figure 13 and described herein as SEQ ID NO: 255 for the heavy chain and SEQ ID NO: 256 for the light chain. The three constructs were used in the 20C2 maturation program using the Fab library methods visualized with phage established in the art. In summary, two libraries were designed to mutate the nine wild-type amino acids of CDR3 of the light chain (kappa) of 20C2 (e.g., Phe-Gln-Gly-Ser-Leu-Val-Pro-Leu-Thr; NO: 60). These libraries were designed LC3-1 and LC3-2 representing the light chain CDR sequences of Xaa-Xaa-Xaa-Xaa-Xaa-Val-Pro-Leu-Thr (SEQ ID NO: 257 and Phe-Gln-Gly- Ser-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO: 258), respectively Biotinylated reverse primers, 20C2LC3-1 (SEQ ID NO: 259) and 20C2LC3-2 (SEQ ID NO: 260), were used in combination with the forward primer 20C2LC3F (SEQ ID NO: 261) to generate the LC3-1 and LC3-2 libraries (see Figure 14) The primers were purified by polyacrylamide gel electrophoresis, while the vector DNA was purified by gel electrophoresis and electroelusion The two light chain libraries were designed to be randomly mutated The final diversities of the three LC3 libraries of 10G5H6 were 4.76 x 108 and 7.45 x 108, respectively (Table 8). 100 clones from the libraries showed 100% diversity of the mutant clones in the designated positions of amino acids.

TABLE 8 * Higher titres are achieved by concentration or phage rescue.

The soluble panning of the two light chain libraries of 20C2 against the high molecular weight bADDL was completed. In summary, four rounds of panoramic visualization were carried out using biotinylated high molecular weight ADDL (bADDL). The first three rounds were carried out using approximately 1.5 μ of the antigen concentration (input = l x 1010 to 1 x 1011). After the termination of the In the third round, the outputs of the two libraries were combined and divided into three groups for analysis with 10 nM, 100 nM and approximately 1.5 μ? of the antigen to increase the requirement of panoramic visualization. As such, a total of 58 exit plates were tested in phage ELISA assays, for example, two plates per library in the first round (a total of four plates), six plates per library in the second round (a total of 12 plates), eight plates for the LC3-1 libraries and 10 plates for the LC3-2 libraries in the third round (a total of 18 plates) and eight plates for each antigen concentration in the fourth round (a total of 24 plates). The panoramic display resulted in 1000 hits, 436 of which were sequenced (Table 9).

TABLE 9 a LC3-1 of 20C2 versus 10% high molecular weight bADDL. b LC3-2 of 20C2 versus 10% high molecular weight bADDL. c LC3-1 of 20C2 + LC3-2 of 20C2 versus 10% high molecular weight bADDL. * Successes by total number of colonies. Sequence and frequency of highly enriched clones are presented in Table 10.

TABLE 10 The Fab fragments from the 109 higher clones based on the enrichment frequency were prepared and a total of 15 clones were converted to the humanized version of IgGl and two clones, 20C2-6 and 20C2-8, were converted to version B humanized IgGl. The KD values for these clones were measured by BIACOREMR using biotin-Apl-20 (Table 11) and bADDL (Table 12) as antigens. Dramatic improvements in affinity were observed compared to 20C2A and 20C2B humanized progenitors, as well as mouse 20C2 antibodies. In particular, low nanomolar to sub-picomolar KDs were achieved with a light chain of CDR3 of the Xaai-Gln-Xaa2-Thr-Arg-Val-Pro-Leu-Thr sequence (SEQ ID NO: 318), where Xaai is Phe or Leu, and Xaai is Ala or Thr. In addition, a comparison between the KD values obtained with BIACOREMR using biotin ß1-20 and bADDL further demonstrates that anti-ADDL antibodies such as 20C2 binds preferably to the multi-dimensional conformations of the ADDLs on the γ monomeric peptides. TABLE 11 TABLE 12 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (10)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An isolated antibody, or fragment thereof, characterized in that it is capable of differentially recognizing a multidimensional conformation of one or more diffusible ligands derived from? Β.

2. A pharmaceutical composition characterized in that it comprises an isolated antibody, or fragment thereof, capable of differentially recognizing a multidimensional conformation of one or more diffusible ligands derived from the? -β, in admixture with a pharmaceutically acceptable carrier.

3. A method for preventing the binding of diffusible ligands derived from? ß to a neuron, characterized in that it comprises contacting the neuron with the antibody according to claim 1, so that the binding of the diffusible ligands derived from the? ß towards the neuron, it is prevented.

4. A method for inhibiting the assembly of the diffusible ligands derived from? Β, characterized in that it comprises contacting a sample containing the ß 1-42 amyloid peptides with the antibody according to claim 1, whereby the assembly of the diffuse ligands derived from ßβ is inhibited.

5. A method for blocking the phosphorylation of the protein na tau in Ser202 / Thr205, characterized in that it comprises contacting a sample containing a tau protein with the antibody according to claim 1, thereby blocking the phosphorylation of the tau protein in Ser202 / Thr205.

6. Use of a pharmaceutical composition according to claim 2, for preparing a medicament for prophylactically or therapeutically treating a disease associated with the diffusible ligands derived from? Β.

7. A method for identifying a therapeutic agent that prevents the binding of the diffuse ligands derived from? -β, to a neuron, characterized in that it comprises contacting a neuron with the diffuse ligands derived from? -β in the presence of an agent and using a antibody according to claim 1 for determining the binding of the diffusible ligands derived from? -β to the neuron, in the presence of the agent.

8. A method for detecting the diffusible ligands derived from? Β, in a sample, characterized because it comprises contacting a sample with the antibody according to claim 1, so that the diffusible ligands derived from ßβ are detected.

9. A method for diagnosing a disease associated with the diffusible ligands derived from ß-ß, characterized in that it comprises contacting a sample with the antibody according to claim 1 so that a disease associated with the diffusible ligands derived from γ is diagnosed. H.H .

10. A kit for detecting the diffusible ligands derived from? ß, characterized in that it comprises the isolated antibody or the fragment thereof according to claim 1.