KR20080068040A - Anti-addl monoclonal antibody and use thereof - Google Patents

Abstract

The present invention relates to antibodies that-differentially-recognize--multi-dimensional conformations-of-AB-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 tau phosphorylation and are there useful in methods for the preventing and treating diseases associated with soluble oligomers of amyloid F 1-42.

Description

Anti-ADDL monoclonal antibody and method of using the same {Anti-ADDL Monoclonal Antibody and Use Thereof}

summary

This invention is a CIP (continuation-in-part) application of PCT Application No. PCT / US2005 / 0038125, filed October 21, 2005.

Background of the Invention

Alzheimer's disease is progressive and degenerative dementia (Terry, et al. (1991) Ann . Neurol . 30: 572-580; Coyle (1987) In: Encyclopedia of Neuroscience , Adelman (ed.), Birkhauser, Boston-Basel-Stuttgart, pp 29-31). In the early stages, Alzheimer's disease presents a serious condition that is primarily unable to form new memories (Selkoe (2002) Science 298: 789-791), which is known to be due to neurotoxin derived from amyloid beta (Aβ). Aβ is an amphiphilic peptide and its abundance is increased by risk factors associated with mutations or Alzheimer's disease. Fibrils formed from Aβ form the center of amyloid plaques, proving that it is a brain with Alzheimer's disease. Similar fibrils produced in vitro are lethal to cultured cranial nerves. These findings indicate that memory loss is the result of neuronal death by fibrillar A β.

Despite strong experimental support for fibrillar Aβ and memory loss, there is a weak correlation between dementia and amyloid plaque burden (katzman (1998) Ann. Neurol . 23: 138-144). In addition, transgenic hAPP mice that developed age-dependent amyloid plaques and most importantly, age-dependent memory dysfunction (Dodart et al. (2002) Nat. Neurosci. 5: 452-457; Kotilinek (2002) J. Neurosci. 22: 6331-6335) show that amnesia can be cured without any change in plaque levels within 24 hours of vaccination of monoclonal antibody against Aβ. This finding is inconsistent with the mechanism that memory loss is dependent on neuronal death caused by amyloid fibrils.

Additional neuroactive molecules formed by Aβ self-assembly have been proposed. Such molecules include soluble Αβ oligomers called Αβ-induced diffuse ligands or ADDLs. The oligomers are metastable and are formed at low concentrations of Aβ 1-42 (Lambert et al. (1998) proc. Natl . Acad . Sci. USA 95: 6448-6453). Aβ oligomers rapidly inhibit long-term potentiation (LTP), a classic experimental paradigm for memory and synaptic plasticity. As such, memory loss results from synaptic disorders by Aβ oligomers, not from fibrillar, prior to neuronal death and synaptic disorders (Hardy & Selkoe (2002) Science 297: 353-356). Soluble oligomers are found in brain tissue, and Alzheimer's disease (Kayed, et al. (2003) Science 300: 486-489; gong, et al. (2003) proc. Natl. Acad. Sci. USA 100: 10417-10422) And hAPP transgenic rat models with Alzheimer's disease (Kotilinek, et al. (2002) J. Neurosci . 22: 6331-6335; Chang, et al. (2003) J. Mol. Neurosci . 20: 305-313). Is elevated at a high rate.

Various methods of treating Alzheimer's disease have been proposed. Persons with strong immune responses to vaccines by vaccine clinical trials exhibit cognitive benefits (Hock, et al. (2003) Neuron 38: 547-554); Frequent inflammation of the central nervous system caused the termination of some experiments (Birmingham & Frantz (2002) Nat. Med. 8: 199-200). As a vaccine replacement, therapeutic antibodies targeting ADDLs without binding to monomers or fibrils have been proposed (Klein (2002) Neurochem. Int. 41: 345-352). ADDLs have been highly antigenic, such as generating oligomer-selective polyclonal antibodies at a concentration of 50 μg / mL in rabbits (Lambert, et al. (2001) J. Neurochem. 79: 595-605). The results obtained in the transgenic mouse model also suggest that antibodies can be successful in reversing memory decline (Dodart, et al. (2002) Nat . Neurosci . 5: 452-457). Thus, for the prevention and treatment of Alzheimer's disease, there is a need in the art for antibodies that selectively treat ADDL. The present invention will satisfy this need.

Summary of the Invention

The present invention relates to an isolated antibody or fragment thereof that can differentially recognize the multidimensional conformation of one or more Aβ-induced diffuse ligands. In particular, the antibody of the present invention may comprise the complementarity determining site of Arg-Xaa ₁ -Leu-Xaa ₂ -Xaa ₃ -Xaa ₄ -Xaa ₅ -Xaa ₆ -Asp-Ala-Met-Asp-Tyr (SEQ ID NO: 9). CDR), where Xaa ₁ Is Gln or Ala; Xaa ₂ Is Ser or Gly; Xaa ₃ Is Pro, Ala, Lys, Arg or Thr; Xaa ₄ Is Lys or Arg; Xaa ₅ Is Gly, Ser or Lys; Xaa ₆ Is Val, Thr, Ile or Arg. In an embodiment, the antibody of the invention is in the form of a mixture with a pharmaceutically acceptable carrier. In another embodiment, the antibody of the invention is in the form of a kit. Another embodiment includes antibodies having heavy and light chain variable region sequences as set forth in SEQ ID NO: 108 and SEQ ID NO: 112. Also provided are antibodies with heavy and light chain sequences as set forth in SEQ ID NO: 138 and SEQ ID NO: 140.

Assembly of Αβ-derived diffuse ligands using antibodies or antibody fragments that prevent Αβ-induced diffuse ligands from binding to neurons and bind to the multidimensional conformation of one or more Αβ-derived diffuse ligands ( Methods of inhibiting assembly are also provided.

The present invention further encompasses methods for the prophylactic or therapeutic treatment of diseases associated with Aβ-induced diffuse ligands using the antibodies of the invention. By administering an antibody of the invention it is possible to prevent the Αβ-induced diffuse ligand from binding to neurons and thereby prevent or treat a disease associated with the Αβ-induced diffuse ligand.

The present invention also relates to methods of identifying therapeutic agents that prevent A [beta] -induced diffuse ligands from binding to neurons. This method involves contacting an Αβ-derived diffuse ligand with neurons in the presence of an agent and using the antibody of the present invention to determine whether the Αβ-derived diffuse ligand binds to the neuron in the presence of the agent. Include.

The present invention also includes methods for detecting Aβ-induced diffuse ligands in a sample and methods for diagnosing diseases associated with Aβ-induced diffuse ligands. Such methods include contacting a sample with an antibody of the invention, such that the Aβ-induced diffuse ligand can be detected and a disease associated with the Aβ-induced diffuse ligand can be diagnosed.

Brief description of the drawings

1 shows nucleic acid sequences corresponding to the variable regions of the heavy chain (FIG. 1A) and the light chain (FIG. 1B) of the murine (anti-ADDL) antibody 20C2. Lowercase letters indicate the leader sequence of the antibody, and uppercase letters indicate the variable region sequence of the antibody. The nucleotides that designate the genetic code of complementarity determining sites (CDRs) are underlined.

FIG. 2 shows a comparison of the variable region amino acid sequences of heavy chains (FIG. 2A) and light chains (FIG. 2B) of murine antibody 20C2 and humanized antibody, Hu20C2 (CDR grafted) and Hu20C2A3 (veneered). The sequences are presented as a comparison between a 20C2 mouse sequence, ie, a sequence that matches the most human genome sequence and a humanized sequence. Sequence differences in frame regions between murine 20C2 and humanized Hu20C2A3 are shown in bold. Sequence differences in the underlined CDR regions between humanized Hu20C2A3 and murine 20C2 are in bold and marked with *. CDRs are underlined.

3 is a nucleic acid sequence showing the variable regions (HCVRs and LCVRs) of the heavy chain (FIGS. 3A and 3B) and the light chain (FIG. 3C) for the humanized anti-ADDL antibody Hu20C2 (CDR grafted). Two humanized versions of the heavy chain of Hu20C2 (HCVRA and HCVRB) were made and differ in one amino acid at position 24. Human amino acids were used in Hu20C2 HCVRA and rat amino acids were used in Hu20C2 HCVRB. Variable region sequences were cloned into heavy and light chain-wide antibody expression vectors.

4 shows the annotated amino acid sequence and the nucleotide sequence of the Hu20C2 humanized antibody in the Fab phage-display vector pFab4. The amino acid sequences corresponding to heavy chain version A (FIG. 4A), heavy chain version B (FIG. 4B) and light chain (FIG. 4C) of the Hu20C2 humanized antibody in Fab phage-display vector pFab4 are italicized and underlined. Part. The nucleotide sequence of heavy chain version A fused with the light chain of Hu20C2 in the pFab4 vector is shown in FIGS. 4D-4E and the sequence encoding the Hu20C2 antibody is shown in lowercase.

5 shows two light chain CDR3 libraries, LC3-1 and LC3-2 (FIG. 5A) and three heavy chain CDR3 libraries, 20C2B-39HC₃, to make Hu20C2 light and heavy chain CDR3s with mature affinity, respectively. -1, 20C2B-39HC₃-2, 20C2B-39HC₃-3 (Fig. 5B) shows the primers and shapes used to prepare. Restriction endonuclease recognition sites used for cloning are indicated in italics. The uppercase letters indicate nucleic acids encoding the variable region chains of the antibody. Nucleic acids encoding CDRs are underlined. Biotin-labeled primers are also indicated.

6 shows a comparison between the amino acid sequence of the human antibody constant region and the IgG2m4 sequence. Starred portions indicate glycosylation sites in Asn297. FcRn binding sites are also indicated. The other sequence between IgG2m4 and IgG2 is underlined.

7 shows amino acids (FIGS. 7A and 7C) and nucleotides (FIGS. 7B and 7D) for the entire IgG2m4 humanized heavy chain (FIGS. 7C and 7D), humanized kappa light chains for the anti-ADDL antibody Hu20C2A3 (FIGS. 7C and 7D). The underlined portions are variable region chains. The rest of the chain is a constant region chain.

FIG. 8, measured by ELISA, shows the interaction between Aβ40 monomer or ADDLs and Hu20C2A3 produced by two different systems, CHO (FIG. 8A) or Pichia (FIG. 8B).

9 shows Hu20C2A3 inhibiting bADDL binding to primary hippocampal neurons.

Figure 10 shows the fluorescent thermal melt analysis of Hu20C2A3.

FIG. 11 is the plasma Aβx-40 level (pM) seen after intravenous injection of Hu20C2A3, irrelevant control or vehicle to APP-YAC mice. Hu20C2A3 was prepared by stable transfection of CHO cells or Pichia. Aβx-40 was measured using 4G8 / G2-10 ELISA. *** Marks are values by Tukey-Kramer HSD post-hoc testing, p <0.001 and Error bars = SEM; N = 6 / group.

Detailed description of the invention

Today, monoclonal antibodies have been produced that differentially recognize the multidimensional molecular structure of Aβ-induced diffuse ligands (ie, ADDLs). The antibody of the present invention is derived from the monoclonal antibody 20C2 of murine. Murine 20C2 is known to show the following features in the art. Murine 20C2 is an IgG1 antibody that binds to both synthetic and endogenous ADDLs that are bound to cultured hippocampal cells. In addition, the antibody can prevent synthetic and endogenous ADDL from binding to cultured cells, alleviate the binding of biotinylated ADDLs (bADDLs) to neurons, and inhibit tau phosphorylation. Prevent. The core linear epitope for 20C2 is Glu-Phe-Arg-His-Asp-Ser (SEQ ID NO: 1), corresponds to amino acid residues 3-8 of Aβ1-42, and is steric It has a structural epitope. The conformational epitope depends on components in residues 17-42 of Aβ, but only when assembled.

Antibodies of the invention are humanized and in embodiments are affinity-matured derivatives of murine 20C2. Like the murine 20C2 antibodies, the antibodies disclosed herein exhibit a high selectivity for the multidimensional conformation of ADDLs even with minimal detection of monomeric Aβ peptides. Antibodies of the invention distinguish endogenous oligomers in cerebral fragments with Alzheimer's disease, preventing bADDLs from binding to neurons. In addition, the antibodies of the present invention are known to produce a significant and positive increase in plasma Aβx-40 levels, which is associated with ultimately lowering brain Aβ. Thus, the antibodies of the present invention can be used to treat ADDL-associated diseases including Alzheimer's disease by inhibiting the binding of ADDL to neurons and the assembly of ADDLs.

Accordingly, the present invention relates to isolated antibodies that differentially recognize one or more multidimensional conformations of ADDLs. Antibodies of the invention are isolated in a substantially absence of other biological macromolecules of the same kind. Thus, an "isolated antibody" is an antibody that is substantially free of other antibodies; However, the molecule may contain additional agents or moieties that do not deleteriously affect the basic properties of the antibody (eg, binding specificity, neutralizing ability, etc.).

Antibodies capable of specific binding to one or more multidimensional conformations of ADDLs bind specific ADDLs derived from the oligomer of Aβ1-42, but according to Western blot analyses, other Aβ such as murine 20C2 Does not cross-react with peptides, i.e., Aβ1-12, Aβ1-28, Aβ1-40 and Aβ12-28; And preferentially binds to ADDLs in solution. Specific binding between the two means an affinity of at least 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , or 10 ¹⁰ M ⁻¹ . Affinities greater than 10 ⁸ M ⁻¹ are expected to result in specific bindings.

In an embodiment, an antibody capable of specifically binding one or more multidimensional conformations of ADDLs is also raised to counter (ie, the animal is immunized by) the multidimensional conformations of ADDLs. In another embodiment, an antibody capable of specifically binding to one or more multidimensional conformations of ADDLs is raised against a low n-mer-forming peptide such as Aβ1-42 [Nle35-Dpro37].

The term "epitope" refers to a site on an antigen to which a B cell or T cell reacts, or a site on the molecule where the corresponding antibody will be produced and bound. For example, one epitope can be recognized by one antibody that matches the epitope.

The linear epitope is one in which the amino acid primary sequence contains the recognized epitope. One linear epitope generally comprises at least three, more commonly at least five, for example about six to ten amino acids in one particular sequence.

In the conformational epitope, in contrast to the linear epitope, the amino acid primary sequence constituting the epitope is not the only element that determines the recognized epitope (e.g., the primary sequence of amino acids determines the epitope Epitope not necessarily recognized by the antibody). In general, the conformational epitope encompasses an increased number of amino acids associated with the linear epitope. Regarding the recognition of the conformational epitope, the antibody recognizes the three-dimensional structure of the peptide or protein. For example, when protein molecules are folded to form a three-dimensional structure, the specific amino acid or polypeptide backbone that forms the conformational epitope is aligned side by side so that the antibody can recognize the epitope. Methods for determining the stereostructure of the epitopes include, but are not limited to, x-ray determination, two-dimensional nuclear magnetic resonance spectroscopy, positioning spin labeling, and electron magnetic resonance spectroscopy. For example, Epitope Mapping Protocols in Methods in Molecular Biology (1996) Vol. 66, Morris (Ed.).

Aβ-induced diffuse ligands or ADDLs preferably consist of an aggregate of fewer than eight or nine amyloid β1-42 peptides and are soluble oligomers of amyloid β1-42 found in association with Alzheimer's disease ( soluble oligomer). This is in contrast to the aggregation mediums that form micelles that lead to fibril formation.

As illustrated below, the antibodies of the invention recognize or bind at least one multidimensional conformation in ADDL. In an embodiment, an antibody of the invention binds to at least two, three, four multidimensional conformations of one ADDL. As shown by SDS-PAGE analysis, the multidimensional conformations of ADDLs surround dimers, trimers, tetramers, pentamers, hexamers, hexamers, octamers, hexamers, depolymers, etc. I'm going. The designation of trimers, tetramers, etc. may vary depending on the analytical method to be used (for example, Bitan, et al. (2005) Amyloid 12: 88-95). definition) is according to the SDS-PAGE assay, as used below. As such, the antibodies of the invention have oligomer-specific properties. In an embodiment, the multidimensional conformation of ADDL is associated with a specific polypeptide structure, which is the conformational epitope recognized by the antibody of the invention. In another embodiment, an antibody of the invention specifically binds to a multidimensional conformation of ADDL, which has a molecular weight in excess of 50 kDa since it has a size range of approximately trimers or tetramers.

In an embodiment, in addition to binding to the multidimensional conformation, the antibodies of the invention bind to selected linear epitopes of amyloid β1-42. The linear epitope of ADDLs is intended as four, five, six or more amino acid residue peptides located at the N-terminal 10, 11, 12, 15 or 20 amino acid residues of amyloid β1-42. In certain embodiments, an antibody of the invention specifically binds to a linear epitope within 1-10, 1-8, 3-10 or 3-8 residues of amyloid β1-42. As an example, the linear epitope of amyloid β1-42 bound to the humanized antibody of the present invention is the amino acid residue Glu-Phe-Arg-His-Asp-Ser (SEQ ID NO: 1).

Antibodies of the invention have similar linear epitopes, whereas such linear epitopes exhibit the binding properties of the antibodies of the invention (ie, the ability to prevent ADDL binding to neurons, the ability to block tau phosphorylation, the ADDL assembly). Ability to inhibit), as well known to those skilled in the art, the linear epitope may only correspond to a portion of the epitope of the antigen (Breitling and Dubel (1999) In: Recombinant Antibodies, John Wiley & Sons, Inc., NY, pg. 115). For example, murine 20C2 is known to bind to an assembly of charge-inverted, truncated Aβ7-42 peptides, which peptides lack a linear epitope (ie amino acid residues 3-8) for 20C2. , The sequences corresponding to residues 7-16 of Aβ are very different. Thus, 20C2 binds to the conformational epitope, including its humanized derivative, and the conformational epitope depends only on elements within residues 17-42 of Aβ when in the multidimensional conformation . Antibodies of the present invention can discriminatively recognize multidimensional ADDLs, thereby differentially preventing binding of ADDL to neurons, differentially preventing tau phosphorylation, and differentially inhibiting ADDL assembly. It can be distinguished from the existing technology in that it is.

Antibodies used in accordance with the present invention are not necessarily limited to monoclonal antibodies, chimeric, human (eg, isolated from B cells), humanized, neutralizing, or bispecific antibodies thereof. (bispecific antibody) or single chain antibodies. In one embodiment the antibody of the invention is a monoclonal antibody. For antibody production, a variety of hosts, such as goats, rabbits, chickens, mice, humans, and the like, can be injected with synthetic or natural ADDLs for immune response. Antibody production methods are well known to those skilled in the art. See, for example, Kohler and Milstein ((1975) Nature 256: 495-497) and Harlow and Lane (Antibodies: A Laboratory Manual (Cold Spring Habor Laboratory, New York (1988)).

Depending on the host species, various adjuvants can be used to boost the immune response. The adjuvant used according to the invention preferably augments the intrinsic response to ADDLs without causing conformational modification of the immunogen, which affects the qualitative form of the response. Particularly suitable adjuvants include 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), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuranyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (E-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP) or 3 De-O-acylated monophosphoryl lipid A (MPL ^TM ; RIBI ImmunoChem Research Inc., Hamilton, MT; GB, which selectively binds to an immune stimulant like other bacterial cell wall constructs 2220211) and oil-in-water emulsions such as squalene or peanut oil. Specific examples of oil-in-water emulsions include 5% squalene, 0.5% TWEEN ^™ 80 and 0.5% SPAN 85 (optionally containing varying amounts of MTP-PE) and use microfluidizers to submicron particles. MF59 formulated. The microfluidizer includes a model 110Y microfluidizer (Microfluidics, Newton, MA); SAF including 10% squalene, 0.4% TWEEN ^™ 80, 5% PLURONIC®-blocked polymer L121 and thr-MDP, and microfluidized into submicron emulsions or vortexed to produce larger particle size emulsions; And RIBI ^™ adjuvant systems including 2% squalene, 0.2% TWEEN ^™ 80 and one or more bacterial cell wall components such as monophosphoryl lipid A, trehalose dimycolate (TDM), cell wall backbone (CWS), and the like. (RAS) (Ribi ImmunoChem, Hamilton, MT).

Other types of adjuvants are ISCOMs (Immunostimulatory Complexes) and ISCOMATRIX®  Saponin adjuvant, including (CSL Ltd., Parkville, Australia). Other suitable adjuvants include fully Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), mineral gels such as aluminum hydroxide, and surface-active substances. Surface-active substances include lyso lecithin, PLURONIC®, polyols, polyanions, peptides, CpG (WO 98/40100), keyhole limpet hemocyanin, dinitrophenol and interleukin (IL-1, IL- 2 and IL-12), macrophage colony stimulating factor (M-CSF) and tumor necrosis factor (TNF). Of the adjuvants used in humans, bacilli Calmette-Guerin (BCG) and Corynebacterium parvum are particularly suitable.

Antibodies to multidimensional conformational ADDL are obtained by immunizing animals with ADDLs. In general, ADDLs can be synthesized or produced by recombinant fragment expression and purification. Synthetic ADDLs can be prepared according to the methods disclosed in the following or US Pat. No. 6,218,506 or US Pat. App. Nos. 60 / 621,776, # 60 / 652,538, 60 / 695,528 and 60 / 695,526. Furthermore, ADDLs may be fused with other proteins, such as keyhole limpet hemocyanin, to make antibodies against chimeric molecules. ADDLs may be steric constitutively pressed to form useful epitopes as described below and may further be associated with the surface. For example, it may be physically bound to or chemically bound to a surface in a manner that allows for the production of conformations recognized by the antibodies of the invention.

Monoclonal antibodies to multi-dimensional conformation of ADDLs can be prepared also by using a technique for the production of antibody molecules by continuous cell lines cultures (continuous cell lines in culture). Such techniques include, but are not necessarily limited to, hybridoma technology, human B cell hybridoma technology, EBV-hybridoma technology, and the like (Kohler, et al. (1975) Nature 256: 495-497; Kozbor, et al. (1985) J. Immunol . Methods 81: 31-42; Cote, et al. (1983) Proc . Natl . Acad . Sci . 80: 2026-2030; Cole, et al. (1984) Mol . Cell Biol . 62: 109-120).

In certain embodiments, an antibody of the invention is humanized. Humanized or chimeric antibodies can be produced by splicing murine antibody genes into human antibody genes to obtain molecules with the appropriate antigenicity and biological activity (Morrison, et al. (1984) Proc. Natl.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. Natl. Acad. Sci. USA 86: 10029-10033; see WO 90/07861). For example, murine antibodies are expressed as Fv or Fab fragments in phage selection vectors. The gene for the light chain (and in the parallel experiment, the gene for the heavy chain) is turned into a library of human antibody genes. At that time, phage antibodies still bound to the antigen will be identified. This method is commonly known as chain shuffling, which produces humanized antibodies that bind to the same epitopes as murine antibodies (Jespers, et al. (1994) Biotechnology NY 12: 899-903). Alternatively, chain shuffling can be performed at the protein level (see Figini, et al. (1994) J. Mol. Biol. 239: 68-78).

Human antibodies can also be obtained by phage-display methods. This may be referred to WO 91/17271 and WO 92/01047. In these methods, phage libraries are made in such a way that each exhibits a different antibody on its outer surface. Antibodies are usually exhibited as Fv or Fab fragments. Fiji display antibodies with the desired specificity are selected by affinity enrichment for ADDLs. Human antibodies against ADDLs can also be obtained from non-human transgenic mammals, which have at least a transgene encoding a human immunoglobulin locus and a fragment of an inactivated resistant immunoglobulin locus. See WO 93/12227 and WO 91/10741. Human antibodies can also be selected by competitive binding experiments to have epitope specificities such as specific murine antibodies. Such antibodies generally retain the useful functional properties of murine 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 ADDLs as an affinity reagent.

As described below, humanized antibodies may be produced by veneering or resurfacing murine antibodies. Veneering involves replacing only surface anchoring site amino acids in the heavy and light chain variable regions of a mouse with the same site of a homologous human antibody sequence. Replacement of murine surface amino acids by human residues at the same position in homologous human sequences has been done to reduce the antigenicity of murine antibodies while maintaining ligand binding. In general, replacement of external residues has little or no effect on internal domains or interdomain contacts. (See US Pat. No. 6,797,492).

Human or humanized antibodies can be designed to have IgG, IgD, IgA, IgM or IgE constant regions and to have isotypes including IgG1, IgG2, IgG3 and IgG4. In certain embodiments, an antibody of the invention is IgG or IgM or a complex thereof. Another embodiment of the invention includes a constant region formed by selective integration of a human IgG4 sequence into a human IgG2 constant region. Representative mutant IgG2 Fc is IgG2m4 and is shown in SEQ ID NO: 140. Antibodies can be expressed as tetramers with two light chains and two heavy chains, separated heavy and light chains, or variable regions of heavy and light chains It can be expressed as single chain antibodies linked through. Methods of producing single chain antibodies are well known to those skilled in the art.

Representative humanized antibody derivatives of murine 20C2 monoclonal antibodies can be obtained by CDR grafting and veneering presented herein. The amino acid sequences for the kappa light chain (ie Hu20C2A3) variable region as well as IgG2M4 heavy chain variable region for humanized 20C2 produced by veneering are shown in Figures 7A and 7C and SEQ ID NOs: 141 and SEQ It is shown as ID NO: 143.

Also considered is a diabody. A diabody separates the binding domains (heavy chains and light chains) of the antibody to which they are bound, and then connects linking moieties that artificially link or bind heavy and light chains in the same polypeptide chain. Refers to engineered antibody constructs prepared by methods that preserve preservation of binding functions (Holliger, et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444; Poljak (1994). Structure 2: 1121-1123). This creates essentially very omitted antibodies with only the variable domains required for antigen binding. Using a linker that is too short to pair between two domains on the same chain, the domain is forced to pair with the complementary domains of the other chain, thus creating two antigen-binding sites. do. These dimeric antibody fragments or diabodies are bivalent or bispecific. Those skilled in the art will appreciate that any method for producing a diabody may be used. The appropriate method is Holliger, et al. (1993) Supra, Poljak (1994) Supra, Zhu, et al. (1996) Biotechnology 14: 192-196 and US Pat. No. 6,492,123, which is incorporated herein by reference.

Isolated antibody fragments of the present invention are also explicitly addressed by the present invention. Fragments include peptide aptamers as well as fragments produced by Fab fragments, F (ab ′) 2 fragments, F (ab ′) fragments, bispecific scFv fragments, Fd fragments and Fab expression libraries. For example, F (ab ′) 2 fragments are produced by pepsin digestion of the antibody molecules of the invention. Fab fragments, on the other hand, are made by reducing the disulfide bridges of F (ab ') 2 fragments. Alternatively, Fab expression libraries may be constructed to quickly and easily recognize monoclonal Fab fragments with the desired specificity (see Huse, et al. (1989) Science 254: 1275-1281). In certain embodiments, antibody fragments of the invention are neutralized antibody fragments that have a variable region binding site. Examples are F (ab ') 2 fragments, F (ab') fragments and Fab fragments. In general, Immunology: Basic Processes (1985), 2nd edition, J. Bellanti (Ed.) Pp. See 95-97.

Peptide aptamers that specifically recognize the multidimensional conformation of ADDLs can be suitably designed or screened in aptamer libraries (eg, provided by Aptanomics SA, Lyon, France). Generally, peptide aptamers are cognitive molecules whose shape is synthesized to conform to the structure of the antibody. Peptide aptamers consist of mutable peptide loops attached to protein scaffolds at both ends. This double structural bond significantly increases the binding affinity of the peptide aptamer to a level comparable to that of the antibody (nanomolecule range).

Exemplary nucleic acid sequences encoding heavy and light chain variable regions for use in the production of antibodies or antibody fragments of the invention are shown in FIGS. 7B and 7D, respectively (ie SEQ ID NOs: 142 and 144). As can be appreciated by the present invention, the heavy chain variable region disclosed herein can be used in combination with any of the light chain variable regions disclosed herein to produce antibodies with modified affinity, dissociation constants, epitopes and the like. have.

Antibodies or antibody fragments of the invention may have additional portions attached thereto. For example, as described in US Pat. No. 4,493,825, microspheres or microparticles can be attached to the antibody or antibody fragment.

In addition, certain embodiments are antibodies or antibodies that have been mutated and selected for increased antigen affinity and neutralizing activity (ie, activity that prevents binding to neurons of ADDLs, or inhibits ADDL assembly), or mutated dissociation constants. Contains fragments. Mutagenesis strains of E. coli (Low, et al. (1996) J. Mol . Biol . 260: 359-368), chain shuffling (Figini, et al. (1994) supra ) and PCR mutations Mutagenesis and the like are established methods of mutating nucleic acid molecules encoding an antibody. In other words, the increased affinity can be selected by contacting large amounts of phage antibodies with a small amount of biotinylated antigen such that the antibodies compete for binding. In this case, the number of antigen molecules should exceed the phage antibody number, but the concentration of the antigen should be somewhat below the dissociation constant. Thus, most phage antibodies with weak affinity remain unbound, while significantly mutated phage antibodies with increased affinity bind to biotinylated antigens. Streptavidin can then support mutated phage antibodies for higher affinity enhancement (Schier, et al. (1996) J. Mol. Biol. 255: 28-43). Exemplary affinity-matured light chain CDR3 amino acid sequences are disclosed herein (see Tables 6 and 7), and in certain embodiments the light chain CDR3 amino acid sequences are Xaa ₁ -Gln-Xaa ₂ -Thr-Arg-Val- Pro-Leu-Thr (SEQ ID NO: 2), the Xaa 'is Phe or Leu, and the Xaa2 is Ala or Thr. Affinity-matured heavy chain CDR3 amino acid sequences are also shown below. An example of a heavy chain CDR3 amino acid sequence is Arg-Gln-Leu-Gly-Thr-Arg-Gly-Thr-Asp-Ala-Met-Asp-Tyr (SEQ ID NO: 3). The invention also includes derivatives of these CDR3s, for example, Arg- Ala- Leu- Ser - Pro- Arg-Ser-Ile-Asp-Ala-Met-Asp-Tyr (SEQ ID NO: 4), Arg -Gln-Leu-Gly- Ala -Arg- Lys - Thr -Asp-Ala-Met-Asp-Tyr (SEQ ID NO: 5), Arg-Gln-Leu-Gly- Pro -Arg- Lys - Arg -Asp- Ala-Met-Asp-Tyr (SEQ ID NO: 6), Arg-Gln-Leu-Gly- Lys - Leu - Lys - Thr -Asp-Ala-Met-Asp-Tyr (SEQ ID NO: 7), or Arg -Gln-Leu-Gly- Arg -Arg-Ser- Val -Asp-Ala-Met-Asp-Tyr (SEQ ID NO: 8), where the other part of Hu20C2A3 heavy chain CDR3 is underlined. In view of this, the present invention specifically relates to the CDR3 amino acid sequence Arg-Xaa ₁ -Leu-Xaa ₂ -Xaa ₃ -Xaa ₄ -Xaa ₅ -Xaa ₆ -Asp-Ala-Met-Asp-Tyr (SEQ ID NO: 9) Has the Xaa ₁ Is Gln or Ala; Xaa ₂ is Ser or Gly; Xaa ₃ Is Pro, Ala, Lys, Arg, or Thr; Xaa ₄ Is Lys or Arg; Xaa ₅ Is Gly, Ser, or Lys; Xaa ₆ Includes anti-ADDL antibodies that are Val, Thr, Ile or Arg.

Other antibody derivatives within the scope of the present invention include humanized antibodies that are identical to the variable regions of Hu20C2A3 except for differences with one amino acid residue in the light chain frame region (eg, Leu-Pro-Val-). Thr-Pro-Gly-Glu-Pro-Ala-Ser, SEQ ID NO: 10).

For therapeutic purposes it is desirable to reduce the dissociation of the antibody from the antigen. To accomplish this, phage antibodies are bound to biotinylated antigens and an excessive amount of unbiotinylated antigen is added. After some time phage antibodies with significantly lower dissociation constants can be harvested with streptavidin (Hawkins, et al. (1992) J. Mol. Biol. 226: 889-96).

Various immunological assays include several methods that can be used for screening to identify antibodies or fragments thereof with the desired specificity for the multidimensional conformation of ADDLs. Numerous protocols (eg, ELISA), latex aggregation, immunofluorescence, kinetics (eg, BIACORE ^™ ) for competitive binding using polyclonal or monoclonal antibodies or fragments thereof Assays are well known. Such immunological assays typically involve the analysis of complex formation between a particular antibody and its cognate antigen. Two-site, monoclonal immunological assays using monoclonal antibodies that respond to two non-interfering epitopes are appropriate, but competitive binding assays can also be used. Such assays can also be used to detect the multidimensional conformation of ADDLs in a sample.

In addition, the antibody or antibody fragment may be treated with other biological activity assays, such as neurons or cultured hippocampal cells, to evaluate the neutralizing or pharmacological activity and potential efficacy of the prophylactic or therapeutic agent. It can also be used to displace a bond or block ADDL assembly. Such assays are described below and are well known in the art.

Antibodies and antibody fragments may be produced and maintained as hybridomas, or alternatively established expression systems, including but not limited to E. coli, yeast (eg, Saccharomyces spp. And Pichia spp.) , Baculovirus, mammalian cells (eg, myeloma, CHO, COS), plants, or transgenic animals (Breitling and Dubel (1999) In: recombinant antibodies, John wiley & Sons, Inc., NY, pp. 119-132). Antibodies and antibody fragments may be linked to any suitable method, including but not limited to, affinity chromatography, immunoglobulin-binding molecules (eg, proteins A, L, G, or H), antibodies, and antibody fragments. Tags (eg, His-tag, FLAG®-tag, Strep tag, C-myc tag). See Breitling and Dubel (1999) supra.

Antibodies and antibody fragments of the invention can be used to diagnose diseases associated with the accumulation of ADDLs, to prevent or prevent ADDLs from binding to neurons, to prevent ADDL assembly, to prevent diseases associated with ADDLs prophylactically or therapeutically. It has a variety of uses, such as handling, identifying therapeutic agents that interfere with the binding of ADDLs to neurons, and preventing phosphorylation of tau protein in Ser202 / Thr205.

Antibodies and antibody fragments of the invention are also useful for methods of preventing or preventing the binding of ADDLs to neurons. The method of the present invention is carried out in a manner that prevents ADDLs from binding to the neurons by contacting them with neurons in vivo or in the laboratory. In certain embodiments, the antibody or antibody fragment of the invention is at least 15%, 20%, 30%, 40%, 50%, 60%, at binding as compared to the binding of ADDLs in the absence of the antibody or antibody fragment. 70%, 80%, 90%, 95%, 97% reduction. The extent to which the antibody can prevent ADDLs from binding to neurons can be measured according to the methods disclosed herein. That is, immunocytochemistry or cellular system alkaline phosphatase assays or other suitable assays. Antibodies particularly useful for reducing neuronal binding of ADDLs include anti-ADDL antibodies, derivatives and antibody fragments having the CDR3 amino acid sequence set forth in SEQ ID NO: 9.

Furthermore, the antibodies and antibody fragments of the present invention are useful in methods of preventing or inhibiting assembly of ADDLs. This method involves contacting an antibody or antibody fragment with a sample containing an amyloid β 1-42 peptide to inhibit ADDL assembly. The extent to which the antibody can block ADDL assembly can be measured according to the methods described below, ie FRET or fluorescence polarization or other suitable method. Particularly useful antibodies for preventing assembly of ADDLs include anti-ADDL antibodies having the CDR3 amino acid sequence set forth in SEQ ID NO: 9. The antibodies disclosed herein are also useful for methods of inhibiting phosphorylation of tau protein at Ser202 / Thr205. This method prevents phosphorylation of the tau protein by blocking the binding of the tau protein to the neuronal cell by contacting the sample containing the tau protein with the antibody or antibody fragment of the present invention. The extent to which the antibody inhibits phosphorylation of tau protein in Ser202 / Thr205 can be measured according to the methods described below or other appropriate methods.

Preventing or reducing the binding of ADDLs to neurons, disrupting the assembly of ADDLs, and preventing phosphorylation of tau protein in Ser202 / Thr205 all prophylactically and therapeutically prevent diseases associated with the accumulation of ADDLs. Find examples of how to handle them. Accordingly, the present invention also includes the use of an antibody or fragment thereof for preventing or treating a disease associated with the accumulation of ADDLs (eg, Alzheimer's or similar memory-related disorders). Elevated levels of Αβ are not necessarily aggregated plaques, but there is evidence to indicate that they are responsible for Alzheimer's associated dementia and concomitant tau abnormalities. Aβ-induced diffuse ligands are directly implicated in neurotoxicity associated with Alzheimer's disease. ADDLs are said to modulate the functional activity associated with high memory and animal-assisted processes in transgenic mice and Alzheimer's disease patients. Thus, eliminating this form of Αβ may reduce the neurotoxicity associated with Alzheimer's disease. As such, treatment with the antibodies of the present invention may reduce ADDL accumulation in the central nervous system and may be effective in treating Alzheimer's disease. Patients who can be treated include those who are currently experiencing symptoms, as well as those who are at risk of disease but do not have symptoms. In Alzheimer's disease, virtually anyone living a long time is at risk of getting it. Therefore, the antibody or antibody fragment of the present invention can be administered prophylactically even to a general person who does not require risk assessment of the subject patient. The method is particularly useful for people with genetic risk of Alzheimer's disease. Such persons include those who have been diagnosed with the disease in their relatives and those whose risk has been measured by genetic or biochemical marker analysis. Genetic markers of risk for Alzheimer's disease include mutations in the APP gene, in particular mutations at position 717, mutations at positions 670 and 671 called the Hardy and Swedish mutations, respectively. Other risk markers are mutations in the presenilin gene, mutations in PS1 and PS2, and ApoE4, and include family history of Alzheimer's disease, hypercholesterolemia, atherosclerosis, and the like. Individuals currently suffering from Alzheimer's disease can be distinguished from characteristic dementia in addition to the presence of the risk factors described above. In addition, there are many diagnostic tests to detect people with Alzheimer's disease. These include CSF tau and Aβ1-42 level measurements. People suffering from Alzheimer's disease can also be diagnosed by the ADRDA criteria or the methods described herein.

In patients without asymptomatic symptoms, treatment can begin at any age (eg, 10, 20, 30 years old). However, it is usually not necessary to start treatment until the patient is 40, 50, 60 or 70 years old. Treatment usually involves long periods of drug use. Treatment can be managed by examining the presence of ADDLs for a long time.

In application to therapy, a pharmaceutical composition or medicament containing the antibody or antibody fragment of the invention may be used to treat symptoms (biochemical, histological or behavioral) in patients suspected of or already suffering from a disease associated with the accumulation of ADDLs. ) And in an amount sufficient or at least to block the treatment of complications and intermediate pathological phenotypes seen in the developmental stage of the disease. In the prophylactic application, a pharmaceutical composition or medicament containing the antibody or antibody fragment of the invention may cause passive immunity in patients susceptible to or at risk for diseases associated with the accumulation of ADDLs, thereby eliminating or reducing the risk or biochemical It is administered in an amount sufficient to attenuate or delay the onset of histologic, behavioral symptoms and complications, and intermediate pathological phenotypes during the course of the disease. In some methods, myocognitive damage is eliminated or reduced when the agent is administered to a patient who has not developed characteristic pathology of Alzheimer's disease. In certain embodiments, the effective amount of the antibody or antibody fragment of the invention results in a minimum of 15%, 20%, 30%, 40%, 50%, Amount to 60%, 70%, 80%, 90%, 95%, or 97%. As such, damage to long term potentiation / memory formation is reduced.

The effective dosage of the composition of the present invention for the treatment of the above-described symptoms depends on many other factors, namely, the method of administration, the pathological condition of the patient, whether the patient is a human or an animal, the other medication administered, and whether the treatment is prophylactic. It depends on whether or not it is enemy. Usually the patient is a human but can also be applied to non-human mammals such as dogs or transformed mammals.

The amount of treatment is generally titrated to optimize safety and effectiveness. The amount for passive immunization with the antibody or antibody fragment is 0.0001-100 mg / kg per host body weight, more generally 0.01-5 mg / kg. For example, the dosage may be 1 mg / kg or 10 mg / kg per body weight or in the range of 1-10 mg / kg. In some methods, two or more antibodies with different binding specificities of the present invention are administered simultaneously, in which case the amount of each antibody administered falls below the indicated amount. Antibodies are usually administered in a variety of ways, with the interval between doses being one week, one month, or one year. Exemplary therapies are followed by subcutaneous administration once every two weeks or at monthly intervals. Subcutaneous administration has been found to advantageously reduce the flu-like symptoms associated with intravenous injection (Lundin, et al. (2002) Blood 100: 768-773). Dosage intervals may be irregular as indicated by specifying blood levels of antibodies to ADDLs within the patient. In some methods, the dosage is adjusted until the plasma antibody concentration is 1-1000 μg / mL, or in other methods 25-300 μg / mL. Alternatively, the antibody or antibody fragment can be administered in a sustained-release formulation, where less frequent administration is required. Dosage and frequency vary depending on the antibody half-life of the patient. In general, human and humanized antibodies have a longer half-life than chimeric or nonhuman antibodies. As noted above, dosage and frequency vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, they are administered in relatively small amounts and at relatively infrequent intervals over a long period of time. Some patients can continue to receive treatment until they die. In therapeutic applications, at relatively short intervals, a relatively large amount is required, until the progression of the disease is reduced or finished, preferably until the patient's symptoms are partially or fully improved. Administered. Thereafter it may be administered in a prophylactic regime.

Antibodies and antibody fragments of the invention may be administered as one component of a pharmaceutical composition or medicament. The pharmaceutical composition or medicament generally comprises an active therapeutic agent and various pharmaceutically acceptable components. Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, PA, 2000. The preferred form depends on the intended dosage form and the therapeutic application. Pharmaceutical compositions are pharmaceutically acceptable, non-toxic carriers or diluents, depending on the desired formulation, but are the excipients commonly used when formulating pharmaceutical compositions for administration to animals or humans. It may include. Diluents are chosen so as not to affect biological activity in the formulation. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution.

The pharmaceutical compositions may also contain large, slow metabolizing proteins or polymers such as polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (e.g. latex functionalized SEPHAROSE ™, agarose, cellulose, And similar materials), polymeric amino acids, amino acid copolymers, and lipid complexes (eg oil droplets or liposomes).

Administration of the pharmaceutical compositions or medicaments of the present invention is not necessarily limited to oral, topical, pulmonary, rectal, subcutaneous, intradermal, nasal, intracranial, intramuscular, intraocular or intraarticular injections, and the like. It can be done through various routes. Other routes are equally effective, but the most typical route of administration is intravenous followed by subcutaneous administration. Intramuscular injections can be placed in the arm or leg muscles. In some methods, the agent is injected directly into a specific tissue where deposits have accumulated, for example intracranial injection. In some embodiments, the antibody or antibody fragment is injected directly into the skull. In other embodiments, the antibody or antibody fragment is administered by a sustained-release composition or a MEDIPAD ^™ instrument.

 In parenteral administration, the antibody or antibody fragment of the invention may be injected into a physiologically acceptable diluent containing a pharmaceutical carrier which is water or oil, saline, glycerol, or a sterile liquid such as ethanol. It may be administered in the form of a solution or suspension in an amount possible. In addition, wetting agents, emulsifiers, surfactants, pH buffers and the like may be included in the composition. Other components of the pharmaceutical composition include components of petroleum, animal, plant and synthetic origin, such as mineral oil, soybean oil, peanut oil. Generally, glycols such as propylene glycol or polyethylene glycol are suitable liquid carriers and are particularly suitable for injection solutions. The antibody can be administered in the form of a depot injection or an implant preparation, which can be formulated in a manner that allows for a gradual reduction of the active ingredient.

Exemplary compositions are formulated in sterile water with a concentration of at least 10 mg / ml in isotonic buffered saline (10 mM histidine, 150 mM sodium chloride, 0.01% (w / v) POLYSORBATE 80, pH 6.0). Antibodies or fragments of antibodies. Exemplary antibody formulations are filled with cold 0.6 ml glass vials, one volume of 3.3 ml solution per vial. Each vial is closed with a TEFLON-coated stopper and sealed with an aluminum cap.

Typically, the composition is prepared injectable as a suspension, even if it is a liquid solution; Solid forms, suspensions, liquid excipients, and the like, suitable for solution before injection can also be prepared. The preparation may be emulsified or encapsulated in liposomes or microparticles such as polylized, polyglycolide, copolymer for enhanced delivery.

In suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; Such suppositories may be made from mixtures comprising the active ingredient in the range of 0.5-10% or more, more preferably 1-2%.

Oral formulations include pharmaceutical additives such as mannitol, lactose, starch, magnesium stearate, saccharin chloride, cellulose, magnesium carbonate and the like. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, progressively reduced formulations or powders and comprise 10-95%, more suitably 25-70% of the active ingredient.

Topical application results in transdermal or intradermal delivery. Topical application can be facilitated by co-administration with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins (Glenn, et al. (1998) Nature 391 : 851). Co-administration can be accomplished by using the components in the form of a mixture or linked molecules obtained by chemical crosslinking or expression as a fusion protein.

Alternatively, intradermal delivery can be accomplished using the dermal route or using a transferosome (Paul, et al. (1995) Eur. J. Immunol. 25: 3521-24; Cevc, et al. (1998) Biochem.Biophys.Acta 1368: 201-15).

Antibodies or antibody fragments of the invention may be optionally administered in combination with other agents that are at least partially effective in the treatment of amyloidosis disease. For example, the antibodies of the invention can be administered with existing palliative therapies for Alzheimer's disease, such as the acetylcholinesterase inhibitors ARICEPT ^™ , EXELON ^™ , and REMINYL ^™ , the NMDA antagonist NAMENDA ^™ . In addition to these approved therapeutic agents, the antibodies of the present invention can be used to provide synergistic / additional benefits to some current approaches, including, without limitation, inhibitors of A [beta] production and accumulation in the development of Alzheimer's disease therapy.

Antibodies and antibody fragments of the invention may also be used to identify ADDLs as therapeutic agents that prevent neurons (eg, hippocampal cells) from binding to downstream events caused by ADDL. Can be applied. Such assays are performed by contacting ADDL to neurons in the presence of an agent and using the antibody or antibody fragment of the invention to determine whether the ADDL has bound to the neurons in the presence of the agent. do. As will be appreciated by those skilled in the art, an agent that prevents the binding of ADDL to neurons may be characterized by the amount of ADDL that binds to the neurons, i. It will reduce the amount detectable in the immunoassay used. Immunoassays suitable for the detection of neuron-binding ADDL are described below.

Agents that can be screened using the methods provided herein include a large number of chemical species and typically are organic molecules, preferably small organic compounds having a molecular weight of at least 100 daltons and less than about 2,500 daltons. The formulations comprise the functional groups necessary for structural interaction with the protein, in particular hydrogen bonding, and generally comprise at least an amine group, a carbonyl group, a hydroxyl group or a carboxyl group. Formulations often include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Agents can also be found among biomolecules, including peptides, antibodies, monosaccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Formulations are obtained from a wide range of resources, including libraries of natural or synthetic products.

Various other reagents such as salts and neutral proteins can be included in the screening assays. Protease inhibitors, nuclease inhibitors, antibacterial agents and the like can also be used to increase the efficiency of the assay. Mixtures of the components may be added in the order in which they provide for the requisite binding.

The formulations identified by the screening assay of the present invention will be beneficial for the treatment of amyloidosis disease and / or tauopathy. In addition, the experimental system used to implement this concept is believed to represent research tools for the evaluation, identification and screening of new drugs associated with amyloid beta induction of tau phosphorylation.

The invention also provides methods for detecting ADDLs using the antibodies or antibody fragments of the invention and for diagnosing diseases associated with the accumulation of ADDLs. Diseases associated with the accumulation of ADDLs are thought to include any disease in which the accumulation of ADDLs results in physiological impairments of long-term strengthening / memory formation. Diseases of this type include, but are not necessarily limited to, Alzheimer's disease and similar memory-related disorders.

In accordance with these methods, a sample obtained from a patient is contacted with an antibody or antibody fragment of the present invention and the antibody or antibody fragment binds to the sample, providing evidence that ADDL is present in the sample. As used herein, a sample is considered to mean a type of bodily fluid or tissue that can be tested using immunoassays. Suitable samples that can be analyzed according to the methods of the invention include, but are not limited to, biopsy samples and sap samples extracted from the brain of a patient (eg, a mammal, such as a human). For in vitro purposes (eg, in assays that monitor oligomer formation), the sample may be a neuronal cell line sample or a neural tissue sample. For therapeutic purposes, a sample may be obtained from an individual suspected of or at risk of having a disease associated with the accumulation of ADDLs, eg, an individual with a family history of disease associated with the accumulation of ADDLs.

Detection of whether the antibody or antibody fragments bound to ADDLs in the sample can be performed using any standard immunoassay (eg, as described below), alternatively, if the antibody fragment is a peptide application If it is an aptamer, the binding can be detected directly by a detectable marker protein (eg, β-galactosidase, GFP or luciferase) fused to an aptamer. The presence or absence of an ADDL-antibody complex is then correlated with the presence or absence of ADDLs in the sample, respectively, which in turn is associated with the presence or absence of a disease associated with the accumulation of ADDLs. One or more antibodies or antibody fragments of the invention can be combined with current non-invasive immuno-based imaging techniques to significantly improve the detection and early diagnosis of diseases associated with the accumulation of ADDLs. Can be used.

The present invention also includes kits having antibodies or antibody fragments to facilitate diagnosis. The kit includes a container containing one or more antibodies or antibody fragments that recognize the multidimensional conformation of the ADDLs, and for detecting antigen-antibody complex formation and for binding to the ADDLs for antibody-antigen complex formation. The use of an antibody for the purpose includes instructions that indicate that the presence or absence of the antibody-antigen complex as a result correlates with the presence or absence of ADDLs in the sample. Examples of containers include multiwell plates capable of detecting ADDLs in multiple samples simultaneously.

The invention can be better understood by the following examples, which are intended for the purpose of illustration of the invention and are not intended to limit the scope of protection by the appended claims.

Example  1: General Materials and Manufacturing Methods

ADDL manufacturing (preparation). ADDLs (Biosource, Camarillo, CA) in F12 medium were prepared from Aβ1-42 according to established methods (Lambert, et al. (2001) supra ). Briefly, Aβ1-42 peptide (American Peptide Co., Sunnyvale, CA or California Peptide Research, Inc., Napa, CA) was weighed to obtain a peptide concentration of 10 mg / mL after HFIP (1, 1, 1, 3, 3, 3-hexafluoro-2-propanol) was placed in a glass vial to hold a sufficient amount. HFIP was added to the dry peptide, the vial was gently swirled to cover and mixed, and the peptide / HFIP solution was left at room temperature for at least 1 hour. Aliquots of peptide solution (50 or 100 μL, 0.5 or 1.0 mg, respectively) were divided into a series of 1.5 mL conical centrifuge tubes. The tube was placed in SPEEDVAC® overnight to remove HFIP. Tubes with a dried peptide film are placed in a sealed container with a capped lid and containing a hygroscopic agent at -70 ° C.

Prior to use, the Aβ1-42 peptide peptide was removed from the -70 ° C reservoir and allowed to warm to room temperature. Fresh DMSO (44 μL / mg peptide membrane; 5 mM) was added and the peptide / DMSO mixture was incubated at the slowest speed for 10 minutes in a vortex mixer. F12 medium (2 mL / mg peptide) was divided into each tube of DMSO / peptide and the tubes were mixed upside down with the lid closed. 100 μM preparation was stored at 2-8 ° C. for 18-20 hours. Samples were centrifuged at 2-8 ° C. for 10 minutes at 14,000 × g speed. Supernatants were transferred to new tubes and stored at 2-8 ° C. until reuse.

Biotinylated ADDL preparations (bADDLs) were prepared using 100% N-terminal biotinylated amyloid beta peptides (American Peptide Company, Sunnyvale, Calif.) In the same manner as the ADDL preparations described above.

Monomers prepared (monomer preparation ) . HFIP dry down preparations of amyloid beta (1-40) peptide (Aβ1-40) were prepared as outlined in Aβ (1-42) peptides. Peptide membranes were dissolved in 2 mL of 25 mM borate buffer (pH 8.5) per mg of peptide, split before use and frozen to -70 ° C.

The primary neurons (primary neurons ) . Primary hippocampal cell cultures were prepared from frozen, dissociated newborn rat hippocampal cells (Cambrex, Corp., East Rutherford, NJ), which cells were thawed to a single well. ) Were plated in 96-well COSTAR® plates at a concentration of 20,000 cells. The cells were left in NEUROBASAL ^™ medium (GIBCO-BRL ^™ , Gaithersburg, MD) free of L-glutamine for 2 weeks and supplemented with B27 (GIBCO-BRL ^™ , Gaithersburg, MD) for binding studies. Was used.

Immunocytochemistry (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 preincubated for 1 hour at room temperature, and the molar ratio of antibody: ADDL before use in hippocampal culture for 21 days was 1: 4. For endogenous ADDLs, human brain protein (prepared as in Lambert, et al. (2001) supra) was incubated with the cells for 1 hour, and the cells were washed, fixed and Became visible.

Lightly fixed and frozen sections (along with 30% at 4 ° C. and 4% paraformaldehyde cryopreserved in 40 μm sucrose) from Alzheimer's disease and control hippocampus were subjected to 1: 1000 in phosphate-buffered saline (PBS). Incubated overnight at 4 ° C. After removing the antibody, the sections were washed three times with PBS and incubated at room temperature with the secondary antibody. The binding was then visualized using DAB (SIGMA ^™ , St. Louis, MO). The sections were then counterstained with hematoxylin, mounted and imaged on a NIKON® ECLIPSE® E600 light microscope with a SPOT ^™ INSIGHT ^™ digital video camera (v. 3.2).

ELISA . Polyclonal anti-ADDLs IgG (M90 / 1; Bethyl Laboratories, Inc., Montgomery, TX) was subjected to IMMULON ^™ 3 REMOVAWELL ^™ at room temperature for 2 hours. Plated at 0.25 mg / well on strip (Dynatech Labs, Chantilly, VA) and the wells blocked with 2% BSA in TBS. Samples diluted with 1% BSA in F12 were added to the wells, allowed to bind for 2 hours at 4 ° C., and washed three times with BSA / TBS at room temperature. Monoclonal antibodies diluted with BSA / TBS were incubated for 90 minutes at room temperature and detected with VECTASTAIN® ABC Kit against murine IgG. HRP labels were visualized with a BIO-RAD® peroxidase substrate and read at 405 nm in a Dynex MRX-TC microplate reader.

Example  2: Isolation of the murine antibody variable region sequence isolation )

The cDNA encoding the variable region of the 20C2 murine antibody was cloned and sequenced, followed by production of the hybrid nucleic acid up to the 5 ′ end of the murine constant region and upstream of the V region of murine. Polymerase chain reaction (PCR) was carried out using specially designed primers. This approach ensures that the murine variable region sequences obtained are complete and accurate. Briefly, mRNA is extracted from a mouse hybridoma cell line using the QIAGEN® OLIGOTEX® Direct mRNA Mini Kit and then converted to cDNA using a first-strand cDNA synthesis kit. CDNA is then used as a template in a PCR reaction to obtain antibody variable region sequences.

To obtain light chain variable region sequences, 11 independent PCR reactions were prepared using 11 light chain 5 ′ PCR primers (MKV-1 to MKV-11) and 3 ′ PCR primers MKC-1 (Table 1), respectively. .

Underlined and italicized sequences represent Xba I and Sac I restriction sites, respectively. W = A or T, M = A or C, K = G or T, Y = C or T, R = A or G.

To obtain the heavy chain variable region sequences, 12 heavy chain 5 ′ PCR primers (MHV-1 to MHV-12) and appropriate isotype specific 3 ′ primers (MHCG-1, MHCG-2A, MHCG- 12 independent PCR reactions were prepared using 2B, MHCG-3) (Table 2).

Underlined and italicized sequences represent Xba I and Sac I restriction sites, respectively. W = A or T, M = A or C, K = G or T, Y = C or T, R = A or G.

Light chain PCR reactions each consisted of 46 μL INVITROGEN ^TM PLATINUM® PCR Super Mix, 1.0 μL of one of the 100 μM 5 ′ primers (MKV-11 to MKV-1), 1.0 μL of the 100 μM 3 ′ primer (MKC-1), And 2.0 μL of hybridoma cDNA. Similar PCR reactions were performed to clone the heavy chain variable region sequences of the mice. The reaction was placed in a DNA thermal cycler and after an initial denaturation step at 97 ° C. for 2 minutes, followed by 30 cycles: 30 seconds at 95 ° C., 45 seconds at 55 ° C., and 90 seconds at 72 ° C. After the last cycle, the final extension step was performed at 72 ° C. for 10 minutes. To determine which PCR reactions yielded results, 5 μL aliquots of each reaction were placed on 1.5% (w / v) agarose / 1X TAE buffer gel containing 0.5 μg / mL ethidium bromide. Separated. Then, PCR output from reactions producing fragments of expected size (420-500 bp) was gel purified at the multicloning site and digested with Xba I and Sac I, plasmid pNEB193 (New England). Ligated into the Xba I and Sac I sites in the multicloning site of Biolabs, Beverly, MA). Alternatively, PCR results can be obtained from INVITROGEN ^™ The TA CLONING® kit was used to directly link into plasmid pCR®2.1. The ligation product was then transformed into XL-1 cells, and an aliquot of the modified E. coli was plated onto an LB agar plate containing 50 μg / mL empicillin ( plated), overlaid with 40 μL of X-Gal stock (50 mg / mL) and 40 μL of IPTG (100 mM) solution for blue / white selection. Plates were incubated overnight at 37 ° C. and potential clones appeared white. DNA from at least 24 independent clones for each PCR result was sequenced on strands using universal forward and reverse primers for pNEB193 pCR®2.1. The resulting sequence was then assembled into contigs to create consensus sequences for the light and heavy chain variable regions of each antibody. Using this approach, the sequence will determine the light and heavy chain antibody variable regions of hybridoma 20C2 (FIGS. 1A-1B). The six complementarity determining regions (CDRs) that form the structure complementary to the antigen are underlined in FIGS. 1A-1B.

Example  3: anti- mouse ADDL  Humanization of antibody variable region sequences humanization )

Murine antibodies light and heavy chain variable region nucleic acids from murine hybridoma cell line 20C2 were humanized using a CDR grafting approach. It will be appreciated by those skilled in the art that humanization of the murine antibody sequence can maximize the therapeutic potential of the antibody by increasing the serum half-life and Fc effector function of the antibody thereby reducing the anti-globulin response. There will be.

Humanization by CDR grafting was performed by selecting human light and heavy chain variable regions from the NCBI protein database with the highest homology with the murine variable regions. The murine variable region sequences were compared with all of the human variable region sequences in the database using the protein-protein basic local alignment search tool (BLAST). Rat CDRs were then linked to human framework regions and preliminary amino acid sequences were analyzed. All differences in the sequence of framework regions between rats and humans were evaluated, particularly whether the differences were part of the canonical sequence for the loop structure or residues located at the VL / VH interface (O'Brien). and Jones (2001) In : Antibody Engineering, Kontermann and Dubel (Eds.), Springer Laboratory Manuals). In addition, framework regions were closely observed for abnormal or unusual amino acids, as compared to the common sequences for human subgroups and potential glycosylation reaction sites. Human residues were chosen where there was an amino acid sequence difference between the framework regions of rats and humans that was neither associated with the base sequence nor located at the VL / VH interface. Where there are differences in major residues, two versions of variable region sequences were produced for evaluation. The CDR grafting strategy only made minimal alterations to the human framework regions so that good antigen binding could be achieved while maintaining the human framework regions that most matched sequences obtained from native human antibodies. The heavy and light chain variable region amino acid sequences of the humanized antibodies as a result of CDR grafting of murine 20C2 are shown in FIGS. 2A and 2B, respectively. This antibody is designated Hu20C2 in the present invention.

The humanized sequence of 20C2 was also designed using a veneering strategy (see, eg, US Pat. No. 6,797,492). Humanization is performed by selecting human light and heavy chain variable regions from the NCBI protein database that exhibits the highest homology for the murine variable regions as well as the closest human antibody germline family. (Kabat, et al. (1991) Sequences of proteins of immunological interest, 5 ^th ed., US Dept. Health and Human Services, NIH, Washington DC). The murine variable region sequences were compared with all human variable region sequences in the database using protein-protein BLAST. The variable region of murine and the human homologous sequence closest to it were modeled as the nearest crystallized human antibody as determined by computer modeling as practiced in the art. Surface maps were drawn from the murine VH and VL sequence models, indicating the solvent accessibility of amino acids in the variable regions of the heavy and light chains of mice. To ensure modeling, these exposed residues were compared with surface accessible residues known as position-by-position (eg, Padlan (1994) Mol . Immunol . 31 (3): 169- 217). According to established methods, scores are assigned to each residue in the designating sequence according to the exposed, most exposed, partially buried, most buried, buried (see See US Pat. No. 6,797,492, which is incorporated herein by reference. The mouse framework residues, which were classified as exposed or most exposed, and homologous human sequences, were replaced with human residues at that location. Designed veneered sequences include murine CDRs, residues adjacent to CDRs, residues known to be associated with the canonical sequence, residues located at the VL / VH interface, and mouse heavy and light chain N Retained residues in the terminal sequence. The N-terminal sequence is known to be contiguous with the CDR surface and is potentially involved in ligand binding. Once the veneered sequence is completed it is remodeled in search of potentially obvious structural issues. A total of 12 and 9 amino acid residues were altered in the heavy and light chain frames, respectively. The heavy and light chain variable region amino acid sequences of the humanized antibodies resulting from veneering murine 20C2 are shown in FIGS. 2A and 2B, respectively. This antibody is designated Hu20C2A3 in the present invention.

In comparison with 20C2, it is noted that light chain replacement resulting in Hu20C2A3, rather than heavy chain replacement, is common with Hu20C2 (Figure 2). In particular, the heavy chain variable region CDR3 is unique to Hu20C2A3.

Once a humanized amino acid sequence is selected, the sequence is reverse-translated to obtain a matching DNA sequence. The DNA sequence is codon-optimized using methods established in the art (Lathe (1985) J. Mol. Biol. 183 (1): 1-12) and flanked for cloning into human antibody expression vectors. It is designed with flanking restriction sites. Nucleotide sequences encoding the light chain variable region and two versions of the heavy chain variable region for Hu20C2 are shown in FIGS. 3A-3C. The two heavy chain variable region versions differ in that one amino acid is replaced at position 24; The heavy chain variable region for version A of Hu20C2 is Phe at position 24 and the heavy chain variable region for version B of Hu20C2 is Leu at position 24.

Example  4: affinity maturation affinity maturation )

Affinity maturation was performed on Hu20C2 antibodies. Nucleic acid molecules encoding only the variable heavy chains or only the light chains of humanized Hu20C2 version A and version B, or the heavy chain version A and the light chain together were cloned in the Fab phage-display vector pFab4. . Nucleic acid sequencing confirmed the sequence and orientation in pFab4. Annotated Hu20C2 Fab sequences in pFab4 are shown in FIGS. 4A-4C, heavy chain version A for SEQ ID NO: 116, heavy chain version B for SEQ ID NO: 117, light chain for SEQ ID NO: It is shown at 118. Nucleotide sequences for heavy chain version A and light chain together in the pFab4 vector are shown in FIGS. 4D-4E. Such constructs are used in Hu20C2 maturation programs using phage-displayed Fab library methods that are well known in the art.

Light chain maturation . Two libraries were designed to mutate nine wild-type amino acids of CDR3 of the light chain of Hu20C2 (ie, Phe-Gln-Gly-Ser-Leu-Val-Pro-Leu-The SEQ ID NO: 39). These libraries are LC3-1 and LC3-2 designated and are Xaa-Xaa-Xaa-Xaa-Xaa-Val-Pro-Leu-Thr (SEQ ID NO: 40) and Phe-Gln-Gly-Ser-Xaa-Xaa- Light chain CDR3 sequence, which is Xaa-Xaa-Xaa (SEQ ID NO: 41). The biotinylated reverse primers, 20C2LC3-1 (SEQ ID NO: 123) and 20C2LC3-2 (SEQ ID NO: 126), were used as forward primers to produce LC3-1 and LC3-2 libraries. forward primer) is used in combination with 20C2LC3F (SEQ ID NO: 120) (see FIG. 5A). Primers are purified by polyacrylamine gel electrophoresis, while vector DNA is purified by gel electrophoresis and electroelution. Two light chain libraries are designed to be randomly mutated. The final diversity of the three 10G5H6 LC ₃ libraries is 4.76 × 10 ⁸ and 7.45 × 10 ^8, respectively (Table 3). Sequencing of about 100 clones obtained from the libraries revealed a diversity of 100% mutant clones at the designed amino acid positions.

Higher titers are obtained by concentration or phage rescue.

Soluble panning of two light chain libraries against high molecular weight bADDL was performed. In brief, a total of four rounds of panning was performed using biotinylated high molecular weight ADDL (bADDL). The first three rounds were performed using about 1.5 μM antigen concentration (input = 1 × 10 ¹⁰ to 1 × 10 ¹¹ ). At the end of the third round, the results of the two libraries were combined to increase panning stringency and divided into three groups, 10 nM, 100 nM, and about 1.5 μM antigens for analysis. As such, all 58 result plates were tested in phage ELISA assay. That is, two plates per library in the first round (all four plates), six plates per library in the second round (all twelve plates), eight in the LC3-1 library in the third round, and three LC3-2 libraries for the 10 plates (18 plates in total) and 8 plates (24 plates in total) for each antigen concentration in the fourth round.

Panning appeared 1000 hits, of which 436 were sequenced (Table 4).

^a 20C2 LC3-1 to high molecular weight 10% bADDL.

^b 20C2 LC3-2 to high molecular weight 10% bADDL.

^c 20C2 LC3-1 + 20C2 LC3-2 to high molecular weight 10% bADDL.

* Indicates hit for total number of colonies.

The sequence and frequency of the highly concentrated clones are shown in Table 5.

Depending on the enrichment frequency, Fab fragments of the top 10 clones were prepared, a total of 15 clones were converted to IgG1 humanized A version, and 20C2-6 and 20C2-8 to IgG1 humanized B version. K _D values for these clones were measured by BIACORE ^™ using biotin-Aβ1-20 (Table 6) and bADDL (Table 7) as antigens. Compared to the murine 20C2 antibody as well as the humanized 20C2A and 20C2B of the parental generation, a marked increase in affinity was observed. In particular, the sequence Xaa ₁ -Gln-Xaa ₂ -Thr-Arg-Val-Pro-Leu-Thr (SEQ ID NO: 2) of the light chain CDR3, Xaa ₁ is Phe or Leu, and Xaa ₂ is determined by Ala or Thr Low nanomolecular weight (nanomolar) for sub-picomolar K _D s was achieved. In addition, comparing bADDL with K _D values obtained by BIACORE ^™ using biotin-Aβ1-20 suggests that anti-ADDL antibodies such as Hu20C2 bind differently to the multidimensional conformation of ADDL than monosaccharide Aβ peptides. do.

Heavy chain maturity . The heavy chain of Hu20C2 is likely to be optimized by producing three libraries covering heavy chain-CDR3 (RQLGLRSIDAMDY; SEQ ID NO: 99). These libraries were designated 20C2B-, respectively, representing heavy chain CDR3 sequences XXXXXRSIDAMDY (SEQ ID NO: 100), RQLGLRSIXXXXX (SEQ ID NO: 101), and RQLGXXXXXAMDY (SEQ ID20C2HC3-1 (SEQ ID NO: 130) NO: 102). 39HC a _{3 -1, 20C2B-39HC 3 -2} , 20C2B-39HC 3 -3. The biotinylated reverse primers, 20C2HC3-1 (SEQ ID NO: 130), 20C2HC3-2 (SEQ ID NO: 133), 20C2HC3-3 (SEQ ID NO: 136), to produce three libraries, were forward primers. Used with 20C2HC3F (SEQ ID NO: 127) (see FIG. 5B). The library contains> 10 ⁸ functional diversity and covers all bindings of amino acids at all positions randomized in each set (see Table 8).

A total of 18 result plates obtained from a total of four rounds of panning were tested in phage ELISA assay. A total of 1235 hits were found, of which 704 were sequenced. Based on the BIACORE ^™ K _D values of the Fab fragments against the single-point BIACORE ^™ 3000 assay as well as the biotinylated-Aβ1-20 and biotinylated ADDL (bADDL) antigens (Table 9), a total of six Fabs Clones were converted to IgG1 and IgG2m4 using CDR grafting or veneered humanization techniques. One of these six Fabs, designated 4a-A3, was isolated from library 20C2B-39HC ₃ -3 and carried three amino acid substitutions (RQLG T R GT DAMDY; SEQ ID NO: 3) to the middle portion of the heavy chain. . As is apparent from FIG. 2B, the heavy chain CDR3 sequence is the heavy chain sequence of Hu20C2A3.

Example  5: IgG2m4  Production of antibodies

IgG2m4 antibody derivatives are prepared to reduce Fc receptor engagement, Clq binding, unwanted cytotoxicity or immunocomplex formation while maintaining the long half-life and pharmacokinetic properties of typical human antibodies. The basic antibody format of IgG2m4 is the same as that of IgG2 and appears to have excellent half-life in the experimental example (Zuckier, et al. (1994) Cancer Suppl . 73: 794-799). The structure of IgG2 is modified to eliminate Clq binding through selective incorporation of IgG4 sequences, while maintaining typical low levels of FcyR binding (Canfield and Morrison (1991) J. Exp . Med . 173: 1483-). 1491). This is possible by using the same cross-over points in the sequence of IgG2 and IgG4, which allows the production of antibodies with natural Fc sequences rather than obtaining artificial mutant sequences. The advantage of using an IgG2m4 antibody of the invention with minimal effector-related activity is the diglycosylated disclosed in Wilcock et al. ((2006) J. Neurosci . 26: 5340-6). Comparable to antibodies.

IgG2m4 forms of human antibody constant regions are formed by selective integration of human IgG4 sequences into standard human IgG2 constant regions, as shown in FIG. 6. Conceptually, IgG2m4 is the result of a pair of chain-swaps in the CH2 region as shown in FIG. Four single mutations were made corresponding to sequences from IgG4. Fc residues mutated in IgG2 include His268Gln, Val309Leu, Ala330Ser, and Pro331Ser, which minimize the potential for neopitope. Specific IgG4 amino acid residues located in the IgG2 constant region are shown in Table 10 along with other alternative residues from the base structure.

Locations marked with * are easy for allelic variation.

^a Hougs, et al. (2001) Immunogenetics 52 (3-4): 242-8.

^b WO 97/11971.

^c Medgyesi, et al. (2004) Eur. J. Immunol. 34: 1127-1135.

^d Tao, et al. (1991) J. Exp. Med. 173: 1025-1028.

^e Armor, et al. (1999) Eur. J. Immunol. 29: 2613.

^f Xu, et al. (1994) J. Biol. Chem. 269: 3469-3474.

^g Canfield and Morrison (1991) J. Exp. Med. 173: 1483.

Human IgG1 / kappa and IgG2m4 / kappa versions of humanized Hu20C2 and Hu20C2A3 antibodies were structured. The completed amino acid sequences of the light and heavy chains of the Hu20C2A3 IgG2m4 antibody are shown in Figures 7A and 7C.

Example  6: humanized anti- ADDL  Binding Affinity and Specificity of Antibodies

Affinity maturation is performed to increase affinity and preferential binding to ADDL. To assess the ADDL binding affinity of the humanized antibody, BIACORE ^™ and titration ELISA were performed as follows. Briefly, streptavidin-coated, sttreptavidin-coated, 96-well microtiter plates (Sigma, St. Louis, Mo.) coated with 10% biotinylated ADDL antigen (1 μM) It became. A series of 2-fold dilution of purified antibody was added to the ADDL captured plate starting at 500 ng / mL and the plate was incubated at 25 ° C. for 2 hours. After washing five times with PBS solution using plate washers (Bio-Tek, Winooski, VA), the polyclonal goat anti-human kappa light chain antibody (Biomeda, Foster City, CA) was 3% nonfat milk blocker. ) Was diluted to 1/2000 and incubated for 1 hour at room temperature. The rabbit anti-goat IgG (H + L) HRP-conjugated (Bethyl Laboratories, Inc., Montgomery, TX) detection antibody is then diluted to 1/2000 and added to a blocking solution at room temperature Incubated for 1 hour. After washing with PBS, HRP substrate, 3, 3 ', 5'5-tetramethylbenzidine (ready-to-use TMB; Sigma, St. Louis, MO) is added and the reaction is 0.5 after 10 minutes. Stopped with NH ₂ SO ₄ . Absorbance was shown at a wavelength of 450 nm on a plate reader (model VICTOR V; Perkin Elmer, Boston, Mass.) And the data was processed using an EXCEL® worksheet. The evaluation deviation between the plates was evaluated within 20%.

K _D of Fab clone A3 was measured by BIACORE ^™ , which was 849 pM for biotinylated-Aβ1-20. K _D of the same Fab clone was 82pM for bADDL, suggesting that A3 Fab preferentially binds to ADDL. When clones were humanized by veneering and when converted to whole IgG1 molecules or whole IgG2m4 molecules (ie Hu20C2A3), the K _D values for Aβ1-20 and ADDL were below the reliable measurement of the BIACORE ^™ instrument. . This represents a significant increase in the binding equilibrium constant of Hu20C2A3 to Aβ1-20 and ADDL compared to Hu20C2.

Hu20C2A3 is a veneered version of clone A3 with IgG2m4 isotype and was expressed in both CHO and Pichia . Two sources of Hu20C2A3 were assessed for their ability to interact with Aβ monomers and ADDL by ELISA. As shown in FIG. 8, Hu20C2A3 produced in CHO (FIG. 8A) or Pichia (FIG. 8B) showed preferential ADDL binding versus Aβ40 monomer binding (6-fold). Coupling Constant (IgGk ₅₀ Value) is determined from these curves, yielding values of 64 pM and 376 pM for ADDL and Aβ monomers for Hu20C2A3 produced in Pichia , respectively, and ADDL and Aβ monomers for Hu20C2A3 produced in CHO cells, respectively. 58 pM and 361 pM values were calculated.

Example  7: Humanized anti- ADDL  To nerve cell using antibody ADDL  Combined blocking

Humanized anti-ADDL antibodies were evaluated for their ability to block ADDL binding to primary hippocampal neurons using the following method. Hu20C2A3 antibody, or PBS as a control, was mixed with bADDL at various molecular ratios and incubated for 1 hour at 37 ° C. in a slow rotator. After preincubation, antibody / bADDL was added to the primary neuronal medium and incubated for another hour at 37 ° C. At the end of the incubation period the bADDL / antibody mixture is removed and the plate washed six times with medium. The cells are then fixed for 10 minutes in 4% paraformaldehyde at room temperature, the solution is removed and fresh fixative is added. So the cells are fixed for 10 minutes again. Cells were permeated by 4% paraformaldehyde containing 0.1% TRITON ^™ X-100 (two at 10 minutes each at room temperature), washed six times in PBS and 10% in PBS for 1 hour at 37 ° C. Treated with BSA. Alkaline phosphatase-conjugated streptavidin (1: 1, 500 in 1% BSA; Molecular Probes, Eugene, OR) is then added to the cells for 1 hour at room temperature. Cells were rinsed six times with PBS, alkaline phosphatase substrate (CDP-STAR® with SAPPHIRE-IITM; Applied Biosystem, Foster City, Calif.) Was added to the cells, and after 30 min incubation, LJL Luminometer (Analyst AD). LJL Biosystems, Sunnyvale, CA). In this analysis, Hu20C2A3 was shown to effectively block bADDL binding to neurons at a sub-stoichiometric antibody ratio (EC ₅₀ = 0.16; FIG. 9) to the peptide.

Blocking bADDL binding indicates that Hu20C2A3 interacts in a biologically related manner with ADDL. To demonstrate that Hu20C2A3 interacts with ADDL related to the conditions of human Alzheimer's disease, the ability of biotinylated Hu20C2A3 against immune-level Aβ containing plaques in human Alzheimer's disease brain tissue was evaluated. Immunohistochemical localization showed vigorous labeling of Aβ in both dense core plaques and spread plaques in human Alzheimer's disease brain tissue. The specificity of this binding manifests itself as a loss of immunoreactivity followed by pre-incubation which increases the amount of ADDL: antibody. Similar to Hu20C2, Hu20C2A3 effectively labels both the dense core and the spread Aβ deposit.

Example  8: Hu20C2A3 of Thermal stability

Evaluation of the protein stability of Hu20C2A3 is performed by SEC-HPLC, fluorescence hot melt analysis, particle size analysis and the like. Fluorescent thermomelt assay reveals that the Fc and Fab unfolding transitions occur at about 70 ° C. and 80 ° C., respectively, consistent with the inherent protein stability that is acceptable (FIG. 10).

Example  9: In vivo Pharmacodynamic ( pharmacodynamic ) And efficacy ( efficacy ) analysis

While the prior art requires continuous administration to reduce measurable brain Aβ, systemic injection of monoclonal anti-Aβ antibodies can dramatically raise plasma levels of Aβ. Shows. This suggests that passive immunity in species with measurable levels of A [beta] results in an increase in plasma A [beta] by changing the balance of A [beta] between the brain and the peripheral compartment. This "peripheral sink" phenomenon ultimately leads to a lowering of brain Aβ. However, cognitive improvement has been observed in animals that have undergone rapid antibody administration prior to significant changes in brain Αβ, indicating that changes in brain Αβ are already taking place before they can be measured using known techniques. . Alternatively, an increase in plasma Αβ may be explained by the stabilization of peripheral Αβ appearing after antibody administration. Regardless, it has been established that early plasma elevation in animal models is a prerequisite for subsequent brain Αβ reduction. Thus, the effect of Hu20C2A3 antibody on plasma Aβ increase has been put to practical use as an indicator of target engagement. After IV infusions of Hu20C2A3 in amounts of 30, 100, and 300 μg / mouse, significant reduction of plasma Aβx-40 was observed, compared to the 4 hour post-injection of the control group of irrelevant antibody (8B4). A clear increase was observed (FIG. 11). The observed increase in plasma Aβx-40 for CHO-derived substances was 491% (30 μg, p > 0.001), 826% (100 μg, p <0.001), and 755% (300 μg, p <0.001) at 8B4 levels. Was. Similarly, the increase in plasma Aβx-40 relative to Pichia -derived material was also increased by 395% (30 μg, p > 0.001), 729% (100 μg, p <0.001), and 838% (300 μg, p <) of 8B4 levels. 0.001).

                         SEQUENCE LISTING <110> Kinney, Gene        Strohl, William R.        An, Zhiqiang   <120> ANTI-ADDL MONOCLONAL ANTIBODY AND USE THEREOF <130> MRK0003WO <150> PCT / US2005 / 038125 <151> 2005-10-21 <160> 153 <170> PatentIn version 3.3 <210> 1 <211> 6 <212> PRT <213> Homo sapiens <400> 1 Glu Phe Arg His Asp Ser 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 peptide <220> <221> MISC_FEATURE (222) (1) .. (1) <223> Xaa denotes Phe or Leu <220> <221> MISC_FEATURE (222) (3) .. (3) <223> Xaa denotes Ala or Thr <400> 2 Xaa Gln Xaa Thr Arg Val Pro Leu Thr 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 peptide <400> 3 Arg Gln Leu Gly Thr Arg Gly Thr Asp Ala Met Asp Tyr 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 peptide <400> 4 Arg Ala Leu Ser Pro Arg Ser Ile Asp Ala Met Asp Tyr 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 peptide <400> 5 Arg Gln Leu Gly Ala Arg Lys Thr Asp Ala Met Asp Tyr 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 peptide <400> 6 Arg Gln Leu Gly Pro Arg Lys Arg Asp Ala Met Asp Tyr 1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 peptide <400> 7 Arg Gln Leu Gly Lys Leu Lys Thr Asp Ala Met Asp Tyr 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 peptide <400> 8 Arg Gln Leu Gly Arg Arg Ser Val Asp Ala Met Asp Tyr 1 5 10 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 peptide <220> <221> MISC_FEATURE (222) (2) .. (2) <223> Xaa denotes Gln or Ala <220> <221> MISC_FEATURE (222) (4) .. (4) <223> Xaa denotes Ser or Gly <220> <221> MISC_FEATURE (222) (5) .. (5) <223> Xaa denotes Pro, Ala, Lys, Arg, or Thr <220> <221> MISC_FEATURE (222) (6) .. (6) <223> Xaa denotes Lys or Arg <220> <221> MISC_FEATURE (222) (7) .. (7) <223> Xaa denotes Gly, Ser, or Lys <220> <221> MISC_FEATURE (222) (8) .. (8) <223> Xaa denotes Val, Thr, Ile or Arg <400> 9 Arg Xaa Leu Xaa Xaa Xaa Xaa Xaa Asp Ala Met Asp Tyr 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 10 Leu Pro Val Thr Pro Gly Glu Pro Ala Ser 1 5 10 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 11 gatctctaga tgaagattgc ctgttaggct gttggtgctg 40 <210> 12 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 12 gatctctaga tggagwcaga cacactcctg ytatgggtg 39 <210> 13 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 13 gatctctaga tgagtgtgct cactcaggtc ctggsgttg 39 <210> 14 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 14 gatctctaga tgaggrcccc tgctcagwtt yttggmwtct tg 42 <210> 15 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 15 gatctctaga tggatttwca ggtgcagatt wtcagcttc 39 <210> 16 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 16 gatctctaga tgaggtkcyy tgytsaycty ctctgrgg 38 <210> 17 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 17 gatctctaga tgggcwtcaa agatggagtc acakwyycwg g 41 <210> 18 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 18 gatctctaga tgtggggayc tktttycmmt ttttcaatg 39 <210> 19 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 19 gatctctaga tggtrtccwc asctcagttc cttg 34 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 20 gatctctaga tgtatatatg tttgttgtct atttct 36 <210> 21 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 21 gatctctaga tggaagcccc agctcagctt ctcttcc 37 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 22 gatcgagctc actggatggt gggaagatgg 30 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 23 gatctctaga tgaaatgcag ctggggcats ttcttc 36 <210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 24 gatctctaga tgggatggag ctrtatcats ytctt 35 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 25 gatctctaga tgaagwtgtg gttaaactgg gttttt 36 <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 26 gatctctaga tgractttgg gytcagcttg rttt 34 <210> 27 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 27 gatctctaga tgggactcca ggcttcaatt tagttttcct t 41 <210> 28 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 28 gatctctaga tggcttgtcy ttrgsgctrc tcttctgc 38 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 29 gatctctaga tggratggag ckggrgtctt tmtctt 36 <210> 30 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 30 gatctctaga tgagagtgct gattcttttg tg 32 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 31 gatctctaga tggmttgggt gtggamcttg cttattcctg 40 <210> 32 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 32 gatctctaga tgggcagact taccattctc attcctg 37 <210> 33 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 33 gatctctaga tggattttgg gctgattttt tttattg 37 <210> 34 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 34 gatctctaga tgatggtgtt aagtcttctg tacctg 36 <210> 35 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 35 gcatcgagct ccagtggata gacagatggg gg 32 <210> 36 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 36 gcatcgagct ccagtggata gaccgatggg gg 32 <210> 37 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 37 gcatcgagct ccagtggatg agctgatggg gg 32 <210> 38 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 38 gcatcgagct ccaagggata gacagatggg gc 32 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 39 Phe Gln Gly Ser Leu Val Pro Leu Thr 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE (222) (1) .. (5) <223> Xaa denotes any amino acid residue <400> 40 Xaa Xaa Xaa Xaa Xaa Val Pro Leu Thr 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE (222) (5) .. (9) <223> Xaa denotes any amino acid residue <400> 41 Phe Gln Gly Ser Xaa Xaa Xaa Xaa Xaa 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 42 Ala Asp Thr Thr His Val Pro Leu Thr 1 5 <210> 43 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 43 Ala His Ser Thr Phe Val Pro Leu Thr 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 44 Ala Gln Ala Ser Phe Val Pro Leu Thr 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 45 Ala Gln Ala Thr Lys Val Pro Leu Thr 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 46 Ala Gln Ser Ser Lys Val Pro Leu Thr 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 47 Ala Gln Ser Thr Leu Val Pro Leu Thr 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 48 Phe Ala Ala Ser Ser Val Pro Leu Thr 1 5 <210> 49 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 49 Phe Glu Ser Thr Tyr Val Pro Leu Thr 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 50 Phe Glu Ser Ser Arg Val Pro Leu Thr 1 5 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 51 Phe Asn Ala Thr Trp Val Pro Leu Thr 1 5 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 52 Phe Gln Ala Ser Arg Val Pro Leu Thr 1 5 <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 53 Phe Gln Ala Thr Arg Val Pro Leu Thr 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 54 Phe Gln Gly Ser Phe Ile Gly Leu Ser 1 5 <210> 55 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 55 Phe Gln Gly Ser Phe Ile Pro Gly Thr 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 56 Phe Gln Gly Ser Phe Leu Pro Pro Ser 1 5 <210> 57 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 57 Phe Gln Gly Ser Phe Leu Pro Gln Leu 1 5 <210> 58 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 58 Phe Gln Gly Ser Leu Phe Pro Pro Val 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 59 Phe Gln Gly Ser Leu Phe Ser Pro Ser 1 5 <210> 60 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 60 Phe Gln Gly Ser Arg Ile Pro Ile Ser 1 5 <210> 61 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 61 Phe Gln Gly Ser Arg Leu Pro Val Ser 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 62 Phe Gln Gly Ser Arg Val Pro Leu Val 1 5 <210> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 63 Phe Gln Ser Ser Phe Val Pro Leu Thr 1 5 <210> 64 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 64 Phe Gln Ser Ser Arg Val Pro Leu Thr 1 5 <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 65 Gly Gln Thr Thr Leu Val Pro Leu Thr 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 66 His Glu Ser Thr Leu Val Pro Leu Thr 1 5 <210> 67 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 67 His Gln Ser Ser Lys Val Pro Leu Thr 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 68 Ile Gln Thr Ser Leu Val Pro Leu Thr 1 5 <210> 69 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 69 Ile Gln Ala Ala Leu Val Pro Leu Thr 1 5 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 70 Leu Gln Ser Ser Phe Val Pro Leu Thr 1 5 <210> 71 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 71 Leu Glu Thr Ser Arg Val Pro Leu Thr 1 5 <210> 72 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 72 Leu Ala Ser Ser His Val Pro Leu Thr 1 5 <210> 73 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 73 Leu Asn Ser Thr Thr Val Pro Leu Thr 1 5 <210> 74 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 74 Leu Gln Ser Lys Ser Val Pro Leu Thr 1 5 <210> 75 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 75 Leu Gln Ser Val Arg Val Pro Leu Thr 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 76 Leu Gln Ser Ser Leu Val Pro Leu Thr 1 5 <210> 77 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 77 Leu Gln Thr Gly Arg Val Pro Leu Thr 1 5 <210> 78 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 78 Leu Gln Thr Ser Phe Val Pro Leu Thr 1 5 <210> 79 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 79 Leu Gln Thr Ser Asn Val Pro Leu Thr 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 80 Leu Gln Thr Thr Arg Val Pro Leu Thr 1 5 <210> 81 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 81 Leu Ser Ser Thr Phe Val Pro Leu Thr 1 5 <210> 82 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 82 Leu Ser Ser Thr His Val Pro Leu Thr 1 5 <210> 83 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 83 Leu Thr Ser Ser Ala Val Pro Leu Thr 1 5 <210> 84 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 84 Leu Val Ser Ser Leu Val Pro Leu Thr 1 5 <210> 85 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 85 Met Glu Thr Ala Asn Val Pro Leu Thr 1 5 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 86 Met Gln Ser Ser Phe Val Pro Leu Thr 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 87 Met Gln Ser Ser Leu Val Pro Leu Thr 1 5 <210> 88 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 88 Met Gln Thr Ser Lys Val Pro Leu Thr 1 5 <210> 89 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 89 Ser Gln Ala Arg Met Val Pro Leu Thr 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 90 Ser Gln Ala Ser Arg Val Pro Leu Thr 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 91 Thr Gln Ser Thr Gln Val Pro Leu Thr 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 92 Val Cys Ala Thr Phe Val Pro Leu Thr 1 5 <210> 93 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 93 Val Gln Ser Ser Ala Val Pro Leu Thr 1 5 <210> 94 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 94 Val Gln Thr Ser Leu Val Pro Leu Thr 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 95 Val Gln Thr Ser Val Val Pro Leu Thr 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 96 Val Gln Thr Thr Ala Val Pro Leu Thr 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 97 Leu Gln Thr Ala Arg Val Pro Leu Thr 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 98 Phe Gln Gly Ser Leu Leu Pro Leu Ser 1 5 <210> 99 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 99 Arg Gln Leu Gly Leu Arg Ser Ile Asp Ala Met Asp Tyr 1 5 10 <210> 100 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE (222) (1) .. (5) <223> Xaa denotes any amino acid residue <400> 100 Xaa Xaa Xaa Xaa Xaa Arg Ser Ile Asp Ala Met Asp Tyr 1 5 10 <210> 101 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE (222) (9) .. (13) <223> Xaa denotes any amino acid residue <400> 101 Arg Gln Leu Gly Leu Arg Ser Ile Xaa Xaa Xaa Xaa Xaa 1 5 10 <210> 102 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE (222) (5) .. (9) <223> Xaa denotes any amino acid residue <400> 102 Arg Gln Leu Gly Xaa Xaa Xaa Xaa Xaa Ala Met Asp Tyr 1 5 10 <210> 103 <211> 459 <212> DNA <213> Mus musculus <400> 103 tgggcagact taccattctc attcctgctg ctgattgtcc ctgcatatgt cttgtcccaa 60 gttactctaa aagagtctgg ccctgggata ttgaagccct cacagaccct cagtctgact 120 tgttctctct ctgggttttc actgagcact tctggtatgg gtgtaggctg gtttcgtcag 180 ccttcaggga agggtctgga gtggctggca cacatttggt gggatgatga taagtcctat 240 aatccatccc tgaagagccg gctcacaatc tccaagtata cctctagaaa ccaggttttc 300 ctcacgatca ccagtgtgga cactgcagat actgccactt actattgtgc tcgaagacaa 360 ctcggactaa gatcaattga tgctatggac tactggggtc aaggaacctc agtcaccgtc 420 tcctcagcca aaacgacacc cccatctgtc tatccactg 459 <210> 104 <211> 435 <212> DNA <213> Mus musculus <400> 104 agattgcctg ttaggctgtt ggtgctgatg ttctggattc ctgcttccac cagtgatgtt 60 ttgatgaccc aaactcctct ctccctgcct gtcagtcttg gagatcaagc ctccatctct 120 tgcagatcta gtcagagcat tctacatagt aatggaaaca cctatttaga gtggtacctg 180 cagaaaccag gccagtctcc aaagctcctg atctacaaag tttccaaccg attttctggg 240 gtcccagaca ggttcagtgg cagtggatca gggacagatt tcacactcaa gatcagcaga 300 gtggaggctg aggatctggg agtttattac tgttttcaag gttcacttgt tccgctcacg 360 ttcggtgctg ggaccaagct ggagctgaaa cgggctgatg ctgcaccaac tgtatccatc 420 ttcccaccat ccagt 435 <210> 105 <211> 123 <212> PRT <213> Mus musculus <400> 105 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Leu Ser Gly Phe Ser Leu Ser Thr Ser             20 25 30 Gly Met Gly Val Gly Trp Phe Arg Gln Pro Ser Gly Lys Gly Leu Glu         35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Ser Tyr Asn Pro Ser     50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Tyr Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Thr Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr                 85 90 95 Cys Ala Arg Arg Gln Leu Gly Leu Arg Ser Ile Asp Ala Met Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser         115 120 <210> 106 <211> 120 <212> PRT <213> Homo sapiens <400> 106 Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser             20 25 30 Gly Met Arg Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu         35 40 45 Trp Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Thr Ser     50 55 60 Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr                 85 90 95 Cys Ala Arg Ile Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 107 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody <400> 107 Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Ser Thr Ser             20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu         35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Ser Tyr Asn Pro Ser     50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr                 85 90 95 Cys Ala Arg Arg Gln Leu Gly Leu Arg Ser Ile Asp Ala Met Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 108 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody <400> 108 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Leu Leu Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Ser Thr Ser             20 25 30 Gly Met Gly Val Gly Trp Phe Arg Gln Pro Pro Gly Lys Gly Leu Glu         35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Ser Tyr Asn Pro Ser     50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Ile Thr Asn Val Asp Pro Val Asp Thr Ala Thr Tyr Tyr                 85 90 95 Cys Ala Arg Arg Gln Leu Gly Thr Arg Gly Thr Asp Ala Met Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 109 <211> 112 <212> PRT <213> Mus musculus <400> 109 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Leu His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly                 85 90 95 Ser Leu Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 110 <210> 110 <211> 112 <212> PRT <213> Homo sapiens <400> 110 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser             20 25 30 Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Ser Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly                 85 90 95 Ile His Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 111 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody <400> 111 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Leu His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Thr                 85 90 95 Thr Arg Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 112 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic antibody <400> 112 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Leu His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Thr                 85 90 95 Thr Arg Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 113 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <400> 113 caggtgaccc tgaaggagtc tggccctgcc ctggtgaagc ccacccagac cctgaccctg 60 acctgcacct tctctggctt cagcctgagc acctctggca tgggcgtggg ctggatccgg 120 cagccccctg gcaaggccct ggagtggctg gcccacatct ggtgggacga cgacaagtcc 180 tacaacccca gcctgaagag ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240 gtgctgacca tgaccaacat ggaccctgtg gacacagcca cctactactg tgcccggcgg 300 cagctgggcc tgcggagcat tgatgccatg gactactggg gccagggcac cacagtgaca 360 gtgtccagc 369 <210> 114 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <400> 114 caggtgaccc tgaaggagtc tggccctgcc ctggtgaagc ccacccagac cctgaccctg 60 acctgcaccc tgtctggctt cagcctgagc acctctggca tgggcgtggg ctggatccgg 120 cagccccctg gcaaggccct ggagtggctg gcccacatct ggtgggacga cgacaagtcc 180 tacaacccca gcctgaagag ccggctgacc atcagcaagg acaccagcaa gaaccaggtg 240 gtgctgacca tgaccaacat ggaccctgtg gacacagcca cctactactg tgcccggcgg 300 cagctgggcc tgcggagcat tgatgccatg gactactggg gccagggcac cacagtgaca 360 gtgtccagc 369 <210> 115 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <400> 115 gatgtggtga tgacccagag ccccctgtcc ctgcctgtga cccctggcga gcctgccagc 60 atctcctgcc ggagctccca gagcatcctg cactccaatg gcaacaccta cctggagtgg 120 tacctgcaga agcctggcca gagcccccag ctgctgatct acaaggtgtc caaccggttc 180 tccggcgtgc ctgaccggtt cagcggctcc ggcagcggca cagacttcac cctgaagatc 240 agccgggtgg aggctgagga tgtgggcgtc tactactgct tccagggcag cctggtgccc 300 ctgacctttg gccagggcac caagctggag atcaag 336 <210> 116 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 116 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Ala Leu Glu Gln Val Thr Leu Lys Glu Ser Gly             20 25 30 Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe         35 40 45 Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg     50 55 60 Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala His Ile Trp Trp Asp 65 70 75 80 Asp Asp Lys Ser Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser                 85 90 95 Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn Met Asp             100 105 110 Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Arg Gln Leu Gly Leu         115 120 125 Arg Ser Ile Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr     130 135 140 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 145 150 155 160 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val                 165 170 175 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala             180 185 190 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly         195 200 205 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly     210 215 220 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 225 230 235 240 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Thr Ser Gly His His His                 245 250 255 His His His Gly Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly             260 265 270 Gly Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln         275 280 285 Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly     290 295 300 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 305 310 315 320 Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr                 325 330 335 Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp             340 345 350 Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala         355 360 365 Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly     370 375 380 Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser 385 390 395 400 Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn                 405 410 415 Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro             420 425 430 Tyr Val Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp         435 440 445 Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala     450 455 460 Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys 465 470 475 480 Glu ser          <210> 117 <211> 481 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 117 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala Ala Leu Glu Gln Val Thr Leu Lys Glu Ser Gly             20 25 30 Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Leu         35 40 45 Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg     50 55 60 Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala His Ile Trp Trp Asp 65 70 75 80 Asp Asp Lys Ser Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser                 85 90 95 Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn Met Asp             100 105 110 Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Arg Gln Leu Gly Leu         115 120 125 Arg Ser Ile Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr     130 135 140 Val Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 145 150 155 160 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys                 165 170 175 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu             180 185 190 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu         195 200 205 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr     210 215 220 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 225 230 235 240 Asp Lys Arg Val Glu Pro Lys Ser Cys Thr Ser Gly His His His His                 245 250 255 His His Gly Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Gly             260 265 270 Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro         275 280 285 Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser     290 295 300 Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu 305 310 315 320 Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu                 325 330 335 Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu             340 345 350 Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr         355 360 365 Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu     370 375 380 Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln 385 390 395 400 Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe                 405 410 415 Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Tyr             420 425 430 Val Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys         435 440 445 Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr     450 455 460 Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu 465 470 475 480 Ser      <210> 118 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 118 Met Lys Tyr Leu Leu Pro Thr Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Ser Arg Asp Val Val Met Thr Gln Ser Pro             20 25 30 Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg         35 40 45 Ser Ser Gln Ser Ile Leu His Ser Asn Gly Asn Thr Tyr Leu Glu Trp     50 55 60 Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val 65 70 75 80 Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser                 85 90 95 Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val             100 105 110 Gly Val Tyr Tyr Cys Phe Gln Gly Ser Leu Val Pro Leu Thr Phe Gly         115 120 125 Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val     130 135 140 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 145 150 155 160 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln                 165 170 175 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val             180 185 190 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu         195 200 205 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu     210 215 220 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 225 230 235 240 Gly glu cys              <210> 119 <211> 5458 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <400> 119 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 60 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 120 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 180 tgagcggata acaatttcac acaagatcta gctattccag agattacgca aattctattt 240 caaggagaca gtcataatga aatacctgct gccgactgca gctgctggtc tgctgctgct 300 ggcggcccag ccggctatgg cttctagaga tgtggtgatg acccagagcc ccctgtccct 360 gcctgtgacc cctggcgagc ctgccagcat ctcctgccgg agctcccaga gcatcctgca 420 ctccaatggc aacacctacc tggagtggta cctgcagaag cctggccaga gcccccagct 480 gctgatctac aaggtgtcca accggttctc cggcgtgcct gaccggttca gcggctccgg 540 cagcggcaca gacttcaccc tgaagatcag ccgggtggag gctgaggatg tgggcgtcta 600 ctactgcttc cagggcagcc tggtgcccct gacctttggc cagggcacca agctggagat 660 caagcgtacg gtggctgcac catctgtctt catcttcccg ccatctgatg agcagttgaa 720 atctggaact gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt 780 acagtggaag gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca 840 ggacagcaag gacagcacct acagcctcag cagcaccctg acgctgagca aagcagacta 900 cgagaaacac aaagtctacg cctgcgaagt cacccatcag ggcctgagct cgcccgtcac 960 aaagagcttc aacaggggag agtgttaaca attgctagaa ttgtgagcgg ataacaattt 1020 cagcaggtcg agttcttgat aacgaggcgt aaaaaatgaa aaagacagct atcgcgattg 1080 cagtggcact ggctggtttc gctaccgtgg cccaggcggc cctcgagcag gtgaccctga 1140 aggagtctgg ccctgccctg gtgaagccca cccagaccct gaccctgacc tgcaccttct 1200 ctggcttcag cctgagcacc tctggcatgg gcgtgggctg gatccggcag ccccctggca 1260 aggccctgga gtggctggcc cacatctggt gggacgacga caagtcctac aaccccagcc 1320 tgaagagccg gctgaccatc agcaaggaca ccagcaagaa ccaggtggtg ctgaccatga 1380 ccaacatgga ccctgtggac acagccacct actactgtgc ccggcggcag ctgggcctgc 1440 ggagcattga tgccatggac tactggggcc agggcaccac agtgacagtg tccagcgcct 1500 ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc tctgggggca 1560 cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg gtgtcgtgga 1620 actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac 1680 tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc cagacctaca 1740 tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt gagcccaaat 1800 cttgtactag tggccaccac caccatcacc atggcggtga acaaaaactc atctcagaag 1860 aggatctggg tggttagcca ttcgtttgtg aatatcaagg ccaatcgtct gacctgcctc 1920 aacctcctgt caatgctggc ggcggctctg gtggtggttc tggtggcggc tctgagggtg 1980 gcggctctga gggtggcggt tctgagggtg gcggctctga gggtggcggt tccggtggcg 2040 gctccggttc cggtgatttt gattatgaaa aaatggcaaa cgctaataag ggggctatga 2100 ccgaaaatgc cgatgaaaac gcgctacagt ctgacgctaa aggcaaactt gattctgtcg 2160 ctactgatta cggtgctgct atcgatggtt tcattggtga cgtttccggc cttgctaatg 2220 gtaatggtgc tactggtgat tttgctggct ctaattccca aatggctcaa gtcggtgacg 2280 gtgataattc acctttaatg aataatttcc gtcaatattt accttctttg cctcagtcgg 2340 ttgaatgtcg cccttatgtc tttggcgctg gtaaaccata tgaattttct attgattgtg 2400 acaaaataaa cttattccgt ggtgtctttg cgtttctttt atatgttgcc acctttatgt 2460 atgtattttc gacgtttgct aacatactgc gtaataagga gtcttaagct agctggccgt 2520 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 2580 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 2640 acagttgcgc agcctgaatg gcgaatggcg attccgttgc aatggctggc ggtaatattg 2700 ttctggatat taccagcaag gccgatagtt tgagttcttc tactcaggca agtgatgtta 2760 ttactaatca aagaagtatt gcgacaacgg ttaatttgcg tgatggacag actcttttac 2820 tcggtggcct cactgattat aaaaacactt ctcaggattc tggcgtaccg ttcctgtcta 2880 aaatcccttt aatcggcctc ctgtttagct cccgctctga ttctaacgag gaaagcacgt 2940 tatacgtgct cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg 3000 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 3060 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 3120 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 3180 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 3240 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 3300 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 3360 Aaaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca 3420 atttaaatat ttgcttatac aatcttcctg tttttggggc ttttctgatt atcaaccggg 3480 gtacatatga ttgacatgct agttttacga ttaccgttca tcgcaggtgg cacttttcgg 3540 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 3600 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 3660 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 3720 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 3780 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 3840 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 3900 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 3960 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 4020 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 4080 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 4140 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 4200 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 4260 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 4320 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 4380 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 4440 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 4500 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 4560 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 4620 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 4680 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 4740 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 4800 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 4860 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 4920 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 4980 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 5040 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 5100 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 5160 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 5220 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 5280 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 5340 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 5400 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagc 5458 <210> 120 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 120 ctatggcttc tagagatgtg gtgatg 26 <210> 121 <211> 398 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <400> 121 agctgctggt ctgctgctgc tggcggccca gccggctatg gcttctagag atgtggtgat 60 gacccagagc cccctgtccc tgcctgtgac ccctggcgag cctgccagca tctcctgccg 120 gagctcccag agcatcctgc actccaatgg caacacctac ctggagtggt acctgcagaa 180 gcctggccag agcccccagc tgctgatcta caaggtgtcc aaccggttct ccggcgtgcc 240 tgaccggttc agcggctccg gcagcggcac agacttcacc ctgaagatca gccgggtgga 300 ggctgaggat gtgggcgtct actactgctt ccagggcagc ctggtgcccc tgacctttgg 360 ccagggcacc aagctggaga tcaagcgtac ggtggctg 398 <210> 122 <211> 398 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <220> <221> misc_feature (222) (329) .. (330) N is a, c, g, or t <220> <221> misc_feature (222) (332) .. (333) N is a, c, g, or t <220> <221> misc_feature 222 (335) .. (336) N is a, c, g, or t <220> <221> misc_feature (338) (338). (339) N is a, c, g, or t <220> <221> misc_feature (222) (341) .. (342) N is a, c, g, or t <400> 122 agctgctggt ctgctgctgc tggcggccca gccggctatg gcttctagag atgtggtgat 60 gacccagagc cccctgtccc tgcctgtgac ccctggcgag cctgccagca tctcctgccg 120 gagctcccag agcatcctgc actccaatgg caacacctac ctggagtggt acctgcagaa 180 gcctggccag agcccccagc tgctgatcta caaggtgtcc aaccggttct ccggcgtgcc 240 tgaccggttc agcggctccg gcagcggcac agacttcacc ctgaagatca gccgggtgga 300 ggctgaggat gtgggcgtct actactgcnn knnknnknnk nnkgtgcccc tgacctttgg 360 ccagggcacc aagctggaga tcaagcgtac ggtggctg 398 <210> 123 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature (222) (57) .. (58) N is a, c, g, or t <220> <221> misc_feature (222) (60) .. (61) N is a, c, g, or t <220> <221> misc_feature (222) (63) .. (64) N is a, c, g, or t <220> <221> misc_feature (222) (66) .. (67) N is a, c, g, or t <220> <221> misc_feature (222) (69) .. (70) N is a, c, g, or t <400> 123 cagccaccgt acgcttgatc tccagcttgg tgccctggcc aaaggtcagg ggcacmnnmn 60 nmnnmnnmnn gcagtagtag ac 82 <210> 124 <211> 398 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <220> <221> misc_feature (222) (341) .. (342) N is a, c, g, or t <220> <221> misc_feature (222) (344) .. (345) N is a, c, g, or t <220> <221> misc_feature (347) (347) .. (348) N is a, c, g, or t <220> <221> misc_feature <222> (350) .. (351) N is a, c, g, or t <220> <221> misc_feature (222) (353) .. (354) N is a, c, g, or t <400> 124 agctgctggt ctgctgctgc tggcggccca gccggctatg gcttctagag atgtggtgat 60 gacccagagc cccctgtccc tgcctgtgac ccctggcgag cctgccagca tctcctgccg 120 gagctcccag agcatcctgc actccaatgg caacacctac ctggagtggt acctgcagaa 180 gcctggccag agcccccagc tgctgatcta caaggtgtcc aaccggttct ccggcgtgcc 240 tgaccggttc agcggctccg gcagcggcac agacttcacc ctgaagatca gccgggtgga 300 ggctgaggat gtgggcgtct actactgctt ccagggcagc nnknnknnkn nknnktttgg 360 ccagggcacc aagctggaga tcaagcgtac ggtggctg 398 <210> 125 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature (222) (45) .. (46) N is a, c, g, or t <220> <221> misc_feature (222) (48) .. (49) N is a, c, g, or t <220> <221> misc_feature (222) (51) .. (52) N is a, c, g, or t <220> <221> misc_feature <222> (54) .. (55) N is a, c, g, or t <220> <221> misc_feature (222) (57) .. (58) N is a, c, g, or t <400> 125 cagccaccgt acgcttgatc tccagcttgg tgccctggcc aaamnnmnnm nnmnnmnngc 60 tgccctggaa gcagtagtag ac 82 <210> 126 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 126 gtttatctcg agcaggtgac cctgaag 27 <210> 127 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <400> 127 ccgtggccca ggcggccctc gagcaggtga ccctgaagga gtctggccct gccctggtga 60 agcccaccca gaccctgacc ctgacctgca ccttctctgg cttcagcctg agcacctctg 120 gcatgggcgt gggctggatc cggcagcccc ctggcaaggc cctggagtgg ctggcccaca 180 tctggtggga cgacgacaag tcctacaacc ccagcctgaa gagccggctg accatcagca 240 aggacaccag caagaaccag gtggtgctga ccatgaccaa catggaccct gtggacagtg 300 cccggcggca gctgggcctg cggagcattg atgccatgga ctactggggc cagggcacca 360 cagtgacagt gtccagcgcc tccaccaagg gcccatcgg 399 <210> 128 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <220> <221> misc_feature (222) (306) .. (307) N is a, c, g, or t <220> <221> misc_feature <222> (309) .. (310) N is a, c, g, or t <220> <221> misc_feature <222> (312) .. (313) N is a, c, g, or t <220> <221> misc_feature (222) (315) .. (316) N is a, c, g, or t <220> <221> misc_feature (222) (318) .. (319) N is a, c, g, or t <400> 128 ccgtggccca ggcggccctc gagcaggtga ccctgaagga gtctggccct gccctggtga 60 agcccaccca gaccctgacc ctgacctgca ccttctctgg cttcagcctg agcacctctg 120 gcatgggcgt gggctggatc cggcagcccc ctggcaaggc cctggagtgg ctggcccaca 180 tctggtggga cgacgacaag tcctacaacc ccagcctgaa gagccggctg accatcagca 240 aggacaccag caagaaccag gtggtgctga ccatgaccaa catggaccct gtggacagtg 300 cccggnnknn knnknnknnk cggagcattg atgccatgga ctactggggc cagggcacca 360 cagtgacagt gtccagcgcc tccaccaagg gcccatcgg 399 <210> 129 <211> 102 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature (222) (81) .. (82) N is a, c, g, or t <220> <221> misc_feature (222) (84) .. (85) N is a, c, g, or t <220> <221> misc_feature (222) (87) .. (88) N is a, c, g, or t <220> <221> misc_feature (222) (90) .. (91) N is a, c, g, or t <220> <221> misc_feature (222) (93) .. (94) N is a, c, g, or t <400> 129 ccgatgggcc cttggtggag gcgctggaca ctgtcactgt ggtgccctgg ccccagtagt 60 ccatggcatc aatgctccgm nnmnnmnnmn nmnnccgggc ac 102 <210> 130 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <220> <221> misc_feature <222> (330) .. (331) N is a, c, g, or t <220> <221> misc_feature <222> (333) .. (334) N is a, c, g, or t <220> <221> misc_feature (222) (336) .. (337) N is a, c, g, or t <220> <221> misc_feature (222) (339) .. (340) N is a, c, g, or t <220> <221> misc_feature (342) (342) .. (343) N is a, c, g, or t <400> 130 ccgtggccca ggcggccctc gagcaggtga ccctgaagga gtctggccct gccctggtga 60 agcccaccca gaccctgacc ctgacctgca ccttctctgg cttcagcctg agcacctctg 120 gcatgggcgt gggctggatc cggcagcccc ctggcaaggc cctggagtgg ctggcccaca 180 tctggtggga cgacgacaag tcctacaacc ccagcctgaa gagccggctg accatcagca 240 aggacaccag caagaaccag gtggtgctga ccatgaccaa catggaccct gtggacagtg 300 cccggcggca gctgggcctg cggagcattn nknnknnknn knnktggggc cagggcacca 360 cagtgacagt gtccagcgcc tccaccaagg gcccatcgg 399 <210> 131 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature (222) (57) .. (58) N is a, c, g, or t <220> <221> misc_feature (222) (60) .. (61) N is a, c, g, or t <220> <221> misc_feature (222) (63) .. (64) N is a, c, g, or t <220> <221> misc_feature (222) (66) .. (67) N is a, c, g, or t <220> <221> misc_feature (222) (69) .. (70) N is a, c, g, or t <400> 131 ccgatgggcc cttggtggag gcgctggaca ctgtcactgt ggtgccctgg ccccamnnmn 60 nmnnmnnmnn aatgctccgc aggcccagct g 91 <210> 132 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <220> <221> misc_feature (222) (318) .. (319) N is a, c, g, or t <220> <221> misc_feature (222) (321) .. (322) N is a, c, g, or t <220> <221> misc_feature <222> (324) .. (325) N is a, c, g, or t <220> <221> misc_feature <222> (327) .. (328) N is a, c, g, or t <220> <221> misc_feature <222> (330) .. (331) N is a, c, g, or t <400> 132 ccgtggccca ggcggccctc gagcaggtga ccctgaagga gtctggccct gccctggtga 60 agcccaccca gaccctgacc ctgacctgca ccttctctgg cttcagcctg agcacctctg 120 gcatgggcgt gggctggatc cggcagcccc ctggcaaggc cctggagtgg ctggcccaca 180 tctggtggga cgacgacaag tcctacaacc ccagcctgaa gagccggctg accatcagca 240 aggacaccag caagaaccag gtggtgctga ccatgaccaa catggaccct gtggacagtg 300 cccggcggca gctgggcnnk nnknnknnkn nkgccatgga ctactggggc cagggcacca 360 cagtgacagt gtccagcgcc tccaccaagg gcccatcgg 399 <210> 133 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature (222) (69) .. (70) N is a, c, g, or t <220> <221> misc_feature (222) (72) .. (73) N is a, c, g, or t <220> <221> misc_feature (222) (75) .. (76) N is a, c, g, or t <220> <221> misc_feature <222> (78) .. (79) N is a, c, g, or t <220> <221> misc_feature (222) (81) .. (82) N is a, c, g, or t <133> 133 ccgatgggcc cttggtggag gcgctggaca ctgtcactgt ggtgccctgg ccccagtagt 60 ccatggcmnn mnnmnnmnnm nngcccagct g 91 <210> 134 <211> 330 <212> PRT <213> Homo sapiens <400> 134 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 135 <211> 326 <212> PRT <213> Homo sapiens <400> 135 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Thr Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro             100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp         115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp     130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn                 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp             180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro         195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu     210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile                 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr             260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys         275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys     290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys                 325 <210> 136 <211> 327 <212> PRT <213> Homo sapiens <400> 136 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro             100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys         115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val     130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe                 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp             180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu         195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg     210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp                 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys             260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser         275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser     290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys                 325 <210> 137 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 137 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Thr Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro             100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp         115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp     130 135 140 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn                 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp             180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro         195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu     210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile                 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr             260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys         275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys     290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys                 325 <210> 138 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 138 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Leu Leu Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser Leu Ser Thr Ser             20 25 30 Gly Met Gly Val Gly Trp Phe Arg Gln Pro Pro Gly Lys Gly Leu Glu         35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Ser Tyr Asn Pro Ser     50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Ile Thr Asn Val Asp Pro Val Asp Thr Ala Thr Tyr Tyr                 85 90 95 Cys Ala Arg Arg Gln Leu Gly Thr Arg Gly Thr Asp Ala Met Asp Tyr             100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser     130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180 185 190 Thr Val Thr Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val         195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys     210 215 220 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser                 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser Gln Glu Asp             260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn         275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val     290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys                 325 330 335 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr             340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr         355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu     370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys                 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu             420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly         435 440 445 Lys      <139> <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid molecule <400> 139 atggaatgga gctgggtctt tctcttcttc ctgtcagtaa ctacaggtgt ccactcgcag 60 gtgaccctga aggagtctgg ccctggcctg ctgaagccca cccagaccct gaccctgacc 120 tgcaccctgt ctggcttcag cctgagcacc tctggcatgg gcgtgggctg gttccggcag 180 ccccctggca agggcctgga gtggctggcc cacatctggt gggacgacga caagtcctac 240 aaccccagcc tgaagagccg gctgaccatc agcaaggaca ccagcaagaa ccaggtggtg 300 ctgaccatca ccaacgtgga ccctgtggac acagccacct actactgtgc ccggcggcag 360 ctgggcacta gggggacgga tgccatggac tactggggcc agggcaccac agtgacagtg 420 tccagcgcat ccaccaaggg cccatccgtc ttccccctgg cgccctgctc caggagcacc 480 tccgagagca cagccgccct gggctgcctg gtcaaggact acttccccga accggtgacg 540 gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 600 tcctcaggac tctactccct cagcagcgtg gtgaccgtga cctccagcaa ctttggcacg 660 cagacctaca cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagacagtt 720 gagcggaaat gctgcgtgga gtgcccacca tgcccagcac ctccagtggc cggaccatca 780 gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc 840 acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg 900 gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt caacagcacg 960 ttccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac 1020 aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaaacc 1080 aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccggga ggagatgacc 1140 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc catgctggac 1260 tccgacggct ccttcttcct ctacagcaag ctaaccgtgg acaagagcag gtggcagcag 1320 gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag 1380 agcctctccc tgtctcctgg taaatga 1407 <210> 140 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 140 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Leu His Ser             20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Thr                 85 90 95 Thr Arg Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu         115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe     130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser                 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu             180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser         195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 141 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 141 atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacaga tgccagatgc 60 gatgtggtga tgacccagac ccccctgtcc ctgcctgtga cccctggcca gcctgccagc 120 atctcctgcc ggagctccca gagcatcctg cactccaatg gcaacaccta cctggagtgg 180 tacctgcaga agcctggcca gagcccccag ctgctgatct acaaggtgtc caaccggttc 240 tccggcgtgc ctgaccggtt cagcggctcc ggcagcggca cagacttcac cctgaagatc 300 agccgggtgg aggctgagga tgtgggcgtc tactactgcc ttcagactac tcgtgtgccc 360 ctgacctttg gccagggcac caagctggag atcaagcgta cggtggctgc accatctgtc 420 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 480 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 540 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 600 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 660 gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgttag 720 <210> 142 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 142 Arg Gln Leu Gly Lys Leu Ala Leu Asp Ala Met Asp Tyr 1 5 10 <210> 143 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 143 Arg Gln Leu Gly Leu Arg Ser Ile Asp Ala Met Asp Tyr 1 5 10 <210> 144 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 144 Arg Gln Leu Gly Gln Arg Gln Thr Asp Ala Met Asp Tyr 1 5 10 <210> 145 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 145 Arg Ala Ile Gln Pro Arg Ser Ile Asp Ala Met Asp Tyr 1 5 10 <210> 146 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 146 Arg Gln Leu Gly Leu Arg Ser Ile Asp Ala His Thr Arg 1 5 10 <210> 147 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 147 Arg Gln Leu Gly Gln Pro Ser Val Asp Ala Met Asp Tyr 1 5 10 <210> 148 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 148 Arg Gln Leu Gly Phe Gln Ser Thr Asp Ala Met Asp Tyr 1 5 10 <210> 149 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 149 Arg Gln Leu Gly Gln Ala Gly His Asp Ala Met Asp Tyr 1 5 10 <210> 150 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 150 Arg Gln Leu Gly Asp Asn Val Ala Asp Ala Met Asp Tyr 1 5 10 <210> 151 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 151 Arg Gln Leu Gly Met Ala Thr Pro Asp Ala Met Asp Tyr 1 5 10 <210> 152 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 152 Arg Gln Leu Gly Ala His Trp Leu Asp Ala Met Asp Tyr 1 5 10 <210> 153 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 153 Arg Gln Leu Gly Pro Glu Pro Gln Asp Ala Met Asp Tyr 1 5 10 -20-  

Claims (12)

An isolated antibody, or fragment thereof, capable of differentially recognizing the multidimensional conformation of one or more Αβ-induced diffuse ligands, wherein the antibody comprises Arg-Xaa ₁ -Leu-Xaa ₂ -Xaa ₃ -Xaa Complementarity Determining Sites (CDRs) of ₄ -Xaa ₅ -Xaa ₆ -Asp-Ala-Met-Asp-Tyr (SEQ ID NO: 9), wherein Xaa ₁ is Gln or Ala; Xaa2 is Ser or Gly; Xaa₃ is Pro, Ala, Lys, Arg or Thr; Xaa ₄ is Lys or Arg; Xaa ₅ is Gly, Ser, or Lys; Xaa ₆ is Val, Thr, Ile or Arg, an isolated antibody or fragment thereof. The isolated antibody or fragment thereof of claim 1, wherein the heavy and light chain variable regions of the antibody comprise the amino acid sequences set forth in SEQ ID NO: 108 and SEQ ID NO: 112, respectively. The isolated antibody or fragment thereof of claim 1, wherein the heavy and light chains of the antibody comprise the amino acid sequences set forth in SEQ ID NO: 138 and SEQ ID NO: 140, respectively. A pharmaceutical composition comprising an isolated antibody or fragment thereof according to claim 1 capable of differentially recognizing the multidimensional conformation of at least one Aβ-induced diffusive ligand in a mixture with a pharmaceutically acceptable carrier. A method of preventing binding of Aβ-induced diffuse ligands to neurons, comprising contacting the antibody according to claim 1 with neurons to prevent the Aβ-induced diffuse ligands from binding to neurons. A method of inhibiting assembly of an Aβ-induced diffuse ligand, characterized by inhibiting assembly of the Aβ-induced diffuse ligand by contacting a sample containing amyloid β 1-42 peptide with the antibody according to claim 1. A method for blocking phosphorylation of tau protein in Ser202 / Thr205, characterized by blocking phosphorylation of tau protein in Ser202 / Thr205 by contacting a sample containing tau protein. A method for preventing or treating a disease associated with Aβ-induced diffuse ligands by administering an effective amount of the pharmaceutical composition according to claim 4. The method of claim 1 for contacting an Αβ-induced diffuse ligand with neurons in the presence of an agent and for determining whether the Αβ-induced diffuse ligand is bound to the neuron in the presence of the agent. A method of identifying a therapeutic agent that prevents binding of Aβ-induced diffuse ligands to neurons, including using the antibody according to the present invention. A method for detecting an Αβ-induced diffuse ligand in a sample comprising contacting the antibody according to claim 1 to the sample such that the Αβ-induced diffuse ligand is detected. A method for diagnosing a disease associated with an Αβ-induced diffuse ligand, comprising contacting the sample according to claim 1 with a sample such that the disease associated with the Αβ-induced diffuse ligand is diagnosed. Aβ-induced diffuse ligand detection kit comprising the isolated antibody of claim 1, or a fragment thereof.

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