TW201139667A - Amyloid-beta binding proteins - Google Patents

Abstract

The present invention relates to amyloid-beta (A β ) binding proteins. Antibodies of the invention have high affinity to A β (20-42) globulomer or any A β form that comprises the globulomer epitope. Method of making and method of using the antibodies of the invention are also provided.

Description

201139667 6. Technical Description: The present invention relates to a powder-β (Αβ) binding protein, a nucleic acid encoding the same, a method for producing the same, and a combination comprising the same And the use of such proteins in the diagnosis, treatment and prevention of conditions such as starch-like degeneration (for example, Alzheimer's disease). [Prior Art] Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive ability and characteristic neuropathological features, including starch-like beta (Αβ) peptide deposition, neurofibrils in several regions of the brain. Oligations and neuronal loss (Hardy and Selkoe, Science 297·. 353, 2002; Mattson, Nature 431: 7004, 2004). Brain-like starch deposition and cognitive impairment, which is very similar to that observed in Alzheimer's disease, is also characteristic of Down's syndrome (2nd dysplasia), which occurs at about 8 births per birth. Among the infants, there is a case where the Αβ peptide is produced by proteolytic treatment of a starch-like precursor protein (App). This treatment is achieved by the action of several proteases called alpha, beta & gamma secretase and produces a number of specific fragments of different lengths. The powder deposits are mainly composed of peptides of 40 or 42 amino acids in length (Αρ4〇, .β42). In addition to human variants, this also includes species other than humans. Other _ milk animals, especially the isoforms of the powder of the temple powder 0 (Bu 42). This protein is easy to aggregate in an aqueous environment and can exist in very different molecular forms. The simple relationship between insoluble protein deposition and the appearance or progression of dementia disorders such as Alzheimer's disease has been confirmed to be unreliable (Terry et al., Ann. Neurol 30: 572-580, 1991; Dickson Et al, Neurobiol. Aging 16: 285-298, 1995). In contrast, synaptic and cognitive sensation appears to be more associated with soluble forms of Αβ(ΐ-42) (Lue et al, Am J. Pathol. 155: 853-862, 1999; McLean et al, Ann Neurol. 46: 860-866, 1999). In the past, multiple strains and monoclonal antibodies against monomeric Αβ( 1-42) have not been shown to produce the desired therapeutic effect in animals and/or humans without causing serious side effects. For example, passive immunization resulting from preclinical studies in very old alpha pρ23 mice that received anti-Aβ (1-42) antibodies to the sputum end of the week for 5 months indicated treatment-related side effects. In particular, these mice showed an increase in the number and severity of microbleeds in mice treated with saline (Pfeifer et al, science 298: 1379, 2002). It also describes a similar increase in bleeding in very old (>24 months) Tg2576 and PDAPP mice (Wilcock et al. 'J Neuroscience 23: 3745-5 1, 2003; Racke et al, J Neuroscience 25: 629-636, 2005). ). In both lines, injection of anti-Aβ (1-42) resulted in a significant increase in microbleeds. W0 2004/067561 mentions a spherical oligomer of Αβ(1-42) peptide ("globulomer j") and a preparation method thereof. WO 2006/094724 mentions non-diffusible globular Αβ (Χ - 38 .... 43) An oligomer wherein X is selected from the group consisting of the numbers 1...24. WO 2004/067561 and WO 2006/094724 further describe the limited proteolysis of globulomers to produce a truncated version of the globulomer' such as Αβ(20-42) or Αβ(12-42) globulomer. WO 2007/064917 describes a recombinant form of amyloprote-like peptide (hereinafter referred to as N-Met Αβ(1-42)) and its globular shape 155263. Doc -4· 201139667 The selection, expression and isolation of the formula. The data indicate the presence of Αβ folding and assembly into Αβ oligomers (which exhibit one or more unique epitopes (hereinafter referred to as globulomer epitopes)) Amyloplast-like independent pathway. Because globulomer epitopes are detected in the brains of AD patients and sputum-transgenic mice, and globulomers specifically bind to neurons and block LTP, globulomers Is a pathologically relevant Αβ conformer. It has been found that soluble Αβ globulomers are essentially composed of P/Q presynaptic calcium. The daughter channel interacts to exert its deleterious effects, and the inhibitor of this interaction is therefore suitable for the treatment of amyloidosis, such as Alzheimer's disease (WO 2008/104385). Selective binding to the Αβ globulomer form The antibodies are described in WO 2007/064972, WO 2007/062852, WO 2008067464 'WO 2008/1 50946 and WO 2008/150949. For example, several individual plants known from WO 2007/062852 and WO 2008/1 50949 Antibodies specifically recognize Αβ(20-42) globulomers. There is a huge unmet therapeutic need to develop biological agents (such as Aβ binding proteins) that prevent or slow disease progression without inducing negative and potentially lethal effects on the human body. The general population lifespan is increased, and this need is particularly pronounced, and with this increase, the number of patients diagnosed with Alzheimer's disease or related conditions is increased each year. In addition, these Αβ-binding proteins will allow for the correct diagnosis of Azhai. Alzheimer's disease in patients with symptoms of Mohs disease, this diagnosis can only be confirmed after necropsy. In addition, Αβ-binding protein will allow clarification of these proteins and The biological characteristics of other biological factors responsible for this debilitating disease. [Abstract] 155263.doc 201139667 The present invention provides a novel set of Aβ binding proteins (or simply referred to as "binding proteins"), CDR-grafted antibodies, humanized antibodies and fragments thereof. It is capable of binding to a soluble Αβ globulomer, such as the Αβ(20-42) globulomer described herein. It should be noted that the binding proteins of the invention may also react (i.e., bind) to the Aβ form other than the Αβ globulomers described herein, which may be present in a starch-like degeneration (such as Alzheimer's disease). ) in the brain of the patient. These Αβ forms may or may not be in the form of oligomeric or globulomers. The 蛋白β-binding form of the binding protein of the present invention includes any Αβ form (hereinafter referred to as "m4D10") m4D10 comprising a murine/mouse monoclonal antibody m4D10 and a reacting globulomer epitope, and its characteristics are described in WO 2007. /062852, which is incorporated herein by reference. These Αβ forms are hereinafter referred to as "targeted Αβ forms". In addition, the present invention also provides compositions for inhibiting the activity of such targeted Αβ forms and providing compositions for treating diseases associated with such targeted Αβ forms, particularly starch-like degeneration, such as Alzheimer's disease, and method. In one aspect, the invention provides a binding protein comprising: at least 90% of the first amino acid sequence SEQ ID NO: 2:

EVQLVESGGGLX, 2QPGGSLRLSCAX24SGFTX29SSYGVH wvrqapgkglewx48x49viwrggridynaafmsrx67tis x71dnskx76tx78ylqmnslraedtavyycarnsdvwgqg TTVTVSS, where X12 is I or V, X24 is A or V, X29 is V or L, X48 is V or L, X49 is S or G, X67 is F or L, and X71 is R or K. X76 is N or S, and 155263.doc 201139667 X78 is L or V; or SEQ ID NO: 3: X^QLQESGPGLVKPSETLSLTCTVSGX^SX^SSYGVHW X37RQPPGKGLEWX48GVIWRGGRIDYNAAFMSRX67TISX71 dtskx76qx78slklssvtaadtavyycarnsdvwgqgttv TVSS, where X1 is Q or E, X27 is G or F, X29 is I or L, X37 is I or V, X48 is I or L, X67 is V or L, X71 is V or K, X76 is N or S, and X78 is F or V; and at least 90°/below. Consistent second amino acid sequence SEQ ID NO: l : dvvmtqx7plslpvtx, 5gqpasisckssqslldidgkty lnwx4Ix42qx44pgqspx50rliylvskldsgvpdrfsgsgs

GTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK R, wherein X7 is S or T, X15 is L or P, X41 is F or L, X42 is Q or L, X44 is R or K, and X5G is R or Q» In another aspect of the present invention, The binding protein described above comprises at least 90%, 91% '92% '93%, 94%, 95%, 96%, 97%, 98% or 99% with an amino acid sequence selected from the group consisting of A first amino acid sequence: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: And SEQ ID NO: 11 In another aspect of the invention, the binding protein described above comprises a first amino acid sequence selected from the group consisting of: 155263.doc 201139667 SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. In another aspect of the invention, the binding protein described above comprises at least 90%, 91%, 92%, 93%, 94%, 95%, and an amino acid sequence selected from the group consisting of 96%, 97%, 98% or 99% of the second amino acid sequence: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. . In another aspect of the invention, the binding protein described above comprises a second amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

In one aspect of the invention, the binding protein described above comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence selected from the group consisting of , 97%, 98% or 99%, such as the first amino acid sequence·· SEQ ID NO: 4, SEQ ID ΝΟ··5, SEQ ID ···6, SEQ ID ΝΟ: 7, SEQ ID ΝΟ: 8. SEQ ID ΝΟ: 9, SEQ ID NO: 10 and SEQ ID NO: ll; and at least 90%, 91%, 92%, 93%, 94 with an amino acid selected from the group consisting of: %, 95%, 96%, 97%, 98% or 99% of the second amino acid sequence: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In another aspect of the invention, the binding protein described above comprises a first amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID 155263. doc 201139667 NO: 11 ; and a second amino acid sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In a particular aspect of the invention 'the binding protein described above comprises and SEQ ID N〇: 6 The amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95. /. 96%, 97%, 98% or 99% of the first amino acid sequence; and at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence as set forth in SEQ ID NO: 14. , 95%, 96%, 97%, 98% or 99% of the second amino acid sequence. In another specific aspect of the invention, the binding protein comprises a first amino acid sequence of SEQ ID NO: 6; and a second amino acid sequence of SEQ ID NO: 14. In a particular aspect of the invention, the binding protein described above comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96 of the amino acid sequence as set forth in SEQ ID NO: %, 97%, 98% or 99% of the first amino acid sequence; and at least 90%, 91%, 92%, 93%, 94%, 95 with the amino acid sequence of SEQ ID NO: % '96%, 97〇/〇, 98% or 99% — the second amino acid sequence. In another specific aspect of the invention, the binding protein described above comprises the first amino acid sequence set forth in SEQ ID NO: 10; and the second amino acid sequence set forth in SEQ ID NO: 14. In one aspect, the binding protein described herein is an antibody. The antibody may be, for example, an immunoglobulin molecule, a disulfide-linked Fv, a monoclonal antibody (mab), a single-chain Fv (scFv), a chimeric antibody, a single domain antibody, a CDR-grafted antibody, a mini-bifunctional antibody, humanization. Antibodies, multispecific antibodies, Fab, dual specific antibodies, dual variable domain (DVD) binding molecules, 155263.doc 201139667

Fab', bispecific antibody, F(ab')2 or Fv. When the binding protein described herein is an antibody, it comprises at least one variable heavy chain corresponding to a first amino acid sequence as defined above, and at least one corresponding to a second amino acid sequence as defined above Light chain. For example, an antibody of the invention comprises (1) at least one variable heavy chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of an amino acid sequence selected from the group consisting of , 97%, 98%, 99% or 100% amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 ' SEQ ID NO: 6 ' SEQ ID NO: 7 'SEQ ID NO: 8 &gt; SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11; and (ii) at least one variable light chain comprising and selected from the group consisting of The amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Consistent amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In a particular aspect of the invention, the antibody of the invention comprises (1) at least one variable heavy bond' comprising at least 90°/ of the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 10. 91%, 92. /0, 93〇/〇, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence, and (ii) at least one variable light chain 'contains with SEQ The amino acid sequence of ID NO: 14 is at least 90%, 91°/. 92%, 93%, 94%, 95. /. , 96%, 97%, 98%, 99% or 100% of the amino acid sequence. The binding protein described herein may additionally (except for the first and second amino acid sequences) comprise another moiety which may be another amino acid sequence or other chemical moiety 155263.doc -10·201139667. For example, an antibody of the invention may comprise a heavy chain immunoglobulin constant domain. The heavy chain immunoglobulin constant domain can be selected from the group consisting of a human IgM constant domain, a human IgG4 constant domain, a human igG1 constant domain, a human IgE constant domain, a human IgG2 constant domain, a human igG3 constant domain, and a human IgA constant domain. . In another aspect, the binding protein of the invention further comprises a heavy bond constant region having an amino acid sequence selected from the group consisting of SEQ ID NO: 25 and SEQ ID NO: 26, additionally comprising having a SEQ ID NO: And a light chain constant region of the amino acid sequence of the group consisting of 27 and SEQ ID NO: 28. In a particular aspect of the invention, the binding protein described herein comprises a variable heavy bond having the amino acid sequence of SEQ id NO. 6 or SEQ ID NO: 10; and the amine of SEQ ID NO. a variable light bond of a base acid sequence; a heavy chain constant region having the amino acid sequence of seq id NO: 25; and a light chain constant region having the amino acid sequence of SEq ID NO: 27. In another specific aspect of the invention, the binding protein described herein comprises the first amino acid sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, and the second amino acid described in SEqIDN: 48 Sequences The binding proteins (eg, antibodies) described herein may additionally comprise a therapeutic agent, a developer, a residue capable of promoting the formation of an immunoadhesion molecule and/or another functional molecule (eg, another peptide or protein). The developer may be radiolabeled (including but not limited to 3h, 14c, 35s, 90y, 99TC, ΠΙΙη, i25l, (1), 177] Lu, 66Ho and 153Sm), enzymes, fluorescent labels, luminescent labels, bioluminescence Label, magnetic label or biotin. The binding proteins of the invention may be glycosylated. According to one aspect of the invention, the glycosylation pattern is a human glycosylation pattern. In another aspect of the invention, the binding protein binds to an Ap form comprising a globulomer epitope responsive to the murine monoclonal antibody m4D10 (i.e., targeting the Αβ form). In particular, the above binding protein binds to a powdered beta (20-42) globulomer as described herein. In one aspect of the invention, the binding proteins described herein are capable of modulating the biological function of Αρ (20-42) globulomer. In another aspect of the invention, the binding proteins described herein are capable of neutralizing Αβ(20-42) globulomer activity. The binding proteins of the invention may exist in crystalline form. In one aspect, the crystal is a drug-free drug controlled release crystal. In another aspect, the in vivo half-life of the crystalline binding protein is greater than its soluble counterpart. In another aspect, the crystallized binding protein retains biological activity after crystallization. The invention also provides isolated nucleic acids encoding any of the binding proteins disclosed herein. Another embodiment provides a vector comprising the nucleic acid. The vector may be selected from the group consisting of pCDNA, pTT (Durocher et al, Nucleic Acids Research 30 (2), 2002), pTT3 (pTT with additional multiple selection sites), pEFBOS (Mizushima and Nagata, Nucleic acids) Research 18 (17), 1990), pBV, pJV and pBJ. In another aspect of the invention, the host cell is transformed with the vector disclosed above. According to an embodiment, the host cell is a prokaryotic cell, including but not limited to E. coli. In a related embodiment, the host cell is a eukaryotic cell selected from the group consisting of a protist cell, an animal cell, a plant cell, and a fungal cell. The animal cell may be selected from the group consisting of mammalian cells, avian cells and insect cells. According to one aspect of the invention, the mammalian cell line is selected from the group consisting of CHO and COS, and the fungal cell is a yeast cell, 155263. Doc • 12- 201139667 Such as Saccharomyces Cerevisiae (*Sacc; zar〇w:); cei cereW&gt;sz.ae), and the insect cell is an insect Sf9 cell. Furthermore, the invention provides a method of making a binding protein disclosed herein comprising culturing any of the host cells disclosed herein in a culture medium under conditions and for a time suitable for making the binding protein. Another embodiment provides a binding protein of the invention made according to the methods disclosed herein. In another embodiment, the invention provides a binding protein made according to the methods disclosed above. The invention also provides pharmaceutical compositions comprising a binding protein (e.g., an antibody) disclosed herein and a pharmaceutically acceptable carrier. An embodiment of the invention provides a composition for the release of a binding protein described herein, wherein the composition comprises a formulation, which in turn comprises a crystal binding protein (eg, a crystalline antibody) disclosed above and a component; A polymeric carrier. In one aspect, the polymeric carrier is a polymer selected from one or more of the group consisting of poly(acrylic acid), poly(cyanoacrylate), poly(amino acid), poly(anhydride), Poly(depsipeptide), poly(ester), poly(lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutyrate), poly(caprolactone), poly(dioxane) Cyclohexanone), poly(ethylene glycol), poly((hydroxypropyl)decyl acrylamide, poly((organo)phosphazene), poly(orthoester), poly(ethylene glycol), Poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymer, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen Protein, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends and copolymers thereof. In another aspect, the ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactose, gelatin, hydroxypropyl 155263.doc • 13· 201139667 cyclodextrin, methoxypolyethylene glycol And polyethylene glycol beta. The present invention is also directed to a method for inhibiting (i.e., reducing) the activity of Αβ(2〇_42) globulomer (or any other dry Αβ form), #include the light Αβ form and the present The binding protein of the invention contacts such that the activity of the targeted Αρ form is inhibited (i.e., reduced). In a particular embodiment, the activity is inhibited in vitro. The method can comprise adding a binding protein of the invention to a sample (eg, a sample derived from an individual (eg, blood, cerebrospinal fluid, serum, tissue, etc.) or a cell culture containing or suspected of containing a targeted Αβ form to inhibit (also That is, to reduce the activity of the Αβ form in the sample. Alternatively, the activity of the targeted Αβ form in the individual can be inhibited (reduced) in vivo. Thus, the present invention further relates to a binding protein described herein for use in inhibition (ie, reducing) the activity of the targeted Αβ form in an individual comprising contacting the Αβ form with a binding protein of the invention such that the activity of the Αβ form is inhibited (ie, decreased). In a related aspect, the invention provides A method of inhibiting (i.e., reducing) the activity of the form in an individual suffering from a disease or condition that is afflicted by the activity of the Αβ-form. The method comprises administering to the individual at least one of the binding proteins disclosed herein. The activity of targeting the Αβ form in an individual is inhibited (ie, decreasing ρ. Thus, the present invention provides a Αρ binding protein as described herein, which is used in Inhibiting (ie, reducing) a targeted Αβ form in an individual suffering from a disease or condition as described herein, wherein administering to the individual at least one binding protein disclosed herein inhibits (ie, reduces) the activity of the Αρ form in the individual In a related aspect, the present invention provides treatment (eg, cure 'inhibition, improvement, delay or prevention of hair #, or 35 prevention of recurrence; see or relapse" or prevention of selection 155263.doc -14 - 201139667 Methods of disease or condition: αΐ anti-trypsin deficiency, C1-inhibitor deficiency angioedema, antithrombin deficiency thromboembolic disease Kuru, Creutzfeld-Jacob disease / Sheep disease, bovine spongiform encephalopathy, Gerstmann_Straussl6r_Scheinker disease, fatality, Immortal disease, Huntington's disease, spinal cord cerebral dysfunction Machado-Joseph atrophy, dentate red globus pallidus atrophy, frontal leaf dementia, sickle cell anemia, unstable hemoglobin inclusion hemolysis, drugs Inclusion body hemolysis, Parkinson's disease, systemic AL-type starch degeneration, nodular AL-induced powder degeneration, systemic AA-type starch degeneration, prostatic halllet degeneration, hemodialysis-based starch degeneration, hereditary (Iceland) cerebral vascular disease, Tylen's disease, familial visceral starch degeneration, familial visceral polyneuropathy, familial visceral starch degeneration, senile systemic amyloidosis, familial amyloid neuropathy胄, familial heart class powder degeneration, Alzheimer's disease, Down syndrome, thyroid medullary cancer and type 2 glucocorticoid disease (T2DM). In a particular embodiment, the disease or condition is a dendritic degeneration, such as Alzheimer's disease or Down's syndrome. In one embodiment, the method comprises the step of administering a -Ap binding protein disclosed herein such that the treatment is achieved. In another embodiment, the invention provides a method of treating an individual afflicted with a disease or condition disclosed herein, comprising the step of administering any of the Ap binding proteins disclosed herein simultaneously or after administration of one or more additional therapeutic agents. Accordingly, the present invention provides a Αβ-binding protein disclosed herein for use in treating an individual suffering from a disease or condition disclosed herein, comprising administering a 155263.doc -15·201139667 or a plurality of other therapeutic agents simultaneously or after administration of the present disclosure. Any of the steps of combining eggs. By way of example, other therapeutic agents are selected from the group of therapeutic agents listed herein. * The binding protein disclosed herein and the pharmaceutical composition comprising the binding protein are administered to an individual by at least one selected from the group consisting of parenteral, dermal, intramuscular, intravenous, intra-articular, intrabronchial, Intra-abdominal, intracapsular, soft-moon, intracavitary, intracavitary, intracranial, lateral ventricle, large intestine, cervix, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic, pericardial, intraperitoneal , intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intracranial, rapid injection, vaginal, rectal, buccal, sublingual, nasal In another embodiment, the invention provides a method of detecting a dry Αβ form in a sample comprising: (1) contacting the sample with a binding protein of the invention, and (π) detecting the (etc. a formation of a complex between the binding protein and an element of the sample, wherein an increase in the formation or formation of the complex relative to the control sample indicates the presence of the Aβ form in the sample. The sample may be obtained from a disease or condition suspected of having the invention disclosed herein. Individual life A sample (eg, whole blood, cerebrospinal fluid, serum, tissue, etc.) or a cell culture containing or suspected of containing the Αρ form. The control sample does not contain the Αβ form or is obtained from a patient who does not have the disease described above. The complex between the binding protein and the sample elements obtained from a patient suspected of having Alzheimer's disease indicates that the disease is diagnosed in the patient. In an alternative embodiment, the targeted Αβ form can be performed in vivo. Detection, for example, by in vivo imaging in an individual. To this end, the invention can be combined with the 155263.doc-16·201139667 protein to bind to the targeted Αβ form and detect the binding protein and the VIII The binding protein of the invention is administered to an individual or a control individual under conditions in which the complex forms between the forms, wherein an increase in complex formation or formation of the individual relative to the control individual indicates the presence of the Aβ form in the individual. The individual may be known or suspected of suffering An individual suffering from a condition or disease that is affected by the activity of the Αβ form. [Embodiment] Unless otherwise defined herein, the scientific terminology used in connection with the present invention will There is a general understanding of the meaning of the term ^ the meaning and scope of the term should be clear, however, if there is any implied ambiguity, the definition provided in this paper takes precedence over any dictionary or foreign definition. In addition, unless the context requires otherwise In addition, the singular terms shall include the plural and the plural terms shall include the singular. In the present application ' unless otherwise stated, the use of "or" means and / or "in addition, the use of the term "including" and other forms, such as "includes" and "included" are not restrictive. Also, the terms such as "element" or "component" are used to encompass the elements and components that comprise a unit and the elements and components that comprise more than one subunit. In general, the nomenclature and techniques used herein for cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and heterogeneous are well known in the art. < Desc/Clms Page number> And technology. The enzymatic reaction and purification techniques are performed as commonly accomplished in the art or as described herein in accordance with the manufacturer's instructions, such as 155263.doc • 17·201139667. The nomenclature used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry, as well as laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients. The present invention relates to Aβ binding proteins, particularly anti-Aβ antibodies or Aβ binding portions thereof, particularly to Aββ binding proteins of Aβ(20-42) globulomers. These Αβ-binding proteins are not only capable of distinguishing other forms of Αβ peptides, especially monomers and fibrils, but also capable of distinguishing 未β globulomers in uncut forms. Accordingly, the present invention relates to Αβ-binding proteins having a binding affinity for Αβ(20-42) globulomers greater than that for Αβ(1-42) globulomers.

The term "Αβ(Χ-Υ)" as used herein refers to the amino acid sequence (including X and oxime) of the amino acid position X to the amino acid position of the human starch β (Αβ) protein, in particular Amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IAT (SEQ ID ΝΟ: 29) amino acid position X to the amino acid position of the amino acid sequence (corresponding to amino acid positions 1 to 43) or any of its naturally occurring A variant, in particular, having at least one amino acid sequence selected from the group consisting of: Α2Τ, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch" ), E22K ("Italian"), D23N ("Iowa"), A42T and A42V, where the number is relative to the beginning of the Αβ peptide, including position X and position Υ or with up to three other amino acid substitutions (both do not prevent the ball) The sequence of the polymer formed). According to one aspect, no other amino acid substitutions are made in the 155263.doc-18.201139667 position of the amino acid 12 or hydrazine (the greater number of which) to the amino acid 42 or hydrazine (the smaller of which). According to another aspect, no other amino acid substitutions are made in the position of the amino acid 20 or X (the greater number of which) to the amino acid 42 or Y (the smaller number). According to another aspect, no other amino acid substitutions are made in the position of the amino acid 20 or X (the greater number of which) to the amino acid 40 or Y (the smaller number thereof). The "other" amino acid substitutions herein are any differences that are not found in nature relative to a typical sequence. More specifically, the term "Αβ(1-42)" as used herein refers to the amino acid sequence (including 1 and 42) of the amino acid position 1 to the amino acid position 42 of the human Αβ protein, in particular Amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQ ID ΝΟ: 30) or any naturally occurring variant thereof, in detail having at least one amino acid sequence selected from the group consisting of: Α2Τ, H6R , D7N, Α21 G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T and A42V, where the number is relative to the Αβ peptide Initially, include 1 and 42 or a sequence with up to three other amino acid substitutions (both do not prevent globulomer formation). According to one aspect, no other amino acid substitutions are present in the amino acid 20 to amino acid 42 position. Similarly, the term "Αβ(1-40)" as used herein refers to the amino acid sequence (including 1 and 40) of the amino acid position 1 to the amino acid position of the human Αβ protein, in particular the amino acid. Sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO: 31) or any naturally occurring variant thereof, in detail having at least one amino acid sequence selected from the group consisting of: A2T, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), 155263.doc -19- 201139667 E22Q ("Dutch"), E22K ("Italian") and D23N ("Iowa"), where the number is relative to the start of the Αβ peptide , including 1 and 40 or a sequence with up to three other amino acid substitutions (both do not prevent globulomer formation). According to one aspect, no other amino acid substitutions are present in the position of amino acid 20 to amino acid 40.

More specifically, the term "Αβ(12-42)" as used herein refers to the amino acid sequence (including 12 and 42) of the amino acid position 12 to the amino acid position 42 of the human Αβ protein, in particular Amino acid sequence VHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQ ID ΝΟ: 32) or any naturally occurring variant thereof, in detail having at least one amino acid sequence selected from the group consisting of: A21G ("Flemish "), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T and A42V, where the number is relative to the beginning of the Αβ peptide, including 12 and 42 or Sequence of up to three other amino acid substitutions (both do not prevent globulomer formation). According to one aspect, no other amino acid substitutions are present in the position of amino acid 20 to amino acid 42. Similarly, the term "Αβ(20-42)" as used herein refers to the amino acid sequence of amino acid position 20 of the human starch-like beta protein to position 42 of the amino acid (including 20 and 42), in detail the amine The base acid sequence F AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO: 33) or any naturally occurring variant thereof, in detail having at least one amino acid sequence selected from the group consisting of: A21G ("Flemish" ), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T and A42V, where the number is relative to the beginning of the Αβ peptide, including 20 and 42 155263.doc -20- 201139667 or a sequence with up to three other amino acid substitutions (both do not prevent globulomer formation). According to one aspect, there are any other amino acid substitutions. The term "Αβ(χ·Υ) globulomer" (Αβ(χ·γ) globular oligo] as used herein refers to a soluble spherical non-covalent association of Αβ(χ_γ) as defined above. It has homogeneity and unique physical characteristics. According to one aspect, the Αβ(Χ·Υ) globulomer is a stable non-fibrillar oligomeric assembly of the ΑΡ(Υ_Υ) peptide, which can be obtained by incubation with an anionic detergent. In contrast to monomers and fibrils, these globulomers are characterized by subunits of the specified number of assemblies (eg, the early assembly form "oligomers" with 4-6 subunits; and late assembly with 12-U subunits) The shape is "oligomer";: W〇2_/〇67561). The globulomer has a 3-dimensional spherical structure ("melted pellets", see Barghorn et al., j Neur〇chem %: Mm, 2005). It can be further characterized by one or more of the following features: • cleavage of the N-terminal amino acid χ23 with a prosthetic protease such as thermolysin or endoprotein/enzyme (1) uC to produce a truncated form of globulomer; _hybrid proteases and antibodies Inaccessible to the c-terminal amino acid 24_γ; • The truncated form of these globulomers retains the 3-dimensional core structure of the globulomer, which is the core epitope of the globulomer configuration Αβ(2〇_γ) The proximity is better. The term "Αβ(χ_γ) globulomer" according to the invention and in particular for the purpose of assessing the binding affinity of the protein of the invention means, inter alia, that by 〇〇 4/〇 67561 (which is incorporated by reference) The product obtained by the process described herein). The method comprises unfolding a Αρ(χ-γ) peptide or a derivative thereof of a natural, recombinant or synthetic Αβ 155263.doc •21 - 201139667 (Χ-Υ) peptide or a derivative thereof. Agent; reduce the role of detergents and continuous cultivation. For the purpose of developing the peptide, a hydrogen bond cleavage agent such as an alcohol (HFIP) can be applied to the protein. When the field action temperature is about 20 to 50 ° C and especially 35 to 4 ° C, the effect time of a few minutes (for example, about 10 to 60 minutes) is sufficient to subsequently dissolve the residue which has evaporated to dryness (for example, a concentrated form). A suitable organic solvent (such as dimethyl sulphate (1) deleted) in which the aqueous buffer is miscible, resulting in a suspension of at least partially unfolded peptide or a derivative thereof, which can then be used as a 'reservoir suspension during the transition period. Store at low temperatures (eg approximately -20 C). Alternatively, the peptide or derivative thereof may be dissolved in a weakly acidic solution (e.g., an aqueous solution), for example, about 1 liter of an aqueous solution of the fertilizer. The insoluble components are removed by centrifugation after a typical incubation time of a few minutes. It is better to have 4 knives and 4 knives. These method steps can be carried out at room temperature (i.e., at a temperature in the range of 2 Torr to 3 Torr). After centrifugation, the supernatant is obtained containing Αβ(χ_γ) peptide or its organism and can be stored at a low temperature (for example, about _2 〇. ()) during the transition period. Subsequent exposure to the detergent involves the peptide or its derivative Polymerization, an intermediate type of oligomer (referred to as oligomer A in WO 2004/067561) is obtained. To this end, a detergent is applied to the at least partially unfolded peptide or derivative thereof until sufficient intermediate oligomers are produced. It is preferred to use an ionic detergent, especially an anionic detergent. According to a particular embodiment, a detergent of formula (1) is used: RX, wherein the group R is an unbranched or branched chain having from 6 to 20, for example from 1 to 14 carbon atoms An alkyl group or an unbranched or branched alkenyl group having 6 to 20, for example, 1 to 14 carbon atoms, the group X being an acidic group or a salt thereof, wherein 155263.doc • 22-201139667 X is selected from For example, _C00_M+, ·δ〇3_Μ+, especially _〇s〇3_m+, and m+ is a hydrogen cation or an inorganic or organic cation selected from, for example, an alkali metal and an alkaline earth metal cation and an ammonium cation. It is preferably a detergent of the formula (1) , where r is an unbranched alkyl group For example, it is advisable to use sodium decyl sulfate (SDS) 1 cinnamic acid, detergent sodium salt of lauric acid creatinine (also known as sark〇syl NL3〇4Gard〇l8) and oleic acid. The action time of the cleaning agent depends, in particular, on whether or not the oligomerized peptide or its derivative is unfolded (and, to what extent). According to the unfolding step, if the peptide or its derivative is treated with a hydrogen bond cleavage agent in advance, That is to say, in particular treated with hexafluoroisopropanol, when the temperature of action is from about 20 to 50 eC and especially from about 35 to 4 (rc, in the range of a few hours, preferably from about 1 to 20 and especially from about 2 to 1 hour A time is sufficient if a less unfolded or substantially unexpanded peptide or derivative thereof is used as a starting point, a relatively long period of action is preferred. If the peptide or derivative thereof has been pre-staged as an alternative to HFIP treatment, for example, as described above Treatment, or the peptide or its derivative directly oligomerizes, when the temperature of action is about 5 to 30 hours, and especially about 丨〇 to 2 hours, from about 35 to 40 C. The action time is sufficient. After incubation, the insoluble components should be separated by centrifugation. A few minutes at 10,000 g. Choose the detergent concentration according to the detergent used. If SDS' is used, it should be used in the range of 〇.〇i to 1% by weight, for example 〇.〇5 to 0_5 weight ❾/. A concentration of about 2% by weight. If lauric acid or oleic acid is used, it is preferred to use a slightly higher concentration, for example, 〇〇5 to 2 weight ❶/, such as 〇·1 to 〇·5 weight. For example, about 〇5 weight 〇/〇. The action of the detergent should occur at a salt concentration that is approximately in the physiological range. Therefore, it should be used especially in the range of 50 to 500 mM, for example, 100 to 200 155263.doc •23 · 201139667 mM range or about 140 mM NaCl concentration. Subsequent reduction of detergent action and continued incubation involves further oligomerization to obtain the Αβ(χ-γ) globulomer of the present invention (referred to as merging polymer B in WO2004/067561) because the composition obtained from the previous step is usually Contains detergents and salt concentrations within the physiological range' so it is desirable to reduce the detergent action and salt concentration. This can be done, for example, by diluting the concentration of detergent and salt advantageously with water or a lower salt concentration buffer (e.g., Tris-HCl, pH 7.3). A dilution factor in the range of about 2 to about ,, preferably in the range of about 3 to 8, and especially about 4, is suitable. It is also possible to reduce the action of the detergent by adding a substance which neutralizes the action of the detergent. Examples of such materials include those capable of complexing a cleaning agent, such as substances capable of stabilizing cells during purification and extraction measures, such as specific E〇/p〇 block copolymers, especially the block under the trade name Pluronic® F 68. Copolymer. Alkoxylation of alkoxylated and especially ethoxylated alkylphenols (such as the Trh〇n8χ series (2 of its Triton® xl00)) at concentrations above or above a certain critical microcell concentration Third octyl phenol, 3·(3_cholestyryl propyl decyl ammonium)-1-propane sulfonate (CHAPS@)) or alkoxylated and especially ethoxylated sorbitan Fatty esters (such as the Tween 8 series, especially Tween® 20) are equivalent. Subsequently, the solution was incubated until sufficient Αβ(Χ-Υ) globulomer of the present invention was produced. When the reaction temperature is from about 2 Torr to 5 Torr and especially from about 35 to 4 (TC, the action time in the range of several hours (for example, in the range of about 1 Torr to 3 Torr or in the range of about 15 to 25 hours) is sufficient. The solution can then be concentrated and the residue which may be present can be separated by centrifugation. Also, it is preferably a few minutes at 10,000 g. After centrifugation, the supernatant is obtained to contain the (XY) globulomer of the present invention. Finally, it can be known per se. By way of example, the AP (X_Y) globulomer of the invention is recovered by filtration, dialysis, precipitation or centrifugation by ultra-155263.doc • 24_201139667. For example, Αβ is separated under denaturing conditions, for example by SDS-PAGE electrophoresis. Χ-Υ) globulomers can produce double bands (eg, ΑβΟ-42) with an apparent molecular weight of 38/48 kDa), and after treatment of globulomers with glutaraldehyde prior to separation, the two bands can be combined Into one. Size exclusion chromatography of the globulomer can produce a single peak (for example, a molecular weight of about 100 kDa corresponding to Αβ( 1-42) globulomer or a glutaraldehyde crosslinked Αβ(1-42) globulomer of about 60 The molecular weight of kDa) starts with Αβ(1-42) peptide, Αβ(12-42) peptide and Αβ(20-42) peptide, and the method is particularly suitable for obtaining Αβ(1-42) globulomer, Αβ (12 - 42) globulomer and Αβ(20-42) globulomer. In a particular embodiment of the invention 'where X is selected from the group consisting of the numbers 2...24 and the Αβ(Χ-Υ) globulomer as defined above is 可β(Ι-Υ) sphere The globulomer obtained by truncating the polymer into a shorter form, wherein the quinone is selected from the group consisting of the numbers 2...24, for example X is 20 or 12, and as defined above, this can be achieved by treatment with an appropriate protease . For example, Αβ(20-42) globulomer can be obtained by proteolytic hydrolysis of thermolysin by Αβ(1-42) globulomer, and can be produced by Αβ(1-42) globulomer. The endoproteinase GluC protein was hydrolyzed to obtain Αβ(12-42) globulomer. When the desired degree of proteolysis is reached, the protease is not activated in a generally known manner. The resulting globulomer can then be isolated according to the procedures already described herein and, if desired, further processed by other processing and purification steps. A detailed description of such methods is disclosed in WO2004/067561, which is incorporated herein by reference. For the purposes of the present invention, Αβ(1-42) globulomers are especially as described below -25· 155263.doc

I Αβ(1-42) globulomer described in 201139667 la; Αβ(20-42) globulomer is especially Αβ(20-42) globulomer as described in Example lb below, and Αβ(12 - 42) The globulomer is especially a Αβ(12-42) globulomer as described in Example lc below. According to one aspect of the invention, the globulomer exhibits affinity for neuronal cells and/or exhibits neuromodulation. According to another aspect of the invention, the globulomer consists of from 丨丨 to 16, for example 12 to 14 Αβ(Χ-Υ) peptides. According to another aspect of the present invention, the term "Αβ(Χ-Υ) globulomer" herein is referred to as a globulomer consisting essentially of Αβ(Χ-Υ) subunits, wherein for example, at least 12 subunits are at least average 11 are Αβ(χ-Υ) type, or 10°/. The following globulomers contain any non-Αβ(Χ-Υ) peptide, or a non-Αβ(Χ-Υ) peptide content below the detection threshold. More specifically, the term "Αβ(1-42) globulomer" as used herein refers to a globulomer consisting essentially of Αβ(ι_42) units as defined above; the term "Αβ(12-42)" herein. "Globomer" means a globulomer consisting essentially of Αβ(12-42) units as defined above; and the term "Αβ(20-42) globulomer" as used herein means substantially as defined above A globulomer composed of β (20-42) units. The term "crosslinked Αβ(Χ-Υ) globulomer" as used herein refers to a conjugated Αβ(Χ-Υ) globulomer as described above, for example by chemical crosslinking, chain crosslinking, pentane A molecule obtained by crosslinking a constituent unit of a dialdehyde cross-linking globulomer. In another aspect of the invention, the cross-linked globulomer is substantially a globulomer in which at least some of the units are joined by covalent bonds rather than by only non-covalent interactions. For the purposes of the present invention, crosslinked Αβ(1_42) globulomers are especially crosslinked Αβ(1·42) oligomers as described in Example Id below. The term "Αβ(Χ-Υ) globulomer derivative" as used herein refers in particular to a globulomer that is attached to a group label that facilitates extraction by a total of 155263.doc •26-201139667. Rongguang group, such as luciferin isothiocyanate, phycoerythrin, Victoria jellyfish fluorescent protein (Aequorea victoria, such as café, Dictyosoma fWescent pr〇tein, or any combination or battalion thereof) Photoactive derivative; | chromophore; chemical hair _, such as luciferase, especially firefly (such as Xie (10) called luciferase, Vibrio fischeri (7) (10) called luciferase, or any combination or chemiluminescence thereof a reactive derivative; an enzymatically active group such as a peroxidase such as horseradish peroxidase, or any enzymatically active derivative thereof; an electron-dense group such as a group containing a heavy metal such as a group having a gold group, a hapten For example, a hapten derived from a strong antigen structure, for example, a peptide sequence predicted by K〇laskaj^T〇nga〇nkar; an aptamer of another molecule; a chelating group such as six groups Amino acid group; mediates other specific proteins The natural or derived protein structure of the white matter interaction, such as a member of the f〇s/jun pair; a magnetic ganglion, such as a ferromagnetic group; or a radioactive group, for example, comprising 1H, 14C, 32P, 35S or 1251 Or a combination thereof; or a conjugate that is linked to a group that promotes inactivation, sequestration, degradation, and/or precipitation by covalent or non-covalent, high affinity interaction, for example, to promote a living organism An internally degraded group (such as a ubiquitin) marker, such as a labeled polymer, such as in vivo assembly; or a globulomer modified by any combination of the above. Such markers and markers are known in the art. a group and a method of attaching it to a protein. The labeling and/or "忒" can be performed before, during or after the globization. In another aspect of the invention, the globulomer derivative can be made by a globulomer The molecule obtained by the labeling and/or labeling reaction. Accordingly, the term "Αβ(χ-Υ) monomer derivative" of 155263.doc •27-201139667 refers in particular to a label or label as described for a globulomer. Αβ monomer. Another in the present invention In the sample, the binding protein described herein binds with high affinity to the Αβ(20-42) globulomer, for example, the dissociation constant (Kd) is at most about ΙΟ·6 Μ; at most about 10-7 M; at most about 1 〇· 8 M ; at most about ι〇·9 μ; up to about 10·丨〇Μ; at most about ίο.丨丨Μ; at most about 1〇-丨2Μ; and at most ι〇π Μ. In one aspect, such as a surface As measured by plasmon resonance, the association rate constant (κ〇η) of the binding protein to Αβ(2〇-42) globulomer described herein is selected from the group consisting of: at least about 102 MV 丨; at least about L〇3 m_Vi; at least about 1〇4 MV; at least about 1〇5 MV; and at least about 1〇6. In another aspect, the dissociation rate constant (Kc) ff of the binding protein for the Αβ (20-42) globulomer is selected from the group consisting of: up to about 10-3, as measured by surface plasmon resonance. S·1 ; at most about 10-4 s·; at most about 1〇·5 one; and at most about 6 s′ In the specific aspect of the present invention, the binding protein described herein is 1×10·9 to lxl0-丨The dissociation constant of 〇M is bound to the Ap(2〇_42) globulomer. In another specific aspect of the present invention, the association rate constant (k〇n) of the binding protein described herein to Αβ(20-42) globulomer is 1 χ 1 〇5 to i χ i 〇6 m ^ In another particular aspect of the invention, the dissociation rate constant of the binding protein described herein for Αβ(20-42) globulomer is 8χΐ〇·5 to 8&gt;&lt;i〇4s, in In another aspect of the invention, the binding affinity of the binding protein described herein to the 峨2〇_42) globulomer is greater than the binding affinity for the Ap(i_42) globulomer. The term "large affinity" is used herein. Refers to the unbound Αβ-binding protein and the unbound Αβ globulomer on the one hand and the Αβ-binding protein globulomer complex on the other hand. I55263.doc •28- 201139667 The balance between the substances is more biased towards the binding protein _ globulomer complex The degree of interaction. Similarly, the term "smaller affinity" as used herein refers to the unbalanced binding of Αβ-binding protein and unbound Ap globulomer on the one hand to the unbalanced Αβ-binding in the human protein-globulomer complex on the other hand. ; 'White and unbound... The degree of interaction of globulomers. The term "larger affinity" is synonymous with "higher affinity" and the term "smaller affinity" is synonymous with: "lower affinity". , η In the relevant aspect of the invention, the binding affinity of the binding protein 本文 42 42 described herein is at least 2 times the binding affinity of the binding protein to the 球 ι 42 42 conjugate (eg, at least 3 or at least 5 times), at least 〇 2 times, such as at least 20 times, up to at least (or at least 10 times), at least (10) times (eg, at least doubling, at least _ times or at least times), and at least i, deleting (eg, At least 2, _ times, at least 3, _ times or at least 5 〇〇〇 times), at least 1 〇, 〇〇〇 times (eg at least from doubled, at least 3 (), _ times or at least 5G, _ times), Or to, 100,000 times. In a further aspect of the invention, the binding protein described herein binds to a Αβ(12_42) globulomer with relatively high affinity, for example, a dissociation constant (Kd) of at most about ι·6Μ; at most about 10-7M. ; up to about 1〇.8m; up to about 1〇9; to: about 10-1° up to about 10-&quot;M; up to about 1〇·12 M; and at most

In the in-situ state, as measured by surface plasmon resonance, the association enthalpy constant D of the binding protein to Αβ(12_42) globulomer described herein is selected from the group consisting of: at least about 102 M-Vl At least about ι〇3 Μ·^·, at least about 104 MV; at least about 1〇5 MV; and at least about ι〇6 Μ·,, in another-like state, as measured by surface plasmon resonance, The dissociation rate constant (koff) of the binding protein pair A 155263.doc •29·201139667 (12-42) globulomer is selected from the group consisting of: up to about 1〇-3 s·1; up to about 1〇·4 S -1 ; up to about 10-5 si; and up to about 10'6 s'1 结合 in the relevant aspect of the invention 'binding affinities of the binding protein described herein to Α β ( 2 〇 42 42) globulomer The binding affinity of the binding protein to the ruthenium (3(12-42) globulomer is about 1-1 to 3-fold. According to one aspect, the Αβ-binding protein of the invention binds to at least one Αβ globulomer as defined above, And having a relatively small affinity for at least one non-spherical form of Αβ. The affinity for Αρ of at least one non-globular polymer form is relatively less than for at least one Αβ globulomer Affinity of the Αβ-binding protein of the present invention includes Αρ-binding protein having a binding affinity for Αβ(2〇_42) globulomer greater than that of Αβ(1-42) monomer. Alternative or other aspects according to the present invention The binding affinity of the Αβ-binding protein to the Αβ(2()_42) globulomer is greater than the binding affinity for the Αβ(1-40) monomer. In particular, the octa-binding protein is a Αβ(20-42) globulomer. The affinity is greater than its affinity for both Αρ(ΐ4〇)& Αβ(1-42) monomers. As used herein, the term "Αρ(χ_γ) monomer" refers to the isolated form of the Αβ(χ_γ) peptide, In particular, it is not in the form of a 非β (Χ-Υ) which is substantially non-covalently interacting with other forms. In particular, Αβ(χ_γ) monomers are generally provided in the form of an aqueous solution. In a particular embodiment of the invention In an embodiment, the aqueous monomer solution contains 0.05% to 0.2%, for example, about 1% ΝΗ4 〇Η. In another specific embodiment of the invention, the monomer, hexahydrate water/liquid mixture contains 0.05% to 0.2%. %, for example, about 0 · 1 % N a Ο Η. When using Nikko γ 丨 田 田 ( (for example, for the determination of the binding partner of the Αβ-binding protein of the present invention Force), can be, from, Α, 'This and dilute the solution in an appropriate manner. This 155263.doc 201139667 outside 'usually should be used within 2 hours after preparation, in detail within 1 hour and especially within 3 〇 minutes More specifically, the term "Aβ (1-40) monomer" as used herein refers to a Αβ(1-40) monomer formulation as described herein, and the term "80(1_42) monomer" herein. By Αβ(1-42) formulation as described herein. The Αβ-binding protein of the present invention is preferably bound to one or two monomers with low affinity such as KD of 1χ 1〇-8 Μ or less affinity, for example, kd is 3&gt;&lt;1〇-8 μ or less affinity, KD is lxlΟ·7 Μ or less affinity, for example 〇 is 3x10 7 Μ or less affinity, or KD is ΙχΙΟ·6 μ or less affinity, eg KD is 3xl〇-5 Μ or less affinity, or KD For lxio·5 Μ or less affinity. According to one aspect of the present invention, the binding affinity of the Αβ-binding protein of the present invention to Αρ(2〇_42) globulomer is at least 2 times, for example at least 3, the binding affinity of the Αβ-binding protein to one or two monomers. 1 or more times, at least 5 times, for example at least 20 times, at least 3 times or at least 5 times, at least 1 times, such as at least 200 times, at least 300 times or at least 5 times, preparing 1 inch 〇〇 times, for example at least 2, 〇 00 times, at least 3,000 times or at least 5 〇〇〇 times at least 1 〇, 〇〇〇 times, such as at least 20, _ times, at least 30, _ times or at least 5 〇, _ times 'Or at least 1 week, double. The Αρ-binding protein of the present invention having an affinity for 非β in a non-spherical form is relatively smaller than the affinity for at least one Αβ globulomer, and additionally includes a binding affinity for Αβ(2〇_42) globulomer greater than Αβ (ΐ-42) Aβ binding protein with binding affinity of fibrils. The binding affinity of the Αβ-binding protein according to the present invention to the Αβ(2().42) globulomer is greater than the binding affinity for the 155263.doc -31-201139667 Αβ(1-40) fibril. According to a particular embodiment, the present invention relates to a Αρ-binding protein having a binding affinity for Αβ(20-42) globulomers greater than that for Αρ(14〇) and Αβ〇-42) fibrils. The term "fibril" as used herein refers to the molecular structure of an assembly comprising non-covalently associated individual ΑρΧ(Υ-Υ) peptides which exhibit a fibril structure in an electron microscope that incorporates Congo red (Cong〇red) The birefringence is then exhibited under polarized light, and its X-ray diffraction pattern is a cross-β structure. In another aspect of the present invention, the fibril is a molecular structure obtainable by a method comprising self-inducing polymerization aggregation suitable for the Αβ peptide in the absence of a detergent, for example, in 〇. i M HC1, Aggregates of 24 units or more or 1 unit or more are formed. It is well known in the art that the method ^ρ(χ_γ) fibrils are preferably used in the form of an aqueous solution. In a particular embodiment of the invention, the Αρ peptide is dissolved in 0.1% νη4οη, which is 20 mM NaH2P〇4, 14〇

NaCM' pH 7.4 1:4 dilution, then adjust the pH to 7.4, incubate the solution for 20 hours at m, then centrifuge at 1 〇〇〇〇g for 1 〇g and resuspend in 20 mM NaH2P〇4, An aqueous fibril solution was prepared by using 140 mM NaC in pH 7.4. The term "Αρ(χ_γ) fibrils" as used herein also refers to the fibrils of the subunits of 3β(Χ Υ), wherein, for example, at least 9〇% of the subunits are of the Αβ(Χ-Υ) type, at least 98% of the subunits. The content of the peptide is less than the detection threshold. More specifically, the term Αβ(1·42) fibril herein refers to Αβ as described in Example 3. 142) Fibril preparation. The Αβ-binding protein of the present invention is preferably bound to one or two fibrils with low affinity, for example, KD is lx10-8 river or less affinity, for example, ^^ is 3&gt;&lt;1〇·8 μ I55263.doc • 32·201139667 or less affinity, heart or less affinity, eg Kd is 3xl〇77 or less affinity, or κ々1χ1()_6 M or lower affinity D, eg KD is 3xl0-5 M Or less affinity, or % is 1&gt;&lt;1〇5 μ or less affinity. According to one aspect of the present invention, the binding affinity of the quinone (3 binding protein to Μ(10) 42) globulomer of the present invention is at least 2 times, for example at least 3 times or at least 5, the binding affinity of the Αρ binding protein for one or both. In multiples, at least 1 times, such as at least 20 times, at least 3 times, or at least 5 times, at least 1 〇, or at least 200 times, at least 3 times, or at least 5 times, at least 2 times. , _ times, at least 3, _ times or at least 5, _ times at least 1 〇, 〇〇〇 times, such as at least 20,000 times, at least 3 inches, 〇〇〇 times or at least 5 times ' or at least 100,000 times . According to a particular embodiment, the invention relates to Aβ binding proteins which have a relatively low affinity for both in the monomeric and fibrillar forms of Aβ relative to at least one Aβ globulomer, in particular a Valley (20-42) globulomer. These Αβ-binding proteins are sometimes referred to as globulomer-specific Αβ-binding proteins. The binding protein of the present invention, such as the humanized antibody 4D10 (4Dl〇hUm), includes a major recognition of the Αβ(20-42) globulomer form rather than the Αβ(ΐ-40) monomer, Αβ(1-42) monomer, A globulomer-specific binding protein of a standard preparation of Αβ-fibrils or sAPP (i.e., a soluble Αβ precursor), which is in contrast to, for example, competing antibodies such as m266 and 3D6. This specificity for globulomers is important 'because the humanized 4D10 specific dry globulomer form of Αβ will: 1) avoid targeting insoluble starch-like deposits, which may cause during immunization with insoluble Αβ Observed inflammatory side effects; 2) Savings reported 155263.doc -33- 201139667 Αβ monomers and APP with predictive physiological functions (Plan et al, J Neurosci 23: 553 1-5535, 2003); and 3) Increase the bioavailability of antibodies as they will not be obscured or inaccessible due to large scale binding to insoluble deposits. PF-4 is a small cytokine belonging to the CXC chemokine family with 70 amino acids and is also known as chemokine (C-X-C motif) ligand 4 (CXCL4). PF-4 is released from activated platelets during platelet aggregation and promotes blood coagulation by mitigating the action of heparin-like molecules. Due to these functions, it is predicted to be involved in wound repair and inflammation (Eismann et al. Blood 76(2): 3 36-44, 1990). PF-4 is commonly found in complexes with proteoglycans and can form complexes with the anticoagulant heparin. Anticoagulant heparin is used in the pharmacology of thrombosis. In treatment, it has a well-described pathological function in heparin-induced thrombocytopenia (HIT), an autoimmune response to the trait of the anticoagulant heparin (Warkentin, N. Engl. J.

Med. 356(9): 891-3, 2007), wherein heparin: PF4 complex is an antigen. PF4 autoantibodies have also been found in patients with thrombosis and who have characteristics similar to HIT but have not previously administered heparin (Warkentin et al, Am j)

Med. 121(7): 632-6, 2008). Heparin-induced thrombocytopenia is characterized by thrombocytopenia (low platelet count) and additional ΗΙτ tend to thrombosis. Given these functions of pF_4 and their association in pathological processes, it can be concluded that administration of binding proteins (eg, antibodies) that display binding (eg, cross-reactivity) to pF_4 present in an individual may affect these PF-4 functions and This leads to a bad (4) effect. The extent and type of such adverse effects may vary, such as the location and size of the epitope on PF-4, and the combination of individual binding proteins, 155263.doc -34-201139667 Strength and species parameters. According to the aspect of the present invention, the binding protein of the present invention shows no binding or low binding to platelet factor 4 (PF_4). This cross-reactivity with PF-4 can be assessed by using a standardized in vitro immunoassay (such as ELISA, dot blotting or BIAe〇re analysis). According to a particular embodiment, the interaction of the binding protein as defined herein with 卯_4 refers to the ratio of the binding protein to the value of the reference anti-PF_4 antibody, which is obtained by the following steps: (1) using human or stone crab macaque plasma at about 1 Approximately 1:3 serial dilutions of :3 16 to approximately 1:3160 (final plasma dilution) were subjected to a sandwich-ELISA (as described in Examples 3.1 and 3.2), (Π) plotted against log-transformed plasma dilution (X-axis) The detected signal &amp; axis), and (iii) the area under the curve (the final plasma dilution of approximately 1:316 to approximately 1:3 160) within the measured range (AUC) , or total peak area). According to a particular embodiment of the invention, the reaction with PF-4 by a sandwich-ELISA assay comprises the following: using a specific amount of the studied binding protein or reference anti-pF_4 antibody or a suitable dilution thereof 'eg 100 μΐ 1 〇pg/ml binding protein or antibody in 100 mM sodium bicarbonate solution (pH 9_6) is coated in each well of the protein adsorption microtiter plate; then the wash plate is 'blocked and washed again; then with the stone crab macaque Or human plasma (eg human blood aggregates with human PF-4) in contact with about 1:3 serial dilutions of about 1:3.16 to about 1:3 160 (final plasma dilution) followed by, for example, one PF-4 Specific antibodies, enzyme-binding secondary antibodies, and colorimetric reactions detect PF_4 bound to each well. As used herein, "reference anti-PF-4 antibody" is an antibody that specifically reacts with ρρ-4 (detailed human (HPF4)), in particular, a monoclonal antibody. The antibody 155263.doc •35·201139667 can be exposed to the antigen by providing an antigen comprising human PF-4, such as human PF-4 having the amino acid sequence EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQL IATLKNGRKICLDLQAPLYKKIIKKLLES (SEQ ID NO: 70), And obtaining an antibody that specifically binds to human PF-4 from the antigenic lineage. The antibody can be purified by affinity using immunogen (human PF-4) as appropriate. The reference anti-PF4 antibody is commercially available, for example, a monoclonal anti-HPF4 antibody, Abeam catalog number: ab49735. According to another specific embodiment, the cross-reaction with PF·4 of a binding protein as defined herein refers to the ratio of the AUC value of the binding protein to the reference anti-PF-4 antibody, which is obtained by the following steps: (1) using human or stone crab macaque The plasma and binding protein is subjected to a sandwich-ELISA (eg, as described in Examples 3.3 and 3.4) with a reference anti-PF-4 antibody at about 1:3 serial dilutions of from about 10 ng/ml to about 10000 ng/ml (final concentration), (as described in Examples 3.3 and 3.4), Ii) plotting the detected signal (y-axis) against the log-transformed concentration of the binding protein or reference anti-PF-4 antibody (X-axis), and (iii) determining such uncurved data within the measured range (about The area under the curve (AUC, or total peak area) of the concentration of the binding protein or reference antibody from 10 ng/ml to about 10000 ng/ml. According to a particular embodiment of the invention, the cross-reaction with PF-4 by a comparative sandwich-ELISA assay comprises the following: a specific amount of antibody-coated protein suitable for capturing the binding protein of interest and the reference anti-PF-4 antibody Adsorb the wells of the microtiter plate, for example, 100 μM sodium bicarbonate (pH 9.6) solution of 1 〇〇μΐ 50 Mg/ml Fc-specific anti-mouse IgG (Sigma catalog number: M3534) per well; Broken and washed again; followed by about 1:3 serial dilutions of the binding protein or reference anti-PF-4 antibody, about 10 155263.doc -36 - 201139667 ng/ml to about 10000 ng/m 最终 (final concentration) Contact; after another washing step, the plate is contacted with, for example, a 1:1 〇 diluted human or stone crab macaque plasma (eg, a human blood group to which human PF 4 is added), followed by, for example, a single pF_4 specific antibody, enzyme binding The secondary antibody and colorimetric reaction detects PF-4 bound to the plate. According to one aspect of the present invention, the cross-linking reaction of the Αβ-binding protein of the present invention with ρρ_4 is the reaction of the reference anti-PF-4 antibody when analyzed by the sandwich-ELISA using the stone crab macaque blood as described herein. For example, at least 1/2, at least 1/5, at least 1/1 〇, at least 1/2 〇 or at least 1/3 〇; and/or when analyzed by a sandwich-ELISA using human plasma as described herein, For example, at least 丨/2, at least 1/5, at least 1/10, at least 1/15 or at least 1/2 〇 of the corresponding cross-reaction of the reference anti-PF-4 antibody. According to another aspect of the invention, the Αβ-binding protein of the present invention and the father of PF_4 are again reacted to the corresponding cross-reaction of the reference anti-pF_4 antibody when analyzed by sandwich-ELISA using the stone crab cynomolgus plasma as described herein. For example at least 1/2, at least 1/5, at least 1/1 〇, at least 1/2 〇, at least K30, to 1/50, at least ι/go or at least and/or when humans are used as described herein The plasma ratio is, for example, at least 1/2, at least 1/5, at least 1/1 〇, at least 1/15, at least 1/2 〇, at least 1/2 of the reference anti-PF_4 antibody. 1/25. According to another aspect of the present invention, the cross-reaction of the Αβ-binding protein of the present invention with PF_4 is a reference anti-pF_4 antibody when analyzed by a sandwich-ELISA and a comparative sandwich-ELISA as described herein using a stone crab cynomolgus mortar. The corresponding cross-reactions are, for example, at least 1/2, at least 1/5, at least 1/1 〇, at least 1/2 〇.155263.doc -37-201139667 or at least 1/30. According to another aspect of the present invention, the cross-reaction of the Αβ-binding protein of the present invention with PF-4 is a reference anti-reference when analyzed by a sandwich-ELIS(R) and Alignment-ELISA using human blood. The corresponding cross-reaction of the PF-4 antibody is, for example, at least 1/2, at least 1/5, at least 1/1 〇, at least 1/2 〇 or at least 1/30. According to another aspect of the invention, the cross-reactivity of the Αβ-binding protein of the present invention with PF-4 is when analyzed by sandwich-ELISA and comparative sandwich-ELISA using the stone crab monkey and human plasma as described herein. For example, the corresponding cross-reaction of the anti-PF_4 antibody is, for example, at least 1/2, at least 1/5', at least 1/1 〇, at least 1/20 or at least 1/30. As used herein, "polypeptide" refers to any polymeric bond of an amino acid. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to the polymeric bond of an amino acid. The term "polypeptide" encompasses polypeptide analogs of natural or artificial proteins, protein fragments and protein sequences. The polypeptide can be a monomeric bite polymer. The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide 'according to its origin or source' which is not related to the natural related components associated with its natural state; substantially free of the same species Other proteins; expressed by cells from different species; or not found in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived will be "isolated" from its natural association. Proteins can also be substantially free of natural association components by the use of protein purification techniques well known in the art. Knives 155263.doc •38- 201139667 As used herein, “recycling” refers to the process of separating a chemical substance (such as a polypeptide) substantially free of natural association components, for example, using protein purification techniques well known in the art. . The term "specifically binds" as used herein with respect to the interaction of an antibody, protein or peptide with a second chemical means that the interaction depends on the particular structure (eg, antigenic determinant or epitope) on the chemical. Exist; for example, an antibody recognizes and binds to a specific protein structure rather than a general protein. If the antibody is specific for the epitope, the presence of an epitope containing an epitope (or free, unlabeled A) in the reaction containing the labeled "purine" and the antibody will reduce binding to the antibody. The amount of eight marks. The term "antibody" as used herein generally refers to any immunoglobulin (Ig) molecule comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) or an essential antigen thereof that retains the Ig knife. Any functional fragment, mutant, variant or derivative that determines the binding characteristics of the base. Such functional fragment, mutant, variant or derivative antibody forms are known in the art. Non-limiting examples thereof are discussed below. As used herein, "full length antibody" refers to four molecules comprising four polypeptide chains (two heavy chains and two light chains). The chains are typically linked to each other via a disulfide bond. Each heavy chain comprises a heavy chain variable region (also referred to herein as a "variable heavy chain" or herein abbreviated as HCVR or VH) and a heavy chain constant region. The key zone contains three domains, CHI, CH2 and CH3. Each light chain comprises a light bond variable region (also referred to herein as "variable light chain", or abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises a domain CL » which can further subdivide the VH and VL regions into hypervariable regions (referred to as complementarity determining regions 155263.doc -39- 201139667 (CDR)), with a more conserved region interspersed For the framework area (FR)). Each of VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), classes (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclasses. The term "antigen-binding portion" (or simply "antibody portion") of an antibody as used herein refers to one or more antibodies that retain the ability to specifically bind to an antigen (eg, Aβ(20-42) globulomer). A fragment, that is, a functional fragment of an antibody. It has been shown that the antigen binding function of an antibody can be performed by one or more fragments of a full length antibody. Such antibody embodiments may also be bispecific, dual specific or multispecific; they specifically bind to two or more different antigens. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment. a divalent fragment comprising two Fab fragments joined by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of a VH and a CH1 domain; (iv) an Fv fragment, which is a single antibody fragment VL and VH domain composition of the arm; (v) dAb fragment (Ward et al, Nature 341: 544-546, 1989; Winter et al, WO 90/05144 A1, incorporated herein by reference), a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by independent genes, they can be joined by a synthetic linker using a recombinant method, enabling preparation of a VL region paired with a VH region to form a monovalent molecule. Single protein chain (referred to as single-chain Fv (scFv); see Example 155263.doc -40·201139667 eg Bird et al, Science 242: 423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988). Such single chain antibodies are also encompassed by the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as bifunctional antibodies, are also contemplated. A minibifunctional antibody is a bivalent, bispecific antibody in which the VH domain and the VL domain are expressed on a single polypeptide chain 'but the linker is too short to be paired between the two domains on the same chain, thereby forcing the The equal domains are paired with complementary domains of another strand and form two antigen binding sites (see, for example, Η 11 iger et al. Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993; Poljak et al. , Structure 2: 1121-1123, 1994). Such antibody binding moieties are known in the art (Kontermann and Dubel ed., Antibody Engineering, Springer-Verlag. New York. page 79, 2001, ISBN 3-540-41354-5). The term "antibody" as used herein also encompasses antibody constructs. The term "antibody construct" as used herein refers to a polypeptide comprising one or more antigen binding portions of the invention linked to a linked polypeptide or immunoglobulin constant domain. A linker polypeptide comprises two or more peptide-linked amino acid residues and is used to link one or more antigen-binding portions. Such linked polypeptides are well known in the art (see, e.g., Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993; Poljak et al, Structure 2: 1121-1123, 1994). An immunoglobulin constant domain refers to a heavy or light chain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art and are presented in Table 1. 155263.doc 41 201139667 Table 1: Sequences of human IgG heavy chain constant domains and light chain constant domains

Protein sequence identifier sequence 123456789012345678901234567890 Ig γ-l constant region SEQ ID NO: 25 ASTKGPSVFFLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK Ig γ-1 constant region mutant of SEQ ID NO: 26 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK g κ constant region SEQ ID NO:27 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC Ig λ constant region SEQ ID NO:28 QPKAAPSVT LFPPSSEELQANKATLVCLISD FYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGS TVEKTVAPTECS Further, a binding protein (e.g., an antibody) of the present invention may be part of a larger immunoadhesive molecule formed by covalent or non-covalent association of a binding protein of the present invention with one or more other proteins or peptides. Such immunoadhesive molecules include the preparation of tetrameric scFv molecules using the streptavidin core region (Kipriyanov et al, Human Antibodies and Hybridomas 6: 93-101, 1995) and the use of cysteine residues, labeled peptides and C-terminals Polyhistidine tag preparation bis-42. 155263.doc 201139667 valence and biotinylated scFv molecules (Kipriyanov et al, Mol. Immunol. 31: 1047-1058, 1994). Antibody fractions such as Fab and F(ab')2 fragments can be prepared from whole antibodies using conventional techniques such as whole antibody papain or pepsin digestion. In addition, antibodies, antibody portions, and immunoadhesive molecules can be obtained using standard recombinant DNA techniques as described herein. As used herein, &quot;isolated antibody&quot; is intended to mean an antibody that is substantially free of other antibodies having different antigenic specificities. However, isolated antibodies that specifically bind to Αβ(2〇_42) globulomers may be cross-reactive with other antigens, such as Αβ globulomers, such as Αβ(12-42) globulomers or other Αβ forms. Further, the isolated antibody may be substantially free of other cellular material and/or chemicals and/or any other dry Αβ form. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may, for example, include amino acid residues not encoded by human germline immunoglobulin sequences in CDRs and, in particular, CDR3 (eg, induced by in vitro random or site-specific mutagenesis or by in vivo somatic cells) Mutation introduced by mutation). However, the term "human antibody" as used herein is not intended to include antibodies that have been ligated into the human framework sequences from CDR sequences derived from the germline of another mammalian species, such as mice. The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, formed or isolated by recombinant means, such as antibodies expressed by recombinant expression vectors transfected into a host cell (further in paragraph hereinafter). Description), antibodies isolated from recombinant human antibody libraries (Hoogenboom, TIB Tech. 15: 62-70, 1997; Azzazy and 155263.doc · 43· 201139667)

Highsmith, Clin. Biochem. 35: 425-445, 2002; Gavilondo JV and Larrick JW (2002) BioTechniques 29: 128-145; Hoogenboom H. and Chames P. (2000) Immunology Today 21: 371-378), Rotation An antibody isolated from an animal (eg, a mouse) that has a human immunoglobulin gene (see, eg, Taylor, LD et al., (1992) Nucl.

Acids Res. 20:6287-6295; Kellermann SA. and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370) or by An antibody that is prepared, expressed, formed, or isolated by any other means of splicing the human immunoglobulin gene sequence to other DNA sequences. The recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibodies are subjected to in vitro mutation induction (or when induced in vivo by somatic mutations in human Ig sequence transgenic animals) and thus V Η and v L of the recombinant antibody The region amino acid sequence is a sequence which is derived from the VH and VL sequences of the human germline and is related to the VH and VL sequences of the human germline, but may not naturally exist in the germline lineage of the human antibody in vivo. An antibody comprising a sequence of heavy and light chain variable regions from one species and a constant region sequence from another species, such as an antibody having a murine heavy chain and a light chain variable region linked to a human constant region. The term "CDR-grafted antibody" refers to an antibody comprising a sequence of heavy and light bond variable regions from one species but wherein the sequence of one or more of the CDR regions of VH and/or VL is replaced by a CDR sequence of another species, such as An antibody having a murine CDR (e.g., CDR3) in which one or more of the murine variable heavy chain and the light 155263.doc-44 - 201139667 chain region have been replaced by human variable heavy and light chain sequences. The terms "Kabat number", "r Kabat definition" and "Kabat mark" are used interchangeably herein. The terms recognized in the art refer to amino acid residues which are variable (i.e., hypervariable) to other amino acid residues in the heavy and light chain variable regions of the antibody or antigen binding portion thereof. Base numbering system (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, Ε·Α. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, US Health And Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from the amino acid position of the CDR1 to 31 to 35', the amino acid position of the CDR2 is from 5 to 65, and the amino acid position of the CDR3 is from 95 to 102. For the light chain variable region, the hypervariable region ranges from 24 to 34 for the cDR12 amino acid, 50 to 56 for the amino acid of the CDR2, and 89 to 97 for the amino acid of the CDR3. The terms "receptor" and "acceptor antibody" as used herein mean at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% amine of one or more framework regions are provided or encoded. An antibody or nucleic acid sequence of a base acid sequence. In some embodiments, the term "r receptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes a constant region. In another embodiment, the term "receptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes one or more of a framework region and a value region. In a particular embodiment, the term "receptor" means providing or encoding at least 8%, such as at least 85%, at least 90%, at least 95%, at least 98, of one or more framework regions. /. Or a human antibody amino acid or nucleic acid sequence of a 1% amino acid sequence. According to this embodiment, the receptor may comprise 155263.doc -45 - 201139667 having at least 1, at least 2, at least 3, at least 4, at least 5 or at least 10 not present in one or more of the human antibodies Amino acid residues at specific positions. The receptor framework region and/or the receptor constant region can be derived, for example, from a germline antibody gene, a mature antibody gene, a functional antibody (eg, an antibody well known in the art, an antibody under development, or a commercially available antibody). . The term "CDR" as used herein refers to a complementarity determining region within a variable sequence of an antibody. There are three CDRs in each of the variable regions of the heavy and light chains, and the three CDRs of each variable region are designated CDR1, CDR2 and CDR3. The term "CDR set" as used herein refers to a group of three CDRs that are capable of binding an antigen in a single variable region. The exact boundaries of these CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides any variable for antibodies The region's definitive residue numbering system, and also provides the exact residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) found that certain sub-portions within the Kabat CDRs use nearly identical peptide backbone configurations even with great diversity at the amino acid sequence level. These sub-portions are designated as LI, L2 and L3 or H1, H2 and H3, where "L" and "Η" represent the light chain region and the heavy chain region, respectively. These regions may be referred to as Chothia CDRs, which have the Kabat CDR Overlapping boundaries. Other boundaries defining CDRs that overlap with Kabat CDRs have been made by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 155263.doc -46-201139667 262(5):7; 32-45 (1996)) description. Other CDR boundary definitions may not strictly follow the above One of the systems will still overlap with the Kabat CDRs, although they may be shortened or prolonged depending on whether a particular residue or group of residues or even the entire CDR does not significantly affect the prediction of antigen binding or the results of an experimental study. The method used may utilize a CDR defined according to any of these systems using a CDR defined by Kabat or Chothia. The term "standard" as used herein refers to a definition as defined by Chothia et al. Mol·Biol. 196:901-907 (1987); Chothia et al., J.

Mol. Biol. According to Chothia et al., a critical portion of the Cdr of many antibodies has almost the same peptide backbone configuration even with great diversity at the amino acid sequence level. Each standard structure primarily specifies a set of peptide backbone torsion angles that cause adjacent segments of the amino acid residue to form a loop. The terms "donor" and "donor antibody" as used herein mean an antibody that provides one or more CDRs. In one embodiment, the donor antibody is an antibody from a species different from the antibody from which the framework region was obtained or produced. In the case of a humanized antibody, the term "donor antibody" refers to a non-human antibody that provides one or more CDRs. The term "framework" or "framework sequence" as used herein refers to the variable sequence minus the remaining sequence of the CDR. Since the exact definition of CDR sequences _J is determined by a system that is not 1*^3, the meaning of the framework sequences follows a correspondingly different interpretation. The six CDRs (CDR-L1, CDR-L2 and CDR-L3 of the light chain, and the CDR-H1, CDR-H2 and CDR-H3 of the heavy chain) also have light chains and heavy errors. The framework region I55263.doc •47· 201139667 is divided into four sub-regions (FR1, FR2, FR3 and FR4) in each chain, wherein CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located in FR3 and FR4. In the case where a specific sub-region is not designated as FR1, FR2, FR3 or FR4, the framework region, when referred to by other names, represents the combined FR in the variable region of a naturally occurring single immunoglobulin chain. One of the FRs used represents one of the four sub-regions, and the FR represents two or more sub-regions of the four sub-regions that make up the framework region. Human heavy-chain and light-linking receptor sequences are known in the art. In one embodiment of the invention, the human heavy and light chain acceptor sequences are selected from the sequences set forth in Tables 2 and 3. In another embodiment, human heavy chain and light link acceptor sequences are selected At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the sequences described in Tables 2 and 3 , at least 97%, at least 98%, or at least 99% of the sequence. Table 2: Relinking Receptor Sequences

SEQ ID NO protein region sequence 123456789012345678901234567890 34 VH3 53 / JH6 FR1_ EVQLVESGGGLIQPGGSLRLSCAASGFTVS 35 VH3_53 / JH6 FR2 ~ WVRQAPGKGLEWVS 36 VH3 53 / JH6 FR3_ RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR 37 VH3 a 53 / JH6 FR4_ WGQGTTVTVSS 38 VH4_59 / JH6 FRl &quot; QVQLQESGPGLVKPSETLSLTCTVSGGSIS 39 VH4_59 / JH6 FR2_ WIRQPPGKGLEWIG 40 VH4_59/JH6 FR3&quot;~ RVTISVDTSKNQFSLKLSSVTAADTAVYYC AR 41 VH4_59/JH6 FR4~ WGQGTTVTVSS • 48 · 155263.doc 201139667

Table 3: Light Link Receptor Sequence SEQ ID NO Protein Region Sequence 123456789012345678901234567890 42 A1/2-30/JK2 FR1 DVVMTQSPLSLPVTLGQPASISC 43 A1/2-30/JK2 FR2 WFQQRPGQSPRRLIY 44 A1/2-30/JK2 FR3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVY YC 45 A1/2 -30/JK2 FR4 FGQGTKLEIKR The term "growth antibody gene" or "gene fragment" as used herein refers to a non-lymphocyte-encoded immunoglobulin that has not undergone genetic rearrangement and mutation to express the maturation process of a particular immunoglobulin. Protein sequence. (See, for example, Shapiro et al, Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et al, Adv Exp Med Biol. 484:13-30 (2001)). One advantage provided by various embodiments of the present invention is based on the recognition that the germline antibody gene is more likely than the mature antibody gene to retain the essential amino acid sequence structural features of the individual in the species, and thus is less useful when used to treat a species. It may be considered to be from a foreign source. The term "critical" residues as used herein refers to certain residues within the variable region that have a greater effect on the binding specificity and/or affinity of the antibody (especially the humanized antibody). Key residues include, but are not limited to, one or more of the following residues: residues adjacent to the CDR, potential glycosylation sites (which may be N- or 0-glycosylation sites), rare residues, a residue capable of interacting with an antigen, a residue capable of interacting with a CDR, a standard residue, a contact residue between a heavy chain variable region and a light chain variable region, a residue in a Vernier region, and a variable weight The Chothia definition of the strand CDR1 overlaps with the Kabat definition of the first heavy chain framework. 155263.doc • 49. Residues in the region of 201139667. The term "humanized antibody" as used herein is immunospecifically bound to the antigen of interest. And comprising a framework (FR) region substantially having an amino acid sequence of a human antibody and an antibody, variant, derivative, analog thereof or substantially having a complementarity determining region (CDR) of an amino acid sequence of a non-human antibody section. The term "substantially" as used herein, in the context of a CDR, refers to an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% of the amino acid sequence of a non-human antibody CDR. CDRs » Humanized antibodies comprise substantially all of at least one and usually two variable domains (Fab, Fab, F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to non-human immunity The CDR regions of the globulin (i.e., the donor antibody) and all or substantially all of the framework regions are the framework regions of the human immunoglobulin consensus sequence. According to one aspect, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. In some embodiments, a humanized antibody comprises a light chain and at least a variable domain of a heavy chain. The antibody may also include the CH1, hinge region, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In a particular embodiment, the humanized antibody contains only humanized variable domains of light and/or heavy bonds. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, but not limited to, IgG 1, IgG2, IgG3, and IgG4. A humanized antibody can comprise a sequence of more than one type or isotype, and a particular constant domain can be selected to optimize the desired effector function using techniques well known in the art. 155263.doc -50- 201139667 The framework regions and CDR regions of humanized antibodies need not correspond exactly to the parent sequence, for example, the donor antibody CDR or consensus framework can be substituted, inserted and/or deleted by at least one amino acid residue. The mutation is induced such that the CDR or framework residues at that position do not correspond to the donor antibody or co-framework. However, in one embodiment, the mutations will not be large-scale. In general, at least 95% &apos; at least 98% or at least 99% of the humanized antibody residues will correspond to the residues of the parent FR and CDR sequences. The term "common framework" as used herein refers to the framework regions of a common immunoglobulin sequence. The term "co-immunoglobulin sequence" as used herein refers to a sequence formed by the most common amino acids (or nucleotides) in the family of related immunoglobulin sequences (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft,

Weinheim, Germany 1987)). In the immunoglobulin family, each position in the consensus sequence is occupied by the most common amino acid in the family at that position. If two amino acids occur equally frequently, either of the common sequences may be included. "Vernier" region as used herein refers to a regulatable CDR structure as described in F00te and Winter (1992, J. Mol. Biol. 224: 487-499, incorporated herein by reference) and fine-tuning with antigen A subset of the framework residues that match. The Vernier region residues form a layer under the CDRs and can have an effect on the structure of the CDRs and antibody affinity. The term "antibody" as used herein also encompasses multivalent binding proteins. The term "multivalent binding protein" is used in the present invention to mean a binding protein comprising two or more antigen binding sites. Multivalent binding proteins are engineered to have two or more antigen binding sites and are generally not naturally occurring. 155263.doc • 51· 201139667 Antibodies. The term "multispecific binding protein" refers to a binding protein capable of binding two or more related or unrelated stems. A dual variable domain (DVD) binding protein as used herein is a binding protein comprising two or more antigen junctions and is a tetravalent or multivalent binding protein. The DVDs may be monospecific, ie capable of binding one antigen; or multispecific, ie capable of binding two or more antigens, comprising two heavy chain DVd polypeptides and two light chain DVD polypeptides. The DVD binding protein is called DVD Ig. Each half of the dvd ig comprises a heavy chain DVD polypeptide and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain, wherein a total of six CDRs are involved in antigen binding per antigen binding site. A DVD binding protein and a method of making a DVD binding protein are described in U.S. Patent Application Serial No. 1 1/507,050, the disclosure of which is incorporated herein by reference. The term "antigenic determinant" includes any polypeptide determinant capable of specifically binding to an immunoglobulin or T cell receptor. In certain embodiments, an epitope determinant comprises a chemically active surface group of a molecule (such as an amino acid, a sugar side chain, a phosphonium group or a sulfonyl group), and in certain embodiments, it may have Features two-dimensional structural features and/or specific charge characteristics. An epitope is a binding protein, particularly an antigenic region to which an antibody binds. In certain embodiments, an antibody is said to specifically bind to an antigen when the binding protein or antibody preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. The binding affinity of the antibodies of the invention can be determined by using standard in vitro immunoassays (such as ELISA, dot blotting or BIAcore analysis (Pharmacia).

Biosensor AB, Uppsala, Sweden and Piscataway, NJ)). For other descriptions see j0nsson, u et al., (1993) Ann Bi〇1 155263.doc -52- 201139667

Clin. 51:19-26; J5nss〇n, U. et al., (1991) Biotechniques 11:620-627; Johnsson, B. et al., (1995) j. m〇I. Rec〇gnit. 8:125 -131; and Johnsson, B. et al., (1991) Anal Biochem. 198:268-277. According to a particular embodiment, the affinity defined herein refers to the value obtained by performing a point collapse method and by means of the measurable method. Determination of binding affinity by point-crushing method according to a specific embodiment of the invention comprises the following: a specific amount of antigen (for example, Αβ(χ-γ) globulomer, Αβ(χ-γ) single as defined above Body or Αβ (Χ-Υ) fibril) or a convenient dilution thereof (for example, diluted in 2 mM NaH2P04, 140 mM NaC Bu pH 7.4, 0.2 mg/ml BSA to, for example, 100 pmol/μΐ, l〇ρηιο1 /μ1, i ρπ1()1/μι, 〇"pmol/μΐ and antigen concentration of 0·01 ριη〇1/μ1) are spotted on the nitrocellulose membrane, followed by blocking the membrane with milk to prevent non-specific binding Washing is followed by contact with the antibody of interest, which is subsequently detected by means of an enzyme-conjugated secondary antibody and a colorimetric reaction; at a given antibody concentration, the amount of bound antibody allows for affinity determination. Thus, the relative affinities of two different antibodies for one target, or the relative affinities of one antibody for two different targets, are herein defined as the use of two antibodies under the conditions of the same conditions at the same conditions. The relationship between the individual amounts of antibodies bound to the target. Unlike the similar method based on the Western blot method, the dot collapse method will determine the affinity of the antibody for the intended target of the natural configuration; unlike the ELISA method, the dot collapse method does not suffer from the difference in affinity between the different targets and the matrix. This allows for a more precise comparison between different targets. The term "surface plasmon resonance" as used herein refers to the use of the BIAcore system (Pharmacia Biosensor AB, Uppsala, by way of example 155263.doc •53·201139667).

Sweden and Piscataway, NJ) extracted the changes in protein concentration in the biosensor matrix to analyze the optical phenomena of immediate biospecific interactions. For further description see J6nsson, U. et al., (1993) Ann. Biol. Clin., 51: 19-26; J0nsson et al., (1991) BioTechniques, 11: 620-627; Johnsson et al., (1995) J. Mol. Recognit., 8: 125-131; and Johnnson et al., (1991) Anal. Biochem., 198: 268-277 ° As used herein, the term "kon" (also known as "Kon", "kon", "Kon") To be understood by such techniques, a binding protein (e.g., an antibody) associates with an antigen to form an association rate constant for, for example, an association complex of an antibody/antigen complex. "Km" is also known as the term "association rate constant" or "," and is used interchangeably herein. This value indicates the rate of binding of a binding protein (eg, an antibody) to its target antigen or the rate of complex formation between a binding protein (eg, an antibody) and an antigen, as shown by the following equation: Antibody ("Ab") + antigen ("Ag ” — Ab_Ag. The term "k〇ff" (also "K〇ff", "k〇ff", "Koff") as used herein is intended to mean that a binding protein (e.g., an antibody) is self-contained as is known in the art. complex. The dissociation rate constant of the dissociation of the substance (e.g., antibody/antigen complex). This value indicates the dissociation of a binding protein (eg, an antibody) from its target antigen or

The dissociation rate of the Ab-Ag complex separated into free antibody and antigen over time is shown by the following equation:

Ab+Ag-Ab-Ag. As used herein, the term "Yi ί ^ 町. Kd" (also known as "Kd" or "KD") is intended to be 155263.doc •54- 201139667 refers to the "equilibrium dissociation constant" and refers to The value obtained at equilibrium or the value obtained by dividing the dissociation rate constant (k〇ff) by the association rate constant (k〇n). The association rate constant (kj, the dissociation rate constant (k (&gt; ff) and the equilibrium dissociation constant (kd) are used to indicate the binding affinity of the binding protein (eg, antibody) to the antigen. The method for determining the association and dissociation rate constants is As is well known in the art, the use of fluorescence-based techniques provides high sensitivity and the ability to examine samples in physiological buffers in equilibrium. Other experimental methods and instruments can be used, such as BIAcore® (Biomolecular Interaction Analysis) assays. (eg available from BIAcore International AB, GE Healthcare company,

Uppsala, the instrument purchased by Sweden). In addition, it can also be used

The KinExA® (Dynamic Exclusion Verification) test purchased by Sapidyne Instniments (B〇ise, Idaho). The term "labeled binding protein" as used herein refers to a binding protein that has been labeled to recognize a binding protein. Similarly, the term "labeled antibody" as used herein refers to a label that has been labeled to provide antibodies. Identifyed antibodies. In one aspect, the label is a detectable label, such as incorporated into a radiolabeled amino acid or with a labeled avidin (eg, containing a fluorescent label that can be detected optically or colorimetrically) The enzymatically active streptavidin) detects a polypeptide linkage having a biotinyl moiety. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (eg, 3Η, 丨4c, 35s, 90Y, 99Te, 丨1丨1, 1 1 Lu, 166h〇, or 153Sm) Fluorescent labeling (eg, FITC, rhodamine, lanthanide metal phosphors), enzymatic labeling (eg, horseradish peroxidase, luciferase, caloriin); chemical 155263 .doc 55- 201139667 light: "the organism's biotinyl; the predetermined polypeptide antigen, the base identified by the second reporter (eg 'the binding of leucine to the sequence, the binding site of the secondary antibody' to the metal, ·, the domain, the epitope tag); and a magnetizer such as a ruthenium chelate. The term "antibody" as used herein also encompasses antibody conjugates. The term "antibody" refers to a binding protein (such as an antibody) that is chemically linked to a second chemical moiety, such as a therapeutic agent. The term "therapeutic agent" as used herein denotes a compound of a "cognitive enhancing drug", a mixture of compounds, a biological macromolecule or an extract prepared from a biological substance, which is intended to improve the cognitive ability of the human brain (ie, thinking, Learning and memory) drugs. Cognitive enhancement drugs act by altering the availability of neurochemical substances (such as neurotransmitters, enzymes, and hormones), improving oxygen supply, by stimulating nerve growth, or inhibiting nerve damage. Examples of cognitive enhancing drugs include compounds that enhance the activity of acetylcholine, such as, but not limited to, acetylcholine receptor agonists (eg, nicotine alpha -7 receptor agonist or ectopic modulator, alpha 4 beta 2 smoke) Alkaline receptor agonist or ectopic modulator), acetaminophen acetal inhibitor (such as donepezil, rivastigmine and gaiantamine), Cholesterol esterase inhibitor, N-mercapto-D-aspartate (NMDA) receptor antagonist (such as memantine), neuroprotective protein (ADNp) agonist, serum 5-HT1A receptor agonist (eg, xaliproden), 5-HT4 receptor agonist, 5-HT6 receptor antagonist, serotonin 1A receptor antagonist, histamine H3 receptor antagonist , calpain inhibitor, vascular endothelial growth factor (VEGF) protein or agonist 155263.doc -56- 201139667 agent, vegetative growth factor, anti-apoptotic compound, ampa type of face acid receptor activator, L Type or N-type calcium channel blockers or modulators, potassium channel blockers, hypoxia-inducible factor (HIF) Agent, HipAmidino-4-ylhydroxylase inhibitor, anti-inflammatory agent, amyloid Αβ-peptide or amyloid plaque inhibitor, τ over-acidification inhibitor, acid-filled diacetate 5 inhibitor (eg Tata Tadalafil, sildenafil, phosphodiesterase 4 inhibitor, monoamine oxidase inhibitor or a pharmaceutically acceptable salt thereof. Specific examples of such cognitive enhancing drugs include, but are not limited to, cholinesterase inhibitors such as donepezil (Aricept®), remexem (Exelon®), garlander (Reminyl®), N-mercapto -D-aspartate antagonist, such as Namenda®. The terms "crystal" and "crystal" as used herein mean a binding protein (eg, an antibody or antigen-binding portion thereof) present in crystalline form. The crystal is a solid form of the substance, which is different from, for example, an amorphous solid or liquid crystalline state. Other forms. A crystal system consists of a regular two-dimensional array of atoms, ions, molecules (e.g., proteins, such as antibodies), or molecular assemblies (e.g., antigen/antibody complexes). These three dimensional arrays are arranged according to specific mathematical relationships well understood in the art. The basic unit or building block that is repeated in the crystal is called an asymmetric unit. The "a unit cell" of the crystal is provided by repeating the asymmetric unit in an arrangement consistent with the well-defined crystallographic symmetry. Crystals are provided by regular translational repeat unit cells in all three dimensions. See Giege, R. and Ducruix, A Barrett,

Crystallization 〇f Nucleic Acids and Proteins,a Practical

Approach, 2nd edition, pp. 20 1-16, 0xford Univershy press, 155263.doc •57- 201139667

New York, New York, (1999). The term "neutralization" as used herein refers to the neutralization of the biological activity of a targeted Ap form when the binding protein specifically binds to the Aβ form. For example, the binding of the binding protein to the Αβ(2〇_42) amino acid region of the globulomer (and/or any other targeted Αβ form) results in a neutralizing antibody that inhibits the globulomer biological activity. According to the aspect of the invention, the neutralizing binding protein binds to the 颂2〇-42) region of the globulomer (and/or any other targeted Αβ form) and reduces the biological activity of the targeted sputum form by at least about 20 %, 4〇%, 6〇%, 8〇%, 85%, or 85〇/. the above. Inhibition of the biological activity targeted to the 0 form by neutralization of the binding protein can be assessed by measuring one or more of the biological activities of the targeted biopsy well known in the art, such as targeting the Αρ form and p/Q-type voltage-controlled interaction of presynaptic calcium channels (eg, binding), inhibition of p/Q-type voltage-gated presynaptic calcium channel activity, and p/Q-type voltage-gated presynaptic calcium Ca + + reflux of the channel, local (eg intracellular) Ca + + concentration, synaptic activity. The term "activity" includes the activity of a binding protein (especially an antibody) to the binding specificity/affinity of an antigen (eg, an Ap (20-42) globulomer (and any other targeting Aβ form); and/or an antibody (eg, The neutralizing potency of targeting the binding of the Ap form to inhibit antibodies that target the biological activity of the Aβ form. This biological activity in the eight-port form involves the interaction of the Αβ form with the P/Q type voltage-gated presynaptic calcium ion channel, which results in inhibition of calcium ion channel activity. The invention also provides an isolated nucleotide sequence encoding a binding protein of the invention. The invention also provides a nucleotide sequence (or a fragment thereof) having the sequence comprising, corresponding to, identical to, hybridized to or complementary At 155263.doc • 58 · 201139667 with at least about 70% of such coding nucleotide sequences (eg, 7〇%, 71%, 72%, 73%, 74%, 75./〇, 76./〇, 77%) , 78./〇 or 79./〇), at least about 80% (eg 80%, 81%, 82%, 83%, 84〇/〇, 85%, 860/〇, 87%, 88% or 89%) ), or at least about 9〇% (eg, 91%, 92%, 93%, 94%, 95%, 96%, 97°/., 98%, 99%, or 100%). sequence. (About the percentage of consistency between 7〇% and 1〇〇% and including all integers (and their fractions) with 100% are considered to be within the scope of the invention.) The sequences may be derived from any source (eg Separation from natural sources, production via semi-synthetic pathways or resynthesis). In particular, the sequences may be isolated or derived from sources other than those described in the Examples (e.g., bacteria, fungi, algae, mice, or humans). For the purposes of the present invention, a "fragment" of a nucleotide sequence is defined as a contiguous sequence having about at least 6, for example at least about 8, at least about a nucleotide, or at least about 15 nucleotides. It corresponds to the region of the specified nucleotide sequence. The term "consistency" refers to the association of two sequences on a nucleotide-by-nucleotide base in a particular comparison window or segment. Thus, identity is defined as the same, corresponding or equivalent degree between the same strand (sense or antisense) of two DNA segments (or two amino acid sequences). By comparing the specific regions of the two optimal alignment sequences, determining the number of positions in the two sequences in which the same base or amino acid is present to generate the number of matching positions, the number of positions divided by the total position in the compared segment The number is multiplied by 1 to calculate the "sequence consistency percentage". Available by Smith &amp;

Waterman Algorithm, Appl. Math. 2: 482, 1981, Needleman &amp; Wunsch Algorithm ’ j· M〇1 Bi〇1 48: 443, 1970, Pearson &amp;

Lipman method, proc· Natl Acad Sci (USA) 85: 2444, 1988 155263.doc • 59- 201139667 and computer programs that perform related algorithms (eg Clustal Macaw J Pileup (http://cmgm.stanford.edu/biochem2 18 /1 lMultiple.pdf f ; Higgins et al., CABIOS. 5L151-153, 1989), FASTDB (Intelligenetics), BLAST (National Center for Biomedical Information; Altschul et al, Nucleic Acids Research 25: 3389-3402, 1997) ' PILEUP (Genetics Computer Group,

The best alignment of the sequences was performed by Madison, WI) or GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, Madison, WI). (See U.S. Patent No. 5,912,120.) For the purposes of the present invention, "complementarity" is defined as the degree of association between two DNA segments. It is determined by measuring the ability of a sense strand of a DN A segment to hybridize to the antisense strand of another DNA segment under appropriate conditions to form a double helix. "Complement" is defined as a sequence that is paired with a given sequence based on the principle of typical base pairing. For example, the sequence A-G-T in one nucleotide strand is "complementary" to the other strand of T-C-A. In the double helix, adenine appears in one strand and thymine appears in the other. Similarly, if a cockroach is found in one strand, the cell is densely defined in the other strand. The greater the association between the nucleotide sequences of the two DNA segments, the greater the ability to form hybrid duplexes between the strands of the two DNA segments. The "similarity" between two amino acid sequences is defined by the presence of a series of identical and conserved amino acid residues in both sequences. The higher the degree of similarity between the two amino acid sequences, the higher, the same or the higher the equivalent between the two sequences. (The "consistency" between two amino acid sequences is defined as the presence of a series of precisely identical or unchanged amino acid residues in the two sequences of 155263.doc -60-201139667.) "Complementarity", "consistency" And the definition of "similarity" is well known to those of ordinary skill. By "encoding" is meant a nucleic acid sequence encoding a polypeptide sequence wherein the polypeptide sequence or portion thereof contains at least 3 amino acids from a polypeptide encoded by the nucleic acid sequence, such as an amino acid of at least 8 amino acids or at least 15 amino acids sequence. The term "polynucleotide" as referred to herein means a polymeric form of two or more nucleotide acids (ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide). The term includes single or double-stranded form of DNA 'but preferably double stranded DNA. The term "isolated polynucleotide" as used herein shall mean a polynucleotide (eg, genomic, CDNA or synthetic source, or some combination thereof) 'in view of its source' the "isolated polynucleoside" "acid" does not associate with the entire polynucleotide or a portion thereof which is found to be "isolated polynucleotide" in nature; is operably linked to polynucleic acid which is not linked thereto in nature; or substantially does not act A portion of the larger sequence exists. The term "vector" as used herein is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of carrier is &quot;plast&quot;, which refers to a circular double-stranded DNA loop that can be joined to other DNA segments. Another type of vector is a viral vector in which other DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which the vectors are introduced (e.g., a bacterial vector having a bacterial origin of replication and a free mammalian vector). Other vectors (e. g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell&apos; and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the performance of genes to which they are operably linked, 155263.doc-61-201139667. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, the expression vectors suitable for use in recombinant DNA technology are usually in plastid form. In this specification, since the plastid is the most common form of the carrier, the "plast" and the "carrier" are used interchangeably. However, the invention is intended to include other forms of expression vectors such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) that serve equivalent functions. Surgery. "Operably linked" means that the components are in a relationship that allows them to function in their intended manner. The control sequence is &quot;operably linked&quot; to the coding sequence such that the performance of the coding sequence is effected in a manner compatible with the control sequence. The sequence of "operably linked" includes expression control sequences contiguous with the relevant genes and expression control sequences that are trans-acting or acting at a distance to control the relevant genes. The term "expression control sequence" as used herein refers to a polynucleic acid sequence necessary to effect expression and processing of the ligated coding sequence. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that modulate the cell; sequences that improve translation efficiency (ie, 〇2" common Sequences; sequences that enhance protein stability; and sequences that increase protein secretion if necessary. The nature of such control sequences varies depending on the host organism; in prokaryotes, promoters, ribosomal binding sites are included in control sequences such as guanidine Point and transcription termination sequence '· in eukaryotes t' such control sequences generally include a promoter and a transcription: a stop sequence. The term "control sequence" is intended to include a component that is necessary for performance and processing' and Other groups I55263.doc-62-201139667, such as leader sequences and fusion partner sequences, may be included as appropriate. "Transformation" as defined herein refers to any process that allows foreign DNA to enter a host cell. Transformation can be carried out under natural or artificial conditions using a variety of methods well known in the art. Transformation can be carried out by any known method of inserting a foreign nucleic acid sequence into a prokaryotic or eukaryotic host cell. The method is based on the host cell selection being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replicating in autonomously replicating plastids or as part of a host chromosome. It also includes cells that transiently express the inserted DNA or RNA for a limited period of time. The term "recombinant host cell" (or simply "host cell") as used herein is intended to mean a cell into which foreign DNA has been introduced. It should be understood that such terms are intended to refer not only to a particular individual cell, but also to the progeny of that cell. Because certain modifications may occur in the passage by mutation or environmental influences, the progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In one aspect, the host cell comprises prokaryotic cells and eukaryotic cells selected from any of the biological worlds. Eukaryotic cells include protists, fungi, plant and animal cells. In another aspect, the host cell includes, but is not limited to, the prokaryotic cell strain Escherichia coli; the mammalian cell line CHO, HEK 293 and COS; the insect cell strain Sf9; and the fungal cell Saccharomyces cerevisiae. Recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation can be performed using standard techniques (eg, electroporation, lipofection) and enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly done in such techniques. This is described in 155263.doc -63- 201139667. The above-described techniques and procedures are generally performed in accordance with the conventional methods well known in the art and as described in the various comprehensive references and more particularly. See for example

Sambrook 人 ' Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, N.Y. (1989)), which is incorporated herein by reference for all purposes. "Transgenic organism" as used in the art and as used herein refers to an organism having cells containing a transgenic gene, wherein the transgenic gene introduced into the organism (or ancestor of the organism) is expressed in the organism. Naturally not expressed as a peptide. A "transgenic gene" is a DNA that stably and operably integrates into the genome of a cell that develops into a transgenic organism, thereby directing the encoded gene product to be expressed in one or more cell types or tissues of the transgenic organism. Construct body. The mouth of the mouth. "Modulation" and "modulation" are used interchangeably and, as used herein, refers to altering or altering the activity of a related molecule (e.g., the biological activity of a targeted sputum form). The adjustment can be to increase or decrease the magnitude of a certain activity or work of the relevant molecule. Exemplary activities and functions of a molecule include, but are not limited to, binding characteristics, enzymatic activity, cellular receptor activation, and signal transduction. 'The term "modulator" as used herein is used to enable a concern or concern ( For example, a compound that targets a change or change in the biological activity of the sputum form. For example, t., the sputum agent can cause a certain activity of the molecule or the value of the power month b compared to that observed in the absence of the regulator. The amount of activity or function is increased or decreased. In certain embodiments, the modulator is an inhibitor that reduces the amount of at least one activity or function of 155263.doc • 64 - 201139667 molecules. "Augmenter" means a modulator that, when contacted with a molecule of interest, causes an increase in the magnitude of an activity or function of the molecule compared to the amount of activity or function observed in the absence of the agonist. The term "antagonist" or "inhibitor" as used herein refers to a quantity that causes an activity or function of a molecule when contacted with a molecule of interest as compared to that observed in the absence of an antagonist. A modulator of reduced activity or function. Specific antagonists of interest include antagonists that block or modulate the biological activity of the targeted sputum form. Antagonists and inhibitors that target the Ap form may include (but T are limited) binding. The binding protein of the invention in the form of Ap(2〇_42) globulomer and any other dry sputum. The dry Αβ form of the antagonist or inhibitor may, for example, reduce the Αβ form to the P/Q type voltage gating process Inhibition of the activity of the pre-laser calcium channel. The term "effective amount" as used herein means sufficient to reduce or ameliorate the severity and/or persistence of a condition or one or more of the conditions (4); prevent the progression of the condition; cause the condition to subside; An amount of a therapy that prevents the prevention or treatment of a condition associated with a condition, or a plurality of symptoms, develops an onset or progression, detects a condition, or enhances or ameliorates another therapy (eg, a prophylactic or therapeutic agent). "Sample" is used in its broadest sense. "Biological sample" as used herein includes, but is not limited to, any amount of material from a living thmg or previously a living thing. Living things include (but not Z) humans, mice, rats, [canines, rabbits, and other animals. These include: (but not limited to, clear, urine, synovial fluid, cells, organs, tissues, bone marrow - Lymph node and spleen. 155263.doc • 65· 201139667 ι. Antibodies of the invention A first particular aspect of the invention provides CDR grafting in combination with Aβ (20-42) globulomers and/or any other targeted Aβ form An antibody or antigen binding portion thereof. A second particular aspect of the invention provides a humanized antibody or antigen binding portion thereof that binds to Aβ (20-42) globulomer and/or any other targeted Aβ form. According to a particular aspect The antibody or portion thereof is an isolated antibody. According to another specific embodiment, the antibody of the invention neutralizes the activity of an Αβ(20-42) globulomer and/or any other crude Αβ form.制备. Methods of Making Anti-Aβ (20·42) Globulomer Antibodies The antibodies of the invention can be made by any of a variety of techniques known in the art. 1. Anti-Aβ (20-42) globulomer monoclonal antibodies using fusion tumor technology Monobody antibodies can be prepared using a variety of techniques known in the art, including the use of fusion tumors, recombinant and phage display techniques, or a combination thereof. For example, monoclonal antibodies can be produced using fusion tumor technology, including those known in the art and taught, for example, in the following literature: Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981) (the references are hereby incorporated by reference in their entirety). The term "monoclonal antibody" as used herein is not limited to antibodies produced by fusion tumor technology. The term "monoclonal antibody" refers to an antibody derived from a single pure line, including any eukaryotic, prokaryotic or purobacterial pure line, rather than the method by which it is produced. Methods for making and screening for specific antibodies using fusion tumor technology are routine and well known in the art 155263.doc • 66-201139667. In one embodiment, the invention provides a method of producing a monoclonal antibody, and an antibody produced by the method, the method comprising culturing a fusion tumor cell secreting an antibody of the invention, wherein the fusion tumor is, for example, by the antigen of the invention The spleen cells isolated from the immunized mice are fused with myeloma cells and then screened for fusion tumors produced by fusion of the fusion tumors capable of binding to the antibody of the present invention. Briefly, mice were immunized with the Ap(2〇_42) globulomer antigen. In a specific embodiment, the antigen is administered with an adjuvant to stimulate an immune response. "These adjuvants include Freund's adjuvant, RIBI (cell wall brewing dipeptide) or ISC〇M (" Immunostimulating complex). Such adjuvants may protect the polypeptide from rapid dispersion by sequestering the polypeptide in a local deposit, or it may contain a substance that stimulates the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is administered, the immunization schedule will involve administration of two or more polypeptides dispersed over several weeks. After immunizing an animal with an Αβ(20-42) globulomer antigen, cells producing antibodies and/or antibodies can be obtained automatically. Serum containing anti-Aβ (20-42) globulomer antibody was obtained by exsanguination or sacrifice of animal animals. The serum can be used as it is in the animal, the immunoglobulin fraction can be obtained from the serum or the anti-Aβ (20-42) globulomer antibody can be purified from the serum. The gold or immunoglobulin obtained in this way is a plurality of strains of serum or immunoglobulin and thus has a different set of characteristics. Once an immune response is detected, e.g., an antibody specific for the antigen Αβ(20-42) globulomer is detected in the serum of the mouse, the spleen is collected and the spleen cells are isolated. The spleen cells are then fused to any suitable myeloma cells, such as cells available from the cell line SP20 of 155263.doc-67-201139667 ATCC, by well-known techniques. The fusion tumor is selected and colonized by limiting dilution. The fusion line is then assayed for cells secreting antibodies capable of binding to Αβ(20-42) globulomer by methods known in the art. Ascites fluid, which generally contains high levels of antibody, can be produced by immunizing mice with a positive fusion tumor. In another embodiment, an antibody can be prepared from an immunized animal to produce an immortalized fusion tumor. Following immunization, the animals are sacrificed and spleen B cells are fused to immortalized myeloma cells as is well known in the art (see, for example, Harlow and Lane, supra). In a specific embodiment, the myeloma cells do not secrete an immunoglobulin polypeptide (a non-secreting cell line). After fusion and antibiotic selection, fusion tumors were screened using Αβ (20-42) globulomers or fractions thereof or cells expressing Αβ(20-42) globulomers. In a specific embodiment, the initial screening is performed using an enzyme-linked immunoassay (ELISA) or a radioimmunoassay (RIA). Examples of ELISA screens are provided in WO 00/37504, which is incorporated herein by reference. Fusion tumors producing anti-Aβ (20.42) globulomer antibodies are selected, selected and screened for the desired characteristics including vigorous fusion tumor growth, high antibody production, and desired antibody characteristics as further discussed below. The fusion tumor can be cultured and expanded in vivo in the same genomic animal, animal lacking the immune system (e.g., nude mice) or cultured and expanded in vitro in cell culture. Methods for selecting, selecting, and amplifying fusion tumors are well known to those of ordinary skill. In a particular embodiment, the fusion tumor is a mouse fusion as described above. In another particular embodiment the &apos;fusion tumor is produced in a non-human, material species such as a rat, sheep, pig, goat, cow or horse. In another embodiment, the fusion tumor is a human fusion tumor, human non-secretory bone rhyme in sputum 155263.doc •68-201139667 Fusion with human cells expressing anti-Αβ(20-42) globulomer antibodies. Antibody fragments that recognize specific epitopes can be produced by known techniques. For example, Fab and F(ab')2 fragments of the invention can be immunoglobulin molecules by using an enzyme such as papain (to produce a Fab fragment) or pepsin (to produce a F(ab')2 fragment). The protein is produced by cleavage. The F(ab')2 fragment contains a variable region, a constant region of the light chain, and a CH1 domain of the heavy chain. 2. Anti-Aβ (20-42) globulomer monoclonal antibody using SLAM. In another aspect of the invention, the use of a single isolated lymphocyte is described in the art as described in the following literature. The procedure of the Cellular Antibody Method (SLAM) produces recombinant antibodies: U.S. Patent No. 5,627,052, PCT Publication WO 92/02551, and Babcock, JS et al., (1996) Proc. iVai/. 5W. C/M 93:7843-7848. In this method, a single cell secreting an antibody of interest (eg, a lymphocyte derived from any of the immunized animals described in Section 1) is screened using an antigen-specific hemolytic plaque assay. , wherein the antigen Αβ(20-42) globulomer or a subunit thereof is conjugated to bromine red blood cells using a linker such as biotin and is used to recognize an antibody secreting a specificity for Αβ(20-42) globulomer Single cell. After identifying the cells secreting the antibody of interest, the heavy and light chain variable region cDNAs are obtained from the cells by reverse transcriptase PCR, and then the specific variable region can be expressed in a suitable immunoglobulin region (eg, human, good) In the case of a region, it can be expressed in a mammalian host cell such as COS or CHO cells. The cells of the antibody of Table Αβ(20-42) globulomer can then be isolated, for example, by panning the transfected cells, such that the 155263.doc-69-201139667 lymphocytes derived from the in vivo selection are used. Host cells transfected with amplified immunoglobulin sequences are subjected to further analysis and selection in vitro. The amplified immunoglobulin sequences can be further manipulated in vitro, such as by the methods of in vitro affinity maturation, such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772. 3. Use of an anti-Aβ (20-42) globulomer monoclonal antibody of a transgenic animal In another embodiment of the invention, the inclusion of some or all humans by Αβ(20-42) globulomer antigen Non-human animals of the immunoglobulin locus are immunized to produce antibodies. In a specific embodiment, the non-human animal is a XENOMOUS transgenic mouse that is an engineered mouse strain comprising a large fragment of a human immunoglobulin locus and a mouse antibody producing a deficiency. See, for example, Green et al, Nature Genetics 7: 13-21 (1994) and U.S. Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO 91/10741, published July 25, 1991, WO 94/02602, published on Feb. 3, 1994, WO 96/34096, published on October 31, 1996, and WO 96/33735, April 1998 WO 98/16654 published on the 23rd, WO 98/24893 published on June 11, 1998, WO 98/50433 published on November 12, 1998, WO 99/45031 published on September 10, 1999, 1999 WO 99/53049, published on October 21, WO 00 09560, published on Feb. 24, 2000, and WO 00/037504, issued on June 29, 2000. XENOMOUSE transgenic mice produce adult human-like lineages of fully human antibodies and produce antigen-specific human monoclonal antibodies. XENOMOUSE transgenic mice contain approximately 80% of the human antibody lineage via the introduction of the human heavy chain locus and the X light chain locus of the 155263.doc-70·201139667 megabase-scale germline configuration YAC fragment. . See Mendez et al, Geweiics 15: 146-156 (1997), Green and Jakobovits J. Exp. Med 188: 483-495 (1998), the disclosure of which is incorporated herein by reference. Library of anti-Αβ(20-42) globulomer monoclonal antibody

Antibodies of the invention can also be prepared using in vitro methods in which antibody libraries are screened to identify antibodies having the desired binding specificity. The method of screening recombinant antibody libraries is well known in the art and includes, for example, Ladner et al. 5, 223, 4, 9; Kang et al., PCT Publication No. W092/18619; Dower et al., PCT Publication No. W091/17271; Winter et al., PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. W092/15679; Breitling et al., PCT Publication No. WO 93/01288; McCafferty et al., PCT Publication No. W092/01047; Garrard et al., PCT Publication No. W092/09690; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246··1275·1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352: 624-628; Gram et al., (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio/Technology 9: 1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al., (1991) PNAS 155263.doc -71 - 201139667 88:7978- 7982; the method described in U.S. Patent Application Publication No. 200301,86,374, the disclosure of which is incorporated herein by reference. The recombinant antibody library may be from an individual immunized with a portion of a Αβ(20-42) globulomer or a Αβ(20·42) globulomer. Alternatively, the recombinant antibody library can be from an untreated individual (i.e., an individual immunized without a Αβ(20-42) globulomer), such as a human antibody library from a human subject immunized without Αβ(20-42) globulomer. . The antibody of the present invention is selected by screening a recombinant antibody library with a peptide comprising a human Αβ(20-42) globulomer, thereby selectively identifying Αβ(20-42) globulomer and distinguishing Αβ(1-42) globulomer, Αβ(1-40) and Αβ(1-42) monomers, Αβ-fibrils and their antibodies to sAPPa. Methods for performing such screening and selection are well known in the art, such as those described in the references in the previous paragraph. In order to select specific binding affinity for Αβ(20-42) globulomer and distinguish Αβ(1-42) globulomer, Αβ(1-40) and Αβ(1·42) monomers, Αβ-fibrils and sAPPa Antibodies of the invention, such as those dissociated from human Αβ (20-42) globulomers at a specific koff rate constant, can be selected using the dot-crush method known in the art to have an antibody with the desired koff rate constant. In order to select specific neutralization activity for Αβ(20-42) globulomers and distinguish between Αβ(1-42) globulomers, Αβ (1-40) and Αβ(1-42) monomers, Αβ-fibrils and Antibodies of the invention of sAPPa, such as those having a particular IC50, can be evaluated for inhibition of Αβ(20-42) globulomer activity using standard methods known in the art. In one aspect, the invention relates to the binding of human Αβ(20-42) globulomers and distinguishes Αβ(1-42) globulomers, Αβ(1-40) and Αβ(1-42) monomers, Αβ - an isolated antibody or antigen binding portion thereof of fibrils and sAPPa. According to the state 155263.doc -72- 201139667, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody. For example, antibodies of the invention can also be produced using a variety of phage display methods known in the art. In phage display, a functional antibody domain is presented on the surface of a phage particle carrying a polynucleotide sequence encoding the same. In particular, the phage can be utilized to present an antigen binding domain that is expressed by a lineage or combinatorial antibody library (e.g., human or murine). The antigen can be used, for example, using a labeled antigen or an antigen bound or captured on a solid surface or bead to select or recognize a phage that exhibits an antigen binding domain that binds to the associated antigen. The phage used in these methods are usually filamentous phage including fd and Ml 3 binding domains expressed by Fb, Fv or disulfide stabilized Fv antibody domain phage recombinantly fused with phage gene III or gene VIII protein. . Examples of phage display methods that can be used to prepare the antibodies of the invention include those disclosed in the following literature: Brinkman et al, J. Immunol. Methods 182: 41-50 (1995); Ames et al, J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al, Eur. J. Immunol. 24:952-958 (1994); Persic et al, Gene 187 9-18 (1997); Burton et al, Advances in Immunology 57:191- 280 (1994); PCT Application No. PCT/GB91/01134; PCT Publication W090/02809; WO91/10737; W092/01047; W092/18619; W093/11236; W095/15982; W095/20401; Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 155263.doc-73-201139667 5, 516, 637; 5, 780, 225; 5, 658, 727; 5, 733, 743 and 5, 969, 108; each of which is incorporated herein by reference in its entirety. As described in the references above, the antibody coding region can be isolated from the phage after selection and used to produce a full antibody comprising a human antibody or any other desired antigen-binding fragment, and is described, for example, in detail below, In any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinant production of Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art, such as PCT Publication W092/22324; Mullinax et al.

BioTechniques 12(6): 864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al, Science 240: 1041-1043 (1988) methods (these references) The literature is incorporated by reference in its entirety. Examples of techniques that can be used to generate single-chain Fvs and antibodies include U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al, PNAS 90:7995-7999 (1993); The technique described in Skerra et al., Science 240: 1038-1040 (1988). Other methods for screening large combinatorial libraries known in the art can be used in place of screening recombinant antibody libraries by phage display to identify dual specific antibodies of the invention. One type of alternative expression system is the expression system for expressing recombinant antibody libraries as RNA-protein fusions as described in the following documents: PCT Publication No. WO 98/31700 by Szostak and Roberts, and Roberts, RW. and Szostak, JW (1997) corpse roc·iVai/. 5W· 155263.doc • 74· 201139667 ί/α 94:12297-12302. In this system, a covalent fusion is formed between the mRNA and its peptide or protein encoded by in vitro translation of the synthetic mRNA having a puromycin (a peptide-based receptor antibiotic) at the 3' end. Thus, 'specific mRNA can be enriched from a complex mixture of mRNAs (eg, combinatorial libraries) based on the characteristics of the encoded peptide or protein (eg, an antibody or portion thereof), such as binding of an antibody or portion thereof to a dual specific antigen . The nucleic acid sequence encoding the antibody or portion thereof recovered from the libraries can be expressed by recombinant means as described above (e.g., in a mammalian host cell) and, in addition, can be initially selected by further rounds Screening of the mutated mRNA-peptide fusions in the sequence or other methods of maturation of the recombinant antibody in vitro as described above allows it to undergo further affinity maturation. In another method, the antibodies of the invention can also be produced using yeast expression methods known in the art. In yeast presentation, the antibody domain is tied to the yeast cell wall using genetic methods and presented on the yeast surface. In particular, the yeast can be utilized to present antigen binding domains that are expressed by a lineage or combinatorial antibody library (eg, human or murine), examples of which can be used to prepare antibodies of the invention, including, by way of citation, The method disclosed in U.S. Patent No. 6,699,658 to Wittrup et al. B. Production of Recombinant Aβ (20-42) Globomer Antibodies The antibodies of the invention can be made by any of a variety of techniques known in the art. For example, from host cell expression 'where will be by standard techniques Expression vectors encoding heavy and light chains are transfected into host cells. The various forms of the term "transfection" are intended to encompass a variety of techniques commonly used to introduce exogenous #DNa into the original 155263.doc-75-201139667 nuclear or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, DEAE-poly Glucose transfection and similar techniques. The antibodies of the invention may be expressed in prokaryotic or eukaryotic host cells. According to a particular aspect of the invention, antibodies are expressed using eukaryotic cells (e.g., mammalian host cells) because such eukaryotic cells (and, in particular, mammalian cells) are more likely than prokaryotic cells to assemble and secrete antibodies that are appropriately folded and immunologically active. . According to one aspect, a mammalian host cell expressing a recombinant antibody of the present invention includes a Chinese hamster ovary (CHO cell) (included in Urlaub and Chasin, (1980) Wi/. in m. t/M 77: 4216-4220 The dhfr-CHO cells are described for use with DHFR detectable markers, such as those described in RJ Kaufman and PA Sharp (1982) Mo/. 5z_〇/_ 159:601-621), NSO myeloma cells, c 〇S cells and SP2 cells. When the recombinant expression vector encoding antibody gene is introduced into a mammalian host cell, the antibody is produced by cultivating the host cell for a period of time sufficient for the antibody to be expressed in the host cell or secreted by the antibody into the culture medium of the growing host cell. The antibody can be recovered from the culture medium using standard protein purification methods. Host cell can also be used to make functional antibody fragments, such as Fab fragments or scFv molecules. It should be understood that variations of the above procedures are within the scope of the invention. For example, it may be desirable to transfect a host cell with DNA encoding a functional fragment of the light chain and/or heavy chain of an antibody of the invention. Recombinant cleavage techniques can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not required for binding to the antigen of interest. Antibodies of the invention also encompass molecules which are represented by such truncated DN A molecules. In addition, the antibody of the present invention can be cross-linked with the second antibody by a standard chemical cross-linking method to produce a heavy chain and a light chain of 155263.doc-76-201139667 the antibody of the present invention and another heavy chain and light chain pair It is concerned with antigen-specific bifunctional antibodies other than antigen. In a specific recombinant expression system of the antibody or antigen-binding portion thereof of the present invention, a recombinant expression vector encoding both an antibody heavy chain and an antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. In recombinant expression vectors, the antibody heavy and light chain genes are each operably linked to a CMV enhancer/AdMLP promoter regulatory element to drive high level transcription of the gene. The recombinant expression vector also carries the DHFR gene, which allows selection of CHO cells that have been transfected with the vector using amidoxime selection/amplification. The selected transfer body host cells are cultured to allow expression of the antibody heavy and light chains and recovery of intact antibodies from the culture medium. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select for metastases, culture host cells, and recover antibodies from the culture medium. The present invention further provides a method of synthesizing the recombinant antibody of the present invention by culturing the host cell of the present invention in a suitable medium until the recombinant antibody of the present invention is synthesized. The method can further comprise isolating the recombinant antibody from the culture medium. 1. Anti-Aβ (20-42) globulomer murine antibody Table 4 shows the amino acid sequence of the VH and VL regions of murine 4D10. Table 4: Amino acid sequence listings in the VH and VL regions Table 4: Amino acid sequence listings in the VH and VL regions

Region of the protein sequence SEQ ID NO 123456789012345678901234567890 23 m4D10_VH QVQLKQSGPSLIQPSQSLSITCTVSGFSLT SYGVHWVRQSPGKGLEWLGVIWRGGRIDYN AAFMSRLSITKDNSKSQVFFKMNSLQADDT AIYYCARNS DVWGTGTTVTVS S 24 m4D10_VL DVVMTQTPLTLSVTIGQPASISCKSSQSLL DIDGKTYLNWLLQRPGQSPKRLIYLVSKLD SGVPDRFTGSGSGTDFTLKISRVEAEDLGV YYCWQGTHFPYTFGGGTKLEIKR * murine light chain and the heavy chain CDR is underlined. 155263.doc •77· 201139667 2. Anti-Aβ (20-42) globulomer chimeric antibody Chimeric antibodies are molecules derived from different animal species in different parts of the antibody, such as having a variable from a murine monoclonal antibody Region and human immunoglobulin constant 疋 region of antibodies. Methods of making chimeric antibodies are known in the art and are discussed in detail herein. See, for example, Morrison, Science 229: 1202 (1985); 〇i et al,

BioTechniques 4:214 (1986); Gillies et al., (1989) J.

Immunol. Methods 125: 191-202; U.S. Patent Nos. 5,307, 7j5; 4,816,567 and 4,816,397. Further, a technique developed by producing a "chimeric antibody" by splicing a gene derived from a mouse antibody molecule having an appropriate antigen specificity with a gene derived from a human antibody molecule having appropriate biological activity can be used (Morrison et al. Human, 1984, Proc. Natl. Acad. Sci·8 1:85 1-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454 The way is incorporated in this article). In one embodiment, the chimeric antibody of the invention is produced by replacing the heavy chain constant region of the murine monoclonal anti-Aβ (20-42) globulomer antibody 4D10 described in WO2007/062852 with a human IgG1 constant region. . 3. Anti-Aβ (20-42) globulomer CDR-grafted antibody The CDR-grafted antibody of the present invention comprises a heavy chain and a light chain variable region sequence derived from a human antibody, wherein one or more CDR regions of VH and/or VL are The CDR sequence replacement of the murine antibody of the invention. The framework sequence from any human antibody 155263.doc •78· 201139667 can be used as a template for CDR transplantation. However, linear substitution on this framework typically results in a loss of binding affinity to the antigen to some extent. The higher the homology of the human antibody to the original murine antibody, the less likely it is to introduce a murine CDR and a human framework into the CDR that may reduce the affinity for deformation. Therefore, 'selecting human variable frameworks that replace the murine variable framework other than CDRs

• The antibody-like variable region framework has, for example, at least 65% sequence identity. Human and murine variable regions other than CDRs have, for example, at least 70%, at least 75% sequence identity, or at least 80% sequence identity. Methods of making chimeric antibodies are known in the art and are discussed in detail herein. (See also EP 239,400; PCT Publication WO 91/09967; US Patent No. 5,225,539; 5,5 3 0,101 and 5,5 85,089); Finishing or Surface Reshaping (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka et al, Protein Engineering 7(6): 805-814 (1994); Roguska et al, PNAS 91: 969-973 (1994)); Chain reorganization (US Patent No. 5,565,352)). Table 5 below illustrates the sequences of the CDR-grafted antibodies (4D10hum antibodies) of the present invention and the CDRs contained therein. 155263.doc -79- 201139667 Table 5: Amino acid sequence listings of VH and VL regions of CDR-grafted antibodies

Region protein sequence SEQ ID NO 123456789012345678901234567890 4 4D10hum_VH.lz EVQLVESGGGLIQPGGSLRLSCAASGFTVS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYN AAFMSRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARNS DVWGQGTTVTVS S 8 17 18 19 4D10hum _VH.2z QVQLQESGPGLVKPSETLSLTCTVSGGSIS SYGVHWIRQPPGKGLEWIGVIWRGGRIDYN AAFMSRVTISVDTSKNQFSLKLSSVTAADT AVYYCARNS DVWGQGTTVTVS S VH 4D10hum CDR-H1 SEQ ID NO: 4,8 of residues 31-35 SYGVH VH 4D10hum CDR-H2 SEQ ID NO: 4, 8 Residue 50-65 VIWRGGRIDYNAAFMS VH 4D10hum CDR-H3 SEQ ID NO: 4, 8 Residue 98-101 NSDV 12 20 21 22 4D10hum_VK.lz DWMTQSPLSLPVTLGQPASISCKSSQSLL DIDGKTYLNWFQQRPGQSPRRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTH FPYT FGQGTKLEIKR VL 4D10hum CDR-L1 SEQ ID NO: 12 Residue 24-39 KSSQSLLDIDGKTYLN VL 4D10hum CDR-L2 SEQ ID NO: 12 Residue 55-61 LVSKLDS VL 4D10hum CDR-L3 SEQ ID NO: 12 Residue 94- 102 WQGTHFPYT * The CDRs in the humanized light and heavy chains are underlined. 4. Anti-Aβ (20-42) globulomer humanized antibody The humanized antibody is an anti-80 155263.doc 201139667 somatic molecule from an antibody that binds to a non-human species of the desired antigen, which has one or more species from a non-human species. Complementarity determining regions (CDRs) and framework regions derived from human immunoglobulin molecules. Human Ig sequences are known to be disclosed, for example, at www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com / ; www. antibodyresource.com/onlinecomp.html ; www.public, iastate. e du/. about, pedro/re search_tools.html ; www.mgen. uni-heidelberg.de/SD/IT/IT.html ; www .whfreeman.com/immunology/CH-05/kuby05.htm ; www.library.thinkquest. org/12429/Immune/Antibody.html ; www.hhmi.org/grants/ lectures/1996/vlab/ ; w ww. path Cam. ac.uk/. about. mrc7/m-ikeimages.html ; www.antibodyresource.com/ ; mcb. harvard.edu/BioLinks/Immuno-logy.html.www.immuno logylink.com/ ; p athb ox Wustl.edu/. about, h center/index.- html ; www.biotech.ufl.edu/_about.hcl/ ; www.pebio. com/pa/3 40913/340913 .html- ; www.nal.usda .gov/ awic/pubs/antibody/ ; www.rn.ehime-u.acjp/. about. yasuhito-/Elisa.html ; www.biodesign.com/table.asp ; www.icnet.uk/axp/facs/ Davies/lin-ks.html ; www. ' biotech.ufl.edu/.ab out. fccl/pr otocol.html ; www.isac- net.org/sites_geo.html ; aximtl. Imt.uni-marburg.de/. ab out. rek/AEP-Start.html ; baserv.uci.kun.nl/. about, jraats/linksl.html ; www.recab.uni-hd.de/immuno. bme .nwu.edu/ ; www.mrc-cpe.cam.ac.uk/imt-doc/pu- 155263.doc •81- 201139667 blic/INTRO.html ; www.ibt.unam.mx/vir/V_mice.html ; imgt.cnusc.fr:8104/ ; www. bio chem.ucl.ac.uk/. about, m art in/abs/i ndex.html ; antibody.bath.ac.uk/ ; abgen.cvm.tamu. Edu/lab/wwwabgen.html ; www.unizh. ch/. about. honegger/AHOsem-inar/SlideO 1 .html ; www. cryst.bbk.ac.uk/.about.ubcg07s/ ; www.nimr.mrc. Ac.uk/ CC/ccaewg/ccaewg.htm ; www.path.cam.ac.uk/. about.mrc7/h, umanisation/TAHHP_html ; www.ibt. un am. mx/vir/structure/s tat_aim.html ; www.biosci. missouri.edu/smithgp/index.html ; www.cryst.bioc.cam. ac. uk/.abo-ut.fmol in a/Web-page s/Pept/sp ottech.html ; www. Jerini.de/frroducts.htm; www.patents.ibm.com/ ibm.html.Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health (1983), each incorporated by reference in its entirety in. Such input sequences can be used to reduce immunogenicity or to reduce, enhance or alter binding, affinity, association rate, dissociation rate, affinity, specificity, half-life, or any other suitable feature known in the art. The framework residues in the human framework regions can be replaced with corresponding residues from the CDR donor antibody to alter, preferably improve antigen binding. Such framework substitutions are recognized by methods well known in the art, for example by establishing a CDR-framework interaction model to identify framework residues important for antigen binding and sequence comparisons to identify abnormal framework residues at specific positions. . (See, for example, Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., 155263.doc • 82 · 201139667

Nature 332: 323 (1988), which is incorporated herein by reference in its entirety. Three-dimensional immunoglobulin models are commonly used and are familiar to those skilled in the art. A computer program that illustrates and displays the possible three-dimensional configuration of the selected candidate immunoglobulin sequence can be utilized. Examinations shown herein permit the analysis of the possible role of residues in the functioning of candidate immunoglobulin sequences, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected from the common and input sequences and combined to achieve desired antibody characteristics, such as increased affinity for the target antigen. In general, CDR residues are directly and to the greatest extent involved in affecting antigen binding. Antibodies can be humanized using a variety of techniques known in the art, such as, but not limited to, Jones et al, Nature 321 : 522 (1986); Verhoeyen et al, Science 239: 1534 (1988); Sims et al, J Immunol. 15 1: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al. Human, J. Immunol. 151:2623 (1993); Padlan, Molecular Immunology 28(4/5): 489-498 (1991); Studnicka et al., Protein Engineering 7(6): 805-814 (1994); Roguska Et al., PNAS 91: 969-973 (1994); PCD Open WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939 'US94/01234, GB89/01334, GB91 /01134, GB92/01755, WO90/14443, WO90/14424, W090/14430 'EP 229246, EP 592,106, EP 5 19,596 'EP 239,400; US Patent Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, Nos. 5, 817, 483, 155263.doc • 83 - 201139667 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352 The techniques described in Nos. 6, 2, 4, 023, 6, 180, 370, 5, 693, 762, 5, 530, 101, 5, 585, 089, 5, 225, 539, 4, 816, 567, each incorporated by reference in its entirety This article (including references cited therein). Table 6 below illustrates the sequences of the humanized antibodies (4D1 Ohum antibodies) of the present invention and the CDRs contained therein. Table 6: Amino acid sequence listings of VH and VL regions of humanized antibodies

Region of the protein sequence SEQ ID NO 123456789012345678901234567890 5 4D10hum_VH.l EVQLVESGGGLVQPGGSLRLSCAASGFTVS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYN AAFMSRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARNS DVWGQGTTVTVS S 6 4D10hum_VH.la EVQLVESGGGLVQPGGSLRLSCAVSGFTLS SYGVHWVRQAPGKGLEWLGVIWRGGRIDYN AAFMSRLTISKDNSKSTVYLQMNSLRAEDT AVYYCARNS DVWGQGTTVTVS S 7 4D10hum_VH.lb EVQLVESGGGLIQPGGSLRLSCAASGFTLS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYN AAFMSRFTISKDNSKNTLYLQMNSLRAEDT AVYYCARNS DVWGQGTTVTVS S 9 4D10hum_VH.2 EVQLQESGPGLVKPSETLSLTCTVSGGSIS SYGVHWIRQPPGKGLEWIGVIWRGGRIDYN AAFMSRVTISVDTSKNQFSLKLSSVTAADT AVYYCARNS DVWGQGTTVTVS S -84. 155263.doc 201139667

Region protein SEQ ID NO sequence 123456789012345678901234567890 10 4D10hum_VH.2a EVQLQESGPGLVKPSETLSLTCTVSGFSLS SYGVHWVRQPPGKGLEWLGVIWRGGRIDYN AAFMSRLTISKDTSKSQVSLKLSSVTAADT AVYYCARNSDWGQGTTVTVSS 11 17 18 19 4D10hum_VH.2b EVQLQESGPGLVKPSETLSLTCTVSGFSLS SYGVHWIRQPPGKGLEWIGVIWRGGRIDYN AAFMSRVTISKDTSKNQFSLKLSSVTAADT AVYYCARNSDVWGQGTTVTVSS VH 4D10hum CDR-H1 SEQ ID NO: 5, 6,7,9,10,11 of 31 residues -35 SYGVH VH 4D10hum CDR-H2 SEQ ID NO: 5 'Residues of 5, 7, 9, 10, 11 50-65 VIWRGGRIDYNAAFMS VH 4D10hum CDR-H3 SEQ ID NO: 5, 6, 7, 9, 10, 11 the residues 98-101 NSDV 13 4D10hum_VK.l DVVMTQTPLSLPVTPGQPASISCKSSQSLL DIDGKTYLNWFLQKPGQSPQRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHFPYTFGQGTKLEIKR 14 4D10hum_VK.la DVVMTQTPLSLPVTPGQPASISCKSSQSLL DIDGKTYLNWLLQKPGQSPQRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHFPYTFGQGTKLEIKR 15 4D10hum_VK.lb DVVMTQTPLSLPVTLGQPASISCKSSQSLL DIDGKTYLNWLLQRPGQSPRRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHFPYTFGQGTKLEIKR 85- 15 5263.doc 201139667

Region of the protein sequence SEQ ID NO 123456789012345678901234567890 DWMTQTPLSLPVTLGQPASISCKSSQSLL 16 4D10hum_VK.lc DIDGKTYLNWFLQKPGQSPRRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHFPYTFGQGTKLEIKR 20 VL 4D10hum CDR-L1 SEQ ID NO: 13, 15, 16 residues of 24-39 KSSQSLLDIDGKTYLN 21 VL 4D10hum CDR-L2 SEQ ID N0: 13 '14, 15, 16 residues 55-61 LVSKLDS 22 VL 4D10hum CDR-L3 SEQ ID NO: residues 94-102 of 13, 14, 15, 16 WQGTHFPYT * CDRs in humanized light and heavy chains are underlined . C. Antibodies and antibody-producing cell lines According to one aspect, the anti-Αβ(20_42) globulomer antibody of the invention or against any other antibody targeting the Αβ form exhibits reduced or neutralized Αβ(20-42) globulomers (and/or any other ability to target the Αβ form) activity. In certain embodiments, the antibody comprises a heavy bond assay region&apos; such as a 1gG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. According to one aspect, the heavy chain constant region is an IgGl heavy chain constant region or an IgG4 heavy chain constant region. According to another aspect, the antibody comprises a light chain constant region: a kappa light chain constant region or a lambda light chain constant region. According to one aspect, the antibody comprises a kappa light chain constant region. The antibody portion can be, for example, a Fab fragment or a single bond Fv fragment. • 86 - 155263.doc 201139667 The replacement of the amino acid residues in the Fc portion to alter the antibody effect is known in the art (Winter et al., U.S. Patent Nos. 5,648,26 and 5,624,821). The Fc portion of the antibody mediates several important effector functions, such as cytokine induction, ADCC, swallowing, complement dependent cytotoxicity (CDC), and half-life/clearance of antibodies and antigen-antibody complexes. Depending on the therapeutic goal, in some cases, these effector functions are required for therapeutic antibodies' but may not be necessary or even harmful in other situations. Certain human IgG isotypes (especially IgG 1 and IgG3) are mediated by FCyR and complement 匸1 q to mediate ADCC and CDC ° neonatal Fc receptors (FcRn) as key components in determining antibody circulating half-life. In another embodiment, at least one amino acid residue in the constant region of the antibody (e.g., the Fc region of the antibody) is replaced such that the effector function of the antibody is altered. One embodiment provides a labeled antibody that derivatizes or binds an antibody of the invention to another functional molecule (e.g., another peptide or protein). For example, a labeled antibody of the invention can be derivatized by functionally linking an antibody of the invention to - or a plurality of other molecular entities (eg, by chemical coupling, gene fusion, non-covalent association, or other means) , or one or more other molecular entities such as another antibody (eg, a bispecific antibody or a minibifunctional antibody), a detectable agent, a pharmaceutical agent, and/or a mediated antibody to another molecule (such as a streptavidin a protein or peptide associated with a prime core region or a polyhistidine tag. Suitable detectable agents that can be used to derivatize the antibodies of the invention include fluorescent compounds. Exemplary fluorescent detectables include fluorescein, isothiocyanate glover: dextran, 5-dimethylamine-1-naphthalene sulfonium, phycoerythrin and the like. Also 155263.doc •87- 201139667 Derivatization of antibodies with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and similar enzymes. When the antibody is derivatized with a detectable enzyme, it is detected by adding an enzyme to generate other reagents that detect the reaction product. For example, when the detectable agent horseradish peroxidase is present, The addition of hydrogen peroxide and diaminobenzidine produces a detectable colored reaction product. Biotin-derived antibodies can also be detected and detected by indirect measurement of avidin or streptavidin binding. Another embodiment of the invention provides a crystalline antibody. According to one aspect, the invention relates to crystals of whole anti-Aβ (2〇-42) globulomer antibodies and fragments thereof as disclosed herein, as well as formulations and compositions comprising such crystals. According to another aspect, the crystalline antibody has a greater in vivo half-life than the antibody soluble counterpart. According to another aspect, the antibody retains biological activity after crystallization. The crystalline antibodies of the present invention can be produced according to the methods disclosed in WO 02/072636, which is known in the art and incorporated herein by reference. Another embodiment of the invention provides a glycosylated antibody, wherein the antibody comprises one or more carbohydrate residues. Initial in vivo protein production can undergo further processing known as post-translational modification. In particular, sugar (glycosyl) residues can be enzymatically added, a process known as glycosylation. The resulting protein having a covalently linked oligosaccharide side chain is referred to as a glycosylated protein or glycoprotein. An antibody is a glycoprotein having one or more carbohydrate residues in the Fc domain as well as in the variable domain. Carbohydrate residues in the Fc domain have an important effect on the effector function of the fc domain, but have minimal effect on the antigen binding or half-life of the antibody (R. Jefferis, corp. rog. 21 (2005), pp. 11_155263.doc-88 - 201139667 1 Page 6) Relatively speaking, the variable domain glycosylation can have an effect on the antigen binding activity of the antibody. Glycosylation in the variable domain may have an adverse effect on antibody binding affinity due to steric hindrance (c〇, M.S. et al., Mol.

Immunol. (1993) 30: 1361-1367) or resulting in increased affinity for antigens (Wallick, SC et al. 'Exp. Med. (1988) 168: 1099-1109; Wright, A. et al.' EMBO J. ( 1991) 10:2717 2723). One aspect of the invention pertains to a glycosylation site mutant in which an O-linked or N-linked glycosylation site of an antibody has been mutated. Those skilled in the art can generate such mutants using standard well-known techniques. The reaction of a glycosylation site that retains biological activity but has increased or decreased binding activity is another object of the invention. In another embodiment, the glycosylation of an antibody of the invention is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modification can be accomplished, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made which result in the elimination of one or more variable region glycosylation sites to eliminate glycosylation at the site. This deglycosylation increases the affinity of the antibody for the antigen. The method is described in further detail in the International Application Publication No. WO 03/016466 A2 and U.S. Patent Nos. 5,714,350 and 6,350,861, the disclosures of each of Alternatively or additionally, an antibody having an increased amount of a reduced fucosylated antibody or a blistering GlcNAc structure of a reduced antibody of the present invention, such as a trehalyl residue, having an altered glycosylation type can be prepared. The glycosylation pattern of 155263.doc-89-201139667 has been shown to enhance the adcc ability of antibodies. Such carbohydrate modification can be achieved, for example, by rendering the antibody in a host cell that is altered by a glycosylation machinery. The cells modified by the glycosylation machinery have been described in the art and can be used as host cells which express the recombinant antibodies of the present invention, thereby producing antibodies having altered glycosylation. See, for example, Shields, R. L. et al., (2002) J. Biol.

Chem. 277:26733-26740; Umana et al., (1999) Nat. Biotech. 17:176-1 and European Patent No. EP 1,176,195; International Application Publication No. WO 03/035835 and WO 99/5434280 The numbers are each incorporated herein by reference in their entirety. Protein glycosylation depends on the amino acid sequence of the associated protein and the host cell that expresses the protein. Different organisms can produce different glycosylation enzymes (e.g., glycosyltransferases and glycosidases) and have different available substrates (nucleoside sugars). Because of these factors, the protein glycosylation pattern and the composition of the glycosyl residues may vary depending on the host system that represents the particular protein. Glycosyl residues suitable for use in the present invention may include, but are not limited to, glucose, galactose, mannose, trehalose, η-ethyl glucosamine, and sialic acid. According to one aspect, the glycosylated antibody comprises a glycosyl residue such that the glycosylation pattern is in human form. It is known to those skilled in the art that different protein glycosylation can produce different protein characteristics. For example, a therapeutic protein produced in a microbial host such as yeast, and which utilizes yeast endogenous pathway glycosylation, may be less potent than the same protein expressed in mammalian cells such as CH0 cell lines. These glycoproteins may also be immunogenic in humans and exhibit reduced in vivo half-life after administration. Specific receptors in humans and other animals recognize specific glycosyl residues and promote rapid clearance of proteins from the bloodstream. Its I55263.doc •90· 201139667 His unfavorable ingredients include protein folding, solubility, sensitivity to protease transport, transport, compartmentalization, secretion, recognition of antigenic or allergenic changes by other proteins or factors. Thus, a physician may prefer a therapeutic protein having a specific saccharlycosylated composition and pattern (e.g., a glycosylation composition and pattern that is identical or at least similar to that produced in a human cell or a species-specific cell of a predetermined individual animal). A glycosylated protein that behaves differently from a glycosylated protein of a host cell can be achieved by genetically modifying the host cell to express a heterologous glycosylation enzyme. Using techniques known in the art, practitioners can produce antibodies that exhibit glycosylation of human proteins. For example, yeast strains have been genetically modified to represent non-naturally occurring glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in such yeast strains are expressed with animal cells (especially human cells) Protein glycosylation is the same protein glycosylation (U.S. Patent Application Publication Nos. 2004018590 and No. 20060137134; and w〇〇5/100584). Another embodiment is directed to an anti-idiotypic (anti-Id) antibody specific for an antibody of the invention. An anti-id antibody is an antibody that recognizes a unique determinant that is normally associated with the antigen binding region of another antibody. Anti-Id can be prepared by immunizing animals with antibodies or their CDR-containing regions. The immunized animal will recognize and respond to the genetic determinant of the immunological antibody and produce an anti-Id antibody. The anti-Id antibody can also be used as an "immunogen" for inducing an immune response in another animal, thereby producing a so-called anti-Id antibody. In addition, those skilled in the art will appreciate that host cell libraries genetically engineered to express various glycosylation enzymes can be used to express related proteins, and 155263.doc-91-201139667 allows members of the library to produce variant sugars. Proteins related to the basic pattern. The physician can then select and isolate related proteins with a particular novel glycosylation pattern. According to another aspect, a protein having a specifically selected novel glycosylation pattern exhibits improved or altered biological properties. Use of D·anti-Αβ(20-42) globulomer antibody The anti-Αβ(20_ 42) globulomer antibody of the present invention or any other twisted Αβ form due to its ability to bind Αβ(20-42) globulomer Antibodies can be used to detect Αβ(2〇_42) globulomers and/or any other target using conventional immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. To the Αβ form (eg in a biological sample such as serum, CSF, brain tissue or plasma). The invention provides a method for detecting a Αβ(20-42) globulomer and/or any other undirected Αβ form in a biological sample, comprising contacting a biological sample with an antibody of the invention, and detecting binding to αΡ(2〇_ 42) an antibody or unbound antibody to a globulomer (and/or any other Αβ-like form), thereby detecting Αβ(20-42) globulomer, and/or any other targeted Αβ form in the biological sample . The antibody is labeled with a detectable substance, either directly or indirectly, for detection of bound or unbound antibodies. Suitable for detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin/biotin and avidin Protein/biotin; examples of suitable fluorescent substances include umbelliferone 'luciferin, luciferin isothiocyanate, rhodamine, dithiazinylamine fluorescein, tannin chloride or algae red Examples of luminescent substances include illuminol '· and suitable examples of radioactive materials include 155263.doc -92- 201139667 3H, 14c, 35S, 90Υ, 99Tc, mIn, 125I, 131I, 丨77Lu, 66h〇 Or 153Sm. In addition to labeled antibodies, the Αβ(20-42) globulomer standard labeled with a detectable substance and the unlabeled anti-Αβ(20_42) globulomer antibody can be assayed in a biological fluid by competitive immunoassay. Αβ(20-42) globulomer and/or any other targeted Αβ form. In this assay, biological samples, labeled Αβ(20-42) globulomer standards and anti-Αβ(20-42) globulomer antibodies were combined and labeled Αβ (20-42) bound to unlabeled antibody was determined. The amount of globulomer standard. Αβ(20-42) globulomer and/or any other amount of Αβ-form in biological samples and labeled Αβ(2〇_ 42) globulomer bound to anti-Αβ(20-42) globulomer antibody The amount of standard is inversely proportional. According to one aspect of the invention, the antibodies of the invention are capable of neutralizing Αβ(20-42) globulomer activity in vitro and in vivo, and/or any other activity in the dry Αβ form. Thus, the antibodies of the invention are useful for inhibiting (i.e., reducing) Αβ(20-42) globulomer activity, and/or for example, in cell cultures containing Αβ(2〇_42) globulomers. Any other activity in the beta form, and/or any other human or other mammalian subject having a Αβ(20-42) globulomer, any of the Αβ forms, and/or cross-reactive with the antibodies of the invention Other dry Αβ forms. In one embodiment, the invention provides a method of inhibiting (ie, reducing) Αβ(20-42) globulomer activity, and/or any other dry Αβ form activity, comprising Αβ(2〇-42) spheres The polymer and/or any other dry Αβ form is contacted with an antibody of the invention such that the Αβ(20-42) globulomer activity and/or any other dry Αβ form activity is inhibited (i.e., reduced). For example, in a cell culture containing or suspected of containing a Αβ(2〇_42) globulomer and/or any other targeted Αβ form of 155263.doc-93·201139667, the antibody of the invention may be added to the culture medium to Inhibition (i.e., reduction) of Αβ(2〇_42) globulomer activity in culture and/or activity of any other Αβ forms. In another embodiment, the invention provides for inhibiting (i.e., reducing) the activity of a targeted Aβ form in an individual, the subject being susceptible to a disease or condition that is compromised by the activity of the Octal form or selected from the group consisting of Disease or condition: αΐ-antitrypsin-deficiency, Cl_inhibitor-deficient angioedema, antithrombin-deficient thromboembolic disease, Kuru_Kuzd_Jack's disease/sheep disease, bovine sponge Brain disease, Jasper _ Stersler _ Sneck disease, fatal familial insomnia, Huntington's disease, spinal cerebellar disorders, Machado _ @瑟缩缩, dentate red nucleus globus pallidus shrinkage, Frontal lobe type cancer, sickle cell anemia, unstable hemoglobin inclusion body hemolysis, drug-induced inclusion body hemolysis, Parkinson's disease, systemic eight! ^ type starch degeneration, nodular incorporation [class hall powder degeneration, Systemic AA starch degeneration, prostatic powder degeneration, hemodialysis starch degeneration, hereditary (Icelandic) cerebral vascular disease, Huntington's disease, familial visceral _ powder degeneration, familial visceral polyneuropathy, family Sexual viscera Powder degeneration, senile systemic class temple powder denatured 'familial class temple powder neuropathy, familial heart like Temple powder degeneration, Azi Hai Mo's disease, Down's syndrome, medullary thyroid cancer, and type 2 diabetes mellitus (T2DM). The present invention provides a method of inhibiting (i.e., reducing) the activity of targeting an Aβ form in an individual suffering from the disease or condition, the method comprising administering to the subject an antibody of the invention such that the activity of the Aβ form is inhibited in the subject (ie, reduce). In the aspect of the invention, the dry Αρ form is a human-transformed form, and the 155263.doc -94·201139667 body is a human individual. Alternatively, the individual can be any non-human mammal that exhibits APP or results in a dry Αβ form that the antibody of the invention can bind to. Further, the individual may be a non-human mammal that has been introduced to the Αβ-form (e.g., by administering a targeted Αβ-form or by expressing sputum or resulting in any other Αβ-form that produces a dry Αβ form). The antibodies of the invention can be administered to a human subject for therapeutic purposes. In addition, an antibody of the invention can be administered to a non-human mammal that exhibits App or any Αβ form that produces a target Αβ form, wherein the antibody can be combined for veterinary therapeutic purposes or as an animal model of human disease. In the latter case, such animal models can be used to assess the therapeutic efficacy of the antibodies of the invention (e.g., test dosing dose schedules). A further embodiment is a method of inhibiting (i.e., reducing) the activity of targeting the Αβ form in an individual suffering from a starch-like degeneration, such as Alzheimer's disease or Down's syndrome. Conditions which are attributable to the activity of the Αβ form include the following diseases and other conditions in which the presence of a targeted Αρ form in an individual who has been shown or suspected to be afflicted with the condition is the cause of the pathophysiology of the condition, or to aggravate the condition The factor. Thus, a condition that is compromised by the activity of the targeted Αβ form is a condition that inhibits (i.e., reduces) the activity of the Αβ form that is expected to alleviate some or all of the symptoms and/or progression of the condition. Such conditions may be confirmed by, for example, an increase in the concentration of the targeted Αβ form in the biological fluid of the individual suffering from the condition (eg, an increase in the concentration of the targeted Α form in the serum, brain tissue, plasma, synovial fluid, etc. of the individual), which may For example, using an anti-Aβ (20_42) globulomer antibody and/or any other antibody targeting the Αβ form as described above or any Αβ form comprising a globulomer epitope responsive to an antibody of the invention Anti-155263.doc •95- 201139667 Body detection. Non-limiting examples of conditions treatable by the antibodies of the invention include those disorders disclosed herein and in the sections below related to pharmaceutical compositions of the antibodies of the invention. In another embodiment, the invention relates to the prevention of A method of progression (eg, deterioration) of a disease condition. The method comprises administering to a subject in need of treatment (e.g., a lactating animal&apos; such as a human) a therapeutically effective amount of any binding protein or antibody as described herein. Alternatively, the method comprises administering to the individual a therapeutically effective amount of any of the proteins as described herein in combination with at least one therapeutic agent in a therapeutically effective amount. In the methods described above for preventing the development or progression of a disorder described herein, one or more biomarkers, diagnostic tests or combinations of biomarkers and diagnostic tests known to those skilled in the art can be used to determine : (丄) whether the individual is at risk of developing one or more of the conditions described herein; or (2) whether the above condition has progressed (eg, worsened) in an individual previously suffering from one or more of the conditions described herein. Individuals at risk of developing the conditions described herein can be identified using one or more of the biomarkers, diagnostic tests or combinations of biomarkers and diagnostic tests known in the art. Similarly, the combination of one or more biomarkers, diagnostic tests, or biomarkers and diagnostic tests known in the art can be used to determine the progression of a disease or condition in an individual who has been identified as having a condition described herein. For example, a combination of one or more biomarkers, neurodevelopment markers or biomarkers or neurodevelopment markers (eg, MRI, etc.) can be used to identify individuals at risk of developing Alzheimer's disease, or The progression of the disease in individuals suffering from Alzheimer's disease. Biomarkable 155263.doc •96· 201139667 Record includes, but is not limited to, β-class starch i-42, τ, phosphorylated τ (ρτ), plasma Αβ antibody, α-antichymotrypsin, class Starch precursor protein, ratio of sputum isoforms in platelets, β-secretase (also known as BACE), CD59, 8-hydroxy-deoxyguanine, glutamate synthase, glial fibrillary acidic protein (GFAP) , GFAP antibody, interleukin-6-receptor complex, vasopressin, melanin transferrin, neuropilin, nitrotyrosine, oxidized cholesterol, sulphatide, synapse Markers, SlOOp, NPS, plasma signaling proteins, and the like, or any combination thereof (see Shaw, L. et al, iVaiwre 2007, 6, 295-303; Borroni, B. et al,

CwreW A/ec/· C/zem. 2007, 14,1171-1178; Phillips, K. et al., Nature Reviews 2006, 5 463-469; Bouwman, FH et al., 2007, 69, 1006-1011; Ray, S. et al., TWziwre Me Mountain cine 2007, 13(11), 1359-1362; Cummings, J. et al., Neurology 2007, 69, 1622-1634.) 0 E. Pharmaceutical Compositions The present invention also provides the present invention. A pharmaceutical composition of an antibody and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising an antibody of the invention is used, but not limited to, to diagnose, detect or monitor a condition; to prevent, treat, treat or ameliorate the condition or one or more symptoms thereof; and/or to study. In a particular embodiment, the composition comprises one or more antibodies of the invention. In another embodiment, the pharmaceutical composition comprises one or more antibodies of the invention and one or more prophylactic or therapeutic agents for treating a condition which is impaired by the activity of the targeted Αβ form, in addition to the antibody of the invention. In another embodiment, a prophylactic or therapeutic agent is known to be or has been or is currently being used to prevent, treat, treat or ameliorate a disease or one or more of its symptoms. According to such embodiments, the composition may further comprise a carrier, diluent or excipient. The antibodies of the invention can be incorporated into pharmaceutical compositions suitable for administration to an individual. Generally, pharmaceutical compositions comprise an antibody of the invention and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Things. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferred to include isotonic agents, e.g., sugars such as mannitol or sorbitol, or sodium chloride. The pharmaceutically acceptable carrier can further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. In another embodiment, the pharmaceutical composition comprises at least one additional therapeutic agent for treating the conditions disclosed herein. Various delivery systems are known and can be used to administer one or more antibodies of the invention or one or more of the antibodies of the invention in combination with a prophylactic or therapeutic agent suitable for the prevention, management, treatment or amelioration of a condition or one or more of its symptoms For example, encapsulated in liposomes, microparticles, microcapsules; recombinant cells which can express antibodies or antibody fragments; receptor-mediated endocytosis (see, for example, Wu &amp; Wu, j Biol. Chem. 262: 4429-4432 ( 1987)); Construction of nucleic acids as part of a retrovirus or other vector. Methods of administering a prophylactic or therapeutic agent of the invention include, but are not limited to, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, intratumoral administration 155263.000 •98· 201139667 and mucosal administration (eg intranasal and oral route). Further, pulmonary administration can be administered, for example, by using an inhaler or nebulizer and a formulation having an aerosol. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication No. WO 92/19244, W097/ No. 32, 572, WO 97/44013, No. W098/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety. In one embodiment, the antibodies, combination therapies, or compositions of the invention of the invention are administered using Alkermes AIR® transpulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.). In a specific embodiment, the prophylactic or therapeutic agent of the present invention is administered intramuscularly, intravenously, intratumorally, orally, intranasally, pulmonaryly or subcutaneously. The prophylactic or therapeutic agent can be administered by any suitable route, for example, by infusion or rapid injection, by transdermal or intradermal mucosa (eg, oral mucosa, rectal and intestinal mucosa, etc.) and can be combined with other bioactive agents. Give it together. The administration can be administered systemically or locally. In a particular embodiment, it may be desirable to topically administer an antibody of the invention to a region in need of treatment; this may be by, for example, but not limited to, local infusion, injection or by means of an implant (the implant is porous or non-invasive) Porous materials include membranes and matrices such as siaUstic membranes, polymers, fibrous matrices (eg, Tissuel 9) or collagen matrices. In one embodiment: an effective amount of one or more of the antibodies of the invention is administered topically to the affected area of the individual to prevent, treat, control and/or modify the condition or symptom thereof. In another embodiment, one of the effective amounts is administered to the affected area of the individual 155263.doc •99·201139667 or a plurality of antibodies of the invention and an effective amount of one or more therapies other than the antibodies of the invention (eg, one or A plurality of prophylactic or therapeutic agents) for preventing, treating, managing and/or ameliorating a condition or one or more symptoms thereof. In another embodiment, the antibody can be delivered by controlled release or sustained release system. In one embodiment, a pump can be used to achieve controlled release or sustained release (see Langer&apos; with the above; Sefton, 1987, CRC Crit. Ref.

Engmed. Eng. 14:20; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, a polymeric material can be used to effect controlled release or sustained release of the therapy of the invention (see, for example, Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Boca Raton, Fla. (1974). Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (ed.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; See Levy et al., 1985, Science 228: 190; During et al, 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Patent No. 5,679,377; U.S. Patent No. 5,912, 015; U.S. Patent No. 5,989, 463; U.S. Patent No. 5,128, 326; PCT Publication No. WO99/15154; and PCT Publication No. WO99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(decyl methacrylate), poly(acrylic acid), poly(ethylene-co-acetic acid) Vinyl ester), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-ethyl 155263.doc •100- 201139667 alkenyl)bicronidone), poly(vinyl alcohol), poly Acrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA) and polyorthoester. In a particular embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable. In another embodiment, the controlled release or sustained release system can be placed adjacent to the prophylactic or therapeutic target, thus requiring only one portion of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release, supra, Vol. 2, p. 115). -138 (1984)) » Controlled Release Systems are discussed in Langer's Review (1990, Science 249: 1527-1533). Sustained release formulations comprising one or more antibodies of the invention can be prepared using any technique known to those skilled in the art. See, e.g., U.S. Patent No. 4,526,938, PCT Publication No. WO 91/05548, PCT Publication WO96/20698 'Ning et al., 1996, "Intratumoral

Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy &amp; Oncology 39:179-1 89 ; Song et al., 1995, "Antibody Mediated Lung

Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science &amp; Technology 50:372-397 » Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro· Int'l. Symp. Control Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759 - 155263.doc -101 - 201139667 760; each of which is incorporated herein by reference in its entirety. In a particular embodiment where the composition of the invention is a nucleic acid encoding an antibody, the nucleic acid can be constructed as part of a suitable nucleic acid expression vector, and for example by using a retroviral vector (see U.S. Patent No. 4,98,286). No.) either by direct injection or by bombardment with microparticles (eg, gene gun; Biolistic, Dupont) or by lipid or cell surface receptors or transfection agents, or by homologous to known cores Box-like peptide linkages (see, for example,

Joliot et al. '1991, proc. Natl Acad. Sci. USA 88:1864- 1868) administered it to make it intracellular, to administer nucleic acids in vivo to promote the performance of the antibodies encoded thereby. Alternatively, the nucleic acid can be introduced into the cell and incorporated into the host cell DNA for expression by homologous recombination. The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, such as intravenous, subcutaneous, intraoral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a particular embodiment, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. Typically, the group for intravenous administration is a solution in sterile isotonic aqueous buffer. The composition may also include a solubilizing agent and a local anesthetic (such as lignoccamne) to reduce pain at the injection site, if desired. If the composition of the present invention is to be administered topically, the composition may be formulated as an ointment, a cream, a transdermal patch #, a lotion, a gel, a shampoo, a spray, an aerosol, and a solution of the emulsion. Forms or other forms familiar to those skilled in the art. See, for example, Remington, Pharmaceutical Sciences and 155263.doc 201139667

Introduction to Pharmaceutical Dosage Forms, 19th Edition,

Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, a viscous to semi-solid or solid form comprising a carrier or one or more excipients compatible with topical application and having a kinetic viscosity greater than that of water is typically employed. Suitable formulations include, but are not limited to, solutions, suspensions, lotions, creams, ointments, powders, liniments, ointments and the like, if necessary sterilized or with additives which affect various properties such as osmotic pressure Mix (eg preservatives, stabilizers, wetting agents, buffers or salts). Other suitable topical dosage forms include sprayable aerosol formulations wherein the active ingredient is, for example, combined with a solid or liquid inert carrier for packaging with a mixture of pressurized volatile materials such as a gas propellant such as freon. Or packaged in a plastic squeeze bottle. If necessary, a moisturizer or a moisturizer may be added to the pharmaceutical composition and the dosage form. Examples of such other ingredients are well known in the art. If the method of the invention comprises intranasal administration of the composition, the composition may be formulated in the form of an aerosol, spray, mist or drops. In particular, the prophylactic or therapeutic agent used in accordance with the present invention may be formulated with a propellant (eg, digas-fluorine, dichlorofluoromethane, dioxane, carbon dioxide, or other suitable gas). The spray formulation of the agent is suitably delivered from a pressurized pack or spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver the metered amount. Capsules and cartridges, e.g., gelatin, containing a powder mixture of the compound and a suitable powder base such as lactose or starch may be formulated for use in an inhaler or insufflator. If the method of the present invention comprises a π-administration, the composition can be formulated as an oral 155263.doc 201139667 syrup-squeeze gelling agent, a gelleap, a solution, a suspension, and the like. The money or capsules can be prepared by conventional means using pharmaceutically acceptable excipients such as binders (for example pregelatinized corn; temple powder, polyethylene etL n phases or (d) a methylcellulose); a filler (such as sugar, microcrystalline cellulose or acid-depleted nitrogen); a lubricant (such as hard moon i magnesium, talc or Shishi stone); a disintegrating agent (such as horse age) Temple powder or ethanol &amp;L sodium sulphate), or a humectant (such as sodium lauryl sulfate). Tablets can be coated by methods well known in the art. The liquid preparation for oral administration can be in the form of, but not limited to, a solution, syrup or suspension, or it can be provided in the form of a dry product which is reconstituted with water or other suitable vehicle. The liquid preparation can be prepared by a conventional method using a pharmaceutically acceptable additive such as a suspension or the like (such as a sorbitol syrup, a cellulose derivative or a hydrogenated edible fat); an emulsifier; (eg lecithin or gum arabic) 'non-aqueous vehicle (eg almond oil, oily ester, ethanol or fractionated vegetable oil), and preservatives (eg p-hydroxybenzoate or propyl sulfonate) Or sorbic acid). The formulation may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents, as appropriate. Formulations for oral administration may be suitably formulated for slow release, controlled release or sustained release of prophylactic or therapeutic agents. The method of the invention may comprise administering a composition formulated with an aerosol, for example, by using an inhaler or a nebulizer. See, for example, U.S. Patent Nos. 6,019,968, 5,985,32, 5,985, 3, 5,934,272, 5,874, 〇64, 5,855,913, 5,290,540, and 4,880,078; WO 92/19244, WO 97/32572, WO 97/44013, 155263.doc • 104-201139667 WO 98/31346 and WO 99/669〇3; The manner of reference is incorporated herein. In a specific embodiment, the antibodies, combination therapies, and/or compositions of the invention are administered using AlkermeS AIR® transpulmonary drug delivery technology (Alkermes, (6), Cambridge, Mass.). The methods of the invention may comprise administering a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection can be provided in unit dosage form with a preservative (for example, in ampoules or in multi-dose containers). The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain such compositions as suspending agents, stabilizing agents and/or dispersing agents. Alternatively, the active ingredient may be in the form of a powder which is reconstituted in a suitable vehicle (for example, sterile pyrogen-free water) before use. The method of the invention may additionally comprise administering a composition formulated as a sump formulation. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition may be formulated with a suitable polymeric or hydrophobic material (for example, an emulsion formulated in an acceptable oil) or with an ion exchange resin; or formulated as a sparingly soluble derivative (eg, formulated as a sparingly soluble salt + this The method of the invention encompasses administering a composition formulated in a neutral or salt form. The pharmaceutically acceptable salt includes a salt formed with an anion such as a salt derived from hydrochloric acid, dish acid, acetic acid, oxalic acid, tartaric acid, and the like; Cationic = salt such as derived from sodium hydroxide, potassium hydroxide, hydroxide, gas oxidized feed, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, Pru a salt of cain or the like. In general, the ingredients of the composition are used alone or in combination with a unit of ingredients (such as in the form of a dried lyophilized powder or a water-free concentrate) in the indicator active agent 155263.doc 201139667 = such as Provided in a bottle or sachet). When the mode of contribution is matched. When the infusion bottle containing no (iv) drug grade water or saline is dispensed and the mode is injection, an injection ampoule may be provided so that the ingredients can be mixed prior to administration. Solid I Water = Two The present invention also provides one or more of the antibody or pharmaceutical combination of the present invention, a non-antibody amount of a sealed container (such as an ampoule or sachet). In one embodiment, one or more of the antibodies or pharmaceutical compositions of the invention are provided in a sealed container in the form of a lyophilized powder or a water-free concentrate and are reconstitutable (eg, with water or saline) to a concentration of Cast. The antibody or pharmaceutical composition of the present invention in the form of a dry:.,,,,,,,, A unit dose of at least μ, at least 5 〇 5 mg or at least 100 mg is provided in a sealed container. The lyophilized antibody or pharmaceutical composition of the invention should be stored at 2t to 8eC. In each device, and the antibody or pharmaceutical composition of the present invention should be within 1 week after recovery, for example, within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, 3 Intra-hours or within hours. In an alternate embodiment, one or more of the antibodies or pharmaceutical compositions of the invention are provided in liquid form in a sealed container indicating the amount and concentration of the antibody. In a further embodiment, the liquid form of the composition administered is at least mg25 mg/ml 'eg at least 〇.5 mg/ml, at least 1 mg/mi, at least 2.5 mg/ml' at least 5 mg/ml, At least 8 mg/nU, at least 1 mg/rrd, to 15 mg/kg 'at least 25 mg/ml, at least 5 mg/ml, at least 75 mg/ml or at least 1 mg/ml for sealed containers in. The liquid form should be stored in its original container at 155263.doc •106·201139667 2C to 8C. The antibodies of the invention can be incorporated into pharmaceutical compositions suitable for parenteral administration. In one aspect, the antibody is prepared to contain an injectable solution of 25 mg of mg per ml of antibody. The injectable solution may consist of a liquid or lyophilized dosage form in a vermiculite or amber vial, an ampoule or a pre-filled syringe. The buffer may be histidine (1-50 mM), optimally 5-10 mM, pH 5.0 to 7 〇 (preferably pH 6.0) ^ Other suitable buffers include, but are not limited to, succinic acid Sodium, sodium citrate, sodium phosphate or potassium phosphate. Solution toxicity can be adjusted using sodium hydride at a concentration of 〇_3 〇〇 mM (150 mM optimal for liquid dosage forms). For the bead dry dosage form, § may include a cryoprotectant, mainly 〇 1 〇% sucrose (preferably 0.5%-1·0%). Other suitable cryoprotectants include trehalose and lactose. Accumulators may be included for lyophilized dosage forms, primarily 1% to 1% mannitol (optimally 2% to 4%). Stabilizers can be used in both liquid and east dry formulations, primarily 1-5 0 mM L-methionine (optimally 5-1 mM). Other suitable builders include glycine, arginine (may be included in 0-0.05%), polysorbate-80 (best 〇.〇〇5%-〇.01%). Other surfactants include, but are not limited to, polysorbate 2 and BRIJ surfactants. The pharmaceutical composition comprising the antibody of the present invention prepared for parenterally administered injectable solution may further comprise an agent suitable for use as an adjuvant, such as an agent for increasing the absorption or dispersion of the antibody. A particularly suitable adjuvant is hyaluronidase, such as Hylenex® (recombinant human hyaluronidase). The addition of hyaluronan to the injectable solution improves the bioavailability of the human after parenteral administration, especially after subcutaneous administration. It also allows for a higher injection site volume (i.e., greater than 丨ml) with less pain and discomfort and the lowest incidence of injection site reactions. (See International Publication No. 155, 263, filed on Dec. No. PCT-A------------------ Forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, lozenges, pills, powders, liposomes, and suppositories. Depending on the intended mode of treatment application. The compositions are in the form of injectable or infusible solutions, such as compositions similar to those used in the passive immunization of humans with other antibodies. In one embodiment, the antibody is administered by intravenous infusion or injection. In another embodiment, the anti-system is administered by intramuscular or subcutaneous injection. Therapeutic compositions are generally sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, lipids or other ordered structures suitable for high drug concentrations. By combining the desired amount of the active compound (i.e., a binding protein of the invention, such as an antibody) in a suitable solvent with one of the above listed ingredients or a combination of such ingredients, followed by filtration sterilization as needed A sterile injectable solution is prepared. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains an inert dispersion medium and the required additional ingredients from the ingredients enumerated above. In the case of a sterile lyophilized powder for the preparation of a sterile injectable solution, the preparation comprises vacuum drying and spray drying which yields the active ingredient plus the powder of any other desired ingredient from its previously sterile filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the desired particle size (in the case of dispersions), and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents (e.g., monostearate and gelatin) which delay absorption in compositions 155263.doc-108-201139667. Antibodies of the invention can be administered by a variety of methods known in the art. For many therapeutic applications, the route of administration/mode can be subcutaneous, intravenous or infusion. As will be appreciated by those skilled in the art, the route of administration and/or mode will vary depending on the desired result. In certain embodiments, the active compound can be prepared with carriers that prevent rapid release of the compound, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly needles, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many of the methods of preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release

Drug Delivery Systems, edited by J.R. R〇binson, Marcel Dekker,

Inc., New York, 1978 0 In certain embodiments, an antibody of the invention can be administered orally, for example, with an inert diluent or an absorbable edible carrier. The antibody (and other components as necessary) may also be enclosed in a hard or soft shell gelatin capsule, compressed into a lozenge or directly incorporated into the individual's diet. For oral therapeutic administration, the antibodies may be combined with excipients and used in the form of ingestible lozenges, buccal tablets, dragees, capsules, elixirs, suspensions, syrups, powders (wafer) ) and its similar dead &gt; In order to administer an antibody of the invention by means other than parenteral administration, it may be desirable to coat the antibody with a material that prevents its inactivation or co-administer the antibody with a material that prevents its inactivation. Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, an antibody of the invention is co-administered and/or co-administered with one or more additional therapeutic agents suitable for treating the condition or disease described herein. For example, the anti-Αβ(2〇-42) globulomer antibody of the present invention can be co-formulated with one or more other antibodies that bind to other targets (for example, antibodies that bind to other soluble antigens or bind to cells of Table 2). / or a total vote. Furthermore, one or more of the antibodies of the invention may be used in combination with two or more of the above therapeutic agents. Such combination therapies may suitably utilize lower doses of the administered therapeutic agent to avoid possible toxicity associated with various monotherapies. Or complications. In certain embodiments, an antibody of the invention can be administered, for example, with σ, with an inert diluent or an assimilable edible carrier. Such media are described, for example, in U.S. Patent Application Serial No. 09/428,082, the disclosure of which is incorporated herein by reference. In a specific embodiment, a nucleic acid sequence comprising a nucleotide sequence encoding an antibody of the invention is administered by means of gene therapy to treat, prevent, manage or ameliorate the condition or one or more symptoms thereof. Gene therapy refers to a therapy performed by administering to a subject an already expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acid produces an antibody of the invention encoded by it that mediates a prophylactic or therapeutic effect. Any method for gene therapy that can be utilized in the art can be used in accordance with the present invention. For a general review of general gene therapy methods, see G〇ldspiel et al., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy 3: 87-95; Tolstoshev, 1993, Ann. Rev.

Pharmacol. Toxicol. 32:573-596 ; Mulligan, Science

260:926-932 (1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIBTECH 155263.doc -110·201139667 11(5):15 5-215 . Methods commonly known in the art of recombinant DNA techniques that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY (1993); and Kriegler, Gene.

Transfer and Expression, A Laboratory Manual, Stockton Press' NY (1990). A detailed description of various methods of gene therapy is disclosed in US2005 0042664 A1, which is incorporated herein by reference. The antibodies of the present invention may be used alone or in combination to treat diseases such as Alzheimer's disease, Down's syndrome, dementia, Parkinson's disease or any other disease or condition associated with the formation of a starch-like protein in the brain. The antibody of the present invention can be used for (7) treatment of a disease. These diseases are caused by changes in the secondary to tertiary structure of the constituent proteins, followed by altered protein aggregation (Hayden et al, JOP. J Pancreas 2005; 6(4): 287-302). In particular, the antibody of the present invention can be used to treat one or more of the following constitutional diseases: α-antitrypsin-deficient, C1-inhibitor-deficient angioedema, antithrombin-deficient thromboembolic disease, library Lu Kuzd-Jack's disease / scrapie, bovine spongiform encephalopathy, Jester Stersler. Sinck's disease, fatal familial insomnia, Huntington's disease, spine cerebellar disorders, Macha More 'Josephic atrophy, dentate red nucleus pallidus atrophy, frontotemporal dementia, sickle cell anemia, unstable hemoglobin inclusion hemolysis, drug-induced inclusion body hemolysis, Parkinson's disease, systemic AL starch Denatured w cross r玍, crusting AL type starch degeneration, systemic AA starch degeneration, pre-J 〗 gland type powder degeneration, blood edge enchantment I powder degeneration, hereditary (Iceland) ★ cerebrovascular disease, Tinton's disease, sudden visceral starch degeneration, Jiaya visceral polyneuropathy, Haiyou visceral dysplasia, senile, π Pingwang's starchy degeneration, familial 195263.doc •111- 201139667 disease, familial heart _ powder degeneration, Alzheimer's disease, Tang = group, thyroid medullary cancer and type 2 diabetes (T2dm). The antibodies of the invention are preferably useful for the treatment of amyloidosis, such as Alzheimer's disease and Down's syndrome. It is to be understood that the antibodies of the invention may be used alone or together with - or a plurality of other agents (eg, therapeutic agents such as small molecules or organisms). Formulations) are used in combination, and the other agents are selected by those skilled in the art for their intended purpose. For example: 'Other therapeutic agents may be "cognitive enhancing drugs," which are drugs that improve the cognitive ability of the brain (ie, thinking, learning, and memory). Cognitive enhancing drugs act by altering the availability of neurochemicals (such as neurotransmitters, enzymes, and hormones), improving oxygen supply, stimulating nerve growth, or inhibiting nerve damage. Examples of cognitive enhancing drugs include compounds that enhance the activity of the B-test, such as, but not limited to, acetylcholine receptor agonists (eg, nicotine "-7 receptor agonist or ectopic modulator," Nicotinic receptor agonists or ectopic modulators), acetylcholinesterase inhibitors (eg donepezil, remexamine and galantamine), acetylcholinesterase inhibitors, N-methyl Hexa-aspartate (NMDA) receptor antagonist (eg, memantine), neuroprotective protein (ADNp) agonist, serotonin 5_HT1A receptor agonist (eg, Zalli Roden), 5 ΗΤ4 receptor agonist, 5·ΗΤ^ receptor antagonist, serotonin 1 Α receptor antagonist, histamine % receptor antagonist, calpain inhibitor, vascular endothelial growth factor (VEGF) protein or agonist , vegetative growth factors, anti-apoptotic compounds, AMPA-type glutamate receptor activators, L- or N-type calcium channel blockers or modulators, potassium channel blockers, hypoxia-inducible factor (HIF) activators , HIF amidoxime 4-hydroxylase inhibition 155263.doc -112- 201139667 agent, anti-inflammatory agent, starch-like Αβ Or an inhibitor of amyloid plaques, an inhibitor of hyperphosphorylation, a phosphodiesterase 5 inhibitor (eg, tadalafil, sildenafil), a phosphodiacetase 4 inhibitor, a monoamine oxidase inhibitor, or a pharmaceutical thereof Salts that are acceptable for the study. Specific examples of such cognitively enhanced drugs include, but are not limited to, cholinesterase inhibitors such as donepezil (Aricepf), lensidine, and galantamine sensitization (10) Methyl aspartate antagonists, such as Namenda®. At least one cognitive enhancing drug can be administered simultaneously with the antibody of the invention or sequentially with the antibody of the invention (and in any order), including current identification Or, in the future, those agents suitable for treating the disease or condition being treated by the antibodies of the invention will be identified. Furthermore, the combinations described herein may have additive or synergistic effects when used in the above materials. The composition is beneficial to the genus, and the remedy for sputum is, for example, an agent which affects the viscosity of the composition. It is to be further understood that the combinations to be included in the present invention are combinations suitable for their intended purpose. The agent is for illustrative purposes and is not intended to be limiting. A combination of parts of the invention may comprise an antibody of the invention and at least one other agent selected from the list below. If combined, the resulting composition may be The predetermined function may also include one or more other agents, such as two or three other agents. The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody of the invention. "Therapeutically effective amount" means an amount effective to achieve the desired therapeutic effect at the required dosage and for a period of time necessary. The therapeutically effective amount of the antibody can be determined by those skilled in the art and can vary depending on such factors as the disease condition, the age, sex and weight of the individual, and the ability of the antibody to elicit the desired response in the individual 155263.doc • 113·201139667. A therapeutically effective amount is also one in which the therapeutic benefit of the antibody exceeds any toxic or detrimental effects. "Prophylactically effective amount" means the amount effective to achieve the desired preventative effect at a necessary dose and for a period of time necessary. By the use of a prophylactic dose to individuals prior to the early stages of the disease or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount. Adjustable administration is fortunate to take the best response (such as treatment or preventive response). For example, a single dose may be administered, and a number of knife openers may be administered over time or depending on the urgency indication of the condition being treated, the dose may be reduced or increased proportionally. It is especially suitable to formulate parenteral compositions as unit dosage forms for ease of administration and uniformity of dosage (iv). A unit dosage form as used herein is intended to mean a physical individual unit for treating an individual of a mammal; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect and desired Pharmaceutical carrier. The specification of the unit dosage form of the invention is defined by the following factors and depends directly on the following factors: (4) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved; and (b) compounding the active compound to achieve the therapeutic sensitivity of the individual The limitations inherent in the technology. An exemplary non-limiting range of therapeutically or prophylactically effective amounts of an antibody of the invention is 0.1-20 mg/kg, such as 1-1 mg/kg. It should be noted that the dose value may vary depending on the type and severity of the condition to be alleviated. It is further understood that for any particular individual, the particular dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the individual administering or supervising the administration of the composition, and the dosage ranges described herein are for illustrative purposes only. It is not intended to limit the scope or implementation of the claimed compositions. 155263.doc • 114· 201139667 It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein are obvious and can be used in a suitable equivalent without departing from the scope of the invention. Or the embodiments disclosed herein are carried out. The present invention will be described in detail with reference to the following examples, which are to be understood by way of example only. EXAMPLES Example 1: Preparation of globulomer &amp; Αβ(1-42) globulomer: Αβ(1-42) synthetic peptide (H_1368, Bachem, Bubend〇rf,

Switzerland) was suspended at 6 mg/ml in ι〇0% ^ sulphur hexafluoropropanol (HFIP) and incubated at 37 ° C under shock! 5 hours to completely dissolve. HFIP is used as a hydrogen bond cleavage agent and is used to eliminate pre-existing structural heterogeneity in the Αβ peptide. HFIP was removed by evaporation in SpeedVac and Αβ(1-42) was resuspended in dimethyl subhard at a concentration of 5 mM and sonicated for 2 sec. HFIP-pretreated Αβ( 1-42) was diluted in discate buffered saline (PBS) (20 mM NaH2P〇4 '140 mM NaCl * pH 7.4) up to 400 μΜ, and 1/10 volume 2% was added. Sodium dodecyl sulfate (SDS) (final concentration of aqueous solution 0.2 ° /. SDS). Incubation at 37 C for 6 hours yielded a 16/20-kDa Αβ( 1-42) globulomer (short form of globular oligomer) intermediate. The 38/48-kDa Αβ(b 42) globulomer was produced by further dilution with 3 Ηβ and incubation at 37 °C for 18 hours. After centrifugation at 3000 g for 20 minutes, the sample was concentrated by ultrafiltration (30-kDa cutoff), dialyzed against 5 mM NaH2P04, 35 mM NaCl 'pH 7.4' at 10, centrifuged for 1 minute, and removed. Package 155263.doc •115· 201139667 Contains a supernatant of 38/48-kDa Αβ(1-42) globulomer. As an alternative to dialysis 38/48-kDa Αβ(1-42) globulomer, it can also be a 9-fold excess (volume) ice-cold sterol/acetic acid solution (33% methanol, 4% acetic acid) at 4 °C. Precipitate in 1 hour. The 38/48-kDa Αβ(1-42) globulomer was then aggregated into granules (10 min at 16200 g), resuspended in 5 mM NaH2P04, 35 mM NaC Bu pH 7.4 and the pH adjusted to 7.4. b) Ap(20-42) globulomer: 1.59 ml of Ap(i_42) globulomer preparation prepared according to Example la with 38 ml buffer (50 mM MES/NaOH, pH 7.4) and 200 μll mg/ml thermophilic bacteria The aqueous protease solution (Roche) is mixed. The reaction mixture was searched for 20 hours at room temperature. Next, 80 μL of 1 mM EDTA aqueous solution, pH 7.4 ' was added and the mixture was additionally adjusted to a SDS content of 0.01% in a 400 μΐ 1% strength SDS solution. The reaction mixture was concentrated to approximately 1 ml via a 15 ml 30 kDa Centriprep tube. The concentrate was mixed with 9 ml buffer (50 mM MES/NaOH, 0.02% SDS 'pH 7.4) and concentrated again to 1 m. Concentrate in a dialysis tube at 6 ° C for 1 L of buffer (5 mM sodium phosphate) , 35 mM NaCl) dialyzed for 16 hours. The dialyzate was adjusted to a SDS content of 0.1% with a 2% aqueous solution of SDS. The sample was centrifuged for 10 minutes at 1 Torr, and the supernatant of Αβ(2〇·42) globulomer was taken out. 〇Αβ(12-42) globulomer:

2 ml of Αβ(1-42) globulomer preparation prepared according to Example la with 38 ml buffer (5 mM sodium phosphate, 35 mM sodium carbonate, pH 7.4) and 150 μΐ 1 mg/ml GluC endonuclease (Roche) mixed aqueous solution. The reaction mixture was stirred at room temperature for 6 hours, followed by an additional 150 μl of 1 mg/ml GluC 155263.doc -116·201139667 endoproteinase (Roche) aqueous solution. The reaction mixture was further stirred at room temperature for 16 hours, followed by the addition of 8 μM 5 M DIFP solution. The reaction mixture was concentrated via a 15 ml 30 kDa Centriprep tube to approximately 1 m of the concentrate and mixed with 9 mi of buffer (5 mM sodium phosphate, 35 mM sodium chloride, pH 7.4) and concentrated again to 1 ml. The concentrate was dialyzed against 1 L of buffer (5 mM sodium citrate, 35 mM NaCl) in a dialysis tube for 16 hours at 6 °C. The dialyzate was adjusted to a SDS content of 0.1% with a 1% aqueous solution of SDS. The sample was centrifuged for 10 minutes under 〇, and the supernatant of Αβ(12_42) globulomer was taken out. d) Crosslinked Αβ(1-42) globulomer: The Αβ(1-42) synthesis (H-1368, Bachem, Bubendorf, Switzerland) was suspended at 6 mg/ml in 100% mu% hexafluoro_2 • Prolaced in propanol (HFIP) and incubated at 37 ° C for 1.5 hours under shaking to completely dissolve. HFIP is used as a hydrogen bond cleavage agent and is used to eliminate pre-existing structural heterogeneity in the Αβ peptide. HFIP was removed by evaporation in SpeedVac and Αβ(12-42) globulomer Αβ( 1-42) was resuspended in 曱 尔 _ at a concentration of 5 mM and sonicated for 20 seconds. HFIP-pretreated Αβ(1-42) was diluted to 400 μM in PBS (20 mM NaH2P04, 140 mM NaC Bu pH 7.4) and 1/10 volume 2°/〇SDS (aqueous solution) was added (final concentration 0.2 % SDS). Incubation at 37 °C for 6 hours yielded an intermediate of l6/20-kDaAp(l_42) globulomer (short form of globulomer oligomer). Further diluted by 3 volumes of water and at 37. (: incubation for 8 hours) yielded 38/48_kDa Αβ(1·42) globulomer. Now incubated with 1 mM pentane at room temperature for 2 hours at room temperature, followed by ethanolamine at room temperature (5 mM) treatment for 30 minutes for cross-linking of 38/48-kDa Αβ(1-42) globulomer. 155263.doc •117· 201139667 Example 2: Production of humanized anti-Αβ(20-42) globulomer antibody , isolation and characterization Example 2.1: Selection of human antibody frameworks The selection of human antibody frameworks is based on the typical structural similarity of human antibodies and amino acid sequence homology. In addition, when based on human VH and Vk germline sequences, amino acids Sequence homology identification is suitable for structural VL and VH framework sequences, considering the retention of the amino acid residues supporting the ring structure and the VH/VL interface and the retention of the valer acid residues in the vernier region. In addition, based on overlapping peptides for multiple MHC I Predictive Affinity of Class and/or MHC Class II Dual Genes, Electron Hybridization Evaluate the immunogenicity of the 4D10 CDRs into VH and VL sequences generated in the VL and VH framework sequences that may be suitable for inclusion. VH and VL are suitable for each Do not share the common sequence of the VH or VL family to further minimize potential immunity Selective back mutation of the murine amino acid residue to retain the amino acid supporting the ring structure and the VH/VL interface. The amino acid sequence alignment is used to determine the individual VH or VL germline genes. The frequency of such back-mutation in the corresponding pool of naturally occurring human VH or VL sequences. Examine the potential N-linked glycosylation sites of the VH and VL sequences generated above (NXS or NXT, where X is in addition to P) Any amino acid.) Example 2·2: Humanization of murine anti-AB (20-42) globulomer antibody 4D10hum_VH.lz (SEQ ID NO: 4): The murine resistance described in Table 4 The heavy chain CDR sequence of Αβ(20-42) globulomer antibody 4D10 was grafted into the acceptor framework of human VH3-53 and JH6 sequences. 4D10hum_VH.l (SEQ ID ΝΟ: 5): The heavy chain CDR sequences of the murine anti-Αβ(20-42) globulomer antibody 4D10 were grafted into the acceptor construct containing the VH3 co-altering I12V human VH3-53 and JH6 sequences 155263.doc-118-201139667. 4D10hum_VH. La (SEQ ID NO: 6): The heavy chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 described in Table 4 were transplanted to contain VH3 to jointly change I12V and The framework restores the human VH3-53 and JH6 sequences of the mutations A24V, V29L, V48L, S49G, F67L, R71K, N76S and L78V in the acceptor framework. 4D10hum_VH.lb (SEQ ID NO: 7): The heavy chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 described in Table 4 were grafted to human VH3- containing the back mutations V29L and R71K. In the acceptor framework of the 53 and JH6 sequences. 4D10hum_VH.2z (SEQ ID NO: 8): The heavy chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 described in Table 4 were transplanted into the acceptor of human VH4-59 and JH6 sequences. In the framework. 4D10hum_VH.2 (SEQ ID NO: 9): The heavy chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 described in Table 4 were transplanted to contain Q1E changes to prevent sputum end germination The acceptor framework of the human VH4-59 and JH6 sequences formed by the acid salt. 4D10hum_VH.2a (SEQ ID ΝΟ: 10): The heavy chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 described in Table 4 were transplanted to contain Q1E changes to prevent guanidine-terminated glutamate The acid salt forms and comprises the acceptor framework of the human VH4-59 and JH6 sequences of the framework back mutations G27F, I29L, I37V, I48L, V67L, V71K, N76S and F78V. 4D10hum_VH.2b (SEQ ID NO: 11): The heavy chain CDR sequences from the murine anti-Aβ (20·42) globulomer antibody 4D10 described in Table 4 were transplanted to 155263.doc • 119-201139667 Contains Q1E changes In the acceptor framework of the human VH4-59 and JH6 sequences preventing the formation of N-terminal pyroglutamate and comprising the framework back mutations G27F, I29L and V71K, 8 4D10hum_VK.lz (SEQ ID NO: 12): The light chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 were grafted into the acceptor framework of the human VKA17/2-30 and Jk2 sequences. 4D10hum_VK.l (SEQ ID NO: 13): The light chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 described in Table 4 were transplanted to contain Vk2 to change S7T, L15P, Q37L, The acceptor framework of the human VKA17/2-30 and Jk2 sequences of R39K and R45Q. 4D10hum_VK.la (SEQ ID NO: 14): The light chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 described in Table 4 were transplanted to contain Vk2 to collectively alter S7T, L15P, Q37L, R39K and R45Q, and the framework of the human VκA17/2-30 and Jk2 sequences that affect the framework of the VL/VH interface back mutation F36L. 4D10hum_VK.lb (SEQ ID NO: 15): The light chain CDR sequences from the murine anti-Aβ (20-42) globulomer antibody 4D10 described in Table 4 were transplanted to contain the 乂&lt;2 co-change 87 Ding and (^37 [the human ¥1^8 17/2-30 and slave 2 sequences in the acceptor framework. 4D10hum_VK.lc (SEQ ID ΝΟ: 16): will be described in Table 4 from the murine anti-Αβ (20-42) The light chain CDR sequence of globulomer antibody 4D10 was transplanted to a sequence comprising: ¥1&lt;2 co-altering 87 butyl, (53 71 ̄ and 1 139 〖 humans 1 &lt; 八 17/2-3 0 and Jk2 sequences In the acceptor framework, some of these VH and Vk back mutations, co-changes or Q1E mutations in 4D10hum_VH.2, 155263.doc-120·201139667 4D10hum_VH.2a or 4D10hum_VH.2b may be removed during subsequent affinity mutations. Example 2.3: Constructing a Humanized Antibody

The electronically constructed humanized antibody described above will be reconstituted using an oligonucleotide. For each variable region cDNA, six oligonucleotides each having 60-80 nucleotides are designed to overlap 20 nucleotides at the ends of each oligonucleotide at 5 and/or 3, respectively. In the adhesion reaction, all six kinds of nucleus acid were combined, cooked, and bonded in the presence of dNTP. Next, dNA polymerase 1 (Klenow fragment (New England Biolabs #M0210, Beverley, ΜΑ.)) will be added to fill a gap of about 4 bp between the overlapping oligonucleotides. PCR will then be performed using two outermost primers containing the overhanging sequence complementary to the multiple selection sites in the modified pBOS vector to amplify the entire variable region gene (Mizushima, S. and Nagata, S., (1990) Nucleic acids

Research Volume 18, No. 17). The PCR product generated from each cDNA assembly will be isolated on an agarose gel and the band corresponding to the predicted variable region cDNA size will be excised and purified. The variable heavy region was inserted in-frame with a cDNA fragment encoding a human IgG1 constant region containing two hinge region amino acid mutations by homologous recombination in bacteria. These mutations are at position 234 (eu') leucine to alanine and at position 23 5 leucine to alanine (Lund et al., 1991, J. Immunol., 147: 2657) β by homology Recombination inserts the variable light chain region into the human kappa constant region in the same frame. The bacterial colony will be isolated and plastid DNA will be extracted; the entire cDNA insert will be sequenced. The correct humanized heavy and light chains corresponding to each antibody will be co-transfected into (: 〇§ cells to transiently produce full-length humanized anti-Αβ globulomer antibodies. Will be agglomerated by protein a agarose 155263.doc •121 - 201139667 Cell chromatography containing recombinant chimeric antibodies was purified by gel chromatography and the bound antibody was lysed by the addition of an acid buffer. The antibody was neutralized and dialyzed into PBS (Dieder Moechars et al., J Biol) Chem 274:6483 -6492 (1999); Ausubel, FM et al., Short Protocols In Molecular Biology (4th ed. 1999) John Wiley &amp; Sons, NY. (ISBN 0-471-32938-X); Lu and Weiner, Cloning and Expression Vectors for Gene Function Analysis (2001) BioTechniques Press. Westborough, MA., p. 298 (ISBN 1-881299-21-X); Kontermann and Dubel et al., Antibody Engineering (2001) Springer-Verlag. New York. Page 790 (ISBN 3-540-41354-5); Old, RW &amp; SB Primrose, Principles of Gene Manipulation: An Introduction To Genetic Engineering (3rd edition 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; Volume 2: page 409 (ISBN 0-632-01318-4); Sambrook, J. et al., Molecular Cloning: A Laboratory Manual (2nd 1989) Cold Spring Harbor Laboratory Press, NY. Volumes 1-3 (ISBN 0-87969-309-6); Winnacker, EL From Genes To Clones: Introduction To Gene Technology (1987) VCH Publishers, NY (Horst Ibelgaufts Translation). Page 634 · (ISBN 0- 89573-614-4); each of which is incorporated herein by reference in its entirety. Although various embodiments and features have been described herein, it will be understood by those skilled in the art that the embodiments and features may be modified without departing from the scope of the invention or the invention as defined in the appended claims. And change. 155263.doc • 122- 201139667 Example 2_4: Expression and purification of humanized antibodies in HEK293 cells The antibody heavy chain encoding SEQ ID NO: 46, SET ID NO: 47 was prepared as described in Example 2.3. A DNA construct of the strand, and an antibody light chain construct encoding the polypeptide of SEQ ID NO: 48. After confirming the DNA by sequencing, all heavy and light chain DNA constructs were expanded in E. coli and DNA was purified using Qiagen Endo Free Plasmid Maxi Prep (catalog number 12362, QIAGEN) according to the manufacturer's protocol. To express the monoclonal antibody 40101111111#1, HEK293 (EBNA) cells were transiently transfected with a plastid encoding the heavy chain of 8 £(^10 1^0:46 and the light chain of SEQ ID NO: 48. The monoclonal antibody 4D10hum#2 was expressed and transiently transfected into HEK293 (EBNA) cells with a plastid encoding the heavy chain of SEQ ID NO: 47 and the light chain of SEQ ID NO: 48. HEK293 was EBNA) cells were shaken in a CO2 incubator (8 〇 / 〇 C02, 125 rpm, 37 ° C) in a culture flask (2 L Corning Cat. No. 43 1198) on a 0.5 L scale in Freestyle 293 medium (invitrogen, Carlsbad CA) Breeding. When the cell culture reaches a density of 1 χ 1 〇 6 cells per ml, the cells are transfected by adding a transfection complex. By first mixing 150 plastids encoding the light chain, 100 pg encoding the heavy chain The plastid and 25 ml Freestyle medium' was followed by the addition of 500 μΐ PEI solution (1 mg/ml (pH 7.0) 25 kDa linear polyethylenimine 'p〇iySCiences Cat. No. 23966) to prepare the transfection complex. The transfection complex was transmixed and incubated for 15 minutes at room temperature 'subsequent addition to cell culture. After transfection Cultures were grown in a CO 2 incubator (8% C02, 125 rpm, 37 ° C). Transfection 155263.doc • 123- 201139667 After 24 hours 'Add 5〇5 to the medium. Trypsin N1 solution ( Organ.Technie, La Courneuve France Cat. No. 19553). After transfection, the cells were granulated by centrifugation (16, 〇〇〇g, 30 minutes), and the supernatant containing the expressed antibody was sterile filtered ( 0·2 μϊη PES filter) and placed at 4 ° C until the start of the purification step. Protein A agarose gel affinity chromatography 'Using Pierce Thermo Scientific reagents and protocols according to the manufacturer's instructions from the supernatant purification Antibodies expressed. The purified 4D10hum antibody was quantified spectrophotometrically at 280 nm for PBS (pH 7) dialyzed solution and analyzed by mass spectrometry and size exclusion chromatography (SEC). Example 2.5: Human Affinity analysis of antibody by surface plasmon resonance (SPR) analysis The interaction of purified humanized antibodies 4D1 Ohum# 1 and 4D10hum#2 with Αβ(20-42) globulomers was evaluated using a BIAcore device. Coupling procedure according to the manufacturer Description (BIAc〇re) Goat anti-human IgG Fc (10, 〇〇〇 RU) was directly immobilized on a CM5 sensor wafer. The respective 4D1 〇hum antibodies were captured on the goat anti-human IgG Fc coated surface of the wafer by injecting 5.0 μΐ 1 pg/ml 4D10hUm antibody solution at a flow rate of 10-15 μΐ/min. The interaction of the soluble Αβ (20-42) globulomer with the 4D1 〇hum antibody on the sensor wafer was examined by injecting a globulomer solution (concentration range: 20-0.3125 nM) at a flow rate of 5 〇 μΐ/min. The association rate was monitored for 5 minutes' and the dissociation rate was monitored for 1 minute. The association rate constant was determined using the manufacturer's software and instructions from the obtained sensor map 155263.doc •124·201139667 (kQn), dissociation rate constant (k.^) and equilibrium dissociation constant (KD). The kinetics and equilibrium constants determined for three different 4D10hum #1 formulations and two different 4D10hum #2 formulations are summarized in Table 7. Table 7 also shows affinity data for antibodies #3, #4 and #5 with chimeric and humanized chains. The heavy chain of antibodies #4 and #5 is identical to 4D10hum#l or #2, and the light chain is a chimera of m4D10 VL (SEQ ID NO: 24) and human Ig kappa constant region (SEQ ID NO: 27). The light chain of antibody #3 is identical to 4D10hum#l or #2, and the heavy chain is a chimera of m4D10 VH (SEQ ID NO: 23) and human Ig γ-l constant region (SEQ ID NO: 25). Table 7: Affinity of 4D10HUM antibody against Αβ(20-42) globulomer antibody antibody batch number experiment k〇n [Μ·1〆] k〇ff [s'1] KD [M] 1 5.22x10s 3.02X10·4 5.78 xlO'10 #1759115 2 5.39χ105 3.61X1CT4 6.71xlO'10 Average 5.31x10s 3.32X10·4 6.25xlO'10 1 4.86x10s 2.81X10·4 5.78X10&quot;10 4D10hum#l #1763976 2 5.13xl05 3.04X10·4 5.93 X10—10 Average 5.00x10s 2.93ΧΚΓ4 5.86xlO'10 1 4.66x10s 2.49X10-4 5.35X10'10 #1773662 2 5.18x10s 2.87X10·4 5.53xlO'10 Average 4.92x10s 2.68X10·4 5.44x1 O'10 1 5.93x10s 2.70X10·4 4.54xlO'10 #1759119 2 5.46xl05 3.32X10·4 6.09xl0'10 4D10hum#2 Average 5.7〇xl05 3.01X10·4 5.32xlO·10 1 5.07x10s 2.68X10*4 5.29x10— 10 #1773659 2 6.86x10s 2.98ΧΚΓ4 4.35x10—10 Average 5.97xl05 2.83X10·4 4.82xl〇·10 155263.doc -125 - 201139667 Table 7 Continued Antibody Experiment k〇n [MV] k〇ff [s'1 KD [M] 4D10#3 1 6.03 χΙΟ5 3.17X10·4 5.25xlO'10 (chimeric heavy chain; light chain and 2 5.22x10s 3.49X1CT4 6.69xl〇·10 4D10hum#l and #2 are the same) Average 5 .63x10s 3.33X10·4 5.97xlO'10 4D10#4 1 4.62x10s 2.94X10·4 6.35xlO·10 (the same as 4D10hum#l; 2 5.06x10s 3.32X10*4 6.57xl〇·10 chimeric light chain) Average 4.84x10s 3.13X10·4 6.46x1 O'10 4D10#5 1 4.94xl05 2.62x1 O'4 5.3〇xlO'10 (The heavy chain is the same as 4D10hum#2; 2 4.72x10s 2.92X10*4 6.19X10'10 Light chain) Average 4.83x10s 2.77X10·4 5.75xlO·]0 Example 2.6: Analysis of antibody selectivity by point-crushing method To characterize the selectivity of a single anti-Aβ (20-42) globulomer antibody, test it Combination with different Αβ forms. To this end, serial dilutions of Αβ(1-42) form in the range of 100 pmol/μΐ to 0.00001 pmol/μΐ in PBS supplemented with 0.2 mg/ml BSA were prepared. 1 μM of each dilution was spotted on a nitrocellulose membrane. Detection was carried out by incubation with the corresponding antibody (0.2 pg/ml) followed by immunostaining with peroxidase-conjugated anti-human IgG and staining reagent BM Blue POD. Αβ standard for point-crushing method 1. Αβ(1-42) globulomer Αβ(1-42) globulomer (by dialysis exchange buffer) was prepared as described in Example la. 2· Αβ(20-42) globulomer 155263.doc -126- 201139667 Preparation of Αβ(20-42) globulomer as described in Example lb

3. Αβ(1-40) monomer, 0.1% NaOH 2.5 mg Ap(l-40) (Bachem Inc., catalog number H-1368) was dissolved in 0.5 ml 0.1% aqueous NaOH solution (fresh preparation) (=5 mg /ml), and immediately shake at room temperature for 30 seconds to obtain a clear solution. Store samples at -20t: until use.

' 4. Αβ(1-42) monomer, 0.1% NaOH 2.5 mg Ap(l-42) (Bachem Inc., Cat. No. H-1368) was dissolved in 0-5 ml 0.1% aqueous NaOH (fresh preparation) (= 5 In mg/ml), and immediately shake at room temperature for 30 seconds to obtain a clear solution. "The sample is stored at -20 ° C until use. 5. Αβ(1-42) fibril Dissolve 1 mg Ap(l-42) (Bachem Inc. Cat. No.: Η-1368) in 500 μΐ 0.1% NH4OH aqueous solution (Eppendorf tube) and stir at room temperature 1 minute. 1 〇〇 μΐ This freshly prepared Αβ(1-42) solution was neutralized with 3 〇 0 μΐ 2 〇 H NaH 2 P04; 140 mM NaCl, pH 7.4. The pH was adjusted to pH 7.4 with 1% HCl. The samples were incubated at 37 ° C for 24 hours and centrifuged (10 minutes at 10000 g). The supernatant was discarded, and the fibrils were pelleted by vortexing for 1 minute with 400 μΐ 20 mM NaH 2 P〇4; 140 mM NaCl, pH 7.4. 6. sAPPa was supplied by Sigma (catalog number S9564; 25 pg in 20 mM NaH2P04; 140 mM NaCl; pH 7.4). sAPPa was diluted to 0.1 mg/ml (= 155263.doc -127-201139667 1 pmol/μΐ) with 20 mM NaH2P〇4, 140 mM NaCl, pH 7.4, 0.2 mg/ml BSA. 7. Αβ(12-42) globulomer Αβ(12-42) globulomer was prepared as described in Example lc. Materials for the point-breaking method: Serial dilutions of Αβ standards in 20 mM NaH2P〇4, 140 niM NaCl, pH 7.4+0.2 mg/ml BSA (see 1. to 7. above) gave the following concentrations: 100 pmol /μΐ, 10 pmol/μΐ, 1 pmol/μΐ, 0.1 pmol/μΐ, 0.01 pmol/μΐ, 0.001 pmol/μΐ, 0.0001 pmol/μΐ and 0.00001 pmol/μΐ

Nitrocellulose: Trans-Blot transfer medium, pure nitrocellulose membrane (0.2 μιη); BIO-RAD anti-human POD: catalog number: 109-035-003 (Jackson Immuno Research) Detection reagent: BM Blue POD Quality, Precipitation, Cat. No.: 11442066001 (Roche) Bovine Serum Albumin, (BSA): Cat. No.: ll926 (Serva) Blocking Reagent: 5% Low Fat Milk TBS Solution Buffer: TBS: 25 mM Tris/HCl Buffer pH 7.5 + 150 mM NaCl TTBS : 25 mM Tris/HCl-buffer pH 7.5 + 150 mM NaCl + 0.05% Tween 20

PBS + 0.2 mg/ml BSA : 20 mM NaH2P04 buffer pH 7.4 + 140 mM NaCl + 0.2 mg/ml BSA Antibody solution I: 20 ml 1% low fat milk TBS solution containing 0.2 Mg/ml antibody 155263.doc • 128 · 201139667 Antibody: humanized monoclonal anti-Aβ antibody 4D10hum#l; 4.7 mg/ml OD 280 nm; antibody solution at -80 °C II: anti-human POD 1:5% in 1% low fat milk TBS solution Dilution point dot collapse procedure: 1) Each of 1 μΐ of 8 different concentrations of Αβ standard (obtained by serial dilution) was spotted on a nitrocellulose membrane at a distance of about 1 cm from each other. 2) Air dry the Αβ standard on the nitrocellulose membrane at room temperature (RT) for at least 10 minutes. (= point collapses) 3) Blocking: The point collapsed ink was incubated with 30 ml of 5% low fat milk TBS solution for 1.5 hours at room temperature. 4) Washing: Discard the blocking solution and incubate the ink at room temperature for 10 minutes with 20 ml TTBS. 5) Antibody solution I: Discard the wash buffer and incubate with the antibody solution I for 2 hours at room temperature. 6) Wash: Discard the antibody solution I and click on the ink to incubate at room temperature with 20 ml TTBS. 1 minute. The washing solution was discarded and the spotted ink was incubated with 20 ml of TTBS for 10 minutes at room temperature. The washing solution was discarded and the spotted ink was incubated with 20 ml of TBS for 10 minutes at room temperature. 7) Antibody solution II: 155263.doc -129- 201139667 Discard the wash buffer and incubate with the antibody solution u for 1 hour at room temperature. 8) Wash: Discard the antibody solution π and click on the ink at room temperature Incubate with 20 ml of TTBS for 10 minutes. Discard the washing solution and incubate the mixture for 10 minutes at room temperature with 2〇 such as TTBS. The washing solution was discarded and the spotted ink was incubated with 20 ml of TBS for 10 minutes at room temperature. 9) Color development: Discard the washing solution. The dot collapse ink was developed with 7.5 ml BM Blue POD for 1 minute. The color development was stopped by crushing the ink with a strong Ηβ wash point. Densitometric analysis based on the density of the breakpoint (GS800 density meter (Bi〇Rad) and set software Quantity one, version 4.5.0 (BioRad)) was used for quantitative evaluation. Only the relative density of the 密度β(20-42) globulomer collapse point of the 光学β(20-42) globule collapse was determined to be greater than 2〇°. The point of collapse. This threshold value for each point of collapse is determined independently. The calculated value indicates the relationship between the recognition of Αβ(20-42) globulomer and the respective Αβ forms of the established antibodies. The dot-cold method was analyzed using a humanized monoclonal anti-Aβ antibody 4D1 Ohum#1. Individual Αβ forms were applied as serial dilutions and incubated with individual antibodies for immune response (1=Αβ(1-42) globulomer; 2=Αβ(20-42) globulomer; 3=Αβ( 1-40) monomer, 0.1% NaOH; 4 = Αβ(1-42) monomer, 〇·1°/.NaOH; 5=Αβ(1-42) fibril preparation; 6=sAPPa(Sigma); The first break point: 1 pmo 1)). The results are summarized in Table 8. 155263.doc •130· 201139667 Table 8: Point-crushing method Quantitative data antigen-antibody: 4D10hum#l Αβ(1-42) globulomer&gt;10000 Αβ(20-42) globulomer 1 Αβ(1-40 Monomer enthalpy, i% NaOH 72000 0.1% Αβ(1-42) monomer NaOH 72000 Αβ(1-42) fibrils&gt;10000 sAPPa &gt;100 Αβ(12-42) globulomer 11 Examples 3: Determination of platelet factor 4 cross-reactions Example 3.1: Cross-reaction with platelet factor 4 in plasma of rock crab macaques by sandwich-ELIS A List of reagents: F96 Cert. Maxisorp NUNC-Immuno plate catalog number 439454 Binding antibody in experiment El: - Humanized monoclonal anti-Aβ antibody 4D10hum#l; 2.36 mg/ml OD 280 nm; stored at -80 °C - humanized monoclonal anti-Aβ antibody 4D10hum#2; 1.74 mg/rtil〇D280 nm; stored at - At 80 ° C • Human/mouse chimeric anti-Αβ monoclonal antibody pure hlG5 wild-type Fc framework (chim hlG5 wt); 0.99 mg/ml OD 280 nm; stored at -80 ° C (used as a positive control) - Affinity purified human polyclonal antibody hlgGl (Chemicon 155263.doc -131 - 201139667 (Millipore), catalog number AG502); 1.00 mg/ml OD 280 nm; stored at -80 °C, (used as a negative control) Reference antibody in the experimental R1: - anti-HPF4 monoclonal antibody; 4.2 mg / ml OD 280 nm; Abeam catalog number: ab49735; stored at -30 ° C (used as positive Control) - anti-Αβ monoclonal antibody pure mlG5; 1.70 mg/ml OD 280 nm; stored at -80 °C - anti-Αβ monoclonal antibody pure m4D10; 8.60 mg/ml OD 280 nm; stored at -80 °C - Monoclonal antibody purely mIgG2a; 7.89 mg/ml OD 280 nm; stored at -80 ° C (used as a negative control) Coating buffer: 100 mM sodium bicarbonate; pH 9.6 Blocking reagent for ELISA; Roche Diagnostics GmbH Cat. No.: 1112589 PBST buffer: 20 mM NaH2P〇4; 140 mM NaCl; 0.05% Tween 20; pH 7.4 PBST + 0.5% BSA buffer: 20 mM NaH2P04; 140 mM NaCl; 0.05% Tween 20; pH 7.4 + 0.50/〇BSA; Serva catalog number 11926 stone crab macaque plasma: EDTA plasma pool from 13 different donors of rock crab macaque; stored at -30 ° C trypsin inhibitor: Sigma catalog number T7902 primary antibody: pRAb-HPF4; 0.5 mg/ml : Abeam Cat. No. ab9561 155263.doc -132- 201139667 Labeling Reagents: Anti-rabbit POD Jackson ImmunoResearch Ltd. Cat. No.: 111-036-045 Staining solution: 42 mM TMB (Roche Diagnostics GmbH Cat. No. 92819060) DMSO solution; 3% H2〇2 aqueous solution; 100 mM sodium acetate, pH 4.9 Stop solution: 2 Method for preparing reagents by sulfonic acid: Binding antibody: The binding antibody is diluted to 10 Mg/ml in coating buffer. Blocking solution: The blocking reagent was dissolved in 100 ml of water to prepare a blocking stock solution, and a 10 ml aliquot was stored at -20 °C. Each plate was diluted 3 ml with 27 ml of water to block the stock solution for blocking. Preparation of blood crab macaque (Macaca fascicularis) blood sputum: stock solution: 2 ml stone crab macaque plasma pool was centrifuged at 10,000 g for 10 minutes. 1.58 ml of the supernatant was removed and diluted with 3.42 ml of PBST + 0.5% BSA buffer (= 1:3.16 dilution). Next, 50 μΐ 10 mg/ml chymotrypsin inhibitor aqueous solution was added. After incubation for 10 minutes at room temperature, the sample was filtered through a 0.22 μηη filter (Millipore Cat. No. SLGS0250S). 155263.doc -133 - 201139667 Serial dilutions of the plasma storage solution of the stone crab macaque: Number of stone crab macaque plasma dilution volume PBST+0.5% BSA buffer volume Stone crab macaque plasma final dilution 1 250 μΐ stock solution 0 ml 1:3.16 2 79 μ1(1) 171 μΐ 1:10 3 79 μΐ(2) 171 μΐ 1:31.6 4 79 μΐ(3) 171 μΐ 1:100 5 79 μΐ (4) 171 μΐ 1:316 6 79 μΐ(5) 171 μΐ 1:1000 7 79 μΐ(6) 171 μΐ 1:3160 8 Ομί 250 μΐ Buffer primary antibody solution only: Primary antibody was diluted to 1 pg/ml in PBST+0.5% BSA buffer. The dilution factor is 1:500. The antibody solution is used immediately. Labeling reagent: Anti-rabbit POD conjugate lyophilizate was reconstituted in 0.5 ml of water. 500 μΐ glycerol was added and a 100 μΐ aliquot was stored at -20 ° C for further use. The concentrated labeling reagent was diluted in PBST buffer. Dilution factor 1:10000 ° Reagent used immediately ° TMB solution: Mix 20 ml of 100 mM sodium acetate, pH 4.9 with 200 μΐ TMB stock solution and 29.5 μΐ 3% peroxide solution. The solution is used immediately. 155263.doc -134· 201139667 Experiment El standard board setting. Stone crab macaque plasma dilution. Note that each sample was run in duplicate. 1 2 3 4 5 6 7 8 9 10 11 12 Positive chimh control G5 wt 4D10hum#l 4D10hum#2 Negative control hlgGl A 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1: 3.16 No No No No B 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 No benefit no C 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1 :31.6 1:31.6 1:31.6 No No No No D 1:100 1:100 1:100 1:100 1:100 1:100 1:100 1:100 No No No E 1:316 1:316 1: 316 1:316 1:316 1:316 1:316 1:316 No No No F 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 No No No G 1: 3160 1:3160 1:3160 1:3160 1:3160 1:3160 1:3160 1:3160 No No No No No No 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 No No No Reference Standard R1 standard board setting. Stone crab macaque plasma dilution. Each sample was run in duplicate. Note 1 1 2 3 4 5 6 7 8 9 10 11 12 Positive control anti-HPF4 mAbmlG5 mAb m4D10 Negative control mIgG2a A 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 None No No No B 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:10 No No No C 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1: 31.6 1:31.6 1:31.6 Benefits None No D 1:100 1:100 1:100 1:100 1:100 1:100 1:100 1:100 No No No E 1:316 1:316 1:316 1:316 1:316 1:316 1:316 1:316 No No No No F 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 No No No No G 1 :3160 1:3160 1:3160 1:3160 1:3160 1:3160 1:3160 1:3160 No No No No No No 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 No No No No 155263.doc -135- 201139667 Program used: 1. Apply 100 μΐ of bound antibody solution per well and incubate overnight at 4 °. 2. Discard the antibody solution and wash each well 3 times with 25 μl of pBST buffer. 3. Add 265 μΐ blocking solution to each well and incubate for 15 hours at room temperature. 4. Discard the blocking solution and wash each well 3 times with 25 μM pBST buffer. 5. After preparing serial dilutions of the stone crab macaque plasma, apply 100 μM of each dilution to each well of the plate. The plates were incubated for 2 hours at room temperature. 6. Discard the plasma dilution of the stone crab macaque and wash the wells 3 times with 25 〇 y pBsT buffer. 7. Add 1 μ μ μ of the antibody solution to each well and incubate for 丨 hours at room temperature. 8. Discard the antibody solution once and wash each well 3 times with 250 μm pbst buffer. 9. Add 200 μΐ labeling solution to the mother well and incubate for 1 hour at room temperature. 1 〇·Discard ^ § solution and wash each well 3 times with 250 μM PBST buffer. 11. Add 1 μ μ〇〇 of the solution to each well. 12. Monitor the color of the plate during color development (5 - 丨 5 minutes at ambient temperature) and when the appropriate color is present, 'stop the reaction by adding 5 〇 μ 1 stop solution per well. 13. Read the absorbance at 450 nm. Data analysis: Logarithmic conversion of plasma dilution factor (X value) using the following equation: x = 1 〇 g (X). The data is captured using a log-transformed X value expressed as a slurry dilution (1: χ) on the X-axis. The 45D45Qnm value of the plasma serial dilutions of each row in the A-G column minus the OD45〇nn^ of the respective PBST blanks in the Η column. Draw the resulting background on the Y-axis 155263.doc -136· 201139667 Correction 〇〇45〇|11 „value. By curve fitting using nonlinear regression “four-parameter logic equation” to “least squares (general) The fit method (equal to the fitting method "S-type dose response (variable slope)") was used to calculate the dilution curve from the data points using the data analysis kit software GraphPad Prism (version 5.03; GraphPad Software Inc.). Curve fitting is performed for the sole purpose of data visualization and not as the basis for any other calculations (ie, area calculation under the curve). The area under the curve (AUC' or total peak area) was determined based on the non-curve fitting data, the log-transformed X value, and the 〇D45〇nm value in the measured range (final plasma dilution 1:3.16 to 1:3160). Use the following calculation settings in the Data Analysis Suite software GraphPadPrism (version 5.03; GraphPad Software Inc.): - The baseline value is set to Y = 〇.〇. - Minimum ridge height: A peak of 1 〇 % which is less than the distance from the minimum value to the maximum value γ is ignored. - Peak direction: By definition, all peaks must be used above the baseline for each individual antibody' using a commercially available anti-HPF4 antibody (Abeam catalog number: ab49735) as a reference antibody for PF4 recognition to calculate the pF4 discrimination factor, where [PF4 discrimination Factor] = [total peak area of anti-HPF4 antibody ab49735] [Total peak area of antibody to be determined] Note. The PF4 discrimination factor was calculated based on the anti-HpF4 antibody auc obtained in the reference experiment because there was no human type of anti-HPF4. The results of the test E1 and the reference experiment ri are shown in Fig. 2A and Fig. 22A and 155263.doc-137·201139667 in Table 9A and Table 9B. Example 3.2: Cross-reaction with platelet factor 4 in human plasma via a sandwich-ELISA assay The same reagent and reagent preparation procedure as in Example 3.1 was used, with the exception of: use of human PF4 (7.3 mg/ml; Molecular Innovation catalog number HPF4) Human plasma stored at -30 ° C (human EDTA plasma pool from 4 different donors; stored at -30 ° C) replaced the stone crab simian plasma. A human plasma stock solution supplemented with HPF4 was prepared as follows. A) Preparation of human plasma dilution: 2 ml human plasma pool was centrifuged at 10,000 g for 10 minutes. Remove 1.58 ml of the supernatant and dilute with 3_42 1111?6 8 butyl + .5 ° / () 8 8 8 (=1: 3.16 dilution). Next, 50 μΐ 10 mg/ml chymotrypsin inhibitor aqueous solution was added. After incubation for 10 minutes at room temperature, the sample was filtered through a 0.22 μηη filter (Millipore catalog number SLGS0250S). B) Preparation of HPF4 stock solution: Add 1 μΐ HPF4 = 73 pg/ml ° C to 99 μΐ PBST+0.5% BS 缓冲 buffer. Prepare human plasma stock solution with 10 ng/ml HPF4: Dilute to 5 ml 1:3.16 A human serum fraction of 0.69 μΐ 73 pg/ml HPF4 was added to human plasma to give a human plasma stock dilution of 10 ng/ml HPF4. 155263.doc -138- 201139667 Serial dilutions, standard plate setup, experimental procedures, and data analysis for sandwich-ELISA using human plasma with HPF4 were similar to the sandwich-ELISA for the use of stone crab macaque plasma in Example 3.1. Said. The bound antibody in Experiment E2: the same as used in Experiment E1 in Example 3.1 The bound antibody in Reference Experiment R2: the same as used in Reference Experiment R1. in Example 3.1. The results of Experiment E2 and Reference Experiment R2 are shown in Figures 1B and 12B and Tables 9A and 9B. Table 9A: Calculation of AUC (or total peak area) from logarithmic transformation data of experiments E1 and E2 depicted in Figures 20A and 20B Positive control mAb mAb Negative control chimhlGSwt2 4D10hum#l 4D10hum#2 hlgGl Stone crab macaque under the curve of the curve 1 1.255 0.042 0.075 0.075 (data from Figure 20A) HPF4/aAp antibody ratio 2 64 36 27 155263.doc -139- 1 The area under the curve is calculated as described in Example 3-1. Table 9: Logarithm of the reference experiments R1 and R2 depicted in Figure 22A and Figure 22Β2 Area under human plasma curve 1 0.949 0.067 0.116 0.113 (data from Figure 20B) HPF4/aAp antibody ratio 2 30 17 18 ” chim hlG5 wt An antibody such as that described in WO 2007/062853, that is, a monoclonal antibody having a binding affinity for Αβ(20-42) globulomers greater than that of Αβ(1·42) globulomer. 201139667 Conversion data calculation AUC (or total peak area) positive control anti-HPF4 mAb mlG5' mAb m4D10 negative control mIgG2a stone crab macaque plasma area under the curve 2 2.681 0.861 0.086 0.005 (from the data in Figure 22A) HPF4 / aAp antibody ratio 1 3 31 517 human plasma curve area 2 1.986 0.311 0.093 0.006 (data from Figure 22B) HPF4/aAp antibody ratio 1 6 21 331 υ mlG5 is an antibody as described in WO 2007/062853', ie the binding affinity for Αβ(20·42) globulomer is greater than Monoclonal antibody to the binding affinity of Αβ(卜42) globulomer. 2) The area under the curve was calculated as described in Example 3.1. Example 3.3: Determination of platelet factor 4 in plasma of stone crab macaque by comparison of sandwich-ELIS Α cross The reagents described in Example 3.1 and the conjugate antibody anti-mouse IgG should be used (Fc specific; produced in goat; Sigma catalog number: M3534; 2.3 mg/ml; stored at -2 °C for reference experiment R3) Medium mouse binding antibody) and anti-human IgG (Fc specific; produced in goat; Sigma catalog number: 12136; 2.2 mg/ml; stored at -20 ° C, used in human E3 in experiment E3 Human/mouse chimeric binding antibodies) Methods for preparing reagents: Blocking solutions, primary antibodies and TMB solutions were prepared as described in Example 3.1. Each of the aligned antibodies was diluted to 10 pg/ml in coating buffer. Binding antibody in Experiment E3: Binding antibody in the same reference experiment R3 as used in Experiment E1 in Example 3.1: same as used in Reference Experiment R1 155263.doc • 140· 201139667 in Example 3.1. Each bound antibody was PBST+0.5% BSA buffer was diluted to 10 pg/ml (stock solution) and serial dilutions were prepared as follows: Volume of antibody dilution PBST + 0.5% Volume of BSA buffer Final antibody concentration 1 250 μΐ stock solution 0 ml 10000 ng/ml 2 79 μ1(1) 171 μΐ 3160 ng/ml 3 79 Μΐ(2) 171 μΐ 1000 ng/ml 4 79μ1(3) 171 μΐ 316 ng/ml 5 79 μΐ(4) 171 μΐ 100 ng/ml 6 79μ1(5) 171 μΐ 31.6 ng/ml 7 79 μΐ(6) 171 μΐ 10 ng/ml 8 0 μΐ 250 μΐ Only buffered stone crab macaque plasma: 400 μΐ stone crab macaque plasma pool was centrifuged at 10,000 g for 10 minutes. The 158 μΐ supernatant was removed and diluted with 684 μΐ PBST + 0.5 % BSA (=1: 3.16 dilution). Next, 10 μΐ 10 mg/ml aqueous solution of the protease was added. After incubation for 10 minutes at room temperature, the sample was filtered through a 0.22 μηη filter (Millipore Cat. No. 31^802508). Subsequently, 500 μl of the 1:3.16 diluted plasma sample was further diluted 1:5.3 ml with 15.3 ml PBST + 0.5% BSA buffer to give a total dilution of 1:100. Labeling reagent: Anti-rabbit POD conjugate lyophilizate was reconstituted in 0.5 ml of water. 500 μΐ glycerol was added and a 100 μΐ aliquot was stored at -20 ° C for further use. The concentrated labeling reagent was diluted in PBST buffer. A dilution factor of 1:5000 〇 reagent is used immediately. 155263.doc -141 - 201139667 Experiment E2 combined antibody plate setup. Binding of antibody binding. Note that each bound antibody at each concentration was performed in duplicate. 1 2 3 4 5 6 7 8 9 10 11 12 Positive control chimhlG5 wt 4D10hum#l 4D10hum#2 Negative control hlgGl A 10000 10000 10000 10000 10000 10000 10000 10000 No no no B 3160 3160 3160 3160 3160 3160 3160 3160 No no No C 1000 1000 1000 1000 1000 1000 1000 1000 No No No D 316 316 316 316 316 316 316 316 No No No E 100 100 100 100 100 100 100 100 No No No F 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 None No or no G 10 10 10 10 10 10 10 10 No no no no Η 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 No or no reference experiment R3 combination antibody plate setting. Binding of antibody binding. Note that each bound antibody at each concentration was performed in duplicate. 1 2 3 4 5 6 7 8 9 10 11 12 Positive control anti-HPF4 mAbmlG5 mAb m4D10 Negative control m!gG2a A 10000 10000 10000 10000 10000 10000 10000 10000 No or no B 3160 3160 3160 3160 3160 3160 3160 3160 Nothing or nothing C 1000 1000 1000 1000 1000 1000 1000 1000 No No No D 316 316 316 316 316 316 316 316 No No No E 100 100 100 100 100 100 100 100 No No No F 31.6 31.6 31.6 31.6 31.6 31.6 31.6 31.6 No No G 10 10 10 10 10 10 10 10 No No No No No No 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 No No No No 155263.doc 142· 201139667 Procedures used: 1. Apply 1〇〇μ丨 to each well The antibody solution (anti-human IgG for Experiment E3; anti-murine 1 § for reference experiment r3) was incubated overnight at 彳7. 2. Discard the antibody solution and wash each well 3 times with 25 μl of PBST buffer. 3. The mother well was added with a 265 μΐ blocking solution and incubated for 2 hours at room temperature. 4. Discard the blocking solution and wash each well 3 times with 25 μl of pBST buffer. 5. After preparing serial dilutions of each bound antibody, apply 100 μM of each of these antibodies to each well in the plate. The plates were incubated for 2 hours at room temperature. 6. Discard the antibody solution and wash each well 3 times with 250 μM PBST buffer. 7. Add 1 μm of rock cynomolgus plasma 1:1 〇〇 dilution to each well and incubate for 2 hours at room temperature. 8. Discard the plasma solution and wash each well 3 times with 25 μM pBST buffer. 9. Add 1 〇〇 μι — antibody solution per well and incubate at room temperature. Ίο. The primary antibody solution was discarded and each well was washed 3 times with 25 μl of PBST buffer. 0 11 • 200 μΐ of labeling reagent was added per well and incubated for 1 hour at room temperature. 12. Discard the labeling reagent and wash each well 3 times with 25 μl of pBST buffer. 13. Add 1 μM TMB solution to each well. 14. Monitor the color of the plate during color development (5 · 15 minutes at ambient temperature) and when the appropriate color appears, stop the solution by adding 5 每 to each well. 15. Read the absorbance at 450 nm. As in Example 3. 丨 夹 针对 使用 石 · e e e e e · e 155 155 155 155 155 155 e e e 155 155 155 155 155 155 155 155 155 155 155 155 e 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 155 Value, and therefore the concentration action curve is calculated. Therefore, the area under the curve was determined based on the uncurved fit data, the logarithmic converted X value, and the 〇D45 〇 nm value in the measured range (final antibody concentration was 1 ng/ml to 10000 ng/ml). The results of Experiment E3 and Reference Experiment R3 are shown in Figures 21A and 23A and Tables 10A and 10B. Example 3.4: Cross-reactivity with platelet factor 4 in human plasma via a comparative sandwich-ELISA assay The same reagent and reagent preparation procedure as in Example 3.3 was used, with the exceptions: Each of the aligned antibodies used in Experiment E4 was coated with buffer. The solution was diluted to 1 μμβ/ml in the solution, and each of the aligned antibodies used in the experiment R4 was diluted to 5 μMg / ml in the coating buffer. Human PF4 (7.3 mg/ml; Molecular Innovation catalog number HPF4; Human plasma stored at -30 ° C (human EDTA plasma pool from 4 different donors; stored at -30 ° C) replaced the stone crab cynomolgus plasma. A human plasma stock solution supplemented with HPF4 was prepared as follows. A) Preparation of human plasma dilution: 4 ml of human plasma pool was centrifuged at 1 〇, 〇〇〇 g for 1 。. 3 16 ml of the supernatant was removed and diluted with 6.84 ml PBST + 0.5% BSA (=1: 3.16 dilution). Next, add 100 μΐ 10 mg/ml aqueous trypsin inhibitor. After incubation for 10 minutes at room temperature, the samples were filtered through a 0.22 μηι filter (Millipore catalog 155263.doc • 144·201139667 SLGS0250S). Subsequently, 5 ml of this 1:3.16 diluted plasma sample was further diluted with 10.8 1111?68 butyl + 0.5% 88 into buffer 1:3.16 to give a total dilution of 1:10. B) Preparation of HPF4 stock solution: Add 1 μΐ HPF4 = 73 pg/ml ° C to 99 μΐ PBST+0.5% BSA buffer. Prepare human plasma stock solution plus 10 ng/ml HPF4: to 12 ml 1:10 A 1.64 μΐ 73 pg/ml HPF4 stock solution was added to the diluted human plasma to produce a human plasma stock dilution supplemented with 10 ng/ml HPF4. The preparation of the conjugated antibody serial dilution, the binding of the antibody plate, the preparation of the blocking solution, the primary antibody, the reagent and the TMB solution were the same as in Example 3.3. Aligned antibody and bound antibody in Experiment E4: Same as used in Experiment E3 in Example 3.3 Aligned antibody and bound antibody in Reference Experiment R4: Same as used in Reference Experiment R4 in Example 3.3. Analytical procedure for alignment sandwich-ELISA using human plasma plus HPF4 (but using 1:10 dilution of human plasma in step 7) and data analysis compared to the use of stone crab cynomolgus plasma in Example 3.3 - ELISA is similar. The results of Experiment E4 and Reference Experiment R4 are shown in Figures 21B and 23B and Tables 10A and 10B. 155263.doc -145- 201139667 Table 10A: AUC (or total peak area) calculated from log-transformed data of experiments E3 and E4 depicted in Figures 21A and 21B Positive control mAb mAb chim 4D10 4D10 Negative control hlG5 wt1 hum#l Hum#2 hlgGl stone crab macaque plasma area under the curve 2 0.290 0.030 0 0 (from the data in Figure 21A) HPF4 / ίΐΑβ anti-Zhao ratio 16 158 &gt; 1583 &gt; 1583 human plasma curve area 2 0.106 0.168 0.051 0.024 (from Figure 21B Data) HPF4/aAp antibody ratio 36 23 75 157 υ chim hlG5 wt is an antibody as described in WO 2007/062853, that is, the binding affinity for Αβ(20-42) globulomer is greater than that of Αβ(1-42) A monoclonal antibody with binding affinity for globulomer. 2) The area under the curve is calculated as described in Example 3.3. 3) For the antibodies 4D10hum#2 and hlgG1, the HPF4 binding activity is too low, so that the calculated AUC is zero. Therefore, the HPF4/aAp antibody ratio cannot be calculated and is indicated as &gt; 158 (the highest ratio achieved by another antibody (4D10hum#l) in this assay). Table 10B: AUC (or total peak area) calculated from logarithmic conversion data of reference experiments R3 and R4 depicted in Figures 21A and 21B. Positive control mAb mAb negative control anti-HPF4 IH1G51 m4D10 mIgG2a stone crab macaque blood area under the curve 2 4.781 0,2768 0,04066 0,01473 (from the data in Figure 23A) HpF4/aAp anti-Zhao ratio 1 17 118 325 Human blood stasis (data from Figure 23B) Area under the curve 2 HPF4/aAp antibody ratio 3.844 1 0.165 23 0.141 27 0.033 118 155263.doc • 146 · 201139667 υ mlG5 is an antibody as described in WO 2007/062853, ie the binding affinity for Αβ(20-42) globulomer is greater than for two Αβ(ι _42) globulomers A single antibody that binds affinity. 2) The area under the curve was calculated as described in Example 33. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the amino acid sequence of the variable light chain of the humanized 4010 antibody comprising the jK2 and νκ 八 17/2-30 framework regions (SEQ ID NO: 1). All CDR regions are underlined. Figure 2 illustrates the amino acid sequence (SEQ ID NO: 2) of the variable heavy chain of the humanized 4D10 antibody comprising the human jH6 (hJH6) and VH3_53 framework regions. All CDR regions are underlined. Figure 3 illustrates the amino acid sequence (SEQ ID NO: 3) of the variable heavy chain of the humanized 4D10 antibody comprising the human JH6 and VH4_59 framework regions. All CDR regions are underlined. Figure 4 illustrates the amino acid sequence (SEQ ID NO: 4) of the variable heavy chain of the humanized 4D10 antibody comprising the human JH6 (hJH6) and VH3_53 framework regions. All CDR regions are underlined. Figure 5 illustrates an amino acid sequence (SEQ ID NO: 5) comprising a variable heavy chain of a humanized 4D10 antibody having a human JH6 and VH3_53 framework region that VH3 collectively alters I12V. All CDR regions are underlined. Figure 6 illustrates the inclusion of a VH3 co-altering I12V and a framework back mutation

The amino acid sequence of the variable heavy chain of the humanized 4D10 antibody of the human JH6 and VH3_53 framework regions of A24V, V29L, V48L, S49G, F67L, R71K, N76S and L78V (SEQ ID NO: 6). All CDR regions are underlined. 155263.doc -147- 201139667 Figure 7 illustrates the amino acid sequence (SEQ ID NO: 7) of the variable heavy chain of the humanized 4D10 antibody comprising the human JH6 and VH3_53 framework regions with framework back mutations V29L and R71K. All CDR regions are underlined. Figure 8 illustrates the amino acid sequence (SEQ ID NO: 8) of the variable heavy chain of the humanized 4D10 antibody comprising the human JH6 and VH4_59 framework regions. All CDR regions are underlined. Figure 9 illustrates the amino acid sequence (SEQ ID NO: 9) of the variable heavy chain of a humanized 4D10 antibody comprising a human JH6 and VH4_59 framework region having a Q1E alteration to prevent N-terminal pyroglutamate formation. All CDR regions are underlined. Figure 10 illustrates a humanized 4D10 antibody comprising a human JH6 and VH4_59 framework region having a Q1E alteration to prevent N-terminal pyroglutamate formation and having framework back mutations G27F, I29L, I37V, I48L, V67L, V71K, N76S and F78V. The amino acid sequence of the variable heavy chain (SEQ ID NO: 10). All CDR regions are underlined. Figure 11 illustrates the amino acid sequence of a variable heavy chain comprising a humanized 4D10 antibody having a Q1E alteration to prevent N-terminal pyroglutamate formation and having the framework back mutations G27F, I29L and V71K of the human JH6 and VH4_59 framework regions ( SEQ ID NO: 11" All CDR regions are underlined. Figure 12 illustrates the amino acid sequence of the variable light bond of the humanized 4D10 antibody comprising the Jk2 and Vk A1 7/2-3 0 framework regions (SEQ ID NO: 12) All CDR regions are underlined. Figure 13 illustrates the amine group of a variable light bond comprising a humanized 4D10 antibody having a Jk2 and Vk A17/2-30 framework region with Vk2 co-altering S7T, L15P, Q37L, R39K and R45Q. Acid sequence (SEQ ID NO: 13). All CDR regions 155263.doc -148· 201139667 are underlined. Figure 14 illustrates the inclusion of Jk2 with Vk2 co-altering S7T, L15P, Q37L, R39K and R45Q, and framework reversion mutation F36L and Vk A17/2-30 The amino acid sequence of the variable light chain of the humanized 4D10 antibody of the framework region (seq ID NO: 14). All CDR regions are underlined. Figure 15 illustrates the inclusion of V7 with a common change of S7T and Q37L. The amino group of the variable light bond of the humanized 4D1 0 antibody of the Jk2 and Vk A17/2-3 0 framework regions Sequence (SEQ ID NO: 15). All CDR regions are underlined. Figure 16 illustrates variable light bonds of humanized 4D10 antibodies comprising Jk2 and Vk A17/2-30 framework regions with Vk2 co-altering S7T, Q37L and R39K Amino acid sequence (SEQ ID NO: 16). All CDR regions are underlined. Figure I7 illustrates murine monoclonal antibody 4D10 (m4D19) and humanized 4D10 antibody comprising human JH6 (hJH6) and VH3-53 framework regions (4D10hum) Alignment of the amino acid sequence of the variable heavy chain. All CDR regions are printed in bold. X at position 12 is I or V 'position 24 X is A or V, and position 29 X is V or L, position X of X is V or L' position 49 X is S or G, position 67 X is F or L, position 71 X is R or K, position 76 X is N or S, and position 78 X is L or V. Figure 18 illustrates the murine monoclonal antibody 4D10 (m4D 19) and the amine of the variable heavy chain of the humanized 4〇1〇 antibody (4D1〇hum) containing the human JH6 and VH4-5 framework regions. The base acid sequence is aligned. All CDR regions are printed in bold. The position 1 is Q or E, the position 27 X is G or F, the position 29 X is I or L·, and the position 37 X is I or V, position X of X is I or L, and position X of X is V or L X of position 71 is V or K, X of position 76 is N or S, and X of position 78 is F or 155263.doc • 149-201139667 v. Figure 19 illustrates the alignment of the murine monoclonal antibody 4D10 (m4D19) with the amino acid sequence of the variable light chain of the humanized 4010 antibody (40101111111) comprising the Jk2 and Vk octa 17/2-30 framework regions. All CDR regions are printed in boldface. X of position 7 is S or T, X of position 15 is L or P, X of position 41 is F or L, X of position 42 is Q or L, X of position 44 is R or K, and X of position 50 Is R or Q. 20A and B show (A) stone crab macaque plasma and (B) human plasma monoclonal antibodies 4D10hum#1 and 4D10hum#2, human/mouse chimeric antibody hlG5 (as determined by sandwich-ELIS A). The positive control) and the human polyclonal antibody hlgGl (negative control) were cross-reactive with platelet factor 4 (PF-4). Binding of PF-4 to immobilized antibodies was detected. 21A and B show humanized monoclonal antibodies 4D10hum#1 and 4D10hum#2, human/mouse chimeric antibody hlG5 (A) in stone crab cynomolgus plasma and (B) human plasma as determined by comparison sandwich-ELISA ( The positive control) and the human polyclonal antibody hlgGl (negative control) were cross-reactive with platelet factor 4 (PF-4). These antibodies were captured on plates by immobilized anti-mouse IgG. Binding of PF-4 to the captured antibody was detected. 22A and B show (A) stone crab macaque plasma and (B) murine monoclonal antibodies m4D10 and mlG5, anti-human PF-4 antibody (positive control) and IgG2a (negative) as determined by sandwich-ELISA. Control platelet factor 4 (PF-4) cross-reacted. Binding of PF-4 to immobilized antibodies was detected. Figure 23 A and B show (A) stone crab macaque plasma and (B) murine monoclonal antibodies m4D10 and mlG5, anti-human 155263.doc •150· 201139667 class PF-4 as determined by comparison sandwich-ELISA The antibody (positive control) and platelet factor 4 (PF-4) of IgG2a (negative control) were cross-reactive. These antibodies were captured on plates by immobilized anti-mouse IgG. Binding of PF-4 to the captured antibody was detected.

Figure 24 illustrates the human JH6 and VH3_53 framework regions comprising a VH3 co-altering I12V and a framework back mutation. A24V, V29L, V48L, S49G, F67L, R71K, N76S and L78V; and Ig γ-l constant region of humanized 4D10 antibody heavy The amino acid sequence of the strand (SEQ ID NO: 46). All CDR regions are underlined. Figure 25 illustrates human JH6 and VH4_59 framework regions comprising a Q1E change to prevent Ν terminal pyroglutamate formation and having framework back mutations G27F, I29L, I37V, I48L, V67L, V71K, N76S and F78V; and Ig γ-l The amino acid sequence of the heavy chain of the humanized 4D10 antibody of the constant region (SEQ ID NO: 47). All CDR regions are underlined. Figure 26 illustrates a variable light chain comprising a Jk2 and Vk A17/2-30 framework region with Vk2 co-altering S7T, L15P, Q37L, R39K and R45Q, and a framework back mutation F36L; and an Ig κ constant region of a humanized 4D10 antibody Amino acid sequence (SEQ ID NO: 48). All CDR regions are underlined. 155263.doc -151 - 201139667 Sequence Listing

&lt;110&gt; American sub-culture company &lt;120&gt; starch-like binding protein &lt;130&gt; 10419PCT &lt;140&gt; 100113264 &lt;141&gt; 2011-04-15 &lt;150&gt;61/324,386;61/373,825; 61/446,624 &lt;151&gt;2010-04-15;2010-08-14; 2011-02-25 &lt;160&gt; 48 &lt;170&gt; Patentln version 3.5 &lt;210&gt; 1 r &lt;211&gt; 113 &lt;212&gt ; PRT &lt; 213 &gt; Synthesis &lt;220&gt;&lt;221&gt; raisc_feature &lt;222&gt; (7).7(7) &lt;223&gt; Kaa may be Ser or Thr &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (15)7, (15) &lt;223&gt; Xaa may be Leu or Pro &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (41) 7. (41)

Xaa may be Phe or Leu &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (42)..(42) &lt;223&gt; Xaa may be Gin or Leu &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (44)., (44) &lt;22 3&gt; Xaa may be or Ly s &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (50) 7. (50) &lt;22$&gt; jiaa For Ai*g or Gin &lt;400&gt; 1

Asp Val Val Met Thr Gin Xaa Pro Leu Ser Leu Pro Val Thr Xaa Gly 15 10 15

Gin Pro Ala Ser lie Ser Cys Lys Ser Scr Gin Ser Leu Leu Asp lie 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Xaa Xaa Gin Xaa Pro Gly Gin Ser 35 40 45

Pro Xaa Arg Leu He Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80

Ser Arg Val GIu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gin Gly 85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu Me Lys 155263 - Sequence Listing.doc 201139667 100 105 110

Arg &lt;210&gt; 2 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (12)..(12) &lt;223&gt; Xaa Is lie or Val &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (24), (24) &lt;223&gt; Xaa may be Ala or Val &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; 29)..(29) &lt;223&gt; Xaa may be Val or Leu &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (48)..(48) &lt;223&gt; Xaa may be Val or Leu &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (49)..(49) &lt;223&gt; Xaa may be Ser or Gly &lt;220&gt;&lt;221&gt; misc.feature &lt;222&gt; (67) ..(67) &lt;223&gt; Xaa may be Phe or Leu &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (71)..(71) &lt;223&gt; j (aa may be human rg or Lys &lt;220&gt;&lt;22i&gt; misc feature &lt;222&gt; (76)..(76) &lt;22^&gt; Xaa may be Asn or Ser &lt;220&gt;&lt;221&gt; misc.feature &lt;222&gt; 78)..(78) &lt;223&gt; Xaa can be Leu or Val &lt;400&gt; 2

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Xaa Gin Pro Gly Gly 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Xaa Ser Gly Phe Thr Xaa Ser Ser Tyr 20 25 30

Gly Val His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Xaa 35 40 45

Xaa Val lie Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60 155263 - Sequence Listing.doc 201 139667

Ser Arg Xaa Thr 11c Ser Xaa Asp Asn Ser Lys Xaa Thr Xaa Tyr Leu 65 70 75 80

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Ding rp Gly Gin Gly Thr Tht Val Thr Val Ser Ser 100 105 no 0 12 3 &lt;21&lt;21&lt;21&lt;21

I12PRTI &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (1)..(1) &lt;223&gt; jCaa may be Gin or Glu &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (27)7 (27) &lt;223&gt; Xaa may be Gly or Phe &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (29)..(29) &lt;223&gt; iCaa is lie or Leu &lt;220&gt;&lt;22]&gt; misc feature &lt;222&gt; (37)..(37) &lt;223&gt; Xaa may be lie or Val &lt;220&gt;&lt;221&gt; misc.feature &lt;222&gt; (48).. (48) &lt;223&gt; 3 (aa may be lie or Leu &lt;220&gt;&lt;22l&gt; misc feature &lt;222&gt; (67)7.(67) &lt;223&gt; Xaa inch is Val or Leu &lt;220&gt;;&lt;221&gt; misc_feature &lt;222&gt; (71)7.(71) &lt;223&gt; Xaa kiss is Val or Lys &lt;220&gt;&lt;22]&gt; miscJeaUire &lt;222&gt; (76)7.(76 &lt;223&gt; Xaa may be Asn or Ser &lt;220&gt;&lt;221&gt; misc feature &lt;222&gt; (78)..(78) &lt;223&gt; Xaa may be Phe or Val &lt;400&gt;

Xaa Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Xaa Ser Xaa Ser Scr Tyr 20 25 30

Gly Val His Ding rp Xaa Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Xaa 155263 - Sequence Listing.doc 201139667 35 40 45

Gly Val lie Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Xaa Thr lie Ser Xaa Asp Thr Ser Lys Xaa Gin Xaa Ser Leu 65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Scr Ser 100 105 110 &lt;210&gt; 4 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;^00&gt;

Giu Val Gin Leu Val Glu Ser Gly Gly Gly Leu lie Gin Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr 20 25 30

Gly Val His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Scr Val lie Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80

Gin Mel Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 100 105 110 &lt;210&gt; 5 &lt;211&gt; 112 &lt;212&gt; m &lt;213&gt; Synthesis &lt;400&gt;

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 15 10 15

Ser Leu Arg Leu Scr Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Tyr 20 25 30

Gly Val His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Va) lie Trp Arg Gly Gly Arg 11c Asp Tyr Asn Ala Ala Phe Met 50 55 60 -4 - 155263 - Sequence Listing.doc 201139667

Ser Arg Phe Thr He Ser Afg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80

Gin Met Asn Ser Leu Arg Ala GIu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Ding rp Gly Gin Gly Thr Thr Vai Thr Val Ser Ser 100 105 110 &lt;210&gt; 6 &lt;211&gt; 112 &lt;212&gt; PR Ding &lt;2i3&gt; Synthesis &lt;400&gt;

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val G3n Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Val Scr Gly Phc Thr Leu Ser Ser Tyr 20 25 30

Giy Val His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45

Gly Val lie Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Leu Utr lie Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu 65 70 75 80

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Vai Ding rp Gly Gin Gly Thr Thr Vai Thr Val Ser Ser 100 105 110 &lt;210&gt; 7 &lt;211&gt; 112 &lt;2I2&gt; PRT &lt;2]3&gt;Synthesis &lt;400&gt;

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu lie Gin Pro Gly Gly ] 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Ser Tyr 20 25 30

Gly Val His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Val lie Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Phe Thr lie Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 155263 - Sequence Listing.doc 20 3 se7sn 6^IA 6Me 9ln A, oo Γ u 6 5 L 8 y5 C9 Γ Ty Γ Ty va Cq A1έ90 ps G, rp Tr va po so A1 Γ csns r8 /5 •o

Th _c s Γ 6 s — o 1 Γ Th va f Th 10&gt;n&gt;12&gt;13&gt; 22 22 1 &lt;&lt;&lt;&lt;&lt; G1 va 2T into &gt;11^8

Le, G5 c s g c 5 SI r0 p s Ly va u u

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Tyr 20 25 30

Gly Val His Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie 35 40 45

Gly Val lie Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phc Ser Leu 65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 100 105 110 &lt;210&gt; 9 &lt;23I&gt; 112 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Glu Val Gin Leu Gin Glu Scr Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser Tyr 20 25 30

Gly Val His Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp He 35 40 45

Gly Val lie Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu 65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 -6- 155263 - Sequence Listing.doc 201139667

Arg Asn Ser Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 100 105 110 &lt;210&gt; 10 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Glu Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30

Gly Val His Trp Val Arg Gin Pro Pro Gly Lys G) y Lea Glu Trp Leu 35 40 45

Gly Val He Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Leu Thr lie Ser Lys Asp Thr Ser Lys Ser Gin Val Scr Leu 65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Trp Gly G]n Gly Thr Thr Val Thr Val Ser Ser 100 105 110 &lt;210&gt; 11 &lt;211&gt; 112 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Glu Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Scr Leu Ser Ser Tyr 20 25 30

Gly Val His Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp 11c 35 40 45

Gly Val lie Ττρ Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Val Thr lie Ser Lys Asp Thr Ser Lys Asn Gin Phe Ser Leu 65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Scr Ser 100 105 110 &lt;210> 12 155263 - Sequence Listing.doc 201139667 &lt;211&gt; 113 &lt;212> PRT &lt;213&gt; Synthesis &lt;400&gt; 12

Asp Val Val Met Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 15 10 15

Gin Pro Ala Ser lie Ser Cys Lys Ser Ser Gin Ser Leu Leu Asp lie 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gin Gin Arg Pro Gly Gin Ser 35 40 45

Pro Arg Arg Leu He Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val G】y Vai Tyr Tyr Cys Trp Gin Gly 85 90 95 rrhr His Phe Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 110

Arg &lt;2i0&gt; 13 &lt;21]&gt; 113 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;4〇〇&gt; 13

Asp Val Val Met Tlir Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 15 10 15

Gin Pro Ala Ser lie Ser Cys Lys Ser Ser Gin Ser Leu Leu Asp Jle 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gin Lys Pro Gly Gin Ser 35 40 45

Pro Gin Arg Leu lie Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gin Gly 85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu 11c Lys 100 105 110

Arg 155263 - Sequence Listing.doc 201139667 &lt;210&gt; 14 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15

Gin Pro Ala Ser lie Ser Cys Lys Ser Ser Gin Ser Leu Leu Asp He 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Ττρ Leu Leu Gin Lys Pro Gly Gin Ser 35 40 45

Pro Gin Arg Leu lie Ding Leu Val Ser Lys Leu Asp Ser G】y Va】Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gin Gly 85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 110

Arg &lt;2]0&gt; 15 &lt;2H&gt; 113 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Asp Vai Val Met Thr Gin Thr Pro I^eu Ser Leu Pro Val Thr Leu Gly 15 10 15

Gin Pro Ala Ser lie Ser Cys Lys Ser Ser Gin Ser Leu Leu Asp lie 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Ding rp Leu Leu Gin Arg Pro Gly Gin Ser 35 40 45

Pro Arg Arg Leu lie Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Glv Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gin Gly 85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 110 -9- 155263 - Sequence Listing.doc 201139667

Arg &lt;210&gt; 16 &lt;211&gt; 113 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt; 16

Asp Val Val Met Thr Gin Thr Pto Leu Ser Leu Pro Val Thr Leu Gly 15 10 15

Gin Pro Ala Ser He Ser Cys Lys Ser Ser Gin Ser Leu Leu Asp ile 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Leu Gin Lys Pro Gly Gin Ser 35 40 45

Pro Arg Arg Leu lie Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Fro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr l.eu Lys Ile 65 70 75 ‘80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gin Gly 85 90 95

Thr His Phe Pro Tyr ITir Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 110

Arg &lt;210&gt; 17 &lt;211&gt; 5 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Ser Tyr Gly Val Hi! &lt;210&gt; 18 &lt;211&gt; 16 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt; 18

Val lie Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met Ser 15 10 15 &lt;230&gt; 19 &lt;211&gt; 4 &lt;212&gt; PRT &lt;2!3&gt; Synthesis &lt;400&gt;

Asn Ser Asp Val 10- 155263 - Sequence Listing.doc 201139667

成2016 £2016 £ 合 205 Γ &gt;&gt;&gt;&gt;&gt;se 0123 o ]n n n Λν s 4 y VV&lt;N/ &lt; LI

As u e 5 LI Γ Ty Γ Th s Ly y G, p so A ] c p s u Le u Le Γ c S5 T into 217 is true &lt;210&gt;&lt;211&gt;&lt;212&gt;&lt;213&gt;&lt;400&gt;

Leu Val Ser Lys Leu Asp Ser &lt;210&gt; 22 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Trp Gin Gly Thr His Phe Pro Tyr Thr &lt;210&gt; 23 &lt;211&gt; 112 &lt;212&gt; PRT &lt;2Π&gt; Synthesis &lt;400&gt; 23

Gin Val Gin Leu Lys Gin Ser Gly Pro Ser Leu lie Gin Pro Ser Gin 15 10 15

Ser Leu Ser lie Thr Cys Thr Val Ser Gly Phe Sei Leu Thr Ser Tyr 20 25 30

Gly Val His Trp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45

Gly Val He Trp Arg Gly Gly Arg lie Asp Tyr Asn Ala Ala Phe Met 50 55 60

Scr Arg Leu Ser He Thr Lys Asp Asn Sex Lys Ser Gin Val Phe Phe 65 70 75 80

Lys Met Asn Ser Leu Gin Ala Asp Asp Thr Ala lie Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser 100 105 110 &lt;210&gt; 24 &lt;21!&gt; 113 &lt;212&gt; PRT &lt;2i3&gt; Synthesis-11 - 155263 - Sequence Listing.doc 201139667 &lt;400&gt; 24

Asp Val Val Met Thr Gin Thr Fro Leu Thr Leu Ser Val Thr lie Gly 15 10 15

Gin Pro Ala Ser He Ser Cys Lys Ser Ser Gin Ser Leu Leu Asp lie 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gin Arg Pro Gly Gin Ser 35 40 45

Pro Lys Arg Leu He Tyr Leu V2I Ser Lys Leu Asp Ser Gly Va] Pro 50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gin Gly 85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys 100 105 110

Arg &lt;210&gt; 25 &lt;211&gt; 330 &lt;2)2&gt; PRT &lt;213&gt; Synthesis &lt;400&gt; 25

Ala Ser Thr Lys Gly Pro Ser Val Pile Phe Leu Ala Pro Ser Ser Lys 15 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 75 80

Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95

Lys Va! 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 lie Ser Arg Thr Pro Glu Vai Thr Cys 130 135 140 •12· 155263-Sequence List.doc 201139667

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 Gin Tyr Asn Scr ΊΉγ Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lvs Val Ser Asn 195 200 205

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220

Gin Pro Arg Glu Pro Gin Val Tyr Thr l^u Pro Pro Ser Arg Giu Glu 225 230 235 240

Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255

Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 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 Ding yT Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 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

Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 &lt;210&gt; 26 &lt;211&gt; 330 &lt;2!2&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 Ί5 80 -13- 155263 - Sequence Listing.doc 201139667

Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys 85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125

Lys Pro Lys Asp Thr Leu N3et lie Scr Arg Thr Pro Glu Val Thr Cys 130 135 140

Val Val Val Asp Val Ser His Glu Asp Fro Glu Val Lys Phe Asn Tr 145 150 155 16i

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175

Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu ]80 J85 190

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220

Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240

Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255

Pro Ser Asp 11c Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Scr Phe Phe 275 280 285

Leu Tyr Ser Lys Uu Tl\r Val Asp Lys Ser Arg Trp Gin Gin 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

Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 &lt;210&gt; 27 &lt;211&gt; 106 &lt;212&gt; FRT &lt;213&gt; Synthesis &lt;400&gt;

Thr Val Ala Ala Pro Ser Val Phe Me Phe Pro Pro Ser Asp Glu Gin 15 10 15 • 14· 155263 - Sequence Listing.doc 201139667

Leu Lys Ser Gly Tin Ala Ser Val Val Cys Leu Leu Asti Aim Phe Tyr 20 25 30

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 35 40 45

Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr 50 55 60

Tyr Ser Leu Scr Scr Thr Leu Thr Leu Scr Lys Ala Asp Tyr Glu Lys 65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Tlir His Gin Giy Leu Ser Ser Pro 85 90 95

Val Thr Lys Scr Phe Asn Arg Gly Glu Cys 100 105 &lt;210&gt; 28 &lt;2]1&gt; 105 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Gin Pro Lys Ala Ala Pro Ser Val Ήίγ Lcu Phe Pro Pro Ser Ser Glu 15 10 15

Glu Leu Gin Ala Asn Lys Ala Thr Leu Val Cys Leu lie Ser Asp Phe 20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gin Ser Asn Asn Lys 50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gin Trp Lys Ser 65 70 75 80

His Arg Ser Tyr Ser Cys Gin Val Thr His Glu Gly Ser Thr Val Glu 85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 &lt;210&gt; 29 &lt;211&gt; 43 &lt;212&gt; PRT &lt;2I3&gt; Synthesis &lt;400&gt;

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 15 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys G]y Ala lie He 20 25 30 •15· 155263-Sequence List.doc 201139667

Gly Leu Met Val Gly Gly Val Val He Ala Thr 35 40 &lt;210&gt; 30 &lt;211&gt; 42 &lt;m&gt; prt &lt;213&gt; Synthesis &lt;400&gt; 30

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 15 10 15

Leu Val Phe Phe Ala Glu Asp Va) Gly Ser Asn Lys Gly Ala lie lie 20 25 30

Gly Leu Met Val Gly Gly Val Val lie Ala 35 40 &lt;210&gt; 31 &lt;21I&gt; 42 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Asp Ala Giu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys 15 10 15

Leu Val Fhe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala lie He 20 25 30 G!y Leu Met Val Gly Gly Val Val lie Ala 35 40 Ding Cheng 3321^b&gt;Λ&gt; ο 1 64 3 21212121 &lt;400&gt; 32

Val His His Gin Lys I-eu Val Phe Phe Ala Glu Asp Val Gly Ser Asn 15 10 15

Lys Gly Ala lie He Gly Leu Met Val Gly Gly Val Val He Ala 20 25 30 &lt;210&gt; 33 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He Gly Leu Met 15 10 15

Val Gly Gly Val Val 丨le Ala 20 &lt;210&gt; 34 &lt;211&gt; 30 16- 155263 - Sequence Listing.doc 201139667 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt; 34

Glu Va! Gin Leu Val Glu Ser Gly Gly Gly Leu lie Gin Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser 20 25 30 &lt;210&gt; 35 &lt;2ll&gt; 14 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 &lt;210&gt; 36 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt; 36

Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin 15 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 nPRT (&gt;&gt; Λ &gt; 01Ζ3 2222 成 &lt;400&gt; 37

Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 1 5 10 T into 3830PR 10 &gt;]1 &gt;12&gt;13&gt; 212 2 2 &lt;400&gt; 38

Gin Val Gin Leu Gin Glu Ser Gly Pro Gly LeO Val Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Va] Ser Gly Gly Ser lie Ser 20 25 30 Ding Cheng 3914PR &gt;&gt;&gt;&gt; 012 3 &lt;21&lt;21&lt;21&lt;21 &lt;400&gt; 39

Trp lie Arg Gin Pro Pro Gly Lys G】y Leu Glu Trp lie G]y 1 5 10 •17· 155263-Sequence List.doc 201139667 T into hr milk ^ book 40'1 b&gt; Λ &gt; .6va 1 z 3 1 «i 1 1J Ob 4 Γ VV &lt;&lt;&lt; A 1

Leuu Γ c s c ph Π 5 Π As sLvlo V4 c s Γ Th p s n* va Γ 6 s 5

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 &lt;210&gt; 41 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 1 5 10

Th5 1e T into al 4223PR · 42f &gt;&gt;&gt;&gt; va lx «-- · « · 11 2 9-22 4 s &lt;v &lt;&lt;&lt;&lt;&lt;&lt;&lt;&lt;&gt;&gt; Al 6 5 LI Γ Th va r0 pu Le Γ Co SI ueo pr Γ cs

Gin Pro Ala Ser lie Ser Cys 20 &lt;210&gt; 43 &lt;212&gt; Ϊ5 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt;

Trp Phe Gin Gin Arg Pro Gly Gin Ser Pro Arg Arg Leu lie Tyr 15 10 15 T into 4432PR and 44 &gt;&gt;&gt;&gt;&gt; ^ ¢51 3 Q II 11 1» 4- fS &lt;2&lt;2&lt;;2&lt;2&lt;4

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15

Leu Lys He Ser Arg Va] CIu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 &lt;210&gt; 45 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt; 45 •18· 155263· Sequence table.doc 201139667

Phe Gly Gin Gly Thr Lys Leu Glu lie Lys Arg 1 5 10 &lt;210&gt; 46 &lt;211&gt; 442 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt; 46

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 15 JO 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Leu Ser Ser Tyr 20 25 30

Gly Val His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45

Gly Val lie Trp Arg Gly Gly Arg He Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Leu Thr lie Ser Lys Asp Asn Ser Lys Ser Thr Va丨 Tyr Leu 65 70 75 80

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Scr 100 105 no

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125

Ser Thr Ser Gly Gly Thr Ala Ala I^u Gly Cys Leu Val Lys Asp Tyr 130 135 140

Phe Pro Glu Pro Val Thr Va) Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160

Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 165 170 175

Leu Ser Ser Va) Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 180 185 190

Tyr IU Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195 200 205

Lys Val Glu Pro Lys Ser Cys Asp Lys TTir His Thr Cys Pro Pro Cys 210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 -19- 155263 - Sequence Listing.doc 201139667 6 s va po s 6 A 2 va va va rp Tr n As Co h 7 p 2 s Ly va u3r0 p P5 s 6 re A 0 Γ ps 5 LV28 Γ Th s Ly 3 A, Π As s'o 1 8 H 2 va u G1 va ym P5 s ΤΑ 2 va Γ y

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Ty 6 ph y GI s 5 y6 L3 d V u L· s cy Γ n u36 Γ €s va , n n As s 5 y5 L3 T Th Me

Asl r o € 7 S3 o Γ rp T u 5 ]V G 3 va pr( n yo 18 G 3 n s Γ c s s

s A 5 s ph40 e £ Γ 6s ly p As r 5 e 9 S3 psu Le va o Γ oo Γ 9 p 3 Γ Th Γ Th s Ly Γ Ty rp Tr r £ A ro el s 4 8 Ly ps va Γ Th u 5 Co L4 s Ly Γ es Γ Ty uu $ y5 Gl41 Γ [y so •1 3 H4 Π As s Hi u Le BM u 5 12 G4 5 Hi c va Γ ε s so y2 C4 Γ es c ph va y oo r 4 p 4 Γ 6s u Le Γ cs u Le Γ 5 e3 s 4 s Ly 2T into 4744, 0 &gt; π &gt; 12 &gt; 13 &gt; 2212121 &lt; 400 &gt; 47

Glu Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Set Glu 15 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30

Gly Val His Trp Val Arg Gin Pro Pro G]y Lys Gly Leu Glu Trp Leu 35 40 45

Gly Val lie Trp Arg Gly Gly Arg He Asp Tyr Asn Ala Ala Phe Met 50 55 60

Ser Arg Leu Thr lie Ser Lys Asp Thr Ser Lys Ser Gin Val Ser Leu 65 70 75 80 · 20· 155263 · Sequence Listing _doc 201139667

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asn Ser Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 100 105 110

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125

Ser Thr Ser Gly Gly Thr Ala Ala Leu G]y Cys Leu Val Lys Asp Tyr Γ30 135 140

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160

Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 165 170 175 ixu Ser Scr Val Val Thr Va] Pro Ser Ser Ser Leu Gly Thr CJln Thr 180 185 190

Tyr lie Cys Asn Val Asn His Lys Pro Scr Asn Thr Lys Val Asp Lys 195 200 205

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pto Cys 210 215 220

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys 245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285

Glu Gin Tyr Asn Ser Thr Tyr Arg Va) Val Ser Val Leu Thr Val Leu 290 295 300

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys Ala Lys Gly 325 330 335

Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 340 345 350

Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365

Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn 370 375 380 • 21 · 155263 - Sequence Listing.doc 201139667

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400

Leu Tyr Scr Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn 405 410 415

Val Phe Scr Cys Ser Val Met His Glu Ala Leu His As His Hisr Thr 420 425 430

Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 &lt;210&gt; 48 &lt;211&gt; 219 &lt;212&gt; PRT &lt;213&gt; Synthesis &lt;400&gt; 48

Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 15 10 15

Gin Pro Ala Ser Me Ser Cys Lys Ser Ser Gin Ser Leu Leu Asp lie 20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gin Lys Pro Gly Gin Ser 35 40 45

Pro Gin Arg Leu lie Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gin Gly 85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 110

Arg Thr Va] Ala Ala Pro Ser Va) Phe He Phe Pro Pro Ser Asp Glu 115 120 125

Gin Leu Lys Ser Gly Thr Ala Ser Va! Val Cys Leu Leu Asn Asn Phe 130 135 140

Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 145 150 155 360

Scr Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Scr Lys Asp Se; 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 Gin Gly Leu Ser Ser 】 95 200 205 -22- 155263 - Sequence Listing.doc 201139667

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys -23- 155263 - Sequence Listing.doc

Claims (1)

201139667 VII. Patent Application Range: 1. A binding protein comprising: at least 90% of the first amino acid sequence with SEQ ID NO: 2 or SEQ ID NO: 3; and at least 90% with SEQ ID NChl 2. A second amino acid sequence according to claim 1, wherein the first amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. 3. The binding protein of claim 1, wherein the second amino acid sequence is at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. 4. The binding protein of any one of claims 1 to 3, comprising: a first amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 &amp; SEQ ID NO: 11; &amp; an amine group selected from the group consisting of The acid sequence is at least 90% such that the second amino acid sequence is SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. 5. The binding protein according to any one of claims 1 to 4, wherein the binding protein is selected from the group consisting of immunoglobulin molecules, disulfide-linked 155263.doc 201139667 Fv, monoclonal antibody, scFv, Chimeric antibodies, single domain antibodies, CDR_grafted antibodies, bifunctional antibodies, humanized antibodies, multispecific antibodies, Fabs, dual specific antibodies, DVDs, Fabs, bispecific antibodies, F(ab')2 and Fv. 6. The binding protein of any one of clause 5, further comprising an immunoglobulin light bond having an amino acid sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID ΝΟ. Fixed area. 7. The binding protein according to any one of items 1 to 6, wherein the binding protein is further encapsulated with an agent selected from the group consisting of an immunoadhesive molecule, a developer, and a therapeutic agent. 8. The binding protein of any one of clauses 1 to 7, wherein the binding protein has a human glycosylation pattern. An isolated nucleic acid encoding the binding protein of any one of claims 8 to 8. A vector comprising the isolated nucleic acid of claim 9. 11. A host cell comprising the vector of claim 1 . A method of producing a binding protein comprising culturing a host cell as claimed in claim 10 in a medium sufficient to produce a binding protein. 13. A binding protein '(iv) is produced according to the method of claim u. A pharmaceutical composition comprising a binding protein according to any one of claims 1 to 8 or 13 and a pharmaceutically acceptable carrier. 15. The pharmaceutical composition of claim 14 which additionally comprises at least one other therapeutic agent. 16. A composition for releasing a binding protein, the composition comprising: 155263.doc 201139667 u) a formulation, wherein the formulation comprises a binding protein according to any one of claims 8 to 8 and a component, wherein the combination The protein is crystallized; and (b) at least one polymeric carrier. 17. A method of treating a disease or condition in a subject selected from the group consisting of: anti-trypsin-deficient, C1-inhibitor-deficient vascular water, swollen, antithrombin-deficient thromboembolic disease, Kuru _ kuru, Creutzfeld-Jacob disease / scrapie, bovine cerebral palsy, Gemmann Straussier_Scheinker disease, lethal familial Insomnia, Huntington's disease, spinal cerebellar dysfunction, Machado-Joseph atrophy, dentate red globus pallidus, frontotemporal dementia, sickle cell anemia, no Stable hemoglobin inclusion body hemolysis, drug-induced inclusion body hemolysis, Parkinson's disease, systemic AL-type starch degeneration, nodular AIj-like starch degeneration, systemic AA-type starch degeneration, prostate-like starch degeneration, blood Dialysis-like starch degeneration, hereditary (Icelandic) cerebral vascular disease, Pretin's disease, familial visceral starch degeneration, familial viscera. Polyneuropathy Familial visceral starch degeneration, senile systemic powdery degeneration, familial amyloid neuropathy, familial cardiac starch I&quot; Alzheimer's disease, Down syndrome, thyroid Medullary Carcinoma and Type 2 Diabetes (T2DM) The method achieves treatment by administering to the individual a binding protein according to any one of claims 1 to 8 or 13. 155263.doc