Anti-Abeta Antibodies

Abstract

Antibodies that bind human beta-amyloid peptide, methods of detecting, measuring and treating amyloidogenic disorders with said antibodies, pharmaceutical compositions comprising the antibodies and methods of manufacture are provided.

Description

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/055,813, filed Jul. 23, 2020, U.S. Provisional Patent Application No. 63/086,589, filed Oct. 1, 2020, U.S. Provisional Patent Application No. 63/187,379, filed May 11, 2021, and U.S. Provisional Patent Application No. 63/219,611, filed Jul. 8, 2021, all of which are incorporated by reference herein in their entirety.

SEQUENCE LISTING STATEMENT

[0002] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Oct. 21, 2021, having the file name "20-1030-US-CON2_Sequence-Listing_ST25.txt" and is 155 kb in size.

FIELD

[0003] The present disclosure relates to anti-Amyloid beta (A.beta.) antibodies as well as compositions and methods of their use.

BACKGROUND

[0004] Alzheimer's disease (AD) is a progressive disease resulting in senile dementia. The disease is generally categorized as late onset, which occurs in old age (65+ years) and early onset, which develops well before the senile period, i.e., between 35 and 60 years. Disease pathology appears to be the same for both types of disease, but abnormalities tend to be more severe and widespread in cases beginning at an earlier age. The disease is characterized by at least two types of lesions in the brain, neurofibrillary tangles and senile plaques. Neurofibrillary tangles are intracellular deposits of microtubule associated tau protein consisting of two filaments twisted about each other in pairs. Senile plaques (i.e., amyloid plaques) are areas of disorganized neuropil up to 150 .mu.m across with extracellular amyloid deposits at the center which are visible by microscopic analysis of sections of brain tissue. The accumulation of amyloid plaques within the brain is also associated with Down's syndrome and other cognitive disorders.

[0005] The principal constituent of the plaques is a peptide termed A.beta. (Abeta) or .beta.-amyloid peptide. A.beta. peptide is a 4-kDa internal fragment of 39-43 amino acids of a larger transmembrane glycoprotein termed amyloid precursor protein (APP). As a result of proteolytic processing of APP by different secretase enzymes, A.beta. is primarily found in both a short form, 40 amino acids in length, and a long form, ranging from 42-43 amino acids in length. Part of the hydrophobic transmembrane domain of APP is found at the carboxy end of A.beta., and may account for the ability of A.beta. to aggregate into plaques, particularly in the case of the long form. Accumulation of amyloid plaques in the brain eventually leads to neuronal cell death. The physical symptoms associated with this type of neural deterioration characterize Alzheimer's disease.

SUMMARY

[0006] The present disclosure relates to antibodies (and antibody fragments) that specifically bind to A.beta., methods of producing such antibodies and antibody fragments and associated nucleic acids, methods of treatment of patients with A.beta.-related neurological disorders, pharmaceutical formulations and compositions of antibodies that show high affinity binding to A.beta. for prophylactic and/or therapeutic use to, for example, treat, reduce the risk of or delay the outset of amyloidogenic disease, prevent, reduce or inhibit markers of amyloidogenic disease, e.g., A.beta. plaques, and improve cognition. The present disclosure further relates to methods of detecting amyloid plaques and measuring the efficacy of treatment in patients being treated for amyloidogenic disease. The disclosure is based, at least in part, on the identification and characterization of monoclonal antibodies that specifically bind to A.beta. peptide and are effective at reducing plaque burden and neutralizing soluble A.beta. species associated with amyloidogenic disorders.

[0007] In various aspects, the disclosure are directed to antibodies or fragments thereof that that specifically binds to A.beta. peptide. The antibodies and fragments include a heavy chain variable region including heavy chain CDR1, CDR2 and CDR3 and a light chain variable region including light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR1, CDR2 and CDR3 and the light chain CDR1, CDR2 and CDR3 are as shown for one of the antibodies in Table 1. In addition, the antibodies or fragments or fragments of the disclosure may have a heavy chain variable region that is as shown for one of the antibodies in Table 1 and may have a light chain variable region that is shown for one of the antibodies in Table 1.

[0008] In various embodiments of the disclosure, the antibodies and fragments thereof include a heavy chain variable region including heavy chain CDR1, CDR2 and CDR3 and a light chain variable region including light chain CDR1, CDR2 and CDR3, wherein [0009] heavy chain CDR1 includes one of SEQ ID NO: 16, 19, or 20, [0010] heavy chain CDR2 includes one of SEQ ID NO: 20, 21, 22 or 23, [0011] heavy chain CDR3 includes one of SEQ ID NO: 18, 24, or 25, [0012] light chain CDR1 includes one of SEQ ID NO: 26, 29, 31, or 32, [0013] light chain CDR2 includes one of SEQ ID NO: 33, 34, 35 or 36, and [0014] light chain CDR3 includes one of SEQ ID NO: 28, 38 or 39.

[0015] The antibody or fragment thereof of the disclosure may include a heavy chain variable region, excluding the CDRs, that is at least 95% or 98% identical an amino acid sequence selected from SEQ ID NO: 3, 4, 5, 6, and 7, and the light chain variable region, excluding the CDRs, that is at least 95% or 98% identical an amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, and 15. In addition, the heavy chain variable region may be selected from SEQ ID NOs: 3, 4, 5, 6, and 7, and the light chain variable region may be selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14 and 15.

[0016] In further embodiments, the disclosure is directed to an antibody or fragment thereof that that specifically binds to A.beta. peptide, including a heavy chain variable region including heavy chain CDR1, CDR2 and CDR3 and a light chain variable region including light chain CDR1, CDR2 and CDR3, having the following amino acid sequences: [0017] heavy chain CDR1 includes amino acid sequence GFTFSNX.sub.1GMS, wherein X.sub.1 is Y or F (SEQ ID NO: 88); [0018] heavy chain CDR2 includes amino acid sequence SX.sub.1RSGSGRTYYSDNVKG, wherein is X.sub.1 is I or V (SEQ ID NO: 89); [0019] heavy chain CDR3 includes amino acid sequence YDHYX.sub.1GX.sub.2SDY, wherein X.sub.1 is S or T and X.sub.2 is S or T (SEQ ID NO: 90); [0020] light chain CDR1 includes amino acid sequence KSSQSLLDYDGKTYLN (SEQ ID NO: 91); [0021] light chain CDR2 includes amino acid sequence X.sub.1VX.sub.2NRDX.sub.3, wherein X.sub.1 is K or R, X.sub.2 is S or T, and X.sub.3 is S or T (SEQ ID NO: 92). [0022] light chain CDR3 includes amino acid sequence WQGTHFPRX.sub.1, wherein X.sub.1 is S or T (SEQ ID NO: 93).

[0023] In addition, the light chain CDR3 may be WQGTHFPRX.sub.1FX.sub.2, wherein X.sub.1 is S or T and X.sub.2 is F or Y (SEQ ID NO: 94).

[0024] Still further, embodiments of the disclosure are directed to an antibody or fragment thereof that specifically binds to A.beta. peptide, including a heavy chain variable region including heavy chain CDR1, CDR2 and CDR3 and a light chain variable region including light chain CDR1, CDR2 and CDR3, having the following amino acid sequences: [0025] heavy chain CDR1 includes amino acid sequence GFTFX.sub.1NX.sub.2GMS, wherein X.sub.1 is S or A, and X.sub.2 is Y or F (SEQ ID NO: 95); [0026] heavy chain CDR2 includes amino acid sequence SX.sub.1RSGX.sub.2X.sub.3RTYYSDNVKG, wherein is X.sub.1 is I or V, X.sub.2 is S or G and X.sub.3 is S or G (SEQ ID NO: 96); [0027] heavy chain CDR3 includes amino acid sequence YDHYX.sub.1GX.sub.2SDY, wherein X.sub.1 is S or T and X.sub.2 is S or T (SEQ ID NO: 90); [0028] light chain CDR1 includes amino acid sequence X.sub.1SSQSLX.sub.2DX.sub.3DGKTYLN, wherein X.sub.1 is K or R, X.sub.2 is V, M or L, and X.sub.3 is Y, T or S (SEQ ID NO: 97); [0029] light chain CDR2 includes amino acid sequence X.sub.1VX.sub.2NRX.sub.3X.sub.4, wherein X.sub.1 is K or R, X.sub.2 is S or T, and X.sub.3 is E or D, and X.sub.4 is S or T (SEQ ID NO: 98). [0030] light chain CDR3 includes amino acid sequence WQGX.sub.1HFPRX.sub.2, wherein X.sub.1 is S or T, and X.sub.2 is S or T (SEQ ID NO: 99).

[0031] The light chain CDR3 may also include WQGTHFPRX.sub.1FX.sub.2X.sub.3, wherein X.sub.1 is S or T, X.sub.2 is S or T and X.sub.3 is F or Y (SEQ ID NO: 100).

[0032] In further aspects of the disclosure, the antibody or fragment thereof of one is humanized, is human IgG1, or may be a full antibody, a chimeric antibody, a CDR-grafted antibody, or a recombinant antibody. Antibody fragments may include a Fab, Fab', F(ab')2, Fabc, or Fv.

[0033] Still further, the antibody or fragment of the disclosure may include a heavy chain constant region including an amino acid sequence at least 95% identical to SEQ ID NO:40, and may include a light chain constant region including an amino acid sequence at least 95% identical to SEQ ID NO:41. The antibody or fragment may specifically bind to an epitope having an amino acid sequence including three or more amino acid positions from amino acids 1-7 of A.beta..

[0034] In additional aspects, the disclosure is directed to a nucleic acid encoding the heavy chain and/or light chain of an antibody as described herein.

[0035] The disclosure is also directed to a pharmaceutical composition including an antibody or fragment thereof as described herein.

[0036] In various embodiments, the disclosure is directed to a method of producing the antibody or fragment thereof as described herein. The method may include (a) culturing cells transformed with nucleic acids encoding the heavy and light chains of the antibody or fragment thereof, so that the cells secrete the antibody or fragment thereof; and (b) purifying the antibody or fragment thereof from cell culture.

[0037] In another aspect, the disclosure is directed to a method of producing a cell line producing the antibody or fragment thereof as described herein. The method may include (a) introducing a vector encoding heavy and light chains of the antibody or fragment thereof and a selectable marker into cells; (b) propagating the cells under conditions to select for cells having increased copy number of the vector; (c) isolating single cells from the selected cells; and (d) banking cells cloned from a single cell selected based on yield of antibody or a fragment thereof. The method may also include propagating the cells under selective conditions and screening for cell lines naturally expressing and secreting at least 100 mg/L/10{circumflex over ( )}6 cells/24 h.

[0038] Addition aspects of the addition include methods of preventing or treating amyloidogenic disease in a patient. The methods include administering an effective dosage of the antibody or fragment as described herein to the patient. The amyloidogenic disease may be systemic amyloidosis, Alzheimer's disease, mature onset diabetes, Parkinson's disease, Huntington's disease, fronto-temporal dementia, Down's syndrome, or mild cognitive impairment.

[0039] When the amyloidogenic disease, the methods of the disclosure may include administering to a patient having the disease the antibody or fragment thereof in a regime effective to treat the disease. In addition, the methods of the disclosure include reducing the risk or delaying the outset of Alzheimer's disease in a patient whose risk of the disease has been determined from a genetic or biochemical marker. The method includes administering to a patient having the disease the antibody or fragment thereof as described herein in a regime effective to reduce the risk or delay the outset of the disease.

[0040] Still further, the disclosure is directed to a method for effecting improvement of cognition in a subject having a condition or disease related amyloidogenic disease. The method include including administering to the subject an effective amount of the antibody or fragment thereof as described herein. The amyloidogenic disease may be systemic amyloidosis, Alzheimer's disease, mature onset diabetes, Parkinson's disease, Huntington's disease, fronto-temporal dementia, Down's syndrome, or mild cognitive impairment.

[0041] Still further, the disclosure is directed to a method for treating Down's syndrome or clinical or pre-clinical Alzheimer's disease in a human subject. The method include including administering to the subject an effective amount of the antibody or fragment thereof as described herein.

[0042] Method of the disclosure also include one or more of inhibiting the formation of amyloid plaque in a human subject, reduce amyloid plaque in the brain of a human subject, inhibiting or reducing amyloid plaque in a subject having or at risk of developing an amyloidogenic disease. The methods include including administering to the subject an effective amount of the antibody or fragment thereof as described herein. In each of these methods, the amyloid plaque may include A.beta..sub.1-42, pyroglutamate species of A.beta. (e.g., A.beta..sub.pE3-42), or a combination thereof.

[0043] In yet another aspect, the disclosure is directed to a method of detecting amyloid plaques in a subject having or at risk of an amyloidogenic disease. The method includes administering to a subject an antibody or fragment as described herein, and detecting the antibody or fragment thereof bound to A.beta. in the subject. The amyloidogenic disease is systemic amyloidosis, Alzheimer's disease, mature onset diabetes, Parkinson's disease, Huntington's disease, fronto-temporal dementia, Down's syndrome, or mild cognitive impairment. In the detection methods, the antibody or fragment thereof may be labeled, for example the with a fluorescent label, a paramagnetic label, or a radioactive label. The radioactive label may be detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0044] A method of measuring efficacy of treatment in a subject being treated for an amyloidogenic disease, including:

[0045] (a) measuring a first level of amyloid plaque in the subject prior to treatment by administering to a subject an antibody or fragment thereof of any one of claims 1-18, and detecting a first amount of the antibody or fragment thereof bound to A.beta. in the subject,

[0046] (b) administering the treatment to the subject,

[0047] (c) measuring a second level of amyloid plaque in the subject after treatment by administering to a subject the antibody or fragment thereof, and detecting the antibody or fragment thereof bound to A.beta. in the subject,

wherein a decrease in the level of amyloid plaque indicates a positive response to treatment.

[0048] Still further, other aspects of the disclosure include a method of measuring efficacy of treatment in a subject being treated for an amyloidogenic disease. The methods includes (a) measuring a first level of amyloid plaque in the subject prior to treatment by administering to a subject an antibody or fragment thereof as described herein, and detecting a first amount of antibody or fragment thereof bound to A.beta. in the subject, (b) administering the treatment to the subject, (c) measuring a second level of amyloid plaque in the subject after treatment by administering to a subject the antibody or fragment thereof, and detecting a second amount of antibody or fragment thereof bound to A.beta. in the subject. No change in the level of amyloid plaque or a small increase in amyloid plaque indicates a positive response to treatment.

[0049] The methods of the disclosure also include reducing, clearing, or promoting clearance of A.beta., or reducing or inhibiting A.beta. accumulation or aggregation, in a human subject. Such methods include administering to the subject an effective regime of the antibody or fragment thereof as described herein. The A.beta. may be present in the subject's brain tissue.

[0050] The methods of the disclosure also include reducing, promoting clearance, or clearing of A.beta. in brain tissue of a subject having or at risk of developing an amyloidogenic disease. Such methods include administering to the subject an effective regime of the antibody or fragment thereof as described herein.

[0051] The methods of the disclosure also include inhibiting or reducing A.beta. accumulation or aggregation in brain tissue of a subject having or at risk of developing an amyloidogenic disease. Such methods include administering to the subject an effective regime of the antibody or fragment thereof as described herein.

[0052] A method of inhibiting A.beta. accumulation or aggregation in brain tissue of a subject having or at risk of developing an amyloidogenic disease, including administering to the subject an effective regime of the antibody of any one of claims 1 to 18, thereby inhibiting A.beta. accumulation or aggregation in brain tissue in the subject. The amyloidogenic disease may be systemic amyloidosis, Alzheimer's disease, mature onset diabetes, Parkinson's disease, Huntington's disease, fronto-temporal dementia, Down's syndrome, or mild cognitive impairment. The A.beta. may be A.beta..sub.1-42, pyroglutamate species of A.beta. (e.g., A.beta..sub.pE3-42), or a combination thereof.

[0053] In each of the foregoing methods of the disclosure, the antibody is administered by peripheral administration, which may be intravenous or subcutaneous administration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 shows an alignment of three different versions of VL that were designed by incorporating human germline framework residues into bapineuzumab (hBP) VL sequence. Canonical or interface residues were not changed.

[0055] FIG. 2 shows competitive ELISA assay graphs for 4918, 4917, 4921, 3818, 49human3, 2931 and bapineuzumab control for IC.sub.50 ratio determination relative to bapineuzumab (hBP).

[0056] FIG. 3 shows competitive ELISA assay graphs for 2926, 2831, 2927, 2726, 2731, 2826 and bapineuzumab control for IC.sub.50 ratio determination relative to bapineuzumab (hBP).

[0057] FIG. 4 shows competitive ELISA assay graphs for 2727, 2931 and bapineuzumab control for IC.sub.50 ratio determination relative to bapineuzumab (hBP).

[0058] FIG. 5A and FIG. 5B show competitive ELISA assay graphs for 2931, 2731 and bapineuzumab (FIG. 5A) and 2726, 2831 and bapineuzumab (FIG. 5B).

[0059] FIGS. 6A-6D show BIAcore sensorgrams of binding of h2726 (FIG. 6A), h2731 (FIG. 6B), h2831 (FIG. 6C) and 2931 (FIG. 6D) to A.beta..sub.1-28 at analyte concentrations from 100 nM to 0.39 nM (2-fold serial dilutions).

[0060] FIG. 7 shows a BIAcore sensorgram comparing binding characteristics of humanized antibodies (PB-0569 (aducanumab), PB-0573 (h2726), PB-0574 (h2731), PB-0575 (h2831), PB-0576 (h2931)) to recombinant Abeta 1-42 (A.beta..sub.1-42) fibrils.

[0061] FIG. 8 shows h2931 binds soluble A.beta. oligomers with high relative affinity.

[0062] FIG. 9 shows graphs evaluating A.beta. fibril binding activity of 2726, 2731, 2831, 2931 versus aducanumab control. Antibody was titrated in a constant concentration of A.beta. fibrils (left panel) or A.beta. fibrils were titrated in a constant concentration of antibody (right panel), both indicating substantially better binding for 2726, 2731, 2831 and 2931 than for aducanumab.

[0063] FIG. 10 shows A.beta. binding in AD brain. Binding to tissue A.beta. pathology appears similar among h2726, h2731, h2831 and h2931 antibodies. Examples of images stained with the four antibodies, h2726, h2731, h2831, h2931, at 0.3 .mu.g/ml show their pattern of staining in two AD brains with different amounts of A.beta. pathology (AD 11-97 and AD 13-75). For each brain, the images are from the same area of the section and show comparatively similar intensity and distribution of pathology with all four antibodies. Staining with aducanumab was always the weakest (Scale bar: 500 .mu.m).

[0064] FIG. 11 shows A.beta. binding in AD brain of controls. Human IgG isotype control antibody produced no staining in AD brains. As shown in these examples, AD sections incubated with human IgG isotype at 1 .mu.g/ml were devoid of any staining (Scale bar: 500 .mu.m).

[0065] FIG. 12 shows quantification of A.beta. binding in AD brain. Quantification of A.beta. pathology staining in AD tissues revealed similar binding between h2726, h2731, h2831 and h2931 antibodies. Section from four AD brains were incubated with the antibodies h2726, h2731, h2831, h2931 as well as aducanumab at the following concentrations: 0.03, 0.1, 0.3, 1, 3 and 9 .mu.g/ml. After imaging of sections, the percent of stained tissue area was determined morphometrically using Halo.RTM. imaging analysis software. Each graph compares measurements in an AD brain obtained with the five antibodies. The four graphs consistently show that the binding profiles of h2726, h2731, h2831, h2931 antibodies are similar. Measurements obtained with aducanumab were significantly lower.

[0066] FIG. 13 shows A.beta. binding in AD brain. hBP binds to tissue A.beta. pathology strongly and in a dose-dependent manner. Images from relatively the same area of the section (Brain AD 13-75) with similar pathology distribution. hBP shows an increase in the amount of staining with concentration, and its binding to A.beta. pathology was stronger than that of BAN2401 or aducanumab at each concentration (Scale bar: 500 .mu.m).

[0067] FIGS. 14A and 14B show individual (FIG. 14A) and pooled (FIG. 14B) results from an ex vivo phagocytosis study of h2931 and aducanumab in APP.PS1 Tg mouse tissue with primary murine microglia. h2931 and aducanumab both demonstrate highly significant reductions in A.beta..sub.1-42 over isotype control.

[0068] FIGS. 15A and 15B show graphs indicating a reduction of soluble oligomer binding to neurites on rat hippocampal neurons with increasing concentration of h2726, h2731, h2831 and h2931 compared to isotype control, and normalized by +/-A.beta. addition. FIG. 15A shows spots per neuron and FIG. 15B shows total spot counts (at 40 fields per well).

[0069] FIG. 16 shows a graph representing the percentage of A.beta. spots per neuron with increasing concentration of 2726, 2731, 2831 and 2931 normalized by +/-A.beta. addition.

[0070] FIG. 17 shows an alignment of bapineuzumab variable heavy chain sequence and four sequences of the disclosure, 2726, 2731, 2831 and 2931. CDRs are in bold.

[0071] FIG. 18 shows an alignment of bapineuzumab light chain sequence and four (variable light chain) sequences of the disclosure, 2726, 2731, 2831 and 2931. CDRs are in bold.

[0072] FIGS. 19A and 19B show a CDR table listing the variable heavy and light chain CDR sequences for antibodies of the disclosure. FIG. 19A refers to heavy chain CDRs and FIG. 19B refers to light chain CDRs.

[0073] FIGS. 20A and 20B show graphs measuring antibody potency for binding heterogenous aggregated A.beta.42 species by competition ELISA. FIG. 20A shows h2931, h2731 and bapineuzumab control, and FIG. 20BA shows h2831, h2726 and bapineuzumab control.

[0074] FIG. 21 shows graphs measuring direct binding and relative affinity of antibodies to fibrillar A.beta.42 by ELISA.

[0075] FIG. 22 shows graphs measuring antibody dose response of A.beta. plaque area binding measured as percent positive tissue by immunohistochemical staining in AD brain.

[0076] FIG. 23 shows quantification of binding of soluble A.beta. to rat hippocampal neurons in the presence of antibody.

[0077] FIG. 24 shows results from an ex vivo phagocytosis study of h2731 in AD tissue with primary murine microglia. h2731 demonstrated highly significant reduction in A.beta..sub.1-42 indicating the antibody robustly promoted phagocytosis and removal of these species.

[0078] FIGS. 25A and 25B confirm the presence of pyroglutamate-3 A.beta. (A .sub.pE3-42) in AD tissue used for ex vivo phagocytosis assays (FIG. 23A) and demonstrates a similar binding pattern for pyroglutamate-3 A.beta. and h2931 (FIGS. 23A and B).

[0079] FIGS. 26A and 26B show results from an ex vivo phagocytosis study of h2931 and h2731 in AD tissue with primary murine microglia. h2931 and h2731 both demonstrate highly significant reductions in pyroglutamate-3 A.beta. (A .sub.pE3-42). indicating that both antibodies robustly promote phagocytosis and removal of these species.

[0080] FIG. 27 shows that h2731 binds the N-terminus of A.beta..sub.1-42 but not A.beta..sub.pE3-42.

[0081] FIGS. 28A and 28B show that antibodies of the present invention induce phagocytosis of A .sub.1-42 protofibrils in THP-1 human monocytes in vitro.

[0082] FIG. 29A and FIG. 29B show the distribution pattern of A .sub.1-XX, as measured by an N-terminal anti-Ab antibody, compared to A .sub.pE3-42 in human AD brain tissue. FIG. 29C shows the quantification of the percent area covered by A .sub.1-XX compared to A .sub.pE3-42 in human AD brain tissue.

[0083] FIG. 30 shows localization of h2731 to A plaques, localization of anti-A .sub.pE3-42 antibody signal to A plaques, and colocalization of h2731 and anti-A .sub.pE3-42 antibody signal to A plaques.

[0084] FIG. 31A and FIG. 31B show that anti-A antibody h2731 promotes A.beta..sub.pE3-42 clearance from AD brain tissue ex vivo in a dose-dependent manner with higher potency than aducanumab.

[0085] FIG. 32A shows the concentration dependence of h2731 and aducanumab clearance of A.beta..sub.pE3-42 from AD brain tissue, and FIG. 32B shows that the effect of h2731 is microglia-dependent.

[0086] FIG. 33 compares predicted CNS exposure of h2731 and aducanumab with repeated dosing.

[0087] FIG. 34 shows that anti-A antibody h2731 promotes clearance of plaques containing A .sub.pE3-42 in AD brain tissue ex vivo.

DETAILED DESCRIPTION OF THE INVENTION

[0088] Monoclonal antibodies (mAbs) targeting the N-terminus of amyloid beta (A.beta.) have been demonstrated clinically to reduce amyloid plaque burden and one such antibody, aducanumab, showed that significant reduction in plaque burden was associated with slowing of cognitive decline in Alzheimer's disease (AD). Preclinical studies have also indicated that monoclonal antibodies (mAbs) targeting N-terminal epitopes of A.beta. elicit an antibody-dependent microglial-mediated A.beta.-plaque clearance and neutralization of soluble toxic A.beta. oligomers both in vitro and in vivo. It is hypothesized that administration of N-terminal A.beta. targeting mAbs slows disease progression via clearance of A.beta. plaques and neutralization of soluble A.beta. aggregates in patients with AD.

[0089] A.beta. antibody bapineuzumab (hBP) is a humanized antibody developed from parental murine antibody 3D6. In accordance with various aspects of the disclosure, a multipronged approach was applied to construct superior antibodies to hBP. Humanness of hBP was analyzed and a determination was made that light chain humanization could be optimized.

[0090] A search was made over the protein sequences in the PDB database [Deshpande et al, 2005] to find structures that would provide a rough structural model of hBP. The crystal structure of hBP fab PDB code 4HIX [Miles, et al., 2013] was utilized for both Vh and Vk structure as it had acceptable resolution and an exact sequence match to hBP Vh and Vk, retaining the same canonical structures for the loops.

[0091] IMGT/DomainGapAlignment was performed for the hBP VL as input sequences. to identify human germ line VK gene sequence IGHV2-30*02 as the closest matched to hBP VL. The frameworks of hBP VL share a high degree of sequence similarity with the corresponding framework regions of IGHV2-30*02. Thus, the framework regions of IGHV2-30*02 VL were chosen as the guidance sequence for further optimization of the hBP framework regions. Additional residues in CDR-L2 that do not make any direct contact with the antigen as per hBP 3D structure were also changed to germline sequence resulting in following changes.

[0092] Three different versions of VL were designed by incorporating human germline framework residues into hBP VL sequence. Canonical or interface residues were not changed. Also, based on structural observation that P15 is located at a turn and the germline gene has Leu at this position, P15L was tested in one version of the variable light chain.

[0093] Based on the 3D structural observations, substitutions at a number of residues in the light chain and heavy chain CDRs and framework were designed. Mutant VL and VH versions were generated and tested for binding in the first round of rational design. Mutations that showed improved binding were combined in the second round of the rational design. Additionally, new mutations guided by further analysis of the structure were also incorporated into the design.

[0094] Accordingly, the disclosure provides antibodies (and antibody fragments), nucleic acids encoding and methods of producing such antibodies and antibody fragments, pharmaceutical compositions, and methods for preventing or treating amyloidogenic disease, reducing the risk or delaying the outset of an amyloidogenic disease, effecting improvement of cognition in an subject having a condition related to amyloidogenic disease, inhibiting the formation of A.beta. plaque in a subject, reducing A.beta. plaque in the brain of a subject, inhibiting or reducing amyloid plaque in a subject at risk of developing an amyloidogenic disease, detecting amyloid plaques, measuring efficacy of a treatment in a subject being treated for an amyloidogenic disease, where amyloidogenic disease comprises Alzheimer's and others as described herein. The disclosure is based, at least in part, on the characterization of a genus of monoclonal antibodies effective at binding beta amyloid protein (A.beta.) (e.g., binding soluble and/or aggregated A.beta.), mediating phagocytosis (e.g., of aggregated A.beta.), reducing plaque burden and/or reducing neuritic dystrophy (e.g., in patient), neutralizing soluble, toxic A.beta. species. The antibodies and fragments of the disclosure exhibit greater binding strength (affinity and/or avidity) for pathologic fibrillar A.beta. than reported current experimental therapies, and high affinity for soluble toxic A.beta. forms. These antibodies may enable more convenient dosing strategies and enhanced patient access.

[0095] Before describing particular aspects of the disclosure in more detail, a number of terms are defined.

Definitions

[0096] The term "antibody" includes intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target. Fragments include separate heavy chains, light chains Fab, Fab', F(ab').sub.2, F(ab)c, Fv and single domain antibodies. Single (variable) domain antibodies include VH regions separated from their VL partners (or vice versa) in conventional antibodies (Ward et al., 1989, Nature 341: 544-546) as well as VH regions (sometimes known as VHH) from species such as Camelidae or cartilaginous fish (e.g., a nurse shark) in which VH regions are not associated with VL regions (see, e.g., WO 9404678). Single domain antibodies in which one chain is separated from its natural partners are sometimes known as Dabs and single domain antibodies from Camelidae or cartilaginous fish are sometimes known as nanobodies. Constant regions or parts of constant regions may or may not be present in single domain antibodies. For example, natural single variable region antibodies from Camelidae include a VHH variable region, and CH2 and CH3 constant regions. Single domain antibodies can be subject of humanization by analogous approaches to conventional antibodies. The Dabs type of antibodies are usually obtained from antibodies of human origin. NANOBODY types of antibody are of Camelidae or shark origin and can be subject to humanization. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes a bispecific antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)).

[0097] An immunoglobulin light or heavy chain variable region (also sometimes referred to herein as a "light chain variable domain" ("VL domain") or "heavy chain variable domain" ("VH domain"), respectively) consists of a "framework" region interrupted by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. The CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain are also sometimes referred to herein, respectively, as CDR-L1, CDR-L2, and CDR-L3; CDRs 1, 2, and 3 of a VH domain are also sometimes referred to herein, respectively, as CDR-H1, CDR-H2, and CDR-H3. When the application discloses a VL sequence with R as the C-terminal residue, the R can alternatively be considered as being the N-terminal residue of the light chain constant region. Thus, the application should also be understood as disclosing the VL sequence without the C-terminal R.

[0098] The assignment of amino acids to each VL and VH domain is in accordance with any conventional definition of CDRs. Conventional definitions include, the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991), the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modelling software; and, the contact definition of Martin et al (bioinfo.org.uk/abs) (see Table A). Kabat provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. When an antibody is said to comprise CDRs by a certain definition of CDRs (e.g., Kabat) that definition specifies the minimum number of CDR residues present in the antibody (i.e., the Kabat CDRs). It does not exclude that other residues falling within another conventional CDR definition but outside the specified definition are also present. For example, an antibody comprising CDRs defined by Kabat includes among other possibilities, an antibody in which the CDRs contain Kabat CDR residues and no other CDR residues, and an antibody in which CDR H1 is a composite Chothia-Kabat CDR H1 and other CDRs contain Kabat CDR residues and no additional CDR residues based on other definitions.

TABLE-US-00001 TABLE A Conventional Definitions of CDRs Using Kabat Numbering Composite of Chothia Loop Kabat Chothia & Kabat AbM Contact L1 L24--L34 L24--L34 L24--L34 L24--L34 L30--L36 L2 L50--L56 L50--L56 L50--L56 L50--L56 L46--L55 L3 L89--L97 L89--L97 L89--L97 L89--L97 L89--L96 H1 H31--H35B H26-H32 . . . H34* H26--H35B* H26--H35B H30--H35B H2 H50--H65 H52--H56 H50--H65 H50--H58 H47--H58 H3 H95--H102 H95--H102 H95--H102 H95--H102 H93--H101 *CDR-H1 by Chothia can end at H32, H33, or H34 (depending on the length of the loop). This is because the Kabat numbering scheme places insertions of extra residues at 35A and 35B, whereas Chothia numbering places them at 31A and 31B. If neither H35A nor H35B (Kabat numbering) is present, the Chothia CDR-H1 loop ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34.

[0099] In some embodiments, the CDRs of the humanized antibodies of the present invention are of a definition selected from the group of Kabat, Chothia, Kabat/Chothia Composite, AbM and Contact.

[0100] One or several amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as a C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position 250 and/or a Leu at position 428 (EU numbering is used in this paragraph for the constant region) for increasing the half-life of an antibody. Substitution at any or all of positions 234, 235, 236 and/or 237 reduce affinity for Fc.gamma. receptors, particularly Fc.gamma.RI receptor (see, e.g., U.S. Pat. No. 6,624,821). An alanine substitution at positions 234, 235, and 237 of human IgG1 can be used for reducing effector functions. Some antibodies have alanine substitution at positions 234, 235 and 237 of human IgG1 for reducing effector functions. Optionally, positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, e.g., U.S. Pat. No. 5,624,821). In some antibodies, a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgG1 is used. In some antibodies, a mutation at one or more of positions 318, 320, and 322 by EU numbering of human IgG1 is used. In some antibodies, positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine. In some antibodies, the isotype is human IgG2 or IgG4.

[0101] The term "humanized immunoglobulin" or "humanized antibody" refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., a "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (i.e., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a heavy chain). The term "humanized variable region" (e.g., "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin or antibody.

[0102] Accordingly, regions or residues of a humanized immunoglobulin or antibody, or of a humanized immunoglobulin or antibody chain, except possibly the CDRs, are substantially identical to the corresponding regions or residues of one or more native human immunoglobulin sequences. The term "corresponding region" or "corresponding residue" refers to a region or residue on a second amino acid or nucleotide sequence which occupies the same (i.e., equivalent) position as a region or residue on a first amino acid or nucleotide sequence, when the first and second sequences are optimally aligned for comparison purposes.

[0103] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. When an epitope is said to be within a range of amino acid residues in a protein (e.g., within residues 1 to 6 of A.beta.), the range is inclusive of the residues defining its borders. Certain residues within the range contribute to the epitope, whereas others may not. The residues that form the epitope may or may not be contiguous with one another. Similarly, when an antibody binds to an epitope found within a particular range of amino acids, the antibody need not contact all the amino acids residues within the range, and the residues of the epitope that are contacted by the antibody may or may not be contiguous with one another. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0104] Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block or compete with the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competitive binding is determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as A.beta.. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more.

[0105] Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody (e.g. 3D6, aducanumab, bapineuzumab) to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2.times., 5.times., 10.times., 20.times. or 100.times.) inhibits binding of the reference antibody by at least 50% but preferably 75%, 90% or 99% as measured in a competitive binding assay. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.

[0106] The epitope of an antibody can also be defined by X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0107] An epitope is also recognized by immunologic cells, for example, B cells and/or T cells. Cellular recognition of an epitope can be determined by in vitro assays that measure antigen-dependent proliferation, as determined by .sup.3H-thymidine incorporation, by cytokine secretion, by antibody secretion, or by antigen-dependent killing (cytotoxic T lymphocyte assay).

[0108] Exemplary epitopes or antigenic determinants can be found within the human amyloid precursor protein (APP) but are preferably found within the A.beta. peptide of APP. Multiple isoforms of APP exist, for example APP.sup.695, APP.sup.751, and APP.sup.770. Amino acids within APP are assigned numbers according to the sequence of the APP.sup.770 isoform (see e.g., GenBank Accession No. P05067, also set forth as SEQ ID NO:85).

[0109] A.beta. (also referred to herein as beta amyloid peptide and A-beta) peptide is a about 4-kDa internal fragment of 39-43 amino acids of APP (A.beta.39, A.beta.40, A.beta.41, A.beta.42, and A.beta.43). A.beta.40, for example, consists of residues 672-711 of APP and A.beta.42 consists of residues 673-713 of APP. As a result of proteolytic processing of APP by different secretase enzymes in vivo or in situ, A.beta. is found in both a "short form", 40 amino acids in length, and a "long form", ranging from 42-43 amino acids in length. Preferred epitopes or antigenic determinants, as described herein, are located within the N-terminus of the A.beta. peptide and include residues within amino acids 1-10 of A.beta., preferably from residues 1-3, 1-4, 1-5, 1-6, 1-7, or 3-7 of A.beta.42. Additional referred epitopes or antigenic determinants include residues 2-4, 5, 6, 7, or 8 of A.beta., residues 3-5, 6, 7, 8, or 9 of A.beta., or residues 4-7, 8, 9, or 10 of A.beta.42.

[0110] "Soluble" or "dissociated" A.beta. refers to A.beta. species that are either monomeric, aggregated, oligomeric, associated or not with other proteins and lipids, which remain in solution (supernatant) after centrifugation at 100,000.times.g. "Insoluble" A.beta. refers to aggregated A.beta. species, amyloid (beta-sheet) or not, that do not remain in solution after 100,000.times.g centrifugation, for example, A.beta. held together by noncovalent bonds. A.beta. (e.g., A.beta.42) is believed to aggregate, at least in part, due to the presence of hydrophobic residues at the C-terminus of the peptide (part of the transmembrane domain of APP). One method to prepare soluble A.beta. is to dissolve lyophilized peptide in neat DMSO with sonication. The resulting solution is centrifuged to remove any insoluble particulates.

[0111] "Specific binding" of an antibody mean that the antibody exhibits appreciable affinity for antigen or a preferred epitope and, preferably, does not exhibit significant cross reactivity. "Appreciable" or preferred binding include binding with an affinity of at least 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 M.sup.-1, or 10.sup.10 M.sup.-1. Affinities greater 10.sup.7 M.sup.-1, preferably greater than 10.sup.8 M.sup.-1 are more preferred. Values intermediate of those set forth herein are also intended to be within the scope of the present disclosure and a preferred binding affinity can be indicated as a range of affinities, for example, 10.sup.6 to 10.sup.10 M.sup.-1, preferably 10.sup.7 to 10.sup.10 M.sup.-1, more preferably 10.sup.8 to 10.sup.10 M.sup.-1. An antibody that "does not exhibit significant cross reactivity" is one that will not appreciably bind to an undesirable entity (e.g., an undesirable proteinaceous entity). For example, an antibody that specifically binds to A.beta. will appreciably bind A.beta. but will not significantly react with non-A.beta. proteins or peptides (e.g., non-A.beta. proteins or peptides included in plaques). An antibody specific for a preferred epitope will, for example, not significantly cross-react with remote epitopes on the same protein or peptide. Specific binding can be determined according to any art-recognized means for determining such binding. Preferably, specific binding is determined according to Scatchard analysis and/or competitive binding assays.

[0112] Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab').sub.2, Fabc, Fv, single chains, and single-chain antibodies.

[0113] The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0114] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the infection and the general state of the patient's own immune system.

[0115] The term "treatment" as used herein, is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.

[0116] The term "amyloidogenic disease" includes any disease associated with (or caused by) the formation or deposition of insoluble amyloid fibrils or amyloid plaques. Exemplary amyloidogenic diseases include, but are not limited to systemic amyloidosis, Alzheimer's disease, mature onset diabetes, Parkinson's disease, Huntington's disease, fronto-temporal dementia, Down's syndrome, mild cognitive impairment, prion-related transmissible spongiform encephalopathies (kuru and Creutzfeldt-Jacob disease in humans and scrapie and BSE in sheep and cattle, respectively), and the like. Different amyloidogenic diseases are defined or characterized by the nature of the polypeptide component of the fibrils deposited. For example, in subjects or patients having Alzheimer's disease, .beta.-amyloid protein (e.g., wild-type, variant, or truncated .beta.-amyloid protein) is the characterizing polypeptide component of the amyloid deposit. Accordingly, Alzheimer's disease is an example of a "disease characterized by deposits of A.beta." or a "disease associated with deposits of A.beta.", e.g., in the brain of a subject or patient. The terms ".beta.-amyloid protein", ".beta.-amyloid peptide", ".beta.-amyloid", "A.beta." and "A.beta. peptide" are used interchangeably herein.

[0117] An individual is at increased risk of a disease if the subject has at least one known risk-factor (e.g., genetic, biochemical, family history, situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor.

[0118] The term "symptom" refers to a subjective evidence of a disease, such as altered gait, as perceived by the patient. A "sign" refers to objective evidence of a disease as observed by a physician.

[0119] Statistical significance means p<0.05.

[0120] Anti-A.beta. Antibodies

[0121] Turning now to various aspects of the disclosure, a first aspect the disclosure is directed to an antibody or fragment thereof that that specifically binds to A.beta. peptide. The antibody or fragment includes the heavy chain CDRs and the light chain CDRs from one of the constructs identified herein as h2726, h2731, h2831, h2931, h2926, h4921, h2828, h2929, h3818G, h2927, h49k3G, h4917G h2727, and h4918G. Particular monoclonal antibodies of the disclosure may bind to an epitope within residues 1-6 of A.beta. (with the first N terminal residue of natural A.beta. designated 1). Some monoclonal antibodies bind to an epitope within amino acids 1-6, some to an epitope within 1-5, and some to an epitope within 1-4. Some antibodies bind to epitopes within amino acids 1-3, 2-5, 3-5, 2-4, 2-5, 2-6, 3-5, or 3-6. When an antibody is said to bind to an epitope within specified residues, such as A.beta. 1-6 for example, what is meant is that the antibody specifically binds to a polypeptide containing the specified residues (i.e., A.beta. 1-6 in this an example); such antibody does not necessarily contact every residue within A.beta. 1-6.

[0122] In another aspect, the antibody or fragment includes a heavy chain variable region having a heavy chain CDR1, CDR2 and CDR3 and a light chain variable region comprising a light chain CDR1, CDR2 and CDR3 from the constructs show in Table 1A.

TABLE-US-00002 TABLE 1A Construct SEQ SEQ ID VH/VL Sequences ID CDR Sequences ID h2726 VH EVQLLESGGGLVQPGGS 3 1 GFTFS NYGMS 16 LRLSCAASGFTFSNYGM 2 SIRSG SGRTY YSDNV KG 20 SWVRQAPGKGLEWVASI 3 YDHYSGSSDY 18 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGSSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTPGE 8 1 KSSQS LLDYD GKTYL N 29 PASISCKSSQSLLDYDG 2 KVSNR DS 33 KTYLNWLLQKPGQSPQR 3 WQGTH FPRT 28 LIYKVSNRDSGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRT FGQGTKVEIK h2731 VH EVQLLESGGGLVQPGGS 3 1 GFTFS NYGMS 16 LRLSCAASGFTFSNYGM 2 SIRSG SGRTY YSDNV KG 20 SWVRQAPGKGLEWVASI 3 YDHYS GSSDY 18 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGSSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTLGE 9 1 KSSQS LLDYD GKTYL N 29 PASISCKSSQSLLDYDG 2 RVTNR DT 34 KTYLNWLLQKPGQSPQR 3 WQGTH FPRS 38 LIYRVTNRDTGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRS FGQGTKVEIK h2831 VH EVQLLESGGGLVQPGGS 4 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG SGRTY YSDNV KG 21 SWVRQAPGKGLEWVASV 3 YDHYS GTSDY 24 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTLGE 9 1 KSSQS LLDYD GKTYL N 29 PASISCKSSQSLLDYDG 2 RVTNR DT 34 KTYLNWLLQKPGQSPQR 3 WQGTH FPRS 38 LIYRVTNRDTGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRS FGQGTKVEIK h2931 VH EVQLLESGGGLVQPGGS 5 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG SGRTY YSDNV KG 21 SWVRQAPGKGLEWVASV 3 YDHYT GTSDY 25 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY TGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTLGE 9 1 KSSQS LLDYD GKTYL N 29 PASISCKSSQSLLDYDG 2 RVTNR DT 34 KTYLNWLLQKPGQSPQR 3 WQGTH FPRS 38 LIYRVTNRDTGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRS FGQGTKVEIK h2926 VH EVQLLESGGGLVQPGGS 5 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG SGRTY YSDNV KG 21 SWVRQAPGKGLEWVASV 3 YDHYT GTSDY 25 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY TGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTPGE 8 1 KSSQS LLDYD GKTYL N 29 PASISCKSSQSLLDYDG 2 KVSNR DS 33 KTYLNWLLQKPGQSPQR 3 WQGTH FPRT 28 LIYKVSNRDSGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRT FGQGTKVEIK h4921G VH EVQLLESGGGLVQPGGS 6 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG GGRTY YSDNV KG 22 SWVRQAPGKGLEWVASV 3 YDHYS GTSDY 24 RSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTLGE 10 1 KSSQS LLDSD GKTYL N 26 PASISCKSSQSLLDSDG 2 RVTNR DT 34 KTYLNWLLQKPGQSPQR 3 WQGTH FPRT 28 LIYRVTNRDTGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRT FGQGTKVEIK h2826 VH EVQLLESGGGLVQPGGS 4 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG SGRTY YSDNV KG 21 SWVRQAPGKGLEWVASV 3 YDHYS GTSDY 24 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTPGE 8 1 KSSQS LLDYD GKTYL N 29 PASISCKSSQSLLDYDG 2 KVSNR DS 33 KTYLNWLLQKPGQSPQR 3 WQGTH FPRT 28 LIYKVSNRDSGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRT FGQGTKVEIK h2929 VH EVQLLESGGGLVQPGGS 5 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRS SGRTY YSDNV KG 21 SWVRQAPGKGLEWVASV 3 YDHYT GTSDY 25 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY TGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTPGE 11 1 RSSQS LVDYD GKTYL N 31 PASISCRSSQSLVDYDG 2 KVSNR DS 33 KTYLNWLLQRPGQSPQR 3 WQGSH FPRS 39 LIYKVSNRDSGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGSHFPRS YGQGTKVEIK h3818G VH EVQLLESGGGLVQPGGS 7 1 GFTFA NYGMS 20 LRLSCAASGFTFANYGM 2 SVRSG GSRTY YSDNV KG 23 SWVRQAPGKGLEWVASV 3 YDHYS GSSDY 18 RSGGSRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGSSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTLGE 12 1 KSSQS LMDTD GKTYL N 32 PASISCKSSQSLMDTDG 2 KVSNR ES 35 KTYLNWLLQKPGQSPQR 3 WQGTH FPRT 28 LIYKVSNRESGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRT FGQGTKVEIK h2927 VH EVQLLESGGGLVQPGGS 5 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG SGRTY YSDNV KG 21 SWVRQAPGKGLEWVASV 3 YDHYT GTSDY 25 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY TGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTPGE 13 1 KSSQS LLDYD GKTYL N 29 PASISCKSSQSLLDYDG 2 KVSNR DS 33 KTYLNWLLQKPGQSPQR 3 WQGTH FPRS 38 LIYKVSNRDSGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRS FGQGTKVEIK h49k3G VH EVQLLESGGGLVQPGGS 6 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG GGRTY YSDNV KG 22 SWVRQAPGKGLEWVASV 3 YDHYS GTSDY 24 RSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTLGE 14 1 KSSQS LLDSD GKTYL N 26 PASISCKSSQSLLDSDG 2 KVSNR DS 33 KTYLNWLLQKPGQSPQR 3 WQGTH FPRT 28 LIYKVSNRDSGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRT FGQGTKVEIK h4917G VH EVQLLESGGGLVQPGGS 6 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG GGRTY YSDNV KG 22 SWVRQAPGKGLEWVASV 3 YDHYS GTSDY 24 RSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTLGE 15 1 KSSQS LLDSD GKTYL N 26 PASISCKSSQSLLDSDG 2 KVTNR ES 36 KTYLNWLLQKPGQSPQR 3 WQGTH FPRS 38 LIYKVTNRESGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRS FGQGTKVEIK h2727 VH EVQLLESGGGLVQPGGS 3 1 GFTFS NYGMS 16 LRLSCAASGFTFSNYGM 2 SIRSG SGRTY YSDNV KG 20 SWVRQAPGKGLEWVASI 3 YDHYS GSSDY 18 RSGSGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGSSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTPGE 13 1 KSSQS LLDYD GKTYL N 29 PASISCKSSQSLLDYDG 2 KVSNR DS 33 KTYLNWLLQKPGQSPQR 3 WQGTH FPRS 38 LIYKVSNRDSGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRS FGQGTKVEIK h4918G VH EVQLLESGGGLVQPGGS 6 1 GFTFS NFGMS 19 LRLSCAASGFTFSNFGM 2 SVRSG GGRTY YSDNV KG 22 SWVRQAPGKGLEWVASV 3 YDHYS GTSDY 24 RSGGGRTYYSDNVKGRF TISRDNSKNTLYLQMNS LRAEDTAVYYCVRYDHY SGTSDYWGQGTLVTVSS VL DVVMTQSPLSLPVTLGE 12 1 KSSQS LMDTD GKTYL N 32 PASISCKSSQSLMDTDG 2 KVSNR ES 25 KTYLNWLLQKPGQSPQR 3 WQGTH FPRT 28 LIYKVSNRESGVPDRFS GSGSGTDFTLKISRVEA EDVGVYYCWQGTHFPRT FGQGTKVEIK

[0123] In another aspect the antibody or fragment of the disclosure includes a heavy chain variable region (VH) as shown for one of the constructs in Table 1. The antibody or fragment may also include light chain variable region (VL) as shown for one of the constructs in Table 1A.

[0124] An alignment of the CDRs for each of the heavy chain and light chain sequences identified in Table 1A and the CDRs from bapineuzumab ("Bapi", "hBP") is show in FIGS. 19A and 19B. In one aspect, the disclosure is directed an antibody or fragment thereof including a heavy chain CDR1, CDR2, and CDR3, wherein CDR1 may be selected from any one of SEQ ID NOS: 16, 19 and 20, wherein CDR2 may be selected from any one of SEQ ID NOS: 17, 20, 21 22, and 23 and wherein CDR3 may be selected from any one of SEQ ID NOS: 18, 24, 25. In addition, the antibody or fragment thereof includes a light chain CDR1, CDR2, and CDR3, wherein CDR1 may be selected from any one of SEQ ID NOS: 26, 29, 31, and 32, wherein CDR2 may be selected from any one of SEQ ID NOS: 27, 33, 34 and 35, and wherein CDR3 may be selected from any one of SEQ ID NOS: 28, 38 and 39. In each of these embodiments, the heavy chain CDRs and the light chain CDRs are not, in combination, simultaneously SEQ ID NOS: 16, 17, 18, 26, 27 and 28.

[0125] Analysis of protein modeling information for the antibodies described above identified two changes in the CDRs that, among others, were the contributors to increased avidity/affinity characteristics of the antibodies of the disclosure: [0126] CDR-L1: S32Y (Ser to Tyr at position 32), and [0127] CDR-H2: G55S (Gly to Ser at position 55)

[0128] Anti-A.beta. antibodies with Tyr at position 32 in CDR-L1 and Ser at position 55 in CDR-H2 that bind the same epitope bound by antibodies listed herein are expected to have the same properties as the listed identified antibodies (See Table 1A and FIG. 19A and FIG. 19B). Antibodies disclosed that do not have Tyr at position 32 in CDR-L1 and Ser at position 55 in CDR-H2 can be modified to possess Tyr at position 32 in CDR-L1 and Ser at position 55 in CDR-H2 and can be expected to confer similar binding properties to such antibodies identified herein.

[0129] Examples of a CDR-L1 with Tyr at position 32 include SEQ NOs: 29 and 31. Examples of a CDR-H2 with Ser at position 55 include SEQ Nos: 20 and 21.

[0130] As examples, antibodies comprising a CDR-L1 with Tyr at position 32 and a CDR-H2 with Ser at position 55 include antibodies with the CDRs of h2726, h2731, h2727, h2826, h2831, h2926, h2927, h2931, h2929 (See Table 1A). Additional such antibodies include antibodies comprising LC CDRs 1, 2, 3 and HC CDRs 1, 2, 3 as set forth in the table below in Table 1B.

TABLE-US-00003 TABLE 1B CDR Sequences SEQ Antibody HC/LC (HC 1, 2, 3; LC 1, 2, 3) ID h2729 HC 1 GFTFS NYGMS 16 2 SIRSG SGRTY YSDNV KG 20 3 YDHYS GSSDY 18 LC 1 RSSQS LVDYD GKTYL N 31 2 KVSNR DS 33 3 WQGSH FPRS 39 h2829 HC 1 GFTFS NFGMS 19 2 SVRSG SGRTY YSDNV KG 21 3 YDHYS GTSDY 24 LC 1 RSSQS LVDYD GKTYL N 31 2 KVSNR DS 33 3 WQGSH FPRS 39 h2827 HC 1 GFTFS NFGMS 19 2 SVRSG SGRTY YSDNV KG 21 3 YDHYS GTSDY 24 LC 1 KSSQS LLDYD GKTYL N 29 2 RVTNR DT 33 3 WQGTH FPRS 38 HC-S55/ HC 1 GFTFS NYGMS 16 LC-Y32 2 SIRSG SGRTY YSDNV KG 20 3 YDHYS GSSDY 18 LC 1 KSSQS LLDYD GKTYL N 29 2 LVSKL DS 27 3 WQGTH FPRT 28

[0131] In view of the binding properties identified for the antibodies identified herein, consensus sequences can be identified that would be expected to provide similar binding properties. For example, in embodiments of the disclosure, antibodies or binding fragments thereof that that specifically bind to A.beta. peptide may include heavy chain variable regions having heavy chain CDR1, CDR2 and CDR3 and a light chain variable regions having light chain CDR1, CDR2 and CDR3, as follows: [0132] heavy chain CDR1 comprises amino acid sequence GFTFSNX.sub.1GMS, wherein X.sub.1 is Y or F (SEQ ID NO: 88); [0133] heavy chain CDR2 comprises amino acid sequence SX.sub.1RSGSGRTYYSDNVKG, wherein is X.sub.1 is I or V (SEQ ID NO: 89); [0134] heavy chain CDR3 comprises amino acid sequence YDHYX.sub.1GX.sub.2SDY, wherein X.sub.1 is S or T and X.sub.2 is S or T (SEQ ID NO: 90); [0135] light chain CDR1 comprises amino acid sequence KSSQSLLDYDGKTYLN (SEQ ID NO: 91); [0136] light chain CDR2 comprises amino acid sequence X.sub.1VX.sub.2NRDX.sub.3, wherein X.sub.1 is K or R, X.sub.2 is S or T, and X.sub.3 is S or T (SEQ ID NO: 92). [0137] light chain CDR3 comprises amino acid sequence WQGTHFPRX.sub.1, wherein X.sub.1 is S or T (SEQ ID NO: 93).

[0138] In some embodiments, the light chain CDR3 comprises WQGTHFPRX.sub.1FX.sub.2, wherein X.sub.1 is S or T and X.sub.2 is F or Y (SEQ ID NO: 94).

[0139] Similar consensus sequences that may be expected to provide binding properties similar to the antibodies described herein include a heavy chain variable region having heavy chain CDR1, CDR2 and CDR3 and a light chain variable region having light chain CDR1, CDR2 and CDR3, as follows: [0140] heavy chain CDR1 comprises amino acid sequence GFTFX.sub.1NX.sub.2GMS, wherein X.sub.1 is S or A, and X.sub.2 is Y or F (SEQ ID NO: 95); [0141] heavy chain CDR2 comprises amino acid sequence SX.sub.1RSGX.sub.2X.sub.3RTYYSDNVKG, wherein is X.sub.1 is I or V, X.sub.2 is S or G and X.sub.3 is S or G (SEQ ID NO: 96); [0142] heavy chain CDR3 comprises amino acid sequence YDHYX.sub.1GX.sub.2SDY, wherein X.sub.1 is S or T and X.sub.2 is S or T (SEQ ID NO: 90); [0143] light chain CDR1 comprises amino acid sequence X.sub.1SSQSLX.sub.2DX.sub.3DGKTYLN, wherein X.sub.1 is K or R, X.sub.2 is V, M or L, and X.sub.3 is Y, T or S (SEQ ID NO: 97); [0144] light chain CDR2 comprises amino acid sequence X.sub.1VX.sub.2NRX.sub.3X.sub.4, wherein X.sub.1 is K or R, X.sub.2 is S or T, and X.sub.3 is E or D, and X.sub.4 is S or T (SEQ ID NO: 98). [0145] light chain CDR3 comprises amino acid sequence WQGX.sub.1HFPRX.sub.2, wherein X.sub.1 is S or T, and X.sub.2 is S or T (SEQ ID NO: 99).

[0146] In some embodiments, the light chain CDR3 comprises WQGTHFPRX.sub.1FX.sub.2X.sub.3, wherein X.sub.1 is S or T, X.sub.2 is S or T and X.sub.3 is F or Y (SEQ ID NO: 100).

[0147] In addition, the light and heavy variable regions may be at least at least 75% identical to the light and heavy chain variable regions show in Table 1A. For example, the light and heavy chain variable regions may be 75% identical, 80%, identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, of 100% identical to VH and/or VL sequences identified in Table 1A. In various aspects, any sequence variation in the VH and VL may be present outside the CDRs so that the VH and VL sequences of the disclosure include the CDRs identified in Table 1A, but the regions of the VH and VL sequences outside of the CDRs may be at least 75% identical to the regions outside the CDRs of the VH and VL sequences in Table 1A.

[0148] For example, the antibody or fragment of the disclosure may include a heavy chain variable region, excluding the CDRS, that is at least 95% identical to one of SEQ ID NOS: 3, 4, 5, 6 and 7, and the light chain variable region, excluding the CDRs, that is at least 95% identical to one of SEQ ID NOS: 8, 9, 10, 11, 12, 13, 14 and 15.

[0149] The antibodies and fragments of the disclosure may also include a heavy chain constant region that is at least 75% identical to SEQ ID NO: 40. For example, the heavy chain constant region may be 75% identical, 80%, identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, of 100% identical to SEQ ID NO: 40.

[0150] The antibodies and fragments of the disclosure may also include a light chain constant region that is at least 75% identical to SEQ ID NO: 41. For example, the light chain constant region may be 75% identical, 80%, identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, of 100% identical to SEQ ID NO: 41.

[0151] A variant antibodies or fragments that are less than 100% identical to the sequences described in Table 1A (plus any constant region) can differ from an anti-A.beta. antibody of Table 1A by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind an A.beta. polypeptide).

[0152] For example, it is possible to introduce mutations only in framework regions of the antibody molecules. Introduced mutations can be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen. These types of mutations can be useful to optimize codon usage, or improve a hybridoma's antibody production. Alternatively, non-neutral missense mutations can alter an antibody's ability to bind antigen. One of skill in the art would be able to design and test mutant molecules with desired properties such as no alteration in antigen binding activity or alteration in binding activity (e.g., improvements in antigen binding activity or change in antibody specificity). Following mutagenesis, the encoded protein can routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecifically bind at least one epitope of an A.beta. polypeptide) can be determined using techniques described herein or by routinely modifying techniques known in the art.

[0153] In each of the foregoing embodiments, the antibody or fragment of the disclosure may be a humanized antibody as described herein. For example, the antibody may be a human IgG1 antibody. In addition, the antibody may a full antibody, a chimeric antibody, a CDR-grafted antibody, or a recombinant antibody. Fragments of the antibody may be a Fab, Fab', F(ab')2, Fabc, or Fv. Fragments are produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.

[0154] The antibody or binding fragments, variant, or derivative disclosed herein can be said to bind to A.beta.) or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5.times.10.sup.-2 sec.sup.-1, 10.sup.-2 sec.sup.-1, 5.times.10.sup.-3 sec.sup.-1 or 10.sup.-3 sec.sup.-1. In certain embodiments, an antibody of the disclosure can be said to bind A.beta. or a fragment or variant thereof with an off rate (k(off)) less than or equal to 5.times.10.sup.4 sec.sup.-1, 10.sup.-4 sec.sup.-1, 5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1, 5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1, 5.times.10.sup.-7 sec.sup.-1 or 10.sup.-7 sec.sup.-1.

[0155] An antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a target polypeptide disclosed herein (e.g., A.beta.) or a fragment or variant thereof with an on rate (k(on)) of greater than or equal to 10.sup.3 M-1 sec-1, 5.times.10.sup.3 M-1 sec-1, 10.sup.4 M-1 sec-1 or 5.times.10.sup.4 M-1 sec-1. In certain embodiments, an antibody of the disclosure can be said to bind a target polypeptide disclosed herein (e.g., A.beta.) or a fragment or variant thereof with an on rate (k(on)) greater than or equal to 10.sup.5 M-1 sec-1, 5.times.10.sup.5 M-1 sec-1, 10.sup.6 M-1 sec-1, or 5.times.10.sup.6 M-1 sec-1 or 10.sup.7 M-1 sec-1.

[0156] Anti-A.beta. antibodies or antigen-binding fragments, variants or derivatives thereof, as described herein can also be described or specified in terms of their binding affinity A.beta.. Binding affinities can include those with a dissociation constant or Kd less than 5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14 M, 10 M, 5.times.10.sup.-15 M, or 10.sup.-15 M.

[0157] Expression of Recombinant Antibodies

[0158] The disclosure is also directed to recombinant polynucleotides encoding antibodies which, when expressed, include the heavy and light chain CDRs of the antibodies of the disclosure. Exemplary polynucleotides, which on expression code for the polypeptide chains comprising the heavy and light chain CDRs of monoclonal antibodies are provided herein (e.g., SEQ ID NO: 42 through SEQ ID NO: 69), which code for the variable light and heavy chain polypeptides, and CDRs thereof, according to SEQ ID NO: 1 through SEQ ID NO: 39. Due to codon degeneracy, other polynucleotide sequences can be readily substituted for those sequences.

[0159] Humanized and human antibodies are typically produced by recombinant expression. Nucleic acids encoding humanized light and heavy chain variable regions may be linked to constant regions are inserted into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the cross-reacting antibodies.

[0160] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences.

[0161] One prokaryotic host useful for cloning the polynucleotides of the present disclosure is E. coli. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.

[0162] Other microbes, such as yeast, are also useful for expression. Saccharomyces is a preferred yeast host, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. Additionally, plants (e.g., rice, tobacco) are useful for expression.

[0163] Mammalian tissue cell culture may also be used to express and produce the polypeptides of the present disclosure (e.g., polynucleotides encoding immunoglobulins or fragments thereof). Eukaryotic cells can be particularly useful because a number of suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, and include CHO cell lines, various Cos cell lines, HeLa cells, preferably, myeloma cell lines, or transformed B-cells or hybridomas. Preferably, the cells are nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like.

[0164] Antibody-coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. Suitable transgenes include coding sequences for light and/or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.

[0165] Vectors containing the polynucleotide sequences of interest (e.g., the heavy and light chain encoding sequences and expression control sequences) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection may be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., supra). For production of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

[0166] When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present disclosure can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns (e.g., Protein A), column chromatography, HPLC purification, gel electrophoresis and the like. Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.

[0167] Increasing the copy number of expression vectors containing polynucleotide sequences of interest is desirable as a way to increase the production of antibodies or antibody fragments. A number of ways to genetically manipulate cells for this purpose and subsequently select the best cells are known in the art. These methods often include an "amplification" step to increase the copy number of the incorporated expression vector to improve the yield obtained for the desired protein. Amplification methods have been previously reported, e.g., by Bebbington and Hentschel (DNA Cloning Volume III (IRL press, 1987)). Any of a number of selectable markers, often in the form of nucleic acid sequences that encode enzymes that are involved in host cell metabolism and are essential for their survival under certain media conditions, can be operably linked to an expression vector, whereby the expression of a desired protein can be promoted upon selection for a selectable marker. Cells selected for a high copy number can be subjected to further amplification methods when the titer of the protein is not acceptably elevated. Such methods can involve subjecting the cells to certain toxic drugs that inhibit the selectable marker (e.g., methotrexate and dihydrofolate reductase, methionine sulphoximine and glutamine synthase, multidrug resistance/adriamycin). Through such inhibition, cell populations with increased levels of expression of this marker may be selected. This often leads to increased expression levels of similarly functionally linked expression cassettes. Vector copy number in individual cells subjected to the amplification method are assessed until a plateau of protein production is reached, preferably at least about 100 mg/ml/10.sup.6 cells/24 hours. Clones that grow through such selection and amplification are subsequently screened for titer/yield to select the best clone and then further evaluated. From such titration and screening, it is common to identify one or a small number of clones for subsequent production of one or more desired proteins and subsequently use it or them alone.

[0168] Pharmaceutical Compositions

[0169] Several methods of preparing and administering anti-A.beta. antibodies, or antigen-binding fragments, variants, or derivatives thereof to a subject in need thereof are known. The route of administration of an anti-A.beta. antibody, or antigen-binding fragment, variant, or derivative thereof, can be, for example, peripheral, oral, central (e.g., intrathecal, intracranial), parenteral, by inhalation or topical.

[0170] As discussed herein, anti-A.beta. antibodies, or antigen-binding fragments, variants, or derivatives thereof can be formulated so as to facilitate administration and promote stability of the active agent. In certain embodiments, pharmaceutical compositions in accordance with the present disclosure comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of an anti-A.beta. antibody, or antigen-binding fragment, variant, or derivative thereof, shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., reduce brain amyloid plaques without affecting vascular amyloid, or minimizes the occurrence of microhemorrhage during chronic dosing of the anti-A.beta. antibody or antigen-binding fragment thereof. In some embodiments, an anti-A.beta. antibody or antigen-binding fragment, variant, or derivative thereof can cross the blood-brain barrier in an effective amount to reduce brain amyloid plaques.

[0171] The pharmaceutical compositions used in this disclosure comprise pharmaceutically acceptable carriers, including, e.g., ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0172] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, isotonic agents can be included, for example, sugars, polyalcohols or salts in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0173] Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions can be administered at specific fixed or variable intervals, e.g., once a day, or on an "as needed" basis.

[0174] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the disclosure can comprise further agents such as dopamine or psychopharmacologic drugs, depending on the intended use of the pharmaceutical composition.

[0175] The amount of an anti-A.beta. antibody, or fragment, variant, or derivative thereof, to be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0176] The term "peripheral administration" as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intranasal, intra-ocular/vitreal, rectal, or vaginal administration. While all these forms of administration are clearly contemplated as being within the scope of the disclosure, an example of a form for administration would be a solution for injection, in particular for subcutaneous, intravenous or intraarterial injection or drip. A suitable pharmaceutical composition for injection can comprise a buffer, a surfactant, optionally a stabilizer agent, etc. Preparations for peripheral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Preservatives and other additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0177] Therapeutic compositions of the disclosure are typically substantially pure from undesired contaminants. This means that the agent is typically at least 50% w/w pure of interfering proteins and other contaminants arising from its production or purification but does not exclude the possibility that the agent is combined with an excess of pharmaceutical acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes monoclonal antibodies (or other therapeutic agents) are at least 60%, 70%, 80%, 90%, 95% or 99% w/w pure of interfering proteins and contaminants from production or purification.

[0178] Treatment Amenable Patients

[0179] The present disclosure is also directed to treatment of Alzheimer's and other amyloidogenic diseases by administration of the antibodies, fragments and pharmaceutical compositions of the disclosure generate a beneficial therapeutic response in a patient (e.g., induction of phagocytosis of A.beta., reduction of plaque burden, inhibition of plaque formation, reduction of neuritic dystrophy, neutralization of soluble, toxic A.beta. species, improving cognitive function, and/or reversing, treating or preventing cognitive decline) in the patient, for example, for the prevention or treatment of an amyloidogenic disease. The disclosure is also directed to use of the disclosed antibodies and fragments in the manufacture of a medicament for the treatment or prevention of an amyloidogenic disease.

[0180] In one aspect, the disclosure provides methods of preventing or treating a disease associated with amyloid deposits of A.beta. in a patient. In one aspect, the amyloid deposits are in the brain or other CNS areas. Such diseases include Alzheimer's disease, Down's syndrome, age-related macular degeneration (AMD), and cognitive impairment. The latter can occur with or without other characteristics of an amyloidogenic disease. Some methods of the disclosure entail administering an effective dosage of an antibody that specifically binds to a component of an amyloid deposit to the patient. Such methods are useful for preventing or treating Alzheimer's disease in human patients

[0181] The methods can be used on both asymptomatic patients and those currently showing symptoms of disease. The antibodies used in such methods can be humanized, human or fragments thereof (e.g., antigen binding fragments) and can be monoclonal or polyclonal, as described herein. In yet another aspect, the disclosure features administering antibodies prepared from a human immunized with A.beta. peptide, which human can be the patient to be treated with antibody.

[0182] In another aspect, the disclosure features administering an antibody with a pharmaceutical carrier as a pharmaceutical composition. Alternatively, the antibody can be administered to a patient by administering a polynucleotide encoding at least one antibody chain. The polynucleotide is expressed to produce the antibody chain in the patient. Optionally, the polynucleotide encodes heavy and light chains of the antibody. The polynucleotide is expressed to produce the heavy and light chains in the patient. In exemplary embodiments, the patient is monitored for level of administered antibody in the blood of the patient.

[0183] Patients amenable to treatment include individuals at risk of disease but not showing symptoms, as well as patients presently showing symptoms. In the case of Alzheimer's disease, potentially anyone who lives long enough is at risk of Alzheimer's disease. Thus, the present methods include administering prophylactically to the general population without the need for any assessment of the risk of the subject patient. The present methods are especially useful for individuals who have a known genetic risk of Alzheimer's disease. Such individuals include those having relatives who have experienced this disease, and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk toward Alzheimer's disease include mutations in the APP gene, particularly mutations at position 717 and positions 670 and 671 referred to as the Hardy and Swedish mutations, respectively. Other markers of risk are mutations in the presenilin genes, PS1 and PS2, and ApoE4, family history of AD, hypercholesterolemia or atherosclerosis. Individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above. In addition, a number of diagnostic tests are available for identifying individuals who have AD. These include measurement of CSF tau and A.beta.42 levels. Elevated tau and decreased A.beta.42 levels signify the presence of AD. Individuals suffering from Alzheimer's disease can also be diagnosed by ADRDA criteria as discussed in the Examples section.

[0184] Treatment in asymptomatic patients can begin at any age (e.g., 10, 20, 30). Usually, however, it is not necessary to begin treatment until a patient reaches 40, 50, 60, or 70. Treatment typically entails multiple dosages over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response falls, a booster dosage is indicated. In the case of potential Down's syndrome patients, treatment can begin antenatally by administering therapeutic agent to the mother or shortly after birth.

[0185] In Vivo Detection

[0186] In another aspect, the disclosure provides methods for detecting amyloid plaques and deposits in a patient having or at risk of developing an amyloidogenic disease. Such methods are useful for diagnosing or confirming amyloidogenic disease or susceptibility to it. For example, the methods can be used in patients with dementia symptoms, wherein observation of abnormal amyloid deposits likely indicates Alzheimer's disease. The methods can also be used in asymptomatic patients. The presence of abnormal deposits of amyloid indicates susceptibility to future symptomatic disease.

[0187] In some embodiments, the method comprises administering to a subject/patient an antibody or fragment thereof of the disclosure and detecting the antibody or fragment thereof bound to A.beta..

[0188] Antibody and/or antibody fragments thereof can be administered by any suitable means that results in delivery to the tissue to be visualized, e.g., administered directly into the brain by intravenous injection into the patient's body or by intracranial injection. Dosage of the antibody and/or fragment thereof can comprise a therapeutic dose, subtherapeutic dose or a serotherapeutic dose. In some embodiments the antibody or fragment thereof is labeled, comprising a fluorescent label, a paramagnetic label, or a radioactive label. The choice of label depends on the means of detection. For example, fluorescent labels are suitable for visual detection. The use of paramagnetic labels is suitable for tomographic detection without surgical intervention. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0189] In another aspect, the disclosure provides methods for measuring the efficacy of treatment in a subject being treated for an amyloidogenic disease. In some embodiments, a first level of amyloid plaque in a subject is measured prior to treatment by administering an antibody or fragment thereof of the disclosure and detecting a first amount of the antibody or fragment thereof bound to A.beta. in the subject. A treatment can then be administered to the subject, followed by measuring a second level of amyloid plaque in the subject, and detecting the antibody or fragment thereof bound to A.beta. in the subject. In some embodiments, a decrease in the level of amyloid plaque indicates a positive response to treatment, and in some embodiments, no change in the level of amyloid plaque or a small increase in amyloid plaque indicates a positive response to treatment. In some embodiments, levels of amyloid plaque can be measured utilizing the methods of detecting amyloid plaques described herein.

[0190] In some embodiments, diagnosis of an amyloidogenic disease can be performed, for example, by comparing the number, size and/or intensity of labeled positions from a measured first level (i.e., baseline) to a subsequent second level of amyloid plaque in a subject. An increase over time indicates disease progression, no change indicates, and fewer or less intense amyloid plaques over time indicates remission.

[0191] Treatment Regimes

[0192] Prophylactic applications: pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, Alzheimer's disease or other amyloidogenic disease in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. Patient susceptibility or risk for developing an amyloidogenic disease can be determined, for example, from a genetic marker, a biochemical marker, unspecified hereditary risk or other means. In therapeutic applications, compositions or mendicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease.

[0193] In some embodiments, administration of agent reduces or eliminates cognitive impairment in patients that have not yet developed characteristic Alzheimer's, or other amyloidogenic disease cognitive pathology. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient immune response has been achieved, where "immune response" or "immunological response" includes the development of a humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their secretion products) response directed against an antigen in a recipient subject. Such a response can be an active response, i.e., induced by administration of immunogen, or a passive response, i.e., induced by administration of immunoglobulin or antibody or primed T-cells.

[0194] In some embodiments, antibody is administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to A.beta. in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 .mu.g/ml and in some methods 25-300 .mu.g/ml. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies.

[0195] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactic effective dose." In this use, the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg/kg per dose, especially 0.5 to 2.5 mg/kg per dose. A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.

[0196] In therapeutic applications, a relatively high dosage (e.g., from about 0.5 to 300 mg/kg of antibody per dose, with dosages of from 5 to 25 mg/kg being more commonly used) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.

[0197] Administration: therapeutic agents can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, intraocular or intramuscular means for prophylactic and/or therapeutic treatment. Intramuscular injection is most typically performed in the arm or leg muscles. In some methods, agents are injected directly into a particular tissue where deposits have accumulated, for example intracranial injection. Intramuscular injection or intravenous infusion are preferred for administration of antibody. In some methods, particular therapeutic antibodies are injected directly into the cranium. In some methods, antibodies are administered as a sustained release composition or device.

[0198] Agents of the disclosure can optionally be administered in combination with other agents that are at least partly effective in treatment of amyloidogenic disease. In the case of Alzheimer's and Down's syndrome, in which amyloid deposits occur in the brain, agents of the disclosure can also be administered in conjunction with other agents that increase passage of the agents of the disclosure across the blood-brain barrier.

[0199] The present disclosure will be more fully described by the following non-limiting examples.

TABLE-US-00004 SEQ ID NO. 40: huIgG1 Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO. 41: huKappa Constant RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 42: h2726_VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTC CTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCG GAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACTACTCCGAT AACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAAT GAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGG GTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC SEQ ID NO. 43: h2726_VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCAT TTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGC TCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGC GTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCG GACAAGGCACCAAGGTCGAGATCAAG SEQ ID NO. 44: h2931_VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAG CTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGG GAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGAC AACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAAT GAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTG GCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA SEQ ID NO. 45: h2931_VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGAT CTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGC TCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGG GTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCG GTCAAGGAACCAAGGTCGAGATCAAG SEQ ID NO. 46: h2731_VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTC CTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCG GAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACTACTCCGAT AACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAAT GAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGG GTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC SEQ ID NO. 47: h2731_VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGAT CTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGC TCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGG GTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCG GTCAAGGAACCAAGGTCGAGATCAAG SEQ ID NO. 48: h2831_VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGGGGATCCCTGCGGCTTTC CTGCGCCGCATCCGGCTTCACCTTTTCAAACTTCGGAATGTCGTGGGTCAGACAGGCCCCGG GAAAGGGTCTGGAATGGGTGGCCTCAGTGCGGTCCGGATCGGGTAGAACCTACTACAGCGAT AACGTGAAGGGCCGGTTCACGATCTCCCGCGACAACTCCAAGAACACCCTGTACTTGCAAAT GAATAGCCTCAGGGCTGAGGATACCGCGGTCTACTACTGTGTGCGCTATGACCACTACACTG GAACTAGCGACTACTGGGGCCAGGGGACCCTCGTGACTGTGTCGTCC SEQ ID NO. 49: h2831_VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGAT CTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGC TCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGG GTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCG GTCAAGGAACCAAGGTCGAGATCAAG SEQ ID NO. 50: h2926_VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAG CTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGG GAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGAC AACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAAT GAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTG GCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA SEQ ID NO. 51: h2926_VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCAT TTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGC TCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGC GTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCG GACAAGGCACCAAGGTCGAGATCAAG SEQ ID NO. 52: h4921G VH (Variable Heavy) Nucleotide Sequence GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTC TTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGG GAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGAC AACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAAT GAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCG GTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC SEQ ID NO. 53: h4921G VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCAT TTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGC TGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACCGCGTGACCAACCGGGATACGGGA GTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGT CGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCG GTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO. 54: h2826 VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGGGGATCCCTGCGGCTTTC CTGCGCCGCATCCGGCTTCACCTTTTCAAACTTCGGAATGTCGTGGGTCAGACAGGCCCCGG GAAAGGGTCTGGAATGGGTGGCCTCAGTGCGGTCCGGATCGGGTAGAACCTACTACAGCGAT AACGTGAAGGGCCGGTTCACGATCTCCCGCGACAACTCCAAGAACACCCTGTACTTGCAAAT GAATAGCCTCAGGGCTGAGGATACCGCGGTCTACTACTGTGTGCGCTATGACCACTACACTG GAACTAGCGACTACTGGGGCCAGGGGACCCTCGTGACTGTGTCGTCC SEQ ID NO. 55: h2826 VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCAT TTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGC TCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGC GTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCG GACAAGGCACCAAGGTCGAGATCAAG SEQ ID NO. 56: h2929 VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAG CTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGG GAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGAC AACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAAT GAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTG GCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA SEQ ID NO. 57: h2929 VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACCCAAAGCCCCCTGTCCCTCCCTGTGACTCCTGGAGAGCCGGCGTCCAT TTCCTGCCGGTCAAGCCAGTCCTTGGTGGACTACGACGGAAAGACCTACCTCAACTGGCTGC TGCAGCGCCCCGGGCAGTCGCCGCAGCGGCTTATCTACAAAGTGTCCAACCGCGACTCGGGC GTGCCGGATAGGTTTTCGGGTTCCGGAAGCGGCACCGACTTCACCCTGAAAATCTCCAGAGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTTCTCACTTCCCACGGTCATATG GCCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO. 58: h3818G VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTCCTGGAGTCCGGCGGTGGACTGGTGCAGCCGGGCGGATCCCTGAGACTGTC CTGCGCCGCGTCGGGCTTTACTTTCGCAAATTACGGCATGAGCTGGGTCAGACAGGCCCCCG GGAAGGGTCTGGAATGGGTGGCCAGCGTCCGGAGCGGGGGATCCCGGACCTATTACTCCGAC AACGTGAAGGGCCGCTTCACCATCTCAAGGGACAACTCCAAGAACACCCTGTACTTGCAAAT GAACAGCCTTCGGGCTGAGGATACTGCCGTGTACTACTGCGTGCGCTACGACCACTACTCCG GATCCTCGGATTACTGGGGACAGGGAACCCTCGTGACCGTGTCATCG SEQ ID NO. 59: h3818G VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCAT TTCGTGCAAGAGCAGCCAGTCCCTGATGGACACCGACGGAAAGACCTACCTTAACTGGCTGC TGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGTCAAACCGGGAGTCCGGA GTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGT CGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCG

GTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO. 60: h2927 VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAG CTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGG GAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGAC AACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAAT GAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTG GCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA SEQ ID NO. 61: h2927 VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCACCGCTCTCCCTCCCTGTGACCCCGGGCGAACCAGCGTCGAT CTCCTGCAAGAGCAGCCAATCATTGCTGGACTACGACGGAAAGACCTATCTTAACTGGCTGC TGCAGAAGCCCGGGCAGAGCCCGCAGCGCCTGATCTACAAAGTGTCCAACAGAGACTCCGGA GTGCCTGATAGGTTCTCGGGTTCCGGCTCCGGTACCGACTTCACTCTGAAAATTTCCCGGGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACCCACTTCCCCCGGTCGTTTG GACAAGGGACCAAGGTCGAGATCAAG SEQ ID NO. 62: h49K3G VH (Variable Heavy) Nucleotide Sequence GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTC TTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGG GAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGAC AACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAAT GAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCG GTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC SEQ ID NO. 63: h49K3G VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCAT TTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGC TGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGTCAAACCGGGATTCCGGA GTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGT CGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCG GTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO. 64: h4917G VH (Variable Heavy) Nucleotide Sequence GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTC TTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGG GAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGAC AACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAAT GAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCG GTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC SEQ ID NO. 65: h4917G VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCAT TTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGC TGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGACCAACCGGGAGTCCGGA GTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGT CGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGTCATTCG GTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO. 66: h2727 VH (Variable Heavy) Nucleotide Sequence GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTC CTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCG GAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACTACTCCGAT AACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAAT GAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGG GTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC SEQ ID NO. 67: h2727 VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCACCGCTCTCCCTCCCTGTGACCCCGGGCGAACCAGCGTCGAT CTCCTGCAAGAGCAGCCAATCATTGCTGGACTACGACGGAAAGACCTATCTTAACTGGCTGC TGCAGAAGCCCGGGCAGAGCCCGCAGCGCCTGATCTACAAAGTGTCCAACAGAGACTCCGGA GTGCCTGATAGGTTCTCGGGTTCCGGCTCCGGTACCGACTTCACTCTGAAAATTTCCCGGGT GGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACCCACTTCCCCCGGTCGTTTG GACAAGGGACCAAGGTCGAGATCAAG SEQ ID NO. 68: h4918G VH (Variable Heavy) Nucleotide Sequence GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTC TTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGG GAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGAC AACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAAT GAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCG GTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC SEQ ID NO. 69: h4918G VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCAT TTCGTGCAAGAGCAGCCAGTCCCTGATGGACACCGACGGAAAGACCTACCTTAACTGGCTGC TGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGTCAAACCGGGAGTCCGGA GTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGT CGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCG GTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO. 70: Aducanumab Heavy Chain: QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTD SVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTIVIVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVIVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO. 71: Aducanumab Light Chain: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 72: Bapineuzumab HC (Heavy Chain) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSD NVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK SEQ ID NO. 73: Bapineuzumab VH (Variable Heavy) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSD NVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS SEQ ID NO: 16: VH CDR1 GFTFSNYGMS SEQ ID NO: 17: VH CDR2 SIRSGGGRTYYSNDYNVKG SEQ ID NO: 18: VH CDR3 YDHYSGSSDY SEQ ID NO. 77: Bapineuzumab LC (Light Chain) DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSILTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 78: Bapineuzumab VL (Variable Light) DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIK SEQ ID NO: 26: VL CDR1 KSSQSLLDSDGKTYLN SEQ ID NO: 27: VL CDR2 LVSSKLDS SEQ ID NO: 28: VL CDR3 WQGTHFPRT SEQ ID NO. 82: Gantenerumab HC amino acid sequence: QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 83: Gantenerumab LC amino acid sequence: DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPAR FSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 84: Amyloid Beta (A.beta.) 1-42: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA SEQ ID NO. 85: Amyloid Beta (AP) Precursor Protein: MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTC IDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVS DALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVC CPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDD EDGDEVEEEAEEPYEEATERTTSIATITTITTESVEEVVREVCSEQAETGPCRAMISRWYFD VTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAMSQSLLKTTQEPLARDPVKLPTTAASTP DAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQH FQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKK

YVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEI QDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQP WHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYE VHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTP EERHLSKMQQNGYENPTYKFFEQMQN SEQ ID NO. 86: huIgG1 Constant Nucleotide Sequence GCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGG AACCGCGGCACTGGGGTGCCTCGTGAAGGACTACTTCCCCGAGCCGGTCACCGTGTCCTGGA ACTCGGGAGCCCTGACCTCCGGAGTGCATACTTTCCCTGCGGTGCTGCAGTCCTCCGGGCTC TACTCGCTGTCAAGCGTGGTCACCGTCCCGAGCTCATCCCTGGGTACTCAGACCTACATTTG CAACGTGAACCACAAACCTTCCAACACCAAGGTCGACAAGAAAGTGGAGCCTAAGAGCTGCG ACAAGACCCACACCTGTCCCCCGTGTCCCGCCCCTGAGCTGCTGGGCGGCCCCAGCGTGTTC CTCTTCCCGCCTAAGCCGAAGGACACTCTGATGATCTCGAGAACCCCTGAAGTGACCTGTGT GGTGGTGGATGTGTCCCACGAGGATCCGGAAGTGAAGTTCAATTGGTACGTGGACGGAGTGG AAGTCCATAACGCCAAGACCAAGCCCCGCGAGGAACAGTACAACTCAACTTACCGGGTGGTG TCAGTGCTGACCGTGCTGCACCAAGATTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTCTC CAACAAGGCGCTGCCGGCCCCCATTGAAAAGACCATCAGCAAGGCTAAGGGCCAGCCCCGGG AACCACAGGTCTACACCTTGCCCCCTTCCCGGGAGGAAATGACCAAGAACCAAGTGTCGCTG ACGTGCCTGGTCAAGGGCTTTTATCCATCTGACATCGCCGTGGAGTGGGAAAGCAACGGCCA GCCGGAAAACAACTACAAGACTACCCCGCCTGTGCTGGACTCCGACGGCTCGTTCTTCCTGT ATTCCAAGCTCACCGTGGATAAGTCCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTG ATGCATGAGGCCCTGCACAACCACTACACTCAGAAATCACTGTCCCTTTCCCCCGGAAAGTA A SEQ ID NO. 87: huKappa Constant Nucleotide Sequence CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGG AACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGA AGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAA AGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGTTAA

EXAMPLES

[0200] The following examples have been included to illustrate modes disclosed herein. Certain aspects of the following examples are described in terms of techniques and procedures found or contemplated by the present co-inventors to work well in the practice disclosed herein. In light of the present disclosure and the general level of skill in the art, those of skill appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations may be employed without departing from the scope of the disclosure.

[0201] "Aducanumab" or "Adu" as used in these experiments refers to an antibody with heavy chain of SEQ ID NO: 70 and light chain of SEQ ID NO: 71, and as set forth in United States patent publication number US 2015/0315267 and PCT publication number WO 2014/089500.

[0202] "BAN-2401" and "gantenerumab" as used in these experiments refer to an antibody with heavy chain of SEQ ID NO: 79 and light chain of SEQ ID NO: 80 as set forth, e.g., in European patent number EP 1960428B1.

[0203] In the following methods, antibody binding profiles to aggregated or fibrillar A.beta. are characterized by ELISA, surface plasmon resonance (SPR) and immunohistochemistry (IHC). The ability to mediate phagocytic plaque clearance is evaluated ex vivo in APP/PS1 transgenic mouse brain as well as AD brain with primary murine microglia by immunofluorescence, ELISA and MSD quantification, and neutralization of A.beta. oligomer neuronal binding is assessed in rat primary hippocampal cultures.

[0204] Results presented herein: relative to other N-terminal A.beta. antibody therapies (bapineuzumab, aducanumab), mAbs of the description exhibited greater apparent affinity for aggregated and fibrillar A.beta. in competition or standard binding ELISAs. The enhanced avidity of mAbs of the disclosure for fibrillar A.beta. was confirmed by SPR equilibrium binding kinetics, indicating 5-11-fold higher avidity than aducanumab due to slower off-rate kinetics. IHC dose response assessments on frozen human AD brain sections showed greater apparent affinity and plaque area binding than aducanumab, regardless of the individual AD donor tissue tested. In ex vivo activity assays, mAbs of the disclosure were shown to significantly facilitate A.beta. plaque reduction by microglial phagocytosis in APP/PS1 mouse tissue and to block soluble A.beta. oligomer binding to rat primary neurons in a concentration-dependent manner. In ex vivo functional assays with human AD brain, mAbs from the description were shown to significantly facilitate clearance of pyroglutamyled A.beta., a post-translationally modified component of senile plaques.

Example 1. A.beta. Antibody Design

[0205] A.beta. antibody bapineuzumab (hBP) is a humanized antibody developed from parental murine antibody 3D6. Here, a multipronged approach was applied to construct superior antibodies to hBP. Humanness of hBP was analyzed and a determination was made that light chain humanization could be optimized.

[0206] A search was made over the protein sequences in the PDB database [Deshpande et al, 2005] to find structures that would provide a rough structural model of hBP. The crystal structure of hBP fab PDB code 4HIX [Miles, et al., 2013] was utilized for both Vh and Vk structure as it had acceptable resolution (2.2 .ANG.) and an exact sequence match to hBP Vh and Vk, retaining the same canonical structures for the loops.

[0207] IMGT/DomainGapAlignment was performed for the hBP VL as input sequences. Human germ line VK gene sequence IGHV2-30*02 is the closest matched to hBP VL. The frameworks of hBP VL share a high degree of sequence similarity with the corresponding framework regions of IGHV2-30*02. Thus, the framework regions of IGHV2-30*02 VL were chosen as the guidance sequence for further optimization of the hBP framework regions. Additionally, three residues in CDR-L2 that do not make any direct contact with the antigen as per hBP 3D structure were also changed to germline sequence resulting in following changes, L50K, K53N and L54R (Kabat).

[0208] Three different versions of VL were designed by incorporating human germline framework residues into hBP VL sequence. Canonical or interface residues were not changed. An alignment of designed VK version designed is shown in FIG. 1.

[0209] Based on structural observation that P15 is located at a turn and the germline gene has Leu at this position, P15L was tested in one version of the variable light chain.

[0210] Based on the 3D structural observations, substitutions at a number of residues in the light chain and heavy chain CDRs and framework were designed. In total thirty-one light chain and thirty-two heavy chain mutant VL and VH versions were generated and tested for binding in the first round of rational design. Mutations that showed improved binding were combined in the second round of the rational design. Additionally, new mutations guided by further analysis of the structure were also incorporated into the design.

[0211] Rational design based mutagenesis was done for following positions within CDR-H1, T28, S30, N31, Y32 and G33 (Kabat). For CDR-H2 positions 151, G53, G54, T57, S60, D61 and N62 were also mutated (Kabat). CDR-H3 positions D96, H97, S99, S100a and Y102 were subjected to rational mutagenesis (Kabat).

[0212] For variable light chain, multiple substitutions were tried at CDR-L1 positions K24, L27c, D27d and S27e (Kabat). Light chain CDR-L2 positions K53 and L54 were subjected to directed and limited mutagenesis (Kabat). CDR-L3 positions were not subjected to substitutions.

[0213] A select few positions in the framework regions were also subjected to rational mutagenesis for heavy chain as well as light chain.

[0214] Fifty-seven additional heavy chain and thirty-three light chain variants were designed and analyzed with assistance of Atum GPSpro software, which analyzes database of human variable heavy and light chains and, based upon computer learning, suggests query sequence-specific changes.

[0215] For the variable heavy domain, a number of substitutions at positions A24, S25, G26, F27, T28, F29, S30, N31, Y32, G33 and M34 were designed and analyzed (Kabat). A majority of these positions were within CDR-H1. Similarly, many of the CDR-H2 residues were subjected to mutagenesis, such as positions A49, S50, 151, R52, S52a, G53, G54, G55, R56, T57, Y58, Y59, S60, D61, N62, V63 and K64 (Kabat). Additionally, multiple substitutions for the amino acids within CDR-H3 were made, for example, positions V93, R94, Y95, D96, H97, Y98, S99, G100, S100a, S100b, D101 and Y102 (Kabat).

[0216] Multiple substitutions were also designed for variable light chain CDR-L1 positions K24, S25, S26, Q27, S27a, L27b, L27c, D27d, S27e, D28, G29, K30, T31, Y32, L33 and N34 (Kabat). For CDR-L2, mutagenesis was performed at positions L50, V51, S52, K53, L54, D55 and S56 (Kabat). The majority of CDR-L3 positions such as Q90, G91, T92, H93, F94, P95, R96 and T97 were also rationally substituted with multiple amino acids (Kabat).

[0217] All variant antibodies resulting from rational as well as GPSpro design were analyzed for expression, melting point (Tm), affinity, and avidity. Eight antibodies from the rational design and six antibodies from the computer learning campaign were selected for further analysis based on the assays mentioned above.

Example 2. IC.sub.50 Ratio Determination by Competitive ELISA Assays

[0218] An assay based on the competition (inhibition) of binding of a labeled antibody to an antigen-coated plate was used to determine IC.sub.50 for antibodies of the disclosure.

[0219] To generate fibrils, A.beta. 1-42 polypeptides, previously treated with HFIP (hexafluoroisopropanol) and dried, were resuspended in DMSO to 5 mM, then further diluted to 100 uM with 10 mM HCl. Samples were incubated at 37.degree. C. for 24 h, and then centrifuged to separate soluble and fibrillar species. The pellet was the resuspended in 1.times.D-PBS to the original volume and sonicated before use.

[0220] Plates were coated with 0.5 mg/ml of fibril A.beta. 42 and blocked, e.g., with 1% BSA/PBS. Seven 3-fold dilutions of hBP starting at 150 .mu.g/ml (75 .mu.g/ml final concentration) and four 3-fold dilutions of test antibody starting at 20 .mu.g/ml (10 .mu.g/ml final concentration) prepared in 0.1% BSA/PBS were added to wells in triplicate, 50 ul per well. 50 ul of hBP-biotin at 0.75 .mu.g/ml (0.35 .mu.g/ml final concentration) prepared in 0.1% BSA/PBS was added to all wells and plates incubated 2 hours at room temperature then washed 3.times. with TTBS. 100 ul of GE Streptavidin HRP diluted 1/10,000 was then added and incubated for 30 minutes. Plates were then washed 6.times. with TTBS. Thermo Fisher o-phenylenediamine dihydrochloride (OPD) substrate was prepared fresh per manufacturers direction, and 100 ul per well was added. The reaction was incubated for 15 minutes and the reaction stopped with 50 ul 2N H.sub.2SO.sub.4. Samples were read 490 nM on Spectromax. FIG. 2, FIG. 3 and FIG. 4 illustrate competitive ELISA assay graphs for 4918, 4917, 4921, 3818, 49human3, 2931 and bapineuzumab control (FIG. 2), 2926, 2831, 2927, 2726, 2731, 2826 and bapineuzumab control (FIG. 3) and 2727, 2929 and bapineuzumab control (FIG. 4). IC.sub.50 for each test antibody are divided by the IC.sub.50 for hBP to yield an half maximal inhibitory concentration (IC.sub.50) ratio. A ratio of less than one indicates better performance than hBP. See Table 2.

TABLE-US-00005 TABLE 2 Competition ELISA on fibrilA.beta.42 IC.sub.50 ratio Antibody (test:hBP) h2931 0.59 h2731 0.61 h2726 0.68 h2831 0.77 h2926 0.99 h4921 1.01 h2826 1.10 h2929 1.16 h3818 1.18 h2927 1.60 h49_hum3 2.16 h49_VK17 2.69 h2727 3.06 h4918 ND hBP 1

Example 3. Monoclonal Antibody Potency Determination by Competitive ELISA

[0221] The binding potency of certain monoclonal antibodies of the disclosure and hBP was measured by their ability to compete with biotinylated-bapineuzumab bound to aggregated A.beta.42 was assessed by competition ELISA. One mg of A.beta. 42 was added to 1 ml of diH2O and was vigorously vortexed and placed on a nutator for 48 hours at room temperature. Plates were coated with 0.5 mg/ml of the heterogeneous A.beta. 42 aggregate mixture and blocked, e.g., with 1% BSA/PBS. Seven 3-fold dilutions of hBP starting at 150 .mu.g/ml (75 .mu.g/ml after dilution with hBP-Biotin) and four 3-fold dilutions of test antibody starting at 20 .mu.g/ml (10 .mu.g/ml after dilution with hBP-Biotin) were added to wells in triplicate, 50 ul per well. 50 ul of hBP-biotin at 0.75 .mu.g/ml (0.35 .mu.g/ml after dilution) was added to all wells and plates incubated 2 hours at room temperature then washed 3.times. with TTBS. 100 ul of GE Streptavidin HRP diluted 1/10,000 was then added and incubated for 30 minutes. Plates were washed six times with TTBS. Thermo Fisher o-phenylenediamine dihydrochloride (OPD) substrate was prepared fresh per manufacturers direction, and 100 ul per well was added. The reaction was incubated for 15 minutes and the reaction stopped with 50 ul 2N H.sub.2SO.sub.4. Samples were read 490 nM on Spectromax. FIG. 5A shows a competitive ELISA assay graph for 2931, 2731 and bapineuzumab control; FIG. 5B shows a competitive ELISA assay graph for 2726, 2831 and bapineuzumab control. FIG. 20A shows a competitive ELISA assay graph for 2931, 2731 and bapineuzumab control (data shown in Table 3, rows 1-2); FIG. 20B shows a competitive ELISA assay graph for 2831, 2726 and bapineuzumab control (data shown in Table 3, rows 4-5). For FIG. 20A and FIG. 20B, curves and resulting IC50 estimations represent nonlinear three-parameter least squares fit of data. Individual points are the average of triplicate samples (coefficient of variation <20%).

TABLE-US-00006 TABLE 3 mAb Bapi h2931 h2731 IC.sub.50 (.mu.g/mL mAb) 15.04 6.901 5.024 mAb Bapi h2726 h2831 IC.sub.50 (.mu.g/mL mAb) 21.83 9.049 9.907

[0222] Results show that antibodies 2931, 2731, 2726, and 2831 showed greater potency than hBP; .about.2-4 lower IC.sub.50 values than hBP.

Example 4. Characterization of Humanized mAbs or Fabs by BIAcore

[0223] To compare the binding characteristics of humanized antibodies or humanized antigen-binding fragments (Fab) to recombinant A.beta..sub.1-42 fibrils, analysis was performed using a BIAcore T200 (GE Life Sciences).

[0224] To generate fibrils, A.beta..sub.1-42 polypeptides, previously treated with HFIP (hexafluoroisopropanol) and dried, were resuspended in DMSO to 5 mM, then further diluted to 100 uM with 10 mM HCl. Samples were incubated at 37.degree. C. for 24 h, and then centrifuged to separate soluble and fibrillar species. The pellet was the resuspended in D-PBS to the original volume and sonicated before use.

[0225] Fibrils were immobilized on sensor chip CMS (GE Healthcare Life Sciences) via amine coupling to a level to ensure a maximum binding of analyte of approximately 100 RU. Various concentrations of antibodies or Fabs (ranging from 1 nM to 100 nM) were passed over the coupled ligand at 30 .mu.L/min in running buffer (HBS+0.05% P-20, 1 mg/mL BSA) for 300 s association time and 1200 s dissociation time. Regeneration of the chip surface was accomplished by 2 short injections of 10 mM Glycine-HCl at pH 1.7. Data was blank-subtracted to both a sensor not containing ligand and 0 nM analyte concentration. Analysis was performed using a global 1:1 fit with BIAcore Insight Evaluation software (v2.0) with bulk refractive index set to zero RU. Off-rate data (k.sub.diss; kd) are shown in Table 4 (Fabs) and Table 6 (antibodies).

[0226] Similar, small dissociation constants can be seen for the h2726, h2731, h2831 and h2931 Fabs and antibodies in comparison to aducanumab, which demonstrated a significantly larger dissociation constant.

TABLE-US-00007 TABLE 4 Injection 1:1 variables binding Apparent Analyte 1 ka kd KD Rmax Solution (1/Ms) (1/s) (M) (RU) h2726 1.29e+5 2.59e-4 2.01e-9 133.5 h2731 1.29e+5 2.89e-4 2.24e-9 134.0 h2831 1.08e+5 2.48e-4 2.31e-9 127.1 h2931 1.23e+5 1.99e-4 1.62e-9 132.0 hBP 1.12e+5 6.00e-4 5.34e-9 116.1

Example 5. Characterization of Humanized mAbs Affinity Apparent by BIAcore

[0227] Determination of binding affinity of anti-A candidates to A .sub.1-28 (Bachem, Torrance, Calif.) was performed using a Biacore T200. Anti-human Fc antibody was immobilized to a CM3 sensor chip (GE Healthcare Life Sciences) via amine coupling and used to capture A antibodies.

[0228] Various concentrations of A .sub.1-28 (analyte, ranging from concentrations of 100 nM down to 0.39 nM, serial diluted 2-fold each dilution step) were passed over the captured ligand at 50 .mu.l/min in running buffer (HBS+0.05% P-20, 1 mg/mL BSA) for 240 s association time and 900 s dissociation time. Data were blank subtracted to both an irrelevant sensor not containing ligand, and buffer runs containing 0 nM analyte concentration. Analysis was performed using a global 1:1 fit with Biacore Evaluation software (v3.0).

[0229] Apparent dissociation constants (KD) are shown in Table 5, where mAbs of the disclosure demonstrated 4-7 nM binding affinity for A .sub.1-28 monomer. Sensorgrams of binding at concentrations from 0.39 nM through 100 nM are shown in FIG. 6A (h2726), FIG. 6B (h2731), FIG. 6C (h2831) and FIG. 6D (h2931).

TABLE-US-00008 TABLE 5 Injection variables Capture Analyte 1 1:1 binding Apparent Rmax Solution Solution ka (1/Ms) kd (1/s) KD (M) (RU) h2726 A.beta..sub.1-28 9.23e+4 5.55e-4 6.01e-9 87.0 h2731 A.beta..sub.1-28 1.19e+5 5.95e-4 5.01e-9 78.3 h2831 A.beta..sub.1-28 7.31e+4 5.08e-4 6.95e-9 88.0 h2931 A.beta..sub.1-28 9.47e+4 4.12e-4 4.35e-9 76.1

Example 6. Characterization of Humanized mAbs Affinity Apparent by BIAcore

[0230] To compare the binding characteristics of humanized antibodies to recombinant A.beta..sub.1-42 fibrils, analysis was performed using a BIAcore T200.

[0231] To generate fibrils, A.beta..sub.1-42 polypeptides, previously treated with HFIP (hexafluoroisopropanol) and dried, were resuspended in DMSO to 5 mM, then further diluted to 100 .mu.M with 10 mM HCl. Samples were incubated at 37.degree. C. for 24 h, and then centrifuged to separate soluble and fibrillar species. The pellet was the resuspended in 1.times.D-PBS to the original volume and sonicated before use.

[0232] Fibrils were immobilized on sensor chip CMS (GE Healthcare Life Sciences) via amine coupling to a level to ensure a maximum binding of analyte of approximately 50 RU. Various concentrations of antibodies (ranging from 0.411 nM to 100 nM) were passed over the coupled ligand at 30 .mu.L/min in running buffer (HBS+0.05% P-20, 1 mg/mL BSA) for 300 s association time and 1200 s dissociation time. Regeneration of the chip surface was accomplished by 2 short injections of 10 mM Glycine-HCl pH 1.7. Data was blank subtracted to both a sensor not containing ligand and 0 nM analyte concentration. Analysis was performed using a global 1:1 fit with BIAcore Insight Evaluation software (v2.0) with bulk refractive index set to zero RU. Apparent dissociation constant (KD) are shown in Table 6 and a comparison sensorgram of binding at 100 nM is shown in FIG. 7.

TABLE-US-00009 TABLE 6 Injection variables 1:1 Immobilized Analyte 1 binding Apparent Rmax ligand Solution ka (1/Ms) kd (1/s) KD (M) (RU) fibril A.beta. 7.5 .mu.g/mL Adu 2.96e+7 1.70e-2 5.74e-10 45.2 Ace4.5 fibril A.beta. 7.5 .mu.g/mL h2726 3.93e+5 2.12e-5 5.40e-11 51.0 Ace4.5 fibril A.beta. 7.5 .mu.g/mL h2731 3.72e+5 2.62e-5 7.04e-11 50.7 Ace4.5 fibril A.beta. 7.5 .mu.g/mL h2831 2.65e+5 2.94e-5 1.11e-10 50.2 Ace4.5 fibril A.beta. 7.5 .mu.g/mL h2931 3.35e+5 2.05e-5 6.12e-11 50.0 Ace4.5

Abeta, amyloid beta, A.beta.; ka, association rate constant; kd, dissociation rate constant; KD, apparent equilibrium dissociation constant; mAb, monoclonal antibody; R.sub.max, maximum response; SPR, surface plasmon resonance.

[0233] The enhanced relative avidity of monoclonal antibodies of the disclosure for fibrillar A.beta. observed by ELISA was confirmed by SPR equilibrium binding kinetics (Table 6), which indicated a 5- to 11-fold greater avidity (apparent KD) than aducanumab.

[0234] This is explained by the different kinetic binding profiles observed in the SPR sensorgram (FIG. 7). Although aducanumab binds A.beta. fibrils at a faster association rate (ka), the much slower dissociation rate (kd) of the monoclonal antibodies of the disclosure resulted in greater measured avidity (i.e., lower KD*) than aducanumab.

Example 7. A.beta. Fibril Binding by ELISA

[0235] The direct binding of certain monoclonal antibodies of the disclosure and aducanumab to A.beta..sub.1-42 and A.beta..sub.pE3-42 fibrils was assessed by ELISA. To generate fibrils, A.beta..sub.1-42 or A.beta..sub.pE3-42 polypeptides, previously treated with HFIP (hexafluoroisopropanol) and dried, were resuspended in DMSO to 5 mM, then further diluted to 100 uM with 10 mM HCl. Samples were incubated at 37.degree. C. for 24 h, and then centrifuged to separate soluble and fibrillar species. The pellet was the resuspended in 1.times.D-PBS to the original volume and sonicated before use.

[0236] 1.0 .mu.g/ml or 2.5 .mu.g/ml of A.beta. fibrils in PBS were coated overnight at room temperature. Plates were blocked 1% BSA/PBS for 1 hour. Antibodies were serially diluted from 10 .mu.g/ml to 4.8 ng/ml in 0.1% BSA-PBS and 0.1% Tween 20 and 100 .mu.l of each dilution was added in duplicate to each antibody and incubated for 2 hrs at room temperature. Plates were washed four times with TBS/Tween 20 and 100 .mu.l of goat anti Human IgG HRP (Jackson ImmunnoResearch Laboratories, Inc, West Grove, Pa. or Invitrogen, Carlsbad, Calif.) at 1/5000 dilution was added to each well and incubated 1 hour at room temperature. Plates were wash six times in TBS/Tween 20, and Thermo Fisher o-phenylenediamine dihydrochloride (OPD) tablets and ThermoFisher substrate buffer were prepared per manufacturer's instructions. 100 ul of substrate was added and incubated 15 min. Reaction was stopped with 50 .mu.l H.sub.2SO.sub.4. Plates were read at 490 nm on a molecular devices spectromax. FIG. 9A, and FIG. 21. For FIG. 21, curves and resulting EC.sub.50 estimations represent nonlinear three-parameter least squares fit of the data (data shown in Table 7).

TABLE-US-00010 TABLE 7 mAb h2726 h2731 h2831 h2931 Adu EC.sub.50 (.mu.g/mL mAb) 0.0359 0.03671 0.04894 0.04495 0.7241

[0237] Plates were coated with dilutions of A.beta. fibrils in PBS from 10 .mu.g/ml to 4.8 ng/ml overnight at room temperature. Plates were blocked 1% BSA/PBS 1 hour. Antibodies at 2 .mu.g/ml in 0.1% BSA/PBS 0.1% Tween 20 were added in duplicate to the appropriate wells and incubated for 2 hrs at room temperature. Plates were washed 4.times. with TBS/Tween 20 and then 100 .mu.l of Jackson Goat anti Human IgG HRP 1/5000 dilution was added to each well and incubated 1 hour at room temperature. Plates were wash six times in TBS/Tween 20, and Thermo Fisher o-phenylenediamine dihydrochloride (OPD) tablets and ThermoFisher substrate buffer were prepared per manufacturer's instructions. 100 .mu.l of substrate was and incubated 15 min. Reaction was stopped with 50 .mu.l H.sub.2SO.sub.4. Plates were read at 490 nm on a molecular devices spectromax. FIG. 9B, right panel.

[0238] Antibodies h2726, h2731, h2831 and h2931, all demonstrated strong affinities to fibrils, with the difference between best and worst performer within 25%. Additionally, these four antibodies all demonstrated significantly greater avidity than aducanumab. For FIG. 21, a 3-fold increase in assay signal (OD490) and a 15 to 20-fold lower estimated EC.sub.50 indicated increased overall binding and relative avidity of h2726, h2731, h2831 and h2931 mAbs to fibrillar A.beta. relative to aducanumab.

Example 8. h2931 Binding of A.beta. Oligomer by ELISA

[0239] The direct binding of h2931 to A.beta. oligomer was assessed by ELISA. To generate oligomers, first lyophilized biotinylated and unlabeled A.beta. (Bachem) were each solubilized at 1 mg/mL in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, Sigma). HFIP was allowed to evaporate from the samples overnight in a fume hood at room temperature. Aliquots were then centrifuged in a speedvac at room temperature to remove all liquid to generate 250 .mu.g aliquots of HFIP films, which were stored at -80.degree. C. until further use.

[0240] Oligomers were prepared by solubilizing 250 .mu.g of biotinylated and unlabeled A.beta. HFIP pellets in dry DMSO (Sigma) to a final concentration of 5 mM. For unlabeled:biotinylated mixtures, samples were combined in a 9:1 ratio (unlabeled:biotinylated) in an sterile 1.5 mL low-binding microcentrifuge tube (Axygen). DMSO-solubilized samples were then diluted to 100 .mu.M with cold phenol-free neurobasal media (Invitrogen) and incubated for 24 hours at 4.degree. C. After incubation, the oligomers were separated from large insoluble material via centrifugation at 14,000 g for 15 minutes. The top 90% of the supernatant was carefully removed and placed in a new sterile low-binding microcentrifuge tube and stored on ice until use.

[0241] 2.5 .mu.g/mL of each preparation in PBS was coated 100 ul per well in Costar ELISA high bind plates overnight at room temperature. Plates were aspirated and then 200 .mu.l of 1% BSA in PBS was added in each well and incubated 1 hour at room temperature. h2931 mAb was made at a starting concentration 10 .mu.g/ml in 0.1% BSA/PBS 0.1% tween 20 buffer and serially diluted seven times (1:2 each time) with the same. The samples were incubated for 2 hours at room temperature. Plates were washed 4 times with TBS.0.1% tween 20. Goat anti-human (H+L) HRP (Jackson Immunoresearch, PA) was diluted 1/5000 in 0.1% BSA/PBS 0.1% tween 20, added at 100 .mu.l/well and incubated 1 hour at room temperature. Plates were washed 4 times and o-phenylenediamine dihydrochloride tablets (ThermoFisher) were prepared as per manufacturer instructions. 100 .mu.l was added per well and incubated for 15 minutes at room temperature. Reactions were stopped by the addition of 50 .mu.l of H.sub.2SO.sub.4, and samples were read at 490 nM on a Molecular Devices SpectroMax. Curves and resulting EC.sub.50 estimations represent nonlinear 3-parameter least-squares fit of data using GraphPad Prism software.

[0242] mAb h2931 was shown to bind soluble oligomers with high relative affinity, with an estimated EC.sub.50 of 23 ng/mL or 0.15 nM. FIG. 8.

Example 9. Anti-A.beta. Antibodies Binding in AD Brain

[0243] Tissue samples. Frozen human AD brain samples were obtained from Banner Sun Health Research Institute, Sun City, Ariz. The tissues are from donors who were confirmed to have high amount of A.beta. pathology and staged according to the Braak system at the provider institution (Table 8). In addition, quality control was performed in-house on all tissue blocks to ascertain their pathology level and distribution.

TABLE-US-00011 TABLE 8 AD donor information Case ID gender Expired_age PMI Braak score AD 13-75 M 77 3.62 VI AD 14-11 M 82 3.98 V AD 15-19 F 83 3.62 V AD 11-97 F 86 2.52 V

[0244] Tissue Sectioning and Fixation. The unfixed frozen brain tissue samples were embedded in Tissue-Tek OCT (Sakura Finetek) in cryomolds dipped in a mixture of 2-methylbutane and dry ice slurry (-60.degree. C.) then stored at -80.degree. C. until sectioning. Serial 10 .mu.m thick cryosections were generated using a Leica 3050S cryostat. The sections were directly thaw-mounted on positively charged glass slides and were stored at -20.degree. C. until use. Prior to immunohistochemistry IHC procedures, the slides were immersed in 10% neutral buffered formalin solution for 10 minutes at 4.degree. C., rinsed in PBS, then incubated for an hour at 37.degree. C. in a glucose oxidase solution (20 mM beta D(+) glucose, 2 mM sodium azide, and 2 units/mL glucose oxidase in 1.times.PBS). The slides were rinsed 3 times for 5 minutes in PBS before they were transferred onto staining racks for processing in an automated stainer.

[0245] Antibody biotinylation. The humanized IgG antibodies were biotinylated using a non-covalent method, by means of incubation with a biotin-conjugated goat anti-human monovalent fab fragment (Jackson ImmunoResearch) in a ratio of 1:4, for 1 hour at room temperature. Unbound excess Fab was absorbed by pre-incubation with human serum for an additional hour before use. The freshly prepared antibodies were then loaded into the stainer for immediate application to tissue sections.

[0246] Immunostaining. The staining was performed in an automated Leica Bond Rx Stainer (Leica Biosystems), using the Bond Research Kit (DS980, Leica Biosystems) and the avidin-biotin amplified immuno-peroxidase detection system. Each biotinylated anti-A.beta. antibody, or a human IgG control, was applied to the sections, at specified concentrations, for one hour and the staining was visualized using the avidin-biotin amplification system (ABC Elite Standard, PK-6100; Vector Laboratories). Hematoxylin counter-staining of nuclei was subsequently applied to sections before dehydration in an ascending series of alcohols, clearing in xylene, cover-slipping, and air-drying.

[0247] Tissue imaging. The stained slides were digitally imaged using a Hamamatsu NanoZoomer 2.0HT slide scanner (Hamamatsu Corporation), and the images were captured in an .ndpi file format using the NanoZoomer Digital Pathology software (NDP.scan, Version 2.7.25). Images included in this report were captured directly from NDP.view and transferred without any enhancement. For morphometry, the digitized slides were analyzed using Halo software (V2.1.1537) to measure the percentage of stained tissue, and the results were plotted using GraphPad Prism 8.

[0248] Results with h2726, h2731, h2831, h2931 and aducanumab. Four humanized anti-A.beta. antibodies of the disclosure, h2726, h2731, h2831 and h2931, as well as aducanumab, were applied to all four AD brains at increasing concentrations: 0.03, 0.1, 0.3, 1, 3 and 9 .mu.g/ml. As shown in FIG. 10 (0.3 .mu.g/mL), the AD brain sections that were incubated with these antibodies exhibited immunopositive structures that are typical for A.beta. pathology in AD. Brains AD 13-75 and AD 14-11 have high density of A.beta. plaques while the pathology in brains AD 11-97 and AD 15-19 was comparatively sparse. In each brain, the staining produced by the four antibodies, h2726, h2731, h2831 and h2931, at a specific concentration, was comparable in intensity and distribution. Staining with aducanumab was the weakest among samples and concentrations. As exemplified in FIG. 11, sections from all four brains that were incubated with control human IgG isotype at 1 or 9 .mu.g/ml had no pathology staining.

[0249] The graphs in FIG. 12 and FIG. 22 are plots of the quantification of staining by the five antibodies in all four AD brains. Measurements of the percentage of tissue surface area that was occupied by the stained pathology confirm that, in each AD brain, the four antibodies, h2726, h2731, h2831, h2931, have similar levels of binding, at all concentrations tested. Correspondingly, the data in Table 9 show that, with each brain, the area under the curve and EC50 values remain comparable for the four antibodies. Values obtained with aducanumab were consistently lower among AD brains throughout the concentration range tested.

[0250] FIG. 22 showed greater plaque area binding (as a percentage positive tissue stained) than aducanumab, notably, at antibody concentrations that are estimated to be clinically relevant exposures in cerebrospinal fluid with 10 mg/kg aducanumab. Similar plaque area staining was observed at the highest concentration tested, suggesting saturation of binding at this level.

TABLE-US-00012 TABLE 9 Area under the curve and half maximal effective concentration (EC.sub.50) h2726 h2731 h2831 h2931 Area Under Curve AD 11-97 50.18 50.58 49.21 47.70 AD 15-19 52.08 52.71 49.73 44.81 AD 13-75 149.3 150.4 138.7 139.1 AD 14-11 149.1 149.2 148.9 134.5 EC50 AD 11-97 0.09163 0.1346 0.1019 0.08893 AD 15-19 0.1356 0.1274 0.1328 0.1330 AD 13-75 0.1615 0.1415 0.2144 0.2273 AD 14-11 0.1325 0.1102 0.1625 0.1691

[0251] Results with Bapineuzumab (hBP)

[0252] Section from brain AD 13-75 were incubated with the humanized antibody hBP as well as aducanumab and BAN2401 at increasing concentrations: 0.03, 0.1, 0.3, 1, 3 and 9 .mu.g/ml. As seen with antibodies h2726, h2731, h2831 and h2931, the level of staining with hBP increased in a dose dependent manner. In addition, hBP staining was stronger than that of aducanumab and BAN2401 at all concentrations tested, as shown in FIG. 13.

Example 10. Ex Vivo Phagocytosis Assays for Determination of (A.beta..sub.1-42 and A.beta..sub.pE3-42) Plaque Clearance

[0253] In the early stages of AD, microglial function is neuroprotective, acting to clear apoptotic cells and pathological protein aggregates, as well as forming a barrier around plaques to restrict their growth and diffusion of synaptotoxic A.beta. oligomers. Ex vivo phagocytosis assays quantitate the antibody-mediated microglial clearance response.

[0254] Primary microglial culture generation: For dissection of neonatal mouse brain tissue, P1 pups are quickly decapitated with sterile scissors. Meninges are removed and forebrain were immediately immersed into 1-5 ml dissection media (e.g., high glucose DMEM with 20% FBS, P/S) on ice until the desired number of pup brains has been dissected. Preferably limit total procedure time to within 10 minutes to minimize cellular damage.

[0255] Tissue was carefully aspirated twice consecutively with new sterile pipettes using a 22G needle, followed by a 25G needle. Sample were centrifuged at 2,500.times.g for five minutes at 4.degree. C. Supernatant was carefully aspirated and 5 ml of fresh growth media was added (high-glucose DMEM, 10% FBS, P/S and 25 ng/ml recombinant mouse GM-CSF) to the cell pellets. The cell pellets are pipetted up and down approximately 10 times with a sterile 10 ml pipette to dissociate the pellets.

[0256] A cell strainer (100 .mu.m pores) was placed onto a fresh 50 ml conical tube and the material was dispensed through the cell strainer into the conical tube. The cell strainer was rinsed with 4-5 ml of fresh media, followed by centrifuging 200.times.g for five minutes at 4.degree. C.

[0257] Cells were plated at a density of two mouse brains per T-75 plastic culture flask. Carefully aspirate supernatant and add 3 ml of fresh growth medium (high-glucose DMEM, 10% FBS, P/S, and 25 ng/ml recombinant mouse GM-CSF) to each cell pellet with 10 ml sterile pipette. Pipette up and down 10 times with a 10 ml pipette to resuspend. Prepare 1 sterile T-75 flask by adding 6 ml of growth medium (high-glucose DMEM, 10% FBS, P/S and 25 ng/ml recombinant mouse granulocyte-monocyte colony-stimulating factor) into each flask, followed by the addition of 6 ml of resuspended cell pellets to obtain 12 ml final in a 5% CO.sub.2 incubator at 37.degree. C.

[0258] Flasks are incubated undisturbed for five days to allow cells to attach. On the fifth day, the culture media was replaced in each flask with 12 ml of fresh growth medium (high-glucose DMEM, 10% FBS, P/S and 25 ng/ml recombinant mouse GM-CSF). Approximately 10% of the mixed cells plated will attach and grow on the plastic surface. The media was changed twice per week (every 3-4 days) to achieve confluence. Such changes are carried out with very carefully without touching the bottom of the flasks where the cells are attached.

[0259] After 7-11 d the flasks were rotated at 200 rpm using a Lab-Line orbital shaker with a 19-mm orbit for 2 h at 37.degree. C. Cell suspensions were centrifuged at 200.times.g and resuspended in assay medium (hybridoma-serum free medium H-SFM [Life Technologies] plus 1% FBS, glutamine, P/S, and 5 ng/ml recombinant mouse GM-CSF).

[0260] Ex vivo assays. Cryostat sections (10 .mu.m in thickness; use the wide blades) of APP/PS1 mouse or human AD brains (postmortem interval, less than 3 h) were `thaw mounted` onto polylysine-coated, round glass coverslips and placed in wells of 24-well tissue culture plates (CT -30 C OT -20 C). Tissue samples can be warmed with thumb in between sections or by reducing OT to -12 C). The coverslips were washed twice with assay medium. Antibodies (control or against A.beta.) were added at a 2.times. concentration 250 .mu.l in assay medium (20 .mu.g/ml final) for 1 h in tissue culture incubator.

[0261] Microglial cells were then seeded at a final density of 800,000 cells/ml (1,600,000 cells/ml stock) in assay medium 250 .mu.l. The cultures were maintained in a humidified incubator at 37.degree. C. in an atmosphere of 5% CO2 for 72 hrs.

[0262] Quantification of total A.beta. (A.beta..sub.1-42). Media was carefully aspirated, followed by washing with ice cold PBS. 100 .mu.l 8M urea was added and tissue resuspended by pipetting and scraped off with pipette tip. Suspension was then frozen at -20.degree. C. until ready for analysis. Suspensions were thawed on ice, centrifuged 16,000.times.g 20 min at 4.degree. C. before dilution and analysis using a V-PLEX Total A.beta.42 Peptide (4G8) Kit (Meso Scale Discovery). Results are shown in FIG. 14A, FIG. 14B and FIG. 24. FIG. 14A and FIG. 24 show A.beta. level per brain section and FIG. 14B shows the same data as a scatter plot per treatment (data for FIG. 14B shown in Table 10; data for FIG. 24 shown in Table 11). h2731, h2931, and aducanumab demonstrated highly significant reductions in A.beta. plaque species over isotype control.

TABLE-US-00013 TABLE 10 mAb Isotype (Avg. pg/mL A.beta..sub.1-42) control h2931 Aducanumab Mean 92619 53113 49501 SD 14801 18239 7961

TABLE-US-00014 TABLE 11 Condition A.beta.1-42 (pg/ml) SD Healthy control 5797.25 2022.51 AD brain + hIgG1 isotype 185138.90 35888.64 AD brain + h2731 101172.05 40194.48

[0263] Quantification of pyroglutamate-3 A.beta. (A.beta..sub.pE3-42). N-terminal truncated and pyroglutamate-modified A.beta. (e.g., A.beta..sub.pE3-42) has been described as a component of mature senile plaques in AD brain (Saido et al., Neuron 14, 1995). It was unknown whether pyroglutamate-modification of N-terminal A.beta. would affect binding of N-terminal antibodies like h2731 and others described herein. Likewise, it was unknown whether these antibodies would have the ability to promote phagocytic-mediated clearance of A.beta..sub.pE3-42.

[0264] The presence of pyroglutamate-3 A.beta. in AD brain used for ex vivo experiments, as well as its similar staining pattern compared to h2931, was confirmed by immunohistochemistry (FIGS. 25A and 25B). To demonstrate removal of pyroglutamate-3 A.beta., a commercial ELISA method was used to measure its removal during ex vivo phagocytosis. Suspensions that were collected following methods above were thawed on ice, centrifuged 16,000.times.g 20 min at 4.degree. C. before dilution and analysis using a commercial ELISA kit (Amyloid Beta N3pE A.beta., IBL America). A.beta..sub.pE3-42 ELISA assay is highly specific to A.beta..sub.pE3-42 when compared to unmodified A.beta..sub.1-42 (data not shown).

[0265] Results are shown in FIG. 26A and FIG. 26B (data shown in Table 12 and Table 13, respectively), which show levels of pyroglutamate-3 A.beta. in brain sections after treatment with indicated antibodies, h2931 in FIG. 26A and h2731 in the FIG. 26B, each compared to a healthy control and compared to AD brain treated with IgG1 isotype control. Sections from different AD brains were used for each treatment. h2731 and h2931 both demonstrate highly significant reductions in pyroglutamate-3 A.beta. over isotype control.

[0266] FIG. 24 and FIG. 26B, taken together, indicate anti-A.beta. antibodies of the present invention (e.g., h2731) promote clearance of both A.beta..sub.1-42 and A.beta..sub.pE3-42 protein when incubated on AD patient brain tissue sections with primary mouse microglia. These results confirm that these antibodies clear both A.beta..sub.1-42 and A.beta..sub.pE3-42 in the human pathology setting.

[0267] The N-terminal-targeted anti-A.beta. antibodies, facilitated abundant microglia-mediated clearance of A.beta. plaque species, including pyroglutamate-modified A.beta., in brain tissue from AD patients. These data support further development of antibodies of the present invention as a subcutaneously administered antibody immunotherapy for Alzheimer's disease.

TABLE-US-00015 TABLE 12 Condition AbpE3-42 (pg/ml) Stdev Healthy control 44.20 6.39 AD brain + hIgG1 isotype 259.42 27.39 AD brain + h2931 62.59 16.16

TABLE-US-00016 TABLE 13 Condition AbpE3-42 (pg/ml) Stdev Healthy control 26.75 34.83 AD brain + hIgG1 isotype 478.91 117.80 AD brain + h2731 153.76 67.59

Example 11. Blocking Oligomers in Hippocampal Binding Assay

[0268] A Binding Assay in Rat Hippocampal Neurons

[0269] E18 primary rat hippocampal neurons were cultured as described by Zago et al. (J. Neurosci 22 Feb. 2012, 32 (8) 2696-2702). Soluble A was pre-incubated with and without antibody on culture DIV14-21 to block neuritic binding to primary neurons.

[0270] Fresh unlabeled, biotinylated or (9:1) unlabeled:biotinylated soluble A was prepared one day prior and incubated overnight at 4.degree. C. The A was spun down @ 14,000 RPM for 15 minutes before use.

[0271] Each dilution of A solution and antibody at (2.times.) of the final treatment concentration in one-half of final treatment volume using NeuroBasal-no phenol red (NB-NPR) or NbActiv4-NPR medium were prepared. After combining, the mixture was mixed 3-4 times then pre-incubated for 30 minutes at 37.degree. C.

[0272] Immediately before binding assay, the neurons were rinsed with pre-warmed NB-NPR at 150 .mu.L/well. The buffer was aspirated and then antibody/A treatment was added to cells at 60 .mu.L/well then incubated for 30-40 minutes at 37.degree. C. under normal incubator conditions (5% CO2; 9% O2).

[0273] The neurons were rinsed twice in 150 .mu.L/well NB-NPR then fixed in 4% paraformaldehyde in 1.times.DPBS for 20 minutes at room temperature.

[0274] The cells were permeabilized in 0.1% Triton X-100 in 1.times.DPBS for 5 minutes and then blocked in 10% normal goat serum (NGS) for 1 hour at room temperature (RT).

[0275] The samples were incubated with microtubule-associated protein 2 (MAP2) and neuronal nuclear protein (NeuN) primary antibodies in 100 .mu.L/well 1.times.DPBS containing 1% BSA+1% NGS overnight at 4.degree. C. On the next day, the samples were rinsed twice in 150 .mu.L/well 1.times.DPBS for 5 minutes each wash. Secondary antibody was added for 1 hour @ room temperature in 100 .mu.L/well 1.times.DPBS+1% BSA+1% NGS.

[0276] High-content imaging (HCl) analysis was performed to quantify soluble A neuritic binding spots using Operetta HCl CLS instrument (Perkin Elmer; modified Neurite Outgrowth algorithm: 40.times. H.sub.2O objective; 25-40 fields per well in microplate format; (n=3) per condition. MAP2 and NeuN neuronal markers were used to each trace neurite tree and count cell body number per optical field (e.g., with microtubule-associated protein 2 (Abcam; Cambridge, UK), and NeuN (EMD Millipore) primary antibodies followed by AlexaFluor (Thermo Fisher Scientific) secondary detection antibodies). Neuritic A spots were detected using various monoclonal and polyclonal A antibodies (e.g., mouse monoclonal anti-A antibody MabN254 (EMD Millipore)) followed by AlexaFluor (Thermo Fisher Scientific) secondary detection antibodies or streptavidin-AF488 for biotinylated A material. FIG. 15A and FIG. 15B show that increasing concentrations of anti-A antibody reduces the number of spots per neuron, indicating activity against A . FIG. 23 shows h2731 effectively blocked the binding of soluble A.beta. aggregates to rat hippocampal synapses (A.beta.42 spots per neuron) in a concentration-dependent manner. The effect of h2731 was detected at molar mAb:A.beta.42 ratios as low as 1:500 (p<0.05) and reached >90% blockade of binding at 1:50 molar ratios (p<0.001) relative to A.beta.42 alone (no mAb preincubation). Data shown in Table 14.

TABLE-US-00017 TABLE 14 Soluble Isotype A.beta. Control h2731 h2731 h2731 h2731 1 .mu.M 1:50 1:1000 1:500 1:100 1:50 Mean (A.beta. spots 76.3 58.7 51.0 39.7 11.3 6.3 per neuron) SD 28.2 12.9 14.9 12.1 4.9 1.5

Example 12. Anti-A Antibody Binding to Native and Modified A Species

[0277] Cryostat sections of human AD brain were thaw-mounted onto poly-D-lysine coated coverslips and placed in 24-well tissue culture plates and incubated with test antibodies for 1 hour at 37.degree. C. 5% CO.sub.2. Primary mouse microglial cells were then seeded at 800,000 cells/ml, and the cultures were maintained at 37.degree. C. 5% CO.sub.2 for 72 hours. Media was carefully aspirated, and sections washed with PBS. The sections were resuspended in 8M urea for quantification by ELISA for A .sub.pE3-42 (Immuno-Biological Laboratories, Minneapolis, Minn.), or MSD for A .sub.1-42 (Meso Scale Diagnostics, Rockland, Md.). The Immuno-Biological Laboratories A .sub.pE3-42 ELISA kit specifically detects the pE3-42 species with no detectable signal for full-length A.beta..

[0278] FIG. 27 demonstrates that h2731 binds with high apparent affinity to the N-terminus of full length A.beta. but not directly to pyroglutamate-modified A.beta. (A.beta..sub.pE3-42). h2731 bound with a half-maximal effective concentration (EC.sub.50) of 8.1 ng/mL (54 pM) to fibrillar A.beta. species with an unmodified N-terminus (A.beta..sub.1-42). h2731 demonstrated no detectable binding to A.beta..sub.pE3-42 up to 100 ng/ml.

Example 13. In Vitro Phagocytic-Mediated Clearance--THP-1 Human Monocyte-Mediated Uptake of A.beta..sub.1-42 Protofibrils

[0279] Synthetic protofibrils of A .sub.1-42 containing an S26C mutation were generated as described in Paranjape et al., ACS Chem. Neurosci. 2012, 3, 302-311. Briefly, A.beta. peptides were dissolved in 100% hexafluoroisopropanol (HFIP) (SigmaAldrich, St. Louis, Mo.) at 1 mM, aliquoted into sterile microcentrifuge tubes, and evaporated uncovered at room temperature overnight in a fume hood. The following day, the aliquots were vacuum-centrifuged to remove any residual HFIP and stored in desiccant at -20.degree. C. Some A.beta. peptides were treated with 100% trifluoroacetic acid and vacuum centrifuged prior to HFIP treatment. A.beta. oligomers and fibrils obtained directly from lyophilized aliquots were prepared by resuspending lyophilized A.beta. peptide aliquots in sterile anhydrous dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, Mo.) at 5 mM. For oligomer preparation the sample was diluted to 100 .mu.M in sterile ice-cold phenol red-free Ham's F-12 cell culture medium with L-glutamine (F-12, Bioworld, Dublin, Ohio) and incubated for 24 hours at 4.degree. C. For fibril preparation, the sample was diluted to 100 .mu.M in 10 mM HCl and incubated for 24 hours at 37.degree. C. A.beta. concentrations in these preparations were based on dry peptide weight.

[0280] Mature protofibrils were conjugated to pHrodo Red Maleimide (Thermo Fisher) before use in in vitro phagocytic-mediated clearance assays.

[0281] Antibodies at concentrations of 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.098, and 0.049 .mu.g/ml were preincubated for 30 min at room-temperature with pHrodo-A .sub.1-42 protofibrils, followed by the addition of THP-1 phagocytic cells. After a 3-hour incubation at 37.degree. C. and 5% CO.sub.2, antibody-mediated phagocytic-mediated clearance was assessed by measuring cellular pHrodo signal via flow cytometry.

[0282] As shown in FIG. 28A and FIG. 28B, anti-A antibodies exhibited A .sub.1-42 protofibril phagocytic activity in a concentration-dependent fashion. These results suggest that antibodies of the present invention may be able to drive A .sub.1-42 clearance in brain tissue.

Example 14. Distribution of Total and Pyroglutamate-Modified A in Brain Tissue from Advanced AD Patients

[0283] Ex vivo IHC methods as described above and herein were conducted on AD brain tissue to determine the distribution of A .sub.1-XX (detected with an N-terminal anti-A antibody) and anti-A .sub.pE3-42.

[0284] Evaluation of A .sub.1-XX and A .sub.pE3-42 confirmed widespread distribution of both species in tissue from patients with advanced stage AD. The distribution pattern (FIG. 29A(1) and FIG. 29A(2) (and magnified FIG. 29B(1) and FIG. 29B(2), respectively)) and quantification (FIG. 29C) of the percent area covered by A .sub.1-XX compared to A .sub.pE3-42 were consistent with prior studies, suggesting that A .sub.pE3-42 represents a relatively smaller pool of modified A intermingled with the unmodified A targeted by N-terminal A antibodies. A .sub.pE3-42 is shown in FIG. 29A(2) and FIG. 29B(2), and intact N-terminal A is shown in FIG. 29A(1) and FIG. 29B(1). Anti-A .sub.pE3-42 antibody did not cross-react with A .sub.1-42 (data not shown).

[0285] The box in FIG. 29A(1) and FIG. 29B(1) show an A plaque with intact N-terminal A and modified A .sub.pE3-42 proximal to blood vessel. Table 15 below reports the quantification of staining in plaques in FIG. 29B(1) and FIG. 29B(2) that is presented as graph in FIG. 29C. The difference between the mean values is statistically significant (p=0.007, paired two-tailed t-test).

TABLE-US-00018 TABLE 15 % Area Stained Antibody (Mean .+-. SD; N = 5) anti-N-terminal A.beta. antibody 12.59 .+-. 4 anti-A.beta..sub.pE3-42 A.beta. antibody 7.17 .+-. 1.8

Example 15. Anti-A Antibody h2731 Colocalizes with A pE3-42 in AD Brain

[0286] Colocalization of h2731 immunostaining and A .sub.pE3-42 was assessed by immunofluorescent microscopy. An N-terminal anti-A antibody (in this case h2731) was pre-conjugated to a Cy3-secondary anti-human antibody (Jackson Laboratories) before application to tissues. A .sub.pE3-42 was detected using a mouse anti-A .sub.pE3-42 antibody with a 488-AlexaFluor-conjugated anti-mouse secondary antibody. Slides were imaged using a Metamorph-assisted IX81 Olympus microscope connected to a Hamamatsu camera (C10600-10B).

[0287] FIG. 30 (panel A) shows localization of h2731 to A plaques; FIG. 30 (panel B) shows localization of anti-A .sub.pE3-42 antibody signal to A plaques; and FIG. 30 (panel C) shows colocalization of h2731 and anti-A .sub.pE3-42 antibody signal to A plaques. Overlapping signal appears more prominent in dense core regions of the plaques.

Example 16. Anti-A Antibodies of the Present Invention Promote A .sub.pE3-42 Clearance from AD Brain Tissue Ex Vivo in a Dose-Dependent Manner with Higher Efficacy than Aducanumab

[0288] Using methods described above and elsewhere herein, the ability of aducanumab and antibodies of the present invention (e.g., h2731) to clear A .sub.pE3-42 protein from AD brain tissue was assessed.

[0289] A physiologically relevant dose-response series of h2731 (3 ng/ml, 10 ng/ml, 30 ng/ml and 100 ng/ml) was incubated with AD patient brain tissue sections and primary mouse microglia for 72 hours. h2731 promoted A .sub.pE3-42 clearance in a concentration-dependent fashion. Results are presented in Table 16 below and FIG. 31A.

TABLE-US-00019 TABLE 16 Ave A.beta..sub.pE3-42 (pg/ml) Antibody Concentration (ng/ml) (n = 4) Stdev hIgG1 isotype 100 524.34 83.36 h2731 3 479.56 129.92 h2731 10 339.06 165.44 h2731 30 229.28 51.16 h2731 100 261.15 60.81

[0290] h2731 robustly promotes clearance of A .sub.pE3-42 from AD patient brain tissue sections by microglial phagocytosis in a concentration-dependent manner and during a relatively short incubation period (72 hours). Thus, the antibodies of the present invention promote ex vivo clearance of A.beta..sub.pE3-42 from an AD patient brain at a concentration range expected to be reached with subcutaneous administration.

[0291] Another series of experiments were conducted comparing h2731 at 25 ng/ml and 75 ng/ml to aducanumab at 25 ng/ml and 225 ng/ml. Results are presented in Table 17 and FIG. 31B.

TABLE-US-00020 TABLE 17 Ave A.beta..sub.pE3-4 (pg/ml) Antibody Concentration (ng/ml) (n = 4) Stdev hIgG1 isotype 225 449.11 58.14 Adu 225 227.30 98.95 Adu 25 247.34 48.06 h2731 75 52.83 25.40 h2731 25 71.31 64.93

[0292] h2731 exhibited superior A.beta..sub.pE3-42 clearance activity when compared to aducanumab, even at 9-fold lower concentrations.

[0293] Another physiologically relevant dose-response series of h2731 and aducanumab (3 ng/ml, 25 ng/ml, and 225 ng/ml) was incubated with AD patient brain tissue sections and primary mouse microglia for 72 hours, both compared to IgG1 isotype control. While both h2731 and aducanumab promoted A .sub.pE3-42 clearance in a concentration-dependent fashion, h2731 again did so significantly more potently, with a p-value of <0.0001 at a 9-fold lower concentration than required for aducanumab to reach a p-value of 0.0005. Results are presented in Table 18 below as well as FIG. 32A.

TABLE-US-00021 TABLE 18 Concentration AbpE3-42 Antibody (ng/ml) (pg/ml) Stdev hIgG1 isotype 225 1.00 6.73 Adu 225 85.97 74.35 Adu 25 146.70 24.30 Adu 3 245.97 41.70 h2731 225 20.60 14.44 h2731 25 41.07 31.15 h2731 3 154.95 35.89

[0294] In order to verify that h2731-mediated ex vivo phagocytosis activity is microglia dependent, a +/- microglia experiment was performed. While microglia alone drive some A .sub.pE3-42 clearance from AD patient tissue sections, clearance is significantly more robust with the combination of h2731 and microglia. h2731 appears to require the presence of microglia for clearance activity, as h2731 alone has shows no activity without microglia. Results are presented in Table 19 and FIG. 32B.

TABLE-US-00022 TABLE 19 Concentration AbpE3-42 Antibody (ng/ml) (pg/ml) Stdev hIgG1 isotype 75 271.79 27.01 h2731 75 263.70 51.28 hIgG1 + Microglia 75 174.58 15.75 h2731 + Microglia 75 58.37 15.53

[0295] The tested antibody concentrations were based on CNS ranges estimated at 0.1% of steady-state plasma minimum and maximum concentrations from modeled pharmacokinetics following monthly administration of 3 mg/kg subcutaneous h2731 (25-75 ng/ml) or 10 mg/kg of intravenous aducanumab (25-225 ng/ml) in humans (FIG. 33).

[0296] Antibodies of the present invention promote ex vivo clearance of A.beta..sub.pE3-42 from an AD patient brain at a concentration range expected to be reached with subcutaneous administration and with greater biological activity than aducanumab.

[0297] Antibody h2731 reduces A.beta..sub.pE3-42 staining in AD brain. FIG. 34 shows that A.beta..sub.pE3-42 (staining indicated by white arrows) was observed in plaques (white triangles) and associated with blood vessels (circular shape in FIG. 34A and FIG. 34C) in AD brain treated with human IgG isotype control antibody (FIG. 34A and FIG. 34B). Treatment with h2731 enhanced microglia-mediated reduction of A.beta..sub.pE3-42 levels as evidenced by the reduction in plaques (FIG. 34C and FIG. 34D). Antibodies of the present invention, as exemplified by h2731, reduce plaques containing A.beta..sub.pE3-42 in tissue.

Example 17. h2731 Target Engagement

[0298] Female APPxPS1 mice expressing a mutant human amyloid precursor protein (hAPP[V717I]) and a mutant human presenilin 1 (hPS1[A246E]) were used to evaluate the ability of h2731 and aducanumab to traverse the blood-brain-barrier subsequent to peripheral administration and bind to amyloid-beta (A ) plaques in the brain. The average age of the animals at the start of the study was 6.7 months. One day prior to drug administration all animals received an injection of an anti-CD4 antibody (20 mg/kg, intravenous) to prevent the formation of anti-drug antibodies in mice receiving h2731 or aducanumab, both of which are fully humanized antibodies. h2731 (3 or 10 mg/kg, subcutaneous, SC) or aducanumab (10 mg/kg, intravenous) were dosed weekly for three weeks and animals were euthanized one week later. Following transcardial perfusion with ice-cold saline, brains were extracted from the mice and flash frozen in 2-methylbutane on dry ice and stored at -80.degree. C.

[0299] Serial sagittal 10 .mu.m thick cryosections were generated using a Leica 3050S cryostat. The sections were directly thaw-mounted on positively charged glass slides and were stored at -20.degree. C. until use. Prior to IHC, the slides were immersed in 10% neutral buffered formalin solution for 10 minutes at 4.degree. C., rinsed in PBS, then incubated for an hour at 37.degree. C. in a glucose oxidase solution (20 mM beta D(+) glucose, 2 mM sodium azide, and 2 units/mL glucose oxidase in 1.times.PBS). The slides were rinsed 3 times for 5 minutes in PBS before they were transferred onto staining racks for processing in an automated stainer. A biotin-SP-conjugated goat anti human IgG (H+L) (Jackson ImmunoResearch Laboratories #109-065-088) was used to detect h2731 or aducanumab in APPxPS1 brain tissue. The staining was performed in an automated Leica Bond Rx Stainer (Leica Biosystems), using the Bond Research Kit (DS980, Leica Biosystems). Hematoxylin counter-staining of nuclei was subsequently applied to sections before dehydration in an ascending series of alcohols, clearing in xylene, cover-slipping, and air-drying. The whole sections were imaged using a NanoZoomer 2.0HT slide scanner (Hamamatsu Corporation, Japan). Morphometric analysis of the digitalized images was carried out using Halo software (V2.1.1537). After delineation of the cerebral cortex as region of interest, the percent of stained tissue area was determined. Data are presented in Table 20.

TABLE-US-00023 TABLE 20 h2731 Aducanumab 3 mg/kg, SC 10 mg/kg, SC 10 mg/kg, IV Plaque Binding 0.070 .+-. 0.025 0.079 .+-. 0.034 0.060 .+-. 0.034 (% ROI) ROI = region of analysis. All data represent mean .+-. SD of n = 5 animals per group

[0300] Reduction in numbers or size of A.beta. plaques in Alzheimer's Disease may correlate with slowing or reversing of disease progression. The ability of the anti-A.beta. antibodies of the present invention to bind to and clear A.beta. in vivo following peripheral administration supports the potential utility of these antibodies as therapeutic agents.

[0301] Thus, the antibodies of the present invention promote microglia-mediated clearance of A.beta..sub.1-42 in brain tissue from patients with AD. Although antibodies of the present invention may not target the pyroglutamate modification directly, they may effectively clear A.beta..sub.pE3-42 at concentrations predicted to be clinically relevant and with higher potency and greater biologic activity than aducanumab, as exemplified by h2731. Clearance of pyroglutamate species by these antibodies may be due to the ability of microglia to recognize opsonized plaques and engulf large particles with diverse content. The antibodies of the present invention may therefore clear other neurotoxic elements co-deposited in plaques by this same mechanism.

[0302] All publications (including GenBank Accession numbers, UniProtKB/Swiss-Prot accession numbers and the like), patents and patent applications cited are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent and patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. In the event of any variance in sequences associated with Genbank and UniProtKB/Swiss-Prot accession numbers and the like, the application refers to the sequences associated with the cited accession numbers as of the effective filing date of the application meaning the actual filing date or earlier date of a priority application disclosing the relevant accession number. Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Sequence CWU 1

1

1021112PRTArtificial SequenceSynthetic peptide 1Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr His Trp Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 1102112PRTArtificial SequenceSynthetic peptide 2Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 1103119PRTArtificial SequenceSynthetic peptide 3Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Arg Ser Gly Ser Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 1154119PRTArtificial SequenceSynthetic peptide 4Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Arg Ser Gly Ser Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Tyr Asp His Tyr Ser Gly Thr Ser Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 1155119PRTArtificial SequenceSynthetic peptide 5Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Arg Ser Gly Ser Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Tyr Asp His Tyr Thr Gly Thr Ser Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 1156119PRTArtificial SequenceSynthetic peptide 6Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Tyr Asp His Tyr Ser Gly Thr Ser Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 1157119PRTArtificial SequenceSynthetic peptide 7Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Val Arg Ser Gly Gly Ser Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 1158112PRTArtificial SequenceSynthetic peptide 8Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Tyr 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 1109112PRTArtificial SequenceSynthetic peptide 9Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Tyr 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Arg Val Thr Asn Arg Asp Thr Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 11010112PRTArtificial SequenceSynthetic peptide 10Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Arg Val Thr Asn Arg Asp Thr Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 11011112PRTArtificial SequenceSynthetic peptide 11Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Asp Tyr 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Ser His Phe Pro Arg Ser Tyr Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 11012112PRTArtificial SequenceSynthetic peptide 12Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Met Asp Thr 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Lys Val Ser Asn Arg Glu Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 11013112PRTArtificial SequenceSynthetic peptide 13Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Tyr 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 11014112PRTArtificial SequenceSynthetic peptide 14Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 11015112PRTArtificial SequenceSynthetic peptide 15Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Lys Val Thr Asn Arg Glu Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 1101610PRTArtificial SequenceSynthetic peptide 16Gly Phe Thr Phe Ser Asn Tyr Gly Met Ser1 5 101719PRTArtificial SequenceSynthetic peptide 17Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asn Asp Tyr Asn1 5 10 15Val Lys Gly1810PRTArtificial SequenceSynthetic peptide 18Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr1 5 101910PRTArtificial SequenceSynthetic peptide 19Gly Phe Thr Phe Ser Asn Phe Gly Met Ser1 5 102017PRTArtificial SequenceSynthetic peptide 20Ser Ile Arg Ser Gly Ser Gly Arg Thr Tyr Tyr Ser Asp Asn Val Lys1 5 10 15Gly2117PRTArtificial SequenceSynthetic peptide 21Ser Val Arg Ser Gly Ser Gly Arg Thr Tyr Tyr Ser Asp Asn Val Lys1 5 10 15Gly2217PRTArtificial SequenceSynthetic peptide 22Ser Val Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val Lys1 5 10 15Gly2317PRTArtificial SequenceSynthetic peptide 23Ser Val Arg Ser Gly Gly Ser Arg Thr Tyr Tyr Ser Asp Asn Val Lys1 5 10 15Gly2410PRTArtificial SequenceSynthetic peptide 24Tyr Asp His Tyr Ser Gly Thr Ser Asp Tyr1 5 102510PRTArtificial SequenceSynthetic peptide 25Tyr Asp His Tyr Thr Gly Thr Ser Asp Tyr1 5 102616PRTArtificial SequenceSynthetic peptide 26Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn1 5 10 15278PRTArtificial SequenceSynthetic peptide 27Leu Val Ser Ser Lys Leu Asp Ser1 5289PRTArtificial SequenceSynthetic peptide 28Trp Gln Gly Thr His Phe Pro Arg Thr1 52916PRTArtificial SequenceSynthetic peptide 29Lys Ser Ser Gln Ser Leu Leu Asp Tyr Asp Gly Lys Thr Tyr Leu Asn1 5 10 15304PRTArtificial SequenceSynthetic peptide 30Ala Gly Ala Gly13116PRTArtificial SequenceSynthetic peptide 31Arg Ser Ser Gln Ser Leu Val Asp Tyr Asp Gly Lys Thr Tyr Leu Asn1 5 10 153216PRTArtificial SequenceSynthetic peptide 32Lys Ser Ser Gln Ser Leu Met Asp Thr Asp Gly Lys Thr Tyr Leu Asn1 5 10 15337PRTArtificial SequenceSynthetic peptide 33Lys Val Ser Asn Arg Asp Ser1 5347PRTArtificial SequenceSynthetic peptide 34Arg Val Thr Asn Arg Asp Thr1 5357PRTArtificial SequenceSynthetic peptide 35Lys Val Ser Asn Arg Glu Ser1 5367PRTArtificial SequenceSynthetic peptide 36Lys Val Thr Asn Arg Glu Ser1 5374PRTArtificial SequenceSynthetic peptide 37Gly Ala Gly Ala1389PRTArtificial SequenceSynthetic peptide 38Trp Gln Gly Thr His Phe Pro Arg Ser1 5399PRTArtificial SequenceSynthetic peptide 39Trp Gln Gly Ser His Phe Pro Arg Ser1 540330PRTArtificial SequenceSynthetic peptide 40Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys

Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 33041107PRTArtificial SequenceSynthetic peptide 41Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10542357PRTArtificial SequenceSynthetic peptide 42Gly Ala Ala Gly Thr Gly Cys Ala Gly 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33562357PRTArtificial SequenceSynthetic peptide 62Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Thr Gly Gly1 5 10 15Ala Gly Thr Cys Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Cys Thr 20 25 30Cys Gly Thr Gly Cys Ala Gly Cys Cys Cys Gly Gly Gly Gly Gly Cys 35 40 45Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Thr Thr 50 55 60Gly Thr Gly Cys Cys Gly Cys Cys Thr Cys Cys Gly Gly Cys Thr Thr65 70 75 80Cys Ala Cys Thr Thr Thr Thr Thr Cys Ala Ala Ala Cys Thr Thr Cys 85 90 95Gly Gly Ala Ala Thr Gly Thr Cys Cys Thr Gly Gly Gly Thr Cys Cys 100 105 110Gly Cys Cys Ala Ala Gly Cys Ala Cys Cys Gly Gly Gly Ala Ala Ala

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Ala Ala Thr Cys Ala Ala Gly 325 330 33566357PRTArtificial SequenceSynthetic peptide 66Gly Ala Ala Gly Thr Gly Cys Ala Gly Cys Thr Thr Cys Thr Gly Gly1 5 10 15Ala Gly Ala Gly Cys Gly Gly Gly Gly Gly Cys Gly Gly Cys Cys Thr 20 25 30Gly Gly Thr Gly Cys Ala Gly Cys Cys Gly Gly Gly Cys Gly Gly Ala 35 40 45Thr Cys Cys Cys Thr Gly Ala Gly Ala Cys Thr Gly Thr Cys Cys Thr 50 55 60Gly Thr Gly Cys Cys Gly Cys Gly Thr Cys Cys Gly Gly Thr Thr Thr65 70 75 80Thr Ala Cys Cys Thr Thr Cys Thr Cys Cys Ala Ala Cys Thr Ala Cys 85 90 95Gly Gly Ala Ala Thr Gly Thr Cys Ala Thr Gly Gly Gly Thr Cys Cys 100 105 110Gly Cys Cys Ala Ala Gly Cys Ala Cys Cys Cys Gly Gly Ala Ala Ala 115 120 125Gly Gly Gly Ala Thr Thr Gly Gly Ala Ala Thr Gly Gly Gly Thr Gly 130 135 140Gly Cys Thr Thr Cys Gly Ala Thr Cys Cys Gly Gly Thr Cys Cys Gly145 150 155 160Gly Cys Thr Cys Gly Gly Gly Ala Cys Gly Gly Ala Cys Cys Thr Ala 165 170 175Cys Thr Ala Cys Thr Cys Cys Gly Ala Thr Ala Ala Cys Gly Thr Cys 180 185 190Ala Ala Gly Gly Gly Cys Ala 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125Gly Gly Gly Thr Cys Thr Gly Gly Ala Ala Thr Gly Gly Gly Thr Cys 130 135 140Gly Cys Cys Ala Gly Cys Gly Thr Gly Cys Gly Gly Thr Cys Cys Gly145 150 155 160Gly Cys Gly Gly Cys Gly Gly Ala Cys Gly Gly Ala Cys Thr Thr Ala 165 170 175Cys Thr Ala Cys Thr Cys Cys Gly Ala Cys Ala Ala Cys Gly Thr Gly 180 185 190Ala Ala Gly Gly Gly Cys Cys Gly Gly Thr Thr Cys Ala Cys Cys Ala 195 200 205Thr Cys Thr Cys Ala Ala Gly Gly Gly Ala Thr Ala Ala Cys Thr Cys 210 215 220Cys Ala Ala Gly Ala Ala Thr Ala Cys Thr Cys Thr Gly Thr Ala Cys225 230 235 240Thr Thr Gly Cys Ala Ala Ala Thr Gly Ala Ala Cys Thr Cys Gly Cys 245 250 255Thr Gly Cys Gly Cys Gly Cys Thr Gly Ala Ala Gly Ala Thr Ala Cys 260 265 270Cys Gly Cys Gly Gly Thr Gly Thr Ala Cys Thr Ala Thr Thr Gly Cys 275 280 285Gly Thr Gly Cys Gly Cys Thr Ala Cys Gly Ala Cys Cys Ala Cys Thr 290 295 300Ala Cys Thr Cys Cys Gly Gly Thr Ala Cys Cys Ala Gly Cys Gly Ala305 310 315 320Cys Thr Ala Cys Thr Gly Gly Gly Gly Ala Cys Ala Gly Gly Gly Ala 325 330 335Ala Cys Cys Cys Thr Thr Gly Thr Gly Ala Cys Cys Gly Thr Gly Thr 340 345 350Cys Gly Ala Gly Cys 35569336PRTArtificial SequenceSynthetic peptide 69Gly Ala Thr Gly Thr Cys Gly Thr Gly Ala Thr Gly Ala Cys Thr Cys1 5 10 15Ala Gly Thr Cys Gly Cys Cys Cys Cys Thr Cys Thr Cys Cys Cys Thr 20 25 30Gly Cys Cys Thr Gly Thr Gly Ala Cys Thr Cys Thr Gly Gly Gly Gly 35 40 45Gly Ala Ala Cys Cys Cys Gly Cys Gly Thr Cys Cys Ala Thr Thr Thr 50 55 60Cys Gly Thr Gly Cys Ala Ala Gly Ala Gly Cys Ala Gly Cys Cys Ala65 70 75 80Gly Thr Cys Cys Cys Thr Gly Ala Thr Gly Gly Ala Cys Ala Cys Cys 85 90 95Gly Ala Cys Gly Gly Ala Ala Ala Gly Ala Cys

Cys Thr Ala Cys Cys 100 105 110Thr Thr Ala Ala Cys Thr Gly Gly Cys Thr Gly Cys Thr Gly Cys Ala 115 120 125Ala Ala Ala Gly Cys Cys Ala Gly Gly Ala Cys Ala Gly Ala Gly Cys 130 135 140Cys Cys Gly Cys Ala Gly Ala Gly Gly Cys Thr Gly Ala Thr Cys Thr145 150 155 160Ala Cys Ala Ala Ala Gly Thr Gly Thr Cys Ala Ala Ala Cys Cys Gly 165 170 175Gly Gly Ala Gly Thr Cys Cys Gly Gly Ala Gly Thr Gly Cys Cys Gly 180 185 190Gly Ala Cys Ala Gly Ala Thr Thr Cys Ala Gly Cys Gly Gly Cys Thr 195 200 205Cys Gly Gly Gly Thr Thr Cys Cys Gly Gly Cys Ala Cys Cys Gly Ala 210 215 220Cys Thr Thr Cys Ala Cys Cys Cys Thr Cys Ala Ala Ala Ala Thr Cys225 230 235 240Thr Cys Cys Cys Gly Cys Gly Thr Cys Gly Ala Gly Gly Cys Cys Gly 245 250 255Ala Gly Gly Ala Cys Gly Thr Gly Gly Gly Cys Gly Thr Gly Thr Ala 260 265 270Thr Thr Ala Cys Thr Gly Thr Thr Gly Gly Cys Ala Gly Gly Gly Ala 275 280 285Ala Cys Cys Cys Ala Cys Thr Thr Thr Cys Cys Thr Cys Gly Gly Ala 290 295 300Cys Cys Thr Thr Cys Gly Gly Thr Cys Ala Ala Gly Gly Gly Ala Cys305 310 315 320Thr Ala Ala Gly Gly Thr Cys Gly Ala Ala Ala Thr Cys Ala Ala Gly 325 330 33570454PRTArtificial SequenceSynthetic peptide 70Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Phe Asp Gly Thr Lys Lys Tyr Tyr Thr Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Arg Gly Ile Gly Ala Arg Arg Gly Pro Tyr Tyr Met Asp 100 105 110Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu225 230 235 240Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310 315 320Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser Pro Gly Lys 45071214PRTArtificial SequenceSynthetic peptide 71Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 21072449PRTArtificial SequenceSynthetic peptide 72Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Lys73119PRTArtificial SequenceSynthetic peptide 73Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115744PRTArtificial SequenceSynthetic peptide 74Ala Ala Ala Ala1754PRTArtificial SequenceSynthetic peptide 75Ala Ala Ala Gly1764PRTArtificial SequenceSynthetic peptide 76Ala Ala Gly Gly177219PRTArtificial SequenceSynthetic peptide 77Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 21578112PRTArtificial SequenceSynthetic peptide 78Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110794PRTArtificial SequenceSynthetic peptide 79Ala Ala Gly Gly1804PRTArtificial SequenceSynthetic peptide 80Ala Gly Gly Gly1814PRTArtificial SequenceSynthetic peptide 81Gly Gly Gly Gly182456PRTArtificial SequenceSynthetic peptide 82Gln Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Asn Ala Ser Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr 100 105 110Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 125Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro145 150 155 160Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185 190Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 195 200 205Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 210 215 220Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala225 230 235 240Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 245 250 255Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275 280 285Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 290 295 300Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln305 310 315 320Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 325 330 335Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 340 345 350Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 355 360 365Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 370 375 380Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr385 390 395 400Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 405 410 415Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 420 425 430Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 435 440 445Ser Leu Ser Leu Ser Pro Gly Lys 450 45583215PRTArtificial SequenceSynthetic peptide 83Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu65 70 75 80Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ile Tyr Asn Met Pro 85 90 95Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105

110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu Cys 210 2158442PRTArtificial SequenceSynthetic peptide 84Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala 35 4085770PRTArtificial SequenceSynthetic peptide 85Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg1 5 10 15Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu65 70 75 80Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu145 150 155 160Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu225 230 235 240Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe305 310 315 320Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 340 345 350Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala385 390 395 400Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala 405 410 415Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440 445Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu465 470 475 480Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe 500 505 510Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser 515 520 525Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser 530 535 540Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp545 550 555 560Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565 570 575Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe 610 615 620Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val625 630 635 640Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp 660 665 670Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 675 680 685Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu705 710 715 720Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val 725 730 735Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met 755 760 765Gln Asn 77086993PRTArtificial SequenceSynthetic peptide 86Gly Cys Cys Ala Gly Cys Ala Cys Thr Ala Ala Gly Gly Gly Gly Cys1 5 10 15Cys Thr Ala Gly Cys Gly Thr Cys Thr Thr Thr Cys Cys Gly Cys Thr 20 25 30Gly Gly Cys Cys Cys Cys Gly Thr Cys Cys Thr Cys Cys Ala Ala Gly 35 40 45Thr Cys Cys Ala Cys Thr Thr Cys Gly Gly Gly Thr Gly Gly Ala Ala 50 55 60Cys Cys Gly Cys Gly Gly Cys Ala Cys Thr Gly Gly Gly Gly Thr Gly65 70 75 80Cys Cys Thr Cys Gly Thr Gly Ala Ala Gly Gly Ala Cys Thr Ala Cys 85 90 95Thr Thr Cys Cys Cys Cys Gly Ala Gly Cys Cys Gly Gly Thr Cys Ala 100 105 110Cys Cys Gly Thr Gly Thr Cys Cys Thr Gly Gly Ala Ala Cys Thr Cys 115 120 125Gly Gly Gly Ala Gly Cys Cys Cys Thr Gly Ala Cys Cys Thr Cys Cys 130 135 140Gly Gly Ala Gly Thr Gly Cys Ala Thr Ala Cys Thr Thr Thr Cys Cys145 150 155 160Cys Thr Gly Cys Gly Gly Thr Gly Cys Thr Gly Cys Ala Gly Thr Cys 165 170 175Cys Thr Cys Cys Gly Gly Gly Cys Thr Cys Thr Ala Cys Thr Cys Gly 180 185 190Cys Thr Gly Thr Cys Ala Ala Gly Cys Gly Thr Gly Gly Thr Cys Ala 195 200 205Cys Cys Gly Thr Cys Cys Cys Gly Ala Gly Cys Thr Cys Ala Thr Cys 210 215 220Cys Cys Thr Gly Gly Gly Thr Ala Cys Thr Cys Ala Gly Ala Cys Cys225 230 235 240Thr Ala Cys Ala Thr Thr Thr Gly Cys Ala Ala Cys Gly Thr Gly Ala 245 250 255Ala Cys Cys Ala Cys Ala Ala Ala Cys Cys Thr Thr Cys Cys Ala Ala 260 265 270Cys Ala Cys Cys Ala Ala Gly Gly Thr Cys Gly Ala Cys Ala Ala Gly 275 280 285Ala Ala Ala Gly Thr Gly Gly Ala Gly Cys Cys Thr Ala Ala Gly Ala 290 295 300Gly Cys Thr Gly Cys Gly Ala Cys Ala Ala Gly Ala Cys Cys Cys Ala305 310 315 320Cys Ala Cys Cys Thr Gly Thr Cys Cys Cys Cys Cys Gly Thr Gly Thr 325 330 335Cys Cys Cys Gly Cys Cys Cys Cys Thr Gly Ala Gly Cys Thr Gly Cys 340 345 350Thr Gly Gly Gly Cys Gly Gly Cys Cys Cys Cys Ala Gly Cys Gly Thr 355 360 365Gly Thr Thr Cys Cys Thr Cys Thr Thr Cys Cys Cys Gly Cys Cys Thr 370 375 380Ala Ala Gly Cys Cys Gly Ala Ala Gly Gly Ala Cys Ala Cys Thr Cys385 390 395 400Thr Gly Ala Thr Gly Ala Thr Cys Thr Cys Gly Ala Gly Ala Ala Cys 405 410 415Cys Cys Cys Thr Gly Ala Ala Gly Thr Gly Ala Cys Cys Thr Gly Thr 420 425 430Gly Thr Gly Gly Thr Gly Gly Thr Gly Gly Ala Thr Gly Thr Gly Thr 435 440 445Cys Cys Cys Ala Cys Gly Ala Gly Gly Ala Thr Cys Cys Gly Gly Ala 450 455 460Ala Gly Thr Gly Ala Ala Gly Thr Thr Cys Ala Ala Thr Thr Gly Gly465 470 475 480Thr Ala Cys Gly Thr Gly Gly Ala Cys Gly Gly Ala Gly Thr Gly Gly 485 490 495Ala Ala Gly Thr Cys Cys Ala Thr Ala Ala Cys Gly Cys Cys Ala Ala 500 505 510Gly Ala Cys Cys Ala Ala Gly Cys Cys Cys Cys Gly Cys Gly Ala Gly 515 520 525Gly Ala Ala Cys Ala Gly Thr Ala Cys Ala Ala Cys Thr Cys Ala Ala 530 535 540Cys Thr Thr Ala Cys Cys Gly Gly Gly Thr Gly Gly Thr Gly Thr Cys545 550 555 560Ala Gly Thr Gly Cys Thr Gly Ala Cys Cys Gly Thr Gly Cys Thr Gly 565 570 575Cys Ala Cys Cys Ala Ala Gly Ala Thr Thr Gly Gly Cys Thr Gly Ala 580 585 590Ala Cys Gly Gly Gly Ala Ala Gly Gly Ala Gly Thr Ala Cys Ala Ala 595 600 605Gly Thr Gly Cys Ala Ala Ala Gly Thr Cys Thr Cys Cys Ala Ala Cys 610 615 620Ala Ala Gly Gly Cys Gly Cys Thr Gly Cys Cys Gly Gly Cys Cys Cys625 630 635 640Cys Cys Ala Thr Thr Gly Ala Ala Ala Ala Gly Ala Cys Cys Ala Thr 645 650 655Cys Ala Gly Cys Ala Ala Gly Gly Cys Thr Ala Ala Gly Gly Gly Cys 660 665 670Cys Ala Gly Cys Cys Cys Cys Gly Gly Gly Ala Ala Cys Cys Ala Cys 675 680 685Ala Gly Gly Thr Cys Thr Ala Cys Ala Cys Cys Thr Thr Gly Cys Cys 690 695 700Cys Cys Cys Thr Thr Cys Cys Cys Gly Gly Gly Ala Gly Gly Ala Ala705 710 715 720Ala Thr Gly Ala Cys Cys Ala Ala Gly Ala Ala Cys Cys Ala Ala Gly 725 730 735Thr Gly Thr Cys Gly Cys Thr Gly Ala Cys Gly Thr Gly Cys Cys Thr 740 745 750Gly Gly Thr Cys Ala Ala Gly Gly Gly Cys Thr Thr Thr Thr Ala Thr 755 760 765Cys Cys Ala Thr Cys Thr Gly Ala Cys Ala Thr Cys Gly Cys Cys Gly 770 775 780Thr Gly Gly Ala Gly Thr Gly Gly Gly Ala Ala Ala Gly Cys Ala Ala785 790 795 800Cys Gly Gly Cys Cys Ala Gly Cys Cys Gly Gly Ala Ala Ala Ala Cys 805 810 815Ala Ala Cys Thr Ala Cys Ala Ala Gly Ala Cys Thr Ala Cys Cys Cys 820 825 830Cys Gly Cys Cys Thr Gly Thr Gly Cys Thr Gly Gly Ala Cys Thr Cys 835 840 845Cys Gly Ala Cys Gly Gly Cys Thr Cys Gly Thr Thr Cys Thr Thr Cys 850 855 860Cys Thr Gly Thr Ala Thr Thr Cys Cys Ala Ala Gly Cys Thr Cys Ala865 870 875 880Cys Cys Gly Thr Gly Gly Ala Thr Ala Ala Gly Thr Cys Cys Ala Gly 885 890 895Ala Thr Gly Gly Cys Ala Gly Cys Ala Gly Gly Gly Cys Ala Ala Thr 900 905 910Gly Thr Gly Thr Thr Cys Ala Gly Cys Thr Gly Cys Ala Gly Cys Gly 915 920 925Thr Gly Ala Thr Gly Cys Ala Thr Gly Ala Gly Gly Cys Cys Cys Thr 930 935 940Gly Cys Ala Cys Ala Ala Cys Cys Ala Cys Thr Ala Cys Ala Cys Thr945 950 955 960Cys Ala Gly Ala Ala Ala Thr Cys Ala Cys Thr Gly Thr Cys Cys Cys 965 970 975Thr Thr Thr Cys Cys Cys Cys Cys Gly Gly Ala Ala Ala Gly Thr Ala 980 985 990Ala87324PRTArtificial SequenceSynthetic peptide 87Cys Gly Ala Ala Cys Thr Gly Thr Gly Gly Cys Thr Gly Cys Ala Cys1 5 10 15Cys Ala Thr Cys Thr Gly Thr Cys Thr Thr Cys Ala Thr Cys Thr Thr 20 25 30Cys Cys Cys Gly Cys Cys Ala Thr Cys Thr Gly Ala Thr Gly Ala Gly 35 40 45Cys Ala Gly Thr Thr Gly Ala Ala Ala Thr Cys Thr Gly Gly Ala Ala 50 55 60Cys Thr Gly Cys Cys Thr Cys Thr Gly Thr Thr Gly Thr Gly Thr Gly65 70 75 80Cys Cys Thr Gly Cys Thr Gly Ala Ala Thr Ala Ala Cys Thr Thr Cys 85 90 95Thr Ala Thr Cys Cys Cys Ala Gly Ala Gly Ala Gly Gly Cys Cys Ala 100 105 110Ala Ala Gly Thr Ala Cys Ala Gly Thr Gly Gly Ala Ala Gly Gly Thr 115 120 125Gly Gly Ala Thr Ala Ala Cys Gly Cys Cys Cys Thr Cys Cys Ala Ala 130 135 140Thr Cys Gly Gly Gly Thr Ala Ala Cys Thr Cys Cys Cys Ala Gly Gly145 150 155 160Ala Gly Ala Gly Thr Gly Thr Cys Ala Cys Ala Gly Ala Gly Cys Ala 165 170 175Gly Gly Ala Cys Ala Gly Cys Ala Ala Gly Gly Ala Cys Ala Gly Cys 180 185 190Ala Cys Cys Thr Ala Cys Ala Gly Cys Cys Thr Cys Ala Gly Cys Ala 195 200 205Gly Cys Ala Cys Cys Cys Thr Gly Ala Cys Gly Cys Thr Gly Ala Gly 210 215 220Cys Ala Ala Ala Gly Cys Ala Gly Ala Cys Thr Ala Cys Gly Ala Gly225 230 235 240Ala Ala Ala Cys Ala Cys Ala Ala Ala Gly Thr Cys Thr Ala Cys Gly 245 250 255Cys Cys Thr Gly Cys Gly Ala Ala Gly Thr Cys Ala Cys Cys Cys Ala 260 265 270Thr Cys Ala Gly Gly Gly Cys Cys Thr Gly Ala Gly Cys Thr Cys Gly 275 280 285Cys Cys Cys Gly Thr Cys Ala Cys Ala Ala Ala Gly Ala Gly Cys Thr 290 295 300Thr Cys Ala Ala Cys Ala Gly Gly Gly Gly Ala Gly Ala Gly Thr Gly305 310 315 320Thr Thr Ala Ala8810PRTArtificial SequenceSynthetic peptidemisc_feature(7)..(7)X = Y of F 88Gly Phe Thr Phe Ser Asn Xaa Gly Met Ser1 5 108917PRTArtificial SequenceSynthetic peptidemisc_feature(2)..(2)X = I or V 89Ser Xaa Arg Ser Gly Ser Gly Arg Thr Tyr Tyr Ser Asp Asn Val Lys1 5 10 15Gly9010PRTArtificial SequenceSynthetic peptidemisc_feature(5)..(5)X = S or Tmisc_feature(7)..(7)X = S or T 90Tyr Asp His Tyr Xaa Gly Xaa Ser Asp Tyr1 5 109116PRTArtificial SequenceSynthetic peptide 91Lys Ser Ser Gln Ser Leu Leu Asp Tyr Asp Gly Lys Thr Tyr Leu Asn1 5 10 15927PRTArtificial SequenceSynthetic peptidemisc_feature(1)..(1)X = K or Rmisc_feature(3)..(3)X = S or Tmisc_feature(7)..(7)X = S or T 92Xaa Val Xaa Asn Arg Asp Xaa1 5939PRTArtificial SequenceSynthetic peptidemisc_feature(9)..(9)X = S or T 93Trp Gln Gly Thr His Phe Pro Arg Xaa1 59411PRTArtificial SequenceSynthetic peptidemisc_feature(9)..(9)X = S or Tmisc_feature(11)..(11)X = F or Y 94Trp Gln Gly Thr His Phe Pro Arg Xaa Phe Xaa1 5 109510PRTArtificial SequenceSynthetic peptidemisc_feature(5)..(5)X = S or Amisc_feature(7)..(7)X = Y or F 95Gly Phe Thr Phe Xaa Asn Xaa Gly Met Ser1 5 109617PRTArtificial SequenceSynthetic peptidemisc_feature(2)..(2)X = I or

Vmisc_feature(6)..(6)X = S or Gmisc_feature(7)..(7)X = S or G 96Ser Xaa Arg Ser Gly Xaa Xaa Arg Thr Tyr Tyr Ser Asp Asn Val Lys1 5 10 15Gly9716PRTArtificial SequenceSynthetic peptidemisc_feature(1)..(1)X = K or Rmisc_feature(7)..(7)X = V, M or Lmisc_feature(9)..(9)X = Y, T or S 97Xaa Ser Ser Gln Ser Leu Xaa Asp Xaa Asp Gly Lys Thr Tyr Leu Asn1 5 10 15987PRTArtificial SequenceSynthetic peptidemisc_feature(1)..(1)X = K or Rmisc_feature(3)..(3)X = S or Tmisc_feature(6)..(6)X = E or Dmisc_feature(7)..(7)X = S or T 98Xaa Val Xaa Asn Arg Xaa Xaa1 5999PRTArtificial SequenceSynthetic peptidemisc_feature(4)..(4)X = S or Tmisc_feature(9)..(9)X = S or T 99Trp Gln Gly Xaa His Phe Pro Arg Xaa1 510012PRTArtificial SequenceSynthetic peptidemisc_feature(9)..(9)X = S or Tmisc_feature(11)..(11)X = S or Tmisc_feature(12)..(12)X = F or Y 100Trp Gln Gly Thr His Phe Pro Arg Xaa Phe Xaa Xaa1 5 10101112PRTArtificial SequenceSynthetic peptide 101Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Arg Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110102112PRTArtificial SequenceSynthetic peptide 102Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110

Claims

1. An antibody or an antigen binding fragment thereof that specifically binds to A.beta. peptide, comprising a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 and a light chain variable region comprising light chain CDR1, CDR2, and CDR3, wherein heavy chain CDR1 comprises the amino acid sequence of SEQ ID NO: 16, heavy chain CDR2 comprises the amino acid sequence of SEQ ID NO: 20, heavy chain CDR3 comprises the amino acid sequence of SEQ ID NO: 18, light chain CDR1 comprises the amino acid sequence of SEQ ID NO: 29, light chain CDR2 comprises the amino acid sequence of SEQ ID NO: 34, and light chain CDR3 comprises the amino acid sequence of SEQ ID NO: 38.

2. The antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain variable region, excluding the CDRs, is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, and 7, and wherein the light chain variable region, excluding the CDRs, is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15.

3. The antibody or antigen binding fragment thereof of claim 2, wherein the heavy chain variable region, excluding the CDRs, is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, and 7, and wherein the light chain variable region, excluding the CDRs, is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15.

4. The antibody or antigen binding fragment thereof of claim 3, wherein the heavy chain variable region, excluding the CDRs, is selected from SEQ ID NOs: 3, 4, 5, 6, and 7, and wherein the light chain variable region, excluding the CDRs, is selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15.

5. The antibody or antigen binding fragment thereof of claim 4, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9.

6. The antibody or antigen binding fragment thereof of claim 5 further comprising a heavy chain constant region comprising an amino acid sequence at least 95% identical to SEQ ID NO: 40.

7. The antibody or antigen binding fragment thereof of claim 5 further comprising a light chain constant region comprising an amino acid sequence at least 95% identical to SEQ ID NO: 41.

8. The antibody or antigen binding fragment thereof of claim 6 further comprising a light chain constant region comprising an amino acid sequence at least 95% identical to SEQ ID NO: 41.

9. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody is a human IgG1.

10. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody is a full antibody, a chimeric antibody, a CDR-grafted antibody, or a recombinant antibody.

11. The antibody or antigen binding fragment thereof of claim 1, wherein the antigen binding fragment is a Fab, Fab', F(ab')2, Fabc, or Fv.

12. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment thereof specifically binds to an epitope having an amino acid sequence including three or more amino acid positions from amino acids 1-7 of A.beta..

13. A pharmaceutical composition, comprising: a pharmaceutically effect amount of the antibody or antigen binding fragment thereof of claim 1; and a pharmaceutically acceptable carrier or diluent.

14. A human IgG1 antibody or an antigen binding fragment thereof that specifically binds to A.beta. peptide, comprising: a) a heavy chain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and b) a light chain comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9.

15. The human IgG1 antibody or antigen binding fragment thereof of claim 14, wherein the antibody is a full antibody, a chimeric antibody, a CDR-grafted antibody, or a recombinant antibody.

16. The human IgG1 antibody or antigen binding fragment thereof of claim 14, wherein the antigen binding fragment is a Fab, Fab', F(ab')2, Fabc, or Fv.

17. A pharmaceutical composition, comprising: a pharmaceutically effective amount of the human IgG1 antibody or antigen binding fragment thereof of claim 14; and a pharmaceutically acceptable carrier or diluent.

18. A human IgG1 antibody that specifically binds to A.beta. peptide, comprising: a) a heavy chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 3, and a constant region comprising the amino acid sequence of SEQ ID NO: 40 with or without the C-terminal lysine; and b) a light chain comprising a variable region comprising the amino acid sequence of SEQ ID NO: 9, and a constant region comprising the amino acid sequence of SEQ ID NO: 41.

19. The human IgG1 antibody of claim 18, wherein the antibody is a full antibody, a chimeric antibody, a CDR-grafted antibody, or a recombinant antibody.

20. A pharmaceutical composition, comprising: a pharmaceutically effective amount of the human IgG1 antibody of claim 18; and a pharmaceutically acceptable carrier or diluent.