Anti-amyloid beta antibodies and methods of using the same

Abstract

To provide antibodies, such as recombinant humanized and monoclonal antibodies, methods of making antibodies, and methods of using antibodies, such as antibodies directed toward and capable of specifically binding to and clearing amyloid-beta (Aβ) plaques in the brain that are suitable for use in the treatment of disorders such as Alzheimer's Disease (AD).SOLUTION: The present invention provides an amino acid sequence of a recombinant humanized IgG1 kappa monoclonal antibody that specifically binds to Aβ (AβpE3), which is suitable for clearing amyloid plaques from the AD brain.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 263,204, filed October 28, 2021, the entire contents of which are incorporated herein by reference.

[0002] Reference to electronic sequence listing The entire sequence listing entitled "ABV21397USO1_ST26.xml", including SEQ ID NO:1 through SEQ ID NO:37, containing the amino acid and DNA sequences disclosed herein, is incorporated herein by reference. The sequence listing has been submitted electronically in XML format. The sequence listing was created on October 27, 2022 and is 22,528 bytes in size.

[0003] This application relates, inter alia, to anti-amyloid beta (Aβ) antibodies and methods of making and using same. [Background technology]

[0004] The amyloid hypothesis of Alzheimer's disease (AD) suggests that an imbalance between Aβ clearance and production leads to the accumulation of the protein in the brain. This initiates several downstream events that ultimately lead to neuronal and synaptic loss, which manifests as the clinical symptoms of AD (Selkoe and Hardy 2016). Recent drug development to treat AD has focused on compounds that reduce both soluble monomeric Aβ and insoluble deposited forms of Aβ, and several mAbs that bind to different forms of Aβ have been tested in the last two decades (van Dyck 2018). The approach of administering anti-Aβ mAbs to treat AD is primarily based on passive immunotherapy, as the mAbs bind to Aβ and stimulate an immune response that helps to clear the protein.

[0005] Historically, mAbs targeting Aβ have not been able to demonstrate efficacy in slowing cognitive and functional decline in AD. However, these studies have provided important data related to safety. Furthermore, these studies have provided insight into the specific forms of Aβ that may need to be targeted to slow AD progression, leading many to believe that Aβ is still a valid target for developing AD therapies (van Dyck 2018); (Aisen, Cummings et al. 2020). Indeed, recent results from a phase 3 study of aducanumab (NCT02484547) and a phase 2 study of donanemab (NCT03367403), both mAbs targeting Aβ, suggest that this class of compounds may be effective in treating AD. Thus, given the scientific support for the amyloid hypothesis of AD and the encouraging signs seen in recent trials of anti-Aβ mAbs, the development of new anti-Aβ mAbs offers further promise in the search for effective AD treatments.

[0006] Anti-Aβ mAbs have been administered to thousands of subjects in numerous clinical trials. Overall, anti-Aβ mAbs are well tolerated. A meta-analysis examining active and passive immunological compounds targeting Aβ, including several mAbs, found no difference from placebo with respect to nearly all adverse events, serious adverse events, and deaths (Penninkilampi, Brothers et al., 2017). One exception to the adverse event data was that subjects receiving anti-Aβ mAbs were more likely to experience amyloid-related imaging abnormalities (ARIA).

[0007] ARIA is a term applied to cerebrovascular abnormalities detected by MRI. ARIA is divided into two categories: ARIA-E describes imaging findings related to vasogenic edema, sulcal effusion, and occasional gyral enlargement, whereas ARIA-H captures MRI signal abnormalities reflecting parenchymal microhemorrhagic and hemosiderin deposition (Sperling, Jack et al., 2011). Both forms have been demonstrated in anti-Aβ mAb studies, but ARIA-E occurs more frequently as a result of anti-Aβ mAb administration (Penninkilampi, Brothers et al., 2017; Greenberg, Bacskai et al., 2020). The exact mechanism(s) of ARIA are unknown but may be related to clearance of amyloid from the cerebral vasculature or may be due to eliciting an inflammatory response at the site of vascular amyloid deposition (Greenberg, Bacskai et al., 2020). Risk factors for ARIA include administration of mAbs targeting the N-terminus of the Aβ peptide, a history of previous microbleeds, carriage of the ε4 allele of the apolipoprotein gene, and high drug doses, although it is unclear whether the increased risk of ARIA is associated with absolute dose levels or with administration of high doses without titration (van Dyck 2018; Aisen, Cummings et al. 2020; Greenberg, Bacskai et al. 2020). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Selkoe and Hardy 2016 [Non-Patent Document 2] van Dyck 2018 [Non-Patent Document 3] Aisen, Cummings et al., 2020 [Non-Patent Document 4] Penninkilampi, Brothers et al., 2017 [Non-Patent Document 5] Sperling, Jack et al., 2011 [Non-Patent Document 6] Greenberg, Bacskai et al., 2020 Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention AC-264713, also known as hu Aβ (AC-264713) [hu IgG1 / K], is a particularly rapidly aggregating, insoluble, aggregated form of Aβ, an N-terminally truncated, pyroglutamic acid modified Aβ (Aβ pE3 AC-264713 is a recombinant humanized immunoglobulin G1 (IgG1) kappa monoclonal antibody (mAb) that binds to human Aβ pE3-42 It selectively binds to fibrils and has a half effective concentration (EC 50 ) is 0.7 nM, and does not bind to full-length Aβ that is not pyroglutamic acid modified.

[0010] Therefore, we have developed a method to remove amyloid plaques from AD brains using Aβ (Aβ pE3 The amino acid sequence of a recombinant humanized IgG1 kappa monoclonal antibody that specifically binds to Aβ is provided. pE3-42 The antibody comprises a variable heavy chain and a variable light chain that contain complementarity determining regions (CDRs) that selectively bind to fibrils. An antibody is also provided that comprises a heavy chain constant region and a light chain constant region that contain a fragment crystallizable region (Fc). These structural elements, encoded by the amino acid sequence of the antibody, result in a pharmaceutical composition that is effective for treating AD in a patient.

[0011] (ii) an anti-human Aβ antibody comprising a variable heavy chain (vH) comprising three CDRs; and (ii) a variable light chain (vL) comprising three CDRs. pE3 An antibody comprising: vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Anti-human Aβ pE3 Antibodies are described herein.

[0012] In certain embodiments, anti-human Aβ pE3 The antibody comprises a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO:8.

[0013] In certain embodiments, anti-human Aβ pE3 The antibody comprises two heavy chains and two light chains, each heavy chain comprising the amino acid sequence set forth as SEQ ID NO:9 and each light chain comprising the amino acid sequence set forth as SEQ ID NO:10.

[0014] In certain embodiments, anti-human Aβ pE3 The antibody comprises an IgG constant region.

[0015] In certain embodiments, anti-human Aβ pE3 The antibody comprises a human heavy chain constant region comprising an Fc portion, the Fc portion being of a human IgG1, IgG2, IgG3, IgG4, or IgM isotype.

[0016] In certain embodiments, anti-human Aβ pE3 The antibody comprises a kappa light chain constant region.

[0017] an anti-human Aβ antibody comprising (i) a variable heavy chain (vH) comprising three CDRs and (ii) a variable light chain (vL) comprising three CDRs; pE3 A composition comprising an antibody, vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Compositions are also provided herein.

[0018] A method for treating a neurodegenerative disorder, comprising: (i) administering to the patient an anti-human Aβ antibody comprising a variable heavy chain (vH) comprising three CDRs; and (ii) a variable light chain (vL) comprising three CDRs. pE3 An antibody comprising: vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Also provided herein is a method comprising administering the antibody to a patient in need thereof.

[0019] In certain embodiments, the neurodegenerative disorder is Alzheimer's disease (AD).

[0020] Anti-human Aβ pE3 A polynucleotide comprising a nucleotide sequence encoding an antibody, the antibody comprising (i) a vH chain comprising three CDRs and (ii) a vL chain comprising three CDRs; vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Polynucleotides are also provided herein.

[0021] Anti-human Aβ pE3 A vector comprising a polynucleotide comprising a nucleotide sequence encoding an antibody, the antibody comprising (i) a vH chain comprising three CDRs and (ii) a vL chain comprising three CDRs; vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Vectors are also provided herein.

[0022] Anti-human Aβ pE3 A eukaryotic host cell transformed with a vector comprising a polynucleotide comprising a nucleotide sequence encoding an antibody, the antibody comprising (i) a vH chain comprising three CDRs and (ii) a vL chain comprising three CDRs; vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Eukaryotic host cells are also provided herein.

[0023] In certain embodiments, the eukaryotic host cell is a mammalian host cell.

[0024] Anti-human Aβ pE3 A method for producing an antibody, comprising the steps of: (a) culturing a mammalian host cell; and (b) producing an anti-human Aβ pE3 Methods including the step of recovering the antibody are also provided herein.

[0025] Anti-human Aβ containing two heavy chains and two light chains pE3 An anti-human Aβ antibody, wherein each heavy chain comprises an amino acid sequence set forth as SEQ ID NO: 37 and each light chain comprises an amino acid sequence set forth as SEQ ID NO: 10. pE3 Further provided in the present invention are antibodies. [Brief description of the drawings]

[0026] [Figure 1] Representative binding of AC-264713 to human AβpE3-42 fibrils. Binding of AC-264713 to human AβpE3-42 fibrils was assessed in a direct binding ELISA. Plates coated with human AβpE3-42 fibrils (50 ng / well) were incubated with serially diluted AC-264713. ELISA was performed in triplicate. A representative figure is shown. Optical density at 450 nm is plotted on the Y-axis. The EC50 value for AC-264713 binding to human AβpE3-42 fibrils is 0.7 nM. [Diagram 2]Figure 1 shows the area of ​​plaques bound by AC-264713 and comparative antibody Ab1 in fresh frozen human AD tissue from the frontal cortex. Tissue from six AD donors was analyzed and data is presented as mean ± SEM. AC-264713 bound to plaques in AD brain tissue in a concentration-dependent manner. The line represents a fit to the data for the six donors combined as described in Example 3. [Diagram 3] AC-264713 stimulates the clearance of amyloid plaques by hiPSC-derived phagocytes from unfixed APPPS1-21 brain tissue. hiPSC-derived phagocytes were co-cultured with APPPS1-21 brain tissue pre-incubated with various concentrations of AC-264713. The remaining amyloid plaques in the tissue were stained and quantified using Thioflavin S. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Dunnett's multiple comparison test. *, p less than 0.05. [Figure 4] Dose-dependent target binding of AC-264713 in APPPS1-21 mice 72 hours after a single intraperitoneal dose. AC-264713 (panel A) and comparative antibody Ab1 (panel D) in the brain were detected using anti-hIgG immunohistochemistry. Total target AβpE3 on adjacent sections was stained using AC-264713 (panel B) or comparative antibody Ab1 (panel E). The % total targets occupied by AC-264713 (panel C) or comparative antibody Ab1 (panel F) at increasing doses was then calculated and shown on the y-axis. *P less than 0.05 (Kruskal-Wallis followed by Dunn's multiple comparison test). [Diagram 5] Correlation of AC-264713 and comparative antibody Ab1 exposure in the brain and dose-dependent target binding in APPPS1-21 mice 72 hours after a single intravenous dose in female mice. Data (signs) showing the correlation between brain exposure and AβpE3 occupancy in individual animals are shown with non-linear fitting (lines). [Figure 6]Figure 1 shows the effect of AC-264713 on microhemorrhage in the APPPS1-21 mouse model, tested using AC-264713 mouse precursor antibody and comparative antibody Ab2. (Panel A) Images for red blood cell quantification. (Panel B) Images for Prussian blue quantification. Data presented as mean ± SEM. Stained images represent findings from the positive control group for microhemorrhage induction. Open arrows in Panel A indicate red blood cells. Arrows in Panel B indicate Prussian blue staining signal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] To promote an understanding of the principles of the present disclosure, reference will now be made to various embodiments, but it is understood that no limitation of the scope of the disclosure is intended thereby, and that modifications and further variations of the disclosure exemplified herein will normally occur to those skilled in the art to which the disclosure pertains.

[0028] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can also include more than one element. The term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ."

[0029] The term "comprise," as used herein, in addition to its ordinary meaning, can also include, and in some embodiments can specifically refer to, the phrases "consist essentially of" and / or "consist of." Thus, the phrase "comprise" can refer to embodiments in which what is claimed as "comprises" a specifically recited element does not include additional elements, as well as to embodiments in which what is claimed as "comprises" a specifically recited element can and / or does include additional elements, or includes additional elements that do not materially affect the basic and novel characteristics of what is claimed. For example, a claim that "comprises" specifically recited elements, e.g., a method, kit, system, etc., also includes, e.g., a method, kit, system, etc. that "consisting of," i.e., when the claimed thing does not include additional elements, and also includes, e.g., a method, kit, system, etc. that "consisting essentially of," i.e., when the claimed thing may include additional elements that do not materially affect the basic and novel characteristics of the claimed thing.

[0030] "About," as used herein, is intended to qualify the numerical value it modifies, indicating that such value varies within a margin of error. When no specific margin of error is given, such as a standard deviation for the mean value given in a chart or table of data, the term "about" should be understood to mean and encompass a range that may be encompassed, for example, ±20%, ±15%, or ±10% of the stated value, and in some embodiments, ±5%, ±3%, or ±2%.

[0031] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0032] Embodiments of the present disclosure relate to antibodies that bind to Aβ in the form of deposits in the brain and stimulate an immune response that helps to clear the protein, and methods of using these antibodies, for example, in the treatment of AD.

[0033] Aβ pE3 is a form of Aβ that appears to aggregate particularly rapidly (Jawhar, Wirths et al., 2011). pE3 Donanemab, which targets Aβ, achieved rapid clearance of plaques from human AD brains and pE3 Considering that Aβ is less prevalent in the cerebral vasculature than other forms of Aβ (Kuo, Emmerling et al., 1997), anti-Aβ agents such as AC-264713 are aimed at achieving rapid plaque clearance with a low risk of ARIA. pE3 Antibodies were produced.

[0034] AC-264713 inhibits Aβ pE3 AC-264713 is a recombinant humanized IgG1 kappa mAb that binds to Aβ pE3 AC-264713 is a murine mAb that binds to. The variable domains of the murine antibody were humanized and then fused with human IgG1 heavy chain and Ig kappa light chain constant regions to create AC-264713. It is understood that humanization of, for example, a murine mAb to provide a humanized mAb, such as an antibody of the present disclosure, can be accomplished by any method known to one of skill in the art, including but not limited to.

[0035] In some embodiments, the antibodies of the present disclosure comprise two variable chains, one heavy chain and one light chain. pE3 There are three CDRs that allow binding to Aβ. There are a total of six different CDRs combined on the variable heavy and variable light chains. Additionally, in one embodiment, the antibody contains a human heavy chain constant region that comprises a human Fc of immunoglobulin class G1 (IgG1). pE3 The antibodies can be fucosylated or non-fucosylated and exhibit in vitro functionality, immune safety and drug-like properties.

[0036] The antibodies according to the present disclosure are directed to human Aβ pE3-42 The antibodies identified in the hybridoma campaign were generated by humanization and down-regulation engineering using fibrillar Aβ antibodies to prioritize antibodies for humanization. pE3-42 The ability of the antibodies to bind to anti-Aβ pE3 The antibody AC-264713 targets human Aβ pE3 and cynomolgus monkey Aβ pE3 Cross-reacts with mouse Aβ pE3 Also, rat Aβ pE3 It does not bind to

[0037] As used herein, the term "antibody" (Ab) refers to an antibody that specifically binds to a specific antigen, e.g., human Aβ. pE3 The term "anti-Aβ" refers to an immunoglobulin molecule that specifically binds to Aβ fibrils. pE3 The antibody is Aβ pE3 The anti-Aβ complex of the present invention binds to and thereby modulates the immune system. pE3Antibodies contain complementarity determining regions (CDRs), also known as hypervariable regions, in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are referred to as framework regions (FRs). As is known in the art, the amino acid portions / boundaries that delineate the hypervariable regions of an antibody may vary depending on the context and the various definitions known in the art. Some portions within the variable domain may be considered hybrid hypervariable portions, in that they may be considered within the hypervariable region under one set of criteria, while being considered outside the hypervariable region under a different set of criteria. One or more of these portions may also be found within an extended hypervariable region. The inventive concept provides antibodies that contain modifications to these hybrid hypervariable portions. Native heavy and light chain variable domains each contain four FR regions, which predominantly adopt a β-sheet configuration and connect with three CDRs, which form loops that connect with, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the target binding site of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987), which can be used to identify and number the CDR sequences in the variable domains.

[0038] The antibodies of the present disclosure may be monoclonal, genetically engineered and / or otherwise modified in nature, including, but not limited to, chimeric antibodies, humanized antibodies, single chain antibodies, and the like. In various embodiments, the antibody comprises all or a portion of an antibody constant region. In some embodiments, the constant region is an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM. In certain embodiments, the antibody comprises an anti-Aβ antibody as described herein. pE3 The antibody comprises an IgG1. pE3In yet another embodiment, the antibody comprises an IgG2 antibody. pE3 The antibody includes IgG4. As used herein, the "constant region" of an antibody includes naturally occurring constant regions, allotypes or variants.

[0039] anti-Aβ pE3 The light chain constant region of the antibody may be a kappa (κ) light chain region or a lambda (λ) region. The λ light chain region may be any one of the known subtypes, e.g., λ1, λ2, λ3, or λ4. In some embodiments, the anti-Aβ pE3 The antibody comprises a kappa (κ) light chain region.

[0040] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies are derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the concepts of the present invention can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant and phage display technologies, or a combination thereof.

[0041] The term "chimeric" antibody, as used herein, refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically chosen from a human immunoglobulin template.

[0042] "Humanized" forms of a non-human (e.g., murine) antibody comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR regions being those of a human immunoglobulin sequence. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.

[0043] The anti-AβpE3 Antibodies include full-length (intact) antibody molecules comprising two full-length light chains and two full-length heavy chains.

[0044] Anti-Aβ containing human IgG4 constant region pE3 The antibody may contain an S228P mutation that has been reported to prevent Fab arm exchange. See, e.g., Silva, JP et al., Journal of Biological Chemistry, 290(9), 5462-5469 (2015).

[0045] For therapeutic and diagnostic uses, Aβ pE3 Anti-Aβ with high affinity pE3 Thus, the present invention provides a method for treating Aβ pE3 In certain embodiments, antibodies that have high binding affinity to anti-Aβ are contemplated. pE3 The antibody is Aβ pE3 with an affinity of at least about 25 nM, but may exhibit higher affinity, e.g., at least about 20 nM, 15 nM, 10 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, or even higher. pE3 with an affinity in the range of about 1 pM to about 10 nM, in the range of about 100 pM to about 10 nM, in the range of about 100 pM to about 1 nM, in the range of about 100 pM to about 2 nM, or an affinity ranging between any of the aforementioned values.

[0046] In some embodiments, the present disclosure provides a monoclonal anti-Aβ antibody having two full-length heavy chains and two full-length light chains that contain two sets of six different complementarity determining regions (CDRs) within two sets of two different variable regions. pE3 An antibody is provided.

[0047] In some embodiments, the antibody is pE3 It is a recombinant, afucosylated, humanized, IgG1 kappa monoclonal antibody that binds to

[0048] In one embodiment, the antibody comprises six CDRs comprising the following sequences: vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (sequence number 6).

[0049] In some embodiments, an antibody of the disclosure comprises a vH CDR1 that comprises or has the amino acid sequence set forth in SEQ ID NO:1; a vH CDR2 that comprises or has the amino acid sequence set forth in SEQ ID NO:2; a vH CDR3 that comprises or has the amino acid sequence set forth in SEQ ID NO:3; a vL CDR1 that comprises or has the amino acid sequence set forth in SEQ ID NO:4; a vL CDR2 that comprises or has the amino acid sequence set forth in SEQ ID NO:5; and a vL CDR3 that comprises or has the amino acid sequence set forth in SEQ ID NO:6.

[0050] In some embodiments, an antibody of the disclosure comprises a heavy chain variable region that comprises or has the amino acid sequence set forth in SEQ ID NO:7, or a sequence with at least 98% sequence identity to SEQ ID NO:7:

[0051] [ka] In some embodiments, an antibody of the disclosure comprises a light chain variable region that comprises or has the amino acid sequence set forth in SEQ ID NO:8, or a sequence with at least 98% sequence identity to SEQ ID NO:8:

[0052] [ka]

[0053] In some embodiments, an antibody of the disclosure comprises a heavy chain that comprises or has an amino acid sequence set forth in SEQ ID NO:9, or a sequence with at least 98% sequence identity to SEQ ID NO:9 (constant region in bold, variable heavy domain underlined, CDRs underlined and bold italicized (as set forth in SEQ ID NOs:1-3, respectively, in order of appearance):

[0054] [ka]

[0055] In some embodiments, an antibody of the disclosure comprises a heavy chain with a C-terminal lysine truncated, e.g., a heavy chain set forth in SEQ ID NO: 9 with the C-terminal lysine truncated / removed.

[0056] In some embodiments, an antibody of the disclosure comprises a light chain that comprises or has an amino acid sequence set forth in SEQ ID NO: 10, or a sequence with at least 98% sequence identity to SEQ ID NO: 10 (constant region in bold, variable heavy domain underlined, CDRs underlined and bold italicized (as set forth in SEQ ID NOs: 4-6, respectively, in order of appearance):

[0057] [ka]

[0058] Embodiments of the inventive concept also encompass nucleic acids encoding the heavy chain sequence of SEQ ID NO: 9 and nucleic acids encoding the light chain sequence of SEQ ID NO: 10. In some embodiments, for example, the mature heavy chain sequence set forth in SEQ ID NO: 9 may be encoded by the nucleic acid sequence set forth in SEQ ID NO: 13 (the constant region is encoded by the bolded sequence, the variable heavy domain is encoded by the underlined sequence, and the CDRs are encoded by the underlined, bold and italicized sequences):

[0059] [ka] TIFF2025016446000007.tif102169

[0060] In some embodiments, for example, the mature light chain sequence set forth in SEQ ID NO: 10 can be encoded by the nucleic acid sequence set forth in SEQ ID NO: 14 (the constant region is encoded by the bolded sequence, the variable light domain is encoded by the underlined sequence, and the CDRs are encoded by the underlined, bold and italicized sequences):

[0061] [ka] TIFF2025016446000009.tif14168

[0062] It will be understood by those skilled in the art that, as a result of the codon degeneracy of the genetic code, nucleic acid sequences different from those set forth in SEQ ID NOs: 13, 14, 15 and 16, which respectively encode the heavy chain sequence of SEQ ID NO: 9, the light chain sequence of SEQ ID NO: 10, the heavy chain sequence of SEQ ID NO: 11 and the light chain sequence of SEQ ID NO: 12, are contemplated without departing from the scope of the inventive concept.

[0063] In some embodiments, the antibody comprises a human heavy chain constant region comprising a human CH1, human hinge, human CH2 and human CH3 domains. In some embodiments, the encoded heavy chain constant region comprises an Fc portion, where the Fc portion is a human IgG1, IgG2, IgG3, IgG4, or IgM isotype. In certain embodiments, the Fc is IgG1 and the allotype is z, non-a. In certain embodiments, the light chain is a kappa light chain.

[0064] anti-Aβ pE3 Polynucleotides encoding antibodies, expression systems and methods for producing antibodies The concept of the present invention is to pE3 Polynucleotide molecules encoding immunoglobulin light and heavy chain genes of antibodies, vectors containing such polynucleotides, and anti-Aβ pE3 This includes host cells capable of producing antibodies.

[0065] The anti-Aβ pE3 Antibodies can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cell and, optionally, secreted into the medium in which the host cell is cultured, from which the antibody can be recovered.

[0066] Such anti-Aβ pE3 To generate a polynucleotide encoding an antibody, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplifying and modifying germline DNA or cDNA encoding the light and heavy chain variable sequences, for example, using the polymerase chain reaction (PCR).

[0067] anti-Aβ pE3 Once DNA fragments encoding antibody-related VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, the VL-encoding DNA fragment or the VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked", when used in this context, is intended to mean that the two DNA fragments are joined in such a way that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0068] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3 and optionally CH4). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but in certain embodiments is IgG1 or IgG4. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0069] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL with another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa constant region or a lambda constant region, but in certain embodiments is a kappa constant region.

[0070] anti-Aβ pE3To express an antibody, DNAs encoding partial or full-length light and heavy chains obtained as described above are inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated to the vector and the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and the expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector.

[0071] The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). pE3 Prior to insertion of the antibody-related light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. pE3 One approach for converting monoclonal antibody-related VH and VL sequences into full-length antibody genes is to use anti-Aβ pE3 The monoclonal antibody-related VH and VL sequences are inserted into an expression vector already encoding the heavy and light chain constant regions, respectively, such that the VH segment is operably linked to a CH segment in the vector, and the VL segment is operably linked to a CL segment in the vector. Additionally, or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a non-immunoglobulin signal peptide). The signal peptide may be a signal peptide derived from a protein.

[0072] The recombinant expression vectors of the disclosure carry, in addition to the antibody chain genes, regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.

[0073] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may have additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selection marker genes. The selection marker gene facilitates selection of host cells into which the vector has been introduced. To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The term "transfection" in its various forms is intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[0074] The antibodies of the present disclosure can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of the antibodies is carried out in eukaryotic cells, such as mammalian host cells, that optimally secrete properly folded and immunologically active antibodies. Exemplary mammalian host cells for expressing recombinant antibodies of the present disclosure include Chinese Hamster Ovary (CHO) cells (including DHFR-CHO cells, as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selection marker, as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow for expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is growing. Antibodies can be recovered from the culture medium using standard protein purification methods. The host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations of the above procedures are within the scope of the present disclosure. For example, host cells can be transfected with an anti-Aβ antibody of the present disclosure. pE3 It may be desirable to transfect DNA encoding either the light chain or the heavy chain of the antibody (but not both).

[0075] Recombinant DNA technology is pE3 Alternatively, truncated DNA molecules may be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antibody. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

[0076] The anti-Aβ pE3For recombinant expression of antibodies, host cells can be co-transfected with two expression vectors of the present disclosure, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. The two vectors can contain identical selectable markers or each can contain a separate selectable marker. Alternatively, a single vector can be used that encodes both heavy and light chain polypeptides.

[0077] anti-Aβ pE3 Once a polynucleotide encoding one or more portions of an antibody is obtained, further modifications or mutations can be introduced into the coding sequence, for example to generate a polynucleotide encoding an antibody having a different CDR sequence, an antibody with reduced affinity for an Fc receptor, for example, an antibody with a LALA mutation, or an antibody of a different subclass. Further modifications and / or mutations that can be introduced into the coding sequence to generate a polynucleotide encoding an antibody can be achieved by any method known to those skilled in the art, including but not limited to, and the generated polynucleotide can be used to encode, for example, an additional anti-Aβ antibody. pE3 Antibodies, e.g., anti-Aβ with reduced affinity for Fc receptors pE3 Antibodies and / or anti-Aβ subclasses pE3 It is understood that the antibody will be used to generate antibodies.

[0078] The anti-Aβ pE3 Antibodies can also be produced by chemical synthesis or by using cell-free platforms.

[0079] anti-Aβ pE3 Antibody purification Once produced by recombinant expression, the polypeptides of the present disclosure may be purified by any method known in the art for purification of proteins.

[0080] anti-Aβ pE3 Once isolated, the antibodies may be further purified.

[0081] composition The antibodies of the present disclosure may be provided as a composition suitable for administration to a subject. In some embodiments, the antibody composition is a pharmaceutical composition comprising an antibody of the present disclosure and a pharma- ceutically acceptable carrier.

[0082] Summary of the Subject Antibody Properties Properties of the subject antibodies, exemplified by, but not limited to, AC-264713, include the following: Human Aβ pE3 High affinity binding to fibrils, e.g., human Aβ, as determined by, e.g., the direct binding ELISA of Example 1 pE3-42 EC on fibrils 50 is approximately 2.5nM or less, 2nM or less, 1.5nM or less, 1nM or less, 0.9nM or less, 0.8nM or less, 0.7nM or less, 0.6nM or less, approximately 0.5nM EC of 0.4nM or less, about 0.3nM or less, about 0.2nM or less, about 0.1nM or less, or about 0.05nM or less, or about 0.05nM to about 2.5nM 50 Any range of values.

[0083] Human Aβ pE3 High specificity binding to fibrils, e.g., human Aβ pE11-40 Does not significantly cross-react with fibrils and is consistent with Aβ from all species, including humans, monkeys, dogs, rabbits, rats, and mice. 1-40 Does not significantly cross-react with

[0084] Favorable immune safety as determined by cytokine release assays.

[0085] In some embodiments, the antibodies of the disclosure are administered at an EC of 0.4 μg / mL into AD brains containing, for example, amyloid plaques. 50 The compound can show dose-dependent binding to AD brain tissue, whereas it does not bind to non-AD human, rat, or cynomolgus monkey brain tissue that is devoid of amyloid plaques.

[0086] Data from studies using the APPPS1-21 mouse model expressing human transgenes for amyloid precursor protein and presenilin 1 revealed that AC-264713 enters the brain and binds to Aβ plaques after a single injection. AC-264713 also binds to amyloid plaques in human brain tissue from donors with AD. Furthermore, in vitro data demonstrated that the fragment crystallizable (Fc) portion of AC-264713 has sufficient effector function to induce hiPSC-derived phagocyte-mediated clearance of plaques. When assessed in an acute microbleed model using APPPS1-21 mice, AC-264713 did not induce microbleeds. Taken together, the data demonstrate that AC-264713 has sufficient effector function, i.e., anti-human Aβ pE3 Evidence that fibrillar monoclonal antibodies clear amyloid plaques from AD brains.

[0087] How to use In embodiments, the methods described herein comprise treating a patient with AD with an anti-Aβ antibody of the present disclosure. pE3 Treatment with antibodies.

[0088] "Inhibiting," "reducing," or any variation of these terms, for example, in the context of treating AD and the development of Aβ plaques associated with AD, encompasses any measurable decrease or complete inhibition to achieve a desired result. For example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99 %, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% There may be a reduction (elimination) of less than, about 85% or less, about 90% or less, about 95% or less, about 99% or less, or at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more, or any range derivable therein, or a reduction in the occurrence of Aβ plaques in patients with AD and treatment of patients with AD. EXAMPLES

[0089] The following examples are provided for illustrative purposes to highlight certain features and characteristics of exemplary embodiments of the inventive concepts described herein.

[0090] Example 1: A panel of identified recombinant hybridoma mAbs that bind to recombinant Aβ protein Using direct binding ELISA, a panel of recombinantly expressed mAbs was identified to bind human Aβ pE3-42 Human Aβ and their ability to bind to fibrils 1-40 , human Aβ 1-42 or human Aβ pE11-40 Because of the 100% sequence identity between humans, monkeys, dogs, and rabbits, binding studies were performed with human Aβ 1-40 and Aβ1-42 were used as representative of these other species. Recombinant human Aβ protein was diluted in 0.2 M carbonate-bicarbonate buffer. Aβ protein was diluted from a 0.1 mM / 0.2 mM stock solution to a working concentration of 1 μg / mL and 50 μL per well (50 ng / well) was added to a 96-well half-area high-binding ELISA plate. The plate was incubated overnight at 4° C. with shaking at 100 rpm. After coating, the plate was washed 4 times with 190 μL / well of 1×PBS using an automated plate washer. The plate was blocked with 190 μL / well of 2% BSA in DPBS and incubated for 1 h at room temperature (RT). Reagents for ELISA were diluted in 0.25% BSA in DPBS.

[0091] [Table 1] TIFF2025016446000011.tif24169

[0092] Dilutions of a panel of mAbs were prepared in a dilution plate from stock solutions to a working 1× initial concentration of either 67 nM or 100 nM, and an 8-point 5× dilution series was performed for each mAb. After incubation for blocking, the plate was washed 4 times with 190 μL / well of 1× PBS using an automated plate washer. A 1:10,000 dilution of anti-mouse IgG-HRP was prepared for detection and 50 μL was added to each well. The plate was incubated for 30 minutes at room temperature. After washing 4 times with 1× PBS, 50 μL of TMB substrate (Life Technology, 002023) was added to each well and the plate was incubated for approximately 5-10 minutes at room temperature until color developed. The reaction was stopped by adding 50 μL per well of 2N sulfuric acid. The plate was read on a plate reader to measure the absorbance at 450 nm (OD 450 ) was obtained. The binding of mAb to Aβ protein (EC 50) was calculated using nonlinear regression (4-parameter dose-response curve model: Y=Bottom+(TopBottom) / (1+10^((LogIC50-X)*Hillslope))) in GraphPad Prism 8.4.3.

[0093] Monoclonal antibody (mAb) AC-233661 inhibits Aβ pE3-42 It showed species specificity and selectivity and was selected for humanization. AC-264713 is a humanized version of AC-233661.

[0094] In some cases, AC-264713 is characterized using comparative antibodies. The amino acid sequences of the antibodies included in the experiments for comparison are listed below:

[0095] [Table 2]

[0096] [Example 2] Binding specificity and epitope identification A. Human Aβ pE3-42 Binding specificity to fibrils AC-264713 human Aβ pE3-42 Binding to fibrils was assessed using the method as described in Example 1, except that anti-human IgG-HRP was used for detection instead of anti-mouse IgG-HRP. pE3-42 EC binding to fibrils 50 The value is 0.7 nM. Figure 1 shows the effect of AC-264713 on human Aβ pE3-42 Representative binding to fibrils is shown.

[0097] B. Epitope Identification Ten biotinylated peptides were generated, listed below, from the N-terminus 3-16 amino acids in length, with each amino acid substituted with a glycine point position change starting from amino acid 3, with biotin at the C-terminus.

[0098] [Table 3]

[0099] The ability of AC-264713 to bind to the peptide was assessed by direct binding ELISA. A decrease in binding indicates that the residue is involved in AC-264713 binding to Aβ. pE3-42 It has been shown to be necessary for binding to fibrils. Recombinant human Aβ biotinylated peptide was diluted in distilled water. Aβ peptide was diluted to a working concentration of 2 μg / mL and 50 μL per well (100 ng / well) was added to a 96-well half-area high-binding ELISA plate. The plate was incubated overnight at 4°C with shaking at 100 rpm. After coating, the plate was washed 4 times with 190 μL / well of 1×PBS using an automated plate washer. The plate was blocked with 190 μL / well of 2% BSA in DPBS and incubated for 1 h at room temperature. Reagents for ELISA were diluted in 0.25% BSA in DPBS.

[0100] Dilutions of AC-264713 were prepared in a dilution plate from the stock solution to a working 1× initial concentration of either 67 nM or 100 nM, and an 8-point 5× dilution series of the mAb was performed. After incubation for blocking, the plate was washed 4 times with 190 μL / well of 1× PBS using an automated plate washer. A 1:10,000 dilution of anti-human IgG-HRP was prepared for AC-264713 detection and 50 μL was added to each well. The plate was incubated for 30 minutes at room temperature. After washing 4 times with PBS, 50 μL of TMB substrate was added to each well and the plate was incubated for approximately 5-10 minutes at room temperature until color developed. The reaction was stopped by adding 50 μL per well of 2N sulfuric acid. The plate was read on a ClarioStar plate reader to measure the absorbance at 450 nm (OD 450 ) was obtained. 50) was calculated using nonlinear regression (4-parameter dose-response curve model: Y=Bottom+(TopBottom) / (1+10^((LogIC50-X)*Hillslope))) in GraphPad Prism 8.4.3. Three independent experiments were performed.

[0101] The results are summarized in the table below.

[0102] [Table 4]

[0103] Analysis of AC-264713 binding to glycine mutant peptides indicates that residues pyro, 3, 4, and 5 are important for binding. Interestingly, AC-264713 binds to E3-16, albeit with a 3-fold reduced binding potency compared to pE3-16, which may be due to spontaneous conversion of glutamic acid to pyroglutamic acid in recombinant Aβ peptide preparations.

[0104] [Example 3] Binding of AC-264713 to amyloid plaques in AD brain tissue Unfixed 20 μm tissue sections were prepared from the cerebral cortex of human cadaveric AD brains using a cryostat and thaw-mounted onto glass slides. The tissue sections were then incubated in Tris-buffered saline (TBS) containing 0.3% H2O2 for 10 min. After three washes with TBS, the sections were incubated in TBS containing 3% milk for 30 min. Biotinylated AC-264713 was then added to the samples and incubated overnight at 4°C. After three washes with TBS, immunoreactivity was visualized by incubation in 1:400 ABC Elite for 1 h, followed by 5 min in DAB.

[0105] Images were acquired from all samples using a slide scanner. The IR of biotinylated AC-264713 in AD samples (n=6) was quantified using the Area Quantification module of HALOTM image analysis software v3.1.1076.423. Using this software, the grey matter was outlined and a "threshold" was determined by an observer blinded to the staining conditions. The threshold was set such that the DAB signal was recognized by the software and background / non-specific staining was excluded from the analysis. Once the appropriate threshold was set, the software measured the percentage of the area of ​​interest that contained positive signal.

[0106] Nonlinear regression analysis was performed using Prism Version 9.1.0 (Graph Pad Software). Variable slope model, IR = Bottom + (X HillSlope )*(Top-Bottom) / (X HillSlope +EC 50 HillSlope ) was used, where Bottom was fixed at zero and Top was fixed at 10% as the typical total area occupied by plaques. Data for all six donors were analyzed with joint data fitting.

[0107] In AD brain tissue (n=6), AC-264713 exerted an EC value of 0.4 μg / ml (confidence interval: 0.1-0.7 μg / ml) on plaques. 50 The EC values ​​of the control antibody Ab1 binding to AD brain tissue were calculated using the EC20 values. 50 was estimated to be 12 μg / ml (Figure 2; confidence interval: 8–17 μg / ml). The Hill coefficients for both AC-264713 and Ab1 were close to 1 (1.5 and 0.8, respectively).

[0108] FIG. 2 shows the area of ​​plaques bound by AC-264713 and control antibody Ab1 in fresh frozen human AD tissue from the frontal cortex.

[0109] Example 4: Stimulated amyloid plaque clearance from unfixed APPPS1-21 brain tissue by hiPSC-derived phagocytes Assay Protocol Unfixed, 20 μm coronal brain tissue sections were prepared from 21-month-old APPPS1-21 mice in a cryostat and thaw-mounted on 12 mm poly-D-lysine-coated coverslips. Dried tissue sections were washed briefly with PBS and incubated overnight at 4°C with AC-264713 at 0 μg / ml or dose a, dose b or dose c (0 μg / ml < dose a < dose b < dose c) in PBS. Immediately before the addition of human induced pluripotent stem cell (hiPSC)-derived phagocytes, tissue sections were washed three times with PBS.

[0110] hiPSC-derived phagocytes were generated as previously described (van Wilgenburg, Browne et al., 2013; Haenseler, Sansom et al., 2017). Briefly, dissociated hiPSCs were aggregated into hemocyte embryoid bodies (EBs) in EB differentiation medium [Revitacell, mTeSR-1 supplemented with 50 ng / ml human VEGF, 50 ng / ml human BMP-4 (Peprotech) and 20 ng / ml SCF (R&D Systems)]. EBs were maintained in T75 culture flasks containing EB maintenance medium [X-VIVO 15 supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin (Thermo-Fisher), 2 mM Glutamax, 55 μM 2-mercaptoethanol, 100 ng / mL recombinant human M-CSF and 25 ng / mL recombinant human IL-3]. Phagocytic cell suspensions were collected from EB flasks and plated directly on antibody-treated APPPS1-21 tissue sections at 0.4 × 10 cells per 0.5 ml in phagocyte culture medium [Advanced DMEM / F12 supplemented with 100 U / ml penicillin / 100 μg / ml streptomycin (Thermo-Fisher), 2 mM Glutamax, 55 μM 2-mercaptoethanol, N-2 supplement (Thermo-Fisher) and 100 ng / ml recombinant human M-CSF]. 6The cells were cultured at a density of 1 / 4 at 37°C / 5% CO2 for 72 hours.

[0111] After co-culture with iPSC-derived phagocytes, tissue sections were fixed in 4% paraformaldehyde and fibrous plaques were labeled with 0.025% Thioflavin S. Fluorescent images were acquired using a slide scanner and analyzed using HALO Image Analysis Software v3.1.1076.423. To measure the area of ​​fibrous plaques, fluorescent images from control-treated tissue sections were thresholded to highlight dense plaques, and tissue sections were subsequently thresholded using the same parameters. Data are reported as the measured area occupied by dense plaques (thresholded area) expressed as a percentage of the total tissue area per section.

[0112] Statistical analysis was performed using Prism Version 8.4.3 (Graph Pad Software). Area measurements of dense plaques were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test to compare the mean values ​​of each AC-264713 treatment group (dose a, dose b, and dose c) with the control group (0 μg / ml). n=6 tissue sections per group. As shown in Figure 3, AC-264713 led to a statistically significant reduction in the percent area of ​​amyloid plaques after co-culture with hiPSC-derived phagocytes at dose a, dose b, or dose c (n=6, adjusted p-values ​​less than 0.0001 for comparison of concentrations a-c with 0 μg / ml).

[0113] Example 5: In vivo AC-264713 exposure and target binding (PK / TB) in APPPS1-21 mice In vivo studies of antibody exposure and target binding in APPPS1-21 animals were conducted to determine brain and serum distribution following AC-264713 administration as well as in vivo plaque binding potential.

[0114] animal APPPS1-21 (B6.Cg-Tg(Thy1-APPSw,Thy1-PSEN1*L166P)21Jckr)) (Radde, Bolmont et al., 2006) aged 8.5-12.5 months were used for the PK / TB study. Original breeding mice were obtained from the laboratory of Mathias Jucker under a license agreement with Fa.Koesler (Germany). Mice were bred at Charles River Laboratories (Sulzfeld, Germany) and sent to the animal facility of AbbVie Deutschland GmbH and aged for the performance of the experiments. Animal health and well-being were managed by a veterinarian. Mice were kept in a temperature- and humidity-controlled room with a 12h:12h dark / light cycle and had free access to water and food.

[0115] In vivo methods For this study, AC-264713 as well as the comparative antibody Ab1 were tested at doses ranging from 1 to 4 and were dosed intravenously via the tail vein. Three female animals were used for each dose. The study time point was 72 hours pa. At the end of the study, blood was drawn by cardiac puncture at 72 hours and serum was isolated. Animals were then perfused with cold PBS with 1000 U / L heparin for 10 minutes, the brains dissected and cut in half mid-sagittally, and the right hemisphere was prepared for immunohistochemistry. The left cortex, the remainder of the forebrain, and the cerebellum were isolated and flash frozen separately in liquid nitrogen, and the left cortex was used for quantification of AC-264713 using ELISA.

[0116] Tissue processing, immunohistochemistry and analysis of target binding The right hemisphere used for immunohistochemistry was drop-fixed in 10% formalin for 24 hours and then switched to 70% ethanol. Tissues were trimmed and samples were dehydrated using a standard ethanol series followed by xylene and paraffin-embedded (ASP300, Leica). Brain hemispheres were randomized based on test article and dose, and four paraffin blocks containing randomized hemispheres were generated. Paraffin sections at 4 μm were prepared and processed in an autostainer using a citrate buffer-based heat-induced antigen retrieval solution and 3,3'-diaminobenzidine (DAB)-based detection. To detect in vivo dosed AC-264713 and the comparative antibody Ab1, biotinylated donkey anti-human F(ab')2 fragment (Jackson Immuno) detecting human IgG(H+L) was used at a concentration of 3.7 μg / mL. Aβ pE3 The target area was determined by applying 0.35 μg / mL of AC-264713 or a control antibody Ab1 at a concentration of 0.26 μg / mL for immunostaining.

[0117] Images were collected with a P1000 scanner. pE3 To quantify binding to Aβ, the area occupied by AC-264713 or the comparative antibody Ab1 was calculated based on the IR of AC-264713 or the comparative antibody Ab1, respectively, in plaques from adjacent areas. pE3The areas were normalized by defining a total target area of ​​1000 nm for each brain. For that, three corresponding sections per brain were analyzed using the Area Quantification module of the HALOTM image analysis software. For each section, the cortex was outlined. Using this software, a "threshold" was determined by an observer blinded to the treatment of the animals. The threshold was set such that positive brown DAB staining of hIgG or AC-264713 or comparative antibody Ab1 immunoreactivity was recognized by the software and background / non-specific staining was excluded from the analysis. Once an appropriate threshold was set, the software measured the percentage of the area of ​​interest (cortex) containing positive immunoreactivity for hIgG or AC-264713 or comparative antibody Ab1. The hIgG IR area in the cortex was then normalized to AC-264713 IR or Ab1 IR, and the values ​​were plotted and analyzed with GraphPadPrism (version 8, GraphPad Software). Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Kruskal-Wallis multiple comparison test).

[0118] Assessment of AC-264713 levels in serum and brain Serum and brain samples were analyzed for AC-264713 and comparative antibody Ab1 using an electrochemiluminescence immunoassay detecting human IgG1. Briefly, serum was diluted with PBS or PBS containing the appropriate concentration of sample matrix to achieve a final concentration of 1% serum in all samples. The dilution factor was selected according to the expected analyte levels. Samples were measured at three dilutions, of which at least two were within the acceptable measurement range. The diluted samples were loaded onto a streptavidin plate pre-coated with biotinylated antibody ab99757 (0.5 μg / mL) that captures human IgG1. The plate was incubated for 1 h, washed with TTBS (Tris Tween buffered saline), and human IgG1 was detected with sulfo-tagged EB89 antibody (1 μg / mL).

[0119] For the analysis of brain samples, the general assay procedure remained the same. Prior to the experiment, brain tissue was homogenized 1:5 in homogenization buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM NaF, 1 mM Na2VO4, 1 mM EDTA pH 7.5, 0.25% Na deoxycholate, 1% NP-40 and 1x protease inhibitor tablet). For analysis, the dilution factors were appropriately adjusted to 1:40 for CSF samples and 1:4 for brain homogenates.

[0120] AC-264713 or Ab1 were used to construct the respective calibration curves, and control samples containing the same concentrations of biological matrix as the test samples were used. For the analysis of serum samples, the calibration curves were based on seven standard points covering a concentration range of 31-7500 ng / mL. For CSF and brain, 11 standard points were used to generate calibration curves covering a concentration range of 0.023-1330 ng / mL. For both analytes, respectively, the LLOQ was 31 ng / mL for serum and 0.152 ng / g for brain.

[0121] Data analysis for in vivo efficacy of target binding Nonlinear regression analysis of normalized IR signal as a function of cortical antibody concentration was performed using Prism Version 9.1.0 (Graph Pad Software). Variable slope model, % area occupied by immunoreactivity [norm.] = Bottom + X * (Top-Bottom) / (X + EC 50 ), where Top is fixed at 100%.

[0122] Image analysis of hIgG stained sections revealed a dose-dependent increase in the test article AC-264713 and the control antibody Ab1 in the cortex and their localization to plaques. pE3 Analysis of immunoreactive plaques and normalization of hIgG IR to Ab1 IR or AC-264713 IR also demonstrated a dose-dependent increase in the area of ​​plaques occupied by AC-264713 as well as the comparator antibody Ab1 (Figure 4).

[0123] All concentrations measured were within the limits of quantification. Concentrations increased in a dose-dependent manner at all doses tested in both cortex and serum, with mean AC-264713 concentrations ranging from 0.0016 to 0.29 μg / g in cortex and 4.5 to 352 μg / mL in serum. Tissue to serum ratios (T / S) ranged from 0.03% to 0.08%.

[0124] Correlating observed brain concentrations in individual animals with target binding quantified by IR (Figure 5), AC-264713 inhibited Aβ pE3 EC of 0.031 μg / g (confidence interval: 0.016 to 0.065 μg / g) 50 In comparison, the comparative antibody Ab1 bound with lower potency and an EC 50 is 0.28 μg / g (confidence interval: 0.13 to 1.2 μg / g).

[0125] [Example 6] Evaluation of AC-264713 in an acute microbleeding model using APPPS1-21 mice In vivo studies were performed to determine whether AC-264713 murine pro-antibody induces microbleeding in an acute microbleeding model using APPPS1-21 mice.

[0126] animal APPPS1-21 mice aged 15-17 months were used for the microbleeding study. Mice were bred at Charles River Laboratories (Wilmington, USA), sent to the AbbVie Bioresearch Center animal facility, aged, and transferred to the Cambridge Research Center (CRC) animal facility to perform the experiments. Mice were housed in a temperature- and humidity-controlled room with a 12h:12h dark / light cycle and had free access to water and food. Animals in the CRC animal facility were group-housed in individually ventilated cages with a 12h:12h light-dark cycle and had free access to food and water. Mice were allowed to acclimate for at least 72 hours before dosing.

[0127] In vivo methods We performed microbleed studies in a previously described model (Janssens, Hermans et al., 2021) with some modifications. At 15 months of age, Aβ pE3 was detectable in the cerebral vasculature, but was not as abundant as other N-terminal Aβ species in APPPS1-21 mice. Mice (n=4, mixed sex) were given a single dose of AC-233661 by intraperitoneal injection. Mouse IgG was used as a negative control (n=4, mixed sex), and a comparison antibody Ab2 with mu IgG2a / k, which causes ARIA in patients with AD, was used as a positive control (n=5). Both negative and positive controls were administered by intraperitoneal injection. Three days after injection or as soon as severe adverse effects were observed (whichever occurred first), mice were euthanized with sodium pentobarbital, perfused with PBS, and brains were rapidly removed.

[0128] Histological analysis for microhemorrhages The right hemisphere of the brain was then drop-fixed in 10% formalin for 24 hours and then switched to PBS containing 30% sucrose. Serial coronal sections 50 μm thick were taken from the rostral to the caudal end of each hemisphere using a freezing sliding microtome.

[0129] For red blood cell quantification, every 12th section was mounted on a glass slide and counterstained with hematoxylin.

[0130] Hemosiderin deposits were stained with Prussian Blue stain. One out of twelve sections were mounted on glass slides. Sections were then rehydrated in PBS and permeabilized in PBS containing 0.25% Triton-X-100. After incubation in 80% ethanol containing saturated NaCl and 0.01 M NaOH, sections were incubated in 80% ethanol containing saturated NaCl, 0.01 M NaOH, and 0.5% Congo Red. Slides were then washed with 80% ethanol and normal saline, followed by incubation in 2% potassium ferrocyanide in 0.12 M HCl. After washing with PBS, slides were dehydrated, coverslipped, and scanned.

[0131] Once scanned using a slide scanner, brain sections were analyzed using the Area Quantification module of HALO™ image analysis software. Using this software, the entire section was outlined and a "threshold" was determined by an observer blinded to the treatment of the animals. The threshold was set such that red blood cells or Prussian blue signal was recognized by the software and background / non-specific staining was excluded from the analysis. Once an appropriate threshold was set, the software measured the percentage of the area of ​​interest (entire section) that contained positive signal. For each animal, the average value was calculated for a series of consecutive sections spaced 600 μm apart.

[0132] The control antibody Ab2 induced significant microhemorrhages within 3 days. In contrast, APPPS1-21 mice treated with AC-264713 mouse precursor antibody remained healthy during this period, with red blood cell and hemosiderin deposits comparable to the negative control mIgG2a / k antibody (Figure 6).

[0133] Summary of the Examples In a direct binding ELISA, AC-264713 binds human Aβ pE3-42 EC of 0.7 nM for fibrils 50 AC-264713 binds to another human pyroglutamic acid-modified Aβ species, Aβ pE11-40AC-264713 also inhibited Aβ from all species, including humans, monkeys, dogs, rabbits, rats, and mice. 1-40 Furthermore, amino acid residues pyro, 3, 4, and 5 of AC-264713 did not cross-react with Abs. pE3-42 Immunostaining of unfixed brain tissue demonstrated that AC-264713 dose-dependently inhibited amyloid plaque-containing AD brains with an EC 50 It was demonstrated that AC-264713 binds to hiPSC-derived phagocytes at 100-fold increase in brain tissue, whereas it does not bind to non-AD human, rat or cynomolgus monkey brain tissue, which does not have amyloid plaques. When AC-264713 was tested at concentrations a-c in an ex vivo phagocytosis assay, AC-264713 activated hiPSC-derived phagocytes to clear plaques. Three days after a single IV injection followed by systemic injection of AC-264713 into APPPS1-21 mice, AC-264713 was detected in brain and serum, with dose-dependent increases in exposure and dose-dependent binding to plaques. In an acute microbleeding model using APPPS1-21 mice, AC-264713 mouse precursor antibody did not induce microbleeding within 3 days after a single IP injection. Taken together, the in vitro and in vivo data demonstrate that AC-264713, an anti-Aβ antibody with sufficient effector function to bind to amyloid plaques in the brain and activate microglial phagocytosis after peripheral administration, is a novel anti-Aβ antibody. pE3 The proposed mode of action of monoclonal antibodies is illustrated. The foregoing demonstrates the suitability of AC-264713 for use in treating Alzheimer's disease by clearing amyloid plaques from the AD brain.

[0134] Exemplary embodiments Various specific embodiments have been illustrated and described, some of which are set forth below, it being understood that various changes can be made without departing from the spirit and scope of the inventive concept.

[0135] 1. An anti-human Aβ antibody comprising: (i) a variable heavy chain (vH) comprising three CDRs; and (ii) a variable light chain (vL) comprising three CDRs. pE3 An antibody comprising: vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Anti-human Aβ pE3 antibody.

[0136] 2. The anti-human Aβ antibody of embodiment 1, comprising a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO: 8. pE3 antibody.

[0137] 3. The anti-human Aβ antibody of embodiment 1, comprising a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth as SEQ ID NO: 10. pE3 antibody.

[0138] 4. The anti-human Aβ antibody of embodiment 1, comprising an IgG constant region. pE3 antibody.

[0139] 5. The anti-human Aβ antibody of embodiment 4, comprising a human heavy chain constant region comprising an Fc portion, wherein the Fc portion is a human IgG1, IgG2, IgG3, IgG4, or IgM isotype. pE3 antibody.

[0140] 6. The anti-human Aβ antibody of embodiment 5, comprising a kappa light chain constant region. pE3 antibody.

[0141] 7. Anti-human Aβ of embodiment 1 pE3 A composition comprising an antibody.

[0142] 8. A method for treating a neurodegenerative disorder, comprising administering to the patient an anti-human Aβ antibody of embodiment 1. pE3The method comprising the step of administering the antibody to a patient in need thereof.

[0143] 9. The method of embodiment 8, wherein the neurodegenerative disorder is Alzheimer's disease (AD).

[0144] 10. Anti-human Aβ pE3 A polynucleotide comprising a nucleotide sequence encoding an antibody, the antibody comprising: (i) a vH chain comprising three CDRs; and (ii) a vL chain comprising three CDRs; vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1), vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO: 2); vH CDR3 is RADDYDVGFAY (SEQ ID NO: 3), vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4), vL CDR2 is AATSLAD (SEQ ID NO:5), vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Polynucleotide.

[0145] 11. A vector comprising the polynucleotide of embodiment 10.

[0146] 12. A eukaryotic host cell transformed with the vector of embodiment 11.

[0147] 13. The eukaryotic host cell of embodiment 12, which is a mammalian host cell.

[0148] 14. Anti-human Aβ pE3 A method for producing an antibody, comprising the steps of: (a) culturing the eukaryotic host cell of embodiment 13; and (b) producing an anti-human Aβ pE3 recovering the antibody.

[0149] 15. An anti-human Aβ antibody comprising two heavy chains and two light chains, each heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 37, and each light chain comprising the amino acid sequence set forth as SEQ ID NO: 10.pE3 antibody.

[0150] Those skilled in the art will readily appreciate that the inventive concept is well adapted to carry out the objects and obtain the ends and advantages mentioned as well as those inherent therein. The specific embodiments described herein are intended to be representative and illustrative and are not intended to limit the scope of the invention. Modifications therein and other uses encompassed within the spirit of the invention as defined by the scope of the claims will be apparent to those skilled in the art.

[0151] References Aisen, P. S., J. Cummings, R. Doody, L. Kramer, S. Salloway, DJ Selkoe, J. Sims, RASperling and B. Vellas (2020). “The Future of Anti-Amyloid Trials.” J Prev Alzheimers Dis 7(3):146–151.

[0152] Greenberg, SM, B. J. Bacskai, M. Hernandez-Guillamon, J. Pruzin, R. Sperling, and S. J. van Veluw (2020). “Cerebral amyloid angiopathy and Alzheimer disease-one peptide, two pathways.” Nat Rev Neurol 16(1):30–42.

[0153] Haenseler, W., S. N. Sansom, J. Buchrieser, S. E. Newey, C. S. Moore, F. J. Nicholls, S. Chintawar, C. Schnell, J. P. Antel, N. D. Allen, M. Z. Cader, R. Wade-Martins, W. S. James, and S. A. Cowley (2017). "A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response." Stem Cell Reports 8(6):1727 - 1742.

[0154] Janssens, J., B. Hermans, M. Vandermeeren, E. Barale-Thomas, M. Borgers, R. Willems, G. Meulders, C. Wintmolders, D. Van den Bulck, A. Bottelbergs, L. Ver Donck, P. Larsen, D. Moechars, W. Edwards, M. Mercken, and B. Van Broeck (2021). "Passive immunotherapy with a novel antibody against 3pE-modified Abeta demonstrates potential for enhanced efficacy and favorable safety in combination with BACE inhibitor treatment in plaque-depositing mice." Neurobiol Dis 154: 105365.

[0155] Jawhar, S., O. Wirths, and T. A. Bayer (2011). "Pyroglutamate amyloid-beta (Abeta): a hatchet man in Alzheimer disease." J Biol Chem 286(45):38825 - 38832.

[0156] Kuo, Y. M., M. R. Emmerling, A. S. Woods, R. J. Cotter, and A. E. Roher (1997). "Isolation, chemical characterization, and quantitation of A beta 3-pyroglutamyl peptide from neuritic plaques and vascular amyloid deposits." Biochem Biophys Res Commun 237(1):188 - 191.

[0157] Penninkilampi, R., H. M. Brothers, and G. D. Eslick (2017). "Safety and Efficacy of Anti-Amyloid-β Immunotherapy in Alzheimer’s Disease: A Systematic Review and Meta-Analysis." J Neuroimmune Pharmacol 12(1):194 - 203.

[0158] Radde, R., T. Bolmont, S. A. Kaeser, J. Coomaraswamy, D. Lindau, L. Stoltze, M. E. Calhoun, F. Jaggi, H. Wolburg, S. Gengler, C. Haass, B. Ghetti, C. Czech, C. Holscher, P. M. Mathews, and M. Jucker (2006). "Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology." EMBO Rep 7(9):940 - 946.

[0159] Selkoe, D. J. and J. Hardy (2016). "The amyloid hypothesis of Alzheimer’s disease at 25 years." EMBO Mol Med 8(6):595 - 608.

[0160] Sperling, R. A., C. R. Jack, Jr., S. E. Black, M. P. Frosch, S. M. Greenberg, B. T. Hyman, P. Scheltens, M. C. Carrillo, W. Thies, M. M. Bednar, R. S. Black, H. R. Brashear, M. Grundman, E. R. Siemers, H. H. Feldman and R. J. Schindler (2011). "Amyloid - related imaging abnormalities in amyloid - modifying therapeutic trials: recommendations from the Alzheimer’s Association Research Roundtable Workgroup.", Alzheimers Dement 7(4):367 - 385.

[0161] van Dyck, C. H. (2018). "Anti - Amyloid - beta Monoclonal Antibodies for Alzheimer’s Disease: Pitfalls and Promise.", Biol Psychiatry 83(4):311 - 319.

[0162] van Dyck, C. H. (2018). "Anti - amyloid - β monoclonal antibodies for Alzheimer’s disease: pitfalls and promise.", Biol Psychiatry 83(4):311 - 319.

[0163] van Wilgenburg, B., C. Browne, J. Vowles, and S. A. Cowley (2013). "Efficient, long-term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions." PLoS One 8(8): e71098.

Claims

1. (i) an anti-human Aβ antibody comprising a variable heavy chain (vH) comprising three CDRs and (ii) a variable light chain (vL) comprising three CDRs. ｐＥ３ An antibody comprising: vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1); vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO:2); vH CDR3 is RADDYDVGFAY (SEQ ID NO:3); vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4); vL CDR2 is AATSLAD (SEQ ID NO:5); vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Anti-human Aβ ｐＥ３ antibody.

2. The anti-human Aβ antibody of claim 1, comprising a heavy chain variable region comprising the amino acid sequence set forth as SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth as SEQ ID NO:

8. ｐＥ３ antibody.

3. The anti-human Aβ antibody of claim 1, comprising two heavy chains and two light chains, each heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 9, and each light chain comprising the amino acid sequence set forth as SEQ ID NO:

10. ｐＥ３ antibody.

4. The anti-human Aβ antibody of claim 1, comprising an IgG constant region. ｐＥ３ antibody.

5. The anti-human Aβ antibody of claim 4, comprising a human heavy chain constant region including an Fc portion, wherein the Fc portion is a human IgG1, IgG2, IgG3, IgG4, or IgM isotype. ｐＥ３ antibody.

6. The anti-human Aβ antibody of claim 5, comprising a kappa light chain constant region. ｐＥ３ antibody.

7. The anti-human Aβ antibody according to claim 1 ｐＥ３ A composition comprising an antibody.

8. A method for treating a neurodegenerative disorder, comprising administering to a subject an anti-human Aβ antibody according to claim 1. ｐＥ３ The method comprising the step of administering the antibody to a patient in need thereof.

9. 9. The method of claim 8, wherein the neurodegenerative disorder is Alzheimer's disease (AD).

10. Anti-human Aβ ｐＥ３ A polynucleotide comprising a nucleotide sequence encoding an antibody, the antibody comprising: (i) a vH chain comprising three CDRs; and (ii) a vL chain comprising three CDRs; vH CDR1 is GFSLSTSGMGVS (SEQ ID NO: 1); vH CDR2 is HIYWDDDKRYNPYMKR (SEQ ID NO:2); vH CDR3 is RADDYDVGFAY (SEQ ID NO:3); vL CDR1 is LASQTIGTWLA (SEQ ID NO: 4); vL CDR2 is AATSLAD (SEQ ID NO:5); vL CDR3 is QQLYSSPFT (SEQ ID NO: 6); Polynucleotide.

11. A vector comprising the polynucleotide of claim 10.

12. A eukaryotic host cell transformed with the vector of claim 11.

13. The eukaryotic host cell of claim 12 which is a mammalian host cell.

14. Anti-human Aβ ｐＥ３ A method for producing an antibody, comprising the steps of: (a) culturing the mammalian host cell of claim 13; and (b) producing an anti-human Aβ ｐＥ３ recovering the antibody.

15. Anti-human Aβ containing two heavy chains and two light chains ｐＥ３ An anti-human Aβ antibody, wherein each heavy chain comprises the amino acid sequence set forth as SEQ ID NO: 37 and each light chain comprises the amino acid sequence set forth as SEQ ID NO:

10. ｐＥ３ antibody.

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