Humanized anti-N-truncated amyloid beta monoclonal antibody

Abstract

The present invention relates to humanized antibodies that bind to amyloid peptides and contain mutations in the variable domains of the heavy and / or light chains that improve binding activity. These antibodies may be useful in the treatment of Alzheimer's disease (AD).

Description

[Technical field]

[0001] Field The present invention relates to humanized antibodies that bind to amyloid peptides. [Background technology]

[0002] background The mouse anti-amyloid beta (Aβ) antibody NT4X-167 was first raised against the Aβ4-40 amyloid peptide and has been reported to specifically bind to the N-truncated amyloid peptides AβpE3-42 and Aβ4-42, but not to the amyloid peptide Aβ1-42 (Antonios et al Acta Neuropathol. Commun. (2013) 6 1 56). Passive immunization with NT4X-167 has been shown to be therapeutically beneficial in Alzheimer's mouse models (Antonios et al Scientific Reports 5 17338; 2015).

[0003] A humanized version of NT4X-167 would be useful for clinical applications, for example, in the treatment of Alzheimer's disease (AD). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Antonios et al Acta Neuropathol.Commun.(2013)6 1 56 [Non-Patent Document 2] Antonios et al Scientific Reports 5 17338;2015 Summary of the Invention

[0005] overview The inventors have unexpectedly discovered that mutation of specific residues within the heavy and / or light chain variable domains improves the binding activity of a humanized version of the NT4X-167 antibody, which may be useful, for example, in the development of candidate molecules for clinical use.

[0006] A first aspect of the present invention is an anti-Aβ antibody comprising a heavy chain variable domain and a light chain variable domain, (a) the heavy chain variable domain (VH domain) comprises SEQ ID NO:2 with no more than four additional alterations, e.g., substitutions, in the framework regions; (b) An anti-Aβ antibody, wherein the light chain variable domain (VK domain) comprises SEQ ID NO:6 with no more than four additional alterations, e.g., substitutions, in the framework regions.

[0007] The anti-Aβ antibody can specifically bind to N-terminal truncated amyloid peptide (AβpE3-x or Aβ4-x). For example, the anti-Aβ antibody can specifically bind to one or more, preferably all, of AβpE3-38, AβpE3-40, AβpE3-14, AβpE3-42, Aβ4-38, Aβ4-40, Aβ4-14, and Aβ4-42.

[0008] Anti-Aβ antibodies do not exhibit specific binding to full-length amyloid peptides or amyloid peptides without N-terminal truncations (Aβ1-x), such as Aβ1-42, Aβ1-38, Aβ1-40, or Aβ1-14.

[0009] Preferably, the heavy chain variable domain (VH domain) of the anti-Aβ antibody comprises SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

[0010] Preferably, the light chain variable domain (VL domain) of the anti-Aβ antibody comprises SEQ ID NO:7 or SEQ ID NO:8.

[0011] A second aspect described herein provides a pharmaceutical composition comprising an antibody of the first aspect and a pharma- ceutically acceptable carrier.

[0012] A third aspect described herein provides a nucleic acid encoding an antibody of the first aspect, or a heavy chain variable domain and / or a light chain variable domain thereof.

[0013] A fourth aspect described herein provides a vector comprising a nucleic acid of the third aspect.

[0014] A fifth aspect described herein provides a host cell comprising a nucleic acid of the third aspect or a vector of the fourth aspect.

[0015] A sixth aspect described herein provides a method of making an antibody according to the first aspect, the method comprising expressing a vector according to the fourth aspect in a host cell culture so that the antibody is produced, and recovering the antibody from the cell culture.

[0016] A seventh aspect described herein provides a method of treating Alzheimer's disease by administering an effective amount of an antibody according to the first aspect or a pharmaceutical composition according to the second aspect to an individual in need of treatment.

[0017] An eighth aspect described herein provides an antibody according to the first aspect or a pharmaceutical composition according to the second aspect for use in a method of treatment of the human or animal body.

[0018] A ninth aspect described herein provides an antibody according to the first aspect or a pharmaceutical composition according to the second aspect for use in a method of treating Alzheimer's disease in an individual.

[0019] [The present invention 1001] 1. An antibody comprising a heavy chain variable domain and a light chain variable domain, (a) the heavy chain variable domain (VH domain) comprises SEQ ID NO:2 with no more than four additional changes in the framework regions; (b) the light chain variable domain (VK domain) comprises SEQ ID NO:6 with no more than four additional changes in the framework regions; The antibody. [The present invention 1002] The antibody of the present invention 1001, which binds to amyloid peptides AβpE3-42 and Aβ4-42, but does not bind to amyloid peptide Aβ1-42. [The present invention 1003] Any of the antibodies of the present invention, wherein the VH domain comprises SEQ ID NO:2 and the VL domain comprises SEQ ID NO:6. [The present invention 1004] The antibody of any of claims 1001 to 1003, wherein the heavy chain variable domain comprises SEQ ID NO:3 with no more than four additional alterations in the framework regions, and optionally, the heavy chain variable domain comprises SEQ ID NO:3. [The present invention 1005] The antibody of any of claims 1001 to 1003, wherein the heavy chain variable domain comprises SEQ ID NO:4 with up to four additional alterations in the framework regions, and optionally, the heavy chain variable domain comprises SEQ ID NO:4. [The present invention 1006] The antibody of any of claims 1001 to 1003, wherein the heavy chain variable domain comprises SEQ ID NO:5 with up to four additional alterations, e.g. substitutions, in the framework regions, and optionally wherein the heavy chain variable domain comprises SEQ ID NO:5. [The present invention 1007] The antibody of any of claims 1001 to 1006, wherein the light chain variable domain comprises SEQ ID NO:7 with up to four additional alterations, e.g. substitutions, in the framework regions, and optionally wherein the light chain variable domain comprises SEQ ID NO:7. [The present invention 1008] The antibody of any of claims 1001 to 1006, wherein the light chain variable domain comprises SEQ ID NO:8 with up to four additional alterations, e.g. substitutions, in the framework regions, and optionally wherein the light chain variable domain comprises SEQ ID NO:8. [The present invention 1009] Any of the antibodies of claims 1001 to 1003, comprising a VH domain of SEQ ID NO:3 and a VK domain of SEQ ID NO:7. [The present invention 1010] Any of the antibodies of claims 1001 to 1003, comprising a VH domain of SEQ ID NO:4 and a VK domain of SEQ ID NO:7. [The present invention 1011] Any of the antibodies of claims 1001 to 1003, comprising a VH domain of SEQ ID NO:5 and a VK domain of SEQ ID NO:8. [The present invention 1012] Any of the antibodies of claims 1001 to 1003, comprising a VH domain of SEQ ID NO:5 and a VK domain of SEQ ID NO:7. [The present invention 1013] A pharmaceutical composition comprising any of the antibodies of the present invention together with a pharma- ceutically acceptable carrier. [The present invention 1014] The antibody of any of claims 1001 to 1012 or the pharmaceutical composition of claim 1014 for use in a method of treatment of the human or animal body. [The present invention 1015] The antibody of any of claims 1001 to 1012 or the pharmaceutical composition of claim 1014 for use in a method for treating Alzheimer's disease in an individual. These and other aspects and embodiments described herein are described in more detail below. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows the binding of mouse NT4X-167 antibody to amyloid peptides. [Diagram 2] FIG. 2 shows binding of murine and chimeric NT4X-167 antibodies to the PSL amyloid peptide. [Diagram 3] FIG. 3 shows binding of mouse NT4X-167, humanized NT4X-167, and chimeric NT4X-167 to AβpE3-42 amyloid peptide: initial version. [Figure 4] FIG. 4 shows the binding of humanized variants to Aβ1-42. [Diagram 5] FIG. 5 shows binding of humanized variants to AβpE3-42. [Figure 6] FIG. 6 shows binding of humanized NT4X-167 antibody to the second round AβpE3-42 peptide: HC-HR version in combination with RKA. [Figure 7] FIG. 7 shows binding of humanized NT4X-167 antibody to second round AβpE3-42 peptide by ELISA. [Figure 8] FIG. 8 shows binding of humanized NT4X-167 antibody to the third round AβpE3-42 peptide: HS-HY version in combination with RKA. [Figure 9] FIG. 9 shows binding of humanized NT4X-167 antibody to round 5 AβpE3-42 peptide: rcNT4XS6A version, rcNT4XS7A version, rcNT4XS8A version in combination with RKA. [Figure 10-1] FIG. 10 shows the thermal stability of binding of humanized rcNT4X_SA antibody, humanized rcNT4X_BA antibody, humanized rcNT4X_TA antibody, humanized rcNT4X_UA antibody, humanized rcNT4X_VA antibody, humanized rcNT4X_WA antibody, and humanized rcNT4X_YA antibody to amyloid peptide AβpE3-42. [Figure 10-2] See description of Figure 10-1. [Figure 11-1] FIG. 11 shows thermal shift analysis of humanized rcNT4X_SA and humanized rcNT4X_S7A antibodies. [Figure 11-2] See description of Figure 11-1. [Figure 12] FIG. 12 shows non-specific protein-protein interactions (cross-interaction chromatography). [Figure 13] FIG. 13 shows non-specific protein-protein interactions (cross-interaction chromatography) of the humanized lead candidate rcNT4XS7A. [Figure 14] FIG. 14 shows purified antibody candidates that were assessed for solubility. [Figure 15-1] FIG. 15 shows an assessment of the serum stability of the binding of humanized rcNT4X_SA and humanized rcNT4X_S7A antibodies to the PSL AβpE3-42 amyloid peptide. [Figure 15-2] See description of Figure 15-1. [Figure 16-1] FIG. 16 shows an assessment of the serum stability of the binding of humanized rcNT4X_SA and humanized rcNT4X_S7A antibodies to Anaspec Aβ4-42 amyloid peptide. [Figure 16-2] See description of Figure 16-1. [Figure 17] FIG. 17 shows the amount of protection from cell death induced by the 4-42 amyloid peptide provided to primary embryonic rat neurons in vitro by humanized rcNT4X_SA and humanized rcNT4X_S7A antibodies. [Figure 18] FIG. 18 shows the amount of protection from cell death induced by (AβGlp3)3-42 amyloid peptide provided to primary embryonic rat neurons in vitro by humanized rcNT4X_SA and humanized rcNT4X_S7A antibodies. [Figure 19] FIG. 19 shows the amount of protection from Aβ1-42 amyloid peptide-induced cell death provided to primary embryonic rat neurons in vitro by humanized rcNT4X_SA and humanized rcNT4X_S7A antibodies. [Figure 20] FIG. 20 shows the amount of protection from Aβ4-42 amyloid peptide-induced cell death provided to human CNS.4U neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A antibodies. [Figure 21]FIG. 21 shows the amount of protection from AβpE3-42 amyloid peptide-induced cell death provided to human CNS.4U neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A antibodies. [Figure 22] FIG. 22 shows the amount of protection from Aβ1-42 amyloid peptide-induced cell death provided to human CNS.4U neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A antibodies. [Figure 23] Figure 23 shows that neuronal loss in the hippocampus of 6-month-old Tg4-42 mice is rescued by both rcNT4X antibodies. Quantification of neurons in CA1 using unbiased stereology. Neuron numbers in the hippocampus of 6-month-old Tg4-42 mice after passive immunization with rcNT4X_SA and rcNT4X_S7A. Tg4-42 mice immunized with rcNT4X antibodies showed significantly more neurons than age-matched mice injected with IgG1. One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons; n=5-6. *p<0.05; ***p<0.001; data presented as mean±SEM. [Figure 24] Figure 24 shows that rcNT4X_S7A has the highest efficacy in rescuing neuronal loss in Tg4-42. Comparison of data from the first NT4X with rcNT4X_SA and rcNT4X_S7A. t-test between NT4X and rcNT4X_S7A. n=5-7. *p<0.05; data presented as mean±SEM. [Diagram 25] Figure 25 shows that there is no significant difference between the MRCT control IgG1 antibody and the IgG2b and PBS control groups. Data from the IgG2ba and PBS groups were taken from Antonios et al. [6]. Neither t-test nor ANOVA showed significant differences. Data presented as mean ± SEM. n=5-7. [Figure 26]Figure 26 shows that passive immunization with rcNT4X_S7A rescues learning defects in Tg4-42 mice. Tg4-42 mice received weekly injections of antibody and IgG1 control antibody (MRCT control) for 12 weeks. Mice were tested at 6 months of age in the Morris water maze. Tg4-42 mice treated with MRCT control antibody showed no preference for the target quadrant in the probe trial, and therefore had impaired spatial reference memory. In contrast, Tg4-42 mice immunized with rcNT4X_S7A antibody did not show learning defects. ***p<0.001; **p<0.01; *p<0.05. n=8 per group. One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons. T, target quadrant, L, left quadrant, R, right quadrant, O, anti-control quadrant. Data presented as mean ± SEM; m = months. [Figure 27]FIG. 27 shows the reduction of cortical plaque burden in immunized 5XFAD mice. Plaque burden analysis of 5XFAD mice immunized with rcNT4X_SA and rcNT4X_S7A compared to IgG1-injected 5XFAD mice. (a) Immunostaining with antibody against pan-Aβ showed that plaque burden was reduced after immunization with rcNT4X_S7A, but not in the group immunized with rcNT4X_SA. (b) Both rcNT4X-treated groups showed a significant reduction in fibrillar Aβ deposits as evidenced by thioflavin S staining. (c) Immunostaining with antibody against Aβ1-x showed that plaque burden was reduced after immunization with rcNT4X_S7A, but not in the group immunized with rcNT4X_SA. (d) Immunostaining with antibodies against pyroglutamic acid Aβ3-x revealed that both rcNT4X antibodies reduced plaque burden, but it was significant only for rcNT4X_S7A antibody and tended to be so for rcNT4X_SA antibody. (e) Immunostaining with antibodies against Aβ4-x revealed that both rcNT4X antibodies reduced plaque burden. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test against control group; n = 7–11; ***p < 0.001, ***p < 0.01, *p < 0.05, data presented as mean ± SEM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description The present invention relates to the discovery that the binding activity of a humanized version of the murine anti-amyloid beta (Aβ) antibody NT4X-167 is significantly improved by mutation of specific residues within the variable domains.

[0022] The anti-Aβ antibodies described herein may comprise a heavy chain variable (VH) domain and a light chain variable (VL) domain. The heavy chain variable domain may comprise SEQ ID NO:2 with up to four additional amino acid mutations, such as substitutions, deletions, or insertions, in the framework regions.

[0023] The antibody can specifically bind to N-terminal truncated amyloid peptides, e.g., pyroglutamic acid (pE) modified amyloid peptides such as AβpE3-38, AβpE3-40, AβpE3-14, and AβpE3-42 (also called AβpE3-x, AβpGlu3-x, Aβ(Glp3)3-x, and p3-x), as well as non-pyroglutamic acid modified amyloid peptides such as Aβ4-38, Aβ4-40, Aβ4-14, and Aβ4-40. Binding can be determined using standard techniques, such as, for example, ELISA or surface plasmon resonance as described below, using the anti-Aβ antibodies described herein in IgG1 format.

[0024] VH may comprise the amino acid sequence of SEQ ID NO:2; or SEQ ID NO:2 having one or more, e.g., two, three, or four, additional amino acid alterations or mutations (e.g., single amino acid substitutions, deletions, or insertions), preferably substitutions, independently in the framework regions, relative to SEQ ID NO:2. The additional amino acid alterations or mutations in the framework regions may be at residues other than 27F, 29L, 63R, and 70V of SEQ ID NO:2, preferably at residues other than 27F, 29L, 63R, 70V, 52BX1, 52CX6, 53X2, 54X3, 55X4, and 56X5.

[0025] The VL domain may comprise the amino acid sequence of SEQ ID NO:6; or SEQ ID NO:6 having one or more, e.g., two, three or four, additional amino acid alterations or mutations (e.g., single amino acid substitutions, deletions or insertions), preferably substitutions, independently in the framework regions, relative to SEQ ID NO:6. The additional amino acid alterations or mutations in the framework regions may be at residues other than 92X7 of SEQ ID NO:6.

[0026] The substitution may be a conservative substitution. For example, the anti-Aβ antibodies described herein may comprise a VH domain of SEQ ID NO:3, 4, or 5, optionally with one, two, three, or four amino acid substitutions in the framework regions. The anti-Aβ antibodies described herein may comprise a VL domain of SEQ ID NO:7 or 8, optionally with one, two, three, or four amino acid substitutions in the framework regions.

[0027] Suitable anti-Aβ antibodies may comprise (i) the VH domain of SEQ ID NO:3 and the VL domain of SEQ ID NO:7, (ii) the VH domain of SEQ ID NO:4 and the VL domain of SEQ ID NO:7, (iii) the VH domain of SEQ ID NO:5 and the VL domain of SEQ ID NO:7, (iv) the VH domain of SEQ ID NO:3 and the VL domain of SEQ ID NO:8, (v) the VH domain of SEQ ID NO:4 and the VL domain of SEQ ID NO:8, and / or (vi) the VH domain of SEQ ID NO:5 and the VL domain of SEQ ID NO:8. Some preferred anti-Aβ antibodies may comprise the VH domain of SEQ ID NO:5 and the VL domain of SEQ ID NO:8.

[0028] The terms "immunoglobulin" and "antibody" may be used interchangeably to refer to any protein that contains an antibody antigen-binding site that has the ability to specifically bind to one or more antigens.

[0029] An "antigen" is an entity (e.g., a proteinaceous entity or peptide) to which an immunoglobulin or antibody (or an antigen-binding fragment thereof) specifically binds. Antigens for the anti-Aβ antibodies described herein can include N-truncated amyloid peptides AβpE3-42 and Aβ4-42.

[0030] Native antibodies are generally heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide linkages between the heavy chains of different antibody isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bridges.

[0031] Antibodies contain globular regions of heavy or light polypeptide chains called "domains". A domain may contain peptide loops, typically three to four loops, stabilized, for example, by beta-pleated sheets and / or intrachain disulfide bonds. Domains are generally referred to as "constant" or "variable" based on the relative lack of sequence variation within the domain of various class members in the case of "constant" domains, or significant variation within the domain of various class members in the case of "variable" domains. "Domains" of antibodies or polypeptides are often referred to interchangeably in the art as "regions" of antibodies or polypeptides.

[0032] The "constant" domain of an antibody light chain is also called the "light chain constant region", "light chain constant domain", "CL" region, or "CL" domain. The "constant" domain of an antibody heavy chain is also called the "heavy chain constant region", "heavy chain constant domain", "CH" region, or "CH" domain. The constant domain of the light chain is aligned with the first constant domain of the heavy chain. The constant domain of the heavy chain, including the tail region of the antibody, is referred to herein as the Fc (fragment crystallizable) domain or Fc region. The Fc region can interact with cell surface Fc receptors and several proteins of the complement system, in a manner that allows antibodies to activate the immune system. The Fc region contains three heavy chain constant domains in each polypeptide chain.

[0033] The "variable" domains of an antibody light chain are also called "light chain variable regions", "light chain variable domains", "VL" regions, or "VL" domains (the "L" here refers to the "light" rather than "lambda" light chain isotype). The "variable" domains of an antibody heavy chain are also called "heavy chain variable regions", "heavy chain variable domains", "VH" regions, or "VH" domains. An intact light chain, for example, has two domains (VL and CL), and an intact heavy chain, for example, has four or five domains (VH, CH1, CH2, and CH3).

[0034] The variable domains of the light and heavy chains contain "hypervariable regions" (HVR or HV), also known as "complementarity determining regions" (CDR), whose sequences are hypervariable and can form structurally defined loops. Generally, antibodies contain six hypervariable regions, interspersed among relatively conserved framework regions (FR); three (H1, H2, H3) in the heavy chain and three (L1, L2, L3) in the light chain. The amino acid sequences of the variable domains in the antibodies described herein are shown below. In these sequences, the CDRs can be easily identified using standard techniques (see, for example, Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242. USDepartment of Health and Human Services). In the Kabat nomenclature, VHCDR1 is located at positions 31-35, VHCDR2 is located at positions 50-65, VHCDR3 is located at positions 95-102, VLCDR1 is located at positions 24-34, VLCDR2 is located at positions 50-56, and VLCDR3 is located at positions 89-97.

[0035] The variable regions of each light / heavy chain pair form an antigen-binding site. The term "antigen-binding site" refers to the site that specifically binds (immunoreacts with) an antigen. The antibodies described herein contain at least one antigen-binding site, and preferably contain two antigen-binding sites. The antigen-binding site that allows binding to a specific epitope is formed from the CDRs of the heavy and light chains aligned by the framework regions. An "antigen-binding region" or "antigen-binding domain" is the region or domain of an antibody that contains the antibody-binding site. The antibodies described herein have at least one antigen-binding site that recognizes the amyloid peptides AβpE3-42 and Aβ4-42.

[0036] Naturally occurring or recombinantly produced antibody chains may be expressed including leader sequences that may be removed during cellular processing to produce mature chains. Mature chains may be recombinantly produced containing non-naturally occurring leader sequences, for example to enhance secretion or alter processing of the particular chain of interest.

[0037] The constant regions of the heavy and light chains of an antibody can exhibit phenotypic variation.Antibody light chains are classified as either kappa (κ) or lambda (λ) based on the amino acid sequence of the light chain constant region, and are approximately 230 residues long.Antibodies described herein comprise a κ light chain (the variable domain of the κ light chain is referred to herein as VK).

[0038] Heavy chains from humans and higher mammals are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), are about 450-600 residues long, and define the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively. There are two subclasses of IgM (H and L), three subclasses of IgA (IgA1, IgA2, and secretory IgA), and four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4). The antibodies described herein are preferably immunoglobulin G (IgG) antibodies. The antibodies described herein are more preferably IgG4 or IgG1 antibodies with minimal effector function.

[0039] The antibodies described herein can contain heavy chains belonging to any of the immunoglobulin isotypes described herein. The antibodies described herein can contain sequences from multiple classes or isotypes.

[0040] The anti-Aβ antibody described herein can exhibit cytotoxic activity. In such antibodies, the constant domain is generally a complement-fixing constant domain, and the class is typically IgG1. Human isotypes IgG1 and IgG4 are exemplary.

[0041] The antibodies described herein may include fragments of whole antibodies. The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain, which preferably retains at least one, and preferably most or all, of the functions normally associated with that portion when present in an intact antibody. Fragments may be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments may also be obtained by recombinant means.

[0042] Fragments of the antibodies described herein can bind to antigens or compete for antigen binding (i.e., specific binding) with intact antibodies (i.e., the intact antibodies from which they are derived). The antibodies described herein bind to amyloid peptides AβpE3-42 and Aβ4-42. Binding fragments are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0043] The antibodies described herein may exist as binding fragments including, but not limited to, Fab, Fab', F(ab')2, chemically linked F(ab')2, monospecific Fab2, bispecific Fab2, trispecific Fab2, monovalent IgG, scFv (single chain variable fragment), di-scFv (bivalent scFv), bispecific diabody, trispecific triabody, scFv-Fc, minibody, or sdAb (single domain antibody) and retain the ability to bind to the amyloid peptides AβpE3-42 and Aβ4-42.

[0044] The antibodies described herein can be part of bispecific or trispecific antibodies. Bispecifics are artificial hybrid antibodies with two different heavy / light chain pairs and two different antigen binding sites; trispecifics are artificial hybrid antibodies with three different heavy / light chain pairs and three different antigen binding sites. Bispecific and trispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Exemplary antibodies described herein can be bispecific antibodies that contain at least two different antigen binding sites.

[0045] Specific binding refers to a situation in which an antibody does not exhibit significant binding to molecules other than the specific epitope on the antigen. This term is also applicable, for example, when an antigen-binding domain is specific to a particular epitope carried by multiple antigens, in which case an antibody carrying the antigen-binding domain will be able to bind to various antigens carrying that epitope.

[0046] The anti-Aβ antibodies described herein, or the nucleic acids encoding such antibodies, will be in an isolated state. The antibodies and nucleic acids will be free or substantially free of naturally associated materials, such as other polypeptides or nucleic acids, with which they are found in their natural environment, or in the environment in which they are prepared (e.g., cell cultures) when such preparation is by recombinant DNA technology carried out in vitro or in vivo. The antibodies and nucleic acids may be formulated with diluents or adjuvants, or may be isolated for practical purposes, for example, the antibodies will usually be mixed with gelatin or other carriers when used to coat microtiter plates for use in immunoassays, or mixed with pharma-ceutically acceptable carriers or diluents when used in diagnosis or therapy.

[0047] Another aspect of the invention provides a nucleic acid encoding an antibody disclosed herein, or a light chain, heavy chain, VH domain, or VL domain thereof. The nucleic acid may encode a heavy chain variable domain (VH domain) comprising SEQ ID NO:2, such as SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, as described above, and / or a light chain variable domain (VK domain) comprising SEQ ID NO:6, such as SEQ ID NO:7 or SEQ ID NO:8. Optionally, the encoded VH and / or VL domain may have up to four additional amino acid mutations in the framework regions.

[0048] Nucleic acids include DNA sequences, and RNA sequences in which the thymine nucleobase is replaced by uracil.

[0049] The antibodies described herein can be produced by recombinant expression. The nucleic acids encoding the light and heavy chain variable regions, optionally linked to constant regions, can be inserted into an expression vector. A vector containing a nucleic acid encoding an antibody described herein is itself an aspect of the present invention. The light and heavy chains can be cloned into the same expression vector or into different expression vectors. The nucleic acids encoding the antibody chains described herein can be operably linked to one or more regulatory sequences in the expression vector that ensure the expression of the antibody chain. Expression regulatory 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 regulatory sequences are eukaryotic promoter systems in vectors that can transform or transfect eukaryotic host cells (e.g., COS cells, CHO cells, or Expi293 cells). Such vectors can be incorporated into a suitable host, whereby the host is maintained under appropriate conditions for high level expression of the nucleotide sequences, and collection and purification of the antibody.

[0050] Aspects of the present invention provide nucleic acid encoding the antibody described herein; vector, preferably expression vector, comprising one or more nucleic acids encoding the antibody described herein; and vector comprising one or more nucleic acids encoding the antibody described herein operably linked to a promoter.Exemplary expression vectors are pHuK and pHuG1, which comprise the nucleotide sequence encoding the antibody described herein in combination with the nucleic acid disclosed herein.Other vectors that provide the nucleotide sequence encoding the constant region of the light and heavy chains of the antibody may also be used.

[0051] The expression vector for use described herein is typically replicable in host organism, either as episome or as part integrated into host chromosomal DNA.Generally, expression vector contains a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to allow detection of cells transformed by desired DNA sequence (see, for example, Itakura et al. US4704362).

[0052] Host cells can be transformed with the expression vector and cultured in conventional nutrient media, as appropriate, for inducing promoters, selecting transformants, and / or amplifying the genes encoding the required sequences. Host cells containing the nucleic acids or vectors described above are provided as an aspect of the invention.

[0053] Another aspect of the present invention provides a method for producing the antibodies described herein, comprising expressing the vectors described herein in a host cell culture so that the antibody is produced, and recovering the antibody from the cell culture. The method may include introducing a vector comprising one or more nucleic acids encoding said antibody or antibody chain into a host cell described herein, growing the host cell culture under conditions that allow expression of the nucleic acids, and recovering the expressed antibody. Any suitable method known in the art may be utilized.

[0054] Suitable microbial host organisms for use in cloning and expressing the nucleic acids and vectors described herein include prokaryotic hosts; bacilli such as Escherichia coli, Bacillus subtilis, and other Enterobacteriaceae such as Salmonella, Serratia, and various species of Pseudomonas. In these prokaryotic hosts, expression vectors can also be made that typically contain expression control sequences (e.g., origin of replication) compatible with the host cell. In addition, there will also be a number of different well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. The promoter will typically have ribosome binding site sequences, etc., to regulate expression, optionally with an operator sequence, for initiation and completion of transcription and translation. Vectors for use in prokaryotic cells may also require an origin of replication component.

[0055] Other microbes, such as yeast, can also be used to express the nucleic acids or vectors described herein. Saccharomyces is a preferred yeast host, and suitable vectors will have the desired expression control sequences (e.g., promoters), origins of replication, termination sequences, etc. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

[0056] In addition to microorganisms, mammalian tissue cell cultures can also be used to express the nucleic acids or vectors described herein and generate antibody polypeptides (e.g., polynucleotides encoding antibodies or fragments thereof) (see, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY 1987). Eukaryotic or mammalian cell hosts containing the nucleic acids or vectors described herein are themselves an aspect of the present invention. Eukaryotic cells are indeed preferred, since a large number of suitable host cell lines capable of secreting heterologous proteins (e.g., intact antibodies) have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, Expi293 cells, ExpiCHO cells, myeloma cell lines, or transformed B cells or hybridomas. The cells can be human or non-human, e.g., non-human mammalian cells. In some preferred embodiments, the cells are Expi293 human cells. The antibodies described herein may be produced in cell lines modified to produce afucosylated proteins, such as the Potelligent® CHOK1SV cell line (BioWa / Lonza), GlymaxX® modified cells (ProBioGen), or the duck embryonic stem cell line EB66 (Valneva). Expression vectors for mammalian cells generally include, but are not limited to, a signal sequence, one or more marker genes, an enhancer element, a promoter, and one or more of the necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like (see, e.g., Co et al., J.Immunol.148:1149 1992).

[0057] The vectors described herein for use in eukaryotic host cells may encode a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature antibody chain or polypeptide. A suitable signal sequence may be heterologous and may be recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, not only mammalian signal sequences are available, but also viral secretory leaders, such as the herpes simplex gD signal.

[0058] Alternatively, the antibody coding sequences described herein can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al., US5741957; Rosen, US5304489; and Meade et al., US5849992). Suitable transgenes include light and / or heavy chain coding sequences operably linked to a promoter and enhancer derived from a mammary gland-specific gene such as casein or beta-lactoglobulin.

[0059] The vectors described herein containing the polynucleotide sequences of interest (e.g., sequences encoding heavy and light chains and expression control sequences) can be transferred into host cells by well-known methods that vary depending on the type of cell host. For example, for prokaryotic cells, calcium chloride transfection is commonly utilized, while for other cell hosts, calcium phosphate treatment, electroporation, lipofection, gene gun, or viral-based transfection can be used (see generally Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 4th ed., 2012). 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 the production of transgenic animals, the transgene can be microinjected into fertilized oocytes, or can be incorporated into the genome of an embryonic stem cell and the nucleus of such a cell transferred into an enucleated oocyte.

[0060] When the heavy and light chains are cloned into separate expression vectors, the vectors are co-transfected to obtain the expression and assembly of an intact antibody as described herein. After expression, whole antibodies, dimers thereof, individual light and heavy chains, or other immunoglobulin types described herein can be purified according to standard techniques in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, (1982)). For the pharmaceutical uses described herein, substantially pure antibodies having at least about 90-95% homogeneity are preferred, with 98-99% or more homogeneity being most preferred. Standard protein purification methods known in the art can be utilized. The following techniques are exemplary of suitable protein purification techniques: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on cation exchange resins such as silica or DEAE, isoelectric focusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration.

[0061] The production of the antibodies described herein can be carried out by any suitable technique, including those described herein and other techniques known in the art. The antibodies described herein can be produced on a commercial scale using methods well established in the art for large-scale production of antibodies. For example, recombinant expression systems such as those described herein can be utilized.

[0062] The antibodies described herein can specifically bind to amyloid peptides AβpE3-42 and Aβ4-42. The antibodies do not or substantially do not specifically bind to amyloid peptides Aβ1-42. The antibodies described herein may also exhibit the following desirable structural, physical, biophysical, and chemical properties (see Examples).

[0063] The affinity of an antibody as described herein is the degree or strength of binding between the antibody and an epitope or antigen. The dissociation constant K d and the affinity constant K a is a quantitative measure of affinity. d is the antibody dissociation rate (k off ) (how quickly it dissociates from the antigen) and the antibody association rate (k on ) (how quickly the antibody binds to the antigen). Antibody-antigen binding is a reversible process, and the rate of the binding reaction is proportional to the concentrations of the reactants. At equilibrium, the rate of [antibody][antigen] complex formation is equal to the rate of dissociation into its components [antibody] + [antigen]. The rate constant is measured using the equilibrium or affinity constant K a (K a =1 / K d ) can be used to define K d The lower the value, the greater the affinity of the antibody for the target. Most antibodies have low micromolar (10 -6 ) to nanomolar (10 -7 ~10 -9 ) range of K d High affinity antibodies generally have a binding affinity in the low nanomolar range (10 -9 ), whereas very high affinity antibodies have a molecular weight of picomolar (10 -12 ) range.

[0064] The antibodies described herein are at least 2×10 2 M -1 s -1 , at least 5 × 10 2 M -1 s -1 , at least 10 3 M -1 s -1 , or at least 5 × 10 3 M -1 s -1 The association rate constant (k on ).

[0065] The antibodies described herein are administered at a concentration of 5×10 -1 s -1 Less than 10 -1 s-1 Less than 5×10 -2 s -1 Less than 10 -2 s -1 Less than or 5×10 -3 s -1 Antibody dissociation (k off ) speed.

[0066] In some embodiments, the antibodies described herein have a nucleotide sequence of at least 10 2 M -1 , at least 5 × 10 2 M -1 , at least 10 3 M -1 , at least 5 × 10 3 M -1 , at least 10 4 M -1 , at least 5 × 10 4 M -1 , at least 10 5 M -1 , at least 5 × 10 5 M -1 , at least 10 6 M -1 , at least 5 × 10 6 M -1 , or at least 10 7 M -1 Affinity constant or K a and binds (e.g., specifically binds) to amyloid peptides AβpE3-42 and Aβ4-42.

[0067] The antibodies described herein are administered at a concentration of 5×10 -2 Less than M, 10 -2 Less than M, 5×10 -3 Less than M, 10 -3 Less than M, 5×10 -4 Less than M, 10 -4 Less than M, 5×10 -5 Less than M, 10 -5 Less than M, 5×10 -6 Less than M, 10 -6 Less than M or 5 x 10 -7 A dissociation constant or K from the amyloid peptides AβpE3-42 and Aβ4-42 of less than Md may have:

[0068] Specific binding of an antibody means that the antibody exhibits an appreciable affinity for a particular antigen or epitope and generally does not exhibit significant cross-reactivity. An antibody that "does not exhibit significant cross-reactivity" is one that does not appreciably bind to undesired entities (e.g., undesired proteinaceous entities). An antibody specific for a particular epitope will not, for example, significantly cross-react with distant epitopes of the same protein or peptide. Specific binding of an antibody as described herein, i.e., k off , k on , K a , and K d may be determined by any art-recognized means for determining such binding.

[0069] The antibody may bind to amyloid peptide Aβ4-42 or AβpE3-42 with a binding affinity of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of that of mouse NT4X-167 antibody to amyloid peptide Aβ4-42 or AβpE3-42, as measured by ELISA. Suitable ELISA techniques are well known in the art. For example, the immobilized amyloid peptide may be contacted with an antibody in IgG1 format and washed one or more times with 0.1% non-ionic detergent, such as polysorbate 20 (Tween 20), to remove unbound antibody. The antibody bound to the immobilized peptide may then be detected using any convenient technique, for example, using a secondary antibody conjugated to a detectable label, such as HRP.

[0070] The antibodies described herein can have high thermal stability, i.e., the antibodies described herein can bind to amyloid peptides AβpE3-42 and Aβ4-42 at temperatures between 30° C. and 85° C., specifically up to 75° C. The antibodies described herein can have a melting temperature between 50° C. and 100° C., specifically 60-80° C., more specifically approximately 66-67° C.

[0071] The antibodies described herein can have low aggregation tendency. Aggregation tendency can be analyzed using standard techniques such as multi-angle light scattering or dynamic light scattering. The antibodies described herein can have low tendency of non-specific protein-protein interactions and good solubility.

[0072] The antibodies described herein can have a low tendency to aggregate when concentrated. The formulations described herein can contain antibodies concentrated to 50-200 mg / ml, e.g., 75-150 mg / ml, preferably 80-120 mg / ml, more preferably 90-110 mg / ml, without forming soluble aggregates in an aqueous solution maintained at physiological pH, e.g., by Dulbecco's PBS, with a preferred concentration being about 100 mg / ml.

[0073] The antibodies described herein may have a low tendency to aggregate when subjected to repeated freezing and thawing or when subjected to temperatures above normal body temperature for extended periods of time, such as 50° C. for 30 days in Dulbecco's PBS.

[0074] The antibodies described herein can have an isoelectric point (pI) of pH 8.6 to pH 9, preferably pH 8.1 to pH 8.7.

[0075] The antibodies described herein can retain binding ability to the amyloid peptides AβpE3-42 and Aβ4-42 after incubation at 37° C. in serum from mice, humans, and / or cynomolgus primates. For example, the antibodies described herein can retain binding ability to AβpE3-42 or Aβ4-42 after incubation for 10-50 days, preferably 20-40 days, more preferably 30 days in serum from mice, humans, and / or cynomolgus monkeys. Antibodies that retain binding ability can exhibit the same or substantially the same binding ability at 37° C. as observed for antibodies not incubated in serum or incubated in a control solution.

[0076] The anti-Aβ antibodies disclosed herein can be non-glycosylated. The Fc region of IgG antibodies retains a highly conserved N-glycosylation site, and glycosylation of Fc fragments is essential for Fc receptor-mediated activity. The N-gly carbohydrate moieties attached to this site are mainly complex-type core fucosylated biantennary structures. In addition, a small proportion of these N-glycans also retain bisecting GlcNAc and α2,6-linked sialic acid residues. Non-glycosylated antibodies can lack one or more carbohydrate moieties, for example, by chemical or enzymatic processes, the absence or mutation of one or more glycosylation sites, or expression in bacteria.

[0077] The anti-Aβ antibodies described herein can be modified to enhance antibody-dependent cellular cytotoxicity (ADCC). ADCC is a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize the bound antibody on a target cell and subsequently cause lysis of the target cell. Such cells can be human cells. ADCC activity of an antibody is generally believed to require the binding of the Fc region of the antibody to an antibody receptor present on the surface of an effector cell, such as, for example, killer cells, natural killer cells, and activated macrophages. By altering (e.g., reducing or eliminating) the fucosylation of carbohydrate structures (i.e., in the Fc region) of a humanized antibody, the ADCC activity of the antibody can be enhanced in vitro, for example, 10-fold or 20-fold or 30-fold or 40-fold or 50-fold or 100-fold or 500-fold or 600-fold or 700-fold or 1000-fold, compared to an unmodified humanized antibody. Because of the increased ADCC activity, such modified antibodies can be used in lower dosages than their unmodified counterparts and generally have fewer or reduced side effects in patients.

[0078] The anti-Aβ antibodies described herein may be used in complement-dependent cytotoxicity (CDC). CDC involves the central innate complement system that acts as an effector of adaptive immunity. The classical CDC pathway is triggered by the binding of an antibody molecule to an antigen on a target cell, and is initiated by the binding of C1q protein to the Fc domain of the bound antibody. The resulting complement cascade activates the membrane attack pathway, resulting in the formation of a membrane attack complex that induces lysis of the target cell. The antibodies described herein may be modified to enhance their ability to induce CDC by any method known in the art, including but not limited to, by altering the protein backbone to contain amino acid residue substitutions in the constant domain of the antibody heavy chain. For examples of IgG1 amino acid substitution combinations used to enhance CDC activity, see Moore et al., mAbs, 2(2), 181-189 (2010). The CDC activity of the modified antibodies described herein may be enhanced, for example, 10-fold or 20-fold or 30-fold or 40-fold or 50-fold or 100-fold or 500-fold or 600-fold or 700-fold or 1000-fold compared to an unmodified humanized antibody.

[0079] The anti-Aβ antibodies can be further modified by chemical modification, for example by PEGylation or incorporation into liposomes, to improve pharmaceutical properties, for example by increasing in vivo half-life.

[0080] The anti-Aβ antibodies described herein can be formulated and / or administered as pharmaceutical compositions containing an active therapeutic antibody agent and a variety of other pharma- ceutically acceptable components (see Remington: The Science and Practice of Pharmacy (22nd ed., Pharmaceutical Press, London, Pa. (2013))). The preferred form depends on the intended mode of administration and therapeutic application. The composition can also contain a pharma- ceutically acceptable non-toxic carrier or diluent, defined as a vehicle commonly used to formulate pharmaceutical compositions for administration to animals or humans, depending on the desired formulation. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like.

[0081] Pharmaceutical compositions containing the anti-Aβ antibodies described herein may also include large slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (such as latex-functionalized Sepharose™, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers may function as immunostimulants (i.e., adjuvants). For parenteral administration, the antibodies or compositions described herein may be administered as injectable dosages of a solution or suspension of the substance with a physiologically acceptable diluent, including a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerol, or ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances, and the like, may be present in the composition. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. The antibodies may be administered in the form of a depot injection or implant preparation, which may be formulated in such a manner as to permit sustained release of the active ingredient.

[0082] The term parenteral, as used herein, includes subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, and intrathecal administration of the antibody or composition described herein. The anti-Aβ antibody or composition described herein may be administered by intranasal or intragastric methods. Typically, the composition is prepared as an injectable liquid, either as a liquid solution or suspension; solid forms suitable for dissolving or suspending in liquid media prior to injection may be prepared. The preparation may be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolide, or copolymers, as previously described, for enhanced adjuvant effect (see Langer, Science 249:1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97 (1997)). The agent of the present invention may be administered in the form of a depot injection or implant preparation, which may be formulated in such a manner as to allow sustained or pulsatile release of the active ingredient.

[0083] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications. Oral formulations contain excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain 10% to 95%, preferably 25% to 70%, of the active ingredient.

[0084] Topical application may result in transdermal or intradermal delivery. Topical administration may be facilitated by co-administration of the agent with cholera toxin or its detoxified derivatives or subunits, or other similar bacterial toxins (see Glenn et al., Nature 391, 851 (1998)). Co-administration may be achieved by using the components as a mixture, or as linked molecules obtained by chemical cross-linking or expression as a fusion protein. Alternatively, transdermal delivery may be achieved using a skin patch or using transferosomes (Paul et al., Eur. J. Immunol. 25:3521 (1995); Cevc et al., Biochem. Biophys. Acta 1368:201-15 (1998)).

[0085] Preferably, the anti-Aβ antibodies described herein, or compositions comprising the anti-Aβ antibodies described herein, may be administered intravenously (IV) or intramuscularly (IM).

[0086] The composition may comprise an anti-Aβ antibody as described herein, a pharma- ceutically acceptable carrier as described herein, and other therapeutic agents, particularly prophylactic or therapeutic agents useful for the prevention, management, or treatment of Alzheimer's disease (AD). Such therapeutic agents may include analgesics, anti-inflammatory drugs, antiviral drugs, drugs that ameliorate fever or elevated body temperature, therapeutic compounds designed to numb pain, such as mouthwashes or sprays that can numb mouth pain, and cognitive enhancing therapeutic agents such as memantine, donepezil, galantamine, and rivastigmine. The compositions described herein may further comprise a composition for rehydrating a subject, for example, by intravenous therapy.

[0087] The compositions described herein may include a nucleic acid, i.e., DNA or RNA, encoding an anti-Aβ antibody described herein, with or without any of the other compositional compounds described above, and any method of delivery of such a nucleic acid. The compositions may themselves include a vector, such as, but not limited to, an expression vector, as described herein, that contains a nucleic acid as described herein.

[0088] The compositions described herein may include a viral vector for use as a nucleic acid delivery system to cells.Suitable viral vector nucleic acid delivery systems include retrovirus systems, adenovirus vectors, viral vectors from the pox family, including vaccinia virus and avian poxvirus, and viral vectors from the alphavirus genus.The nucleic acid encoding the antibody described herein, or the vector containing it, can be packaged into liposomes for delivery to individuals or cells, which can be incorporated into the compositions described.The vector and nucleic acid encoding the antibody can be adsorbed to or associated with particulate carriers.

[0089] The compositions described herein may include gene therapy vectors containing nucleotide sequences encoding the antibodies described herein, or naked antibody polypeptide chains according to the invention. The compositions may include such vectors or polypeptides in combination with the antibodies described herein and other composition components as described above.

[0090] The antibodies described herein may be used in a kit. The term "kit" is used in reference to a combination of reagents and other materials that facilitate sample analysis. In some embodiments, the immunoassay kits described herein include a suitable antigen, a binding agent that includes a detectable group, and a detection reagent. A system for amplifying the signal generated by the detectable group may or may not be included in the kit. In addition, in other embodiments, the kit includes components such as, but not limited to, a device for sample collection, sample tubes, holders, trays, racks, dishes, plates, instructions for the user of the kit, solutions or other chemical reagents, and samples used for standardization, normalization, and / or control samples.

[0091] The kit may contain at least one antibody as described herein. The kit may optionally contain the composition as described herein in one or more containers, together with one or more other prophylactic or therapeutic agents useful for the prevention, management, or treatment of Alzheimer's disease (AD). If the composition containing the components for administration is not formulated for delivery via the digestive tract, such as oral delivery, a device that can deliver the kit components through some other route, such as a syringe, may be included. The kit may further include instructions for the prevention, treatment, management, or amelioration of AD, as well as dosage information for side effects and methods of administration.

[0092] The present invention also provides diagnostic kits. The antibodies described herein may be useful for monitoring, diagnosing, or providing prognosis for the onset or progression of AD, and may be used in suitable kits for such purposes. The antibodies described herein may be used in diagnostic kits to detect the presence of N-truncated amyloid peptides, such as AβpE3-42 or Aβ4-42, in a sample of bodily fluid obtained from an individual, where the individual may be a mammal, such as a human or a non-human primate or laboratory animal, including mouse, rat, and rabbit. A sample of bodily fluid, such as, but not limited to, blood, serum, or cerebrospinal fluid (CSF), is taken from an individual and tested for the presence of N-truncated amyloid peptides using the antibodies described herein. Measurement of amyloid peptide levels in the blood of an individual using the antibodies described herein can provide information regarding the susceptibility, risk of developing, diagnosis, or prognosis of AD in the individual, or the appropriate administration schedule or dosage of the antibodies or compositions described herein for treating the individual. The diagnostic method is generally carried out in vitro. A method for detecting the presence of N-truncated amyloid peptides in a sample from an individual may include contacting the sample with an anti-Aβ antibody described herein and determining binding of the antibody to one or more peptides in the sample.

[0093] Kits useful for such diagnosis may contain the antibodies described herein coupled to a detectable substance, including, but not limited to: various enzymes for use in assays including EIA and ELISA, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; particles such as latex beads or bacteria for use in agglutination tests; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, luciferin, and aequorin; and photoactivators, such as, but not limited to, iodine ( 131 I, 125 I, 123 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 115 In, 113 In, 112 In, 111 In), and technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), Molybdenum ( 99 Mo), Xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, and 117 Radioactive materials such as Sn; positron-emitting metals using various positron emission tomography, non-radioactive paramagnetic metal ions, and radiolabeled or specific radioisotope-conjugated molecules. Detectable labels that can be easily measured can be conjugated to the antibodies described herein and used in the diagnosis of diseases described herein. Detectable substances can be coupled or conjugated to antibodies directly or indirectly through intermediates (such as linkers known in the art) using techniques known in the art. Metal ions that can be conjugated to antibodies for use as diagnostic agents are known in the art (see, for example, US474900).

[0094] Detection of the antigen by using any of the methods or detectable substances described above, using the antibodies described herein in the kits described herein, can give a positive result for the presence of the amyloid peptides AβpE3-42 or Aβ4-42 and can diagnose an individual as having AD or provide prognostic information regarding an individual having or at risk of AD. Such an individual can then require and / or receive treatment for AD as described herein.

[0095] Aspects of the present invention relate, inter alia, to the treatment of Alzheimer's disease (AD) and other diseases and disorders associated with AD, as well as to the treatment of other neurological diseases characterized by soluble amyloid. Aspects of the present invention also relate to methods of treatment, including prevention of AD, by administering an effective amount of an antibody or composition described herein to an individual in need of treatment. The antibody or composition described herein, preferably a pharmaceutical composition (e.g., a composition comprising an antibody described herein, a pharma- ceutically acceptable excipient, and optionally an additional therapeutic agent), may be for use in a method of treatment of the human or animal body. The antibody or composition described herein, preferably a pharmaceutical composition, may be for use in a method of treatment of the human or animal body, where the treatment is a therapeutic or prophylactic treatment of AD in an individual.

[0096] The aforementioned treatment methods can include administering to an individual an antibody or composition described herein (e.g., a composition comprising an antibody described herein, a pharma- ceutically acceptable excipient, and, optionally, an additional therapeutic agent) under conditions that produce a beneficial therapeutic response in the individual, e.g., for the prevention or treatment of AD.

[0097] Such individuals may be suffering from AD. The methods of treatment described herein may be used in both asymptomatic patients and patients currently showing symptoms of AD. The antibodies described herein may be administered prophylactically to individuals who do not have AD. The antibodies described herein may be administered to individuals who do not have or do not show symptoms of AD. The antibodies described herein may be administered to individuals who have or are suspected of having AD. Individuals who may receive treatment include individuals who are at risk or susceptible to AD but do not show symptoms, and individuals suspected of having AD, as well as individuals currently showing symptoms. The antibodies described herein may be administered prophylactically to the general population without the need to assess the risk of the subject individual. In some embodiments, individuals suitable for the treatment described herein may include individuals who have early onset AD or one or more symptoms thereof, and individuals in whom amyloid peptides are detected in a sample of a body fluid, such as CSF.

[0098] The terms "treat", "treating" or "treatment" (or grammatical equivalents) mean that the severity of an individual's condition is reduced or at least partially improved or ameliorated, and / or some alleviation, reduction or diminution of at least one clinical symptom is achieved, and / or the progression of the condition is inhibited or slowed, and / or the onset of a disease or disorder is prevented or delayed.

[0099] The antibody described herein that can be used in the method of treating AD can be any of the sequences and formats described herein that specifically bind to N-truncated amyloid peptides AβpE3-42 and / or Aβ4-42. The antibody used for the method of treatment described herein can be a fragment of the antibody described herein, e.g., an antigen-binding fragment. The antibody described herein can be administered to an individual with AD.

[0100] The antibody described herein can be administered to an individual in need of treatment with a pharmaceutical carrier or pharmaceutical composition, or in any of the compositions described herein. Alternatively, the antibody can be administered to an individual by administering a polynucleotide that encodes at least one antibody chain. The polynucleotide is expressed to produce the antibody chain in the patient. Optionally, the polynucleotide encodes the heavy and light chains of the antibody. The polynucleotide is expressed to produce the heavy and light chains in the individual.

[0101] The antibodies described herein can be used in a method of preventing or treating AD, comprising administering an effective dosage of an antibody described herein to a patient. As used herein, an "effective amount" or "effective dosage" or "sufficient amount" of a therapeutic antibody described herein (or grammatical equivalents) refers to an amount of an antibody or composition described herein that is effective to produce a desired effect, optionally a therapeutic effect, (i.e., by administration of a therapeutically effective amount). For example, an "effective amount" or "effective dosage" or "sufficient amount" can be an amount such that the severity of an individual's condition, e.g., AD, is reduced or at least partially improved or ameliorated, and / or some alleviation, reduction, or reduction of at least one clinical symptom is achieved, and / or the progression of AD is inhibited or delayed, and / or the onset of AD is prevented or delayed.

[0102] The terms "patient," "individual," or "subject" include humans and other mammalian subjects who receive either prophylactic or therapeutic treatment with one or more agents (e.g., immunotherapeutic agents or antibodies) described herein. Mammalian subjects include primates, e.g., non-human primates. Mammalian subjects also include laboratory animals commonly used in research, such as, but not limited to, rabbits, and rodents such as rats and mice.

[0103] The amount of the antibody or composition described herein that is suitable for achieving therapeutic or prophylactic treatment is defined as an effective dose, for example, a therapeutically effective dose or a prophylactically effective dose. In both prophylactic and therapeutic treatment regimes, the reagent can be administered several times until a sufficient immune response is achieved. The term "immune response" or "immunological response" includes the development of humoral (antibody-mediated) and / or cellular (antigen-specific T cells or their secretory products-mediated) responses directed against an antigen in the recipient subject. Typically, the immune response is monitored, and repeated administration is performed if the immune response begins to decrease.

[0104] The effective dose of the compositions described herein for treating the above conditions varies depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drug treatments administered, and whether the treatment is preventive or therapeutic.Generally, the patient is a human, but non-human mammals, such as non-human primates, rabbits, rats, and mice, including transgenic mammals, can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

[0105] For passive immunization with the antibodies described herein, the dosage is in the range of about 0.01-100 mg / kg, more typically 0.1-50 mg / kg, of the host's body weight. For example, the dosage can be at least 1 mg / kg body weight, or at least 10 mg / kg body weight, or within the range of 1-100 mg / kg. In another example, the dosage can be at least 0.5 mg / kg body weight, or at least 50 mg / kg body weight, or within the range of 0.5-50 mg / kg, preferably at least 5 mg / kg. In a preferred example, the dosage can be about 50 mg / kg.

[0106] The methods described herein may include administration of the antibody to a subject as a single dose, two doses, or multiple doses. The dose of the antibody may be about 100 μg / kg to 100 mg / kg of the patient's body weight, about 300 μg / kg to 60 mg / kg of the patient's body weight, or about 10 mg / kg to 50 mg / kg of the patient's body weight. The subject may be administered such doses daily, every other day, weekly, or by other schedules determined by empirical analysis. Treatment may include administration as multiple doses over an extended period of time, for example, for at least six months. Further treatment regimes may include administration once every two weeks, or once a month, or once every three to six months. Exemplary dosing schedules include 1 to 20 mg / kg or 15 mg / kg on consecutive days, 30 mg / kg on alternate days, or 60 mg / kg every week.

[0107] The antibodies described herein may be administered at multiple time points. The interval between single doses may be weekly, monthly, or yearly. The intervals may be irregular as indicated by measuring blood levels of anti-Aβ antibodies in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of 1-1000 μg / ml, in some methods 25-300 μg / ml. Alternatively, the antibodies described herein may be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency will vary depending on the half-life of the antibody in the patient. In general, humanized antibodies exhibit a longer half-life than chimeric and non-human antibodies.

[0108] Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the antibodies or cocktails thereof described herein are administered to a patient not yet in a disease state to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose." In this use, the exact amount again depends on the patient's health and general immunity, but generally ranges from 0.1 to 25 mg per dose, specifically 0.5 to 2.5 mg per dose. A relatively low dose is administered at relatively infrequent intervals over a long period of time.

[0109] Doses of nucleic acids encoding the antibodies described herein range from about 10 ng-1 g, 100 ng-100 mg, 1 μg-10 mg, or 30-300 μg DNA per patient. Doses of infectious viral vectors vary from 10-100 virions or more per dose.

[0110] The antibodies and compositions described herein may be administered for therapeutic and / or prophylactic treatment by parenteral, topical, intravenous, oral, gastric, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, or intramuscular methods described herein. Intramuscular injection or intravenous infusion is preferred for administration of antibodies.

[0111] Other aspects and embodiments described herein provide for the above aspects and embodiments in which the term "comprising" is replaced with the term "consisting of," as well as the above aspects and embodiments in which the term "comprising" is replaced with the term "consisting essentially of."

[0112] It should be understood that the present application discloses all combinations of any of the above aspects and embodiments with each other unless the context requires otherwise. Similarly, the present application discloses all combinations of preferred and / or optional features alone or together with any of the other aspects unless the context requires otherwise.

[0113] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to those of skill in the art upon reference to this disclosure and, accordingly, are encompassed within the scope described herein.

[0114] All documents and sequence database entries mentioned herein are incorporated herein by reference in their entirety for all purposes.

[0115] The antibody residue positions described herein are numbered according to the scheme set forth in Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242. USDepartment of Health and Human Services. Where appropriate, the position of substitution may be described relative to the residues according to Kabat numbering that are invariant within the immunoglobulin sequence. Another antibody numbering scheme is described in Honegger, A and Pluckthun A. (2001). J. Mol. Biol 309, 657-67.

[0116] "And / or," as used herein, should be understood as specifically disclosing each of the two specified features or components, either with or without the other. For example, "A and / or B" should be understood as specifically disclosing (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein. EXAMPLES

[0117] experiment material and method 1. RNA Preparation from Hybridoma Cells Frozen pellets of mouse hybridoma cells NT4X-167 stored at −80°C were supplied by Thomas Bayer and processed for RNA isolation using the Qiagen RNeasy kit according to the manufacturer's protocol.

[0118] 2. First strand cDNA synthesis NT4X-167 RNA (approximately 26 μg) was reverse transcribed to generate cDNA using the GE Life Sciences First Strand cDNA Synthesis Kit according to the manufacturer's protocol, which was repeated twice to generate three independent NT4X-167 cDNA products (rounds 1, 2, and 3) to detect and avoid reverse transcriptase-induced cDNA mutations.

[0119] 3. cDNA Sequencing NT4X-167 cDNA was amplified by PCR in three separate reactions. Immunoglobulin cDNA was PCR amplified with kappa light chain primers + MKC mix or heavy chain primers + MHC mix using Phusion Flash High-Fidelity PCR Master Mix. The result of each PCR reaction was a single amplification product that was purified using a QIAquick PCR purification kit and sequenced in both directions (by GATC Biotech) using M13 forward and M13 reverse primers to obtain three independent sets of sequence information for each immunoglobulin chain.

[0120] 4. VK and VH NT4X-167 DNA sequences The consensus DNA sequence of the NT4X-167 VK PCR product was named NT4X-167 VK, and the consensus DNA sequence of the NT4X-167 VH PCR product was named NT4X-167 VH, and are shown in SEQ ID NOs 9 to 12, respectively. Germline analysis of the NT4X-167 sequence indicates that the kappa light chain is mouse MKV4 and the heavy chain is mouse MHV7.

[0121] 5. Construction of Chimeric NT4X-167 Expression Vector Construction of chimeric expression vectors involves cloning the amplified variable regions into IgG / κ vectors (pHuK and pHuG1) using ligase-independent cloning (LIC). Vectors (pCMV modified) were digested with BfuA1 (BspM1) and then compatible overhangs were generated by T4 DNA polymerase 3'-5' exonuclease activity (+dATP). Antibody sequences were generated by first amplifying the variable regions by PCR from NT4X-167 cDNA with primers containing the 3' end (forward primer) of the leader sequence (most of the sequence is present in the vector) or the beginning (reverse primer) of the constant region (IgG1 or κ) followed by the beginning of the variable region (in each direction). Complementary overhangs were generated in the PCR products by T4 DNA polymerase + dTTP treatment. Vectors and inserts were incubated at RT, transformed into chemically competent TOP10 bacteria and plated on kanamycin plates. Several clones were isolated and colonies were screened by PCR using primers HCMVi and HuG1 LIC Rev for VH or HuK LIC Rev for VK. Clones generating PCR products of the correct size were selected, miniprepped using a QIAGEN kit, and sequenced using the same primers.

[0122] 6. Generation of Chimeric Antibodies Expi293 suspension cells growing in Expi293 transfection medium and antibiotics were co-transfected with cNT4X-167 VH.pHuG1 and cNT4X-167 VK.pHuK (1 μg DNA each) using ExpiFectamine 293 reagent. Cells were grown in 1 mL growth medium for 5 days. Up to 81 μg / mL of chimeric NT4X-167 antibody was measured in the conditioned medium by ELISA.

[0123] 7. Amyloid peptides Amyloid peptides Aβ1-42, AβpE3-42, and 4-42 were purchased from Peptide Speciality Laboratories (PSL) or California peptides.

[0124] 8. Transgenic Mice The transgenic homozygous mouse lines Tg4-42hom (hereafter referred to as Tg4-42) and 5XFAD used in this study have been described previously [1, 2].

[0125] 10. Passive Immunization The potential therapeutic effects of reverse cloned (rc) humanized NT4X (rcNT4X_SA and rcNT4X_S7A) antibodies were studied in Tg4-42 and 5XFAD mice using passive immunization. Passive immunization was performed by intraperitoneal injection and compared with a control group using an antibody of the same immunoglobulin class as both rcNT4X antibodies (IgG1, MRCT control antibody).

[0126] Male and female Tg4-42 mice were immunized by injection of 10 mg / kg body weight of antibody diluted in sterile PBS (pH 7.4). Mice received weekly injections starting at 3 months of age. Each mouse received a total of 12 injections. Behavioral testing was performed between the 10th and 11th injections. Animals were sacrificed after the last injection. A control group received an intraperitoneal injection of IgG1 MRCT control antibody (10 mg / kg body weight). Animals were sacrificed after the last injection at 6 months of age.

[0127] Six-week-old female 5XFAD mice received weekly injections of rcNT4X_SA and rcNT4X_S7A (10 mg / kg body weight diluted in sterile PBS) or MRCT control (IgG1; 10 mg / kg body weight diluted in sterile PBS). Each mouse received a total of 12 intraperitoneal injections. Animals were sacrificed after the last injection at 18 weeks of age.

[0128] The control group was treated similarly to the treatment group.

[0129] 11. Spatial reference memory using the Morris water maze Spatial reference memory was assessed in Tg4-42 mice using the Morris water maze [3] as previously described [2].

[0130] 12. Quantifying Neuron Number Using Unbiased Stereological Analysis Stereological analysis was performed as previously described [2, 4]. In cresyl violet-stained sections, the hippocampal cell layer CA1 (bregma -1.22 to -3.52 mm) was outlined and analyzed by a stereological workstation (Olympus BX51 with a motorized specimen stage for automated sampling), StereoInvestigator 7 (MicroBrightField, Williston, USA), and a 100x oil immersion lens (NA = 1.35).

[0131] 13. Immunohistochemistry and Histology Mouse tissue samples were processed as previously described [5]. For plaque load staining, the following antibodies were used: antibody 1-57 (pyroglutamic Aβ3-x, 1:5000, mouse monoclonal [5]), antibody 80C2 (anti-Aβ1-X, Synaptic Systems Göttingen, 1:500, monoclonal mouse), polyclonal antibody 24311 (anti-pan Aβ, 1:500, rabbit [2]), and polyclonal antibody 029 (anti-Aβ4-x; 1:500; guinea pig). Biotinylated secondary anti-rabbit and secondary anti-mouse antibodies (1:200) were purchased from DAKO. Staining was visualized using the ABC method with the Vectastain kit (Vector Laboratories) and diaminobenzidine as chromogen. Counterstaining was performed with hematoxylin. For DAPI staining, sections were deparaffinized, washed with PBS, and then incubated in 4',6-diamidin-2'-phenylindole (DAPI, 1 μg / ml) for 1 min. For Thioflavin S fluorescent staining, tissue sections were deparaffinized, rehydrated, washed twice with deionized water, treated with 1% (w / v) Thioflavin S in aqueous solution, and counterstained in 1% (w / v) aqueous solution of 4',6-diamidin-2-phenylindole. Embedding was performed with aqueous fluorescent mounting medium (DAKO).

[0132] 14. Quantification of Aβ levels Plaque burden was quantified in immunized 5XFAD mice. For each animal, three paraffin-embedded sections were separated from each other by at least 40 μm. Relative plaque burden was assessed in the cortex using an Olympus BX-51 microscope equipped with an Olympus DP-50 camera and ImageJ software (NIH, USA). Representative pictures at 20x magnification were systematically acquired. Images were binarized to 8-bit black and white images using ImageJ, and a fixed intensity threshold was applied to define DAB staining. The percentage of area covered by DAB staining was also calculated as 1 mm 2 Measurements were also made of the number of particles per unit area and the average size of the particles.

[0133] 15.Statistical analysis Differences between groups were tested by one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons, ANOVA followed by Dunnett's multiple comparisons, or Student's t test, as indicated. All data are presented as mean ± standard error of the mean (SEM), as indicated. All statistics were calculated using GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego, CA, USA).

[0134] 16. Research approval Animal experiments were approved by the local animal protection authorities (Niedersachisches Landesamt fur Verbraucherschutz und Lebensmittelsicherheit) under approval number 17 / 2447. Experiments were carried out in accordance with the approved protocol.

[0135] 17.ELISA Each well of a 94-well MaxiSorp plate (Nunc) was coated with a 50 μL aliquot of amyloid peptide 1-42, pE3-42, or 4-42 at 200 ng / mL in PBS and incubated overnight at 4° C. The wells were washed three times with PBS-T (0.1% Tween 20) and blocked with 150 μL per well of 5% milk in PBS / 0.05% Tween 20. The wells were then incubated for 1 h at RT with shaking and washed three times with PBS-T (0.1% Tween 20). 50 μl of primary antibody serially diluted in 1% milk PBS / 0.05% Tween 20 was added to the wells of the assay plate using a 3-fold dilution series starting at approximately 100 μg / mL. The incubation and washing steps were then repeated. Anti-human kappa chain HRP (Sigma A7164-1mL) was diluted 4,000-fold in PBS / 1% milk / 0.05% Tween 20 and 50 μL was added to each well. The incubation and washing steps were repeated, then 75 μL of K-Blue substrate (Neogen) was added per well and incubated for 5-10 min at RT. The reaction was stopped by adding 50 μl of RED STOP solution (Neogen) to each well and the optical density was read at 650 nm.

[0136] result Generation of a chimeric version of the NT4X-167 antibody Binding of the amyloid peptides Aβ1-42, AβpE3-42, and 4-42 to the chimeric NT4X-167 antibody was measured by ELISA and compared to the original murine NT4X-167 antibody. The chimeric NT4X-167 antibody showed comparable EC values ​​in the ELISA assay to that of the murine NT4X-167 antibody (Figure 1). 50 At this value, it bound to the AβpE3-42 peptide, but not to the Aβ1-42 or 4-42 peptides (FIG. 2).

[0137] To further characterize the binding of the murine and chimeric NT4X-167 antibodies to amyloid peptides, SPR analysis was performed using a Biacore T200 (GE Healthcare). The chimeric NT4X-167 antibody showed an apparent K D It bound to AβpE3-42 and 4-42 peptides, but not to Aβ1-42, at 100 ng / mL. The NT4X-167 sequence was used to design a humanized version of the anti-NT4X-167 antibody.

[0138] Design of NT4X-167 humanized antibody variants Human VH and VK cDNA database International Immunogenetics Database2009 9 and Kabat Database Release 5 of Sequences of Proteins of Immunological Interest (Last updated November 17, 1999) 8 The protein sequences of were used in a Kabat alignment to compile a database of human immunoglobulin sequences. Our database contains 10,406 VH sequences and 2,894 VK sequences.

[0139] Molecular model of NT4X-167 A homology model of the mouse NT4X-167 antibody variable region was calculated. As determined by Blast analysis of the Accelrys antibody pdb structural database, the atomic coordinates of 2DQU_L.pdb and 1WEJ_H.pdb were the sequence templates with the highest scores for VL and VH, respectively, and the atomic coordinates of 1YNL_LH.pdb were the overall (interface) sequence template with the highest scores. These templates were used to generate 20 initial models; the top-scoring models were refined by modeling each CDR loop with the five best loop templates. The 20 final models were used to determine the consensus of residues within 4 Å of the CDR loops.

[0140] Human Framework Selection The human VH and VK databases were interrogated with the NT4X-167 VH and VK protein sequences using various selection criteria. FW residues that were within 4 Å of the CDR residues (Kabat definition) in the structure of the murine NT4X-167 antibody were identified and termed "4 Å adjacent residues."

[0141] Humanized sequences and incomplete sequences were removed from the analysis. Sequence AF062228 was selected as a human heavy chain donor candidate. This sequence has a high score in sequence identity and similarity and does not have somatic mutations from germline. AF062228 has eight 4 Å adjacent residue changes (Tables 1 and 2).

[0142] Similarly, sequence AY942002 was selected as a human kappa light chain donor candidate (Table 4). AY942004, AF054661, and AF113887 were rejected due to the number of somatic mutations in the framework. AJ698329 was rejected due to a G→Q change in framework 4. All other sequences examined were highly similar, but AY942002 showed better framework identity and similarity with NT4X_VK. AY942002 has no somatic mutations from the germline and has three possible 4 Å adjacent residue changes (Table 4).

[0143] NT4X-167 RHA and RHB Design With the appropriate human frameworks identified, synthetic protein and DNA sequences can be designed. The initial design of the humanized version of NT4X-167 is to graft CDRs 1, 2, and 3 from NT4X-167 VH to the acceptor FW of AF062228, thus creating variant NT4X-167 RHA. Eight 4 Å adjacent residues are then backmutated to their mouse equivalents, thereby creating variant NT4X-167 RHB, one at a time in the following variants: the sequences were assembled in silico and named NT4X-167 RHA to NT4X-167 RHJ. Tables 1-3 compare the VH protein sequences of mouse NT4X-167 and the humanized version of NT4X-167. All humanized variants were cloned into pMoG1 and pMoK vectors to allow for the purification of antibodies from these constructs for in vivo studies in multiple mouse models (5XFAD and Tg4-42). The final lead humanized candidates will be cloned into pHuG4 and pHuK vectors.

[0144] Design of NT4X-167 RKA and NT4X-167 RKB The framework from AY942002 was used to design the DNA and protein for the humanized construct. Shown are CDRs 1, 2, and 3 from NT4X-167 VK grafted into the acceptor FW of AY942002 to generate the initial version of humanized NT4X-167 RKA. There are three mismatched 4 Å adjacent residues in NT4X-167 RKA, which were backmutated to the equivalent mouse residues in variant NT4X-167 RKB. These residues were backmutated one at a time in the following variants: sequences were assembled in silico and named NT4X-167 RKA to NT4X-167 RKE (Table 4).

[0145] Generation of NT4X-167 humanized antibody The genes for NT4X-167 HA, HB, KA, and KB were synthesized by GenScript. The native human framework sequences AF062228 and AY942002, heavy and light chains, respectively, and the native mouse CDR sequences were assembled in silico and named NT4X-167 RHA to NT4X-167 RHJ and NT4X-167 RKA to NT4X-167 RKE. To use codons preferentially utilized by human cells, sequences for RHA / RHB and RKA / RKB were optimized by silent mutagenesis and synthesized using software algorithms dedicated to GenScript. The RKA / RKB and RHA / RHB constructs were PCR amplified with primers specific for the expression vector plus insert (previously described for the chimeric version) and inserted into pMoK and pMoG1, respectively, in a ligase-independent cloning reaction and used to transform TOP10 bacteria. Version HA was subsequently modified by PCR mutagenesis to obtain other humanized variants, which are annotated in Table 4.

[0146] Clones were sequenced and plasmid DNA was prepared using QIAGEN Plasmid Miniprep or Qiagen Plasmid Maxiprep kits. The sequences of the expression constructs (HA, HB, KA, and KB) are shown in SEQ ID NOs: 13-20. Expression plasmid preparations encoding (humanized or chimeric) VH and VK were used to transfect Expi293 cells and cultured in serum-free medium for 5-7 days, after which conditioned medium containing secreted antibody was harvested.

[0147] Antibody expression The concentration of IgG1κ antibody in Expi293 cell conditioned medium was measured by ELISA. Most of the antibody was produced at good expression levels.

[0148] Antigen binding by early versions (round 1 and round 2) of the humanized NT4X-167 antibody The data shown in Figure 3 show the binding of the RHA / RHB heavy chain combined with the RKA / RKB light chain version of the humanized NT4X-167 antibody to the amyloid peptides Aβ1-42 and AβpE3-42. No difference in binding could be observed between the versions containing the RKA or RKB versions of the κ light chain, implying that the back mutations introduced in the KB are not essential for binding. Version RHA showed no evidence of binding to the amyloid peptide, while the humanized version containing the RHB version bound to AβpE3-42. Taking this data into account, and subsequently employing only the KA light chain, a further version of the humanized heavy chain was synthesized using the Stratagene mutagenesis kit QuikChange Lightning Site-Directed Mutagenesis kit (Stratagene) to generate version NT4X RHC~RHJ (Table 1). The results of the binding ELISA using the humanized version RHC~RHJ to the AβpE3-42 peptide are shown in Figure 5. Humanized versions RHB / RKA and RHB / RKB bound to the AβpE3-42 peptide, whereas the other humanized versions showed no evidence of binding, and a third round of humanized variants was synthesized.

[0149] Antigen binding by round 3 and 4 of humanized NT4X-167 antibody The third round of humanized NT4X-167 heavy chain variants RHK-RHR were generated (Table 2). The RHK-RHR variants were obtained using a Stratagene mutagenesis kit (QuikChange Lightning Site-Directed Mutagenesis kit).

[0150] The RHC-RHR heavy chain was combined with the RKA version of the light chain and a binding ELISA was performed using the AβpE3-42 peptide (Figure 6). Humanized versions RHB, RHM, RHN, RHO, and RHR combined with RKA bound the AβpE3-42 peptide, with RHB / RKA, RHM / RKA, RHN / RKA, RHO / RKA, and RHR / RKA being the optimal binders. Four of the eight key heavy chain CDR framework residues in RHP (arginine backmutated to valine by SDM), RHQ (valine backmutated to phenylalanine), RHK (phenylalanine backmutated to glycine), and RHL (leucine backmutated to isoleucine) showed reduced binding, suggesting that these four residues should be maintained as murine residues for full binding. Further humanized variants were generated in which all four murine residues, represented by RHP, RHQ, RHK, and RHL, were incorporated, while the four key framework residues, represented by RHM, RHN, RHO, and RHR, were maintained as human framework residues, generating versions RHS, RHT, RHU, RHV, RHW, RHX, and RHY (Table 2). Peptide binding ELISA using AβpE3-42 showed that variant RHS~RHY combined with RKA had a similar PSL AβpE3-42 binding profile as RHB / RKA (Figure 8). The RHS / RKA variant is preferred due to the number of "human" key CDR framework residues it contains (lower immunogenicity).

[0151] Antigen binding by humanized NT4X-167 antibodies SA, S6A, S7A, and S8A Further variants of RHS / KA(SA) were generated to achieve >85% identity with human germline. IMGT Domain Gap analysis was performed on the SA version to identify residues that could be mutated to increase the % identity with human germline. The RKA light chain had 89.6% identity with human germline IGKV1-39*01 and IGKJ4*01 sequences. However, the RHS heavy chain had 79.4% identity with human germline IGHV4-4*08 sequence. Germline analysis of the RHS sequence identified six residues that could be backmutated to human germline and combined with RKA to generate versions RHS6, RHS7, and RHS8. Peptide binding ELISA using AβpE3-42 showed that variant RHS7RKA(S7A) was the best humanization candidate, with 84.5% identity with human germline.

[0152] Antigen binding by additional variants generated based on the crystal structure of the NT4X-167 antibody To increase the % identity with human germline, further variants of RHS7 were generated based on the crystal structure of pE3-14 peptide bound to mouse NT4X FAB. The crystal structure highlighted F67, Y68, and I39 as possible amino acids that could be changed without affecting peptide binding. Therefore, additional variants F67Y, Y68N, I39W, and a combination of F67Y and Y68N were generated (Table 2) and investigated for binding to Aβ1-42, AβpE3-42, and 4-42. RHS71 with the F67Y mutation retained equivalent binding properties to the parent S7A heavy chain variant and was therefore selected as a potential heavy chain humanized variant in combination with the RKA light chain. Since the affinities of these antibodies were in the nM range, we wanted to investigate whether it would be possible to predict which amino acids could be mutated based on the crystal structure and Schrodinger modeling prediction software to increase the affinity of the RHS71 / RKA antibody. Five additional variants of the heavy chain were generated containing the following mutations: S53M, S53H, R100H, L103R, and L103H (Table 4b). In addition, five variants of the light chain were also generated: RKF (N92W), RKG (N92Y), RKH (N92H), RKI (L94R), and RKJ (L94H). The sequences of the light chain variants are shown in Table 4. The binding of these additional humanized variants to Aβ1-42, AβpE3-42, and 4-42 was investigated by ELISA (Figure 9) and Biacore. Of all the variants tested, RHS71 (containing the F67Y mutation in the heavy chain) combined with RKH (containing the N92H mutation) showed a 2-fold improvement in Biacore.

[0153] Thermal stability of humanized candidate antibodies BA, SA, TA, UA, VA, WA, and YA at high temperatures The goal of this experiment is to measure the EC of each candidate by subjecting them to elevated temperatures ranging from 30°C to 85°C for 10 minutes, cooling them to 4°C, and 80The aim of this study was to test the thermal stability of the humanized antibodies when used in an ELISA assay at concentrations of 1000 mM NaCl, 100 mM MgCl, 1 ... and 100 mM MgCl. All humanized versions were stable up to 75° C. and appeared to retain their ability to bind the AβpE3-42 peptide, with reduced binding to the peptide at 75° C.

[0154] Determination of the Tm (melting temperature) of humanized candidate antibodies NT4X-167-SA and NT4X-167-S7A To determine the melting temperature of the lead candidate antibodies NT4X-167-SA and NT4X-167-S7A, the antibodies were tested in a thermal shift assay. Samples were incubated with a fluorescent dye (Sypro Orange) in a qPCR thermal cycler for 71 cycles with an increment of 1°C per cycle. The Tm for the humanized antibody was calculated to be 66-67°C.

[0155] Aggregation of humanized candidate antibodies NT4X-167-SA and NT4X-167-S7A The samples were injected into a size-exclusion column at 0.4 mL / min in an HPLC system and analyzed by multi-angle light scattering to determine absolute molar mass and check for aggregation (see FIG. 10). The profiles showed no signs of aggregation and had average molecular weights of about 133.98 kDa for NT4X-167_SA and 129.92 kDa for NT4X-167_S7A, which is the expected range for IgG monomers in this analytical setup. The antibodies are monodisperse (Mw / Mn<1.05). The mass recovery was 100% (calculated mass over injected mass), indicating good protein recovery and that the samples do not appear to contain insoluble aggregates that would not adsorb to the column and would be retained by the guard column. Overall, the data suggest that there are no aggregation issues for the humanized antibodies NT4X-167_SA and NT4X-167_S7A.

[0156] Nonspecific Protein-Protein Interactions (CICs) Cross-interaction chromatography using bulk purified human polyclonal IgG is a technique for monitoring nonspecific protein-protein interactions and can be used to distinguish between soluble and insoluble antibodies (Section 8.19). An elevated retention index (k') indicates a tendency for self-interaction and low solubility. The humanized NT4X-167 RHS / RKA antibody, RHB / RKA antibody, and RHS7 / RKA antibody (cloned as MoG1K) show retention indices below 0.2, indicating a low tendency for nonspecific interactions and good solubility (Figure 11).

[0157] Soluble humanized NT4X-167 candidate antibodies RHS / RKA and RHS7 / RKA Humanized NT4X-167 antibodies RHS / RKA (SA) and RHS7 / RKA (S7A) were concentrated using solvent absorption concentrators (MWCO 7500 kDa) and the concentration was measured at set intervals. The antibodies were concentrated to >50 mg / mL without obvious precipitation.

[0158] Freeze / thaw stress analysis of humanized NT4X-167 candidate antibodies RHS / RKA and RHS7 / RKA Samples of purified candidate antibodies were subjected to 10 cycles of 15 min at -80°C followed by 15 min thawing at room temperature. Samples were then analyzed by SEC-MALS to check for aggregation (Figures 12 and 13). The data suggest that freeze / thaw does not cause aggregation in the humanized NT4X-167 antibody.

[0159] Heat-induced stress analysis of humanized NT4X-167 candidate antibodies RHS / RKA and RHS7 / RKA Samples of purified candidate antibodies were exposed to (a) 4°C, (b) 25°C, (c) 37°C, and (d) 50°C for 30 days. Samples were then analyzed by SEC-MALS to check for aggregation (Figure 14). Overall, the data suggests that there is no aggregation problem with the humanized NT4X-167 antibody.

[0160] Serum stability assessment of humanized NT4X-167 candidate antibodies RHS / RKA and RHS7 / RKA Purified samples of humanized NT4X-167 antibodies RHS / RKA and RHS7 / RKA were incubated in mouse, human, and cynomolgus sera. The binding ability of the antibodies after incubation was measured by binding ELISA with AβpE3-42 and 4-42 peptides. The binding of NT4X-167 humanized antibodies incubated in three different sera was compared with that of antibodies not incubated and antibodies incubated in PBS. The ELISA assay showed that the binding of antibodies incubated in serum with AβpE3-42 and 4-42 peptides was very similar to that of antibodies incubated in PBS and antibodies not incubated. Thus, NT4X-167 humanized antibodies RHS / RKA and RHS7 / RKA retained their binding ability after being incubated in mouse, human, and cynomolgus sera for 30 days.

[0161] Humanized NT4X-167 was shown to be capable of binding amyloid peptide 4-42, AβpE3-42, but not Aβ1-42. The humanized antibody also showed protection against neuronal cell death in rat and human neurons. The antibody was modified and expressed as a fully humanized antibody without significant loss of binding potency. Experiments with chimeric antibodies consisting of mouse variable regions on human constant regions showed potency similar to or improved to that of the mouse antibody in binding ELISA or kinetic studies using Biacore (Figures 1 and 2).

[0162] Initial experiments showed that fully humanized NT4X-167, i.e., NT4X-167 without framework mutations to introduce mouse 4 Å proximal residues, did not bind AβpE3-42 peptide as well as the chimeric positive control antibody, whereas the version containing the full set of mutations bound comparably to the chimeric positive control. This reduced binding was isolated to the fully humanized heavy chain. However, we unexpectedly found that the introduction of specific back mutations allowed us to generate two lead candidate antibodies, NT4X RHS / RKA(SA) and NT4X RHS7 / RKA(S7A). These lead candidates were cloned not only into the initial MoG1 vector, but also into the HuG1K and HuG4K vectors. Both candidates showed excellent binding, expression, thermostability, affinity, and functional activity.

[0163] In vitro cell assays Neuroprotection by humanized antibodies NT4X_SA and NT4X_S7A in rat and human primary cortical cultures The humanized antibodies NT4X_SA and NT4X_S7A were found to retain the property of the original murine NT4X antibody to protect against cell death in rat neurons induced by N-truncated amyloid peptides (4-42 and pyroGul3-42; Figures 17 and 18), but not against the amyloid full-length peptide Aβ1-42 (Figure 19). All three antibodies are highly equipotent against the 4-42 peptide, but murine NT4X is slightly more potent against pyro3-42 than either SA or S7A, and S7A is slightly more potent than SA.

[0164] The humanized antibodies NT4X_SA and NT4X_S7A were found to retain the properties of the original mouse NT4X antibody, which protects against cell death induced by N-truncated amyloid peptides (4-42 and pyroGul3-42; Figures 20 and 21) in human neurons, but not against the amyloid full-length peptide Aβ1-42 (Figure 22). All three humanized antibodies are more potent than the original mouse NT4X mouse antibody in protecting against cell death by the 4-42 peptide, but the N92H humanized antibody is more potent than pyro3-42. None of the NT4X antibodies or the humanized versions protect human neurons against death induced by the Aβ1-42 amyloid peptide. The results are generally consistent with rat neurons, with some increase in potency against pyro3-42 in human neurons.

[0165] In vivo studies in transgenic mouse models Alzheimer's treatment with rcNT4X_SA and rcNT4X_S7A in mouse models 5XFAD and Tg4-42 Tg4-42 mice expressing Aβ4-42 were immunized for 12 weeks, starting at 12 weeks of age. We demonstrated that rcNT4X_SA and rcNT4X_S7A rescued CA1 neuron loss in the hippocampus of Tg4-42, with rcNT4X_S7A being more effective in treatment. rcNT4X_S7A was further tested in the Morris water maze test for spatial reference memory performance. The spatial reference memory deficit of Tg4-42 at 6 months of age was completely rescued.

[0166] 5XFAD mice were immunized for 12 weeks starting at 6 weeks of age. The effect on plaque load was analyzed in the cortex. rcNT4X SA reduced plaques stained by thioflavin and N-terminal specific antibodies against pyroglutamic Aβ3-X and Aβ4-X. No effect was seen with antibodies against pan-Aβ and Aβ1-X. In contrast to mice immunized with rcNT4X_S7A, which showed significant plaque reduction by all staining assays (plaques stained by thioflavin or antibodies recognizing Aβ1-X, pyroglutamic Aβ3-X, Aβ4-X, and pan-Aβ were significantly reduced).

[0167] rcNT4X_SA and rcNT4X_S7A rescue neuronal loss and memory impairment in Tg4-42 mice CA1 neuronal loss in the hippocampus of Tg4-42 mice is significant at 4 months of age [6]. Therefore, we started 12 weeks of passive immunization at 3 months. Tg4-42 mice immunized with rcNT4X_SA and rcNT4X_S7A showed significantly more neurons compared to the IgG1 control group. The significance level was higher in the rcNT4X_S7A group (Figure 23). Immunization of Tg4-42 with rcNT4X_S7A was significantly more potent compared to NT4X (Figure 24). There was no difference in neuronal number between the initial NT4X and rcNT4X_SA. Data from immunization with rcNT4X_SA and rcNT4X_S7A are plotted against the initial mouse NT4X antibody immunization. Control groups injected with IgG1, IgG2b, and PBS were not significantly different (Figure 25). Data from IgG2ba and PBS taken from Antonios et al. [6]. Passive immunization with rcNT4X_S7A completely rescued spatial reference memory deficits in Tg4-42 mice tested by Morris water maze (FIG. 26).

[0168] rcNT4X_SA and rcNT4X_S7A reduce plaque burden in 5XFAD mice 5XFAD mice were treated between 6 and 18 weeks of age. Passive immunization with both rcNT4X antibodies reduced plaque burden for different Aβ species compared to isotype control IgG1 antibody. rcNT4X significantly reduced plaques staining for pyroglutamic Aβ3-x, Aβ4-x, and thioflavin. No effect was detected in Aβ1-x and pan-Aβ positive plaques. The plaque reduction effect of rcNT4X_S7A was significantly altered, as plaques positive for pyroglutamic Aβ3-x, Aβ4-x, and thioflavin as well as plaques positive for Aβ1-x and pan-Aβ were reduced (Figure 27).

[0169] Tg4-42 mice develop severe hippocampal neuronal loss and spatial reference memory deficits [2, 6]. The Tg4-42 model represents the first mouse model that exclusively expresses N-truncated Aβ4-42. At 6 months of age, this model features significant spatial reference memory loss assessed by the Morris water maze test, and a large amount of degenerated CA1 neurons in the hippocampus of Tg4-42 mice, which could be rescued by passive immunization with the antibody NT4X [6]. In this study, we used a novel humanized version of the NT4X antibody cloned in a mouse IgG1 backbone. Passive immunization with rcNT4X_S7A for 12 weeks, starting at 3 months of age, also rescued spatial reference memory deficits in Tg4-42 mice. Furthermore, the number of CA1 neurons in the hippocampus was significantly rescued compared to the group of Tg4-42 animals treated with IgG1. Interestingly, when comparing the treatment effects between NT4X, rcNT4X_SA, and rcNT4X_S7A, Tg4-42 mice exposed to rcNT4X_S7A showed significantly higher efficacy compared to NT4X. Therefore, we assume that rcNT4X_S7A has the highest efficacy among the different NT4X versions.

[0170] array TIFF0007610515000001.tif7161SEQ ID NO:1 RHA sequence

[0171] TIFF0007610515000002.tif7169SEQ ID NO:2: RHA sequences having 27F, 29L, 63R, and 70V, and 52BX1, 53X2, 54X3, 55X4, 56X5, and 52CX6, where X1 is D or N, X2 is A, N, or P, X3 is A or S, X4 is F or L, X5 is I or K, and X6 is F or Y.

[0172] TIFF0007610515000003.tif7161SEQ ID NO:3 RHS sequence

[0173] TIFF0007610515000004.tif7161SEQ ID NO:4 RHS7 sequence

[0174] TIFF0007610515000005.tif7161SEQ ID NO:5 RHS71 sequence

[0175] TIFF0007610515000006.tif7161SEQ ID NO:6 92 RKA sequence having X7 [X7 is N, H, Y, or W]

[0176] TIFF0007610515000007.tif7161SEQ ID NO:7 RKA sequence

[0177] TIFF0007610515000008.tif7161SEQ ID NO:8 RKH sequence

[0178] TIFF0007610515000009.tif25542SEQ ID NO: 9 and 10: NT4X-167 kappa light chain variable region protein and DNA sequences

[0179] TIFF0007610515000010.tif25542SEQ ID NO: 11 and 12: Protein and DNA sequences of the NT4X-167 heavy chain variable region

[0180] TIFF0007610515000011.tif25592SEQ ID NO: 13 and 14: NT4X-167 HA protein and DNA sequences

[0181] TIFF0007610515000012.tif25592SEQ ID NO: 15 and 16: NT4X-167 HB protein and DNA sequences

[0182] TIFF0007610515000013.tif25586SEQ ID NO: 17 and 18: NT4X-167 KA protein and DNA sequences

[0183] TIFF0007610515000014.tif25586SEQ ID NO: 19 and 20: NT4X-167 KB protein and DNA sequences

[0184] table Table 1. NT4X-167 heavy chain humanization strategy TIFF0007610515000015.tif25579CDR is in the box. The underlined residues indicate the back-translation into the murine residues. Bolded residues indicate key FW residues that are retained as human to increase % human identity.

[0185] Table 1 shows NT4X_VH(SEQ ID NO:12), AF062228(SEQ ID NO:21), NT4X RHA(SEQ ID NO:22), NT4X RHB(SEQ ID NO:23), NT4X RHC(SEQ ID NO:24), NT4X RHD(SEQ ID NO:25), NT4X RHE(SEQ ID NO:26), NT4X RHF(SEQ ID NO:27), NT4X RHG(SEQ ID NO:28), NT4X RHH(SEQ ID NO:29), NT4X RHI(SEQ ID NO:30), NT4X RHJ(SEQ ID NO:31), NT4X RHK(SEQ ID NO:32), NT4X RHL(SEQ ID NO:33), NT4X RHM(SEQ ID NO:34), NT4X RHN(SEQ ID The sequences of NT4X RHO (SEQ ID NO:35), NT4X RHP (SEQ ID NO:37), NT4X RHQ (SEQ ID NO:38), NT4X RHR (SEQ ID NO:39), NT4X RHS (SEQ ID NO:40), and NT4X RHT (SEQ ID NO:41) are aligned.

[0186] Table 2: NT4X-167 heavy chain humanization strategy - Further heavy chain versions increasing % human identity and affinity TIFF0007610515000016.tif25578CDR is in the box. The underlined residues indicate the back-translation into the murine residues. Bold residues indicate key FW residues that are conserved in humans. The dotted underlined residues indicate the KABAT CDRs that were mutated to the human FW residues selected (considering the IMGT CDRs). Wavy underlined residues indicate further mutations to increase % human identity. Double underlined residues indicate affinity maturation mutations.

[0187] Table 2 shows NT4X_VH(SEQ ID NO:12), AF062228(SEQ ID NO:21), NT4X RHA(SEQ ID NO:22), NT4X RHB(SEQ ID NO:23), NT4X RHS(SEQ ID NO:40), NT4X RHS2(SEQ ID NO:42), NT4X RHS3(SEQ ID NO:43), NT4X RHS4(SEQ ID NO:44), NT4X RHS5(SEQ ID NO:45), NT4X * RHS6 (SEQ ID NO:46), NT4X * RHS7 (SEQ ID NO: 47), NT4X * RHS8 (SEQ ID NO: 48), NT4X * RHS71(F67Y)(SEQ ID NO:49), NT4X * RHS72(Y68N)(SEQ ID NO:50), NT4X * RHS73(F67Y / Y68N)(SEQ ID NO:51), NT4X * RHS74(I39W)(SEQ ID NO:52), NT4X * RHS81(S53M)(SEQ ID NO:53), NT4X * RHS82(S53H)(SEQ ID NO:54), NT4X * RHS83(R100H)(SEQ ID NO:55), NT4X * RHS84(L103R)(SEQ ID NO:56), and NT4X * This is an alignment of the sequence of RHS85(L103H) (SEQ ID NO:57).

[0188] Table 3: NT4X-167 Heavy Chain Humanized RHS Version % Human Identity TIFF0007610515000017.tif113128

[0189] Table 4. NT4X-167 κ light chain humanization strategy TIFF0007610515000018.tif25478CDR is in the box. The underlined residues indicate the back-translation into the murine residues. Bolded residues indicate affinity maturation mutations.

[0190] Table 4 shows the sequence alignment of NT4X_VK (SEQ ID NO:10), AY942002 (SEQ ID NO:58), NT4X RKA (SEQ ID NO:59), NT4X RKB (SEQ ID NO:60), NT4X RKC (SEQ ID NO:61), NT4X RKD (SEQ ID NO:62), NT4X RKE (SEQ ID NO:63), NT4X RKF(N92W) (SEQ ID NO:64), NT4X RKG(N92Y) (SEQ ID NO:65), NT4X RKH(N92H) (SEQ ID NO:66), NT4X RKI(L94R) (SEQ ID NO:67), and NT4X RKJ(L94H) (SEQ ID NO:68).

[0191] (Table 5) TIFF0007610515000019.tif57147

[0192] Table 6: Heavy chain amino acid sequences (see Figure 7) TIFF0007610515000020.tif247119

[0193] Table 6 shows the sequences of NT4X_VH (SEQ ID NO:12), AF062228 (SEQ ID NO:21), NT4X RHA (SEQ ID NO:22), NT4X RHB (SEQ ID NO:23), NT4X RHK (SEQ ID NO:32), NT4X RHL (SEQ ID NO:33), NT4X RHM (SEQ ID NO:34), NT4X RHN (SEQ ID NO:35), NT4X RHO (SEQ ID NO:36), NT4X RHP (SEQ ID NO:37), NT4X RHQ (SEQ ID NO:38), and NT4X RHR (SEQ ID NO:39).

[0194] reference TIFF0007610515000021.tif68166

[0195] Additional Statement of the Invention The following numbered statements of the invention are part of this specification.

[0196] 1. An antibody comprising a heavy chain variable domain and a light chain variable domain, (a) the heavy chain variable domain (VH domain) comprises SEQ ID NO:2 with no more than four additional alterations, e.g., substitutions, in the framework regions; (b) An antibody in which the light chain variable domain (VK domain) comprises SEQ ID NO:6 with no more than four additional alterations, e.g., substitutions, in the framework regions.

[0197] 2. An antibody according to statement 1, which binds to amyloid peptides AβpE3-42 and Aβ4-42, but does not bind to amyloid peptide Aβ1-42.

[0198] 3. An antibody according to any of the preceding statements that binds to the amyloid peptide AβpE3-42 with a binding affinity of at least 85% of the binding affinity of the murine NT4X-167 antibody to the amyloid peptide AβpE3-42 as measured by ELISA.

[0199] 4. The antibody according to any of the preceding statements, wherein the VH domain comprises SEQ ID NO:2.

[0200] 5. The antibody according to any of the preceding statements, wherein the VL domain comprises SEQ ID NO:6.

[0201] 6. The antibody according to any of the preceding statements, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X1 (Kabat 52B position) is D.

[0202] 7. The antibody according to any of statements 1 to 5, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X1 (Kabat 52B position) is N.

[0203] 8. The antibody according to any of the preceding statements, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X2 (Kabat position 53) is A.

[0204] 9. The antibody according to any of statements 1 to 7, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X2 (Kabat position 53) is P.

[0205] 10. The antibody according to any of the preceding statements, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X3 (Kabat position 54) is A.

[0206] 11. The antibody according to any of statements 1 to 9, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X3 (Kabat position 54) is S.

[0207] 12. The antibody according to any of the preceding statements, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X4 (Kabat position 55) is F.

[0208] 13. The antibody according to any of statements 1 to 11, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X4 (Kabat position 55) is L.

[0209] 14. The antibody according to any of the preceding statements, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X5 (Kabat position 56) is I.

[0210] 15. The antibody according to any of statements 1 to 13, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X5 (Kabat position 56) is K.

[0211] 16. The antibody according to any of the preceding statements, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X6 (Kabat 52C position) is F.

[0212] 17. The antibody according to any of statements 1 to 15, wherein the heavy chain variable domain comprises SEQ ID NO:2, in which X6 (Kabat 52C position) is Y.

[0213] 18. An antibody according to any of statements 1-5, wherein the heavy chain variable domain comprises SEQ ID NO:3 with no more than four additional substitutions in the framework regions.

[0214] 19. The antibody according to statement 18, wherein the heavy chain variable domain comprises SEQ ID NO:3.

[0215] 20. An antibody according to any of statements 1-5, wherein the heavy chain variable domain comprises SEQ ID NO:4 with no more than four additional alterations, e.g., substitutions, in the framework regions.

[0216] 21. The antibody according to statement 20, wherein the heavy chain variable domain comprises SEQ ID NO:4.

[0217] 22. An antibody according to any of statements 1-5, wherein the heavy chain variable domain comprises SEQ ID NO:5 with no more than four additional alterations, e.g., substitutions, in the framework regions.

[0218] 23. The antibody according to statement 22, wherein the heavy chain variable domain (VH domain) comprises SEQ ID NO:5.

[0219] 24. The antibody according to any of the preceding statements, wherein the light chain variable domain comprises SEQ ID NO:6, in which X7 (Kabat position 92) is N.

[0220] 25. The antibody according to any of statements 1 to 23, wherein the light chain variable domain comprises SEQ ID NO:6, in which X7 (Kabat position 92) is H.

[0221] 26. The antibody according to any of statements 1 to 23, wherein the light chain variable domain comprises SEQ ID NO:6, in which X7 (Kabat position 92) is Y.

[0222] 27. The antibody according to any of statements 1 to 23, wherein the light chain variable domain comprises SEQ ID NO:6, in which X7 (Kabat position 92) is W.

[0223] 28. An antibody according to any of statements 1-25, wherein the light chain variable domain comprises SEQ ID NO:7 with no more than four additional alterations, e.g. substitutions, in the framework regions.

[0224] 29. The antibody according to statement 28, wherein the light chain variable domain comprises SEQ ID NO:7.

[0225] 30. An antibody according to any of statements 1-23 and 25, wherein the light chain variable domain comprises SEQ ID NO:8 with no more than four additional alterations, e.g., substitutions, in the framework regions.

[0226] 31. The antibody according to statement 30, wherein the light chain variable domain comprises SEQ ID NO:8.

[0227] 32. An antibody according to any of statements 1 to 5, comprising a VH domain of SEQ ID NO:3 and a VK domain of SEQ ID NO:7.

[0228] 33. An antibody according to any of statements 1 to 5, comprising a VH domain of SEQ ID NO:4 and a VK domain of SEQ ID NO:7.

[0229] 34. An antibody according to any of statements 1 to 5, comprising a VH domain of SEQ ID NO:5 and a VK domain of SEQ ID NO:8.

[0230] 35. An antibody according to any of statements 1 to 5, comprising a VH domain of SEQ ID NO:5 and a VK domain of SEQ ID NO:7.

[0231] 36. A pharmaceutical composition comprising an antibody according to any of the preceding statements together with a pharma- ceutically acceptable carrier.

[0232] 37. A nucleic acid encoding the antibody of any one of claims 1 to 35.

[0233] 38. A vector comprising the nucleic acid of statement 37 operably linked to a promoter.

[0234] 39. A host cell comprising the nucleic acid of statement 37 or the vector of statement 38.

[0235] 40. A method for producing an antibody according to any of statements 1 to 35, comprising expressing in a host cell culture a vector according to statement 36 so as to produce the antibody; and recovering the antibody from the cell culture.

[0236] 41. A method for treating or preventing Alzheimer's disease by administering to an individual in need of treatment an effective amount of an antibody according to any of statements 1-35 or a pharmaceutical composition of statement 36.

[0237] 42. An antibody according to any of statements 1 to 35 or a pharmaceutical composition of statement 36 for use in a method of treatment of the human or animal body.

[0238] 43. An antibody according to any of statements 1 to 35 or a pharmaceutical composition of statement 36 for use in a method of treating Alzheimer's disease in an individual.

Claims

1. 1. An antibody comprising a heavy chain variable domain and a light chain variable domain, (a) the heavy chain variable domain (VH domain) comprises the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; (b) the light chain variable domain (VK domain) comprises the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:8; Binds to amyloid peptides AβpE3-42 and Aβ4-42, but does not bind to amyloid peptides Aβ1-42; The antibody.

2. The antibody of claim 1, wherein the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO:

3.

3. The antibody of claim 1, wherein the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO:

4.

4. The antibody of claim 1, wherein the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO:

5.

5. The antibody of any one of claims 1 to 4, wherein the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO:

7.

6. The antibody of any one of claims 1 to 4, wherein the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO:

8.

7. The antibody of claim 1, comprising a VH domain having the amino acid sequence shown in SEQ ID NO:3 and a VK domain having the amino acid sequence shown in SEQ ID NO:

7.

8. The antibody of claim 1, comprising a VH domain having the amino acid sequence shown in SEQ ID NO:4 and a VK domain having the amino acid sequence shown in SEQ ID NO:

7.

9. The antibody of claim 1, comprising a VH domain having the amino acid sequence shown in SEQ ID NO:5 and a VK domain having the amino acid sequence shown in SEQ ID NO:

8.

10. The antibody of claim 1, comprising a VH domain comprising the amino acid sequence shown in SEQ ID NO:5 and a VK domain comprising the amino acid sequence shown in SEQ ID NO:

7.

11. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 10 together with a pharma- ceutically acceptable carrier.

12. An antibody according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11 for use in a method of treatment of the human or animal body.

13. An antibody according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11 for use in a method for the treatment of Alzheimer's disease in an individual.

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