Humanized anti-N truncated amyloid-β monoclonal antibody

Abstract

The present invention relates to a humanized antibody that binds amyloid peptides and contains mutations in the variable domains of the heavy and / or light chains, which mutations improve the binding activity. The antibody can be used for the treatment of Alzheimer's disease (AD).

Description

Technical Field

[0001] The present invention relates to a humanized antibody that binds amyloid peptides. Background Art

[0002] The murine anti-amyloid β (Aβ) antibody NT4X-167 was originally generated against the Aβ4-40 amyloid peptide and was 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 shown therapeutic benefits in a mouse model of Alzheimer's disease (Antonios et al Scientific Reports 5 17338; 2015).

[0003] The humanized version of NT4X-167 can be used for clinical applications, such as for the treatment of Alzheimer's disease (AD). Summary of the Invention

[0004] The inventors have unexpectedly found that by mutating certain residues within the heavy and / or light chain variable domains, the binding activity of the humanized version of the NT4X-167 antibody can be improved. For example, this may be useful in the development of candidate molecules for clinical applications.

[0005] A first aspect of the present invention provides an anti-Aβ antibody comprising a heavy chain variable domain and a light chain variable domain, wherein

[0006] a) the heavy chain variable domain (VH domain) comprises SEQ ID NO:2 having four or fewer additional alterations such as substitutions in the framework region, and

[0007] b) the light chain variable domain (VK domain) comprises SEQ ID NO:6 optionally having up to four or fewer additional alterations such as substitutions in the framework region.

[0008] The anti-Aβ antibody can specifically bind to N-terminally truncated amyloid peptides (AβpE3-x or Aβ4-x). For example, the anti-Aβ antibody can specifically bind to one or more of AβpE3-38, AβpE3-40, AβpE3-14, AβpE3-42, Aβ4-38, Aβ4-40, Aβ4-14, and Aβ4-42, preferably all.

[0009] The anti-Aβ antibody may not show specific binding to the full-length amyloid peptide or amyloid peptides without N-terminal truncation (Aβ1-x) such as Aβ1-42, Aβ1-38, Aβ1-40, or Aβ1-14.

[0010] 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.

[0011] Preferably, the light chain variable domain (VL domain) of the anti-Aβ antibody comprises SEQ ID NO:7, or SEQ ID NO:8.

[0012] The second aspect described herein provides a pharmaceutical composition comprising the antibody of the first aspect and a pharmaceutically acceptable carrier.

[0013] The third aspect described herein provides a nucleic acid encoding the antibody of the first aspect or its heavy chain variable domain and / or light chain variable domain.

[0014] The fourth aspect described herein provides a vector comprising the nucleic acid of the third aspect.

[0015] The fifth aspect described herein provides a host cell comprising the nucleic acid of the third aspect or the vector of the fourth aspect.

[0016] The sixth aspect described herein provides a method for preparing the antibody according to the first aspect, the method comprising expressing the vector according to the fourth aspect in a host cell culture to produce the antibody; and recovering the antibody from the cell culture.

[0017] The seventh aspect described herein provides a method for treating Alzheimer's disease by administering to an individual in need thereof an effective amount of the antibody according to the first aspect or the pharmaceutical composition according to the second aspect.

[0018] The eighth aspect described herein provides the antibody according to the first aspect or the pharmaceutical composition according to the second aspect for use in a method for treating the human or animal body.

[0019] The ninth aspect described herein provides the antibody according to the first aspect or the pharmaceutical composition according to the second aspect for use in a method for treating Alzheimer's disease in an individual.

[0020] These and other aspects and embodiments described herein are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shows the binding of murine NT4X-167 antibody to amyloid peptides.

[0022] Figure 2 Shows the binding of murine and chimeric NT4X-167 antibodies to PSL amyloid peptides.

[0023] Figure 3 Shows the binding of murine, humanized, and chimeric NT4X-167 to AβpE3-42 amyloid peptide: initial version.

[0024] Figure 4 Shows the binding of the humanized variant to Aβ1-42.

[0025] Figure 5 Shows the binding of the humanized variant to AβpE3-42.

[0026] Figure 6 Shows the binding of the second round of humanized NT4X-167 antibody to AβpE3-42 peptide: HC to HR version binding RKA.

[0027] Figure 7 Shows the binding of the second round of humanized NT4X-167 antibody to AβpE3-42 peptide by ELISA.

[0028] Figure 8 Shows the binding of the third round of humanized NT4X-167 antibody to AβpE3-42 peptide: HS to HY version binding RKA.

[0029] Figure 9 Shows the binding of the fifth round of humanized NT4X-167 antibody to AβpE3-42 peptide: rcNT4XS6A, rcNT4XS7A, rcNT4XS8A versions binding RKA.

[0030] Figure 10 Shows the thermal stability of humanized rcNT4X_SA, BA, TA, UA, VA, WA, YA antibodies that bind to amyloid peptide AβpE3-42.

[0031] Figure 11 Shows the thermal shift analysis of humanized rcNT4X_SA and rcNT4X_S7A antibodies.

[0032] Figure 12 Shows non-specific protein-protein interactions (cross-interaction chromatography).

[0033] Figure 13 Shows the non-specific protein-protein interactions (cross-interaction chromatography) of the humanized lead candidate rcNT4XS7A.

[0034] Figure 14 Shows the purified antibody candidates for solubility assessment.

[0035] Figure 15Shows the serum stability assessment of humanized rcNT4X_SA and rcNT4X_S7A antibodies that bind to PSLAβpE3-42 amyloid peptide.

[0036] Figure 16 Shows the serum stability assessment of humanized rcNT4X_SA and rcNT4X_S7A antibodies that bind to Anaspec Aβ4-42 amyloid peptide.

[0037] Figure 17 Shows the amount of protection provided to primary embryonic rat neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A (antibodies from cell death induced by 4-42 amyloid peptide).

[0038] Figure 18 Shows the amount of protection provided to primary embryonic rat neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A (antibodies from cell death induced by (AβGlp3)3-42 amyloid peptide).

[0039] Figure 19 Shows the amount of protection provided to primary embryonic rat neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A (antibodies from cell death induced by Aβ1-42 amyloid peptide).

[0040] Figure 20 Shows the amount of protection provided to human CNS.4U neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A (antibodies from cell death induced by Aβ4-42 amyloid peptide).

[0041] Figure 21 Shows the amount of protection provided to human CNS.4U neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A (antibodies from cell death induced by AβpE3-42 amyloid peptide).

[0042] Figure 22 Shows the amount of protection provided to human CNS.4U neurons in vitro by humanized rcNT4X_SA and rcNT4X_S7A (antibodies from cell death induced by Aβ1-42 amyloid peptide).

[0043] Figure 23It is shown that both rcNT4X antibodies can rescue the loss of hippocampal neurons in 6-month-old Tg4-42 mice. Neurons in CA1 were quantified using unbiased stereology. The number of neurons 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 ANOVA, followed by Bonferroni multiple comparisons; n = 5 - 6. *p < 0.05; ***p < 0.001; Data are represented as mean ± S.E.M.

[0044] Figure 24 It is shown that rcNT4X_S7A has the highest potency in rescuing neuronal loss in Tg4-42. Data comparison of original NT4X with rcNT4X_SA and rcNT4X_S7A. T-test between NT4X and rcNT4X_S7A. n = 5 - 7. *p < 0.05; Data are represented as mean ± S.E.M.

[0045] Figure 25 It is shown that there is no significant difference between MRCT control IgG1 antibodies compared to IgG2b and PBS control groups. Data for IgG2ba and PBS groups are from Antonios et al. 6 . Neither t-test nor ANOVA showed significant differences. Data are represented as mean ± S.E.M. n = 5 - 7.

[0046] Figure 26 It is shown that passive immunization with rcNT4X_S7A rescues the learning deficit in Tg4-42 mice. Tg4-42 mice were injected weekly with antibodies and IgG1 control antibody (MRCT control) for 12 weeks. 6-month-old mice were tested in the Morris water maze. In Tg4-42 mice treated with MRCT control antibody, spatial reference memory was impaired as they did not show preference for the target quadrant during the exploration trial. In contrast, Tg4-42 mice immunized with rcNT4X_S7A antibody showed no learning deficit. ***p < 0.001; **p < 0.01; *p < 0.05. n = 8 per group. One-way ANOVA, followed by Bonferroni multiple comparisons. T target quadrant, L left quadrant, R right quadrant, O opposite quadrant. Data are represented as mean ± S.E.M; m = months.

[0047] Figure 27Shows reduced cortical plaque burden in immunized 5XFAD mice. Plaque burden analysis of rcNT4X_SA- and rcNT4X_S7A-immunized 5XFAD mice compared to IgG1-injected 5XFAD mice. (a) Immunostaining with anti-pan-Aβ antibody shows reduced plaque burden after rcNT4X_S7A immunization, but not in the rcNT4X_SA immunization group. (b) Thioflavin S staining confirms that both rcNT4X-treated groups show significantly reduced fibrillar Aβ deposition. (c) Immunostaining with anti-Aβ1-x antibody shows reduced plaque burden after rcNT4X_S7A immunization, but not in the rcNT4X_SA immunization group. (d) Immunostaining with anti-pyroglutamate Aβ3-x antibody shows reduced plaque burden for both rcNT4X antibodies, however this is only significant for the rcNT4X_S7A antibody and trends for the rcNT4X_SA antibody (e) Immunostaining with anti-Aβ4-x antibody shows reduced plaque burden for both rcNT4X antibodies. One-way ANOVA, then Dunnett's multiple comparison test relative to the control group; n = 7-11; ***p < 0.001, **p < 0.01, *p < 0.05 Data are represented as mean ± S.E.M. DETAILED DESCRIPTION

[0048] The present invention relates to the discovery that the binding activity of a humanized version of the murine anti-amyloid (Aβ) antibody NT4X-167 is significantly improved by mutations of certain residues within the variable domain.

[0049] The anti-Aβ antibody 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 having four or fewer additional amino acid mutations such as substitutions, deletions or insertions in the framework region.

[0050] The antibody may specifically bind to N-terminally truncated amyloid peptides, such as pyroglutamate (pE)-modified amyloid peptides (also known as AβpE3-x, AβpGlu3-x, Aβ(Glp3)3-x and p3-x), such as AβpE3-38, AβpE3-40, AβpE3-14 and AβpE3-42, as well as non-pyroglutamate modified amyloid peptides, such as Aβ4-38, Aβ4-40, Aβ4-14 and Aβ4-40. Binding can be determined, for example, using standard techniques such as ELISA or surface plasmon resonance as described below, using the anti-Aβ antibody described herein in IgG1 form.

[0051] The VH may comprise SEQ ID NO:2; or have, independently in the framework region, 1 or more, such as 2, 3, or 4 or more other amino acid alterations or mutations (such as single amino acid substitutions, deletions, or insertions), preferably a substituted SEQ ID NO:2 amino acid sequence, relative to SEQ ID NO:2. The other amino acid alterations or mutations in the framework region 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, 52BX 1 , 52CX 6 , 53X 2 , 54X 3 , 55X 4 and 56X 5 at residues other than.

[0052] The VL domain may have SEQ ID NO:6; or have, independently in the framework region, 1 or more, such as 2, 3, or 4 or more amino acid alterations or mutations (such as single amino acid substitutions, deletions, or insertions), preferably a substituted SEQ ID NO:6 amino acid sequence, relative to SEQ ID NO:6. The other amino acid alterations or mutations in the framework region may be at residues other than 92X of SEQ ID NO:6 7 at residues other than.

[0053] 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 having 1, 2, 3, or 4 amino acid substitutions in the framework region. The anti-Aβ antibodies described herein may comprise a VL domain of SEQ ID NO:7 or 8 optionally having 1, 2, 3, or 4 amino acid substitutions in the framework region.

[0054] Suitable anti-Aβ antibodies may comprise (i) a VH domain of SEQ ID NO:3 and a VL domain of SEQ ID NO:7, (ii) a VH domain of SEQ ID NO:4 and a VL domain of SEQ ID NO:7, (iii) a VH domain of SEQ ID NO:5 and a VL domain of SEQ ID NO:7, (iv) a VH domain of SEQ ID NO:3 and a VL domain of SEQ ID NO:8, (v) a VH domain of SEQ ID NO:4 and a VL domain of SEQ ID NO:8, and / or (vi) a VH domain of SEQ ID NO:5 and a VL domain of SEQ ID NO:8. Some preferred anti-Aβ antibodies may comprise a VH domain of SEQ ID NO:5 and a VL domain of SEQ ID NO:8.

[0055] The terms “immunoglobulin” and “antibody” are used interchangeably and refer to any protein that contains an antibody antigen-binding site having the ability to specifically bind one or more antigens.

[0056] An “antigen” is an entity (e.g., a protein entity or a peptide) to which an immunoglobulin or antibody (or an antigen-binding fragment thereof) specifically binds. The antigens of the anti-Aβ antibodies described herein can include N-truncated amyloid peptide AβpE3-42 and Aβ4-42.

[0057] Natural antibodies are generally heterotetrameric glycoproteins of approximately 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 a covalent disulfide bond, with a different number of disulfide bonds between the heavy chains of different antibody isotypes. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges.

[0058] Antibodies contain spherical regions of heavy or light chain polypeptides called “domains”. Domains can contain peptide loops, typically 3 to 4 loops, which can be stabilized, for example, by β-sheets and / or intra-chain disulfide bonds. Domains are generally referred to as “constant” or “variable” based on the relatively lack of sequence variation within the domain for members of each class in the case of “constant” domains, or the significant variation within the domain for members of each class in the case of “variable” domains. An antibody or polypeptide “domain” is generally interchangeably referred to in the art as an antibody or polypeptide “region”.

[0059] The “constant” domain of an antibody light chain can be referred to as the “light chain constant region”, the “light chain constant domain”, the “CL” region, or the “CL” domain. The “constant” domain of an antibody heavy chain can be referred to as the “heavy chain constant region”, the “heavy chain constant domain”, the “CH” region, or the “CH” domain. The constant domain of the light chain aligns with the first constant domain of the heavy chain.

[0060] The constant domain of the heavy chain that contains 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 some proteins of the complement system, by which method the antibody can activate the immune system. The Fc region contains three heavy chain constant domains in each polypeptide chain.

[0061] The "variable" domain of an antibody light chain may be referred to as the "light chain variable region", "light chain variable domain", "VL" region, or "VL" domain (where "L" here refers to "light" rather than the light chain isotype "λ"). The "variable" domain of an antibody heavy chain may be referred to as the "heavy chain variable region", "heavy chain variable domain", "VH" region, or "VH" domain. A complete light chain has, for example, two domains (VL and CL), and a complete heavy chain has, for example, four or five domains (VH, CH1, CH2, and CH3).

[0062] The light and heavy chain variable domains include "hypervariable regions" (HVR or HV), also known as "complementary determining regions" (CDR), which are highly variable in sequence and may form loops with defined structures. Generally, an antibody contains six hypervariable regions; three in the heavy chain (H1, H2, H3) and three in the light chain (L1, L2, L3) interspersed between relatively conserved framework regions (FR). In the antibodies described herein, the amino acid sequences of the variable domains are shown below. The CDRs in these sequences can be readily identified using standard techniques (see, for example, Kabat, E.A., Wu, T.T., Perry, H.M., Gottesmann, K.S & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91 - 3242. U.S. Department of Health and Human Services). In the Kabat nomenclature, VH CDR1 is located at positions 31 - 35, VH CDR2 is located at positions 50 - 65, VH CDR3 is located at positions 95 - 102, VL CDR1 is located at positions 24 - 34, VL CDR2 is located at positions 50 - 56, and VL CDR3 is located at positions 89 - 97.

[0063] The variable regions of each light / heavy chain pair form the antigen - binding site. The term "antigen - binding site" refers to the site that specifically binds to an antigen (immune reaction). The antibodies described herein contain at least one antigen - binding site, preferably containing two antigen - binding sites. The antigen - binding site is formed by the heavy and light chain CDRs and is aligned by the framework regions so as to be able to bind to a specific epitope. The "antigen - binding region" or "antigen - binding domain" is the antibody region or domain that includes the antibody - binding site. The antibodies described herein have at least one antigen - binding site that recognizes amyloid peptides AβpE3 - 42 and Aβ4 - 42.

[0064] Naturally occurring or recombinantly produced antibody chains can be expressed with a leader sequence that is removed during cellular processing to yield the mature chain. The mature chain can also be recombinantly produced and can contain a non-naturally occurring leader sequence, for example to enhance secretion or alter processing of a particular chain of interest.

[0065] The constant regions of the heavy and light chains of an antibody may exhibit phenotypic variation. Based on the amino acid sequence of the light chain constant region, antibody light chains are classified as kappa (κ) or lambda (λ), and are approximately 230 residues in length. The antibodies described herein contain a κ light chain (the variable domain of the κ light chain is referred to herein as VK).

[0066] Heavy chains from humans and higher mammals are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), are approximately 450 - 600 residues in length, and define the antibody isotypes 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 antibodies or IgG1 antibodies with minimal effector function.

[0067] The antibodies described herein can contain a heavy chain belonging to any immunoglobulin isotype described herein. The antibodies described herein can contain sequences from more than one type or isotype.

[0068] The anti-Aβ antibodies described herein can exhibit cytotoxic activity. In such antibodies, the constant domain is typically a complement-fixing constant domain and the class is typically IgG1. Human isotypes IgG1 and IgG4 are exemplary.

[0069] The antibodies described herein can contain fragments of a full antibody. The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than the full or complete antibody or antibody chain, where the portion preferably retains at least one, preferably most or all of the functions that are typically associated with that portion when present in the full antibody. Fragments can be obtained by chemical or enzymatic treatment of the full or complete antibody or antibody chain. Fragments can also be obtained by recombinant means.

[0070] Fragments of the antibodies described herein can bind antigen or compete with the full antibody (i.e., the full antibody from which they are derived) for antigen binding (i.e., specific binding). The antibodies described herein bind amyloid peptide AβpE3 - 42 and Aβ4 - 42. The binding fragments are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the full immunoglobulin.

[0071] The antibodies described herein may exist in the form of binding fragments, including but not limited to Fab, Fab', F(ab') 2 , chemically linked F(ab') 2 , monospecific Fab 2 , bispecific Fab 2 , trispecific Fab 2 , 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 amyloid peptides AβpE3-42 and Aβ4-42.

[0072] The antibodies described herein may be part of bispecific or trispecific antibodies. Bispecific antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different antigen-binding sites; trispecific antibodies are artificial hybrid antibodies having 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 ligation of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Exemplary antibodies described herein may be bispecific antibodies comprising at least two different antigen-binding sites.

[0073] Specific binding refers to a situation in which the antibody does not show any significant binding to molecules other than its specific epitope on the antigen. The term also applies, for example, to cases where an antigen-binding domain is specific for a particular epitope carried by many antigens, in which case an antibody carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope.

[0074] The anti-Aβ antibodies described herein or nucleic acids encoding such antibodies will be in isolated form. The antibodies and nucleic acids will be free or substantially free of substances with which they are naturally associated, such as other polypeptides or nucleic acids found in their natural environment or in the environment (e.g., cell culture) in which they are prepared (when such preparation is by recombinant DNA techniques practiced in vitro or in vivo). The antibodies and nucleic acids may be formulated with diluents or adjuvants, but are still isolated for practical purposes, e.g., an antibody is usually mixed with gelatin or other carriers when used to coat microtiter plates for immunoassays, or with pharmaceutically acceptable carriers or diluents when used for diagnosis or therapy.

[0075] Another aspect of the present invention provides a nucleic acid encoding an antibody or its light chain, heavy chain, VH domain, or VL domain as disclosed herein. As described above, the nucleic acid can encode, for example, 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 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 domain and / or VL domain can have up to four additional amino acid mutations in the framework region.

[0076] The nucleic acid can include DNA and RNA sequences, in which the thymine nucleobase is replaced by uracil.

[0077] The antibodies described herein can be produced by recombinant expression. Nucleic acids encoding the light and heavy chain variable regions optionally linked to constant regions as described above can be inserted into an expression vector. A vector comprising a nucleic acid encoding an antibody described herein is itself an aspect of the present invention. The light and heavy chains can be cloned in the same or different expression vectors. Nucleic acids encoding the antibody chains described herein can be operably linked to one or more control sequences in one or more expression vectors that ensure the expression of the antibody chains. Expression control sequences include, but are not limited to, promoters (e.g., native or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells (e.g., COS, CHO, or Expi293 cells). Such vectors can be incorporated into a suitable host and maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of the antibody.

[0078] Aspects of the present invention provide a nucleic acid encoding an antibody described herein; a vector, preferably an expression vector, comprising one or more nucleic acids encoding an antibody described herein; and a vector comprising one or more nucleic acids encoding an antibody described herein operably linked to a promoter. Exemplary expression vectors are pHuK and pHuG1, which in combination with the nucleic acids disclosed herein contain nucleotide sequences encoding an antibody described herein. Other vectors providing nucleotide sequences encoding the constant regions of the antibody light and heavy chains can also be used.

[0079] Expression vectors as used herein can generally replicate in a host organism either as an episome or as an integral part of the host chromosomal DNA. Typically, the expression vector contains a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequence (see, e.g., Itakura et al. US4704362).

[0080] Host cells can be transformed with the expression vector and cultured in a conventional nutrient medium suitable for inducing the promoter, selecting transformants, and / or amplifying the gene encoding the desired sequence. In one aspect of the invention, there are provided host cells containing the above-described nucleic acid or vector.

[0081] Another aspect of the invention provides a method for preparing the antibodies described herein, the method comprising expressing the vector described herein in a host cell culture to produce the antibodies and recovering the antibodies from the cell culture. The method can include transferring a vector containing one or more nucleic acids encoding an antibody or antibody chain as described above into a host cell as described herein, growing the host cell culture under conditions permitting expression of the one or more nucleic acids, and recovering the expressed antibodies. Any suitable method known in the art can be employed.

[0082] Microbial host organisms suitable for cloning and expressing the nucleic acids and vectors described herein include prokaryotic hosts; Escherichia coli, bacilli such as Bacillus subtilis and other Enterobacteriaceae such as Salmonella, Serratia and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which generally contain expression control sequences (e.g., origin of replication) compatible with the host cell. Additionally, there will be many various well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system or the promoter system of bacteriophage λ. The promoter will generally optionally control expression with an operator sequence and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation. Vectors for prokaryotic cells may also require an origin of replication component.

[0083] Other microorganisms, such as yeast, can also be used to express the nucleic acids or vectors described herein. Yeast of the genus Saccharomyces is a preferred yeast host and has a suitable vector having an expression control sequence (such as a promoter), an origin of replication, a termination sequence, and other required sequences of a similar nature. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, in particular, the promoters of alcohol dehydrogenase, iso-cytochrome C, and the enzymes responsible for maltose and galactose utilization.

[0084] In addition to microorganisms, mammalian tissue cell cultures can also be used to express the nucleic acids or vectors described herein and to produce antibody polypeptides (e.g., polynucleotides encoding antibodies or fragments thereof (see, for example, Winnacker, From Genes to Clones, VCH Publishers, N.Y. 1987)). Eukaryotic or mammalian cell hosts containing the nucleic acids or vectors described herein are in themselves an aspect of the present invention. Eukaryotic cells are actually preferred because many suitable host cell lines capable of secreting heterologous proteins (such as 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 cells or non-human cells, e.g., non-human mammalian cells. In some preferred embodiments, the cells are Expi293 human cells. The antibodies described herein can be produced in cell lines engineered to produce fucosylated proteins, such as the CHOK1SV cell line (BioWa / Lonza), engineered cells (ProBioGen), or the duck embryonic stem cell line EB66 (Valneva). Expression vectors for mammalian cells generally include, but are not limited to, one or more of the following: signal sequences, one or more marker genes, enhancer elements, promoters, and necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, etc. (see, for example, Co et al., J. Immunol. 148:1149 1992).

[0085] The vectors described herein for eukaryotic host cells can also encode a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature antibody chain or polypeptide. Suitable signal sequences can be heterologous and can be recognized and processed by the host cell (i.e., cleaved by signal peptidase). In mammalian cell expression, mammalian signal sequences as well as viral secretory leader sequences, such as the herpes simplex gD signal, are available.

[0086] Alternatively, the antibody-encoding 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 the coding sequences of the light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes such as casein or β-lactoglobulin.

[0087] The vectors described herein containing the polynucleotide sequence of interest (e.g., the heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, gene gun, or virus-based transfection can be used for other cell hosts. (See generally Green and Sambrook, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 4th ed., 2012)). Other methods for transforming 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 a fertilized oocyte or can be incorporated into the genome of embryonic stem cells, and the nucleus of such cells can be transferred into an enucleated oocyte.

[0088] When the heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of the complete antibodies described herein. Once expressed, the complete antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms described herein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, etc. (see generally, Scopes, Protein Purification (Springer-Verlag, N.Y., (1982))). For the pharmaceutical uses described herein, a substantially pure antibody of at least about 90 to 95% homogeneity is preferred, and most preferably a substantially pure antibody of 98 to 99% or higher homogeneity. Standard protein purification methods known in the art can be employed. The following procedures are examples of suitable protein purification procedures: fractionation on an immunoaffinity column or ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or on a cation exchange resin (e.g., DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration.

[0089] The antibodies described herein can be produced by any suitable technique including the techniques described herein and other techniques known in the art. The antibodies described herein can be produced on a commercial scale using methods recognized in the art for large-scale production of antibodies. For example, recombinant expression systems such as those described herein can be employed.

[0090] The antibodies described herein can specifically bind amyloid peptides AβpE3-42 and Aβ4-42. The antibodies may not show or may show substantially no specific binding to amyloid peptide Aβ1-42. The antibodies described herein can also exhibit the desired structural, physical, biophysical, and chemical properties described below and as described with reference to the examples.

[0091] The affinity of the antibodies described herein is the degree or strength with which the antibody binds to an epitope or antigen. The dissociation constant K d and the association constant K a are quantitative measures of affinity. K d is the ratio of the rate at which the antibody dissociates (k off ), the rate at which it dissociates from the antigen, to the rate at which the antibody associates (k on ), the rate at which it binds to the antigen. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentration of the reactants. At equilibrium, the rate of formation of the [antibody][antigen] complex is equal to the rate of dissociation into its components [antibody] + [antigen]. Measurement of the reaction rate constants can be used to define the equilibrium or affinity constant K a (K a = 1 / K d ). The smaller the K d value, the greater the affinity of the antibody for its target. The K d values of most antibodies are in the range of low micromolar (10 -6 ) to nanomolar (10 -7 to 10 -9 ). High-affinity antibodies are generally considered to be in the low nanomolar range (10 -9 ), while very high-affinity antibodies are in the picomolar (10 -12 ) range.

[0092] The antibodies described herein can have 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 -1association rate constant (k on ).

[0093] The antibodies described herein may have a dissociation (k -1 s -1 ), less than 10 -1 s -1 ), less than 5×10 -2 s -1 ), less than 10 -2 s -1 ), or less than 5×10 -3 s -1 dissociation rate of the antibody (k off ).

[0094] In some embodiments, the antibodies described herein are 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 -11 affinity constant or K a binding (e.g., specific binding) to amyloid peptides AβpE3-42 and Aβ4-42.

[0095] The dissociation constant or K d of the antibodies described herein with amyloid peptides AβpE3-42 and Aβ4-42 can be less than 5×10 - 2 M, less than 10 -2 M, less than 5×10 -3 M, less than 10 -3 M, less than 5×10 -4 M, less than 10 -4 M, less than 5×10 -5 M, less than 10 -5 M, less than 5×10 -6 M, less than 10 -6M or less than 5×10 -7 M,

[0096] Specific binding of an antibody means that the antibody exhibits a significant affinity for a specific antigen or epitope and generally does not exhibit significant cross-reactivity. An antibody that "does not exhibit significant cross-reactivity" is an antibody that does not significantly bind to an undesired entity (e.g., an undesired protein entity). An antibody specific for a particular epitope, for example, will not exhibit significant cross-reactivity with a distal epitope on the same protein or peptide. Specific binding of the antibodies described herein, i.e., k off 、k on 、K a and K d , can be determined by any means recognized in the art for determining such binding.

[0097] The antibodies described herein can bind amyloid peptide Aβ4-42 or AβpE3-42 with a binding affinity that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the binding affinity of the murine NT4X-167 antibody for amyloid peptide Aβ4-42 or AβpE3-42, as measured by ELISA. Suitable ELISA techniques are well known in the art. For example, immobilized amyloid peptide in the form of IgG1 can be contacted with the antibody and washed one or more times in 0.1% non-ionic detergent (such as polysorbate 20 (Tween 20)) to remove unbound antibody. Then any convenient technique can be used to detect the antibody bound to the immobilized peptide, for example, using a secondary antibody conjugated to a detectable label such as HRP.

[0098] The antibodies described herein can be heat stable, i.e., the antibodies described herein can bind amyloid peptides AβpE3-42 and Aβ4-42 at temperatures from 30°C to 85°C, particularly up to 75°C. The melting temperature of the antibodies described herein can be from 50°C to 100°C, specifically from 60°C to 80°C, more specifically in the vicinity of 66 - 67°C.

[0099] The antibodies described herein may have a low tendency to aggregate. Standard techniques such as multi-angle light scattering or dynamic light scattering can be used to analyze the aggregation tendency. The antibodies described herein may have a lower tendency for non-specific protein-protein interactions and good solubility.

[0100] When concentrated, the antibodies described herein may have a low tendency to aggregate. The formulations described herein may contain antibodies concentrated to 50 - 200 mg / ml, such as 75 - 150 mg / ml, preferably 80 - 120 mg / ml and more preferably 90 - 110 mg / ml, with a preferred concentration of about 100 mg / ml, without forming soluble aggregates in an aqueous solution maintained at physiological pH, such as by Dulbecco’s PBS.

[0101] When the antibodies described herein are subjected to repeated freeze - thaw cycles or extended temperatures above normal body temperature, they may have a low tendency to aggregate. For example, the extended temperature is 30 days at 50 °C in Dulbecco’s PBS.

[0102] The isoelectric point (pI) of the antibodies described herein may be from pH 8.6 to pH 9, preferably from pH 8.1 to pH 8.7.

[0103] After incubation in the sera of mice, humans, and / or cynomolgus primates at 37 °C, the antibodies described herein may retain the ability to bind to amyloid peptides AβpE3 - 42 and Aβ4 - 42. For example, after incubation in mouse, human, and / or cynomolgus monkey sera for 10 to 50 days, preferably 20 - 40 days, and more preferably 30 days, the antibodies described herein may retain the ability to bind to AβpE3 - 42 or Aβ4 - 42. The antibodies that retain the binding ability may exhibit the same or substantially the same binding ability as that observed for antibodies not incubated in sera or incubated in a control solution at 37 °C.

[0104] The anti - Aβ antibodies disclosed herein may be non - glycosylated. The Fc region of IgG antibodies has highly conserved N - glycosylation sites, and glycosylation of the Fc fragment is crucial for Fc receptor - mediated activities. The N - glycosyl carbohydrate moieties attached to this site are mainly complex - type fucosylated biantennary structures. Additionally, a small amount of these N - glycans also carry bisecting GlcNAc and α - 2,6 - linked sialic acid residues. Due to, for example, chemical or enzymatic processes, deletion or mutation of one or more glycosylation sites, or expression in bacteria, non - glycosylated antibodies may lack one or more carbohydrate moieties.

[0105] The anti-Aβ antibodies described herein can be modified to enhance their antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is a cell-mediated response in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize an antibody bound to a target cell and subsequently cause lysis of the target cell. Such cells can be human cells. It is generally believed that the ADCC activity of an antibody requires the binding of the Fc region of the antibody to an antibody receptor present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages. By altering the fucosylation of the carbohydrate structure of a humanized antibody (e.g., reducing or eliminating) (i.e., in the Fc region), the ADCC activity of the antibody can be increased, 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 in vitro relative to the unmodified humanized antibody. Due to the increased ADCC activity, such modified antibodies can be used at lower doses than their unmodified counterparts and generally have fewer or reduced side effects in patients.

[0106] The anti-Aβ antibodies described herein can be used for complement-dependent cytotoxicity (CDC). CDC involves the central innate complement system, which acts as an effector of adaptive immunity. The classical CDC pathway is triggered by an antibody molecule bound to an antigen on a target cell and initiated by the binding of C1q protein to the Fc domain of the bound antibody. The resulting complement cascade activates the membrane attack pathway, leading to the formation of a membrane attack complex that induces lysis of the target cell. The antibodies described herein can be modified by any method known in the art to enhance their ability to trigger CDC, such as, but not limited to, engineering the protein backbone to contain amino acid residue substitutions in the constant domain of the antibody heavy chain. For examples of combinations of IgG1 amino acid substitutions for enhancing CDC activity, see Moore et al., mAbs, 2(2), 181-189 (2010). The CDC activity of the modified antibodies described herein can be increased, 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 relative to the unmodified humanized antibody.

[0107] The anti-Aβ antibodies can be further modified by chemical modification, such as by PEGylation or by incorporation into liposomes, to improve their pharmaceutical properties, for example, by increasing the in vivo half-life.

[0108] The anti-Aβ antibodies described herein can be formulated and / or administered as a pharmaceutical composition, which comprises an active therapeutic antibody reagent and a variety of other pharmaceutically 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. Depending on the desired formulation, the composition may also contain a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the composition. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution, and Hank's solution. Additionally, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0109] The pharmaceutical composition comprising the anti-Aβ antibody described herein may also contain large, slowly metabolized macromolecules, such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex-functionalized Sepharose TM , agarose, cellulose, etc.), polyamino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Additionally, these carriers can be used as immunostimulants (i.e., adjuvants).

[0110] For parenteral administration, the antibodies or compositions described herein can be administered with a pharmaceutical carrier in an injectable dose as a solution or suspension of the substance in a physiologically acceptable diluent, which pharmaceutical carrier can be a sterile liquid, such as water for injection, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances, etc., can be present in the composition. Other components of the pharmaceutical composition are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, and mineral oil. Generally, diols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, especially for injectable solutions. The antibodies can be administered in the form of depot injections or implant formulations, which can be formulated in a manner that allows for slow release of the active ingredient.

[0111] As used herein, the term parenteral includes subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, and intrathecal administration of the antibodies or compositions described herein. The anti-Aβ antibodies or compositions described herein can also be administered by nasal or gastric methods.

[0112] Typically, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, a liquid vehicle prior to injection may also be prepared. As noted above, the formulations may also be emulsified or encapsulated in liposomes or microparticles, such as polylactides, polyglycolides or copolymers, to enhance adjuvant effects (see Langer, Science 249:1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97 (1997)). The agents of the invention may be administered in the form of depot injections or implant formulations which may be formulated to allow for sustained or pulsed release of the active ingredient.

[0113] Additional formulations suitable for other modes of administration include oral, intranasal and pulmonary formulations, suppositories and transdermal administration. Oral formulations include excipients such as pharmaceutical grade 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 from 10% to 95%, preferably from 25% to 70%, of the active ingredient.

[0114] Topical application may result in transdermal or intradermal delivery. Transdermal delivery may be facilitated by co-administering the agent with cholera toxin or a detoxified derivative or subunit thereof or other similar bacterial toxins (see Glenn et al., Nature 391, 851 (1998)). Co-administration may be achieved by administering these components as a mixture or as a conjugate molecule obtained by chemical cross-linking or expressed as a fusion protein. Alternatively, transdermal delivery may be achieved using a skin patch or using transferosomes (Paule et al., Eur. J. Immunol. 25:3521 (1995); Cevc et al., Biochem. Biophys. Acta 1368:201-15 (1998)).

[0115] Preferably, the anti-Aβ antibodies or compositions comprising the anti-Aβ antibodies described herein may be administered intravenously (IV) or intramuscularly (IM).

[0116] The composition may comprise an anti-Aβ antibody described herein, a pharmaceutically acceptable carrier described herein and other therapeutic agents, particularly prophylactic or therapeutic agents useful for the prevention, control or treatment of Alzheimer's disease (AD). Such therapeutic agents may include analgesics, anti-inflammatory agents, antiviral agents, agents for ameliorating fever or elevated body temperature, therapeutic compounds designed to numb pain, such as mouthwashes or sprays that numb oral pain and cognitive enhancement therapies, such as memantine, donepezil, galantamine and rivastigmine. The compositions described herein may additionally comprise compositions for hydrating a subject, for example by intravenous therapy.

[0117] The compositions described herein may comprise nucleic acids encoding the anti-Aβ antibodies described herein, i.e., DNA or RNA, and any method of delivering such nucleic acids with or without any other composition compounds discussed above. The compositions may also comprise a vector, such as but not limited to the expression vectors described herein, which itself comprises the nucleic acids described herein.

[0118] The compositions described herein may comprise viral vectors that serve as nucleic acid delivery systems for entry into cells. Suitable viral vector nucleic acid delivery systems include retroviral systems, adenoviral vectors, viral vectors from the poxvirus family, including vaccinia virus and avian poxviruses, and viral vectors from the alphavirus genus. The nucleic acids encoding the antibodies described herein, or vectors containing them, can be packaged into liposomes for delivery to an individual or cell, and can be incorporated into the compositions described. The vectors and nucleic acids encoding the antibodies can also be adsorbed onto or bound to particulate carriers.

[0119] The compositions described herein may comprise gene therapy vectors that contain nucleotide sequences encoding the antibodies described herein or the naked antibody polypeptide chains according to the invention. The compositions may comprise such vectors or polypeptides in combination with the antibodies and any other composition components described herein.

[0120] The antibodies described herein can be used in a kit. The term "kit" refers to the combined use of reagents and other materials that facilitate the analysis of a sample. In some embodiments, the immunoassay kits described herein include a suitable antigen, a binding agent comprising a detectable moiety, and a detection reagent. A system for amplifying the signal generated by the detectable moiety may or may not be included in the kit. Additionally, in other embodiments, the kit includes, but is not limited to, components such as a device for sample collection, sample tubes, racks, trays, shelves, culture dishes, plates, instructions provided to the kit user, solutions or other chemical reagents, and samples and / or control samples for standardization and normalization.

[0121] The kit may comprise at least one of the antibodies described herein. The kit may contain the compositions described herein in one or more containers, optionally together with one or more other prophylactic or therapeutic agents for the prevention, control, 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 capable of delivering the kit components via some other route, such as a syringe, may be included. The kit may further include instructions for the prevention, treatment, control, or improvement of AD and for the side effects and dosage information of the administration method.

[0122] The present invention also provides a diagnostic kit. The antibodies described herein can be used to monitor, diagnose, or provide a prognosis for the development or progression of AD, and can be used in kits suitable for such purposes. The antibodies described herein can be used in a diagnostic kit to detect the presence of N-truncated amyloid peptides (such as AβpE3-42 or Aβ4-42) in a body fluid sample taken from an individual, where the individual can be a human or a mammal, such as a non-human primate or an experimental animal, including mice, rats, and rabbits. A body fluid sample is obtained from an individual, such as but not limited to blood, serum, or cerebrospinal fluid (CSF), and the presence of N-truncated amyloid peptides is tested using the antibodies described herein. Measuring the level of amyloid peptides in an individual's blood using the antibodies described herein can provide information regarding the susceptibility, risk of onset, diagnosis, or prognosis of AD in the individual, or a suitable administration protocol or dosage of the antibodies or compositions described herein for treating the individual. Diagnostic methods are generally performed in vitro. A method for detecting the presence of N-truncated amyloid peptides in a sample from an individual can include contacting the sample with an anti-Aβ antibody described herein, and determining the binding of the antibody to one or more peptides in the sample.

[0123] The kit that can be used for the above diagnosis can contain the antibodies described herein conjugated to a detectable substance, and the detectable substances include but are not limited to: various enzymes for assays including EIA and ELISA, such as but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; cofactors, such as but not limited to streptavidin / biotin and avidin / biotin; particles for agglutination tests, such as latex beads or bacteria; fluorescent substances, 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; radioactive substances, 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, 188 Re, 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 Sn; A positron-emitting metal using various positron emission tomography, non-radioactive paramagnetic metal ions, and molecules radiolabeled or conjugated to specific radioisotopes. Any detectable label that can be readily measured can be conjugated to the antibodies described herein and used to diagnose the diseases described herein. The detectable substance can be directly coupled or conjugated to the antibody or indirectly coupled or conjugated through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. Metal ions that can be conjugated to antibodies for use as diagnostics are known in the art (see, for example, US474900).

[0124] Using the antibodies described herein in the kits described herein, a positive result for the presence of amyloid peptide AβpE3-42 or Aβ4-42 can be obtained by antigen detection using any of the methods described above or the detectable substances described above, and an individual can be diagnosed as having AD or provided with prognostic information regarding an individual having or at risk of having AD. As described herein, such an individual may subsequently require and / or receive treatment for AD.

[0125] Aspects of the invention particularly relate to the treatment of Alzheimer's disease (AD) and other AD-related diseases and disorders, as well as other neurological diseases characterized by soluble amyloid proteins. Aspects of the invention also relate to methods of treating (including preventing) AD by administering to an individual in need thereof an effective amount of an antibody or composition described herein. The antibodies or compositions described herein, preferably pharmaceutical compositions (e.g., compositions comprising an antibody described herein, a pharmaceutically acceptable excipient, and optionally an additional therapeutic agent), can be used in methods of treating the human or animal body. The antibodies or compositions described herein, preferably pharmaceutical compositions, can be used in methods of treating the human or animal body, wherein the treatment is a therapeutic or prophylactic treatment of AD in an individual.

[0126] The above-mentioned methods of treatment can include administering to an individual an antibody or composition described herein (e.g., a composition comprising an antibody described herein, a pharmaceutically acceptable excipient, and an optional additional therapeutic agent) under conditions that result in a beneficial therapeutic response in the individual, e.g., for preventing or treating AD.

[0127] Such an individual may have AD. The methods of treatment described herein can be used in asymptomatic patients and patients currently exhibiting symptoms of AD. The antibodies described herein can be administered prophylactically to individuals not having AD. The antibodies described herein can be administered to individuals not having or not exhibiting symptoms of AD. The antibodies described herein can be administered to individuals having or appearing to have AD. Individuals amenable to treatment include individuals at risk of or predisposed to AD but not exhibiting symptoms and individuals suspected of having AD, as well as individuals currently exhibiting symptoms. The antibodies described herein can be administered prophylactically to the general population without any assessment of the risk to individual subjects. In some embodiments, individuals suitable for treatment described herein can include individuals having early-onset AD or one or more of its symptoms, as well as individuals in whom amyloid peptides are detected in a body fluid sample such as CSF.

[0128] The terms "treat", "treating", or "treatment" (or grammatically equivalent terms) refer to a reduction in the severity of an individual's condition or at least partial improvement or remission and / or achieving some alleviation, slowing, or reduction of at least one clinical symptom and / or inhibition or delay of disease progression, and / or prevention or delay of the onset of a disease or disorder.

[0129] The antibodies described herein that can be used in methods of treating AD can be antibodies of any sequence and format described herein that specifically bind to N-truncated amyloid peptide AβpE3-42 and / or Aβ4-42. The antibodies for use in the methods of treatment described herein can be fragments of the antibodies described herein, e.g., antigen-binding fragments. The antibodies described herein can be administered to individuals having AD.

[0130] The antibodies described herein can be administered to an individual in need of treatment with a drug carrier or a pharmaceutical composition or any composition described herein. Alternatively, the antibody can be administered to an individual by administering a polynucleotide encoding at least one antibody chain. The polynucleotide is expressed to produce the antibody chain(s) 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.

[0131] The antibodies described herein can be used in a method for preventing or treating AD, the method comprising administering to a patient an effective dose of the antibody described herein. As used herein, an “effective amount” or “effective dose” or “sufficient amount” (or a grammatically equivalent term) of a therapeutic antibody described herein refers to an amount of the antibody or composition described herein that effectively produces the desired effect, which is optionally a therapeutic effect (i.e., by administering a therapeutically effective amount). For example, an “effective amount” or “effective dose” or “sufficient amount” can be an amount such that the severity of the condition of the individual, such as AD, is reduced or at least partially improved or alleviated and / or at least some reduction, mitigation, or decrease 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.

[0132] The terms “patient,” “individual,” or “subject” include human and other mammalian subjects that receive prophylactic or therapeutic treatment with one or more of the reagents described herein (e.g., an immunotherapeutic agent or an antibody). Mammalian subjects include primates, such as 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.

[0133] The amount of the antibody or composition described herein sufficient to effect a therapeutic or prophylactic treatment is defined as an effective dose, e.g., a therapeutically or prophylactically effective dose. In prophylactic and therapeutic treatment regimens, the reagent can be administered in several doses until a sufficient immune response is obtained. The term “immune response” or “immunological response” includes the development of a humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secreted products) response to an antigen in a recipient subject. Typically, the immune response is monitored and repeated doses are given if the immune response begins to wane.

[0134] The effective dose of the composition described herein for treating the above-mentioned condition varies depending on many different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Generally, the patient is human, but non-human mammals, such as non-human primates, rabbits, rats, and mice, including transgenic mammals, can also be treated. The therapeutic dose needs to be titrated to optimize safety and efficacy.

[0135] For passive immunization with the antibodies described herein, the dosage ranges from about 0.01 to 100 mg / kg of the host body weight, more typically from 0.1 to 50 mg / kg. For example, the dosage can be at least 1 mg / kg body weight or at least 10 mg / kg body weight or in 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 in 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.

[0136] The methods described herein can include administering the antibodies to a subject in a single dose, two doses, or multiple doses. The dosage of the antibodies can be about 100 μg / kg to 100 mg / kg of the patient body weight, about 300 μg / kg to 60 mg / kg of the patient body weight, or about 10 mg / kg to 50 mg / kg of the patient body weight. Such dosages can be administered to the subject daily, every other day, weekly, or according to any other schedule determined by empirical analysis. The treatment can include administration in multiple doses over an extended period, such as at least six months. Other treatment regimens can include administration once every two weeks, once a month, or once every 3 to 6 months. Exemplary dosage regimens include 1 - 20 mg / kg or 15 mg / kg for several consecutive days, 30 mg / kg every other day, or 60 mg / kg weekly.

[0137] The antibodies described herein can be administered in a variety of circumstances. The time interval between single doses can be weekly, monthly, or annually. The time interval can also be irregular, as indicated by measuring the blood level 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, and in some methods, 25 - 300 μg / ml. Alternatively, the antibodies described herein can be administered as a sustained - release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half - life of the antibody in the patient. Generally, humanized antibodies exhibit longer half - lives than chimeric and non - human antibodies.

[0138] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a composition or a mixture thereof containing the antibodies described herein is administered to a patient who is not yet in a diseased state to enhance the patient's resistance. This amount is defined as a "prophylactically effective dose". In this use, the exact dosage again depends on the patient's health and systemic immunity, but generally ranges from 0.1 to 25 mg per dose, particularly from 0.5 to 2.5 mg per dose. Relatively low doses are administered at relatively infrequent time intervals over a long period.

[0139] The dosage range of the nucleic acid encoding the antibodies described herein is from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg or 30 - 300 μg DNA per patient. The dosage of the infectious viral vector is 10 - 100 or even more viral particles per dose.

[0140] As described herein, the antibodies and compositions described herein can be administered by parenteral, topical, intravenous, oral, gastric, subcutaneous, intra - arterial, intracranial, intraperitoneal, intranasal or intramuscular methods for therapeutic and / or prophylactic treatment. Intramuscular injection or intravenous infusion is preferred for the administration of antibodies.

[0141] Other aspects and embodiments described herein provide the term "comprising" replaced by the term "consisting of" for the above - mentioned aspects and embodiments, and provide the term "comprising" replaced by the term "consisting essentially of" for the above - mentioned aspects and embodiments.

[0142] It should be understood that, unless the context otherwise requires, this application discloses all combinations of any one of the above - mentioned aspects and embodiments with each other. Similarly, unless the context otherwise requires, this application discloses all combinations of the preferred and / or optional features, either alone or in combination with any other aspect.

[0143] After reading this disclosure, modifications of the above - mentioned embodiments, other embodiments and their variations will be apparent to those skilled in the art, and thus, these are all within the scope described herein.

[0144] All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

[0145] The positions of the antibody residues described herein are numbered according to the scheme set forth in Kabat, E.A., Wu, T.T., Perry, H.M., Gottesmann, K.S & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication no. 91 - 3242. U.S. Department of Health and Human Services. Where appropriate, the positions of substitutions can be described relative to the invariant Kabat - numbered residues in the immunoglobulin sequence. Alternative antibody numbering schemes are described in Honegger, A and Plückthun A. (2001) J. Mol. Biol 309, 657 - 67.

[0146] As used herein, "and / or" shall be understood to mean the specific disclosure of each of two designated features or components with or without the other. For example, "A and / or B" shall be regarded as the specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were listed separately herein.

[0147] Experiment

[0148] Materials and Methods

[0149] 1. Preparation of RNA from hybridoma cells.

[0150] Thomas Bayer provided a frozen precipitate of mouse hybridoma cells NT4X-167 stored at -80 °C and processed using the Qiagen Rneasy kit according to the manufacturer's protocol to isolate RNA.

[0151] 2. First-strand cDNA synthesis

[0152] NT4X-167 RNA (approximately 26 μg) was reverse transcribed using the GE Life Sciences First Strand cDNA Synthesis kit according to the manufacturer's protocol to generate cDNA. This operation was repeated twice to generate 3 independent NT4X-167 cDNA products (rounds 1, 2, and 3) to detect and avoid cDNA mutations induced by reverse transcriptase.

[0153] 3. cDNA sequencing

[0154] NT4X-167 cDNA was amplified by PCR in 3 separate reactions. Immunoglobulin cDNA was PCR amplified using the PhusionFlash High-Fidelity PCR Master Mix with either κ light chain primers plus MKC or heavy chain primers plus MHC mixtures. The result of each PCR reaction was a single amplified product, which was purified using the QIAquick PCR Purification kit and sequenced in both directions (by GATC Biotech) using M13-forward and M13-reverse primers to obtain three sets of independent sequence information for each immunoglobulin chain.

[0155] 4. VK and VH NT4X-167 DNA sequence

[0156] The consensus DNA sequences of the NT4X-167VK PCR products were named NT4X-167VK, and the consensus DNA sequences of the NT4X-167VH PCR products were named NT4X-167VH, which are shown in SEQ ID NO: 9-12 respectively. Germline analysis of the NT4X-167 sequences indicated that the κ light chain was murine MKV4 and the heavy chain was murine MHV7.

[0157] 5. Construction of chimeric NT4X-167 expression vector

[0158] Construction of the chimeric expression vectors required cloning the amplified variable regions into the IgG / κ vectors (pHuK and pHuG1) using ligation-independent cloning (LIC). The vectors (pCMV modified) were digested with BfuA1 (BspM1), and then compatible overhangs were generated using the 3'-5' exonuclease activity of T4 DNA polymerase (+dATP). The variable regions were first amplified by PCR of the NT4X-167 cDNA, and primers containing the 3' end of the leader sequence (present in most of the sequences in the vector) – forward primers – or the start of the constant region (IgG1 or κ) – reverse primers – followed by the start of the variable region (in each direction) were used to generate the antibody sequences. Complementary overhangs were generated in the PCR products by treatment with T4 DNA polymerase +dTTP. The vectors and inserts were incubated at room temperature, transformed into chemically competent TOP10 bacteria, and plated on kanamycin plates. Several clones were isolated, and colonies were screened by PCR using the primers HCMVi and HuG1 LIC Rev for VH or HuK LIC Rev for VK. Clones that produced PCR products of the correct size were selected, miniprepped using the QIAGEN kit, and sequenced using the same primers.

[0159] 6. Generation of chimeric antibodies

[0160] Expi293 suspension cells grown in Expi293 transfection medium and antibiotics were co-transfected with cNT4X-167VH.pHuG1 and cNT4X-167VK.pHuK (1 μg DNA each) using ExpiFectamine 293 reagent. The cells were grown in 1 mL of growth medium for 5 days. Chimeric NT4X-167 antibodies up to 81 μg / mL were measured in the conditioned medium by ELISA.

[0161] 7. Amyloid peptides

[0162] Amyloid peptides, Aβ1-42, AβpE3-42, and 4-42 were purchased from Peptide Speciality Laboratories (PSL) or California peptides.

[0163] 8. Transgenic mice

[0164] The transgenic homozygous mouse lines Tg4-42hom (hereinafter referred to as Tg4-42) and 5XFAD used in this study have been described previously 1 , 2 .

[0165] 10. Passive immunization

[0166] The potential therapeutic effects of reverse-cloned (rc) humanized NT4X (rcNT4X_SA and rcNT4X_S7A) antibodies were investigated in Tg4-42 and 5XFAD mice using passive immunization. Passive immunization was performed by intraperitoneal injection, and antibodies of the same immunoglobulin class as the two rcNT4X antibodies (IgG1, MRCT control antibody) were used for comparison with the control group.

[0167] Male and female Tg4-42 mice were immunized by injecting 10 mg / kg body weight of the antibody diluted in sterile PBS (pH 7.4). Mice received injections weekly starting at three months of age. Each mouse received a total of 12 injections. Behavioral tests were performed between the 10th and 11th injections. Animals were sacrificed after the last injection. The control group received intraperitoneal injection of IgG1 MRCT control antibody (10 mg / kg body weight). Animals were sacrificed at six months of age after the last injection.

[0168] 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 at 18 weeks of age after the last injection.

[0169] The control group was treated in the same manner as the treatment group.

[0170] 11. Spatial reference memory in Morris water maze

[0171] As previously described, the Morris water maze 3 was used to evaluate the spatial reference memory of Tg4-42 mice 2 .

[0172] 12. Quantification of neuron number using unbiased stereology

[0173] Stereological analysis was performed as previously described 2 , 4. Define the CA1 hippocampal cell layer (from -1.22 to -3.52 mm anterior to bregma) on cresyl violet-stained sections, and analyze using a stereology workstation (Olympus Bx51, with a motorized sample stage for automatic sampling), StereoInvestigator 7 (MicroBrightField, Williston, USA), and a 100x oil immersion objective (NA = 1.35).

[0174] 13. Immunohistochemistry and histology

[0175] Process mouse tissue samples as described above 5 . For plaque load staining, use the following antibodies: Antibody 1-57 (pyroglutamate Aβ3-x, 1:5000, mouse monoclonal 5 ), Antibody 80C2 (anti-Aβ1-X, Synaptic 1:500, mouse monoclonal), polyclonal antibody 24311 (anti-pan-Aβ, 1:500, rabbit 2 ), and polyclonal antibody 029 (anti-Aβ4-x; 1:500; guinea pig). Biotinylated anti-rabbit and anti-mouse secondary antibodies (1:200) were purchased from DAKO. Use the Vectastain kit (Vector Laboratories), use diaminobenzidine as the chromogen, and visualize the staining using the ABC method. Counterstain with hematoxylin. For DAPI staining, deparaffinize the sections and wash in PBS, then incubate in 4’,6-diamidino-2-phenylindole (DAPI, 1 μg / ml) for 1 min. For thioflavin S, deparaffinize and rehydrate the fluorescently stained tissue sections, wash twice in deionized water treated with 1% (w / v) aqueous solution of thioflavin S, and counterstain in 1% (w / v) aqueous solution of 4’,6-diamidino-2-phenylindole. Mount in an aqueous fluorescence mounting medium (DAKO).

[0176] 14. Quantification of Aβ load

[0177] Quantify the plaque load in 5XFAD mice. For each animal, three paraffin-embedded sections are at least 40 μm apart from each other. Evaluate the relative plaque load 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 are taken systematically. Use ImageJ to binarize the pictures into 8-bit black and white pictures, and apply a fixed intensity threshold to define DAB staining. Measure the percentage of the area covered by DAB staining, as well as the number of grains per mm 2 and the average size of the grains.

[0178] 15. Statistical analysis

[0179] Differences between groups were tested using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons, ANOVA followed by Dunnett multiple comparisons, or the indicated student’s t-test. All data were presented as the indicated mean ± standard error of the mean (SEM). All statistics were calculated using GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego, CA, USA).

[0180] 16. Research approval

[0181] Animal experiments were approved by the local animal protection authority ( Landesamt für Verbraucherschutz und Lebensmittelsicherheit) with approval number 17 / 2447. Experiments were conducted according to the approved protocol.

[0182] 17. ELISA

[0183] Each well of a 96-well MaxiSorp plate (Nunc) was coated with 50 μL aliquots of 1-42, pE3-42, or 4-42 amyloid peptides 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 of a 5% milk solution in PBS / 0.05% Tween 20 per well. The wells were then incubated with shaking for 1 h at room temperature and washed three times with PBS-T (0.1% Tween 20). Fifty microliters of primary antibody serially diluted in 1% milk PBS / 0.05% Tween 20, starting at approximately 100 μg / mL, was added to the wells of the assay plate. The incubation and washing steps were then repeated. Anti-human κ-chain HRP (Sigma A7164-1 mL) 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 to each well and incubated for 5–10 min at room temperature. The reaction was terminated by adding 50 μL of RED STOP solution (Neogen) to each well, and the optical density was read at 650 nm.

[0184] Results

[0185] Generation of chimeric versions of NT4X-167 antibody

[0186] The binding of 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. In the ELISA assay, the chimeric NT4X-167 antibody bound to the AβpE3-42 peptide and did not bind to Aβ1-42 or 4-42 peptides with an EC Figure 1 value comparable to that of the murine NT4X-167 antibody ( 50 ). Figure 2 )

[0187] To further characterize the binding of murine and chimeric NT4X-167 antibodies to amyloid peptides, SPR analysis was performed using a Biacore T200 (GE Healthcare). The chimeric NT4X-167 antibody bound to AβpE3-42 and 4-42 peptides but did not bind to Aβ1-42 with an apparent K D value comparable to that of the original murine NT4X-167 antibody. A humanized version of the anti-NT4X-167 antibody was designed using the NT4X-167 sequence.

[0188] Design of NT4X-167 humanized antibody variants

[0189] Human VH and VK cDNA databases

[0190] from the International ImMunoGeneTics database 2009 9 and the Kabat database version 5 of the protein sequences of human and murine immunoglobulins and the protein sequences of immunological significance (latest update 17-Nov-1999) 8 were used to compile the database for human immunoglobulin sequences in the Kabat alignment. Our database contains 10,406 VH and 2,894 VK sequences.

[0191] Molecular model of NT4X-167

[0192] The homology model of the variable region of the murine NT4X-167 antibody was calculated. As determined by Blast analysis of the Accelrys antibody pdb structure database, the atomic coordinates of 2DQU_L.pdb and 1WEJ_H.pdb were the highest scoring sequence templates for VL and VH, respectively, and the atomic coordinates of 1YNL_LH.pdb were the highest scoring overall (interface) sequence template. These templates were used to generate 20 initial models; the highest scoring model was refined by modeling each CDR loop using its 5 best loop templates. The consensus of the residues within the CDR loops was determined using the twenty final models.

[0193] Human framework selection​

[0194] Query the human VH and VK databases with NT4X-167VH and VK protein sequences using various selection criteria. Identify the FW residues within the CDR residues (Kabat definition) in the mouse NT4X-167 antibody structure and name them " adjacent residues". Adjacent residues

[0195] Remove humanized sequences and incomplete sequences from the analysis. Select the sequence AF062228 as a human heavy chain donor candidate. This sequence scored highly in sequence identity and similarity and had no somatic mutations from its germline. AF062228 had 8 adjacent residue changes (Tables 1 and 2).

[0196] Similarly, the sequence AY942002 was selected as a human κ 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 viewed were very similar, but AY942002 showed better framework identity and similarity to NT4X_VK. AY942002 had no somatic mutations in its germline and had three potential adjacent residue changes (Table 4).

[0197] Design of NT4X-167RHA and RHB

[0198] Since a suitable human framework has been identified, synthetic protein and DNA sequences can be designed. The initial design of the humanized version of NT4X-167 was to transplant CDR 1, 2, and 3 from NT4X-167VH into the recipient FW of AF062228, thus creating variant NT4X-167RHA. Then, the 8 adjacent residues were back-mutated to the mouse equivalent residues, thus creating variant NT4X-167RHB, and one mutation at a time in the following variants: The sequences were assembled on the computer and designated as NT4X-167RHA to NT4X-167RHJ. Tables 1-3 compare the mouse and humanized versions of the NT4X-167VH protein sequence. All humanized variants were cloned into the pMoG1 and pMoK vectors such that antibodies could be purified from these constructs for in vivo studies in many mouse models (5XFAD and Tg4-42). The final lead humanized candidate will be cloned into the pHuG4 and pHuK vectors.

[0199] Design of NT4X-167RKA and NT4X-167RKB

[0200] The framework of AY942002 is used for the DNA and protein of designing humanized constructs. It shows that CDR 1, 2, and 3 from NT4X-167VK are joined to the acceptor FW of AY942002 to generate the initial version of humanized NT4X-167RKA. There are 3 unmatched adjacent residues in NT4X-167RKA, and these residues are back-mutated to the equivalent mouse residues in variant NT4X-167RKB. These residues are back-mutated one by one in the following variants: the sequences are assembled on the computer and designated as NT4X-167RKA to NT4X-167RKE (Table 4).

[0201] Production of NT4X-167 humanized antibody

[0202] The genes of NT4X-167HA, HB, KA, and KB were synthesized by GenScript. The natural human framework sequences AF062228 and AY942002 are the heavy and light chains respectively, and the natural mouse CDR sequences are assembled on the computer and designated as NT4X-167RHA to NT4X-167RHJ and NT4X-167RKA to NT4X-167RKE respectively. Using GenScript's proprietary software algorithm, the sequences of RHA / RHB and RKA / RKB were optimized by silent mutagenesis to use the codons preferentially used and synthesized by human cells. The RKA / RKB and RHA / RHB constructs were PCR amplified with specific primers to the expression vector + insert (as described previously for the chimeric version), and inserted into pMoK and pMoG1 respectively in a ligation-independent cloning reaction, and used to transform TOP10 bacteria. Subsequently, version HA was modified by PCR mutagenesis to obtain other humanized variants annotated in Table 4.

[0203] The clones were sequenced, and plasmid DNA was prepared using the QIAGEN Plasmid Miniprep Kit or Qiagen Plasmid Maxiprep Kit. The expression construct sequences (HA, HB, KA, and KB) are shown as SEQ ID NO:13-20. The expression plasmid preparations encoding (humanized or chimeric) VH and VK were used to transfect Expi293 cells, cultured in serum-free medium for 5-7 days, and then the conditioned medium containing the secreted antibody was harvested.

[0204] Antibody expression

[0205] The concentration of IgG 1 κ antibody in the Expi293 cell conditioned medium was measured by ELISA. Most antibodies were produced at good expression levels.

[0206] Antigen binding of initial versions (round 1 and round 2) of humanized NT4X-167 antibody

[0207] Figure 3 The data shown in Figure 3 show the binding of the RHA / RHB heavy chains in combination with the RKA / RKB light chain versions of the humanized NT4X-167 antibody to amyloid peptides Aβ1-42 and AβpE3-42. No difference was observed in the binding between the versions containing the κ light chain in the RKA or RKB versions, meaning that the reverse mutations introduced in KB are not essential for binding. Version RHA did not show evidence of binding to amyloid peptides, nor did the humanized versions containing the RHB version that binds to AβpE3-42. Considering this data, additional versions of the humanized heavy chain were synthesized using only the KA light chain forward and the Stratagene mutagenesis kit QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene), generating versions NT4X RHC-RHJ (Table 1). The results of the binding ELISA using the humanized versions RHC-RHJ against the AβpE3-42 peptide are as Figure 5 shown. The humanized versions RHB / RKA and RHB / RKB bind to the AβpE3-42 peptide, while the other humanized versions did not show signs of binding, and a third round of humanized variants was synthesized.

[0208] Antigen binding of third and fourth round humanized NT4X-167 antibodies

[0209] A third round of humanized NT4X-167 heavy chain variants, RHK to RHR, was generated (Table 2). The RHK-RHR variants were obtained using the Stratagene mutagenesis kit (QuikChange Lightning Site-Directed Mutagenesis Kit).

[0210] The RHC-RHR heavy chains were combined with the light chain of the RKA version and binding ELISA was performed using the AβpE3-42 peptide ( Figure 6)。The humanized versions RHB, RHM, RHN, RHO, and RHR in combination with RKA bind to the AβpE3-42 peptide, where RHB / RKA, RHM / RKA, RHN / RKA, RHO / RKA, and RHR / RKA are the best binders. Four of the eight key heavy-chain CDR framework residues in RHP (arginine reverse mutated to valine by SDM), RHQ (valine reverse mutated to phenylalanine), RHK (phenylalanine reverse mutated to glycine), and RHL (leucine reverse mutated to isoleucine) showed reduced binding, indicating that these four residues should be retained as mouse residues for full binding. Other humanized variants were generated in which all four mouse residues represented by RHP, RHQ, RHK, and RHL were incorporated, and the four key framework residues represented by RHM, RHN, RHO, and RHR were maintained as human framework residues to generate versions RHS, RHT, RHU, RHV, RHW, RHX, and RHY (Table 2). Peptide-binding ELISA using AβpE3-42 showed that variants RHS to RHY had PSL AβpE3-42 binding characteristics similar to RHB / RKA ( Figure 8 )。In terms of the number of "human" key CDR framework residues it contains, the RHS / RKA variant is preferred (lower immunogenicity).

[0211] Antigen binding of humanized SA, S6A, S7A and S8A NT4X-167 antibodies

[0212] Other variants of RHS / KA (SA) were generated to achieve >85% identity with the human germline. IMGT domain gap analysis was performed on the SA version to identify residues that could be mutated to increase the percentage of identity with the human germline. The RKA light chain has 89.6% identity with the human germline IGKV1-39*01 and IGKJ4*01 sequences. However, the RHS heavy chain has 79.4% identity with the human germline IGHV4-4*08 sequence. Germline analysis of the RHS sequence identified six residues that could be reverse mutated to the human germline and, in combination with RKA, generated versions RHS6, RHS7, and RHS8. Peptide-binding ELISA using AβpE3-42 showed that variant RHS7RKA (S7A) is the best humanized candidate and has 84.5% identity with the human germline.

[0213] Antigen binding of additional variants generated based on the crystal structure of NT4X-167 antibody

[0214] Based on the crystal structure of the pE3-14 peptide in complex with murine NT4X FAB, additional variants of RHS7 were generated to increase the percentage identity with the human germline. The crystal structure highlighted F67, Y68, and I39 as potential amino acids that could be altered without affecting peptide binding. Accordingly, additional variants of F67Y, Y68N, I39W, and the combined F67Y and Y68N were generated (Table 2) and studied for binding to Aβ1-42, AβpE3-42, and 4-42. RHS71 with the F67Y mutation retained binding characteristics equivalent to the parental S7A heavy chain variant and was thus selected as a potential heavy chain humanized variant to be combined with the RKA light chain. Since the affinities of these antibodies were in the nM range, we wanted to investigate whether it was possible to predict, based on the crystal structure and Schrodinger modeling prediction software, the amino acids that we could mutate to increase the affinity of the RHS71 / RKA antibody. Five additional variants of the heavy chain were generated with the following mutations: S53M, S53H, R100H, L103R, and L103H (Table 4b). Additionally, five variants of the light chain were 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 studied by ELISA( Figure 9 ) and Biacore. Among all the variants tested, the combination of RHS71 (which contains the F67Y mutation in the heavy chain) and RKH (which contains the N92H mutation) showed a two-fold improvement on Biacore.

[0215] Thermal stability of humanized BA, SA, TA, UA, VA, WA, YA candidate antibodies to high temperature

[0216] The purpose of this experiment was to test the thermal stability of the humanized antibodies when subjected to higher temperatures, from 30 °C to 85 °C for 10 minutes, cooled to 4 °C, and used in ELISA assays at each candidate EC 80 concentration. All humanized versions were stable, retaining their ability to bind to the AβpE3-42 peptide until the binding decreased at 75 °C.

[0217] Determination of Tm (melting temperature) of humanized NT4X-167-SA and NT4X-167-S7A candidate antibodies

[0218] To determine the melting temperatures of the lead candidate antibodies NT4X-167-SA and NT4X-167-S7A, these antibodies were tested in a thermal shift assay. In a qPCR thermocycler, the samples were incubated with a fluorescent dye (Sypro Orange) for 71 cycles, increasing 1 °C per cycle. The Tm of the humanized antibodies was calculated to be 66 - 67 °C.

[0219] Aggregation of humanized NT4X-167-SA and NT4X-167-S7A candidate antibodies

[0220] The sample was injected into the size exclusion chromatography column of the HPLC system at 0.4 mL / min and analyzed by multi-angle light scattering to determine the absolute molar mass and to check for aggregation (see Figure 10 ). The chromatogram did not show signs of aggregation. The average molecular weight of NT4X-167_SA was approximately 133.98 kDa and that of NT4X-167_S7A was approximately 129.92 kDa, which is the expected range for IgG monomers in this analytical setup. The antibody was monodisperse (Mw / Mn < 1.05). The mass recovery was 100% (the calculated mass was higher than the injected mass), indicating good protein recovery, and the sample did not appear to adhere to the column or contain insoluble aggregates that would be retained by the guard column. Overall, the data indicate that the humanized NT4X-167_SA and NT4X-167_S7A antibodies do not have aggregation problems.

[0221] Non-specific protein-protein interaction (CIC)

[0222] Cross-interaction chromatography using large amounts of purified human polyclonal IgG is a technique for monitoring non-specific protein-protein interactions and can be used to distinguish soluble and insoluble antibodies (Section 8.19). A higher retention index (k') indicates a tendency for self-interaction and low solubility. The humanized NT4X-167RHS / RKA, RHB / RKA, and RHS7 / RKA antibodies (cloned as MoG1K) showed a retention index below 0.2, indicating a lower tendency for non-specific interactions and good solubility ( Figure 11 ).

[0223] Solubility of humanized NT4X-167RHS / RKA and RHS7 / RKA candidate antibodies

[0224] The humanized NT4X-167RHS / RKA (SA) and RHS7 / RKA (S7A) antibodies were concentrated using a solvent absorption concentrator (MWCO 7500 kDa), and the concentration was measured at certain time intervals. The antibody was concentrated to >50 mg / mL with no obvious precipitation.

[0225] Freeze / thaw stress analysis of humanized NT4X-167RHS / RKA and RHS7 / RKA candidate antibodies

[0226] Samples of the purified candidate antibodies were subjected to 10 cycles of 15 minutes at -80 °C and then melted at room temperature for 15 minutes. The samples were then analyzed by SEC-MALS to check for aggregation ( Figure 12 and 13 ). The data indicate that freeze / thaw does not cause aggregation in the humanized NT4X-167 antibody.

[0227] Thermal-induced stress analysis of humanized NT4X-167RHS / RKA and RHS7 / RKA candidate antibodies

[0228] The purified candidate antibody samples were exposed at a) 4 °C, b) 25 °C, c) 37 °C, and d) 50 °C for 30 days. The samples were then analyzed by SEC-MALS to examine aggregation ( Figure 14 ). Overall, the data indicate no aggregation issues in the humanized NT4X-167 antibody.

[0229] Serum stability assessment of humanized NT4X-167RHS / RKA and RHS7 / RKA candidate antibodies

[0230] The purified samples of humanized NT4X-167RHS / RKA and RHS7 / RKA antibodies were incubated in mouse, human, and cynomolgus monkey sera. The binding ability of the antibodies after incubation was measured by binding ELISA to AβpE3-42 and 4-42 peptides. The binding of the NT4X-167 humanized antibody that had been incubated in 3 different sera was compared to the antibody binding that had not undergone any incubation and the antibody that had been incubated in PBS. The ELISA assay showed that the serum-incubated antibodies bound to AβpE3-42 and 4-42 peptides very similarly to the PBS-incubated and non-incubated antibodies. Thus, the humanized NT4X-167RHS / RKA and RHS7 / RKA antibodies retained their binding ability after 30 days of incubation in mouse, human, and cynomolgus monkey sera.

[0231] The humanized NT4X-167 was shown to be able to bind amyloid peptide 4-42, AβpE3-42 and not bind Aβ1-42. The humanized antibody also showed a protective effect against neuronal cell death in rat and human neurons. The antibody was engineered and expressed as a fully humanized antibody without significant loss of binding potency. Experiments using chimeric antibodies consisting of murine variable regions on human constant regions showed similar or improved titers in binding ELISA or kinetic studies using Biacore ( Figure 1 and 2 ).

[0232] Initial experiments showed that the fully humanized NT4X-167, without introducing murine framework mutations of neighboring residues, did not bind to the AβpE3-42 peptide nor to the chimeric positive control antibody, but the mutated version with the complete set of binding had the same binding as the chimeric positive control. This reduction in binding was isolated to the fully humanized heavy chain. However, we unexpectedly found that the introduction of specific back mutations enabled us to generate two lead candidate antibodies, NT4X RHS / RKA (SA) and NT4XRHS7 / RKA (S7A). These lead candidates have also been cloned into HuG1K and HuG4K vectors as well as the initial MoG1 vector. Both candidates showed excellent binding, expression, thermal stability, affinity, and functional activity.

[0233] In vitro cell assays

[0234] Neuron protection by NT4X_SA and NT4X_S7A humanized antibodies in rat and human primary cortical cultures

[0235] It was found that the humanized antibodies NT4X_SA and NT4X_S7A retained the ability of the original murine NT4X antibody to protect against N-truncated amyloid peptides (4-42 and pyroGul3-42; Figure 17 and 18 )-induced cell death in rat neurons, but not against the full-length amyloid peptide Aβ1-42 ( Figure 19 ). All three antibodies were of comparable potency against the 4-42 peptide, but murine NT4X was slightly more potent than SA or S7A against pyro3-42, and S7A was slightly more potent than SA.

[0236] It was found that the humanized antibodies NT4X_SA and NT4X_S7A retained the ability of the original murine NT4X antibody to protect against N-truncated amyloid peptides (4-42 and pyroGul3-42; Figure 20 and 21 )-induced cell death, but not against the full-length amyloid peptide Aβ1-42 in human neurons ( Figure 22 ). All three humanized antibodies were more effective than the original murine NT4X antibody in protecting against 4-42 peptide-induced cell death, whereas the N92H humanized antibody was more effective against pyro 3-42. Neither the NT4X antibody nor the humanized versions protected human neurons from Aβ1-42 amyloid peptide-induced death. The results were largely consistent with those in rat neurons, with an increase in potency against pyro3-42 in human neurons.

[0237] In vivo testing in transgenic mouse models

[0238] Treatment of Alzheimer's disease using rcNT4X_SA and rcNT4X_S7A in 5XFAD and Tg4-42 mouse models

[0239] 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 a higher therapeutic effect for rcNT4X_S7A. rcNT4X_S7A was additionally tested for spatial reference memory performance in the Morris water maze test. The spatial reference memory deficit in Tg4-42 at six months of age was completely rescued.

[0240] 5XFAD mice were immunized for 12 weeks starting at 6 weeks of age. The effect on plaque burden was analyzed in the cortex. rcNT4XSA reduced thioflavin-stained plaques and N-terminal specific antibodies against pyroglutamate Aβ3-X and Aβ4-X. No effect was seen with antibodies against pan-Aβ and Aβ1-X. Compared to mice immunized with rcNT4X_S7A, significant plaque reduction was shown in all staining assays: plaques stained with thioflavin or antibodies recognizing Aβ1-X, pyroglutamate Aβ3-X, Aβ4-X, and pan-Aβ were significantly reduced.

[0241] rcNT4X_SA and rcNT4X_S7A rescue neuronal loss and memory decline in Tg4-42 mice

[0242] Loss of CA1 neurons in the hippocampus of Tg4-42 mice was evident at 4 months of age 6 . Therefore, we initiated passive immunotherapy starting at three months for 12 weeks. Compared to the IgG1 control group, Tg4-42 mice immunized with rcNT4X_SA and rcNT4X_S7A showed significantly more neurons. The rcNT4X_S7A group had a higher significance level ( Figure 23 ). Compared to NT4X, the potency of immunizing Tg4-42 with rcNT4X_S7A was significantly higher ( Figure 24 ). There was no difference in the number of neurons between the original NT4X and rcNT4X_SA. The data on immunization with rcNT4X_SA and rcNT4X_S7A were plotted relative to immunization with the original murine NT4X antibody. There were no significant differences in the control groups injected with IgG1, IgG2b, and PBS ( Figure 25 ). The data on IgG2ba and PBS were from Antonios et al. 6 . Passive immunization with rcNT4X_S7A completely rescued the spatial reference memory deficit in Tg4-42 mice in the Morris water maze test ( Figure 26 ).

[0243] rcNT4X_SA and rcNT4X_S7A reduce plaque burden in 5XFAD mice

[0244] 5XFAD mice were treated between 6 and 18 weeks of age. Passive immunization with the two rcNT4X antibodies reduced the plaque burden of different Aβ species compared to the isotype control IgG1 antibody. rcNT4X significantly reduced plaques stained for pyroglutamate Aβ3-x, Aβ4-x, and thioflavin. No effect was detected in Aβ1-x and pan-Aβ positive plaques. The plaque-reducing effect of rcNT4X_S7A was significantly altered as plaques were positive for pyroglutamate Aβ3-x, Aβ4-x, and thioflavin, but the positivity for Aβ1-x and pan-Aβ was reduced ( Figure 27 ).

[0245] Tg4-42 mice exhibit severe hippocampal neuronal loss and spatial reference memory deficits 2 , 6 . The Tg4-42 model represents the first mouse model expressing only the N-truncated Aβ4-42. At six months of age, this model is characterized by a substantial loss of spatial reference memory evaluated by the Morris water maze test and a large number of degenerated CA1 neurons in the hippocampus of Tg4-42 mice, which can be rescued by passive immunization with the antibody NT4X 6 . In this study, we used a novel humanized version of the NT4X antibody cloned on the murine IgG1 backbone. Twelve-week rcNT4X_S7A passive immunization starting from three months of age also rescued the spatial reference memory deficits in Tg4-42 mice. Moreover, compared with the group of Tg4-42 animals treated with IgG1, the number of CA1 neurons in the hippocampus was significantly rescued. Interestingly, comparing the treatment effects of NT4X, rcNT4X_SA, and rcNT4X_S7A, Tg4-42 mice exposed to rcNT4X_S7A were significantly higher than NT4X. Therefore, we hypothesize that rcNT4X_S7A has the highest potency among different versions of NT4X.

[0246] Sequence

[0247]

[0248] SEQ ID NO:1 RHA sequence

[0249]

[0250] SEQ ID NO:2: RHA sequence with 27F, 29L, 63R, 70V, and 52BX 1 , 53X 2 , 54X 3 , 55X 4 , 56X 5 and 52CX 6 where X 1 is D or N, X 2 is A, N, or P, X 3 is A or S, X 4 is F or L, X 5 is I or K, and X 6 is F or Y.

[0251]

[0252] SEQ ID NO:3 RHS sequence

[0253]

[0254] SEQ ID NO:4 RHS7 sequence

[0255]

[0256] SEQ ID NO:5 RHS71 sequence

[0257]

[0258] SEQ ID NO:6 with 92X 7 RKA sequence, where X 7 is N, H, Y or W

[0259]

[0260] SEQ ID NO:7 RKA sequence

[0261]

[0262] SEQ ID NO:8 RKH sequence

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274] References

[0275] 1. Oakley, H., et al J Neurosci 2006, 26, 10129 - 10140

[0276] 2. Bouter, Y., et al Acta Neuropathol 2013, 126, 189 - 205

[0277] 3. Morris, R. J Neurosci Methods 1984, 11, 47 - 60.

[0278] 4. Jawhar, S., et al Neurobiol Aging 2012, 33, 196.e129–196.e140.

[0279] 5. Wirths, O., et al J Neural Transm 2010, 117, 85 - 96

[0280] 6. Antonios, G., et al Scientific reports 2015, 5, 17338. doi:10.1038 / srep17338

[0281] 7. Wittnam, J. L., et al J Biol Chem 2012, 287, 8154 - 8162

[0282] 8. Kabat, E. A., et al. Sequences of Proteins of Immunological Interest. 5ed. NIH National Technical Information Service. (1991) 1 - 3242.

[0283] 9. Lefranc, M.-P., et al.. Nucl. Acids Res. (2015) 43(D1): D413 - D422. doi:10.1093 / nar / gku1056

[0284] Other statements of the present invention:

[0285] The following numbered statements of the invention are part of the specification;

[0286] 1. An antibody, said antibody comprising a heavy chain variable domain and a light chain variable domain, wherein

[0287] a) the heavy chain variable domain (VH domain) comprises SEQ ID NO:2 having four or fewer additional alterations such as substitutions in the framework region, and

[0288] b) The light chain variable domain (VK domain) comprises SEQ ID NO:6 having four or fewer additional alterations such as substitutions in the framework regions.

[0289] 2. The antibody according to claim 1, wherein the antibody binds amyloid peptide AβpE3-42 and Aβ4-42 and does not bind amyloid peptide Aβ1-42.

[0290] 3. The antibody according to any one of the preceding claims, wherein the antibody binds amyloid peptide AβpE3-42 with a binding affinity of at least 85% of the binding affinity of the murine NT4X-167 antibody for amyloid peptide AβpE3-42, as measured by ELISA.

[0291] 4. The antibody according to any one of the preceding claims, wherein the VH domain comprises SEQ ID NO:2.

[0292] 5. The antibody according to any one of the preceding claims, wherein the VL domain comprises SEQ ID NO:6.

[0293] 6. The antibody according to any one of the preceding claims, wherein the heavy chain variable domain comprises SEQ ID NO:2, wherein X1 (Kabat position 52B) is D.

[0294] 7. The antibody according to any one of claims 1 to 5, wherein the heavy chain variable domain comprises SEQ ID NO:2, wherein X1 (Kabat position 52B) is N.

[0295] 8. The antibody according to any one of the preceding claims, wherein the heavy chain variable domain comprises SEQ ID NO:2, wherein X2 (Kabat position 53) is A.

[0296] 9. The antibody according to any one of claims 1 to 7, wherein the heavy chain variable domain comprises SEQ ID NO:2, wherein X2 (Kabat position 53) is P.

[0297] 10. The antibody according to any one of the preceding claims, wherein the heavy chain variable domain comprises SEQ ID NO:2, wherein X3 (Kabat position 54) is A.

[0298] 11. The antibody according to any one of claims 1 to 9, wherein the heavy chain variable domain comprises SEQ ID NO:2, wherein X3 (Kabat position 54) is S.

[0299] 12. The antibody according to any one of the foregoing statements, wherein the heavy chain variable domain comprises SEQ ID NO: 2, wherein X4 (Kabat position 55) is F.

[0300] 13. The antibody according to any one of statements 1 to 11, wherein the heavy chain variable domain comprises SEQ ID NO: 2, wherein X4 (Kabat position 55) is L.

[0301] 14. The antibody according to any one of the foregoing statements, wherein the heavy chain variable domain comprises SEQ ID NO: 2, wherein X5 (Kabat position 56) is I.

[0302] 15. The antibody according to any one of statements 1 to 13, wherein the heavy chain variable domain comprises SEQ ID NO: 2, wherein X5 (Kabat position 56) is K.

[0303] 16. The antibody according to any one of the foregoing statements, wherein the heavy chain variable domain comprises SEQ ID NO: 2, wherein X6 (Kabat position 52C) is F.

[0304] 17. The antibody according to any one of statements 1 to 15, wherein the heavy chain variable domain comprises SEQ ID NO: 2, wherein X6 (Kabat position 52C) is Y.

[0305] 18. The antibody according to any one of statements 1 to 5, wherein the heavy chain variable domain comprises SEQ ID NO: 3 having four or fewer additional substitutions in the framework region.

[0306] 19. The antibody according to statement 18, wherein the heavy chain variable domain comprises SEQ ID NO: 3

[0307] 20. The antibody according to any one of statements 1 to 5, wherein the heavy chain variable domain comprises SEQ ID NO: 4 having four or fewer additional alterations such as substitutions in the framework region.

[0308] 21. The antibody according to statement 20, wherein the heavy chain variable domain comprises SEQ ID NO: 4.

[0309] 22. The antibody according to any one of statements 1 to 5, wherein the heavy chain variable domain comprises SEQ ID NO: 5 having four or fewer additional alterations such as substitutions in the framework region.

[0310] 23. The antibody according to statement 22, wherein the heavy chain variable domain (VH domain) comprises SEQ ID NO: 5.

[0311] 24. An antibody according to any one of the foregoing statements, wherein the light chain variable domain comprises SEQ ID NO: 6, wherein X7 (Kabat position 92) is N.

[0312] 25. An antibody according to any one of statements 1 to 23, wherein the light chain variable domain comprises SEQ ID NO: 6, wherein X7 (Kabat position 92) is H.

[0313] 26. An antibody according to any one of statements 1 to 23, wherein the light chain variable domain comprises SEQ ID NO: 6, wherein X7 (Kabat position 92) is Y.

[0314] 27. An antibody according to any one of statements 1 to 23, wherein the light chain variable domain comprises SEQ ID NO: 6, wherein X7 (Kabat position 92) is W.

[0315] 28. An antibody according to any one of statements 1 to 25, wherein the light chain variable domain comprises SEQ ID NO: 7 having four or fewer additional alterations such as substitutions in the framework region

[0316] 29. The antibody according to statement 28, wherein the light chain variable domain comprises SEQ ID NO: 7.

[0317] 30. An antibody according to any one of statements 1 to 23 and 25, wherein the light chain variable domain comprises SEQ ID NO: 8 having four or fewer additional alterations such as substitutions in the framework region

[0318] 31. The antibody according to statement 30, wherein the light chain variable domain comprises SEQ ID NO: 8.

[0319] 32. An antibody according to any one of statements 1 to 5, comprising the VH domain of SEQ ID NO: 3 and the VK domain of SEQ ID NO: 7.

[0320] 33. An antibody according to any one of statements 1 to 5, comprising the VH domain of SEQ ID NO: 4 and the VK domain of SEQ ID NO: 7.

[0321] 34. An antibody according to any one of statements 1 to 5, comprising the VH domain of SEQ ID NO: 5 and the VK domain of SEQ ID NO: 8.

[0322] 35. An antibody according to any one of claims 1 to 5, comprising the VH domain of SEQ ID NO:5 and the VK domain of SEQ ID NO:8.

[0323] 36. A pharmaceutical composition comprising the antibody according to any one of the foregoing claims and a pharmaceutically acceptable carrier.

[0324] 37. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 35.

[0325] 38. A vector comprising the nucleic acid according to claim 37 operably linked to a promoter.

[0326] 39. A host cell comprising the nucleic acid according to claim 37 or the vector according to claim 38.

[0327] 40. A method for preparing the antibody according to any one of claims 1 to 35, the method comprising expressing the vector according to claim 36 in a host cell culture to produce the antibody; and recovering the antibody from the cell culture.

[0328] 41. A method for treating or preventing Alzheimer's disease by administering to an individual in need thereof an effective amount of the antibody according to any one of claims 1 to 35 or the pharmaceutical composition according to claim 36.

[0329] 42. The antibody according to any one of claims 1 to 35 or the pharmaceutical composition according to claim 36 for use in a method of treating a human or animal body.

[0330] 43. The antibody according to any one of claims 1 to 35 or the pharmaceutical composition according to claim 36 for use in a method of treating Alzheimer's disease in an individual. Sequence Listing <110> Georg-August-Universität Göttingen Stiftung öffentlichen Rechts der Medizinischen Fakultät <120> Humanized Antibody <130> 30A-147 078 <160> 68 <170> PatentIn version 3.5 <210> 1 <211> 116 <212> PRT <213> Artificial <220> <223> RHA sequence <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 2 <211> 116 <212> PRT <213> Artificial <220> <223> RHA sequence with 27F, 29L, 63R and 70V as well as 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. <220> <221> MISC_FEATURE <222> (58)..(58) <223> 52BX1, where Xaa is Asp or Asn <220> <221> MISC_FEATURE <222> (59)..(59) <223> 52CX6, where Xaa is Phe or Tyr <220> <221> MISC_FEATURE <222> (61)..(61) <223> 53X2, where Xaa is Ala, Asn or Pro <220> <221> MISC_FEATURE <222> (62)..(62) <223> 54X3, where Xaa is Ala or Ser <220> <221> MISC_FEATURE <222> (63)..(63) <223> 55X4, where Xaa is Phe or Leu <220> <221> MISC_FEATURE <222> (64)..(64) <223> 56X5, where Xaa is Ile or Lys <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Xaa Xaa Tyr Xaa Xaa Xaa Xaa 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 3 <211> 116 <212> PRT <213> Artificial <220> <223> RHS sequence <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 4 <211> 116 <212> PRT <213> Artificial <220> <223> RHS7 sequence <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Phe Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 5 <211> 116 <212> PRT <213> Artificial <220> <223> RHS71 sequence <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Tyr Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 6 <211> 107 <212> PRT <213> Artificial <220> <223> Has an RKA sequence of 92X7, where X7 is N, H, Y or W <220> <221> MISC_FEATURE <222> (92)..(92) <223> 92X7, where Xaa is Asn, His, Tyr or Trp <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Xaa Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 107 <212> PRT <213> Artificial <220> <223> RKA sequence <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 8 <211> 107 <212> PRT <213> Artificial <220> <223> RKH sequence <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 321 <212> DNA <213> artificial <220> <223> NT4X-167 kappa light chain variable region <220> <221> CDS <222> (1)..(321) <400> 9 gat atc cag atg aca cag act aca tcc tcc ctg tct gcc tct ctg gga 48 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 gac aga gtc acc atc agt tgc agg gca agt cag gac att agc aat tat 96 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 tta aac tgg tat cag cag aaa cca gat gga act gtt aaa ctc ctg atc 144 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 tac tac aca tca aga tta cac tca gga gtc cca tca agg ttc agt ggc 192 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt ggg tct gga aca gat tat tct ctc acc att agc aac ctg gag caa 240 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 gaa gat att gcc act tac ttt tgc caa cag ggt aat acg ctt cct ccg 288 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 acg ttc ggt gga ggc acc aag ctg gaa atc aaa 321 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 10 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 348 <212> DNA <213> artificial <220> <223> NT4X-167 heavy chain variable region <220> <221> CDS <222> (1)..(348) <400> 11 cag gtg cag ctg aag cag tca gga cct ggc cta gtg cag ccc tca cag 48 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 agc ctg tcc atc acc tgc aca gtc tct ggt ttc tca tta act agc tat 96 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 ggt ata cac tgg gtt cgc cag tct cca gga aag ggt ctg gag tgg ctg 144 Gly Ile His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 gga gtg atg tgg agt ggt gga atc aca gac ttt tat gca gct ttc ata 192 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 tcc aga ctg agc atc agc agg gac atc tcc aag agc caa gtt ttc ttt 240 Ser Arg Leu Ser Ile Ser Arg Asp Ile Ser Lys Ser Gln Val Phe Phe 65 70 75 80 aaa atg aac agt ctg caa gct gat gac aca gcc ata tac tac tgt gcc 288 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 aga ggg agt cgc tat gct ttg gac tac tgg ggt caa ggc acc tca gtc 336 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 tcc gtc tcc tca 348 Ser Val Ser Ser 115 <210> 12 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 12 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Ser Arg Asp Ile Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Ser Val Ser Ser 115 <210> 13 <211> 348 <212> DNA <213> artificial <220> <223> NT4X-167 HA <220> <221> CDS <222> (1)..(348) <400> 13 cag gtg cag ctg cag gag agc gga ccc gga ctg gtg aag ccc tcc gag 48 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 acc ctg agc ctg acc tgc acc gtg agc gga ggc agc atc agc agc tac 96 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 ggc atc cac tgg att aga cag cct cct ggc aag ggc ctg gag tgg atc 144 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 ggc gtg atg tgg agc ggc ggc atc acc gat ttc tac gcc gcc ttc atc 192 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 agc agg gtg acc atc agc gtg gac acc agc aag aac cag ttc agc ctg 240 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 aag ctg agc agc gtg aca gct gcc gac acc gcc gtg tac tac tgc gcc 288 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 agg ggc agc aga tac gcc ctg gac tac tgg ggc caa ggc acc ctg gtg 336 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 acc gtg agc agc 348 Thr Val Ser Ser 115 <210> 14 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 14 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 15 <211> 348 <212> DNA <213> artificial <220> <223> NT4X-167 HB <220> <221> CDS <222> (1)..(348) <400> 15 cag gtg cag ctg cag gaa agc gga ccc ggc ctg gtg aag cct agc gag 48 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 acc ctg agc ctg acc tgc acc gtg agc ggc ttc agc ctg acc agc tac 96 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 ggc atc cac tgg atc agg cag cct cct ggc aag ggc ctg gaa tgg ctg 144 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 ggc gtg atg tgg tcc ggc ggc atc acc gac ttc tac gcc gcc ttc atc 192 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 agc agg ctg acc atc agc agg gac acc agc aag aac cag gtg agc ctg 240 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 aag atg agc agc gtg acc gcc gcc gat aca gcc gtg tac tac tgc gcc 288 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 agg ggc tcc aga tac gcc ctg gac tac tgg gga cag ggc acc ctg gtg 336 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 acc gtg agc agc 348 Thr Val Ser Ser 115 <210> 16 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 16 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 17 <211> 321 <212> DNA <213> 人工 <220> <223> NT4X-167 KA <220> <221> CDS <222> (1)..(321) <400> 17 gac atc cag atg acc caa agc cct agc agc ctg agc gcc agc gtg gga 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac agg gtg acc atc acc tgc agg gcc agc cag gac atc agc aac tac 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 ctg aac tgg tac cag cag aag ccc ggc aag gcc ccc aag ctg ctg atc 144 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 tac tac acc agc agg ctg cac agc ggc gtg cct agc agg ttc agc gga 192 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agc ggc agc ggc acc gac ttc acc ctg acc atc agc agc ctg cag ccc 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gag gac ttc gcc acc tac tac tgc cag cag ggc aac acc ctg cct cct 288 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 acc ttt ggc ggc ggc acc aag ctg gag atc aag 321 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 18 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 321 <212> DNA <213> Artificial <220> <223> NT4X-167 KB <220> <221> CDS <222> (1)..(321) <400> 19 gac atc cag atg acc cag agc cct agc agc ctg agc gct agc gtg ggc 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac agg gtg acc atc acc tgc agg gcc agc cag gac atc agc aac tac 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 ctg aac tgg tac cag cag aaa ccc ggc gga gcc ccc aag ctg ctg atc 144 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Pro Lys Leu Leu Ile 35 40 45 tac tac acc agc aga ctg cac agc ggc gtg ccc agc aga ttt agc ggc 192 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agc ggc agc ggc acc gat tac acc ctg acc atc agc agc ctg cag ccc 240 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gag gac ttc gcc acc tac ttc tgc cag cag ggc aac acc ctg cct cct 288 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 acc ttt ggc ggc ggc acc aag ctg gag atc aag 321 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 20 <211> 107 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 21 <211> 123 <212> PRT <213> Artificial <220> <223> AF062228 <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Asn Tyr Asp Phe Trp Ser Gly Tyr Ser Asn Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHA <400> 22 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 23 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHB <400> 23 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 24 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHC <400> 24 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 25 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHD <400> 25 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 26 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHE <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 27 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHF <400> 27 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 28 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHG <400> 28 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 29 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHH <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 30 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHI <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 31 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHJ <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 32 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHK <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 33 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHL <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Ile Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 34 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHM <400> 34 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 35 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHN <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 36 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHO <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 37 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHP <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 38 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHQ <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 39 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHR <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Leu Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 40 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHS <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 41 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHT <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 42 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHS2 <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 43 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHS3 <400> 43 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 44 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHS4 <400> 44 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 45 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X RHS5 <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 46 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS6 <400> 46 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asp Phe Tyr Ala Ala Phe Ile 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 47 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS7 <400> 47 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Phe Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 48 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS8 <400> 48 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Phe Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 49 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS71(F67Y) <400> 49 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Tyr Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 50 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS72(Y68N) <400> 50 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Phe Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 51 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS73(F67Y / Y68N) <400> 51 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 52 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS74(I39W) <400> 52 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Phe Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 53 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS81(S53M) <400> 53 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Tyr Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 54 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS82(S53H) <400> 54 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp His Gly Gly Ile Thr Asn Tyr Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 55 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS83(R100H) <400> 55 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Tyr Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 56 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS84(L103R) <400> 56 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Tyr Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 57 <211> 116 <212> PRT <213> Artificial <220> <223> NT4X*RHS85(L103H) <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Met Trp Ser Gly Gly Ile Thr Asn Tyr Tyr Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Ser Arg Tyr Ala His Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 58 <211> 107 <212> PRT <213> Artificial <220> <223> AY942002 <400> 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 59 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKA <400> 59 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 60 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKB <400> 60 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 61 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKC <400> 61 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 62 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKD <400> 62 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 63 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKE <400> 63 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 64 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKF(N92W) <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Trp Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 65 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKG(N92Y) <400> 65 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 66 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKH(N92H) <400> 66 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 67 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKI(L94R) <400> 67 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Arg Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 68 <211> 107 <212> PRT <213> Artificial <220> <223> NT4X RKJ(L94H) <400> 68 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr His Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105

Claims

1. An antibody comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain is a VH domain and the light chain variable domain is a VK domain, wherein a) the amino acid sequence of the VH domain is as shown in SEQ ID NO: 3 and the amino acid sequence of the VK domain is as shown in SEQ ID NO: 7; or b) the amino acid sequence of the VH domain is as shown in SEQ ID NO: 4 and the amino acid sequence of the VK domain is as shown in SEQ ID NO: 7; or c) the amino acid sequence of the VH domain is as shown in SEQ ID NO: 5 and the amino acid sequence of the VK domain is as shown in SEQ ID NO: 8; or d) the amino acid sequence of the VH domain is as shown in SEQ ID NO: 5 and the amino acid sequence of the VK domain is as shown in SEQ ID NO:

7.

2. The antibody according to claim 1, wherein the amino acid sequence of the VH domain is as shown in SEQ ID NO: 3 and the amino acid sequence of the VK domain is as shown in SEQ ID NO:

7.

3. The antibody according to claim 1, wherein the amino acid sequence of the VH domain is as shown in SEQ ID NO: 4 and the amino acid sequence of the VK domain is as shown in SEQ ID NO:

7.

4. The antibody according to claim 1, wherein the amino acid sequence of the VH domain is as shown in SEQ ID NO: 5 and the amino acid sequence of the VK domain is as shown in SEQ ID NO:

8.

5. The antibody according to claim 1, wherein the amino acid sequence of the VH domain is as shown in SEQ ID NO: 5 and the amino acid sequence of the VK domain is as shown in SEQ ID NO:

7.

6. A pharmaceutical composition comprising the antibody according to any one of the preceding claims and a pharmaceutically acceptable carrier.

7. Use of the antibody according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating Alzheimer's disease in an individual.

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