Improved Aβ protofibril binding antibodies

Abstract

Improved Aβ protofibril binding antibodies. The present invention relates to amyloid beta peptide (Aβ), more specifically to antibodies that bind to Aβ protofibrils and their use in the medical and / or prophylactic treatment of Alzheimer's disease and other conditions associated with Aβ protein aggregation. In addition, the present invention may relate to the diagnosis of these diseases and to monitoring disease progression by using the antibodies of the present invention. In addition, the present invention may relate to veterinary uses of the antibodies of the present invention.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201580036189.2 and invention name “Improved Aβ protofibril binding antibodies”. The parent application is the PCT international patent application PCT / EP2015 / 065633 filed on July 8, 2015, which entered the Chinese national phase. Technical Field

[0002] The present invention relates to amyloid beta peptide (Aβ), more specifically to antibodies that bind to Aβ protofibrils and their use in the medical and / or prophylactic treatment of Alzheimer's disease and other conditions associated with Aβ protein aggregation. In addition, the present invention may relate to the diagnosis of these diseases and monitoring disease progression by using the antibodies of the present invention. In addition, the present invention may relate to veterinary uses of the antibodies of the present invention. Background Art

[0003] Alzheimer's disease (AD) belongs to a group of neurodegenerative diseases and causes cognitive, memory and behavioral impairments. Hallmarks of AD include extracellular amyloid plaques, intraneuronal neurofibrillary tangles, neuronal dysfunction and eventual brain atrophy. The risk of developing AD increases with age, and the number of people reaching advanced age increases, with the disease having an increasing impact on the quality of life of the elderly. In addition, society is faced with an accelerating increase in its costs.

[0004] Although the disease is known for many years and several treatment suggestions have been made, even today, there is no currently available effective disease-modifying therapy, and at most only medicines for symptomatic treatment can be provided. A large number of studies have been carried out on the mechanism behind the disease. In short, for some reason, A β begins to gather and, through several intermediate forms, eventually produces insoluble fibril / plaque deposits in the brain. It was believed in the early days that the plaque therefore affected neurons and the signals transmitted therefrom, but the results of extensive research now show that the soluble, aggregated intermediate forms of A β are most likely the main cause of the disease and the symptoms observed in patients.

[0005] In the cascade from Aβ monomers to insoluble Aβ fibrils, one such intermediate form is the soluble high molecular weight Aβ protofibrils, which were first described by Walsh in The Journal of Biological Chemistry in 1997 (Vol. 272 ​​(35) p. 22364-72). The importance of Aβ protofibrils for the development of AD was established by a team of scientists led by Lars Lannfelt at Uppsala University in their studies of the Arctic mutation, an E693G mutation in the amyloid precursor protein (APP) that causes increased formation of Aβ protofibrils. Families carrying this mutation in northern Sweden were found to develop severe Alzheimer's disease early in life, and the discovery of this combination provided a basic idea for new treatments. Therefore, Aβ protofibrils were identified as being closely related to the disease and an important target for treatment. Based on their studies of Aβ peptides containing the Arctic mutation, Lannfelt et al. were able to generate sufficient amounts of Aβ protofibrils (Arctic as well as wild-type) in vitro for immunization and for subsequent selection of antibodies with high affinity for Aβ protofibrils compared to other species in the Aβ system. Examples of methods for generating Aβ protofibrils and antibodies that bind to these protofibrils are disclosed in WO02 / 03911 and WO2005 / 123775.

[0006] Of particular importance is the development of the mouse monoclonal antibody mAb158 (an antibody that binds to Aβ protofibrils disclosed in EP2004688), which comprises the following CDR sequences:

[0007] The high affinity and selectivity of the humanized form of mAb158 (BAN2401) makes it a very important candidate for the treatment and / or prevention of Alzheimer's disease in particular and is currently undergoing clinical trials in preparation for use as a pharmaceutical product. The characterization of BAN2401 is described in EP2004688.

[0008] The efficacy of antibodies depends on several pharmacokinetic and pharmacodynamic factors, see, for example, Deng et al., Expert Opin. Drug Metab. Toxicol 8(2)(2012): p. 141-160; Boswell et al., Bioconjugate Chem. 21(2010): p. 2153-2163; Konterman, Current Opinion in Biotechnology 22(2011): p. 1-9 and Igawa et al., mAbs 3: 3(2011): p. 243-252. Among them, extended serum half-life with enhanced systemic exposure generally provides considerable potential for improving significant therapeutic value. It also provides the opportunity to reduce the dose, which is of great systemic significance. Summary of the invention

[0009] The present invention provides antibodies that bind to Aβ protofibrils and their use in the treatment and / or prophylactic treatment of Alzheimer's disease and other disorders associated with Aβ protein aggregation. In addition, the present invention may relate to antibodies for diagnosing such diseases, and their use in monitoring the disease progression of these diseases, as well as veterinary uses of the antibodies. It has been found that, surprisingly, the half-life and exposure of the humanized antibody based on mAb158 (i.e., BAN2401) are significantly enhanced, for example, by about two times or more, mainly by introducing one or more mutations in certain positions of the variable light chain of the BAN2401 antibody, i.e., 17, 79 and / or 82 (Kabat positions 17, 74 and 77), respectively, see also Fig. 9 , where these positions are referred to as x1, x2, and x3. In BAN2401, the amino acid at position 17 (Kabat position 17) is A, the amino acid at position 79 (Kabat position 74) is R, and the amino acid at position 82 (Kabat position 77) is R. Kabat numbering is according to Kabat et al., Sequences of Proteins of Immunological Interest, 1991 (NIH Publication No. 91-3242).

[0010] Optionally, further improvement of the antibodies according to the invention can be achieved by combining each mutation providing increased half-life with one or more adjacent mutations, i.e. 13, 21, 81 and / or 84 (Kabat positions 13, 21, 76 and 79) of the variable light chain of the antibody, see also Fig. 9 , where these positions are called y 1-4 ; 37, 38 and / or 40 of the variable heavy chain of an antibody (Kabat positions 37, 38 and 40), see also Fig.10 , where these positions are called z1-3 , for further improvement in immunological significance, i.e., low immunogenicity. Compared to the BAN2401 sequence, when x1 is mutated, mutations y1 and / or y2 may be introduced, and when x2 and / or x3 are mutated, mutations y3 and / or y4 may be introduced. In addition, mutation z may be introduced 1-3 .

[0011] The present invention is as follows:

[0012] [1] An antibody or antigen-binding fragment thereof having affinity for Aβ protofibrils, wherein the antibody or antigen-binding fragment thereof has a variable light chain according to SEQ ID NO: 8, wherein

[0013] x1 is selected from A, D, E and Q, or a functional analog thereof;

[0014] x2 is selected from R, T, K, A and G, or a functional analog thereof;

[0015] x3 is selected from R, S, C, G and N, or a functional analog thereof;

[0016] y1 is selected from V and A;

[0017] y2 is selected from I and V;

[0018] y3 is selected from S and Q;

[0019] y4 is selected from E and D; and optionally

[0020] The variable heavy chain according to SEQ ID NO: 14, wherein

[0021] z1 is selected from V and I;

[0022] z2 is selected from R and Q; and

[0023] z3 is selected from A, N and T;

[0024] The combination of x1=A, x2=R and x3=R is excluded.

[0025] [2] The antibody or antigen-binding fragment according to [1], wherein

[0026] x1 is selected from A, D, E and Q;

[0027] x2 is selected from R, T, K, A and G;

[0028] x3 is selected from R, S, C, G and N;

[0029] y1 is selected from V and A;

[0030] y2 is selected from I and V;

[0031] y3 is selected from S and Q;

[0032] y4 is selected from E and D; and

[0033] The variable heavy chain according to SEQ ID NO: 14, wherein

[0034] z1 is selected from V and I;

[0035] z2 is selected from R and Q; and

[0036] z3 is selected from A, N and T;

[0037] The combination of x1=A, x2=R and x3=R is excluded.

[0038] [3] The antibody or antigen-binding fragment according to [1] or [2], wherein the light chain a comprises a combination of mutations selected from the following:

[0039] x1 and (y1 and / or y2);

[0040] x1 and (y1 and / or y2) and x2 and (y3 and / or y4);

[0041] x1 and (y1 and / or y2) and x2 and x3 and (y3 and / or y4);

[0042] x1 and (y1 and / or y2) and x3 and (y3 and / or y4);

[0043] x2(y3 and / or y4);

[0044] x2 and x3 and (y3 and / or y4); and

[0045] x3 and (y3 and / or y4);

[0046] The combination of x1=A, x2=R and x3=R is excluded.

[0047] [4] The antibody or antigen-binding fragment according to any one of [1] to [3], wherein the variable light chain comprises one or more mutations selected from the group consisting of:

[0048] x1 is D and (y1 and / or y2);

[0049] x1 is D and (y1 and / or y2), x2 is T and (y3 and / or y4);

[0050] x1 is D and (y1 and / or y2), x2 is T, x3 is S and (y3 and / or y4);

[0051] x1 is D and (y1 and / or y2) and x3 is S and (y3 and / or y4);

[0052] x2 is T(y3 and / or y4);

[0053] x2 is T and x3 is S and (y3 and / or y4); and

[0054] x3 is S and (y3 and / or y4);

[0055] Where y1 is V or A and y2 is V or I, excluding the combination of y1=V and y2=I;

[0056] y3 is S or Q and y4 is E or D, excluding the combination of y3=S and y4=E.

[0057] [5] The antibody or antigen-binding fragment according to any one of [1] to [4], wherein

[0058] x1 is D;

[0059] x2 is T;

[0060] x3 is R;

[0061] y1 is selected from V and A;

[0062] y2 is selected from V and I;

[0063] y3 is selected from Q and S;

[0064] y4 is selected from D and E;

[0065] z1 is V;

[0066] z2 is R; and

[0067] z3 is selected from N, T and A;

[0068] Combinations in which y1 is V, y2 is I, y3 is S, y4 is E, z1 is V, z2 is R and z3 is A are excluded.

[0069] [6] The antibody or antigen-binding fragment thereof according to [1], comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0070] [7] The antibody or antigen-binding fragment thereof according to [1], comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0071] [8] The antibody or antigen-binding fragment thereof according to [1], comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0072] [9] The antibody or antigen-binding fragment thereof according to [1], comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0073]

[10] The antibody or antigen-binding fragment thereof according to [1], comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0074]

[11] The antibody or antigen-binding fragment thereof according to [1], comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0075]

[12] The antibody or antigen-binding fragment thereof according to [1], comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0076]

[13] The antibody or antigen-binding fragment thereof according to [1], comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0077]

[14] The antibody or antigen-binding fragment according to any one of [1] to

[13] , wherein the antibody or the antigen-binding fragment comprises an IgG heavy chain constant region.

[0078]

[15] The antibody according to any one of [1] to

[14] , which is used for medical treatment.

[0079]

[16] The antibody according to any one of [1] to

[14] , which is used for treating and / or preventing Alzheimer's disease and other disorders associated with Aβ protein aggregation.

[0080]

[17] The antibody used according to

[16] , wherein the other diseases associated with Aβ protein aggregation are selected from traumatic brain injury (TBI), Lewy body dementia (BD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathies, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD).

[0081]

[18] Use of the antibody according to any one of [1] to

[14] for the preparation of a medicament for treating and / or preventing Alzheimer's disease and other disorders associated with Aβ protein aggregation.

[0082]

[19] The use according to

[18] , wherein the other conditions associated with Aβ protein aggregation are selected from traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD).

[0083]

[20] A method for reducing the amount of Aβ protofibrils in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment according to [1] to

[14] .

[0084]

[21] A method for treating and / or preventing Alzheimer's disease in a subject suffering from or at risk of developing the disease, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment according to [1] to

[14] .

[0085]

[22] A method for treating and / or preventing traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD) in a subject suffering from or at risk of developing the disease, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment according to [1] to

[14] .

[0086]

[23] A method for measuring the amount of Aβ protofibrils and / or aggregated Aβ proteins in humans, comprising contacting the human tissue or body fluid with the antibody or antigen-binding fragment according to [1] to

[14] in vivo or in vitro, and measuring the amount of the antibody or antigen-binding fragment bound to the Aβ protofibrils and / or aggregated Aβ proteins.

[0087]

[24] A method for diagnosing Alzheimer's disease in a human suffering from or at risk of developing the disease, comprising contacting a tissue or body fluid of the human with the antibody or antigen-binding fragment or fragment thereof according to [1] to

[14] in vivo or in vitro, and measuring the amount of the antibody bound to aggregated Aβ protein.

[0088]

[25] A method for diagnosing any of traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD) in a human suffering from or at risk of developing the disease, comprising contacting a tissue or body fluid of the human with the antibody or antigen-binding fragment or fragment thereof according to [1] to

[14] in vivo or in vitro, and measuring the amount of the antibody bound to aggregated Aβ protein.

[0089]

[26] A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of [1] to

[14] , and a pharmaceutically acceptable excipient and / or diluent.

[0090]

[27] The antibody according to any one of [1] to

[14] , which is for veterinary use. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Analysis of binding and selectivity for Aβ protofibrils compared to Aβ monomers for A17D, A17D / R79T, A17D / R82S, and A17D / R79T / R82S compared to BAN2401 (control) is provided. Inhibition of binding of Aβ1-42 protofibrils in solution is indicated by open circles and open squares, and inhibition of binding of Aβ1-40 monomers in solution is indicated by closed circles and closed squares.

[0092] Figure 2Plasma drug exposure of BAN2401 and antibodies of the present invention in mice presented in time relative to concentration graphs is provided. The figure shows plasma levels collected at time points of 0.5 hours, 2 days, 7 days, 15 days and 29 days after administration after a single intravenous injection of BAN2401, A17D, A17D / R79T and A17D / R79T / R82S; and plasma levels collected at time points of 0.5 hours, 2 days, 7 days, 14 days and 28 days after administration after a single intravenous injection of A17D / R82S. A17D / R82S is not included in the same PK study as other antibodies shown herein, but is given in a separate case. However, except for two plasma sampling time points, the same research design is used for these two separate studies. All plasma samples are analyzed by ELISA in the same case to avoid inter-assay variation. Plasma drug concentrations expressed in μg / ml are shown on the y-axis (logarithmic scale), and the time after administration expressed in hours (h) is shown on the x-axis. Mean group values ​​are shown with error bars indicating standard deviation. Mean AUC was calculated by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight) 0-inf The values ​​and terminal half-lives are shown in Table 1.

[0093] Figure 3 Plasma drug exposure of BAN2401 and antibodies of the present invention in rats presented in time relative to concentration graphs is provided. The figure shows plasma levels collected at time points of 0.5 hours, 2 hours, 7 hours, 24 hours, 2 days, 4 days, 7 days, 14 days and 29 days after administration after a single intravenous injection of BAN2401, A17D, A17D / R79T and A17D / R79T / R82S. Plasma drug concentrations expressed in μg / ml are shown on the y-axis (logarithmic scale), and the time after administration expressed in hours (h) is shown on the x-axis. The average group values ​​shown are with error bars indicating standard deviations. The average AUC is calculated by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight) 0-inf The values ​​and mean terminal half-lives are shown in Table 2.

[0094] Figure 4Data are provided for analysis of binding and selectivity of Aβ protofibrils compared to Aβ monomers for deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8) compared to BAN2401. Inhibition of binding of Aβ1-42 protofibrils in solution is indicated by open circles and open squares, and inhibition of binding of Aβ1-40 monomers in solution is indicated by closed circles and closed squares. A) A17D / R79T_DI 1, B) A17D / R79T_DI 2, C) A17D / R79T_DI 3, D) A17D / R79T_DI 4, E) A17D / R79T_DI 5, F) A17D / R79T_DI 6, G) A17D / R79T_DI 7, H) A17D / R79T_DI 8.

[0095] Figure 5 Plasma drug exposure of BAN2401 and antibodies of the present invention in mice presented in time relative to concentration graphs is provided. Plasma levels collected at time points of 0.5 hours, 2 days, 7 days, 14 days and 28 days after administration after a single intravenous injection of BAN2401, A17D / R79T and 8 deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8). Plasma drug concentrations expressed in μg / ml are shown on the y-axis (logarithmic scale), and the time after administration expressed in hours (h) is shown on the x-axis. The average group values ​​shown are with error bars indicating standard deviations. Average AUC was calculated by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight) 0-inf The values ​​and mean terminal half-lives are shown in Table 6.

[0096] Figure 6 Plasma drug exposure of BAN2401 and mutants in rats presented with time relative to concentration graphs is provided. Plasma levels collected at time points of 0.5 hours, 2 days, 7 days, 14 days and 28 days after administration after a single intravenous injection of BAN2401, A17D / R79T and 8 deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8). Plasma drug concentrations represented by μg / ml are displayed on the y-axis (logarithmic scale), and the time after administration represented by hours (h) is displayed on the x-axis. The average group values ​​displayed are with error bars indicating standard deviations. Average AUC is calculated by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight) 0-inf The values ​​and mean terminal half-lives are shown in Table 7.

[0097] Figure 7Dose-corrected plasma drug exposure for BAN2401, A17D / R79T_DI 3, A17D / R79T_DI 4, and A17D / R79T_DI 8 in cynomolgus monkeys presented as a time versus concentration graph is provided. Antibody plasma levels collected at time points of 5 minutes, 1 hour, 2 hours, 8 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, 21 days, and 28 days after administration of a single intravenous infusion of BAN2401, and antibody plasma levels collected at time points of 5 minutes, 2 hours, 8 hours, 24 hours, 3 days, 7 days, 14 days, 21 days, and 28 days after administration of a single intravenous infusion of A17D / R79T_DI 3, A17D / R79T_DI 4, and A17D / R79T_DI 8. Compared to BAN2401 (5 mg / kg), the antibodies of the present invention were administered to monkeys at different doses (10 mg / kg) in different studies. Therefore, the plasma exposure graphs have been dose-adjusted. Dose-corrected plasma drug concentrations expressed in μg / ml per mg / kg injected dose are shown on the y-axis (logarithmic scale), and time after administration expressed in hours (h) is shown on the x-axis. The average group values ​​are shown with error bars indicating standard deviations. The dose-adjusted mean AUC was calculated by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight) 0-inf The values ​​and terminal half-lives are shown in Table 8.

[0098] Figure 8 Amino acid sequences related to the present invention are provided.

[0099] Fig. 9 A table is provided on two pages, in which the VL-CDR1-3 sequences of the amino acid sequences of the light chain variable chains are in grey. BAN2401: SEQ ID NO: 7. New antibodies having a light chain variable chain according to the invention: SEQ ID NO: 8. Specific examples of such variable light chains: i): SEQ ID NO: 9; ii): SEQ ID NO: 10; iii): SEQ ID NO: 11; and iv): SEQ ID NO: 12.

[0100] Fig.10 A table divided into three pages is provided, in which the amino acid sequence of the heavy chain variable chain VH-CDR1-3 sequence is in grey. BAN2401: SEQ ID NO: 13. A novel antibody having a heavy chain variable chain according to the invention: SEQ ID NO: 14. Specific examples of such variable heavy chains: i): SEQ ID NO: 15; ii): SEQ ID NO: 16; iii): SEQ ID NO: 17; and iv): SEQ ID NO: 18.

[0101] Fig.11 A simple allometric scaling of the central clearance (CL) of BAN2401, A17D / R79T_DI 3, A17D / R79T_DI 4 and A17D / R79T_DI 8 for preclinical species including mice, rats and cynomolgus monkeys is provided. The CL of different species is plotted against their weight (diamonds) respectively. The regression line has been extrapolated to indicate the CL in a male weighing 70 kg. For BAN2401, the measured true central CL is represented by an open square and deviates from the linear regression line based on the CL of BAN2401 in mice, rats and cynomolgus monkeys. In contrast to the excellent linear correlation of the CL of the antibodies of the present invention, BAN2401 shows a poor linear correlation of CL, which indicates an uncertain half-life prediction. DETAILED DESCRIPTION

[0102] Mutations A17D, R79T and R82S represent positions in the BAN2401 antibody where the amino acids in positions 17, 79 and 82 are mutated in the variable light chain.

[0103] "BAN2401" refers to a humanized monoclonal antibody of mouse antibody mAb158, comprising a variable light chain having an amino acid sequence as shown in SEQ ID NO: 7 and a variable heavy chain as shown in SEQ ID NO: 13. Both BAN2401 and mAb158 and their features, including VL-CDR1-3 and VH-CDR1-3, are described in EP2004688. BAN2401 is not included in the present invention.

[0104] The following abbreviations mean:

[0105] BAN2401: an antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO:7; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:13.

[0106] A17D: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO:19; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:13.

[0107] A17D / R79T: an antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO:20; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:13.

[0108] A17D / R79T / R82S: an antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO:21; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:13.

[0109] A17D / R82S: an antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO:22; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:13.

[0110] A17D / R79T_DI 1: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0111] A17D / R79T_DI 2: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0112] A17D / R79T_DI 3: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0113] A17D / R79T_DI 4: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0114] A17D / R79T_DI 5: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0115] A17D / R79T_DI 6: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0116] A17D / R79T_DI 7: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0117] A17D / R79T_DI 8: An antibody comprising: a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0118] The antibody or antigen-binding fragment thereof according to the present invention comprises: amino acid A, D, E, Q or a functional analogue at position 17 (Kabat position 17) of the light chain variable chain, amino acid R, T, K, A, G or a functional analogue at position 79 (Kabat position 74) and amino acid R, S, C, G, N or a functional analogue at position 82 (Kabat position 77). Functional analogues are amino acids that provide a lower pI value compared to A (position 17) and respectively to R (positions 79 and 82) without negatively affecting binding to the antigen.

[0119] The amino acid sequence in the present disclosure is as follows:

[0120] SEQ ID NO: 1: Variable heavy chain VH-CDR1 of BAN2401.

[0121] SEQ ID NO: 2: Variable heavy chain VH-CDR2 of BAN2401.

[0122] SEQ ID NO: 3: Variable heavy chain VH-CDR3 of BAN2401.

[0123] SEQ ID NO: 4: Variable light chain VL-CDR1 of BAN2401.

[0124] SEQ ID NO: 5: Variable light chain VL-CDR2 of BAN2401.

[0125] SEQ ID NO: 6: Variable light chain VL-CDR3 of BAN2401.

[0126] SEQ ID NO: 7: Variable light chain of BAN2401.

[0127] SEQ ID NO: 8: Common variable light chain sequence in the antibodies of the present invention.

[0128] SEQ ID NO: 9: Specific variable light chain sequence in the antibody of the present invention.

[0129] SEQ ID NO: 10: Specific variable light chain sequence in the antibody of the present invention.

[0130] SEQ ID NO: 11: Specific variable light chain sequence in the antibody of the present invention.

[0131] SEQ ID NO: 12: Specific variable light chain sequence in the antibody of the present invention.

[0132] SEQ ID NO: 13: Variable heavy chain of BAN2401.

[0133] SEQ ID NO: 14: Common variable heavy chain sequence in the antibodies of the present invention.

[0134] SEQ ID NO: 15: Specific variable heavy chain sequence in the antibody of the present invention.

[0135] SEQ ID NO: 16: Specific variable heavy chain sequence in the antibody of the present invention.

[0136] SEQ ID NO: 17: Specific variable heavy chain sequence in the antibody of the present invention.

[0137] SEQ ID NO: 18: Specific variable heavy chain sequence in the antibody of the present invention.

[0138] SEQ ID NO: 19: Specific variable light chain sequence in the antibody of the present invention.

[0139] SEQ ID NO: 20: Specific variable light chain sequence in the antibody of the present invention.

[0140] SEQ ID NO: 21: Specific variable light chain sequence in the antibody of the present invention.

[0141] SEQ ID NO: 22: Specific variable light chain sequence in the antibody of the present invention.

[0142] SEQ ID NO: 23: Amino acid sequence of the human IgG1 constant region contained in the antibody of the present invention.

[0143] SEQ ID NO: 24: Amino acid sequence of the human kappa chain constant region contained in the antibody of the present invention.

[0144] The variable light chain of BAN2401 (SEQ ID NO: 7) and the antibodies of the present invention comprises three CDR-sequences (VL-CDR1-3):

[0145] VL-CDR1:RSSQSIVHSNGNTYLE SEQ ID NO:4

[0146] VL-CDR2: KVSNRFS SEQ ID NO: 5

[0147] VL-CDR3: FQGSHVPPT SEQ ID NO: 6,

[0148] And the variable heavy chain of BAN2401 (SEQ ID NO: 13) and the antibody of the present invention comprises three CDR-sequences (VH-CDR1-3):

[0149] VH-CDR1: SFGMH SEQ ID NO: 1

[0150] VH-CDR2:YISSGSSTIYYGDTVKG SEQ ID NO:2

[0151] VH-CDR3: EGGYYYGRSYYTMDY SEQ ID NO: 3.

[0152] According to one aspect of the present invention, there is provided an antibody that binds to Aβ protofibrils, which has the following CDR sequence combination:

[0153]

[0154] And comprises a variable light chain having SEQ ID NO: 8, wherein x1 is A, D, E, Q or a functional analogue, x2 is R, T, K, A, G or a functional analogue, and x3 is R, S, C, G, N or a functional analogue, excluding the combination of x1=A, x2=R and x3=R.

[0155] Functional analogs are amino acids that provide lower pi values ​​compared to A (x1) and respectively to R (x2 and x3) without negatively affecting binding to the antigen.

[0156] The variable heavy chain has the amino acid sequence shown in SEQ ID NO: 14, which comprises any combination of z1, z2 and z3, and wherein

[0157] z1 is selected from V and I;

[0158] z2 is selected from R and Q; and

[0159] z3 is selected from A, N and T;

[0160] The combination of z1=V, z2=R and z3=A is excluded.

[0161] It should be noted that based on the present teachings, the identification of functional analogs of the specific amino acids defined above at each position can be easily carried out by a person skilled in the art, since methods for introducing amino acids at specific positions according to the present invention are disclosed, as well as methods for testing the resulting products, for example with respect to affinity for Aβ protofibrils and other important characteristics, see below.

[0162] Therefore, in one aspect of the present invention, there is provided an antibody or antigen-binding fragment thereof having affinity for Aβ protofibrils, wherein the antibody or antigen-binding fragment thereof has a variable light chain according to SEQ ID NO: 8, wherein

[0163] x1 is selected from A, D, E and Q, or a functional analog thereof;

[0164] x2 is selected from R, T, K, A and G, or a functional analog thereof;

[0165] x3 is selected from R, S, C, G and N, or a functional analog thereof;

[0166] y1 is selected from V and A;

[0167] y2 is selected from I and V;

[0168] y3 is selected from S and Q;

[0169] y4 is selected from E and D; and optionally

[0170] The variable heavy chain according to SEQ ID NO: 14, wherein

[0171] z1 is selected from V and I;

[0172] z2 is selected from R and Q; and

[0173] z3 is selected from A, N and T;

[0174] The combination of x1=A, x2=R and x3=R is excluded.

[0175] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein

[0176] x1 is selected from A, D, E and Q;

[0177] x2 is selected from R, T, K, A and G;

[0178] x3 is selected from R, S, C, G and N;

[0179] y1 is selected from V and A;

[0180] y2 is selected from I and V;

[0181] y3 is selected from S and Q;

[0182] y4 is selected from E and D; and

[0183] The variable heavy chain according to SEQ ID NO: 14, wherein

[0184] z1 is selected from V and I;

[0185] z2 is selected from R and Q; and

[0186] z3 is selected from A, N and T;

[0187] The combination of x1=A, x2=R and x3=R is excluded.

[0188] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the light chain comprises a combination of mutations selected from the group consisting of:

[0189] x1 and (y1 and / or y2);

[0190] x1 and (y1 and / or y2) and x2 and (y3 and / or y4);

[0191] x1 and (y1 and / or y2) and x2 and x3 and (y3 and / or y4);

[0192] x1 and (y1 and / or y2) and x3 and (y3 and / or y4);

[0193] x2(y3 and / or y4);

[0194] x2 and x3 and (y3 and / or y4); and

[0195] x3 and (y3 and / or y4);

[0196] The combination of x1=A, x2=R and x3=R is excluded.

[0197] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the variable light chain comprises one or more mutations selected from the group consisting of:

[0198] x1 is D and (y1 and / or y2);

[0199] x1 is D and (y1 and / or y2), x2 is T and (y3 and / or y4);

[0200] x1 is D and (y1 and / or y2), x2 is T, x3 is S and (y3 and / or y4);

[0201] x1 is D and (y1 and / or y2) and x3 is S and (y3 and / or y4);

[0202] x2 is T(y3 and / or y4);

[0203] x2 is T and x3 is S and (y3 and / or y4); and

[0204] x3 is S and (y3 and / or y4);

[0205] Where y1 is V or A, and y2 is V or I, excluding the combination of y1=V and y2=I;

[0206] y3 is S or Q, and y4 is E or D, excluding the combination of y3=S and y4=E.

[0207] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided wherein y1 is A, y2 is V, y3 is Q and y4 is D.

[0208] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein:

[0209] x1 is D;

[0210] x2 is T;

[0211] x3 is R;

[0212] y1 is selected from V and A;

[0213] y2 is selected from V and I;

[0214] y3 is selected from Q and S;

[0215] y4 is selected from D and E;

[0216] z1 is V;

[0217] z2 is R; and

[0218] z3 is selected from N, T and A;

[0219] The combination in which y1 is V, y2 is I, y3 is S, y4 is E, z1 is V, z2 is R and z3 is A is excluded.

[0220] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein:

[0221] x1 is D;

[0222] x2 is T;

[0223] x3 is R;

[0224] y1 is V;

[0225] y2 is V;

[0226] y3 is Q;

[0227] y4 is E;

[0228] z1 is V;

[0229] z2 is R; and

[0230] z3 is N.

[0231] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein x1 is D;

[0232] x2 is T;

[0233] x3 is R;

[0234] y1 is V;

[0235] y2 is V;

[0236] y3 is S;

[0237] y4 is D;

[0238] z1 is V;

[0239] z2 is R; and

[0240] z3 is N.

[0241] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein:

[0242] x1 is D;

[0243] x2 is T;

[0244] x3 is R;

[0245] y1 is A;

[0246] y2 is 1;

[0247] y3 is Q;

[0248] y4 is E;

[0249] z1 is V;

[0250] z2 is R; and

[0251] z3 is N.

[0252] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein:

[0253] x1 is D;

[0254] x2 is T;

[0255] x3 is R;

[0256] y1 is A;

[0257] y2 is 1;

[0258] y3 is S;

[0259] y4 is D;

[0260] z1 is V;

[0261] z2 is R; and

[0262] z3 is N.

[0263] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein:

[0264] x1 is D;

[0265] x2 is T;

[0266] x3 is R;

[0267] y1 is V;

[0268] y2 is V;

[0269] y3 is Q;

[0270] y4 is E;

[0271] z1 is V;

[0272] z2 is R; and

[0273] z3 is T.

[0274] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein:

[0275] x1 is D;

[0276] x2 is T;

[0277] x3 is R;

[0278] y1 is V;

[0279] y2 is V;

[0280] y3 is S;

[0281] y4 is D;

[0282] z1 is V;

[0283] z2 is R; and

[0284] z3 is T.

[0285] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein:

[0286] x1 is D;

[0287] x2 is T;

[0288] x3 is R;

[0289] y1 is A;

[0290] y2 is 1;

[0291] y3 is Q;

[0292] y4 is E;

[0293] z1 is V;

[0294] z2 is R; and

[0295] z3 is T.

[0296] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein:

[0297] x1 is D;

[0298] x2 is T;

[0299] x3 is R;

[0300] y1 is A;

[0301] y2 is 1;

[0302] y3 is S;

[0303] y4 is D;

[0304] z1 is V;

[0305] z2 is R; and

[0306] z3 is T.

[0307] According to one aspect of the invention, each mutation x1-x3 is combined with one or more further mutations y1-y4:

[0308] When x1 is not A, introduce mutations y1 and / or y2;

[0309] When x2 is not R and / or x3 is not R, introducing mutations y3 and / or y4;

[0310] A variable light chain is provided, comprising the following mutant combinations compared to SEQ ID NO: 7:

[0311] x1 and (y1 and / or y2); or

[0312] x1 and (y1 and / or y2) and x2 and (y3 and / or y4); or

[0313] x1 and (y1 and / or y2) and x2 and x3 and (y3 and / or y4); or

[0314] x1 and (y1 and / or y2) and x3 and (y3 and / or y4); or

[0315] x2 and (y3 and / or y4); or

[0316] x2 and x3 and (y3 and / or y4); or

[0317] x3 and (y3 and / or y4);

[0318] where the parameters x and y are as defined above.

[0319] In one embodiment, the light chain of the antibody according to the present invention (SEQ ID NO: 8) or an antigen-binding fragment thereof comprises only one or more of the mutations X1-x3 (A17D (x1), R79T (x2) and R82S (x3)) in the light chain variable chain (using N-terminal numbering):

[0320] A17D; or

[0321] A17D and R79T; or

[0322] A17D and R79T and R82S; or

[0323] A17D and R82S; or

[0324] R79T; or

[0325] R79T and R82S; or

[0326] R82S;

[0327] wherein yl is V, y2 is I, y3 is S and y4 is E (unchanged from SEQ ID NO: 7).

[0328] According to another embodiment, mutations y1-y4 are introduced to provide any one of the following combinations:

[0329] A17D and (y1 and / or y2);

[0330] A17D and (y1 and / or y2) and R79T and (y3 and / or y4);

[0331] A17D and (y1 and / or y2) and R79T and R82S and (y3 and / or y4);

[0332] A17D and (y1 and / or y2) and R82S (y3 and / or y4);

[0333] R79T (y3 and / or y4);

[0334] R79T and R82S and (y3 and / or y4);

[0335] R82S and (y3 and / or y4);

[0336] Where y1 is V or A, and y2 is V or I, excluding the combination of y1=V and y2=I,

[0337] y3 is S or Q, and y4 is E or D, excluding the combination of y3=S and y4=E.

[0338] Other specific combinations are disclosed in SEQ ID NOs: 9-12.

[0339] The variable heavy chain (VH) of the antibody according to the present invention has the amino acid sequence SEQ ID NO: 14, wherein z1 is V or I, z2 is R or Q, and z3 is A, N or T, such as SEQ ID NO: 15-18.

[0340] In one embodiment of this aspect, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain selected from the amino acid sequence shown in any one of SEQ ID NOs: 9-12.

[0341] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable heavy chain selected from the amino acid sequence shown in any one of SEQ ID NOs: 15-18.

[0342] In one embodiment of this aspect, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain selected from the amino acid sequence shown in any one of SEQ ID NOs: 9-12; and a variable heavy chain selected from the amino acid sequence shown in any one of SEQ ID NOs: 15-18.

[0343] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the amino acid sequence shown in SEQ ID NO:9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:15.

[0344] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0345] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0346] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0347] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the amino acid sequence shown in SEQ ID NO:9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:16.

[0348] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0349] In one embodiment of this aspect, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0350] In one embodiment of this aspect, an antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0351] In one embodiment, the antibody or antigen-binding fragment according to the present invention comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE constant regions or any allelic variant thereof, as discussed in Kabat et al. (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), which is included herein by reference. Any such sequence may be used in the present invention. In a more preferred embodiment, the antibody heavy chain constant region is IgG1. The amino acid sequence of the human IgG1 constant region is known in the art and is shown in SEQ ID NO: 23.

[0352] In one embodiment, the antibody or antigen-binding fragment according to the present invention comprises a light chain constant region selected from κ- and λ-chain constant regions or any allelic variant thereof, as discussed in Kabat et al. (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), which is included herein by reference. Any such sequence may be used in the present invention. In a more preferred embodiment, the antibody light chain constant region is κ. The amino acid sequence of the human κ chain constant region is known in the art and is shown in SEQ ID: 24.

[0353] In one embodiment, the antigen binding fragment according to the present invention is a Fab fragment, or a F(ab')2 fragment or a single chain Fv fragment.

[0354] The antibodies or antigen-binding fragments according to the present invention may comprise any combination of variable light and heavy chains as defined above.

[0355] According to one aspect of the present invention, there is provided an improved antibody or antigen binding fragment having a high affinity for Aβ protofibrils for treatment, for example, by administering one or more antibodies or antigen binding fragments according to the present invention to patients suffering from Alzheimer's disease and other conditions associated with Aβ protein aggregation or the risk of these diseases, other conditions associated with Aβ protein aggregation such as traumatic brain injury (TBI), dementia with Lewy bodies (DLB), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD). Suitable dosages can vary over a wide range, for example, 0.01 to 100 mg / kg / agent, depending on the medical indication and patient status, route of administration, for example, intravenous, subcutaneous infusion or by topical administration, in addition to selected frequencies, for example, single doses, daily, weekly, quarterly or even less frequent administration.

[0356] In one aspect, an antibody or antigen-binding fragment thereof of the invention is provided for use in therapeutic treatment.

[0357] In one aspect, antibodies or antigen binding fragments thereof of the present invention for treating and / or preventing Alzheimer's disease and other disorders associated with Aβ protein aggregation are provided. Typically, such other disorders can be selected from traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, and Parkinson's disease dementia (PDD).

[0358] In one aspect, the invention provides an antibody or antigen-binding fragment thereof for use in the preparation of a medicament for treating and / or preventing Alzheimer's disease and other conditions associated with Aβ protein aggregation. Typically, such other conditions may be selected from traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, and Parkinson's disease dementia (PDD).

[0359] In one aspect, a method of reducing the amount of A[beta] protofibrils in a human is provided, comprising administering to the human a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention.

[0360] In one aspect, a method for treating and / or preventing the disease in a subject suffering from Alzheimer's disease and other disorders associated with Aβ protein aggregation or a risk of the disease is provided, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention to the subject. Typically, such other disorders may be selected from traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, and Parkinson's disease dementia (PDD).

[0361] A "subject" is typically a mammal, such as a human. Other mammals represent mammals for which veterinary use / treatment / prevention is to be applied.

[0362] In one aspect, a method for measuring the amount of Aβ protofibrils and / or aggregated Aβ proteins in a human can be provided, comprising contacting the tissue or body fluid of the human with a labeled antibody or antigen-binding fragment thereof of the present invention in vivo or in vitro, and measuring the amount of the antibody or antigen-binding fragment that binds to the Aβ protofibrils and / or aggregated Aβ proteins. The antibody or antigen-binding fragment can be labeled with a radioligand such as I 131 , C 11 , C 14 , H 3 、F 18 or Gallium 68 Labels are used for detection purposes but are not limited to these radioactive isotopes.

[0363] In one aspect, a method for diagnosing the disease in a person suffering from Alzheimer's disease and other conditions associated with Aβ protein aggregation or at risk of developing the disease can be provided, wherein the other conditions associated with Aβ protein aggregation are such as traumatic brain injury (TBI), dementia with Lewy bodies (DLB), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, and Parkinson's disease dementia (PDD), the method comprising contacting the tissue or body fluid of the person in vivo or in vitro with an antibody or antigen-binding fragment thereof of the present invention, and measuring the amount of the antibody or antigen-binding fragment bound to the aggregated protein. Typically, the other conditions associated with Aβ protein aggregation can be selected from traumatic brain injury (TBI), dementia with Lewy bodies (DLB), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, and Parkinson's disease dementia (PDD). Typically, the analysis is performed in vivo or in vitro (in a sample taken from a patient) by contacting a human body fluid or tissue with a preparation of one or more antibodies or antigen-binding fragments of the present invention and measuring the amount of antibody or antigen-binding fragment bound to Aβ protofibrils. Quantification of protofibrils will be used for diagnosis of the above-mentioned diseases, follow-up of various treatments, and development of new drugs. Optionally, the antibodies or antigen-binding fragments thereof in such preparations will be labeled with reagents that will be detected and measured by any technique known in the art, such as by ELISA, Biacore analysis, and / or using SPECT, PET, MRI imaging. The antibodies or antigen-binding fragments can be labeled with radioligands such as I 131 , C 11 , C 14 , H 3 、F 18 or Gallium 68 Labels are used for detection purposes but are not limited to these radioactive isotopes.

[0364] According to another aspect of the present invention, a pharmaceutical composition comprising an effective amount of one or more antibodies or antigen-binding fragments thereof according to the present invention is prepared. The medical composition comprising the antibody according to the present invention may also comprise other components known for such preparations, such as buffers, components for preservation and stability, in addition to an effective amount of the antibody.

[0365] In another aspect, the antibodies of the invention may be provided for veterinary use. Typically, the veterinary use comprises treating and / or preventing a disorder associated with Aβ protein aggregation.

[0366] According to another aspect of the invention, treatment may be provided using antibodies according to the invention in combination with symptomatic treatments such as acetylcholinesterase inhibitors, NMDA antagonists and 5HT6 inhibitors.

[0367] Combinations with other disease-modifying therapies, such as γ-secretase inhibitors (GSIs), γ-secretase modulators (GSMs), β-secretase (BACE) inhibitors, BACE modulators, vaccines, other antibodies, drugs targeting tau or neuroinflammatory progression, antihypertensive drugs, etc., provide additional possibilities for effective treatment.

[0368] Combination with nutritional products may offer additional possibilities for effective treatment.

[0369] In one aspect, a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable excipient and / or diluent is provided, the composition may further comprise an additional therapeutic agent. Typically, the additional therapeutic agent may be selected from acetylcholinesterase inhibitors, NMDA antagonists, 5HT6 inhibitors, GSI, GSM, BACE inhibitors, BACE regulators, vaccines, other antibodies, drugs targeting tau or neuroinflammatory progression, antihypertensive drugs and nutritional products. The composition may be provided in a single dose or sequential dose.

[0370] The present invention will be illustrated by means of a number of non-limiting examples:

[0371] Example 1. Antibody Generation and Methods Used

[0372] Reference antibody generation

[0373] The reference antibody BAN2401 was generated according to the method described previously in EP2004688.

[0374] Production of Antibodies of the Invention

[0375] Use CHOK1SV GS and CHOK1SV GS-KO Xceed respectively TM Expression system (Lonza) in Chinese Hamster Ovary (CHO) cells by transient and / or stable production of antibodies of the invention. TM Transient transfection of the expression system generated the following mutants: A17D, A17D / R79T, and A17D / R79T / R82S. TMTransient and stable transfection of the expression system generated the following mutants: A17D / R79T_DI 1, A17D / R79T_DI 2, A17D / R79T_DI 3, A17D / R79T_DI 4, A17D / R79T_DI 5, A17D / R79T_DI 6, A17D / R79T_DI 7, and A17D / R79T_DI 8. The A17D / R82S mutant was generated by stable transfection using the CHOK1SV GS expression system.

[0376] The sequences of the light and heavy chain coding regions of the mutants were synthesized by using conventional methods.

[0377] For CHOK1SV GS-KO Xeed TM Transient transfection in the expression system, the light chain coding region was subcloned into the pXC-17.4 vector and the heavy chain coding region was subcloned into the pXC-18.4 vector. The expression culture was harvested 6 days after transfection and clarified by centrifugation and sterile filtration. The clarified cell culture supernatant was purified using protein A chromatography. The eluted antibody was obtained in PBS (pH 7.4). The product was further purified by preparative size exclusion chromatography (SEC) to remove aggregates. The collected monomer peaks were then analyzed by analytical SEC, and it was determined that the aggregate level was less than 2% for all products.

[0378] Basically according to the manufacturer's recommendations in CHOK1SV GS-KO Xeed TM Stable expression was performed in the system. In brief, two vectors (pXC-17.4 and pXC-18.4) containing the light chain and the heavy chain were connected to a double gene vector containing these two genes. CHOK1SV GS-KO cells were transfected by electroporation with the linearized double gene vector. Screening of clones and productivity screening of dead cultures were analyzed by ELISA. Production was performed with CD-CHO culture medium supplemented with 15% Feed A and 15% Feed B (Life Technologies). Supernatants were harvested by centrifugation and sterile filtration. The clarified cell culture supernatant was purified using protein G chromatography and the buffer was exchanged into Dulbecco PBS (Gibco).

[0379] For stable transfection using the CHOK1SV GS expression system (Lonza), the heavy chain gene was connected to the pEE6.4 vector and the light chain gene was connected to the pEE12.4 vector. The two vectors were connected to form a double gene vector. CHOK1SV cells were transfected by electroporation with the linearized double gene vector. In essence, transfection was performed according to the manufacturer's recommendations. Screening of clones and productivity screening of dead cultures were analyzed by ELISA. Production was performed using CD-CHO culture medium supplemented with 15% nutrient A and 15% nutrient B (Life Technologies). The supernatant was harvested by centrifugation and sterile filtration. The clarified cell culture supernatant was purified using protein G chromatography and the buffer was exchanged into Dulbecco PBS (Gibco). The quality analysis of the products was performed by size exclusion HPLC and SDS-PAGE using the purified material of all the mutants produced.

[0380] Target binding analysis by inhibition ELISA

[0381] The binding properties of the antibodies of the present invention compared to BAN2401 to Aβ protofibrils and Aβ monomers were analyzed using inhibition ELISA, wherein the antibodies were pre-incubated with Aβ protofibrils or Aβ monomers in solution and then transferred to an ELISA plate coated with Aβ, as described in Tucker et al. J Alzheimers Dis. 2015; 43(2): 575-88. doi: 10.3233 / JAD-140741. PubMed PMID: 25096615 and the references cited therein.

[0382] Pharmacokinetic studies in wild-type mice

[0383] 8-10 week old female C57BL / 6 mice were divided into groups and injected with a single intravenous (iv) dose of 10 mg / kg of BAN2401 or the antibody of the invention. Plasma from all animals was collected at different time points from 30 minutes to 29 days after injection and used to measure antibody concentrations and subsequently calculate pharmacokinetic (PK) parameters. Mice were sacrificed at the terminal plasma collection time point.

[0384] Pharmacokinetic studies in rats

[0385] 8-week-old female Sprague Dawley rats were grouped and injected intravenously with a single dose of 10 mg / kg of BAN2401 or an antibody of the invention. Plasma from all animals was collected at different time points from 30 minutes to 29 days after injection and used to measure antibody concentrations and subsequently calculate PK parameters. Rats were sacrificed at the terminal plasma collection time point.

[0386] Pharmacokinetic studies in monkeys

[0387] Male cynomolgus monkeys were divided into groups (N=3) and given a single intravenous infusion of 5 mg / kg BAN2401 or 10 mg / kg of the antibody of the present invention. The serum of all animals was collected at different time points from 5 minutes to 28 days after injection and used to measure the antibody concentration. The serum levels of BAN2401 and the antibody of the present invention were determined by ELISA. Biotinylated Aβ1-42 protofibrils were added to a microplate fixed with avidin for coating. After blocking, the monkey serum samples were added to the wells. After washing away any unbound substances, alkaline phosphatase (AP)-labeled goat anti-human IgG was added to the wells. After washing to remove any unbound reagents, p-nitrophenyl phosphate (used as a substrate for AP) was added to the wells. The reaction was terminated with sodium hydroxide solution, and the absorbance was measured at 405 nm and 492 nm. The absorbance at 492 nm was subtracted from the absorbance at 405 nm. The values ​​were converted to concentrations by means of a standard curve and used for subsequent calculation of PK parameters.

[0388] Direct ELISA for measuring Aβ antibodies

[0389] The levels of BAN2401 and the antibodies of the invention were measured in cell culture media, purified antibody products, and plasma collected in mouse and rat PK studies by direct ELISA for measuring anti-Aβ antibodies. The samples were serially diluted and incubated in microtiter plate wells coated with Aβ1-40 to allow BAN2401 and the antibodies of the invention to bind. Goat anti-human IgG conjugated with horseradish peroxidase (HRP) was used as the detection antibody, and TMB (substrate for HRP) was added. The reaction was stopped by adding 2M H2SO4, which resulted in a yellow color when measured at 450nm. The method was used in a quantitative manner, where OD 450 The values ​​were converted to concentrations using a standard curve.

[0390] Calculation of PK parameters by non-compartmental analysis

[0391] Individual terminal half-life calculations were performed using a non-compartmental model with Phoenix WinNonLin 6.3 software (Pharsight). The area under the curve (AUC) was calculated using the lin-up log-down method with Phoenix-WinNonLin. 0-inf ) were calculated. Group means and standard deviations of AUC and terminal half-life were calculated using GraphPad Prism (v 6.04).

[0392] Statistical analysis

[0393] Statistical analysis of the group means of the individually determined terminal half-life and AUC was performed using GraphPad Prism software (v.6.04). One-way ANOVA followed by Bonferroni post-test multiple comparisons was used in the study. Tests were performed at the following significance levels *P < 0.05, **P < 0.01, ***P < 0.001 and ***P < 0.0001.

[0394] Example 2. Target Binding Characteristics

[0395] Binding of Aβ protofibrils retained in A17D, A17D / R79T, A17D / R82S and A17D / R79T / R82S compared to BAN2401

[0396] As described in Example 1 (inhibition ELISA), the target binding profile of the antibodies of the invention was analyzed by inhibition ELISA after BAN2401 (control). The results are shown in Figure 1 In, analysis of the binding and selectivity of A17D, A17D / R79T, A17D / R82S and A17D / R79T / R82S and BAN2401 to Aβ protofibrils compared to Aβ monomers is shown. The results show that the binding and selectivity of the antibodies of the invention (A17D, A17D / R79T, A17D / R82S and A17D / R79T / R82S) for binding to Aβ protofibrils are retained compared to the binding to Aβ monomers.

[0397] Example 3. Pharmacokinetic profile of antibodies in mice

[0398] Pharmacokinetics of BAN2401, A17D, A17D / R79T, A17D / R82S, and A17D / R79T / R82S in wild-type mice

[0399] To study the pharmacokinetic profile of the antibodies of the invention in wild-type mice, 8-10 week old C57BL / 6 female mice were administered 10 mg / kg BAN2401 (N=8), A17D (N=7), A17D / R79T (N=6), A17D / R82S (N=8) or A17D / R79T / R82S (N=7) with a single intravenous injection. The animals were bled after 0.5 hours, 2 days, 7 days, 14 or 15 days and 28 or 29 days, and the levels of BAN2401 and the antibodies of the invention were analyzed by ELISA using Aβ1-40 for capture as described in Example 1 and detected by HRP-conjugated goat anti-human IgG (direct ELISA). A17D / R82S was administered in a study initiated separately from the other antibodies. However, plasma samples from two different studies were analyzed simultaneously by ELISA to avoid inter-assay variation.

[0400] like Figure 2 As shown in the time versus concentration graphs in Table 1 and the calculated PK parameters shown in Table 1, the PK profiles of the antibodies of the invention (A17D, A17D / R79T, A17D / R82S, and A17D / R79T / R82S) differ significantly from those of BAN2401, especially within the first 48 hours after injection. While a decrease in the area under the curve (AUC) was observed for A17D compared to BAN2401, 0-inf ) measured by 6-fold increase in exposure, A17D / R79T, A17D / R82S and A17D / R79T / R82S AUC increased 10-11 times (Table 1). In addition, compared with BAN2401, the terminal plasma half-life of the antibodies of the present invention was significantly prolonged (Table 1).

[0401] Table 1. Plasma PK parameters of BAN2401, A17D, A17D / R79T, A17D / R82S and A17D / R79T / R82S in mice. Half-life and AUC of all antibodies were calculated individually for all animals by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight). 0-inf Statistical analysis was performed by one-way ANOVA followed by Bonferroni post hoc multiple comparisons. 0-inf The values ​​and mean terminal half-life are shown in the table. The terminal half-life and AUC between BAN2401 and mutants are shown in the table 0-inf There was a statistically significant difference between the two groups, where ** indicates P < 0.001 and **** indicates P < 0.0001.

[0402]

[0403] Example 4. Pharmacokinetic profile of antibodies in rats

[0404] Pharmacokinetics of BAN2401, A17D, A17D / R79T, and A17D / R79T / R82S in rats

[0405] To study the pharmacokinetic profile of the antibodies of the invention in rats, 8-week-old female Sprague Dawley rats were injected intravenously with a single 10 mg / kg BAN2401 (N=3), A17D (N=4), A17D / R79T (N=6), or A17D / R79T / R82S (N=5). The animals were bled at 0.5 hours, 2 hours, 7 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, and 29 days after injection. The levels of BAN2401 and the antibodies of the invention were analyzed by ELISA (direct ELISA) using Aβ1-40 for capture and detection with HRP-conjugated goat anti-human IgG as described in Example 1. A rapid decrease in the levels of BAN2401 in the plasma of wild-type mice during the first 48 hours after administration resulted in low exposure ( Figure 2 ) was not seen in rats, whereas BAN2401 and the antibodies of the present invention exhibited similar PK profiles ( Figure 3 ). Plasma half-life and AUC calculated individually for all rats in the study 0-inf Statistical analysis of the values ​​showed that the AUC between BAN2401 and A17D or A17D / R79T / R82S 0-inf There were no significant differences in AUC or terminal half-life (Table 2). However, A17D / R79T showed a significantly higher AUC than BAN2401. 0-inf There was no statistical difference in the terminal half-life between the two (Table 2).

[0406] Table 2. Plasma PK parameters of BAN2401, A17D, A17D / R79T and A17D / R79T / R82S in rats. Half-life and AUC of all antibodies were calculated individually for all animals by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight). 0-inf The values ​​were statistically analyzed by one-way ANOVA followed by Bonferroni post hoc multiple comparisons. The average AUCs of BAN2401, A17D, A17D / R79T, and A17D / R79T / R82S were calculated. 0-inf The values ​​and mean terminal half-life are shown in the table. The terminal half-life and AUC between BAN2401 and mutants are shown in the table 0-inf There was no statistical difference, where ** indicates P < 0.01.

[0407]

[0408] Example 5. Deimmunization

[0409] Ex vivo whole protein T cell assays for BAN2401, A17D, A17D / R79T, and A17D / R79T / R82S

[0410] To assess whether the mutations introduced in the antibodies of the invention lead to an increased risk of immunogenic responses in humans, A17D, A17D / R79T and A17D / R79T / R82S were analyzed after BAN2401 by an ex vivo whole protein T cell activation assay. TM The time course T cell assay measures the ability of antibodies to induce CD4+ T cell responses (Antitope Ltd, Cambridge, UK). Samples were tested on CD8+ depleted peripheral blood mononucleated cells (PBMCs) from a group of 25 healthy donors (donors 1-25, Table 3) with broad HLA diversity. The ability of antibodies to induce CD4+ T cell responses was measured by proliferation and IL-2 secretion.

[0411] The results of the study showed that the overall potential risk of immunogenicity was low for BAN2401 and borderline low for A17D, with 8% and 12% of donors responding positive, respectively (Table 3). Analysis of A17D / R79T and A17D / R79T / R82S showed a somewhat unexpectedly higher risk of immunogenicity, as the combined frequencies of proliferation and IL-2 secretion were 24% and 20% of the study cohort, respectively.

[0412] Table 3.Summary of healthy donor T cell proliferation and IL-2 ELISpot responses for BAN2401, A17D, A17D / R79T, and A17D / R79T / R82S. Proliferation-positive T cell responses are indicated by "P" and positive ELISpot responses are indicated by "E". Borderline responses (*) are indicated. Correlations are expressed as the percentage of proliferation responses that were also positive in the ELISpot assay. Both proliferation and ELISpot assays must be positive to be considered a response for a donor. Humanized A33 (Welt S et al., Clin Cancer Res. 2003 Apr;9(4):1347-53., 2003), a therapeutic antibody control with a known mean immunogenicity of 32% in the clinic, typically stimulates 20-30% of donors to respond positively in the T cell proliferation assay. Phytohaemagglutinin (PHA) and Keyhole Limpet Hemocyanin (KLH) are useful antigens used as positive controls.

[0413]

[0414]

[0415] In silico T cell epitope screening of BAN2401

[0416] To further address the immunogenicity risk caused by mutations and find relevant positions for deimmunization, in silico T cell epitope screening was performed on BAN2401, A17D, A17D / R79T, A17D / R82S, and A17D / R79T / R82S. The variable region sequences of BAN2401, A17D, A17D / R79T, A17D / R82S, and A17D / R79T / R82S were provided to Antitope Ltd (Cambridge, UK) for in silico analysis using its proprietary in silico technology iTope. TM and TCED TM Non-germline promiscuous MHC class II binding sequences were identified in both the heavy and light chains of the antibodies analyzed. TCED TM BLAST search analysis revealed a two-part match to a previously identified epitope in a database of peptides with known ex vivo immunogenicity.

[0417] Ex vivo T cell epitope mapping of BAN2401, A17D, A17D / R79T, A17D / R82S, and A17D / R79T / R82S

[0418] To verify the immunogenicity risk caused by novel mutations and identify sites of deimmunization, EpiScreen TM T cell epitope mapping technology (Antitope Ltd, Cambridge, UK) evaluated the presence of CD4+ T cell epitopes from 44 peptides (15-mers) from the variable regions of BAN2401, A17D, A17D / R79T, A17D / R82S and A17D / R79T / R82S. Such peptides were selected to cover all potential T cell epitopes identified by computer simulation screening and to cover two regions (including peptides with or without mutations) in the A17D substitution region and the R79T and R82S substitution regions.

[0419] For the Episcreen selected from the previous section TM Peptides tested in a panel of 11 human donors for full antibody analysis. 3 The proliferation assay of [H]-thymidine incorporation measured the T cell response of each donor to each peptide. The results identified the presence of three potential T cell epitopes in the sequence (epitopes 1, 5 and 8). "Epitope 1", which is present in the heavy chain, is considered weak. "Epitope 5", which includes the A17D mutation, is weak, and no donor response was observed for the relevant peptides of the wild-type BAN2401 sequence. "Epitope 8" is the most critical epitope based on the frequency of T cell responses and was identified in the peptides of antibodies A17D / R79T, A17D / R82S and A17D / R79T / R82S, but not in wild-type BAN2401 or A17D.

[0420] Data from ex vivo T cell epitope mapping support the conclusions from the whole antibody time course T cell assay that A17D / R79T and A17D / R79T / R82S are associated with an increased overall risk of immunogenicity. In addition, A17D / R82S (not included in the whole antibody T cell assay) also appears to have an increased risk of immunogenicity due to the R82S mutation in "epitope 8".

[0421] Deimmunization of peptides identified as potential T cell epitopes by ex vivo T cell epitope mapping

[0422] Typically, deimmunization is achieved by changing a single amino acid at one of the important anchor positions of a potential class II MHC binding peptide (p1, p4, p6, p7, and p9 of the 9-mer). Specific deimmunization mutations in the three epitopes (epitopes 1, 5, and 8) identified in the above-mentioned ex vivo peptide mapping are selected. Substitutions are selected based on the reduction in the expected binding affinity of the peptide to the binding pocket of the class II MHC molecule. Two deimmunization substitutions are tested for each peptide that has been indicated as potentially immunogenic, and the Episcreen TMThese peptides were analyzed together with the original peptides of BAN2401, A17D, A17D / R79T, A17D / R82S, and A17D / R79T / R82S by peptide mapping technique.

[0423] Use Measurement 3 [H]-Thymidine incorporation proliferation assay measures T cell responses of each donor to each peptide. The results of this study confirm previous findings, but all epitopes (epitopes 1, 5, and 8) were considered weak in this study (Table 4). Nevertheless, compared with the original peptide, all deimmunization substitutions successfully reduced the immunogenicity risk of peptides covering three epitopes, in which there were no or few T cell responders (Table 4). This shows that the selected substitutions are effective and deimmunization is successful.

[0424] Table 4. Using EpiScreen TM Deimmunization results at the peptide level for epitopes identified as immunogenic by T cell epitope mapping. Peptides derived from BAN2401 and antibodies of the invention identified as immunogenic are shown, as well as the same peptides with deimmunizing substitutions introduced (underlined). Half-life improving mutations are shown in bold. All deimmunized peptides were tested in an ex vivo T cell assay, and the results are shown in the rightmost column (donor response frequency).

[0425]

[0426] *) Non-deimmunized antibody

[0427] Design and production of deimmunized variants of A17D / R79T

[0428] Eight deimmunized variants of A17D / R79T were designed based on the results from the above T cell epitope mapping and peptide deimmunization. In all three epitopes identified as potentially immunogenic, deimmunization mutations that showed reduced immunogenicity in ex vivo T cell epitope mapping were introduced in the combinations shown in Table 5.

[0429] Table 5. Summary of 8 deimmunized variants of the double mutant A17D / R79T. For a particular antibody, deimmunized mutations of epitopes 1, 5 and 8 selected and functionally validated at the peptide level in an ex vivo T cell assay are shown. N-terminal numbering is used. VH = variable heavy chain, VL = variable light chain.

[0430]

[0431] Example 6. Target binding characteristics of deimmunized antibodies

[0432] Retained Aβ protofibril binding in eight deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8) compared to BAN2401

[0433] The target binding profiles of eight deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8) were analyzed after BAN2401 (control) by the inhibition method described in Example 1 (inhibition ELISA). The results are shown in Figure 4 In which is shown an analysis of the binding and selectivity to Aβ protofibrils compared to Aβ monomers for A17D / R79T_DI 1-8 and BAN2401. The results show that in the antibodies of the present invention (A17D / R79T_DI 1-8), the binding and selectivity to Aβ protofibrils are retained compared to the binding to Aβ monomers.

[0434] Example 7. Pharmacokinetic profile of deimmunized antibodies in mice

[0435] Pharmacokinetic study of BAN2401, A17D / R79T, and eight deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8) in wild-type mice

[0436] To compare the PK profiles of 8 deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8) with those of BAN2401 and A17D / R79T in wild-type mice, 8-10 week old C57BL / 6 female mice were grouped (N=5) and injected intravenously with a single 10 mg / kg antibody. The animals were bled after 0.5 hours, 2 days, 7 days, 14 days, and 28 days, and the levels of BAN2401 and the antibodies of the invention were analyzed by capturing with Aβ1-40 as described in Example 1 and detecting with an ELISA (direct ELISA) using a goat anti-human IgG coupled to HRP. Figure 5 As shown in the time versus concentration graph in , the PK profile of the deimmunized mutant of A17D / R79T (A17D / R79T_DI 1-8) is similar to that of A17D / R79T and different from that of BAN2401. The results showed that the AUC of A17D / R79T and the deimmunized variant (A17D / R79T_DI 1-8) were significantly higher than those of BAN2401. 0-inf Both the PK and terminal half-lives were significantly enhanced (Table 6). Thus, all deimmunized variants showed improved PK profiles compared to BAN2401 and showed a PK profile similar to that of A17D / R79T, with increased exposure and prolonged elimination half-life.

[0437] Table 6. Plasma PK parameters of BAN2401, A17D / R79T and deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8) in mice. The half-life and AUC of all antibodies were calculated individually for all animals by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight). 0-inf Statistical analysis was performed by one-way ANOVA followed by Bonferroni post hoc multiple comparisons. 0-inf The terminal half-life and AUC between BAN2401 and mutants are shown in the table. 0-inf There was statistically significant difference between the two groups, where * indicates P < 0.05, **P < 0.01, **P < 0.001 and ***P < 0.0001.

[0438]

[0439] Example 8. Pharmacokinetic profile of deimmunized antibodies in rats

[0440] Pharmacokinetic profiles of BAN2401, A17D / R79T, and eight deimmunized variants of A17D / R79T (A17D / R79TDI 1-8) in rats

[0441] To compare the PK profiles of 8 deimmunized variants of A17D / R79T (A17D / R79T_DI1-8) with those of BAN2401 and A17D / R79T in rats, 8-week-old female Sprague Dawley rats were grouped (N=5) and injected intravenously with a single 10 mg / kg antibody. The animals were bled after 0.5 hours, 2 days, 7 days, 14 days, and 28 days, and the levels of BAN2401 and antibodies of the invention were analyzed by ELISA using Aβ1-40 for capture and HRP-coupled goat anti-human IgG detection (Example 1, direct ELISA).

[0442] like Figure 6 As shown in the Figure, BAN2401, A17D / R79T and deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8) showed quite similar PK profiles in rats. The results showed that in rats, A17D / R79T_DI 1 had a significantly longer terminal half-life than BAN2401 (p < 0.05), while the AUC of A17D / R79T, A17D / R79T_DI 1 and A17D / R79T_DI 4 were significantly lower than those of BAN2401. 0-inf Significantly greater than BAN2401 (p<0.01) (Table 7).

[0443] Table 7. Plasma PK parameters of BAN2401, A17D / R79T and deimmunized variants of A17D / R79T (A17D / R79T_DI 1-8) in rats. The half-life and AUC of all antibodies were calculated individually for all animals by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight). 0-inf , and statistical analysis was performed by one-way ANOVA followed by Bonferroni test followed by multiple comparisons. The mean AUC and mean terminal half-life are shown in the table. The table shows the statistical differences in terminal half-life and AUC between BAN2401 and the antibodies of the present invention, where * indicates P < 0.05, and **P < 0.01.

[0444]

[0445] Example 9. Pharmacokinetic profile of deimmunized antibodies in monkeys

[0446] Pharmacokinetic profiles of BAN2401, A17D / R79T_DI 3, A17D / R79T_DI 4, and A17D / R79T_DI 8 in cynomolgus monkeys.

[0447] To compare the PK profiles of A17D / R79T_DI 3, A17D / R79T_DI 4 and A17D / R79T_DI 8 with that of BAN2401 in monkeys, male cynomolgus monkeys were divided into groups (N=3) and given a single intravenous infusion of 5 mg / kg BAN2401 or 10 mg / kg of the antibody of the invention. Blood was collected from monkeys injected with BAN2401 at 5 minutes, 1 hour, 2 hours, 8 hours, 24 hours, 2 days, 4 days, 7 days, 14 days, 21 days and 28 days after administration, while blood was collected from monkeys injected with the antibody of the invention at 5 minutes, 2 hours, 8 hours, 24 hours, 3 days, 7 days, 14 days, 21 days and 28 days. The plasma concentrations of the administered antibodies were analyzed by ELISA as described in Example 1 (direct ELISA). The PK profiles are shown as Figure 7 The time versus concentration plots for all antibodies are shown in Table 1, while the half-life and AUC 0-inf Calculated by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight) (Table 8).The PK parameters from BAN2401 shown in Table 8 are results from a separate study in which BAN2401 was administered by intravenous infusion at a dose of 5 mg / kg BAN2401.

[0448] Table 8Plasma PK parameters of BAN2401, A17D / R79T_DI 3, A17D / R79T_DI 4 and A17D / R79T_DI 8 in cynomolgus monkeys. The terminal half-life and AUC of all antibodies were calculated by non-compartmental analysis using Phoenix WinNonlin 6.3 (Pharsight). 0-inf The PK parameters for BAN2401 presented here are from a separate study in which BAN2401 was administered by intravenous infusion at a dose of 5 mg / kg BAN2401. To simplify the AUC comparison between BAN2401 and the antibodies of the invention, the dose-adjusted mean AUC 0-inf and mean terminal half-life values ​​are shown in the table.

[0449]

[0450] Example 10. Simple allometric scaling - mouse, rat and monkey to human

[0451] To predict the human half-life of the antibodies of the invention (shown here as A17D / R79T_DI 3, A17D / R79T_DI 4, and A17D / R79T_DI 8), a 2-compartment model and simple allometric scaling were performed. The 2-compartment model was applied to the plasma PK profiles of BAN2401 and the antibodies of the invention using Phoenix WinNonlin 6.3 (Pharsight) to estimate the clearance (CL) and volume (V) values ​​in mice, rats, and monkeys. The CL and V values ​​were plotted against body weight, and simple allometric scaling was applied (Deng et al. mAbs 2011: 3 (1): p. 61-66. doi: 10.4161 / mabs.3.1.13799). The terminal half-life was calculated as follows:

[0452]

[0453] In a simple allometric scale of volume or clearance, the estimated parameter values ​​in preclinical species are plotted against body weight. The constants and exponents from the regression line are used to predict V and CL in a person weighing 70 kg. Both the exponent of the regression line (which should preferably be about 0.85 for clearance and about 1 for volume) and adherence are confidence measures for estimating a particular parameter in a person.

[0454] like Fig.11As shown, BAN2401 has a poor linear correlation in the central CL between the species tested, leading to indeterminate simple allometric scaling. The deviation from exponential (which should be close to 0.85) suggests that the poor correlation is affected by the high clearance of BAN2401 in mice. This leads to an underestimate of the expected CL in humans and therefore an overestimation of the predicted terminal half-life. The terminal half-life of BAN2401 was estimated to be 41 days, which deviates considerably from the half-life measured in the clinic (5-7 days). The actual CL of BAN2401 has been estimated at 41 days. Fig.11 In contrast to the poor linear correlation of BAN2401 between mice, rats and cynomolgus monkeys, the antibodies of the present invention (A17D / R79T_DI 3, A17D / R79T_DI 4 and A17D / R79T_DI 8) showed excellent correlation ( Fig.11 ). For A17D / R79T_DI 3, A17D / R79T_DI 4 and A17D / R79T_DI 8, the terminal half-lives calculated based on the predicted human PK parameters were 13, 15 and 20 days in humans, respectively. For BAN2401 and the antibodies of the invention, simple allometric scaling of volume (V) showed good linear correlation and an exponent of about 1 (not shown).

[0455] The antibodies of the present invention show a linear correlation of CL between species, with a more reliable allometric scale, and they behave like other therapeutic IgG1s, such as bevacizumab, omalizumab and trastuzumab (as described by Deng et al., Expert Opin. Drug Metab. Toxicol 8(2) (2012): p. 141-160), which have a half-life of about 15 to 30 days in humans. In contrast to BAN2401, which has a less than ideal PK profile in mice and humans that deviates from many other therapeutic IgG1s, the antibodies of the present invention have been engineered to result in a normalized PK profile in mice. They show a PK profile and half-life similar to other therapeutic IgG1s in mice, rats and cynomolgus monkeys, with a half-life of about 15 to 30 days in humans. This finding suggests that the extended half-life of the antibodies of the present invention in mice will translate to humans.

[0456] The present invention also relates to the following embodiments (which correspond to the claims of the original application):

[0457] 1. An antibody or antigen-binding fragment thereof having affinity for Aβ protofibrils, wherein the antibody or antigen-binding fragment thereof has a variable light chain according to SEQ ID NO: 8, wherein

[0458] x1 is selected from A, D, E and Q, or a functional analog thereof;

[0459] x2 is selected from R, T, K, A and G, or a functional analog thereof;

[0460] x3 is selected from R, S, C, G and N, or a functional analog thereof;

[0461] y1 is selected from V and A;

[0462] y2 is selected from I and V;

[0463] y3 is selected from S and Q;

[0464] y4 is selected from E and D; and optionally

[0465] The variable heavy chain according to SEQ ID NO: 14, wherein

[0466] z1 is selected from V and I;

[0467] z2 is selected from R and Q; and

[0468] z3 is selected from A, N and T;

[0469] The combination of x1=A, x2=R and x3=R is excluded.

[0470] 2. The antibody or antigen-binding fragment according to embodiment 1, wherein

[0471] x1 is selected from A, D, E and Q;

[0472] x2 is selected from R, T, K, A and G;

[0473] x3 is selected from R, S, C, G and N;

[0474] y1 is selected from V and A;

[0475] y2 is selected from I and V;

[0476] y3 is selected from S and Q;

[0477] y4 is selected from E and D; and

[0478] The variable heavy chain according to SEQ ID NO: 14, wherein

[0479] z1 is selected from V and I;

[0480] z2 is selected from R and Q; and

[0481] z3 is selected from A, N and T;

[0482] The combination of x1=A, x2=R and x3=R is excluded.

[0483] 3. The antibody or antigen-binding fragment according to embodiment 1 or 2, wherein the light chain a comprises a combination of mutations selected from the group consisting of:

[0484] x1 and (y1 and / or y2);

[0485] x1 and (y1 and / or y2) and x2 and (y3 and / or y4);

[0486] x1 and (y1 and / or y2) and x2 and x3 and (y3 and / or y4);

[0487] x1 and (y1 and / or y2) and x3 and (y3 and / or y4);

[0488] x2(y3 and / or y4);

[0489] x2 and x3 and (y3 and / or y4); and

[0490] x3 and (y3 and / or y4);

[0491] The combination of x1=A, x2=R and x3=R is excluded.

[0492] 4. The antibody or antigen-binding fragment of any one of embodiments 1 to 3, wherein the variable light chain comprises one or more mutations selected from:

[0493] x1 is D and (y1 and / or y2);

[0494] x1 is D and (y1 and / or y2), x2 is T and (y3 and / or y4);

[0495] x1 is D and (y1 and / or y2), x2 is T, x3 is S and (y3 and / or y4);

[0496] x1 is D and (y1 and / or y2) and x3 is S and (y3 and / or y4);

[0497] x2 is T(y3 and / or y4);

[0498] x2 is T and x3 is S and (y3 and / or y4); and

[0499] x3 is S and (y3 and / or y4);

[0500] Where y1 is V or A and y2 is V or I, excluding the combination of y1=V and y2=I;

[0501] y3 is S or Q and y4 is E or D, excluding the combination of y3=S and y4=E.

[0502] 5. The antibody or antigen-binding fragment of any one of embodiments 1 to 4, wherein

[0503] x1 is D;

[0504] x2 is T;

[0505] x3 is R;

[0506] y1 is selected from V and A;

[0507] y2 is selected from V and I;

[0508] y3 is selected from Q and S;

[0509] y4 is selected from D and E;

[0510] z1 is V;

[0511] z2 is R; and

[0512] z3 is selected from N, T and A;

[0513] Combinations in which y1 is V, y2 is I, y3 is S, y4 is E, z1 is V, z2 is R and z3 is A are excluded.

[0514] 6. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0515] 7. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0516] 8. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0517] 9. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0518] 10. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15.

[0519] 11. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0520] 12. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0521] 13. The antibody or antigen-binding fragment thereof according to embodiment 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO: 16.

[0522] 14. The antibody or antigen-binding fragment of any one of embodiments 1 to 13, wherein the antibody or the antigen-binding fragment comprises an IgG heavy chain constant region.

[0523] 15. The antibody according to any one of embodiments 1 to 14 for use in medicine.

[0524] 16. The antibody according to any one of embodiments 1 to 14 for use in the treatment and / or prevention of Alzheimer's disease and other disorders associated with Aβ protein aggregation.

[0525] 17. The antibody used according to embodiment 16, wherein the other disorders associated with Aβ protein aggregation are selected from traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD).

[0526] 18. Use of the antibody according to any one of embodiments 1 to 14 in the preparation of a medicament for the treatment and / or prevention of Alzheimer's disease and other disorders associated with Aβ protein aggregation.

[0527] 19. The use according to embodiment 18, wherein the other conditions associated with Aβ protein aggregation are selected from traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD).

[0528] 20. A method of reducing the amount of A[beta] protofibrils in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment of embodiments 1 to 14.

[0529] 21. A method for treating and / or preventing Alzheimer's disease in a subject suffering from or at risk of developing the disease, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment according to embodiments 1 to 14.

[0530] 22. A method for treating and / or preventing traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, and Parkinson's disease dementia (PDD) in a subject suffering from or at risk of developing the disease, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment according to embodiments 1 to 14.

[0531] 23. A method for measuring the amount of Aβ protofibrils and / or aggregated Aβ proteins in humans, comprising contacting the human's tissue or body fluids with the antibody or antigen-binding fragment according to embodiments 1 to 14 in vivo or in vitro, and measuring the amount of the antibody or antigen-binding fragment bound to the Aβ protofibrils and / or aggregated Aβ proteins.

[0532] 24. A method for diagnosing Alzheimer's disease in a human suffering from or at risk of developing the disease, comprising contacting a tissue or body fluid of the human with an antibody or antigen-binding fragment or fragment thereof according to embodiments 1 to 14 in vivo or in vitro, and measuring the amount of the antibody bound to aggregated Aβ protein.

[0533] 25. A method for diagnosing any of traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, tauopathy, systemic amyloidosis, atherosclerosis, and Parkinson's disease dementia (PDD) in a human suffering from or at risk of developing the disease, comprising contacting a tissue or body fluid of the human with an antibody or antigen-binding fragment or fragment thereof according to embodiments 1 to 14 in vivo or in vitro, and measuring the amount of the antibody bound to aggregated Aβ protein.

[0534] 26. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of embodiments 1 to 14, and a pharmaceutically acceptable excipient and / or diluent.

[0535] 27. The antibody according to any one of embodiments 1 to 14 for veterinary use. Sequence Listing <110> BioArctic Neuroscience AB Eisai R&D Management Co., Ltd. <120> Improved Aβ protofibril binding antibodies <130> P141879WO00 <140> PCT <141> 2015-07-08 <150> US 62 / 022952 <151> 2014-07-10 <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 1 Ser Phe Gly Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 2 Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val Lys 1 5 10 15 Gly <210> 3 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 3 Glu Gly Gly Tyr Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp Tyr 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 4 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 5 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 6 Phe Gln Gly Ser His Val Pro Pro Thr 1 5 <210> 7 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 7 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Ala Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 8 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <220> <221> MISC_FEATURE <222> (13) <223> Xaa can be Val or Ala <220> <221> MISC_FEATURE <222> (17) <223> Xaa can be Ala, Asp, Glu, Gln or a functional analog thereof <220> <221> MISC_FEATURE <222> (21)..(21) <223> Xaa can be Ile or Val <220> <221> MISC_FEATURE <222> (79)..(79) <223> Xaa can be Arg, Thr, Lys, Ala, Gly or a functional analog thereof <220> <221> MISC_FEATURE <222> (81)..(81) <223> Xaa can be Ser or Gln <220> <221> MISC_FEATURE <222> (82)..(82) <223> Xaa can be Arg, Ser, Cys, Gly, Asn or a functional analog thereof <220> <221> MISC_FEATURE <222> (84)..(84) <223> Xaa can be Glu or Asp <400> 8 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Xaa Thr Pro Gly 1 5 10 15 Xaa Pro Ala Ser Xaa Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Xaa Ile 65 70 75 80 Xaa Xaa Val Xaa Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 9 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Asp Pro Ala Ser Val Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Gln Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 10 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 10 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Asp Pro Ala Ser Val Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Arg Val Asp Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 11 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Ala Thr Pro Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Gln Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 12 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 12 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Ala Thr Pro Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Arg Val Asp Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 13 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 14 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <220> <221> MISC_FEATURE <222> (37)..(37) <223> Xaa can be Val or Ile <220> <221> MISC_FEATURE <222> (38)..(38) <223> Xaa can be Arg or Gln <220> <221> MISC_FEATURE <222> (40)..(40) <223> Xaa can be Ala, Asn or Thr <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Xaa Xaa Gln Xaa Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 15 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Asn Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 16 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 17 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 18 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Gly Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly Tyr Tyr Tyr Gly Arg Ser Tyr Tyr Thr Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 19 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 19 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 20 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 20 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> twenty one <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> twenty one Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Ser Ser Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> twenty two <211> 112 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> twenty two Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Asp Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Ser Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 23 <211> 330 <212> PRT <213> Homo sapiens <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 24 <211> 107 <212> PRT <213> Homo sapiens <400> 24 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims

1. An antibody or antigen-binding fragment thereof having affinity for Aβ protofibrils, wherein the antibody or antigen-binding fragment thereof has a variable light chain according to the following amino acid sequence, and The variable heavy chain according to the following amino acid sequence, EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWZ1Z2QZ3PGKGLEWVAYISSGSSTIYYGDTVKGR FTISRDNAKNSLFLQMSSLRAEDTAVYYCAREGGYYYGRSYYTMDYWGQGTTV TVSS, in x1 is D; x2 is T; x3 is R; y1 is selected from V and A; y2 is selected from V and I; y3 is selected from Q and S; y4 is selected from D and E; z1 is V; z2 is R; and z3 is selected from N, T and A; Combinations in which y1 is V, y2 is I, y3 is S, y4 is E, z1 is V, z2 is R and z3 is A are excluded.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:

16.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:

15.

4. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:

15.

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:

15.

6. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 12; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:

15.

7. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 9; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:

16.

8. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 10; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:

16.

9. The antibody or antigen-binding fragment thereof according to claim 1, comprising a variable light chain comprising the amino acid sequence shown in SEQ ID NO: 11; and a variable heavy chain comprising the amino acid sequence shown in SEQ ID NO:

16.

10. The antibody or antigen-binding fragment of any one of claims 1 to 9, wherein the antibody or the antigen-binding fragment comprises an IgG heavy chain constant region.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 for use in medical treatment.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, for use in the treatment and / or prevention of Alzheimer's disease and other disorders associated with Aβ protein aggregation, wherein the other disorders associated with Aβ protein aggregation are selected from Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD).

13. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 in the preparation of a medicament for treating and / or preventing Alzheimer's disease and other disorders associated with Aβ protein aggregation, wherein the other disorders associated with Aβ protein aggregation are selected from Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD).

14. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 10 for the preparation of a medicament for treating and / or preventing Alzheimer's disease in a subject suffering from or at risk of developing Alzheimer's disease.

15. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 10 for the preparation of a medicament for treating and / or preventing traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD) in a subject suffering from or at risk of developing said disease.

16. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 10 in the preparation of a diagnostic agent for measuring the amount of Aβ protofibrils and / or aggregated Aβ protein in humans, wherein the measurement comprises contacting the human tissue or body fluid with the antibody or antigen-binding fragment in vivo or in vitro, and measuring the amount of the antibody or antigen-binding fragment bound to the Aβ protofibrils and / or aggregated Aβ protein.

17. Use of a reagent for measuring the amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 that binds to aggregated Aβ protein in the preparation of a diagnostic agent for diagnosing Alzheimer's disease in a human suffering from or at risk of developing the disease, wherein the diagnosis comprises contacting the human tissue or body fluid with the antibody or antigen-binding fragment or fragment thereof according to claims 1 to 10 in vivo or in vitro, and measuring the amount of the antibody bound to aggregated Aβ protein.

18. Use of a reagent for measuring the amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 that binds to aggregated Aβ protein in the preparation of a diagnostic agent for diagnosing any of traumatic brain injury (TBI), Lewy body dementia (LBD), Down syndrome (DS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia, systemic amyloidosis, atherosclerosis and Parkinson's disease dementia (PDD) in a human suffering from or at risk of developing the disease, wherein the diagnosis comprises contacting the human tissue or body fluid with the antibody or antigen-binding fragment or fragment thereof according to claims 1 to 10 in vivo or in vitro, and measuring the amount of the antibody bound to aggregated Aβ protein.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 10, and a pharmaceutically acceptable excipient and / or diluent.

Images