Methods of treating neurological disorders with anti-A [beta] antibodies

Abstract

Antibodies that bind to human beta amyloid peptide, methods of detecting, measuring, and treating amyloid-producing disorders using the antibodies, pharmaceutical compositions comprising the antibodies, and methods of making are provided.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 481,631, filed January 26, 2023; U.S. Provisional Application Serial No. 63 / 597,868, filed November 10, 2023; and U.S. Provisional Application Serial No. 63 / 597,861, filed November 10, 2023, and U.S. Provisional Application Serial No. 63 / 618,045, filed January 5, 2024, each of which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] The sequence list in computer-readable form is submitted electronically with this application and is incorporated herein by reference in its entirety. The sequence list is contained in a file created on January 4, 2024, with the filename "20-1030-WO2_SeqList" and a size of 124,150 bytes. Technical Field

[0005] This disclosure relates to anti-amyloid β (Aβ) antibodies and compositions thereof, as well as methods of using them. Background Technology

[0006] Alzheimer's disease (AD) is a progressive disease that causes age-related dementia. It is generally classified as either late-onset (occurring in old age, 65+) or early-onset (fully developed before old age, between 35 and 60 years). The pathology of these two types appears to be similar, but in cases starting at an earlier age, the abnormalities are often more severe and widespread. The disease is characterized by at least two types of lesions in the brain: neurofibrillary tangles and senile plaques. Neurofibrillary tangles are intracellular deposits of microtubule-associated tau protein, which consists of two filaments intertwined in pairs. Senile plaques (amyloid plaques) are anastomosing neurofibrillary networks up to 150 μm in diameter, visible through microscopic analysis of brain tissue sections, with extracellular amyloid protein deposits at their center. The accumulation of intracerebral amyloid plaques is also associated with Down syndrome and other cognitive disorders.

[0007] The main building blocks of plaques are peptides called amyloid β (Aβ or Abeta), or β-amyloid peptide. Aβ peptide is a 4-kDa intracellular fragment of a larger transmembrane glycoprotein called amyloid precursor protein (APP), consisting of 39 to 43 amino acids. Due to the proteolytic processing of APP by different secretases, Aβ is primarily found in both short (40 amino acids) and long (42 to 43 amino acids) forms. A portion of the hydrophobic transmembrane domain of APP is found at the carboxyl terminus of Aβ and may explain the ability of Aβ to aggregate into plaques, particularly in the long form. The accumulation of amyloid plaques in the brain ultimately leads to neuronal cell death. The physical symptoms associated with this type of neurodegeneration characterize Alzheimer's disease.

[0008] Monoclonal antibodies (mAbs) targeting amyloid beta have been clinically demonstrated to reduce amyloid plaque burden in patients. The FDA has granted accelerated approval to the anti-amyloid beta antibodies aducanumab and lecanemab. FDA approval of these antibodies was based on the antibodies demonstrating a reduction in amyloid beta on PET imaging, which was identified as a surrogate endpoint that could reasonably predict clinical benefit. Indeed, clinical trials of anti-amyloid beta antibodies, including aducanumab and lecanemab, have shown that reduced plaque burden is associated with a slowing of cognitive decline in Alzheimer's disease. However, these treatments have limited efficacy and / or treatment-related side effects. Furthermore, these antibodies require intravenous administration and / or frequent high-dose subcutaneous administration, which places a burden on patients and caregivers. Therefore, there remains a need for effective and safe anti-amyloid beta antibodies for the treatment of Alzheimer's disease, particularly those effective when administered subcutaneously as part of a relatively infrequent dosing regimen. Summary of the Invention

[0009] This disclosure relates to antibodies (and antibody fragments) that specifically bind to Aβ, methods for generating such antibodies and antibody fragments and associated nucleic acids, methods for treating patients with Aβ-related neurological disorders, pharmaceutical formulations and compositions exhibiting antibodies that bind with high affinity for Aβ for prophylactic and / or therapeutic purposes, such as treating amyloidogenic diseases, reducing their risk or delaying their onset, preventing, reducing or inhibiting markers of amyloidogenic diseases (e.g., amyloid plaques), and improving cognition. This disclosure further relates to methods for detecting amyloid plaques and measuring the efficacy of treatment in patients undergoing treatment for amyloidogenic diseases. This disclosure is at least in part based on the identification and characterization of monoclonal antibodies that specifically bind to Aβ peptides and effectively reduce plaque burden and neutralize soluble Aβ species associated with amyloidogenic diseases.

[0010] In one aspect, this disclosure provides methods for treating Alzheimer's disease in subjects with this need. In one aspect, this disclosure provides a method for treating Alzheimer's disease in subjects, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof approximately every 3-5 weeks. In another aspect, this disclosure provides a method for reducing amyloid plaques in subjects, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof approximately every 3-5 weeks. In yet another aspect, this disclosure provides a method for converting a subject from amyloid-positive to amyloid-negative, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof approximately every 3-5 weeks. This method also includes administering to the subject about 65 mg to about 200 mg of an antibody or an antigen-binding fragment thereof approximately every 3-5 weeks.

[0011] In one aspect, this disclosure provides a method for treating Alzheimer's disease in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof about every 4 weeks. In another aspect, this disclosure provides a method for reducing amyloid plaques in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof about every 4 weeks. In yet another aspect, this disclosure provides a method for converting a subject from amyloid-positive to amyloid-negative, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof about every 4 weeks.

[0012] In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment binds to an epitope located at the N-terminus of the Aβ peptide, and the epitope comprises at least one amino acid selected from amino acids 1-10 of the Aβ peptide. In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment binds to an epitope containing at least one amino acid selected from amino acids 1-7 of the Aβ peptide.

[0013] In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment binds to amyloid β with an apparent KD of about 5 nM or less. 1-42 Protofibril binding. In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment binds to amyloid β at an apparent KD of about 1 nM or less. 1-42 Primary fibrillary binding. In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment binds to amyloid β with an apparent KD of about 10 nM or less. 1-28 Monomer combination.

[0014] In some embodiments, the method includes administering about 20 mg to about 100 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In some embodiments, the method includes administering about 100 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In some embodiments, the method includes administering about 45 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In some embodiments, the method includes administering about 70 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In some embodiments, the method includes administering about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In some embodiments, the anti-amyloid β antibody is administered approximately every 4 weeks.

[0015] In some embodiments, the anti-amyloid beta antibody or its antigen-binding fragment is administered as a pharmaceutical composition comprising the anti-amyloid beta antibody or its antigen-binding fragment and a pharmaceutically acceptable diluent. In some embodiments, a pharmaceutically effective amount of the anti-amyloid beta antibody or its antigen-binding fragment comprises about 45 mg. In some embodiments, a pharmaceutically effective amount of the anti-amyloid beta antibody or its antigen-binding fragment comprises about 70 mg. In some embodiments, a pharmaceutically effective amount of the anti-amyloid beta antibody or its antigen-binding fragment comprises about 200 mg. In some embodiments, administration is performed approximately every 4 weeks.

[0016] In some implementations, administration is intravenous or subcutaneous. In some implementations, administration is subcutaneous.

[0017] In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, CDR2, and CDR3, and the light chain variable region comprises light chain CDR1, CDR2, and CDR3, wherein...

[0018] The heavy chain CDR1 contains an amino acid sequence of one of SEQ ID NO: 16, 19, or 20.

[0019] The heavy chain CDR2 contains an amino acid sequence of one of SEQ ID NO: 20, 21, 22, or 23.

[0020] The heavy chain CDR3 contains an amino acid sequence of one of SEQ ID NO: 18, 24, or 25.

[0021] The light chain CDR1 contains an amino acid sequence of one of SEQ ID NO: 26, 29, 31, or 32.

[0022] The light chain CDR2 contains an amino acid sequence of one of SEQ ID NO: 33, 34, 35, or 36, and

[0023] The light chain CDR3 contains an amino acid sequence of one of SEQ ID NO: 28, 38 or 39.

[0024] In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment includes

[0025] The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 16.

[0026] The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 20.

[0027] The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 18.

[0028] The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 29.

[0029] The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 34, and

[0030] The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 38.

[0031] In some embodiments, the heavy chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, and the light chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 9. In one embodiment of the first aspect, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9.

[0032] In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment includes

[0033] The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 16.

[0034] The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 20.

[0035] The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 18.

[0036] The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 29.

[0037] The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 33, and

[0038] The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 28.

[0039] In some embodiments, the heavy chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, and the light chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0040] In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment includes

[0041] The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 19.

[0042] The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 21.

[0043] The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 24.

[0044] The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 29.

[0045] The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 34, and

[0046] The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 38.

[0047] In some embodiments, the heavy chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 4, and the light chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 4, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9.

[0048] In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment includes

[0049] The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO: 19.

[0050] The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO: 21.

[0051] The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO: 25.

[0052] The light chain CDR1 contains the amino acid sequence of SEQ ID NO: 29.

[0053] The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 34, and

[0054] The light chain CDR3 contains the amino acid sequence of SEQ ID NO: 38.

[0055] In some embodiments, the heavy chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, and the light chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9.

[0056] In some embodiments, the anti-amyloid β antibody is humanized IgG1. In one embodiment of the first aspect, the anti-amyloid β antibody is a holoantibody, a chimeric antibody, a CDR transplanted antibody, or a recombinant antibody.

[0057] In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, the heavy chain constant region comprising at least 95% of the same amino acid sequence as SEQ ID NO: 40, and the light chain constant region comprising at least 95% of the same amino acid sequence as SEQ ID NO: 41.

[0058] In some embodiments, the anti-amyloid β antibody comprises a heavy chain of SEQ ID NO: 101 with or without a C-terminal lysine, and a light chain of SEQ ID NO: 102. In some embodiments, the anti-amyloid β antibody is h2731.

[0059] Therefore, in various aspects, this disclosure relates to methods utilizing antibodies or fragments thereof that specifically bind to the Aβ peptide. In some embodiments, the antibody and fragment include a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 are as shown in one of the antibodies in Table 1. Furthermore, the antibodies or fragments of this disclosure may have a heavy chain variable region as shown in one of the antibodies in Table 1, and may also have a light chain variable region as shown in one of the antibodies in Table 1.

[0060] In another aspect, this disclosure provides a method for treating Alzheimer's disease in a subject, the method comprising subcutaneously administering about 20 mg to about 200 mg of an anti-amyloid β antibody to the subject approximately every 4 weeks; the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102. In another aspect, this disclosure provides a method for treating Alzheimer's disease in a subject, the method comprising subcutaneously administering about 45 mg of an anti-amyloid β antibody to the subject approximately every 4 weeks; the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102. In another aspect, this disclosure provides a method for treating Alzheimer's disease in a subject, the method comprising subcutaneously administering approximately 70 mg of an anti-amyloid β antibody to the subject approximately every 4 weeks; the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102. In another aspect, this disclosure provides a method for treating Alzheimer's disease in a subject, the method comprising subcutaneously administering approximately 200 mg of an anti-amyloid β antibody to the subject approximately every 4 weeks; the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102.

[0061] In another aspect, this disclosure provides a method for reducing amyloid plaques in a subject, the method comprising subcutaneously administering about 20 mg to about 200 mg of an anti-amyloid β antibody to the subject approximately every 4 weeks; the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102. In another aspect, this disclosure provides a method for reducing amyloid plaques in a subject, the method comprising subcutaneously administering about 45 mg of an anti-amyloid β antibody to the subject approximately every 4 weeks; the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102. In another aspect, this disclosure provides a method for reducing amyloid plaques in a subject, the method comprising subcutaneously administering approximately 70 mg of an anti-amyloid β antibody to the subject approximately every 4 weeks; the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102. In another aspect, this disclosure provides a method for reducing amyloid plaques in a subject, the method comprising subcutaneously administering approximately 200 mg of an anti-amyloid β antibody to the subject approximately every 4 weeks; the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102.

[0062] On the other hand, this disclosure provides a method for treating Alzheimer's disease in a subject, comprising subcutaneously administering to the subject a concentration of C sufficient to achieve approximately 20 μg / mL to approximately 40 μg / mL. ave The dose of anti-Aβ antibody, comprising a heavy chain of SEQ ID NO: 101 with or without a C-terminal lysine, and a light chain of SEQ ID NO: 102.

[0063] On the other hand, this disclosure provides a method for treating Alzheimer's disease in a subject, comprising subcutaneously administering to the subject an AUC sufficient to achieve approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL. 0-tau The dose of anti-Aβ antibody, comprising a heavy chain of SEQ ID NO: 101 with or without a C-terminal lysine, and a light chain of SEQ ID NO: 102.

[0064] In some implementations, the maximum concentration (C0) of anti-amyloid β antibody or its antigen-binding fragment in the subject within the dosing interval is specified. maxThe concentration is approximately 30 µg / mL to approximately 60 µg / mL. In some embodiments, the C-cell concentration of the anti-amyloid β antibody or its antigen-binding fragment in the subject is... max The value is approximately 50 µg / mL to approximately 60 µg / mL. In some embodiments, the C-value of the anti-amyloid β antibody or its antigen-binding fragment in the subject is... max The value does not exceed approximately 60 µg / mL. In some implementations, C max The value is serum C max Value. In some implementations, C max The value is plasma C max value.

[0065] In some implementations, the average concentration (C0) of the anti-amyloid β antibody or its antigen-binding fragment within the dosing interval is... ave The concentration (value) is approximately 20 µg / mL to approximately 40 µg / mL. In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment in the subject's C... ave The value is approximately 30 µg / mL to approximately 40 µg / mL. In some embodiments, the anti-amyloid β antibody or its antigen-binding fragment in the subject's C... ave The value does not exceed approximately 40 µg / mL. In some implementations, C ave The value is serum C ave Value. In some implementations, C ave The value is plasma C ave value.

[0066] In some implementations, the area under the concentration-time curve (AUC) of the dosing interval of the anti-amyloid β antibody or its antigen-binding fragment in the subject is specified. 0-tau The AUC (value) is approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL. In some embodiments, the AUC of the anti-amyloid β antibody or its antigen-binding fragment in the subject is... 0-tau The value is approximately 20,000 hr*ug / mL to approximately 30,000 hr*ug / mL. In some embodiments, the AUC of the anti-amyloid β antibody or its antigen-binding fragment in the subject is... 0-tau The value does not exceed approximately 30,000 hr*ug / mL. In some implementations, the AUC 0-tau The value is the serum AUC. 0-tau Value. In some implementations, AUC 0-tau The value is the plasma AUC. 0-tau value.

[0067] In some embodiments, amyloid plaques (i.e., cerebral amyloid β plaques) are reduced in the subject. In some embodiments, the method further includes reducing amyloid plaques in the subject.

[0068] In some embodiments, the reduction of cerebral amyloid β plaques includes a reduction of at least about 30 centiloids to about 70 centiloids. In some embodiments, the reduction of cerebral amyloid β plaques includes a reduction of about 45 centiloids to about 80 centiloids. In some embodiments, the reduction of cerebral amyloid β plaques includes a reduction of about 50 centiloids to about 85 centiloids.

[0069] In some embodiments, the reduction of cerebral amyloid β plaques includes a reduction of at least about 40% to about 90%. In some embodiments, the reduction of cerebral amyloid β plaques includes a reduction of about 60% to about 100%. In some embodiments, the reduction of cerebral amyloid β plaques includes a reduction of about 65% to about 100%.

[0070] In some embodiments, the reduction in cerebral amyloid plaques is a reduction compared to baseline (e.g., pre-treatment values). In some embodiments, the reduction in cerebral amyloid β plaques is a reduction compared to the level before administration of anti-amyloid β antibody in the subject.

[0071] In some embodiments, reduction in cerebral amyloid β plaques is achieved after 6 months of treatment. In some embodiments, reduction in cerebral amyloid β plaques is achieved after approximately 12 months of treatment. In some embodiments, reduction in cerebral amyloid β plaques is achieved after approximately 18 months of treatment.

[0072] In some implementations, the reduction of brain amyloid β plaques is assessed by positron emission tomography (PET).

[0073] In some implementations, the subject is switched from amyloid-positive to amyloid-negative. In some implementations, the treatment includes increasing the probability of the subject switching from amyloid-positive to amyloid-negative.

[0074] In some embodiments, the treatment includes increasing the probability of a subject transitioning from amyloid-positive to amyloid-negative to about 10% to about 40%. In some embodiments, the treatment includes increasing the probability of a subject transitioning from amyloid-positive to amyloid-negative to about 30% to about 60%. In some embodiments, the treatment includes increasing the probability of a subject transitioning from amyloid-positive to amyloid-negative to about 40% to about 80%.

[0075] In some implementations, the probability of a subject transitioning from amyloid-positive to amyloid-negative is approximately 6 months after treatment. In some implementations, the probability of a subject transitioning from amyloid-positive to amyloid-negative is approximately 12 months after treatment. In some implementations, the probability of a subject transitioning from amyloid-positive to amyloid-negative is approximately 18 months after treatment.

[0076] In some implementations, the treatment includes slowing, stopping, or reversing the decline in cognitive function. In some implementations, the treatment includes slowing the decline in cognitive function. In some implementations, cognitive function is measured using at least one of CRD-SB, ADAS-Cog14, ADCOMS, and ADCS MCI-ADL. In some implementations, cognitive function is measured using ADCOMS.

[0077] In another aspect, this disclosure provides a method for modulating biomarkers in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody approximately every 3-5 weeks, the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102. In another aspect, this disclosure provides a method for increasing the Aβ 42 / 40 ratio in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody approximately every 3-5 weeks, the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or lacking a C-terminal lysine, and a light chain of SEQ ID NO: 102. On the other hand, this disclosure provides a method for reducing the amount of phosphorylated-tau in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody about every 3 to 5 weeks, the anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO: 102.

[0078] In some embodiments, administration is intravenous or subcutaneous. In some embodiments, administration is a subcutaneous injection. In some embodiments, administration is a single subcutaneous injection.

[0079] In some embodiments, the biomarkers in the subject are adjusted. In some embodiments, the biomarkers in the subject are adjusted compared to baseline. In some embodiments, the method further includes detecting the biomarkers in samples collected from the subject. In some embodiments, the method further includes quantifying the biomarkers in samples collected from the subject.

[0080] In some embodiments, the biomarker includes the Aβ 42 / 40 ratio in the subject. In some embodiments, the Aβ 42 / 40 ratio in the subject is [not specified]. In some embodiments, the Aβ 42 / 40 ratio in the subject is increased by at least 25%. In some embodiments, the Aβ 42 / 40 ratio in the subject is increased by at least 50%. In some embodiments, the Aβ 42 / 40 ratio in the subject is increased by about 25% to about 100%. In some embodiments, the Aβ 42 / 40 ratio in the subject is increased by about 50% to about 100%.

[0081] In some embodiments, the biomarker comprises a phosphorylated-tau (phospho-tau) value. In some embodiments, the phosphorylated-tau value includes at least one of the following: p181-tau, p212-tau, p217-tau, p231-tau, and p235-tau. In some embodiments, the phosphorylated-tau value includes a p181-tau value. In some embodiments, the phosphorylated-tau value includes a p212-tau value. In some embodiments, the phosphorylated-tau value includes a p217-tau value. In some embodiments, the phosphorylated-tau value includes a p231-tau value. In some embodiments, the phosphorylated-tau value includes a p235-tau value. In some embodiments, the phosphorylated-tau value is reduced. In some embodiments, the phosphorylated-tau value is reduced by at least about 10%. In some embodiments, the phosphorylated-tau value is reduced by about 10% to about 30%. In some implementations, the phosphorylation-tau value is reduced by about 20% to about 30%.

[0082] In some embodiments, the sample includes blood or a portion thereof collected from the subject. In some embodiments, the sample includes plasma collected from the subject. In some embodiments, the sample contains serum collected from the subject. In some embodiments, the sample includes cerebrospinal fluid (“CSF”) collected from the subject.

[0083] In some embodiments, the method includes a risk of less than about 45% ARIA-E. In some embodiments, the method includes a risk of about 25% to about 45% ARIA-E. In some embodiments, the method includes a risk of less than about 75% ARIA-E. In some embodiments, the method includes a risk of about 50% to about 75% ARIA-E. In some embodiments, the method includes a risk of less than about 15% symptomatic ARIA-E. In some embodiments, the method includes a risk of less than about 30% symptomatic ARIA-E. In some embodiments, the risk of ARIA-E is the risk of severe ARIA-E. In some embodiments, the risk of ARIA-E is the risk approximately 6 months after treatment. In some embodiments, the risk of ARIA-E is the risk approximately 12 months after treatment. In some embodiments, the risk of ARIA-E is the risk approximately 18 months after treatment. In some embodiments, the subject does not experience symptomatic ARIA-E during treatment.

[0084] In some embodiments, the method includes a risk of less than about 35% for ARIA-H. In some embodiments, the method includes a risk of about 10% to about 35% for ARIA-H. In some embodiments, the risk of ARIA-H is the risk of severe ARIA-H. In some embodiments, the risk of ARIA-H is the risk approximately 6 months after treatment. In some embodiments, the subject does not experience symptomatic ARIA-H during treatment.

[0085] In some implementations, ARIA is evaluated using magnetic resonance imaging (“MRI”).

[0086] In some embodiments, the subject is an APOE4 homozygous subject. In some embodiments, the subject is an APOE4 heterozygous subject or an APOE4 non-carrier. In some embodiments, the method further includes determining the subject's APOE4 status prior to administration.

[0087] In some implementations, the treatment lasts for at least 6 months. In some implementations, the treatment lasts for at least 12 months. In some implementations, the treatment lasts for at least 18 months.

[0088] In some embodiments, administration is performed using a syringe. In other embodiments, administration is performed using an auto-injector.

[0089] In some implementations, the subjects are mammals. In some implementations, the subjects are humans.

[0090] In various aspects, this disclosure relates to pharmaceutical compositions comprising an anti-amyloid β antibody or antigen-binding fragment as described herein, for the treatment of a subject with Alzheimer's disease. In various aspects, treatment comprises administering to the subject about 20 mg to about 200 mg of the antibody or its antigen-binding fragment approximately every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration. In embodiments, the pharmaceutical composition comprises pharmaceutically acceptable excipients for administration, including, for example, subcutaneous administration.

[0091] In various aspects, this disclosure relates to pharmaceutical compositions comprising an anti-amyloid β antibody or an antigen-binding fragment thereof for reducing amyloid plaques in a subject. In various aspects, treatment of a subject comprises administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or its antigen-binding fragment approximately every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration. In embodiments, the pharmaceutical composition comprises pharmaceutically acceptable excipients for administration, including, for example, subcutaneous administration.

[0092] In various aspects, this disclosure relates to pharmaceutical compositions comprising an anti-amyloid β antibody or antigen-binding fragment as described herein, for converting a subject from amyloid-positive to amyloid-negative. In various aspects, treatment of the subject comprises administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or its antigen-binding fragment approximately every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration. In embodiments, the pharmaceutical composition comprises pharmaceutically acceptable excipients for administration, including, for example, subcutaneous administration.

[0093] In various embodiments of the pharmaceutical composition, administration includes, for example, administering about 45 mg of anti-amyloid β antibody subcutaneously to the subject about once every 4 weeks, administering about 70 mg of anti-amyloid β antibody subcutaneously to the subject about once every 4 weeks, or administering about 200 mg of anti-amyloid β antibody subcutaneously to the subject about once every 4 weeks.

[0094] In various aspects, this disclosure relates to the use of anti-amyloid β antibodies or antigen-binding fragments as described herein in the preparation of a medicament for treating a subject with Alzheimer's disease. In various aspects, the medicament is used to administer approximately 20 mg to approximately 200 mg of the anti-amyloid β antibody or antigen-binding fragment to the subject approximately every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration.

[0095] In various aspects, this disclosure relates to the use of anti-amyloid β antibodies or antigen-binding fragments as described herein in the preparation of medicaments for reducing amyloid plaques in subjects. In various aspects, the medicament is used to administer to a subject about 20 mg to about 200 mg of an anti-amyloid β antibody or its antigen-binding fragment once every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration.

[0096] In various aspects, this disclosure relates to the use of anti-amyloid β antibodies or antigen-binding fragments as described herein in the preparation of medicaments for converting a subject from amyloid-positive to amyloid-negative. In various aspects, the medicament is used to administer to a subject about 20 mg to about 200 mg of an anti-amyloid β antibody or its antigen-binding fragment once every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration.

[0097] In embodiments of the use of anti-amyloid β antibody or its antigen-binding fragment, administration includes, for example, administering subcutaneously to a subject about 45 mg of anti-amyloid β antibody or its binding fragment about once every 4 weeks, administering subcutaneously to a subject about 70 mg of anti-amyloid β antibody or its binding fragment about once every 4 weeks, or administering subcutaneously to a subject about 200 mg of anti-amyloid β antibody or its binding fragment about once every 4 weeks. Attached Figure Description

[0098] Figure 1 This shows a comparison of three different versions of VL, which were designed by incorporating human germline framework residues into the bapineuzumab (hBP) VL sequence. Canonical or interface residues remained unchanged.

[0099] Figure 2 The IC50 values ​​for 4918, 4917, 4921, 3818, 493, 2931, and bapizumab (hBP) are shown as a control. 50 Competitive ELISA assay for ratio determination.

[0100] Figure 3 The IC50 values ​​for 2926, 2831, 2927, 2726, 2731, 2826, and bapizumab (hBP) are shown as a control. 50 Competitive ELISA assay for ratio determination.

[0101] Figure 4 The IC50 values ​​for 2727, 2931, and bapizumab (hBP) are shown as a control. 50 Competitive ELISA assay for ratio determination.

[0102] Figure 5A and Figure 5B Showing 2931, 2731 and bapizumab ( Figure 5A ), as well as 2726, 2831 and bavicilium ( Figure 5B Competitive ELISA assay diagram.

[0103] Figures 6A-6D The results show that h2726 (...) at analyte concentrations ranging from 100 nM to 0.39 nM (2-fold serial dilutions) Figure 6A h2731 Figure 6B h2831 Figure 6C ) and 2931 ( Figure 6D ) bind to Aβ 1-28 BIAcore sensor image.

[0104] Figure 7 The comparison showed that humanized antibodies (PB-0569 (aducanumab), PB-0573 (h2726), PB-0574 (h2731), PB-0575 (h2831), PB-0576 (h2931)) were effective against recombinant amyloid β1-42 (Aβ). 1-42 BIAcore sensor image of the binding properties of fibrils.

[0105] Figure 8 The h2931 exhibits high relative affinity for binding soluble Aβ oligomers.

[0106] Figure 9 The graph shows the Aβ fibril binding activity of 2726, 2731, 2831, and 2931 compared to adunatumumab. Titration of the antibody in a constant concentration of Aβ fibrils (left inset) or in a constant concentration of antibody in a constant concentration of Aβ fibrils (right inset) both indicated that the binding of 2726, 2731, 2831, and 2931 was substantially superior to adunatumumab.

[0107] Figure 10 This image shows Aβ binding in AD brains. Binding to tissue Aβ pathology appears similar among the h2726, h2731, h2831, and h2931 antibodies. Images stained with the four antibodies h2726, h2731, h2831, and h2931 at 0.3 µg / ml show staining patterns in two AD brains (AD 11–97 and AD 13–75) with varying amounts of Aβ pathology. For each brain, images are from the same area of ​​the slices and show relatively similar pathological intensity and distribution for all four antibodies. Staining with aducanamab is consistently the weakest (scale bar: 500 µm).

[0108] Figure 11 This shows Aβ binding in the control AD ​​brain. Human IgG isotype control antibody does not produce staining in the AD brain. As shown in these examples, AD sections incubated with 1 µg / ml human IgG isotype showed no staining (scale bar: 500 µm).

[0109] Figure 12 This demonstrates the quantification of Aβ binding in the AD brain. Quantification of Aβ pathological staining in AD tissue reveals similar binding between the h2726, h2731, h2831, and h2931 antibodies. Sections from four AD brains were incubated with the following concentrations of antibodies h2726, h2731, h2831, h2931, and aducunabumab: 0.03, 0.1, 0.3, 1, 3, and 9 µg / ml. After section imaging, the percentage of stained tissue area was determined morphometrically using Halo® imaging analysis software. The figures compare measurements obtained in the AD brain using the five antibodies. Four Figure 1 The binding profiles of the h2726, h2731, h2831, and h2931 antibodies were similar. Measurements obtained with aducanamab were significantly lower.

[0110] Figure 13 The image shows Aβ binding in the brain of patients with Alzheimer's disease (AD). hBP binds strongly and in a dose-dependent manner to tissue Aβ pathology. Images are taken from relatively identical areas of sections with similar pathological distributions (brain AD 13 to 75). hBP staining shows increasing intensity with increasing concentration, and its binding to Aβ pathology is stronger than that of BAN2401 or adunatumab at all concentrations (scale bar: 500 µm).

[0111] Figure 14A and 14B Individuals showing the in vitro phagocytosis of h2931 and adunatumab in APP.PS1Tg mouse tissues with primary murine microglia ( Figure 14A ) and mergers Figure 14B Results: Both h2931 and adunatumab demonstrated superior performance compared to the isotype control Aβ. 1-42 The height has been significantly reduced.

[0112] Figure 15A and 15B The following figure shows that, compared to the isotype control, with increasing concentrations of h2726, h2731, h2831, and h2931, the binding of soluble oligomers to neurites on rat hippocampal neurons decreased, normalized by + / - Aβ addition. Figure 15A Showing spots for each neuron and Figure 15B Display the total spot count (40 fields of view per well).

[0113] Figure 16 The graph shows the percentage of Aβ spots normalized by adding + / - Aβ to each neuron at increasing concentrations of 2726, 2731, 2831, and 2931.

[0114] Figure 17 The image shows the alignment of the bapizumab variable heavy chain sequence with four sequences of this disclosure, 2726, 2731, 2831, and 2931. CDRs are shown in bold.

[0115] Figure 18 The image shows an alignment of the bavicilium light chain sequence with the four (variable light chain) sequences of 2726, 2731, 2831, and 2931 of this disclosure. CDRs are shown in bold.

[0116] Figure 19A and 19B The table shows the CDRs of the variable heavy and light chain CDR sequences of the antibodies disclosed herein. Figure 19A Heavy chain CDR and Figure 19B Refers to light chain CDRs.

[0117] Figure 20A and 20B The graph shows the antibody titers of Aβ42 species bound to heterogeneous aggregates measured by competitive ELISA. Figure 20A Showing the comparisons of h2931, h2731, and bapizumab, and... Figure 20B A shows the control group of h2831, h2726, and bavicilium.

[0118] Figure 21 The graph shows the direct binding and relative affinity of the antibody to fibrillary Aβ42 as measured by ELISA.

[0119] Figure 22 A graph showing the antibody dose response that measures the area of ​​Aβ plaque binding (which is measured as the percentage of positive tissue in the AD brain by immunohistochemical staining).

[0120] Figure 23 This study demonstrates the quantification of the binding of soluble Aβ to rat hippocampal neurons in the presence of antibodies.

[0121] Figure 24 This study presents the results of an in vitro phagocytic activity study of h2731 in AD tissues containing primary mouse microglia. h2731 demonstrates Aβ 1-42 The significant reduction in the level indicates that the antibody robustly promotes phagocytosis and removal of these species.

[0122] Figure 25A and 25BPyroglutamate-3 Aβ (Aβ) was confirmed to be present in AD tissues used for in vitro phagocytosis assays. pE3-42 ) ( Figure 23 A) and demonstrates a similar binding pattern between pyroglutamic acid-3 Aβ and h2931 ( Figure 23 A and B).

[0123] Figure 26A and 26B This study presents the results of in vitro phagocytosis studies of h2931 and h2731 in AD tissues containing primary mouse microglia. Both h2931 and h2731 demonstrate the efficacy of pyroglutamate-3 Aβ (Aβ...). pE3-42 The significant reduction in the height of the antibodies indicates that both antibodies robustly promote phagocytosis and removal of these species.

[0124] Figure 27 The display shows h2731 combined with Aβ 1-42 The N-terminus, but does not bind to Aβ. pE3-42 .

[0125] Figure 28A and 28B The antibody of this invention demonstrates its ability to induce Aβ phagocytosis in THP-1 human monocytes in vitro. 1-42 Primary fibers.

[0126] Figure 29A and Figure 29B Display of Aβ in AD brain tissue pE3-42 In comparison, Aβ as measured by N-terminal anti-Ab antibody 1-XX The distribution pattern. Figure 29C Display of Aβ in human AD brain tissue pE3-42 In comparison, Aβ 1-XX Quantification of the percentage of area covered.

[0127] Figure 30 This shows the localization of h2731 on Aβ plaques and its anti-Aβ effect. pE3-42 Antibody signaling localization of Aβ plaques, and h2731 and anti-Aβ pE3-42 Colocalization of antibody signals to Aβ plaques.

[0128] Figure 31A and Figure 31B The anti-Aβ antibody h2731 was shown to promote the ex vivo Aβ uptake from AD brain tissue in a dose-dependent manner. pE3-42 Its efficacy in clearing the virus is higher than that of adunatumumab.

[0129] Figure 32A Show Aβ pE3-42 Concentration-dependent clearance of h2731 and adunazaab from AD brain tissue, and Figure 32BThe effects of h2731 are microglia-dependent.

[0130] Figure 33 Compare the predicted CNS exposure of h2731 and adunatumab under repeated dosing.

[0131] Figure 34 The anti-Aβ antibody h2731 showed that it promoted the presence of Aβ in AD brain tissue. pE3-42 In vitro removal of plaques.

[0132] Figure 35 This is a schematic diagram of the clinical trial plan for a single-dose escalation study of h2731 in healthy volunteers and Alzheimer's disease subjects. It shows some inclusion criteria, dosage, and a timeline for some assessments.

[0133] Figure 36 This is a schematic diagram of the clinical trial plan for a multi-level dose escalation study of h2731 in Alzheimer's disease subjects. It shows some inclusion criteria, dosage, and a timeline for some assessments.

[0134] Figure 37 Details of open-label extended studies of h2731 in some Alzheimer's disease subjects enrolled in either the single-escalation dose study of Example 19 or the multiple-escalation dose study of Example 20 are shown. Some dosages and timelines for some assessments are displayed. Detailed Implementation

[0135] Monoclonal antibodies (mAbs) targeting the N-terminus of amyloid β (Aβ) have been clinically demonstrated to reduce amyloid plaque burden, and one such antibody, adunatumab, has shown that a significant reduction in plaque burden is associated with a slowing of cognitive decline in Alzheimer's disease (AD). Preclinical studies have also indicated that monoclonal antibodies (mAbs) targeting the N-terminal epitope of Aβ induce antibody-dependent microglia-mediated clearance of Aβ plaques and neutralization of soluble toxic Aβ oligomers both in vitro and in vivo. It is hypothesized that administration of mAbs targeting the N-terminal Aβ slows disease progression in AD patients via the clearance of Aβ plaques and the neutralization of soluble Aβ aggregates.

[0136] The Aβ antibody bapizumab (hBP) is a humanized antibody developed from parental mouse antibody 3D6. According to various aspects of this disclosure, a multi-pronged approach is employed to construct superior antibodies against hBP. The humanity of hBP is analyzed, and optimizable light chain humanization is determined.

[0137] The protein sequences in the PDB database [Deshpande et al., 2005] were searched to find structures that would provide a rough structural model of hBP. The crystal structure of hBP fab PDB code 4HIX [Miles et al., 2013] was used for the Vh and Vk structures because it has acceptable resolution and an accurate sequence matching hBP Vh and Vk, preserving the same canonical structure for the loop.

[0138] IMGT / DomainGapAlignment was performed on hBP VL as the input sequence to identify the human germline VK gene sequence IGHV2-30*02 as the closest match to hBP VL. The frame region of hBP VL shares high sequence similarity with the corresponding frame region of IGHV2-30*02. Therefore, the frame region of IGHV2-30*02 VL was selected as the guide sequence for further optimization of the hBP frame region. Based on the hBP 3D structure, additional residues in CDR-L2 that do not directly contact the antigen were also changed to germline sequences, resulting in the following changes.

[0139] Three different versions of the VL were designed by incorporating human germline framework residues into the hBP VL sequence. The canonical or interface residues remained unchanged. Furthermore, P15L was tested in one version of the variable light chain based on structural observations that P15 is located at a turn and that the germline gene has Leu at that position.

[0140] Based on 3D structural observations, substitutions were designed at numerous residues in the light and heavy chain CDRs and framework. In the first round of rational design, mutant VL and VH versions were generated and their binding was tested. In the second round of rational design, mutations that improved binding were combined. Furthermore, new structure-guided mutations will be incorporated into the design through further analysis.

[0141] Therefore, this disclosure provides antibodies (and antibody fragments), nucleic acids encoding such antibodies and antibody fragments, methods for producing such antibodies and antibody fragments, pharmaceutical compositions, and methods for: preventing or treating amyloidosis, reducing the risk of amyloidosis or delaying the onset of amyloidosis, achieving cognitive improvement in subjects with conditions associated with amyloidosis, inhibiting the formation of Aβ plaques in subjects, reducing Aβ plaques in the brains of subjects, inhibiting or reducing amyloid plaques in subjects at risk of developing amyloidosis, detecting amyloid plaques, and measuring therapeutic efficacy in subjects undergoing treatment for amyloidosis, wherein amyloidosis includes Alzheimer's disease and other diseases as described herein. This disclosure is based, at least in part, on the characterization of monoclonal antibodies that effectively bind β-amyloid (Aβ) (e.g., bind soluble and / or aggregated Aβ), mediate (e.g., aggregated Aβ) phagocytosis, reduce plaque burden and / or reduce neuritic dystrophy (e.g., in patients), and neutralize soluble toxic Aβ species. The antibodies and fragments disclosed herein exhibit greater binding strength (affinity and / or affinity) to pathological fibrillary Aβ and higher affinity for soluble toxic Aβ forms than reported current experimental therapies. These antibodies enable more convenient dosing strategies and enhanced patient accessibility.

[0142] Before describing specific aspects of this disclosure in more detail, several terms are defined.

[0143] definition

[0144] The term "antibody" includes both the complete antibody and its binding fragments. Typically, the fragment competes with the complete antibody from which it is derived for specific binding to the target. Fragments include individual heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Fv, and single-domain antibodies. Single-domain antibodies comprise the VH region (sometimes called VHH) of a conventional antibody, separated from its VL partner (or vice versa) (Ward et al., 1989, Nature 341: 544-546), as well as the VH region (sometimes called VHH) from species in which the VH region does not associate with the VL region (such as camelids or cartilaginous fishes such as nurse sharks) (see, for example, WO 9404678). Single-domain antibodies in which one chain is separated from its natural partner are sometimes called Dabs, and single-domain antibodies from camelids or cartilaginous fishes are sometimes called nanobodies. Constant regions or portions of constant regions may or may not be present in single-domain antibodies. For example, naturally occurring monovariable domain antibodies from camelids contain a VHH variable region and CH2 and CH3 constant regions. Monovariable antibodies can be humanized using methods similar to those used for conventional antibodies. Dab-type antibodies are typically obtained from human-derived antibodies. Nanobody-type antibodies are derived from camelids or sharks and can be humanized. Fragments can be produced using recombinant DNA technology or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies with two distinct heavy / light chain pairs and two distinct binding sites (see, for example, Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)).

[0145] The variable regions of the immunoglobulin light or heavy chains (sometimes referred to herein as “light chain variable domains” (“VL domains”) or “heavy chain variable domains” (“VH domains”), respectively) consist of “framework” regions interrupted by three “complementarity-determining regions” or “CDRs”. The framework regions are used to align CDRs for specific binding to epitopes of the antigen. The CDRs contain the antibody amino acid residues primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of the VL domain are sometimes referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively; and CDRs 1, 2, and 3 of the VH domain are sometimes referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively. When this application discloses a VL sequence with R as a C-terminal residue, this R can be alternatively considered as an N-terminal residue of the light chain constant region. Therefore, this application should also be understood as disclosing a VL sequence without C-terminal R.

[0146] The amino acid assignments of the VL and VH domains conform to any conventional definition of a CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 & 1991), the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); and the complex of the Chothia-Kabat CDR, where CDR-H1 is a complex of the Chothia and Kabat CDRs; Oxford The AbM definition used by Molecular's antibody modeling software; and the contact definition by Martin et al. (bioinfo.org.uk / abs) (see Table A). Kabat provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or different light chains are assigned the same number. When an antibody is said to contain a CDR according to a definition of a CDR (e.g., Kabat), that definition specifies the minimum number of CDR residues present in the antibody (i.e., Kabat CDRs). It is not excluded that there may be other residues that fall within another conventional CDR definition but outside the specified definition. For example, antibodies containing CDRs as defined by Kabat are particularly likely to include antibodies in which the CDRs contain Kabat CDR residues but no other CDR residues, and antibodies in which CDR H1 is the complex Chothia-Kabat CDR H1 and the other CDRs contain Kabat CDR residues but no other CDR residues based on other definitions.

[0147] Table A

[0148]

[0149] *Chothia's CDR-H1 can end at H32, H33, or H34 (depending on the ring length). This is because the Kabat numbering scheme places the insertion of additional residues at 35A and 35B, while Chothia numbering places them at 31A and 31B. If neither H35A nor H35B (Kabat numbering) is present, the Chothia CDR-H1 ring ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34.

[0150] In some embodiments, the CDR of the humanized antibody of the present invention has the definition of the group consisting of Kabat, Chothia, Kabat / Chothia complex, AbM and Contact.

[0151] One or more amino acids (such as the C-terminal lysine of the heavy chain) at the amino or carboxyl terminus of the light and / or heavy chains may be deleted or derivatized in a portion or all of the molecule. Substitutions may be made in the constant region to reduce or increase effector functions, such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (EU designations are used for constant regions in this paragraph) to increase the half-life of the antibody. Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce affinity for Fcγ receptors, particularly the FcγRI receptor (see, for example, US 6,624,821). Alanine substitutions at positions 234, 235, and 237 of human IgG1 can be used to reduce effector function. Some antibodies have alanine substitutions at positions 234, 235, and 237 of human IgG1 to reduce effector function. Optionally, positions 234, 236, and / or 237 of human IgG2 are substituted with alanine, and position 235 is substituted with glutamine (see, for example, US 5,624,821). In some antibodies, mutations are used at one or more of the EU numbers 241, 264, 265, 270, 296, 297, 322, 329, and 331 of human IgG1. In some antibodies, mutations are used at one or more of positions 318, 320, and 322 of the EU number in human IgG1. In some antibodies, positions 234 and / or 235 are substituted with alanine and / or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine. In some antibodies, the isotype is human IgG2 or IgG4. For example, the C-terminal lysine in the heavy chain constant region of the antibody heavy chain described herein is optional, and sequences with or without C-terminal lysine can be considered.

[0152] The terms "humanized immunoglobulin" or "humanized antibody" refer to an immunoglobulin or antibody containing at least one human immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The terms "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refer to an immunoglobulin or antibody chain (i.e., a light chain or a heavy chain, respectively) having a variable region comprising a variable framework region substantially derived from a human immunoglobulin or antibody and a complementarity-determining region (CDR) substantially derived from a non-human immunoglobulin or antibody (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs), and further comprising a constant region (e.g., at least one constant region or a portion thereof for the light chain, and preferably three constant regions for the heavy chain). The term “humanized variable region” (e.g., “humanized light chain variable region” or “humanized heavy chain variable region”) refers to a variable region comprising a variable frame region substantially derived from human immunoglobulins or antibodies and a complementarity-determining region (CDR) substantially derived from non-human immunoglobulins or antibodies. As used herein, “excluding CDR” means the portion of an antibody that does not include CDR amino acids, such as the frame region and antibody constant region.

[0153] Therefore, regions or residues of humanized immunoglobulins or antibodies, or regions or residues of humanized immunoglobulin or antibody chains (except for possible CDRs), are substantially identical to corresponding regions or residues of one or more natural human immunoglobulin sequences. The terms "corresponding region" or "corresponding residue" refer to a region or residue on the second amino acid or nucleotide sequence that occupies the same (i.e., equivalent) position as a region or residue on the first amino acid or nucleotide sequence when the first and second sequences are optimally aligned for comparative purposes.

[0154] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an antibody binds. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of one or more proteins. Epitopes formed from adjacent amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least three, and more commonly, at least five or eight to ten amino acids in a unique spatial conformation. When an epitope is referred to as being within a range of amino acid residues in a protein (e.g., within residues 1 to 6 of Aβ), this range includes residues that define its boundaries. Some residues within this range contribute to the epitope, while others may not. The residues forming an epitope may be adjacent to each other or may not be adjacent to each other. Similarly, when an antibody binds to an epitope found within a specific amino acid range, the antibody does not need to contact all amino acid residues within that range, and the residues of the epitope that the antibody contacts may be adjacent to each other or may not be adjacent to each other. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, Methods in Molecular Biology, Vol. 66, ed. Glenn E. Morris (1996).

[0155] Antibodies that recognize the same epitope can be identified in a simple immunoassay that shows the ability of one antibody to block another antibody from binding to a target antigen or to compete with another antibody for binding to a target antigen—a competitive binding assay. Competitive binding is determined in an assay in which an immunoglobulin-inhibiting reference antibody specifically binds to a common antigen (such as Aβ) under which the assay is performed. Many types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using I-125 label (also see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directly labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such assays involve using purified antigens bound to a solid surface or cell carrying either the unlabeled test immunoglobulin or the labeled reference immunoglobulin. Competitive inhibition is determined by measuring the amount of label bound to the solid surface or cell in the presence of the test immunoglobulin. Typically, an excess of the test immunoglobulin is present. Typically, when an excess of the competitive antibody is present, it inhibits the specific binding of the reference antibody to the common antigen by at least 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, or more.

[0156] Competition between antibodies is determined by an assay in which the test antibody inhibits the specific binding of a reference antibody (e.g., 3D6, adunatumab, bapizumab) to a common antigen (see, for example, Junghans et al., Cancer Res. 50:1495, 1990). If an excess of the test antibody (e.g., at least 2×, 5×, 10×, 20×, or 100×) inhibits the binding of the reference antibody by at least 50%, but preferably 75%, 90%, or 99%, the test antibody competes with the reference antibody, as determined in a competitive binding assay. Antibodies identified by the competitive assay (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody to cause steric hindrance.

[0157] Antibody epitopes can also be determined by identifying contact residues through X-ray crystallography of antibodies that bind to their antigens. Alternatively, if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, then the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, then the two antibodies have overlapping epitopes.

[0158] Epitopes are also recognized by immune cells (such as B cells and / or T cells). Cellular recognition of epitopes can be determined by in vitro assays that measure antigen-dependent proliferation, such as through... 3 H-thymidine incorporation is determined through cytokine secretion, antibody secretion, or antigen-dependent killing (cytotoxic T lymphocyte assay).

[0159] Exemplary epitopes or antigenic determinants may be present within human amyloid precursor protein (APP), but are preferably present within the Aβ peptide of APP. Multiple isoforms of APP exist, such as APP... 695 APP 751 and APP 770 The amino acids in the app are based on the app's... 770 The sequence number of the same type (see, for example, GenBank accession number P05067, also shown as SEQ ID NO:85).

[0160] Aβ (also referred to herein as β-amyloid peptide and amyloid β (A-beta)) peptide is an approximately 4-kDa internal fragment of APP consisting of 39 to 43 amino acids (Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43). Aβ40 is composed, for example, of residues 672 to 711 of APP, and Aβ42 is composed of residues 673 to 713 of APP. Due to the in vivo or in situ proteolytic processing of APP by various secretases, Aβ exists in both a "short form" (40 amino acids in length) and a "long form" (42 to 43 amino acids in length). The preferred epitope or antigenic determinant, as described herein, is located within the N-terminus of the Aβ peptide and comprises amino acids 1 to 10 of Aβ, preferably residues 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, or 3 to 7 of Aβ42. Additional epitopes or antigenic determinants include residues 2 to 4, 5, 6, 7, or 8 of Aβ; residues 3 to 5, 6, 7, 8, or 9 of Aβ; or residues 4 to 7, 8, 9, or 10 of Aβ42.

[0161] "Soluble" or "dissociated" Aβ refers to monomeric, aggregated, oligomeric, and non-associative Aβ species that remain in solution (supernatant) after centrifugation at 100,000 × g. "Soluble" Aβ refers to aggregated Aβ species, whether or not they are amyloid (β-sheets), that do not remain in solution after centrifugation at 100,000 × g, such as Aβ held together by non-covalent bonds. It is believed that Aβ (e.g., Aβ42) aggregation is at least partly due to the presence of hydrophobic residues at the C-terminus of the peptide (the transmembrane domain of APP). One method for preparing soluble Aβ is to dissolve the lyophilized peptide in pure DMSO using sonication. The resulting solution is centrifuged to remove any insoluble particles.

[0162] "Specific binding" of an antibody means that the antibody exhibits considerable affinity for the antigen or preferred epitope and preferably does not exhibit significant cross-reactivity. "Perceptible" or preferred binding includes binding at a concentration of at least 10... 6 10 7 10 8 10 9 M -1 Or 10 10 M -1 A combination of affinity. Greater than 10 7 M -1 Preferably greater than 10 8 M -1 The affinity is preferred. Values ​​between those described herein are also intended to be within the scope of this disclosure, and the preferred binding affinity can be expressed as a range of affinity, such as 10. 6 Up to 10 10 M -1 Preferably 107 Up to 10 10 M -1 More preferably 10 8 Up to 10 10 M -1 An antibody that “does not exhibit significant cross-reactivity” is one that does not imperceptibly bind to an undesirable entity (e.g., an undesirable protein entity). For example, an antibody that specifically binds to Aβ will imperceptibly bind to Aβ but will not significantly react with non-Aβ proteins or peptides (e.g., non-Aβ proteins or peptides contained in a plaque). For example, an antibody specific to a preferred epitope will not significantly cross-react with distant epitopes on the same protein or peptide. Specific binding can be determined according to any art-recognized means for measuring such binding. Preferably, specific binding is determined according to Scatchard analysis and / or competitive binding assays.

[0163] Binding fragments are generated through recombinant DNA technology or through enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, Fabc, Fv, single-chain, and single-chain antibodies.

[0164] The term "patient" includes individuals and other mammalian subjects receiving preventative or therapeutic treatment. In some implementations, the terms "individual" and "patient" are used interchangeably.

[0165] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutic effective dose" is defined as an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The effective dose for this purpose will depend on the severity of the infection and the general state of the patient's autoimmune system.

[0166] As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or to an isolated tissue or cell line from a patient suffering from a disease, symptoms of a disease, or a predisposition to a disease, wherein the purpose is to cure, heal, reduce, alleviate, alter, remedy, improve, enhance, or influence the disease, symptoms of a disease, or a predisposition to a disease.

[0167] The term "amyloidogenic disease" includes any disease associated with (or caused by) the formation or deposition of insoluble amyloid fibrils or amyloid plaques. Exemplary amyloidogenic diseases include, but are not limited to, systemic amyloidosis, Alzheimer's disease, adult-onset diabetes mellitus, Parkinson's disease, Huntington's disease, frontotemporal dementia, Down syndrome, mild cognitive impairment, prion-associated infectious spongiform encephalopathy (kuru disease and Creutzfeldt-Jacob disease in humans, scrapie in sheep and bovine encephalopathy, and BSE, respectively). Different amyloidogenic diseases are defined or characterized by the nature of the polypeptide components of the deposited fibrils. For example, in subjects or patients with Alzheimer's disease, β-amyloid (e.g., wild-type, variant, or truncated β-amyloid) is the characteristic polypeptide component of amyloid deposition. Therefore, Alzheimer's disease is an example of a "disease characterized by Aβ deposition" or "disease associated with Aβ deposition" in the brain of a subject or patient, for example. The terms "β-amyloid protein," "β-amyloid peptide," "β-amyloid protein," "Aβ," and "Aβ peptide" are used interchangeably herein.

[0168] If a subject has at least one known risk factor (e.g., genetic, biochemical, family history, situational exposure), and placing an individual under that risk factor would result in a statistically significant higher risk of developing the disease than placing an individual without that risk factor, then that individual is at increased risk of developing the disease.

[0169] The term "symptoms" refers to subjective evidence of disease perceived by the patient, such as changes in gait. "Signs" refers to objective evidence of disease observed by the physician.

[0170] Statistical significance means p < 0.05.

[0171] "Half-life (t1 / 2)" refers to the time required for the concentration of an antigen-binding polypeptide to reach half of its original value. The serum half-life of a protein can be measured via pharmacokinetic studies according to the method described by Kim et al. (Eur. J. of Immuno. 24: 542, 1994). According to this method, a radiolabeled protein is intravenously injected into mice, and its plasma concentration is measured periodically as a function of time, for example, from approximately 3 minutes to approximately 72 hours after injection. Other methods for pharmacokinetic analysis and molecular half-life determination are well known to those skilled in the art. Details can be found in Kenneth, A et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic analysis: A Practical Approach (1996).

[0172] "Clearance (CL)" refers to the volume of plasma from which protein is irreversibly cleared per unit time. Clearance is calculated as dose / AUC (AUC: area under the curve or area under the plasma drug concentration-time curve). Clearance can also be calculated by dividing the drug elimination rate by the drug's plasma concentration (Clearance = CL * Concentration).

[0173] Mean residence time (MRT) is the average time an antigen-binding peptide remains in the body before irreversible elimination. It is calculated as MRT = AUMC / AUC.

[0174] "Steady-state concentration" (Css) is the concentration reached when the drug elimination rate becomes equal to the drug administration rate due to continuous drug administration. Css fluctuates between peak and trough levels and is measured in micrograms per ml.

[0175] As used herein, “baseline” is a parameter value prior to or at the time of administration of the pharmaceutical composition of the present invention, including, for example, values ​​of a given biomarker or subject status prior to the first administration of the antibody disclosed herein.

[0176] "Amyloid negative" means that the subject does not have brain amyloid β plaques that can be observed using positron emission tomography ("PET"), and includes, but is not limited to, subjects with zero centiloid values.

[0177] "Amyloid positive" means that the subject has β-amyloid plaques in the brain that can be observed using PET.

[0178] As used herein, “ARIA risk” or “ARIA risk” refers to the probability that a subject will develop an amyloid-related imaging abnormality observable on MRI. Total ARIA risk includes the risk of developing observable ARIA-E and / or observable ARIA-H. In contrast, “ARIA-E risk” refers only to the probability that a subject will develop observable ARIA-E on MRI, regardless of whether the subject develops observable ARIA-H, and “ARIA-H risk” refers only to the probability that a subject will develop observable ARIA-E on MRI, regardless of whether the subject develops observable ARIA-E. ARIA risk can be assessed at baseline (before administration of the anti-amyloid beta antibody of this disclosure) or alternatively during or after treatment.

[0179] Treatment plan

[0180] Prophylactic use: The pharmaceutical composition or drug is administered in an amount sufficient to eliminate or reduce the risk of disease, alleviate the severity of disease, or delay the onset of disease (including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during the development of the disease) to patients susceptible to Alzheimer's disease or other amyloidosis-related diseases, or who are otherwise at risk for Alzheimer's disease or other amyloidosis-related diseases. Patient susceptibility or risk of developing an amyloidosis-related disease can be determined, for example, from genetic markers, biochemical markers, unspecified genetic risks, or other means. In therapeutic use: The composition or drug is administered in an amount sufficient to cure or at least partially prevent the symptoms (biochemical, histological, and / or behavioral) (including its complications and intermediate pathological phenotypes present during the development of the disease) to patients susceptible to or already suffering from the disease.

[0181] In some implementations, the administration of the agent reduces or eliminates cognitive impairment in patients who have not yet developed characteristic Alzheimer's disease or other amyloid-producing cognitive pathologies. An amount sufficient to achieve therapeutic or preventative treatment is defined as a therapeutic or preventative effective dose. In both preventative and therapeutic regimens, the agent is typically administered in several doses until a sufficient immune response has been achieved, where "immune response" or "immunological response" includes the development of an antigen-targeted (antibody-mediated) humoral and / or cellular (mediated by antigen-specific T cells or their secretory products) response in the recipient subject. Such a response can be an active response, induced by the administration of an immunogen, or a passive response, induced by the administration of immunoglobulins or antibodies or sensitized T cells.

[0182] In some implementations, the antibody is administered under various conditions. The interval between single doses can be weekly, monthly, or annually. In some implementations, a single dose may be administered approximately once or twice a week, approximately once or twice every two weeks, approximately once or twice every three weeks, approximately once or twice every four weeks, approximately once or twice every five weeks, or approximately once or twice every six weeks. In one particular implementation, the antibody is administered subcutaneously approximately every four weeks.

[0183] The intervals can also be irregular (as indicated by measuring the patient's blood Aβ antibody levels). In some methods, the dose is adjusted to achieve plasma antibody concentrations of 1 to 1000 μg / ml, and in others, 25 to 300 μg / ml. Alternatively, the antibody can be administered as a sustained-release formulation, in which case a less frequent administration is required. The dose and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies.

[0184] In some implementations, patients with Alzheimer's disease who are given a pharmaceutically effective amount of an anti-Aβ antibody (or its antigen-binding fragment) as described herein are treated by a reduction in amyloid plaque burden as measured by PET imaging.

[0185] In another embodiment, the antibody may be administered in a fixed dose with each application. For example, a fixed dose of about 65 mg, about 70 mg, about 195 mg, or about 200 mg may be administered to an individual in a single dose. In one particular embodiment, about 70 mg of antibody is administered subcutaneously about every four weeks. In one particular embodiment, about 200 mg of antibody is administered subcutaneously about every four weeks.

[0186] In another specific embodiment, an individual is given a subcutaneous dose of approximately 45 mg of h2731 approximately every four weeks. In another specific embodiment, an individual is given a subcutaneous dose of approximately 70 mg of h2731 approximately every four weeks. In another specific embodiment, an individual is given a subcutaneous dose of approximately 200 mg of h2731 approximately every four weeks.

[0187] In another specific embodiment, an individual is given a subcutaneous dose of approximately 45 mg of h2726 approximately every four weeks. In another specific embodiment, an individual is given a subcutaneous dose of approximately 70 mg of h2726 approximately every four weeks. In another specific embodiment, an individual is given a subcutaneous dose of approximately 200 mg of h2726 approximately every four weeks.

[0188] In another specific embodiment, an individual is given a subcutaneous dose of approximately 45 mg of h2726 approximately every four weeks. In another specific embodiment, an individual is given a subcutaneous dose of approximately 70 mg of h2831 approximately every four weeks. In another specific embodiment, an individual is given a subcutaneous dose of approximately 200 mg of h2831 approximately every four weeks.

[0189] In another specific embodiment, an individual is given a subcutaneous dose of approximately 45 mg of h2726 approximately every four weeks. In another specific embodiment, an individual is given a subcutaneous dose of approximately 70 mg of h2931 approximately every four weeks. In another specific embodiment, an individual is given a subcutaneous dose of approximately 200 mg of h2931 approximately every four weeks.

[0190] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, a composition containing the antibody or a mixture thereof of the present invention is administered to a patient who is not yet in a disease state to enhance the patient's resistance. This amount is defined as the "preventive effective dose." In this use, the precise amount again depends on the patient's health status and general immunity. Relatively low doses are administered at relatively infrequent intervals over a longer period. Some patients continue treatment for the rest of their lives.

[0191] In therapeutic applications, relatively high doses at relatively short intervals are sometimes required until disease progression is reduced or terminated, and preferably until the patient shows partial or complete improvement in disease symptoms. Afterward, a prophylactic regimen can be administered to the patient.

[0192] Administration: For prophylactic and / or therapeutic treatment, the therapeutic agent can be administered via parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, intraocular, or intramuscular means. Intramuscular injection is most commonly performed in the muscles of the arm or leg. In some methods, the agent is injected directly into a specific tissue where a deposit has accumulated, such as intracranial injection. For antibody administration, intramuscular injection or intravenous infusion is preferred. In some methods, a specific therapeutic antibody is injected directly into the skull. In some methods, the antibody is administered as a sustained-release composition or device. In some embodiments, an auto-injector device is used to administer the antibody subcutaneously.

[0193] Dosage regimen

[0194] This disclosure provides methods for treating neurological conditions (e.g., Alzheimer's disease) including administering a composition comprising an anti-amyloid beta antibody. For example, in some embodiments, this disclosure provides a method for treating Alzheimer's disease in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid beta antibody or an antigen-binding fragment thereof about every 3-5 weeks. In some embodiments, this disclosure provides a method for treating Alzheimer's disease in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid beta antibody or an antigen-binding fragment thereof about every 4 weeks. In example embodiments, the administration is subcutaneous.

[0195] This disclosure also provides methods for reducing amyloid plaques in subjects. For example, in some embodiments, this disclosure provides a method for reducing amyloid plaques in subjects comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof about every 3-5 weeks. In some embodiments, this disclosure provides a method for reducing amyloid plaques in subjects comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof about every 4 weeks. In example embodiments, the administration is subcutaneous.

[0196] This disclosure also provides methods for converting a subject from amyloid-positive to amyloid-negative. For example, in some embodiments, this disclosure provides a method for converting a subject from amyloid-positive to amyloid-negative by administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof approximately every 3-5 weeks. In some embodiments, this disclosure provides a method for converting a subject from amyloid-positive to amyloid-negative by administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof approximately every 4 weeks. In example embodiments, the administration is subcutaneous.

[0197] In one example implementation, the method includes administering approximately 45 mg of an anti-amyloid beta antibody or an antigen-binding fragment thereof to a subject approximately every 4 weeks. In another example implementation, the method includes administering approximately 70 mg of an anti-amyloid beta antibody or an antigen-binding fragment thereof to a subject approximately every 4 weeks. In yet another example implementation, the method includes administering approximately 200 mg of an anti-amyloid beta antibody or an antigen-binding fragment thereof to a subject approximately every 4 weeks. In the example implementation, the administration is subcutaneous.

[0198] In some embodiments, the method includes administering a pharmaceutically effective amount of an anti-amyloid β antibody or its antigen-binding fragment to a subject. For example, in some embodiments, the method includes administering up to about 200 mg to a subject (e.g., up to about 180 mg, up to about 160 mg, up to about 140 mg, up to about 120 mg, up to about 100 mg, up to about 70 mg, or up to about 50 mg). In some embodiments, the method includes administering to a subject about 20 mg to about 200 mg (e.g., about 30 mg to about 200 mg, about 40 mg to about 200 mg, about 50 mg to about 200 mg, about 60 mg to about 200 mg, about 70 mg to about 200 mg, about 80 mg to about 200 mg, about 90 mg to about 200 mg, about 100 mg to about 200 mg, about 120 mg to about 200 mg, about 140 mg to about 200 mg, about 160 mg to about 200 mg, about 180 mg to about 200 mg, or about 190 mg to about 200 mg). For example, in some embodiments, the method includes administering to a subject about 160 mg to about 200 mg (e.g., about 170 mg to about 200 mg, about 180 mg to about 200 mg, or about 190 mg to about 200 mg) of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0199] In some embodiments, the method includes administering to a subject about 20 mg to about 140 mg (e.g., about 30 mg to about 140 mg, about 40 mg to about 140 mg, about 50 mg to about 140 mg, about 60 mg to about 140 mg, about 70 mg to about 140 mg, about 80 mg to about 140 mg, about 90 mg to about 140 mg, about 100 mg to about 140 mg, about 110 mg to about 140 mg, about 120 mg to about 140 mg, or about 130 mg to about 140 mg) of an anti-amyloid β antibody or an antigen-binding fragment thereof. In some embodiments, the method includes administering to a subject about 20 mg to about 120 mg (e.g., about 30 mg to about 120 mg, about 40 mg to about 120 mg, about 50 mg to about 120 mg, about 60 mg to about 120 mg, about 70 mg to about 120 mg, about 80 mg to about 120 mg, about 90 mg to about 120 mg, about 100 mg to about 120 mg, or about 110 mg to about 120 mg). In some embodiments, the method includes administering to a subject about 20 mg to about 100 mg (e.g., about 30 mg to about 100 mg, about 40 mg to about 100 mg, about 50 mg to about 100 mg, about 60 mg to about 100 mg, about 70 mg to about 100 mg, about 80 mg to about 100 mg, or about 90 mg to about 100 mg). In some embodiments, the method includes administering to a subject about 20 mg to about 80 mg (e.g., about 30 mg to about 80 mg, about 40 mg to about 80 mg, about 50 mg to about 80 mg, about 60 mg to about 80 mg, or about 70 mg to about 80 mg). In some embodiments, the method includes administering to a subject about 20 mg to about 60 mg (e.g., about 30 mg to about 60 mg, about 40 mg to about 60 mg, or about 50 mg to about 60 mg).

[0200] In an example embodiment, the method includes administering to a subject about 40 mg to about 50 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In an example embodiment, the method includes administering to a subject about 65 mg to about 75 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In an example embodiment, the method includes administering to a subject about 195 mg to about 205 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0201] In some implementations, the method includes administering to a subject about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, or about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In some implementations, the method includes administering to a subject about 45 mg, about 70 mg, or about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0202] In an example implementation, the method includes administering approximately 45 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof to a subject. In an example implementation, the method includes administering approximately 70 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof to a subject. In an example implementation, the method includes administering approximately 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof to a subject.

[0203] In some embodiments, the method includes administering an anti-amyloid beta antibody or an antigen-binding fragment thereof approximately every 4 weeks. In some embodiments, the method includes administering an anti-amyloid beta antibody or an antigen-binding fragment thereof approximately monthly. In some embodiments, the method includes administering an anti-amyloid beta antibody or an antigen-binding fragment thereof approximately every 28 days.

[0204] In some embodiments, the method includes administering an anti-amyloid beta antibody or its antigen-binding fragment as a single dose (e.g., a single subcutaneous injection) approximately every 3-5 weeks. In some embodiments, the method includes administering an anti-amyloid beta antibody or its antigen-binding fragment as a single dose approximately every 4 weeks. In some embodiments, the method includes administering an anti-amyloid beta antibody or its antigen-binding fragment as a single dose approximately every 4 weeks. In some embodiments, the method includes administering an anti-amyloid beta antibody or its antigen-binding fragment as a single dose approximately once a month. In some embodiments, the method includes administering an anti-amyloid beta antibody or its antigen-binding fragment as a single dose approximately every 28 days.

[0205] In some embodiments, the method includes administering an anti-amyloid β antibody or its antigen-binding fragment in an injectable form. In some embodiments, the method includes administering an anti-amyloid β antibody or its antigen-binding fragment in a parenteral injection form. In some embodiments, the method includes administering the anti-amyloid β antibody or its antigen-binding fragment via intravenous or subcutaneous injection. In exemplary embodiments, the method includes administering an anti-amyloid β antibody or its antigen-binding fragment in a subcutaneous injection form. In some embodiments, the method includes administering the anti-amyloid β antibody or its antigen-binding fragment via a syringe. In some embodiments, the method includes administering the anti-amyloid β antibody or its antigen-binding fragment via an auto-injector.

[0206] Anti-Aβ antibody

[0207] Anti-amyloid β antibodies or fragments comprise heavy chain CDRs and light chain CDRs from one of the constructs identified herein as h2726, h2731, h2831, h2931, h2926, h4921, h2828, h2929, h3818G, h2927, h49k3G, h4917G, h2727, and h4918G. Certain monoclonal antibodies disclosed herein can bind to epitopes within residues 1-6 of Aβ (where the first N-terminal residue of native Aβ is designated as 1). Some monoclonal antibodies bind to epitopes within amino acids 1-6, some bind to epitopes within amino acids 1-5, and some bind to epitopes within amino acids 1-4. Some antibodies bind to epitopes within amino acids 1-3, 2-5, 3-5, 2-4, 2-5, 2-6, 3-5, or 3-6. When an antibody is referred to, for example, as binding to an epitope within specific residues (such as Aβ 1-6), it means that the antibody specifically binds to a polypeptide containing at least one of the specific residues (i.e., at least one amino acid selected from Aβ amino acids 1-6 in this example); such antibodies do not necessarily contact every residue within Aβ 1-6. In some embodiments, the antibody binds to an epitope containing at least one amino acid selected from amino acids 1-10 of the Aβ peptide. On the other hand, the antibody binds to an epitope containing at least one amino acid selected from amino acids 1-7 of the Aβ peptide. Additional amino acids of the epitope may be outside the region of amino acids 1-10 or amino acids 1-7.

[0208] On the other hand, the anti-amyloid β antibody or fragment contains a heavy chain variable region and a light chain variable region, the heavy chain variable region having heavy chain CDR1, CDR2 and CDR3 from the constructs shown in Table 1A, and the light chain variable region having light chain CDR1, CDR2 and CDR3 from the constructs shown in Table 1A.

[0209] Table 1A

[0210]

[0211]

[0212]

[0213]

[0214] On the other hand, the anti-amyloid β antibody or fragment of this disclosure includes a heavy chain variable region (VH) as shown in one of the constructs in Table 1. The anti-amyloid β antibody or fragment may also include a light chain variable region (VL) as shown in one of the constructs in Table 1A.

[0215] The comparison of the CDRs of each of the heavy and light chain sequences identified in Table 1A with the CDRs from bapizumab (“Bapi”, “hBP”) is shown in Table 1A. Figure 19A and 19B In one aspect, this disclosure relates to antibodies or fragments thereof comprising heavy chains CDR1, CDR2, and CDR3, wherein CDR1 is selected from any one of SEQ ID NO: 16, 19, and 20, wherein CDR2 is selected from any one of SEQ ID NO: 17, 20, 21, 22, and 23, and wherein CDR3 is selected from any one of SEQ ID NO: 18, 24, and 25. Additionally, anti-amyloid β antibodies or fragments thereof comprise light chains CDR1, CDR2, and CDR3, wherein CDR1 is selected from any one of SEQ ID NO: 26, 29, 31, and 32, wherein CDR2 is selected from any one of SEQ ID NO: 27, 33, 34, and 35, and wherein CDR3 is selected from any one of SEQ ID NO: 28, 38, and 39. In each of these embodiments, the heavy chain CDR and the light chain CDR are not simultaneously SEQ ID NO: 16, 17, 18, 26, 27 and 28 in combination.

[0216] Protein modeling information analysis of the above antibodies identified two changes in CDR, which in particular resulted in an increase in the affinity / affinity characteristics of the antibodies disclosed in this invention:

[0217] CDR-L1: S32Y (at position 32, Ser becomes Tyr), and

[0218] CDR-H2: G55S (at position 55, Gly becomes Ser)

[0219] Anti-Aβ antibodies that bind to the same epitopes as those listed in this paper, having Tyr at position 32 in CDR-L1 and Ser at position 55 in CDR-H2, are expected to have the same properties as the identified antibodies listed (see Table 1A and...). Figure 19Aand Figure 19B The disclosed antibody, which does not have Tyr at position 32 in CDR-L1 and Ser at position 55 in CDR-H2, can be modified to have Tyr at position 32 in CDR-L1 and Ser at position 55 in CDR-H2, and can be expected to impart similar binding properties to such antibodies identified herein.

[0220] Examples of CDR-L1 with Tyr at position 32 include SEQ NO: 29 and 31. Examples of CDR-H2 with Ser at position 55 include SEQ NO: 20 and 21.

[0221] As an example, antibodies containing CDR-L1 with Tyr at position 32 and CDR-H2 with Ser at position 55 include antibodies containing CDRs h2726, h2731, h2727, h2826, h2831, h2926, h2927, h2931, and h2929 (see Table 1A). Additional such antibodies include those containing LC CDRs 1, 2, and 3 and HC CDRs 1, 2, and 3 as described in Table 1B below.

[0222] Table 1B

[0223]

[0224] Given the binding properties identified for the antibodies identified herein, common sequences that are expected to provide similar binding properties can be identified. For example, in embodiments of this disclosure, an antibody or its binding fragment that specifically binds to the Aβ peptide may comprise a heavy chain variable region having heavy chain CDR1, CDR2, and CDR3 and a light chain variable region having light chain CDR1, CDR2, and CDR3, as follows:

[0225] The heavy chain CDR1 contains the amino acid sequence GFTFSNX1GMS, where X1 is Y or F (SEQ ID NO: 88);

[0226] The heavy chain CDR2 contains the amino acid sequence SX1RSGSGRTYYSDNVKG, where X1 is I or V (SEQ ID NO: 89);

[0227] The heavy chain CDR3 contains the amino acid sequence YDHYX1GX2SDY, where X1 is S or T and X2 is S or T (SEQ ID NO:90);

[0228] The light chain CDR1 contains the amino acid sequence KSSQSLLDYDGKTYLN (SEQ ID NO: 91);

[0229] The light chain CDR2 contains the amino acid sequence X1VX2NRDX3, where X1 is K or R, X2 is S or T, and X3 is S or T (SEQ ID NO: 92).

[0230] The light chain CDR3 contains the amino acid sequence WQGTHFPRX1, where X1 is S or T (SEQ ID NO: 93).

[0231] In some implementations, the light chain CDR3 comprises WQGTHFPRX1FX2, wherein X1 is S or T and X2 is F or Y (SEQ ID NO: 94).

[0232] Similar common sequences that are expected to provide binding properties similar to those of the antibodies described herein include heavy chain variable regions having heavy chain CDR1, CDR2, and CDR3, and light chain variable regions having light chain CDR1, CDR2, and CDR3, as follows:

[0233] The heavy chain CDR1 contains the amino acid sequence GFTFX1NX2GMS, where X1 is S or A and X2 is Y or F (SEQ ID NO:95);

[0234] The heavy chain CDR2 contains the amino acid sequence SX1RSGX2X3RTYYSDNVKG, where X1 is I or V, X2 is S or G and X3 is S or G (SEQ ID NO: 96);

[0235] The heavy chain CDR3 contains the amino acid sequence YDHYX1GX2SDY, where X1 is S or T and X2 is S or T (SEQ ID NO:90);

[0236] The light chain CDR1 contains the amino acid sequence X1SSQSLX2DX3DGKTYLN, where X1 is K or R, X2 is V, M or L, and X3 is Y, T or S (SEQ ID NO: 97).

[0237] The light chain CDR2 contains the amino acid sequence X1VX2NRX3X4, where X1 is K or R, X2 is S or T, X3 is E or D, and X4 is S or T (SEQ ID NO: 98).

[0238] The light chain CDR3 contains the amino acid sequence WQGX1HFPRX2, where X1 is S or T and X2 is S or T (SEQ ID NO:99).

[0239] In some implementations, the light chain CDR3 comprises WQGTHFPRX1FX2X3, wherein X1 is S or T, X2 is S or T and X3 is F or Y (SEQ ID NO: 100).

[0240] Furthermore, the light and heavy chain variable regions can have at least 75% identity with the light and heavy chain variable regions shown in Table 1A. For example, the light and heavy chain variable regions can have 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH and / or VL sequences identified in Table 1A. In all respects, any sequence variation in VH and VL can exist outside the CDR (e.g., regions excluding the CDR), such that the VH and VL sequences of this disclosure include the CDRs identified in Table 1A, but the regions of the VH and VL sequences excluding the CDRs can have at least 75% identity with the regions of the VH and VL sequences excluding the CDRs in Table 1A.

[0241] For example, the anti-amyloid β antibody or fragment disclosed herein may include a heavy chain variable region (excluding CDR) having at least 95% identity with one of SEQ ID NO: 3, 4, 5, 6 and 7 and a light chain variable region (excluding CDR) having at least 95% identity with one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14 and 15.

[0242] The antibodies and fragments disclosed herein may also contain a heavy chain constant region having at least 75% identity with SEQ ID NO: 40. For example, the heavy chain constant region may have 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 40.

[0243] The antibodies and fragments disclosed herein may also contain a light chain constant region having at least 75% identity with SEQ ID NO:41. For example, the light chain constant region may have 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:41.

[0244] Variant antibodies or fragments having less than 100% identity with the sequences described in Table 1A (plus any constant regions) may differ from the anti-Aβ antibodies in Table 1A by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6 to 10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. "Conserved amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced along all or part of the coding sequence, such as through saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind Aβ peptides).

[0245] For example, mutations may be introduced only in the framework regions of the antibody molecule (e.g., in regions excluding CDRs). The introduced mutations may be silent or neutral missense mutations, i.e., those with no or minimal effect on the antibody's ability to bind antigens. These types of mutations can be used to optimize codon selection or improve antibody production in hybridomas. Alternatively, non-neutral missense mutations may alter the antibody's ability to bind antigens. Those skilled in the art will be able to design and test mutant molecules with desired properties (such as no change in antigen-binding activity or altered binding activity (e.g., improved antigen-binding activity or changes in antibody specificity)). After mutagenesis, the encoded protein can routinely express its encoded protein function and / or biological activity (e.g., the ability to immune-specifically bind at least one epitope of the Aβ polypeptide), which can be determined using the techniques described herein or by conventional modification techniques known in the art. The anti-amyloid β antibody h2731 has several physicochemical properties that make it suitable for use in the methods of this disclosure, including, for example, those described in Tables 2 and 3. Additional properties, including pharmacokinetic parameters, are discussed herein.

[0246] Table 2A

[0247]

[0248] Table 2A provides information on h2731 binding to various amyloid β types (including Aβ). 1-42 fibrils, Aβ 1-42 Aggregates and AβpE3-42 IC of fibrils 50 Value and / or EC 50 Value data. See, for example, US Patent No. 11,440,953.

[0249] The IC50 of h2731 was determined using a competitive (inhibition) assay based on the binding of labeled antibodies to an antigen-coated plate. 50 Then the IC 50 In addition to the IC50 of bapizumab (hBP) 50 The half-maximum inhibitory concentration (IC50) was obtained. 50 The ratio is shown in column 1 of Table 2A. A ratio of 0.61 indicates that h2731 binds to Aβ. 1-42 In terms of fibrils, h2731 exhibits better performance than hBP. Using similar competitive assays, h2731 demonstrated an IC50 of 5.024 µg / mL. 50 The value (column 2 of Table 2A) is higher than the IC observed for hBP. 50 The value is several times lower.

[0250] h2731 and Aβ were also evaluated using ELISA. 1-42 and Aβ pE3-42 Direct binding of fibrils (columns 3 and 4 of Table 2A), for h2731 against Aβ 1-42 The fibrils provided an EC50 value of 36.71 ng / mL. h2731 exhibited a strong affinity for fibrils, significantly higher than that of aducunabumab. Furthermore, there was a 3-fold increase in the measured signal (OD490) and a 15- to 20-fold decrease in the estimated EC50. 50 This indicates that h2731 has increased overall binding and relative affinity to fibrils Aβ compared to adunatumab.

[0251] However, although h2731 binds to the N-terminus of full-length Aβ with high apparent affinity, it does not bind to pyroglutamic acid-modified Aβ (Aβ... pE3-42 ) Specific binding. h2731 binds to unmodified N-terminal Aβ fibrils at a half-maximal effective concentration (EC50) of 8.1 ng / mL (54 pM). 1-42 () Combination. h2731 did not show any interaction with Aβ at concentrations up to 100 ng / ml. pE3-42 Detectable binding.

[0252] Table 2B

[0253]

[0254] Aβ: Amyloid-β, Aβ; ka: Binding rate constant; kd: Dissociation rate constant; KD: Apparent equilibrium dissociation constant; mAb: Monoclonal antibody; Rmax : Maximum response; SPR: Surface plasmon resonance.

[0255] Table 2B provides additional amyloid-β binding data for h2731, including information on h2731 and recombinant Aβ. 1-42 fibrils and A β1-28 Data on monomer binding kinetics. As shown in column 2 of Table 3A, h2731 binds to Aβ. 1-42 fibrils and Aβ 1-28 The monomers have association constants of 3.72 x 10⁻⁶. 5 M -1 s -1 and 1.19 x 10 5 M -1 s -1 Although adunatumab binds to Aβ fibrils at a faster binding rate (ka), the much slower dissociation rate (kd) of h2731 of this disclosure results in a greater measured affinity (i.e., a lower KD*) than adunatumab.

[0256] The dissociation rate data (kd) of h2731 are shown in column 3 of Table 3A. This column provides the dissociation rate data of h2731 for Aβ. 1-42 fibrils and Aβ 1-28 The monomers are 2.62 x 10 -5 s -1 and 5.95 x 10 -4 s -1 The kd value. The enhanced relative affinity of h2731 of this disclosure for fibrils Aβ, observed by ELISA, was confirmed by SPR equilibrium binding kinetics (column 4 of Table 3A), indicating an affinity (apparent KD) 5 to 11 times higher than that of adunatumumab, including for Aβ. 1-28 4-7 nM binding affinity of monomers.

[0257] In additional embodiments, the methods of this disclosure may utilize one or more of several different anti-amyloid β antibodies or fragments thereof. In particular, antibodies suitable for use in the methods of this disclosure have the physicochemical and pharmacological properties discussed herein, allowing for therapeutically effective administration via once-monthly subcutaneous application. Additional exemplary anti-amyloid β antibodies suitable for use in the methods of this disclosure include those in US Patent No. 11,440,953, which is incorporated herein by reference in its entirety.

[0258] In each of the foregoing embodiments, the antibody or fragment of this disclosure may be a humanized antibody as described herein. For example, the antibody may be a human IgG1 antibody. Furthermore, the antibody may be a whole antibody, a chimeric antibody, a CDR-transplanted antibody, or a recombinant antibody. The fragment of the antibody may be Fab, Fab', F(ab')2, Fac, or Fv. The fragment is generated by recombinant DNA technology or by enzymatic or chemical separation of intact immunoglobulins.

[0259] The antibodies or binding fragments, variants or derivatives disclosed herein may be referred to as having a value of less than or equal to 5 × 10⁻⁶. -2 Second -1 10 -2 Second -1 5×10 -3 Second -1 or 10 -3 Second -1 The dissociation rate (k(off)) binds to Aβ or its fragments or variants. In some embodiments, the antibody of this disclosure may be called at a dissociation rate of less than or equal to 5 × 10⁻⁶. 4 Second -1 10 -4 Second -1 5×10 -5 Second -1 , or 10 -5 Second -1 5×10 -6 Second -1 10 -6 Second -1 5×10 -7 Second -1 or 10 -7 Second -1 The dissociation rate (k(off)) of Aβ or its fragments or variants.

[0260] The antibodies or antigen-binding fragments, variants, or derivatives disclosed herein may be referred to as having a value greater than or equal to 10. 3 M-1 second-1, 5×10 3 M-1 seconds - 1, 10 4 M-1 seconds - 1 or 5×10 4 The M-1 second-1 binding rate (k(on)) binds to the target peptide (e.g., Aβ) or a fragment or variant thereof disclosed herein. In some embodiments, the antibody of this disclosure may be called at a rate greater than or equal to 10 5 M-1 seconds - 1, 5×10 5 M-1 seconds - 1, 10 6 M-1 seconds - 1, or 5 × 10 6 M-1 seconds -1 or 10 7The binding rate (k(on)) of M-1 seconds-1 binds to the target peptide (e.g., Aβ) or its fragments or variants disclosed herein.

[0261] Anti-Aβ antibodies or their antigen-binding fragments, variants, or derivatives described herein may also be described or specified in relation to their binding affinity for Aβ. Binding affinity may include a value less than 5 × 10⁻⁶. -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M or 10 -15 The dissociation constant of M or those of Kd.

[0262] The disclosed pharmaceutical agent may optionally be administered in combination with other pharmaceutical agents that are at least partially effective in treating amyloidosis. In the cases of Alzheimer's disease and Down syndrome, where amyloid deposition occurs in the brain, the disclosed pharmaceutical agent may also be administered in combination with other pharmaceutical agents that increase the cross-linking of the disclosed pharmaceutical agent across the blood-brain barrier.

[0263] Expression of recombinant antibodies

[0264] This disclosure also relates to recombinant polynucleotides encoding antibodies that, upon expression, contain the heavy chain CDR and light chain CDR of the antibodies of this disclosure. Exemplary polynucleotides are provided herein that, upon expression, encode polypeptide chains containing the heavy chain CDR and light chain CDR of monoclonal antibodies (e.g., SEQ ID NO: 42 to SEQ ID NO: 69), which encode variable light chain and heavy chain polypeptides and their CDRs according to SEQ ID NO: 3 to SEQ ID NO: 39. Due to codon degeneracy, these sequences can be readily substituted with other polynucleotide sequences.

[0265] Humanized antibodies and human antibodies are typically produced through recombinant expression. Nucleic acids encoding variable regions of a humanized light chain and heavy chain, which can be linked to constant regions, are inserted into an expression vector. These light and heavy chains can be cloned into the same or different expression vectors. DNA segments encoding immunoglobulin chains are control sequences operatively linked into the expression vector to ensure the expression of immunoglobulin peptides. Expression control sequences include, but are not limited to, promoters (e.g., natively associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system within a vector capable of transforming or transfecting eukaryotic host cells. Once the vector has been integrated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of cross-reactive antibodies.

[0266] These expression vectors typically replicate in the host organism either as an episome or as part of the host's chromosomal DNA. Often, the expression vectors contain selection markers (such as ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to allow detection of these cells transformed with the desired DNA sequence.

[0267] One prokaryotic host suitable for the polynucleotides disclosed in the clone is *Escherichia coli*. Other suitable microbial hosts include bacilli, such as *Bacillus subtilus*, and other Enterobacteriaceae, such as *Salmonella*, *Serratia*, and various *Pseudomonas* species. Expression vectors can also be prepared in these prokaryotic hosts, which will typically contain expression control sequences (e.g., origin of replication) compatible with the host cell. Furthermore, any number of well-known promoters will be available, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or promoter systems derived from bacteriophage λ. These promoters typically control expression (optionally along with an operon sequence) and have ribosome binding site sequences, etc., for initiating and completing transcription and translation.

[0268] Other microorganisms, such as yeast, can also be used for expression. *Saccharomyces* is a preferred yeast host, possessing suitable vectors with the desired expression control sequences (e.g., promoters), origin of replication, and termination sequences. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters particularly include those derived from alcohol dehydrogenases, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose. Furthermore, plants (e.g., rice, tobacco) can be used for expression.

[0269] Mammalian tissue cell cultures can also be used to express and produce the polypeptides of this disclosure (e.g., polynucleotides encoding immunoglobulins or fragments thereof). Eukaryotic cells can be particularly useful because many suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, including CHO cell lines, various Cos cell lines, HeLa cells (preferably, myeloma cell lines), or transformed B cells or hybridomas. Preferably, the cells are non-human. The cell expression vector may contain expression control sequences, such as origin of replication, promoters, and enhancers, as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenoviruses, bovine papillomaviruses, cytomegaloviruses, etc.

[0270] Antibody-coding sequences can be incorporated into transgenic organisms to introduce them into the genome of transgenic animals and subsequently expressed in their milk. Suitable transgenic organisms include coding sequences for light and / or heavy chains that are operatively linked to promoters and enhancers from mammary gland-specific genes such as casein or β-lactoglobulin.

[0271] Vectors containing polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells using well-known methods that vary depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipid transfection, biolistics, or virus-based transfection can be used for other cell hosts. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., ibid.). For the generation of transgenic animals, transgenes can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells, and the nuclei of such cells can be transferred into enucleated oocytes.

[0272] When the heavy and light chains are cloned onto separate expression vectors, the vectors are co-transfected to achieve expression and assembly of the complete immunoglobulin. Once expressed, the complete antibody, its dimer, the individual light and heavy chains, or other immunoglobulin forms of this disclosure can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column (e.g., Protein A), column chromatography, HPLC purification, gel electrophoresis, etc. For pharmaceutical applications, substantially pure immunoglobulins are preferably at least about 90% to 95% homogeneous, and most preferably 98% to 99% or higher homogeneity.

[0273] Increasing the copy number of an expression vector containing a polynucleotide sequence of interest is desirable as a means of increasing the production of antibodies or antibody fragments. Various methods for genetically manipulating cells for this purpose and subsequently selecting the optimal cells are known in the art. These methods typically involve an "amplification" step to increase the copy number of the incorporated expression vector to improve the yield of the desired protein. Amplification methods have been reported in the past, such as those by Bebbington and Hentschel (DNACloning Volume III (IRL press, 1987)). Any of a number of selectable markers (usually in the form of nucleic acid sequences encoding enzymes involved in host cell metabolism and essential for their survival under certain culture conditions) can be operatively ligated to the expression vector, thereby promoting the expression of the desired protein upon selection of the selectable marker. When the protein titer does not increase acceptablely, cells selected for high copy numbers can be subjected to further amplification methods. Such methods may involve subjecting cells to toxic drugs that inhibit certain inhibitory markers (e.g., methotrexate and dihydrofolate reductase, methionine sulfoxide and glutamine synthase, multidrug resistance / doxorubicin). Through this inhibition, cell populations with increased expression levels of the marker can be selected. This typically leads to increased expression levels of expression cassettes linked to similar functions. The vector copy number in individual cells subjected to the amplification method is assessed until a plateau in protein production is reached, preferably at least about 100 mg / ml / 10 6 Cells / 24 hours. Subsequently, clones selected through this process and amplified are assessed based on titer / yield to select the best clones for further evaluation. From this titration and screening, typically one or a small number of clones are identified for the subsequent production of one or more desired proteins, which are then used alone.

[0274] Pharmaceutical Composition

[0275] In some embodiments, the anti-Aβ antibody or a fragment thereof is administered as part of a pharmaceutical composition. Several methods are known for preparing pharmaceutical compositions comprising an anti-Aβ antibody or an antigen-binding fragment thereof, a variant thereof, or a derivative thereof for use in a subject with this need. In some embodiments, the anti-Aβ antibody or an antigen-binding fragment thereof is formulated for parenteral administration. In an exemplary embodiment, the anti-Aβ antibody or an antigen-binding fragment thereof is formulated for subcutaneous injection.

[0276] For the purposes of this disclosure, a pharmaceutically effective amount of an anti-Aβ antibody or its antigen-binding fragment, variant, or derivative means an amount sufficient to achieve effective binding to a target and provide benefits during prolonged administration of the anti-Aβ antibody or its antigen-binding fragment, such benefits include, for example, reduction of cerebral amyloid plaques without affecting vascular amyloid, or minimization of microbleeds. In some embodiments, the anti-Aβ antibody or its antigen-binding fragment, variant, or derivative crosses the blood-brain barrier in an effective amount to reduce cerebral amyloid plaques.

[0277] The amount of anti-Aβ antibody or its fragments, variants, or derivatives to be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific administration regimen. Dosing regimens can also be adjusted to provide the best expected response (e.g., a therapeutic or prophylactic response).

[0278] This disclosure provides several pharmaceutically effective amounts of anti-Aβ antibodies or antigen-binding fragments thereof (e.g., about 20 mg to about 200 mg, and additional amounts and / or ranges disclosed herein). Therefore, this disclosure provides the use of pharmaceutical compositions comprising these amounts in the methods disclosed herein. Such pharmaceutically effective amounts may be administered as a single dose, multiple doses, or by infusion over a defined time period. In example embodiments, these pharmaceutical compositions are administered as a single dose. In example embodiments, these pharmaceutical compositions are administered as a single subcutaneous injection.

[0279] For example, in some aspects, this disclosure provides a method of treating a subject with Alzheimer's disease, comprising administering to the subject, about every 3-5 weeks, a pharmaceutical composition comprising about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0280] In some aspects, this disclosure provides a method for reducing amyloid plaques in a subject, comprising administering to the subject, about every 3-5 weeks, a pharmaceutical composition comprising about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0281] In some aspects, this disclosure provides a method for converting a subject from amyloid-positive to amyloid-negative, comprising administering to the subject, approximately every 3-5 weeks, a pharmaceutical composition comprising approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0282] For example, in some aspects, this disclosure provides a method of treating a subject with Alzheimer's disease, comprising administering to the subject, about every 3-5 weeks, a pharmaceutical composition comprising about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0283] In some aspects, this disclosure provides a method for reducing amyloid plaques in a subject, comprising administering to the subject, about every 3-5 weeks, a pharmaceutical composition comprising about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0284] In some aspects, this disclosure provides a method for converting a subject from amyloid-positive to amyloid-negative, comprising administering to the subject, approximately every 3-5 weeks, a pharmaceutical composition comprising approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0285] In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising about 40 mg to about 50 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In some embodiments, the method includes administering to a subject a pharmaceutical composition comprising about 65 mg to about 75 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In an example embodiment, the method includes administering to a subject a pharmaceutical composition comprising about 195 mg to about 205 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof.

[0286] In example embodiments, the method includes administering to a subject a pharmaceutical composition comprising about 45 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In example embodiments, the method includes administering to a subject a pharmaceutical composition comprising about 70 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In example embodiments, the method includes administering to a subject a pharmaceutical composition comprising about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof. In example embodiments, these pharmaceutical compositions are administered as a single subcutaneous injection approximately every 3-5 weeks (e.g., approximately every 4 weeks).

[0287] Several methods are known for preparing anti-Aβ antibodies or their antigen-binding fragments, variants, or derivatives and administering them to subjects in need. Routes of administration for anti-Aβ antibodies or their antigen-binding fragments, variants, or derivatives may include, for example, peripheral, oral, central (e.g., intrathecal, intracranial), parenteral, inhalation, or topical administration.

[0288] As discussed herein, anti-Aβ antibodies or their antigen-binding fragments, variants, or derivatives can be formulated to facilitate the administration and stability of the active agent. In some embodiments, the pharmaceutical compositions according to this disclosure comprise pharmaceutically acceptable, non-toxic, sterile carriers such as physiological saline, non-toxic buffers, preservatives, etc. For the purposes of this application, a pharmaceutically effective amount of an anti-Aβ antibody or its antigen-binding fragments, variants, or derivatives should be considered sufficient to achieve effective binding to the target and to provide a benefit, such as reducing brain amyloid plaques without affecting vascular amyloid, or minimizing the occurrence of microbleeds. In some embodiments, an anti-Aβ antibody or its antigen-binding fragments, variants, or derivatives can cross the blood-brain barrier in an effective amount to reduce brain amyloid plaques.

[0289] The pharmaceutical compositions used in this disclosure comprise pharmaceutically acceptable carriers, including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates), glycine, sorbic acid, potassium sorbate, a mixture of glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0290] The ability to prevent microbial contamination can be achieved through various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, isotonic agents, such as sugars, polyols, or salts, can be included in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0291] Parenteral preparations can be a single bolus dose, an infusion or loading bolus dose, followed by a maintenance dose. These compositions can be administered at specific fixed or variable intervals, such as once daily, or "as needed".

[0292] Formulations intended for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffer media. Parenteral carriers include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's dextrose, or non-volatile oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, for example. Furthermore, the pharmaceutical compositions of this disclosure may contain other pharmaceutical agents, such as dopamine or psychopharmacological drugs, depending on the intended use of the pharmaceutical composition.

[0293] The amount of anti-Aβ antibody or its fragments, variants, or derivatives to be combined with carrier materials to produce a single dosage form will vary depending on the host being treated and the specific administration regimen. The composition can be administered as a single dose, multiple doses, or by infusion over an established time period. Dosing regimens can also be adjusted to provide the best expected response (e.g., therapeutic or prophylactic response).

[0294] As used herein, the term "peripheral administration" includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intranasal, intraocular / vitreous, rectal, or vaginal administration. While all these forms of administration are clearly considered to be within the scope of this disclosure, examples of administration forms will be solutions for injection, particularly for subcutaneous, intravenous, or intra-arterial injection or infusion. Suitable pharmaceutical compositions for injection may contain buffers, surfactants, optionally stabilizers, etc. Formulations for peripheral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Preservatives and other additives, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0295] The therapeutic compositions disclosed herein are generally substantially pure and free of undesirable contaminants. This means that the agent is generally at least 50% w / w pure interfering protein and other contaminants generated or purified therefrom, but the possibility of combining the agent with excess pharmaceutically acceptable carriers or other mediators intended to facilitate its use is not excluded. Sometimes, monoclonal antibodies (or other therapeutic agents) are at least 60%, 70%, 80%, 90%, 95%, or 99% w / w pure interfering protein and contaminants generated or purified therefrom.

[0296] Pharmacokinetic endpoints

[0297] This disclosure provides a dosing regimen for anti-amyloid β antibodies designed to achieve a drug exposure profile in subjects suitable for clearing amyloid plaques and / or treating neurodegenerative diseases (e.g., Alzheimer's disease). To this end, this disclosure provides a method for administering anti-amyloid β antibodies to achieve specific pharmacokinetic endpoints in subjects, including values ​​of, for example, the following parameters suitable for plaque clearance and / or disease treatment: mean concentration over the dosing interval (C0). ave Steady-state concentration (C) within the dosing interval ss ), the maximum concentration within the dosing interval (C) max The area under the concentration-time curve (AUC) from zero to infinity. 0-∞ and the area under the concentration-time curve (AUC) within the dosing interval 0-tau In various implementation schemes, these pharmacokinetic endpoints can be assessed in a variety of bodily fluids collected from subjects, including, for example, whole blood, serum, plasma, and / or CSF.

[0298] Maximum drug concentration (C) max )

[0299] Therefore, in another aspect, this disclosure provides a method for treating Alzheimer's disease in a subject, comprising subcutaneously administering to the subject a concentration of C sufficient to achieve approximately 30 μg / mL to approximately 60 μg / mL. max Value (steady-state C) max The disclosure provides a dose of anti-Aβ antibody at a concentration of approximately 30 μg / mL to approximately 60 μg / mL. In another aspect, this disclosure provides a method for reducing amyloid plaques in a subject, comprising subcutaneously administering to the subject a C-type antibody sufficient to achieve a concentration of approximately 30 μg / mL to approximately 60 μg / mL. max The disclosure provides a dose of anti-Aβ antibody. In another aspect, it provides a method for converting a subject from amyloid-positive to amyloid-negative, comprising subcutaneously administering to the subject a dose sufficient to achieve approximately 30 μg / mL to approximately 60 μg / mL of C. max The anti-Aβ antibody is a dose of [value missing]. In an example embodiment, the anti-Aβ antibody comprises a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO: 102.

[0300] In some embodiments, the treatment includes achieving approximately 30 μg / mL to approximately 60 μg / mL of an anti-amyloid β antibody or its antigen-binding fragment in the subject (e.g., approximately 35 μg / mL to approximately 60 μg / mL, approximately 40 μg / mL to approximately 60 μg / mL, approximately 45 μg / mL to approximately 60 μg / mL, approximately 30 μg / mL to approximately 55 μg / mL, approximately 35 μg / mL to approximately 55 μg / mL, approximately 30 μg / mL to approximately 50 μg / mL, or approximately 35 μg / mL to approximately 50 μg / mL). max In some implementations, C max The value is the steady-state serum C max Value. In some implementations, C max The value is the steady-state plasma C max value.

[0301] Average drug concentration (C) ave )

[0302] On the other hand, this disclosure provides a method for treating Alzheimer's disease in a subject, comprising subcutaneously administering to the subject serum C levels sufficient to achieve approximately 20 μg / mL to approximately 40 μg / mL. ave The disclosure provides a dose of anti-Aβ antibody. In another aspect, this disclosure provides a method for reducing amyloid plaques in a subject, comprising subcutaneously administering to the subject a serum C level sufficient to achieve approximately 20 μg / mL to approximately 40 μg / mL. ave The disclosure provides a dose of anti-Aβ antibody. In another aspect, it provides a method for converting a subject from amyloid-positive to amyloid-negative, comprising subcutaneously administering to the subject a serum C antibody at a concentration sufficient to achieve approximately 20 μg / mL to approximately 40 μg / mL. ave The anti-Aβ antibody is a dose of [value missing]. In an example embodiment, the anti-Aβ antibody comprises a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO: 102.

[0303] In some embodiments, the treatment includes achieving approximately 20 µg / mL to approximately 40 µg / mL of an anti-amyloid β antibody or its antigen-binding fragment in the subject (e.g., approximately 23 µg / mL to approximately 40 µg / mL, approximately 25 µg / mL to approximately 40 µg / mL, approximately 28 µg / mL to approximately 40 µg / mL, approximately 30 µg / mL to approximately 40 µg / mL, approximately 35 µg / mL to approximately 40 µg / mL, approximately 20 µg / mL to approximately 38 µg / mL, approximately 23 µg / mL to approximately 38 µg / mL, approximately 25 µg / mL to approximately 38 µg / mL, approximately 28 µg / mL to approximately 38 µg / mL, approximately 20 µg / mL to approximately 35 µg / mL, approximately 20 µg / mL to approximately 30 µg / mL, or approximately 25 µg / mL to approximately 30 µg / mL). max In some implementations, C ave The value is the steady-state serum C ave Value. In some implementations, C ave The value is the steady-state plasma C. ave value.

[0304] Area under the concentration-time curve (AUC) of the dosing interval 0-tau )

[0305] On the other hand, this disclosure provides a method for treating Alzheimer's disease in a subject, comprising subcutaneously administering to the subject an area under the dosing interval concentration-time curve (AUC) sufficient to achieve approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL. 0-tau The disclosure provides a dose of anti-Aβ antibody sufficient to achieve an AUC of approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL. In another aspect, the disclosure provides a method for reducing amyloid plaques in a subject, comprising subcutaneously administering to the subject an AUC sufficient to achieve an AUC of approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL. 0-tau The disclosure provides a dose of anti-Aβ antibody. In another aspect, this disclosure provides a method for converting a subject from amyloid-positive to amyloid-negative, comprising subcutaneously administering to the subject an AUC sufficient to achieve approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL. 0-tau The anti-Aβ antibody is a dose of [value missing]. In an example embodiment, the anti-Aβ antibody comprises a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO: 102.

[0306] In some implementations, the treatment includes achieving an AUC of approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL in the subject (e.g., 16,000 hr*ug / mL to approximately 30,000 hr*ug / mL, 18,000 hr*ug / mL to approximately 30,000 hr*ug / mL, 20,000 hr*ug / mL to approximately 30,000 hr*ug / mL, 22,000 hr*ug / mL to approximately 30,000 hr*ug / mL, or 25,000 hr*ug / mL to approximately 30,000 hr*ug / mL). 0-tau Value (steady-state AUC) 0-tau (Value). In some embodiments, the treatment includes achieving an AUC of about 15,000 hr*ug / mL to about 25,000 hr*ug / mL (e.g., 16,000 hr*ug / mL to about 25,000 hr*ug / mL, 18,000 hr*ug / mL to about 25,000 hr*ug / mL, 20,000 hr*ug / mL to about 25,000 hr*ug / mL or 22,000 hr*ug / mL to about 25,000 hr*ug / mL) in the subject. 0-tau Value (steady-state AUC) 0-tau (Value). In some implementations, AUC 0-tau The value is the steady-state serum AUC. 0-tau Value. In some implementations, AUC 0-tau The value is the steady-state plasma AUC. 0-tau value.

[0307] Amyloid plaque clearance rate

[0308] This disclosure further provides the use of anti-amyloid-β antibodies for reducing amyloid plaques in subjects. Reduction of amyloid plaques has been shown to be associated with a slowing of cognitive decline during treatment with anti-amyloid-β antibodies. See, for example, M. Shi, et al., Impact of Anti-amyloid-β Monoclonal Antibodies on the Pathology and Clinical Profile of Alzheimer's Disease: A Focus on Aducanumab and Lecanemab. 14 Front. Aging Neurosci. 1 (2022); CH van Dyck, et al., Lecanemab in Early Alzheimer's Disease 388 N. Engl. J. Med. 9 (2023). In fact, based on plaque reduction data from clinical trials, the FDA granted accelerated approval to aducanumab and lecanemab.

[0309] Therefore, in another aspect, this disclosure provides a method for treating Alzheimer's disease in subjects with amyloid plaques, the method comprising:

[0310] (a) Administering to a subject approximately every 4 weeks a composition comprising approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102; and

[0311] (b) Reduce amyloid plaques in subjects.

[0312] Detection of cerebral amyloid plaques is performed using methods known to those skilled in the art. In some embodiments, the method further includes assessing amyloid by positron emission tomography (PET) imaging. In some embodiments, amyloid reduction is determined by PET. PET imaging agents are known to those skilled in the art and include 18F-florbetapir, florbetaben F18, and flutemetamol F18. In some embodiments, amyloid plaques measured by PET are quantified using a composite standardized uptake ratio (SUVR). In some embodiments, amyloid plaques measured by PET are calculated using a Centiloid scale. In some embodiments, changes in amyloid plaque load are measured by changes in SUVR over time. In some embodiments, changes in amyloid plaque load are measured by changes in Centiloid over time. See Navitsky M, Joshi AD, Kennedy I, et al., Standardization of amyloid quantitation with florbetapir standardized uptake value ratios to the Centiloid scale, Alzheimers Dement 2018 14:1565-71 and Oshi AD, Pontecorvo MJ, Lu M, et al., A Semiautomated Method for Quantification of F 18 Florbetapir PETImages, J Nucl Med. 2015;56(11):1736-41.

[0313] Therefore, in another aspect, this disclosure provides a method for reducing amyloid plaques in subjects, the method comprising:

[0314] (a) Administering to a subject approximately every 4 weeks a composition comprising approximately 20 mg to approximately 200 mg (e.g., approximately 45 mg, approximately 70 mg, approximately 150 mg, or approximately 200 mg) of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102; and

[0315] (b) Reduce amyloid plaques in subjects, as measured by PET.

[0316] On the other hand, this disclosure provides a method for treating Alzheimer's disease in subjects, comprising:

[0317] (a) Observe the first amyloid plaque value obtained from the subject's first PET scan;

[0318] (b) Approximately every 3-5 weeks, a composition comprising approximately 20 mg to approximately 200 mg (e.g., approximately 45 mg, approximately 70 mg, approximately 150 mg, or approximately 200 mg) of an anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102; or

[0319] (c) Observe the second amyloid plaque values ​​obtained from the subject's second PET scan; and

[0320] (d) The first amyloid plaque value was compared with the second amyloid plaque value to observe a reduction in amyloid plaques in the subjects.

[0321] On the other hand, this disclosure provides a method for treating Alzheimer's disease in subjects with amyloid plaques, the method comprising:

[0322] (a) The subject undergoes a first PET scan to observe the first amyloid plaque value;

[0323] (b) The subject is given a composition comprising about 20 mg to about 200 mg (e.g., about 45 mg, about 70 mg, about 150 mg or about 200 mg) of an anti-amyloid β antibody approximately every 3 to 5 weeks; the anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101 and a light chain of SEQ ID NO: 102;

[0324] (c) Perform a second PET scan on the subject to observe the second amyloid plaque value; and

[0325] (d) The first amyloid plaque value was compared with the second amyloid plaque value to observe a reduction in amyloid plaques in the subjects.

[0326] On the other hand, this disclosure provides a method for reducing amyloid plaques in subjects, comprising:

[0327] (a) Observe the first amyloid plaque value obtained from the subject's first PET scan;

[0328] (b) Approximately every 3-5 weeks, a composition comprising approximately 20 mg to approximately 200 mg (e.g., approximately 45 mg, approximately 70 mg, approximately 150 mg, or approximately 200 mg) of an anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102; or

[0329] (c) Observe the second amyloid plaque values ​​obtained from the subject's second PET scan; and

[0330] (d) The first amyloid plaque value was compared with the second amyloid plaque value to observe a reduction in amyloid plaques in the subjects.

[0331] On the other hand, this disclosure provides a method for reducing amyloid plaques in subjects with amyloid plaques, the method comprising:

[0332] (a) The subject undergoes a first PET scan to observe the first amyloid plaque value;

[0333] (b) The subject is given a composition comprising about 20 mg to about 200 mg (e.g., about 45 mg, about 70 mg, about 150 mg or about 200 mg) of an anti-amyloid β antibody approximately every 3 to 5 weeks; the anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101 and a light chain of SEQ ID NO: 102;

[0334] (c) Perform a second PET scan on the subject to observe the second amyloid plaque value; and

[0335] (d) The first amyloid plaque value was compared with the second amyloid plaque value to observe a reduction in amyloid plaques in the subjects.

[0336] In some implementations, the treatment includes reducing amyloid-β plaques (i.e., brain amyloid-β plaques) in the subject. In some implementations, the treatment results in a reduction of brain amyloid-β plaques in the subject. In some implementations, the subject achieves a reduction in brain amyloid-β plaques as assessed by PET.

[0337] In some embodiments, the treatment results in a reduction in cerebral amyloid beta plaques in the patient. In some embodiments, the patient achieves a reduction in cerebral amyloid beta plaques as assessed by positron emission tomography (PET). In some embodiments, the reduction in cerebral amyloid beta plaques includes a reduction by at least about 10 centiloids (e.g., at least about 15 centiloids, at least about 20 centiloids, at least about 25 centiloids, or at least about 30 centiloids) compared to baseline. In some embodiments, the reduction in cerebral amyloid beta plaques includes a reduction by at least about 30 centiloids (e.g., at least about 35 centiloids, at least about 40 centiloids, at least about 45 centiloids, or at least about 50 centiloids) compared to baseline. In some embodiments, the reduction in cerebral amyloid beta plaques occurs after about 6 months of treatment (e.g., after about 24 weeks of treatment). In some embodiments, the reduction in cerebral amyloid beta plaques is achieved after about 12 months of treatment. In some implementations, reduction of cerebral amyloid β plaques is achieved after approximately 18 months of treatment.

[0338] In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of at least about 10 centiloids (e.g., at least about 15 centiloids, at least about 20 centiloids, at least about 25 centiloids, or at least about 30 centiloids). In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of at least about 30 centiloids (e.g., at least about 35 centiloids, at least about 40 centiloids, at least about 45 centiloids, at least about 50 centiloids, at least about 55 centiloids, or at least about 60 centiloids) after 6 months of treatment. In some embodiments, reduction of cerebral amyloid β plaques is achieved after about 6 months of treatment. In some embodiments, reduction of cerebral amyloid β plaques is achieved after about 12 months of treatment. In some embodiments, reduction of cerebral amyloid β plaques is achieved after about 18 months of treatment.

[0339] In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of about 10 centiloids to about 90 centiloids (e.g., about 20 centiloids to about 90 centiloids, about 30 centiloids to about 90 centiloids, about 40 centiloids to about 90 centiloids, or about 50 centiloids to about 90 centiloids). In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of about 10 centiloids to about 80 centiloids (e.g., about 20 centiloids to about 80 centiloids, about 30 centiloids to about 80 centiloids, about 40 centiloids to about 80 centiloids, or about 50 centiloids to about 80 centiloids). In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of approximately 10 centiloids to approximately 70 centiloids (e.g., approximately 20 centiloids to approximately 70 centiloids, approximately 30 centiloids to approximately 70 centiloids, approximately 40 centiloids to approximately 70 centiloids, or approximately 50 centiloids to approximately 70 centiloids). In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of approximately 10 centiloids to approximately 60 centiloids (e.g., approximately 20 centiloids to approximately 60 centiloids, approximately 30 centiloids to approximately 60 centiloids, approximately 40 centiloids to approximately 60 centiloids, or approximately 50 centiloids to approximately 60 centiloids). In some embodiments, reduction of cerebral amyloid β plaques is achieved after approximately 6 months of treatment. In some embodiments, reduction of cerebral amyloid β plaques is achieved after approximately 12 months of treatment. In some implementations, reduction of cerebral amyloid β plaques is achieved after approximately 18 months of treatment.

[0340] In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of at least about 10 centiloids (e.g., at least about 15 centiloids, at least about 20 centiloids, at least about 25 centiloids, or at least about 30 centiloids) after about 6 months of treatment. In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of at least about 30 centiloids (e.g., at least about 35 centiloids, at least about 40 centiloids, at least about 45 centiloids, or at least about 50 centiloids) after about 6 months of treatment. In some embodiments, the reduction of cerebral amyloid β plaques includes a reduction of approximately 10 centiloids, approximately 15 centiloids, approximately 20 centiloids, approximately 25 centiloids, approximately 30 centiloids, approximately 35 centiloids, approximately 40 centiloids, approximately 45 centiloids, approximately 50 centiloids, approximately 55 centiloids, approximately 60 centiloids, approximately 65 centiloids, approximately 70 centiloids, approximately 75 centiloids, approximately 80 centiloids, approximately 85 centiloids, approximately 90 centiloids, or approximately 95 centiloids. In some embodiments, the reduction of cerebral amyloid β plaques is achieved after approximately 6 months of treatment. In some embodiments, the reduction of cerebral amyloid β plaques is achieved after approximately 12 months of treatment. In some embodiments, the reduction of cerebral amyloid β plaques is achieved after approximately 18 months of treatment.

[0341] In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of approximately 10 centiloids to approximately 80 centiloids after approximately 6 months of treatment (e.g., approximately 20 centiloids to approximately 80 centiloids, approximately 30 centiloids to approximately 80 centiloids, approximately 10 centiloids to approximately 60 centiloids, or approximately 10 centiloids to approximately 50 centiloids). In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of approximately 30 centiloids to approximately 70 centiloids after approximately 6 months of treatment (e.g., approximately 40 centiloids to approximately 70 centiloids, approximately 50 centiloids to approximately 70 centiloids, approximately 30 centiloids to approximately 60 centiloids, or approximately 30 centiloids to approximately 50 centiloids).

[0342] In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of at least about 25 centiloids (e.g., at least about 35 centiloids, at least about 40 centiloids, at least about 45 centiloids, or at least about 50 centiloids) after about 12 months of treatment. In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of at least about 40 centiloids (e.g., at least about 45 centiloids, at least about 50 centiloids, at least about 55 centiloids, or at least about 60 centiloids) after about 12 months of treatment.

[0343] In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of approximately 20 centiloids to approximately 90 centiloids after approximately 12 months of treatment (e.g., approximately 30 centiloids to approximately 90 centiloids, approximately 40 centiloids to approximately 90 centiloids, approximately 20 centiloids to approximately 80 centiloids, or approximately 20 centiloids to approximately 70 centiloids). In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of approximately 45 centiloids to approximately 80 centiloids after approximately 12 months of treatment (e.g., approximately 50 centiloids to approximately 80 centiloids, approximately 55 centiloids to approximately 80 centiloids, approximately 45 centiloids to approximately 75 centiloids, or approximately 45 centiloids to approximately 70 centiloids).

[0344] In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of at least about 35 centiloids (e.g., at least about 40 centiloids, at least about 45 centiloids, at least about 50 centiloids, or at least about 55 centiloids) after about 18 months of treatment. In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of at least about 50 centiloids (e.g., at least about 55 centiloids, at least about 60 centiloids, at least about 65 centiloids, or at least about 70 centiloids) after about 18 months of treatment.

[0345] In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of approximately 30 centiloids to approximately 100 centiloids after approximately 18 months of treatment (e.g., approximately 40 centiloids to approximately 100 centiloids, approximately 50 centiloids to approximately 100 centiloids, approximately 30 centiloids to approximately 95 centiloids, or approximately 30 centiloids to approximately 90 centiloids). In some embodiments, reduction of cerebral amyloid β plaques includes a reduction of approximately 50 centiloids to approximately 85 centiloids after approximately 18 months of treatment (e.g., approximately 65 centiloids to approximately 85 centiloids, approximately 70 centiloids to approximately 85 centiloids, approximately 50 centiloids to approximately 80 centiloids, or approximately 50 centiloids to approximately 75 centiloids).

[0346] In some implementations, the treatment resulting in a reduction of cerebral amyloid β plaques in patients includes a reduction of at least 20% (e.g., at least 22%, at least 25%, at least 27%, at least 30%, at least 32%, at least 35%, or at least 37%) compared to baseline, and a reduction of at least 40% (e.g., at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, or at least 50%) compared to baseline.

[0347] In some embodiments, the treatment includes a reduction in cerebral amyloid beta plaques. In some embodiments, the reduction in cerebral amyloid beta plaques includes a reduction of at least about 30% (e.g., at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or at least about 70%). In some embodiments, the reduction in cerebral amyloid beta plaques includes a reduction of about 30% to about 100% (e.g., about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, or about 70% to about 100%). In some embodiments, the reduction in cerebral amyloid beta plaques in a subject includes a reduction of about 30% to about 90% (e.g., about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, or about 70% to about 90%). In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of about 30% to about 80% (e.g., about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%). In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of about 30% to about 70% (e.g., about 40% to about 70% or about 50% to about 70%). In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In some embodiments, the reduction in cerebral amyloid β plaques is achieved after about 6 months of treatment. In some embodiments, the reduction in cerebral amyloid β plaques is achieved after about 12 months of treatment. In some implementations, reduction of cerebral amyloid β plaques is achieved after approximately 18 months of treatment.

[0348] In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of at least about 20% (e.g., at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%) after about 6 months of treatment. In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of about 20% to about 90% (e.g., about 30% to about 90%, about 40% to about 90%, or about 50% to about 90%) after about 6 months of treatment. In some embodiments, the reduction in cerebral amyloid β plaques in the subject includes a reduction of about 30% to about 70% (e.g., about 35% to about 70%, about 40% to about 70%, about 30% to about 65%, or about 30% to about 60%) after about 6 months of treatment.

[0349] In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of at least about 30% (e.g., at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60%) after about 12 months of treatment. In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of about 30% to about 100% (e.g., about 40% to about 100%, about 50% to about 100%, or about 50% to about 90%) after about 12 months of treatment. In some embodiments, the reduction in cerebral amyloid β plaques in the subject includes a reduction of about 60% to about 100% (e.g., about 65% to about 100%, about 70% to about 100%, about 65% to about 95%, or about 65% to about 90%) after about 12 months of treatment.

[0350] In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of at least about 40% (e.g., at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or at least about 70%) after about 18 months of treatment. In some embodiments, the reduction in cerebral amyloid β plaques includes a reduction of about 40% to about 100% (e.g., about 50% to about 100%, about 60% to about 100%, or about 50% to about 90%) after about 18 months of treatment. In some embodiments, the reduction in cerebral amyloid β plaques in the subject includes a reduction of about 65% to about 100% (e.g., about 70% to about 100%, about 75% to about 100%, about 65% to about 95%, or about 65% to about 90%) after about 18 months of treatment.

[0351] In some embodiments, the treatment results in a reduction of cerebral amyloid β plaques in the patient, including a reduction of at least 0.05 PET Standard Update Value Ratio (“SUVr”) units compared to baseline (e.g., at least 0.10 PET SUVr units, at least 0.15 PET SUVr units, at least 0.20 PET SUVr units, or at least 0.25 PET SUVr units). In some embodiments, the reduction of cerebral amyloid β plaques includes a reduction of at least 0.25 PET SUVr units compared to baseline (e.g., at least 0.30 PET SUVr units, at least 0.35 PET SUVr units, or at least 0.40 PET SUVr units, or at least 0.45 PET SUVr). In some implementations, reduction of cerebral amyloid β plaques includes a reduction of at least 0.50 PET SUVr units (e.g., at least 0.55 PET SUVr units, at least 0.60 PET SUVr units, or at least 0.65 PET SUVr units or at least 0.70 PET SUVr units) compared to baseline.

[0352] In some embodiments, reduction in cerebral amyloid β plaques occurs after approximately 6 months of treatment (e.g., approximately 24 weeks of treatment). In some embodiments, reduction in cerebral amyloid β plaques occurs after approximately 12 months of treatment (e.g., approximately 48 weeks of treatment). In some embodiments, reduction in cerebral amyloid β plaques occurs after approximately 18 months of treatment (e.g., approximately 72 weeks of treatment).

[0353] In some implementations, the treatment results in the patient achieving an amyloid-negative state. In other implementations, the patient is converted from an amyloid-positive state to an amyloid-negative state.

[0354] On the other hand, this disclosure provides a method for converting a subject from amyloid-positive to amyloid-negative, the method comprising:

[0355] (a) Administering to a subject approximately every 4 weeks a composition comprising approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102; and

[0356] (b) Reduce amyloid plaques in subjects to make them amyloid-negative, as measured by PET.

[0357] In some embodiments, the treatment includes a transition of at least about 10% (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 45%, or at least about 50%) of the subjects from an amyloid-positive status to an amyloid-negative status. In some embodiments, the treatment includes a transition of about 10% to about 90% (e.g., about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 10% to about 80%, about 10% to about 70%, or about 10% to about 60%) of the subjects from an amyloid-positive status to an amyloid-negative status. In some embodiments, the treatment includes a transition of about 30% to about 80% (e.g., about 40% to about 80%, about 50% to about 80%, about 30% to about 70%, or about 30% to about 60%) of the subjects from an amyloid-positive status to an amyloid-negative status. In some embodiments, amyloid-positive and / or amyloid-negative status is assessed by PET. In some embodiments, amyloid-negative status is achieved after approximately 6 months (e.g., approximately 24 weeks) of treatment. In some embodiments, amyloid-negative status is achieved after approximately 12 months (e.g., approximately 48 weeks) of treatment. In some embodiments, amyloid-negative status is achieved after approximately 18 months (e.g., approximately 72 weeks) of treatment.

[0358] In some embodiments, the treatment includes at least about 5% (e.g., at least about 10%, at least about 15%, at least about 20%, or at least about 25%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 6 months. In some embodiments, the treatment includes about 5% to about 50% (e.g., about 10% to about 50%, about 20% to about 50%, about 10% to about 45%, or about 10% to about 40%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 6 months. In some embodiments, the treatment includes about 10% to about 40% (e.g., about 15% to about 40%, about 20% to about 40%, about 10% to about 35%, or about 10% to about 30%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 6 months.

[0359] In some embodiments, the treatment includes at least about 15% (e.g., at least about 20%, at least about 25%, at least about 30%, or at least about 35%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 12 months. In some embodiments, the treatment includes about 15% to about 80% (e.g., about 20% to about 80%, about 30% to about 80%, about 15% to about 75%, or about 15% to about 70%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 12 months. In some embodiments, the treatment includes about 30% to about 60% (e.g., about 35% to about 60%, about 40% to about 60%, about 30% to about 55%, or about 30% to about 50%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 12 months.

[0360] In some embodiments, the treatment includes at least about 25% (e.g., at least about 30%, at least about 35%, at least about 40%, or at least about 45%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 18 months. In some embodiments, the treatment includes about 25% to about 100% (e.g., about 30% to about 100%, about 40% to about 100%, about 25% to about 95%, or about 25% to about 90%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 18 months. In some embodiments, the treatment includes about 40% to about 80% (e.g., about 45% to about 80%, about 50% to about 80%, about 40% to about 75%, or about 40% to about 70%) of the subjects transitioning from an amyloid-positive to an amyloid-negative status after about 18 months.

[0361] In some implementations, cerebral amyloid β plaques in subjects are measured approximately 3 months, 6 months, 12 months, and / or 18 months after the start of treatment. In some implementations, cerebral amyloid β plaques in subjects are measured approximately monthly, approximately every 3 months, approximately every 6 months, or approximately annually after the start of treatment.

[0362] cognitive decline

[0363] Cognitive decline is a hallmark of neurodegenerative diseases, including Alzheimer's disease. The initial stages of cognitive decline in Alzheimer's may manifest as forgetfulness. However, as the disease progresses, the effects of cognitive decline more broadly impact a patient's life and behavior, affecting executive function, language skills, and visuospatial processing. This, in turn, can lead to problems with decision-making, problem-solving, and independent living. Ultimately, Alzheimer's disease results in significant cognitive decline and dementia, leading to impaired retention of basic memory and simple daily activities.

[0364] Recent evidence demonstrates a link between reduced amyloid plaque buildup in the brain and a slowing of cognitive decline in Alzheimer's disease patients. See, for example, Y. Zhang et al., Amyloid β-based therapy for Alzheimer's disease: challenges, successes and future, 8 Signal Transduction and Targeted Therapy 248 (2023). For instance, a phase 2 clinical trial of donanemab showed both a reduction in amyloid plaque buildup in the brain and a slowing of cognitive decline. More recently, a phase 3 clinical trial of lencanemab also reported both a reduction in amyloid plaque buildup in the brain and a slowing of cognitive decline. Considering this recent clinical evidence, a causal link between clearing amyloid plaques and slowing cognitive decline is emerging in patients.

[0365] In some embodiments, treating Alzheimer's disease may include slowing, stopping, and / or reversing the decline in a subject's cognitive abilities. In some embodiments, the treatment includes slowing, stopping, and / or reversing the decline in cognitive function. In another embodiment, the treatment includes reducing (e.g., slowing or stopping) the decline in cognitive function. In another embodiment, the treatment includes slowing the decline in cognitive function. In another embodiment, the treatment includes stopping the decline in cognitive function. In yet another embodiment, the treatment includes reversing the decline in cognitive function.

[0366] Various cognitive assessment tools are available and can be used in conjunction with the methods of this disclosure, including, for example, the Mini-Mental State Examination (MMSE), the Alzheimer's Disease Comprehensive Score (ADCOMS), the Alzheimer's Disease Assessment Scale-Cognition (ADAS-COG) (including, for example, the 14-item Alzheimer's Disease Assessment Scale-Cognition (ADAS-Cog14)), Activities of Daily Living with Mild Cognitive Impairment (ADCS-ADL-MCI), Impressions Based on Clinician Interviews (CIBI), the Neurological Test Battery (NTB), the Dementia Disability Assessment (DAD), the Clinical Dementia Rating-sum of boxes (CDR-SB), and the Neuropsychiatric Inventory (NPI). In some embodiments, treatment includes slowing, stopping, and / or reversing cognitive decline as assessed using one of these cognitive assessment tools. In another embodiment, the methods of this disclosure further include monitoring the subject using at least one of these cognitive assessment tools.

[0367] In some embodiments, cognitive function is measured using at least one of the following: CRD-SB, ADAS-Cog14, ADCOMS, and ADCS MCI-ADL. In some embodiments, CRD-SB is used to measure cognitive function. In some embodiments, ADAS-Cog14 is used to measure cognitive function. In some embodiments, ADCOMS is used to measure cognitive function. In some embodiments, ADCS MCI-ADL is used to measure cognitive function.

[0368] In some embodiments, cognitive function is measured on multiple occasions, such as before administration and at week 4, week 16, 6 months, and / or 1 year after administration. In some embodiments, cognitive function is measured approximately 3 months, 6 months, 12 months, and / or 18 months after initiation of treatment. In some embodiments, cognitive function is measured approximately monthly, approximately every 3 months, approximately every 6 months, or approximately annually after initiation of treatment.

[0369] Biomarker regulation

[0370] In another embodiment, this disclosure provides a method for modulating biomarkers in a subject, comprising administering to the subject, approximately every 3-5 weeks, a composition comprising approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102. In another embodiment, this disclosure provides a method for modulating biomarkers in a subject, comprising administering to the subject, approximately every 3-5 weeks (e.g., approximately every 2 weeks), a composition comprising approximately 100 mg to approximately 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102. In another embodiment, this disclosure provides a method for modulating biomarkers in a subject, comprising administering to the subject, about once every 4 weeks (e.g., about once every 2 weeks), a composition comprising about 100 mg to about 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102.

[0371] amyloid β ratio

[0372] In some implementations, the biomarker includes the Aβ 42 / 40 ratio in the subject. Aβ has been proven... 42 / Aβ 40 Ratios (e.g., CSF and / or blood amyloid beta)42 With amyloid β 40 The ratio of Aβ was associated with well-established AD indicators (including amyloid PET and CSF biomarkers), where lower Aβ levels were associated with lower Aβ levels. 42 / Aβ 40 A ratio (e.g., Aβ42 / 40 ratio <0.150) corresponds to a higher amyloid plaque burden. See, for example, C. Delaby, et al., The Aβ1–42 / Aβ1–40 ratio in CSF is more strongly associated to tau markers and clinical progression than Aβ1–42 alone, 14 Alzheimer's Research & Therapy 20 (2022); X. Chang, et al., A Review of Application of Aβ42 / 40 Ratio in Diagnosis and Prognosis of Alzheimer's Disease. 90 J. Alzheimer's Disease 495 (2002). For example, total plasma Aβ 42 / Aβ 40 The ratio has proven valuable in identifying individuals with mild cognitive impairment (MCI), in predicting progression to dementia, and in detecting underlying AD pathology revealed by FDG-PET, amyloid-PET, and CSF biomarkers. See, for example, V. Perez-Grijalba et al., Plasma Aβ42 / 40 Ratio Detects Early Stages of Alzheimer's Disease and Correlates with CSF and Neuroimaging Biomarkers in the AB255 Study. 6 J. Prevention of Alzheimer's Disease 34, (2019). Therefore, Aβ 42 / Aβ 40 Changes in the ratio can be used to track individuals throughout the treatment process, for example, those with Aβ. 42 / Aβ 40 An increase in the ratio indicates a reduction in amyloid plaque burden during treatment.

[0373] Furthermore, an emerging trend in Alzheimer's disease (AD) treatment is shifting the target population from individuals with dementia or MCI to cognitively healthy individuals at risk of AD. This at-risk population faces challenges in determining whether β-amyloid (Aβ) positivity is a primary criterion for clinical trial eligibility. Using this metric, the screening failure rate (SRF) in this population rises to >70%. See, for example, JD Doecke et al., Total Aβ. 42 / Aβ 40 ratio in plasmapredicts amyloid-PET status, independent of clinical AD diagnosis. 94Neurology 1580 (2020). Aβ 42 / Aβ 40 The ratio can be used to identify the group and track them throughout the treatment process (e.g., preventative treatment).

[0374] Therefore, this disclosure provides a method for regulating the Aβ 42 / 40 ratio in a subject, comprising administering to the subject every 3-5 weeks a composition comprising about 20 mg to about 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102. In some embodiments, regulation includes increasing the Aβ 42 / 40 ratio in the subject.

[0375] In another aspect, this disclosure provides a method for increasing the Aβ 42 / 40 ratio in a subject, comprising administering to the subject, about every 3-5 weeks, a composition comprising about 20 mg to about 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102.

[0376] In another aspect, this disclosure provides a method for increasing the Aβ 42 / 40 ratio in a subject, comprising administering to the subject, about every 3-5 weeks (e.g., about every 2 weeks), a composition comprising about 100 mg to about 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102.

[0377] A method for increasing the Aβ 42 / 40 ratio in subjects, the method comprising:

[0378] (a) Administering to the subject approximately every 3-5 weeks a composition comprising approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102; and

[0379] (b) Determine the Aβ 42 / 40 ratio value derived from samples collected from the subject, wherein the Aβ 42 / 40 ratio value indicates an increase in the Aβ 42 / 40 ratio in the subject.

[0380] A method for increasing the Aβ 42 / 40 ratio in subjects, the method comprising:

[0381] (a) The subject is administered a composition comprising approximately 100 mg to approximately 200 mg of an anti-amyloid β antibody, which comprises a heavy chain having or lacking a C-terminal lysine residue of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102, approximately every 3-5 weeks; and

[0382] (b) Determine the Aβ 42 / 40 ratio value derived from samples collected from the subjects, wherein the Aβ 42 / 40 ratio value indicates an increase in the Aβ 42 / 40 ratio in the subjects.

[0383] In some embodiments, the Aβ 42 / 40 ratio is increased by at least about 10% (e.g., about 15%, about 20%, about 25%, about 30%, about 35%, or about 40%). In some embodiments, the Aβ 42 / 40 ratio is increased by about 10% to about 150% (e.g., about 20% to about 150%, about 30% to about 150%, about 10% to about 120%, about 20% to about 120%, about 30% to about 120%, about 10% to about 100%, or about 20% to about 100%). In some embodiments, the Aβ 42 / 40 ratio is increased by about 25% to about 100% (e.g., about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 25% to about 90%, about 25% to about 80%, about 35% to about 90%, or about 35% to about 80%). In some implementations, the Aβ 42 / 40 ratio value is increased compared to the baseline.

[0384] Phosphorylation-tau

[0385] Phosphorylated tau (p-tau) species have become some of the most promising biomarkers for Alzheimer's disease. See, for example, S. Janelidze et al., Head-to-head comparison of 10 plasma phospho-tau assays in prodromal Alzheimer's disease. 19 Brain 1591-1601 (2023). Hyperphosphorylation of tau is a hallmark of Alzheimer's disease pathology, leading to the self-aggregation of p-tau bundles in affected subjects. See, for example, C.-X. Gong, K. Iqbal, Hyperphosphorylation of Microtubule-Associated Protein Tau: A Promising Therapeutic Target for Alzheimer Disease, 15 Current Med. Chem. 2331 (2009). Phosphorylated-tau levels (e.g., in blood) are associated with amyloid-β (Aβ) pathology and disease severity, as well as with established cerebrospinal fluid (CSF) and neuroimaging biomarkers. See, for example, Kac, PR, et al., Diagnostic value of serum versus plasma phospho-tau for Alzheimer's disease. 14 Alz Res Therapy 65 (2022). Phosphorylated-tau further distinguishes biomarker-positive AD dementia from other dementias and Aβ-negative controls, thus demonstrating specificity for AD relative to non-AD neurodegenerative diseases. Therefore, p-tau levels can provide information for clinical diagnosis and eligibility for treatments, including therapy with anti-amyloid-β antibodies. Furthermore, p-tau species have the potential to help expand global accessibility to AD diagnosis because p-tau species can be measured in blood samples, unlike CSF, which do not require lumbar puncture. See, for example, F. Gonzalez-Ortiz et al., Plasma phospho-tau in Alzheimer's disease: towards diagnostic and therapeutic trial applications. 18 Mol. Neurodegeneration 18 (2023).

[0386] Several phosphorylated tau species have been identified as biomarkers for Alzheimer's disease, including p181-tau, p212-tau, p217-tau, p231-tau, and p235-tau. For example, plasma levels of p-tau181, p-tau217, and p-tau231 have been shown to be associated with in vivo pathological markers and autopsy-verified diagnoses. Several of these p-tau species are highly accurate in detecting cerebral amyloidosis and predicting whether patients will progress to cognitive impairment and neurodegeneration. Furthermore, changes in p-tau levels in subjects undergoing anti-amyloid beta therapy have been shown to correlate with amyloid clearance rates. See, for example, F. Gonzalez-Ortiz (2023).

[0387] In some aspects, this disclosure provides a method for regulating the amount of phosphorylated-tau in a subject, comprising administering to the subject, about every 3-5 weeks, a composition comprising about 20 mg to about 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102. In some aspects, this disclosure provides a method for regulating the amount of phosphorylated-tau in a subject, comprising administering to the subject, about every 3-5 weeks (e.g., about every 2 weeks), a composition comprising about 100 mg to about 200 mg of an anti-amyloid β antibody, the anti-amyloid β antibody comprising a heavy chain having or lacking a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO: 102. In some embodiments, regulation includes increasing the amount of phosphorylated-tau in the subject.

[0388] Therefore, in some embodiments, the biomarker includes a phosphorylation-tau value. In some embodiments, the phosphorylation-tau value includes at least one of the following: p181-tau, p212-tau, p217-tau, p231-tau, and p235-tau. In some embodiments, the phosphorylation-tau value includes a p181-tau value. In some embodiments, the phosphorylation-tau value includes a p212-tau value. In some embodiments, the phosphorylation-tau value includes a p217-tau value. In some embodiments, the phosphorylation-tau value includes a p231-tau value. In some embodiments, the phosphorylation-tau value includes a p235-tau value.

[0389] In some embodiments, the phosphorylated-tau value is reduced by about 5% to about 50% (e.g., about 10% to about 50%, about 15% to about 50%, about 20% to about 50%, about 10% to about 45%, about 10% to about 40%, or about 10% to about 35%). In some embodiments, the phosphorylated-tau value is reduced by about 10% to about 30% (e.g., about 15% to about 30%, about 20% to about 30%, about 10% to about 25%, about 15% to about 25%, or about 20% to about 30%). In some embodiments, the phosphorylated-tau value is reduced compared to a baseline.

[0390] In some embodiments, the p181-tau value is reduced by about 5% to about 50% (e.g., about 10% to about 50%, about 15% to about 50%, about 20% to about 50%, about 10% to about 45%, about 10% to about 40%, or about 10% to about 35%). In some embodiments, the p181-tau value is reduced by about 10% to about 30% (e.g., about 15% to about 30%, about 20% to about 30%, about 10% to about 25%, about 15% to about 25%, or about 20% to about 30%). In some embodiments, the p181-tau value is reduced compared to a baseline.

[0391] In some embodiments, the p217-tau value is reduced by about 5% to about 50% (e.g., about 10% to about 50%, about 15% to about 50%, about 20% to about 50%, about 10% to about 45%, about 10% to about 40%, or about 10% to about 35%). In some embodiments, the p217-tau value is reduced by about 10% to about 30% (e.g., about 15% to about 30%, about 20% to about 30%, about 10% to about 25%, about 15% to about 25%, or about 20% to about 30%). In some embodiments, the p217-tau value is reduced compared to a baseline.

[0392] Amyloid-associated imaging abnormalities (ARIA)

[0393] Treatment with beta-amyloid-targeted passive immunotherapy (including, for example, aduhelm, lencanemab, donepemab, and gantenerumab) is accompanied by a significant risk of amyloid-related imaging abnormalities (ARIA). See, for example, M. Filippi, et al., Amyloid-Related Imaging Abnormalities and β-Amyloid–Targeting Antibodies A Systematic Review. 79 JAMA Neurol. 291 (2022). AIRA is the most common side effect of anti-β-amyloid antibodies and can be classified into ARIA-E (cerebral edema, involving disruption of the tight endothelial junctions of the blood-brain barrier and subsequent fluid accumulation) and ARIA-H (cerebral microbleeds (mH), small hemorrhages in the brain that are often accompanied by hemosiderinosis). ARIA-E may be associated with acute neuroinflammation and overwhelming of the perivascular clearance system, while ARIA-H may be associated with vascular amyloid clearance and subsequent small vessel weakening and / or rupture. See, for example, H. Hampel, et al., Amyloid-related imaging abnormalities (ARIA): radiological, biological and clinical characteristics, 146 Brain 4414 (2023).

[0394] Although often asymptomatic and detected only by MRI, ARIA can be symptomatic in certain cases. For example, ARIA has been shown to include a number of side effects such as headache, worsening confusion, dizziness, visual disturbances, nausea, and seizures. Furthermore, at least one death related to ARIA-E during adanulumab treatment and at least one death due to ARIA-H during donepemab treatment have been reported to date. See, for example, C.G. Withington and R.S. Curner, Amyloid-Related Imaging Abnormalities With Anti-amyloid Antibodies for the Treatment of Dementia Due to Alzheimer's Disease. 13 Frontiers in Neurology 1 (2022). The risk of ARIA-H increases with age and cerebrovascular disease, and the rate of ARIA is generally higher in homozygous ApoE4 patients (ApoE4 carriers) compared to ApoE4 non-carriers or ApoE4 heterozygous patients. In addition, an increased risk of ARIA-E was observed at the start of treatment, corresponding to higher doses and >4 microbleeds on baseline MRI.

[0395] In some embodiments, the treatment includes an ARIA-E risk of less than about 70% (e.g., less than about 65%, less than about 60%, less than about 55%, or less than about 50%). In some embodiments, the treatment includes an ARIA-E risk of less than about 45% (e.g., less than about 40%, less than about 35%, less than about 30%, less than about 25%, or less than about 20%). In some embodiments, the treatment includes an ARIA-E risk of less than about 15% (e.g., less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%).

[0396] In some embodiments, the treatment results in less than about 45% (e.g., less than about 40%, less than about 35%, less than about 30%, less than about 25%, or less than about 20%) of subjects experiencing symptomatic ARIA-E. In some embodiments, the treatment results in less than about 15% (e.g., less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%) of subjects experiencing symptomatic ARIA-E. In some embodiments, the treatment results in less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5%) of subjects experiencing symptomatic ARIA-E.

[0397] In some implementations, the risk of ARIA-E is the risk of severe ARIA-E. In some implementations, the risk of ARIA-E is the risk of moderate+ ARIA-E. In some implementations, the risk of ARIA-E is the risk of moderate ARIA-E. In some implementations, the risk of ARIA-E is the risk of mild+ ARIA-E. In some implementations, the risk of ARIA-E is the risk of mild ARIA-E. In some implementations, the risk of ARIA-E includes the risk of hyperintensity of FLAIR signals at more than one location, where each FLAIR location has a range of 5-10 cm. In some implementations, the risk of ARIA-E includes the risk of hyperintensity of FLAIR signals at one location, where the FLAIR location has a range of 5-10 cm. In some implementations, the risk of ARIA-E includes the risk of hyperintensity of FLAIR signals at more than one location, where each FLAIR location has a range of less than 5 cm, and where each FLAIR location is confined to the sulci, cortex, and / or subcortical white matter. In some implementations, the risks of ARIA-E include the risk of high FLAIR signal intensity at a location where the FLAIR location has a range of less than 5 cm and where the FLAIR location is confined to the sulcus, cortex and / or subcortical white matter.

[0398] In some implementations, the patient is a homozygous APOE4 patient. In some implementations, the treatment includes an ARIA-E risk of less than about 70% (e.g., less than about 65%, less than about 60%, less than about 55%, or less than about 50%) in a homozygous APOE4 patient. In some implementations, the treatment includes an ARIA-E risk of less than about 40% (e.g., less than about 35%, less than about 30%, or less than about 25%) in a homozygous APOE4 patient.

[0399] In some implementations, the subject is APOE4 homozygous and the treatment includes a risk of ARIA-E less than about 75% (e.g., less than about 70%, less than about 65%, less than about 60%, less than about 55%, or less than about 50%) in APOE4 homozygous subjects. In some implementations, the subject is APOE4 homozygous and the treatment includes a risk of symptomatic ARIA-E less than about 30% (e.g., less than about 25%, less than about 20%, or less than about 15%) in APOE4 homozygous subjects.

[0400] In some implementations, the patient is either APOE4 heterozygous or APOE4 negative. In some implementations, treatment includes an ARIA-E risk of less than about 40% (e.g., less than about 35%, less than about 30%, less than about 25%, or less than about 20%) in APOE4 heterozygous or APOE4 negative patients. In some implementations, treatment includes an ARIA-E risk of less than about 15% (e.g., less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%) in APOE4 heterozygous or APOE4 negative patients.

[0401] In some embodiments, the subject is an APOE4 heterozygous subject or an APOE4 negative subject, and the treatment includes an ARIA-E risk of less than about 45% (e.g., less than about 40%, less than about 35%, less than about 30%, less than about 25%, or less than about 20%) in APOE4 heterozygous or APOE4 negative subjects. In some embodiments, the subject is an APOE4 heterozygous subject or an APOE4 negative subject, and the treatment includes a symptomatic ARIA-E risk of less than about 15% (e.g., less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%) in APOE4 heterozygous or APOE4 negative subjects. In some embodiments, the ARIA-E risk is a post-treatment risk of about 6 months (e.g., about 24 weeks). In some embodiments, the ARIA-E risk is a post-treatment risk of about 12 months (e.g., about 48 weeks). In some embodiments, the ARIA-E risk is a post-treatment risk of about 18 months (e.g., about 72 weeks).

[0402] In some implementations, the treatment includes an ARIA-H risk of less than about 25% (e.g., less than about 22%, less than about 20%, less than about 18%, or less than about 16%). In some implementations, the treatment includes an ARIA-H risk of less than about 15% (e.g., less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%).

[0403] In some implementations, the risk of ARIA-H is the risk of severe ARIA-H. In some implementations, the risk of ARIA-H is the risk of moderate ARIA-H. In some implementations, the risk of ARIA-H is the risk of mild ARIA-H. In some implementations, the risk of ARIA-H includes the risk of ≤4 new microbleeding events and / or the risk of ≤1 superficial siderosis lesion area. In some implementations, the risk of ARIA-H includes the risk of ≤9 new microbleeding events and / or the risk of ≤2 superficial siderosis lesion areas.

[0404] In some implementations, the risk of ARIA-H is a post-treatment risk of approximately 6 months (e.g., approximately 24 weeks). In some implementations, the risk of ARIA-H is a post-treatment risk of approximately 12 months (e.g., approximately 48 weeks). In some implementations, the risk of ARIA-H is a post-treatment risk of approximately 18 months (e.g., approximately 72 weeks).

[0405] In some embodiments, the treatment results in less than about 45% (e.g., less than about 40%, less than about 35%, less than about 30%, less than about 25%, or less than about 20%) of subjects experiencing ARIA-E. In some embodiments, the treatment results in less than about 15% (e.g., less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%) of subjects experiencing ARIA-E. In some embodiments, the treatment results in less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5%) of subjects experiencing ARIA-E.

[0406] In some implementations, the treatment resulted in fewer than about 15% (e.g., fewer than about 14%, fewer than about 13%, fewer than about 12%, fewer than about 11%, fewer than about 10%, fewer than about 9%, fewer than about 8%, fewer than about 7%, fewer than about 6%, or fewer than about 5%) of the subjects experiencing symptomatic ARIA-E.

[0407] In some implementations, the treatment results in less than about 25% (e.g., less than about 22%, less than about 20%, less than about 18%, or less than about 16%) of subjects experiencing ARIA-H. In some implementations, the treatment results in less than about 15% (e.g., less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5%) of subjects experiencing ARIA-H.

[0408] In some embodiments, ARIA-E and / or ARIA-H levels (e.g., the percentage of subjects experiencing ARIA-E and / or ARIA-E) are ARIA-E and / or ARIA-H levels after approximately 6 months (e.g., approximately 24 weeks) of treatment. In some embodiments, ARIA-E and / or ARIA-H levels are ARIA-E and / or ARIA-H levels after approximately 12 months (e.g., approximately 48 weeks) of treatment. In some embodiments, ARIA-E and / or ARIA-H levels are ARIA-E and / or ARIA-H levels after approximately 18 months (e.g., approximately 72 weeks) of treatment.

[0409] In some implementations, the patient does not experience symptomatic ARIA, as assessed by magnetic resonance imaging (MRI). In some implementations, the patient does not experience symptomatic ARIA-E, as assessed by MRI. In some implementations, the patient does not experience symptomatic ARIA-H, as assessed by MRI. In some implementations, the patient is APOE4 heterozygous or APOE4 negative. In some implementations, the patient is APOE4 homozygous.

[0410] Treatment Amenable Patient

[0411] This disclosure also relates to the treatment of Alzheimer's disease and other amyloidosis by administering the antibodies, fragments, and pharmaceutical compositions of this disclosure, producing a beneficial therapeutic response in patients (e.g., inducing Aβ phagocytosis, reducing plaque burden, inhibiting plaque formation, reducing neurotrophic dystrophy, neutralizing soluble toxic Aβ species, improving cognitive function, and / or reversing, treating, or preventing cognitive decline), for example, for the prevention or treatment of amyloidosis. This disclosure also relates to the use of the disclosed antibodies and fragments in the preparation of medicaments for the treatment or prevention of amyloidosis.

[0412] In one aspect, this disclosure provides methods for preventing or treating diseases associated with Aβ amyloid deposition in patients. In another aspect, this amyloid protein is deposited in the brain or other CNS regions. Such diseases include Alzheimer's disease, Down syndrome, age-related macular degeneration (AMD), and cognitive impairment. The latter may occur with or without other features of amyloid-producing diseases. Some methods of this disclosure require administering an effective dose of an antibody that specifically binds to the components of the amyloid deposition to the patient. Such methods can be used for the prevention or treatment of Alzheimer's disease in human patients.

[0413] The method can be used in asymptomatic patients and those currently exhibiting symptoms of disease. The antibody used in such a method can be a humanized antibody, a human antibody, or a fragment thereof (e.g., an antigen-binding fragment), and can be a monoclonal or polyclonal antibody as described herein. In another aspect, this disclosure is characterized by administering an antibody prepared from a person immunized with an Aβ peptide, who may be a patient to be treated with the antibody.

[0414] In another aspect, this disclosure is characterized by administering the antibody together with a drug carrier as a pharmaceutical composition. Alternatively, the antibody can be administered to a patient by administering a polynucleotide encoding at least one antibody chain. The polynucleotide is expressed to generate the antibody chain in the patient. Optionally, the polynucleotide encodes both the heavy and light chains of the antibody. The polynucleotide is expressed to generate both the heavy and light chains in the patient. In an exemplary embodiment, the level of the administered antibody in the patient's blood is monitored.

[0415] Suitable patients for treatment include individuals at risk of the disease but asymptomatic, as well as those currently showing symptoms. In the case of Alzheimer's disease, anyone who lives long enough is potentially at risk of developing the disease. Therefore, this approach involves prophylactic administration to the general population without any assessment of the risk in the subject patients. This approach is particularly useful for individuals with a known genetic risk of developing Alzheimer's disease. Such individuals include those with relatives who have had the disease and those whose risk has been determined by analyzing genetic or biochemical markers. Genetic markers of Alzheimer's risk include mutations in the APP gene, particularly at positions 717 and 670 and 671, known as Hardy and Swedish mutations, respectively. Other risk markers are mutations in the prosensory receptor genes PS1 and PS2, as well as ApoE4, a family history of AD, hypercholesterolemia, or atherosclerosis. Individuals currently diagnosed with Alzheimer's disease can be identified by the presence of characteristic dementia and the risk factors described above. Furthermore, many diagnostic tests are available for identifying individuals with AD. These include measuring CSF tau and Aβ42 concentrations. Elevated tau and decreased Aβ42 concentrations indicate the presence of AD. Individuals with Alzheimer's disease can also be diagnosed using the ADRDA criteria discussed in Example 1.

[0416] Treatment for asymptomatic patients can begin at any age (e.g., 10, 20, 30 years). However, treatment is usually not required before the patient reaches 40, 50, 60, or 70 years of age. Treatment typically requires multiple doses over a period of time. Treatment can be monitored by measuring antibody levels over time. If the response declines, a booster dose is indicated. In the case of a patient with underlying Down syndrome, treatment can be initiated prenatally or shortly after birth by administering a therapeutic agent to the mother.

[0417] APOE4 status

[0418] Subjects carrying the apolipoprotein E ε4 allele (APOE4) are at increased risk of ARIA during treatment with anti-amyloid therapy. See, for example, C. Dagostin et al., Efficacy of anti-amyloid-β monoclonal antibody therapy in early Alzheimer's disease: a systematic review and meta-analysis. Neological Sciences (2023). Subjects homozygous for APOE4 are at the highest risk, partly due to their increased load of β-amyloid protein accumulating in brain microvessels. Furthermore, ongoing studies have shown that APOE4 carriers exhibit greater perivascular Aβ clearance during treatment, leading to greater vascular permeability and extravasation of fluid and erythrocytes, resulting in a higher ARIA-E and ARIA-H ratio. See, for example, MC. Dagostin et al., Efficacy of anti-amyloid-β monoclonal antibody therapy in early Alzheimer's disease: a systematic review and meta-analysis. Neological Sciences (2023).

[0419] In some implementations, the subject is an APOE4 heterozygous subject or an APOE4 negative subject. In some implementations, the subject is an APOE4 homozygous subject. In some implementations, the subject is an APOE4 heterozygous subject. In some implementations, the subject is an APOE4 negative subject (non-carrier).

[0420] In vivo testing

[0421] On the other hand, this disclosure provides methods for detecting amyloid plaques and deposits in patients with amyloidosis or at risk of developing amyloidosis. Such methods can be used to diagnose or confirm amyloidosis or susceptibility to it. For example, the methods can be used in patients with dementia symptoms, where the observation of abnormal amyloid deposits may indicate Alzheimer's disease. The methods can also be used in asymptomatic patients. The presence of abnormal amyloid deposits indicates susceptibility to future symptomatic diseases.

[0422] In some implementations, the method includes administering the disclosed antibody or a fragment thereof to a subject / patient and detecting the antibody or a fragment thereof bound to Aβ.

[0423] Antibodies and / or their antibody fragments can be administered by any suitable means leading to delivery to the tissue to be visualized, such as intravenous injection into the patient's body or direct administration to the brain via intracranial injection. The dosage of antibodies and / or their fragments can include therapeutic, subtherapeutic, or supratherapeutic doses. In some embodiments, the antibodies or their fragments are labeled, including with fluorescent, paramagnetic, or radioactive markers. The choice of marker depends on the detection method. For example, fluorescent markers are suitable for visual detection. The use of paramagnetic markers is suitable for computed tomography detection without surgical intervention. In some embodiments, radioactive markers are detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0424] On the other hand, this disclosure provides a method for measuring the therapeutic efficacy in a subject undergoing treatment for amyloidosis. In some embodiments, prior to treatment by administration of an antibody or fragment thereof of this disclosure, a first level of amyloid plaques in the subject is measured and a first amount of antibody or fragment thereof bound to Aβ is detected in the subject. Treatment may then be administered to the subject, followed by measurement of a second level of amyloid plaques in the subject and detection of antibody or fragment thereof bound to Aβ in the subject. In some embodiments, a decrease in amyloid plaque concentration indicates a positive response to treatment, and in some embodiments, no change in amyloid plaque concentration or a slight increase in amyloid plaques indicates a positive response to treatment. In some embodiments, the level of amyloid plaques can be measured using methods for detecting amyloid plaques as described herein.

[0425] In some implementations, the diagnosis of amyloidosis can be made, for example, by comparing the number, size, and / or intensity of marker sites from a first level (i.e., baseline) with a second level of amyloid plaques subsequently observed in the subject. Increases over time indicate disease progression, no change indicates disease progression, and randomly shifting fewer or lower intensity amyloid plaques indicate remission.

[0426] Detection of cerebral amyloid plaques is performed using methods known to those skilled in the art. In some embodiments, amyloid plaque burden is measured in patients using positron emission tomography (PET) imaging. PET imaging agents are known to those skilled in the art and include 18F-florbetapir, florbetaben F18, and flutemetamol F18. In some embodiments, amyloid plaques measured by PET are quantified using a composite standardized uptake ratio (SUVR). In some embodiments, amyloid plaques measured by PET are calculated using a Centiloid scale. In some embodiments, changes in amyloid plaque burden are measured by changes in SUVR over time. In some embodiments, changes in amyloid plaque burden are measured by changes in Centiloid over time. See Navitsky M, Joshi AD, Kennedy I, et al., Standardization of amyloid quantitation with florbetapir standardized uptakevalue ratios to the Centiloid scale, Alzheimers Dement 2018 14:1565-71 and OshiAD, Pontecorvo MJ, Lu M, et al., A Semiautomated Method for Quantification of F18 Florbetapir PET Images, J Nucl Med. 2015;56(11):1736-41.

[0427] use

[0428] In various aspects, this disclosure relates to pharmaceutical compositions comprising an anti-amyloid beta antibody or an antigen-binding fragment as described herein, for the treatment of a subject with Alzheimer's disease. In various aspects, treatment comprises administering to the subject about 20 mg to about 200 mg of the antibody or its antigen-binding fragment approximately every 3-5 weeks. In various aspects, intermediate doses as described herein may be administered subcutaneously.

[0429] In various aspects, this disclosure relates to pharmaceutical compositions comprising an anti-amyloid β antibody or an antigen-binding fragment thereof for reducing amyloid plaques in a subject. In various aspects, treatment of a subject comprises administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or its antigen-binding fragment approximately every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration.

[0430] In all respects, this disclosure relates to pharmaceutical compositions comprising an anti-amyloid β antibody or an antigen-binding fragment as described herein, for the purpose of converting a subject from amyloid-positive to amyloid-negative. In all respects, treatment of the subject comprises administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody or its antigen-binding fragment approximately every 3-5 weeks. In all respects, intermediate doses as described herein may be administered subcutaneously.

[0431] In embodiments of the various pharmaceutical compositions, administration includes, for example, subcutaneous administration of about 45 mg of anti-amyloid β antibody to a subject about every 4 weeks, subcutaneous administration of about 70 mg of anti-amyloid β antibody to a subject about every 4 weeks, or subcutaneous administration of about 200 mg of anti-amyloid β antibody to a subject about every 4 weeks. In embodiments, the pharmaceutical composition includes a pharmaceutically acceptable excipient for administration (including, for example, subcutaneous administration).

[0432] In various aspects, this disclosure relates to the use of anti-amyloid β antibodies or antigen-binding fragments as described herein in the preparation of a medicament for treating a subject with Alzheimer's disease. In various aspects, the medicament is used to administer approximately 20 mg to approximately 200 mg of the anti-amyloid β antibody or antigen-binding fragment to the subject approximately every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration.

[0433] In various aspects, this disclosure relates to the use of anti-amyloid β antibodies or antigen-binding fragments as described herein in the preparation of medicaments for reducing amyloid plaques in subjects. In various aspects, the medicament is used to administer to a subject about 20 mg to about 200 mg of an anti-amyloid β antibody or its antigen-binding fragment once every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration.

[0434] In various aspects, this disclosure relates to the use of anti-amyloid β antibodies or antigen-binding fragments as described herein in the preparation of medicaments for converting a subject from amyloid-positive to amyloid-negative. In various aspects, the medicament is used to administer to a subject about 20 mg to about 200 mg of an anti-amyloid β antibody or its antigen-binding fragment once every 3-5 weeks. In various aspects, intermediate doses as described herein may be used for subcutaneous administration.

[0435] In embodiments of the use of anti-amyloid β antibody or its antigen-binding fragment, administration includes, for example, administering subcutaneously to a subject about 45 mg of anti-amyloid β antibody or its binding fragment about once every 4 weeks, administering subcutaneously to a subject about 70 mg of anti-amyloid β antibody or its binding fragment about once every 4 weeks, or administering subcutaneously to a subject about 200 mg of anti-amyloid β antibody or its binding fragment about once every 4 weeks.

[0436] This disclosure will be described more fully through the following non-limiting embodiments.

[0437] SEQ ID NO: 40: huIgG1 constant region

[0438] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0439] SEQ ID NO: 41: huκ constant region

[0440] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0441] SEQ ID NO: 42: Nucleotide sequence of h2726_VH (Variable heavy chain)

[0442] GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTCCTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCGGAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACTACTCCGATAACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGGGTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC

[0443] SEQ ID NO: 43: Nucleotide sequence of h2726_VL (Variable light chain)

[0444] GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGCGTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCGGACAAGGCACCAAGGTCGAGATCAAG

[0445] SEQ ID NO: 44: Nucleotide sequence of h2931_VH (Variable heavy chain)

[0446] GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAGCTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGGGAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGACAACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTGGCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA

[0447] SEQ ID NO: 45: h2931_VL (Variable Light Chain) Nucleotide Sequence

[0448] GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGATCTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGGGTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCGGTCAAGGAACCAAGGTCGAGATCAAG

[0449] SEQ ID NO: 46: h2731_VH (Variable Heavy Chain) Nucleotide Sequence

[0450] GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTCCTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCGGAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACTACTCCGATAACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGGGTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC

[0451] SEQ ID NO: 47: h2731_VL (Variable Light Chain) nucleotide sequence

[0452] GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGATCTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGGGTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCGGTCAAGGAACCAAGGTCGAGATCAAG

[0453] SEQ ID NO: 48: h2831_VH (Variable Heavy Chain) nucleotide sequence

[0454] GAAGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGGGGATCCCTGCGGCTTTCCTGCGCCGCATCCGGCTTCACCTTTTCAAACTTCGGAATGTCGTGGGTCAGACAGGCCCCGGGAAAGGGTCTGGAATGGGTGGCCTCAGTGCGGTCCGGATCGGGTAGAACCTACTACAGCGATAACGTGAAGGGCCGGTTCACGATCTCCCGCGACAACTCCAAGAACACCCTGTACTTGCAAATGAATAGCCTCAGGGCTGAGGATACCGCGGTCTACTACTGTGTGCGCTATGACCACTACACTGGAACTAGCGACTACTGGGGCCAGGGGACCCTCGTGACTGTGTCGTCC

[0455] SEQ ID NO: 49: h2831_VL (Variable Light Chain) nucleotide sequence

[0456] GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGATCTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGGGTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCGGTCAAGGAACCAAGGTCGAGATCAAG

[0457] SEQ ID NO: 50: h2926_VH (Variable Heavy Chain) nucleotide sequence

[0458] GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAGCTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGGGAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGACAACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTGGCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA

[0459] SEQ ID NO: 51: h2926_VL (Variable Light Chain) Nucleotide Sequence

[0460] GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGCGTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCGGACAAGGCACCAAGGTCGAGATCAAG

[0461] SEQ ID NO: 52: h4921G VH (Variable Heavy Chain) Nucleotide Sequence

[0462] GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTCTTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGGGAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGACAACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAATGAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCGGTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC

[0463] SEQ ID NO: 53: h4921G VL (Variable Light Chain) nucleotide sequence

[0464] GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACCGCGTGACCAACCGGGATACGGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCGGTCAAGGGACTAAGGTCGAAATCAAG

[0465] SEQ ID NO: 54: h2826 VH (Variable Heavy Chain) nucleotide sequence

[0466] GAAGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGGGGATCCCTGCGGCTTTCCTGCGCCGCATCCGGCTTCACCTTTTCAAACTTCGGAATGTCGTGGGTCAGACAGGCCCCGGGAAAGGGTCTGGAATGGGTGGCCTCAGTGCGGTCCGGATCGGGTAGAACCTACTACAGCGATAACGTGAAGGGCCGGTTCACGATCTCCCGCGACAACTCCAAGAACACCCTGTACTTGCAAATGAATAGCCTCAGGGCTGAGGATACCGCGGTCTACTACTGTGTGCGCTATGACCACTACACTGGAACTAGCGACTACTGGGGCCAGGGGACCCTCGTGACTGTGTCGTCC

[0467] SEQ ID NO: 55: h2826 VL (Variable Light Chain) nucleotide sequence

[0468] GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGCGTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCGGACAAGGCACCAAGGTCGAGATCAAG

[0469] SEQ ID NO: 56: h2929 VH (Variable Heavy Chain) nucleotide sequence

[0470] GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAGCTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGGGAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGACAACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTGGCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA

[0471] SEQ ID NO: 5: h2929 VL (Variable Light Chain) nucleotide sequence

[0472] GATGTCGTGATGACCCAAAGCCCCCTGTCCCTCCCTGTGACTCCTGGAGAGCCGGCGTCCATTTCCTGCCGGTCAAGCCAGTCCTTGGTGGACTACGACGGAAAGACCTACCTCAACTGGCTGCTGCAGCGCCCCGGGCAGTCGCCGCAGCGGCTTATCTACAAAGTGTCCAACCGCGACTCGGGCGTGCCGGATAGGTTTTCGGGTTCCGGAAGCGGCACCGACTTCACCCTGAAAATCTCCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTTCTCACTTCCCACGGTCATATGGCCAAGGGACTAAGGTCGAAATCAAG

[0473] SEQ ID NO: 58: h3818G VH (Variable Heavy Chain) nucleotide sequence

[0474] It should be noted that there may be some inaccuracies in the translation due to the complexity and potential ambiguity of the biological sequence content. It is recommended to double-check with relevant biological experts or reference materials for more accurate understanding.GAAGTGCAGCTCCTGGAGTCCGGCGGTGGACTGGTGCAGCCGGGCGGATCCCTGAGACTGTCCTGCGCCGCGTCGGGCTTTACTTTCGCAAATTACGGCATGAGCTGGGTCAGACAGGCCCCCGGGAAGGGTCTGGAATGGGTGGCCAGCGTCCGGAGCGGGGGATCCCGGACCTATTACTCCGACAACGTGAAGGGCCGCTTCACCATCTCAAGGGACAACTCCAAGAACACCCTGTACTTGCAAATGAACAGCCTTCGGGCTGAGGATACTGCCGTGTACTACTGCGTGCGCTACGACCACTACTCCGGATCCTCGGATTACTGGGGACAGGGAACCCTCGTGACCGTGTCATCG

[0475] SEQ ID NO: 59: h3818G VL (Variable Light Chain) nucleotide sequence

[0476] GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGATGGACACCGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGTCAAACCGGGAGTCCGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCGGTCAAGGGACTAAGGTCGAAATCAAG

[0477] SEQ ID NO: 60: h2927 VH (Variable Heavy Chain) nucleotide sequence

[0478] GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAGCTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGGGAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGACAACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTGGCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA

[0479] SEQ ID NO: 61: h2927 VL (Variable Light Chain) nucleotide sequence

[0480] GATGTCGTGATGACTCAGTCACCGCTCTCCCTCCCTGTGACCCCGGGCGAACCAGCGTCGATCTCCTGCAAGAGCAGCCAATCATTGCTGGACTACGACGGAAAGACCTATCTTAACTGGCTGCTGCAGAAGCCCGGGCAGAGCCCGCAGCGCCTGATCTACAAAGTGTCCAACAGAGACTCCGGAGTGCCTGATAGGTTCTCGGGTTCCGGCTCCGGTACCGACTTCACTCTGAAAATTTCCCGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACCCACTTCCCCCGGTCGTTTGGACAAGGGACCAAGGTCGAGATCAAG

[0481] SEQ ID NO: 62: h49K3G VH (Variable Heavy Chain) nucleotide sequence

[0482] GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTCTTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGGGAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGACAACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAATGAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCGGTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC

[0483] SEQ ID NO: 63: h49K3G VL (Variable Light Chain) nucleotide sequence

[0484] GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGTCAAACCGGGATTCCGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCGGTCAAGGGACTAAGGTCGAAATCAAG

[0485] SEQ ID NO: 64: h4917G VH (Variable Heavy Chain) nucleotide sequence

[0486] GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTCTTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGGGAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGACAACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAATGAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCGGTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC

[0487] SEQ ID NO: 65: h4917G VL (Variable Light Chain) nucleotide sequence

[0488] GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGACCAACCGGGAGTCCGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGTCATTCGGTCAAGGGACTAAGGTCGAAATCAAG

[0489] SEQ ID NO: 66: h2727 VH (Variable Heavy Chain) nucleotide sequence

[0490] GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTCCTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCGGAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACTACTCCGATAACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGGGTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC

[0491] SEQ ID NO: 67: h2727 VL (Variable Light Chain) nucleotide sequence

[0492] GATGTCGTGATGACTCAGTCACCGCTCTCCCTCCCTGTGACCCCGGGCGAACCAGCGTCGATCTCCTGCAAGAGCAGCCAATCATTGCTGGACTACGACGGAAAGACCTATCTTAACTGGCTGCTGCAGAAGCCCGGGCAGAGCCCGCAGCGCCTGATCTACAAAGTGTCCAACAGAGACTCCGGAGTGCCTGATAGGTTCTCGGGTTCCGGCTCCGGTACCGACTTCACTCTGAAAATTTCCCGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACCCACTTCCCCCGGTCGTTTGGACAAGGGACCAAGGTCGAGATCAAG

[0493] SEQ ID NO: 68: h4918G VH (Variable Heavy Chain) nucleotide sequence

[0494] GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTCTTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGGGAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGACAACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAATGAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCGGTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC

[0495] SEQ ID NO: 69: h4918G VL (Variable Light Chain) nucleotide sequence

[0496] GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGATGGACACCGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGTCAAACCGGGAGTCCGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCGGTCAAGGGACTAAGGTCGAAATCAAG

[0497] SEQ ID NO: 70: Aducanumab heavy chain:

[0498] QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0499] SEQ ID NO: 71: Adunamab light chain:

[0500] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0501] SEQ ID NO: 72: Bapineuzumab HC (heavy chain)

[0502] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0503] SEQ ID NO: 73: VH (Variable Heavy Chain) of Bapilizumab

[0504] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS

[0505] SEQ ID NO: 16: VH CDR1 GFTFSNYGMS

[0506] SEQ ID NO: 17: VH CDR2 SIRSGGGRTYYSNDYNVKG

[0507] SEQ ID NO: 18: VH CDR3 YDHYSGSSDY

[0508] SEQ ID NO: 77: LC (Light Chain) of Bapilizumab

[0509] DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0510] SEQ ID NO: 78: Bavizumab VL (variable light chain)

[0511] DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIK

[0512] SEQ ID NO: 26: VL CDR1KSSQSLLDSDGKTYLN

[0513] SEQ ID NO: 27: VL CDR2LVSKLDS

[0514] SEQ ID NO: 28: VL CDR3WQGTHFPRT

[0515] SEQ ID NO: 82: Gantingluumab HC amino acid sequence:

[0516] QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0517] SEQ ID NO 83: Light chain amino acid sequence of gengtinglumab:

[0518] DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0519] SEQ ID NO: 84: Amyloid-β (Aβ) 1-42:

[0520] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

[0521] SEQ ID NO: 85: Amyloid-β (Aβ) precursor protein:

[0522] MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAMSQSLLKTTQEPLARDPVKLPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN

[0523] SEQ ID NO: 86: Nucleotide sequence of the huIgG1 constant region

[0524] GCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCGTGAAGGACTACTTCCCCGAGCCGGTCACCGTGTCCTGGAACTCGGGAGCCCTGACCTCCGGAGTGCATACTTTCCCTGCGGTGCTGCAGTCCTCCGGGCTCTACTCGCTGTCAAGCGTGGTCACCGTCCCGAGCTCATCCCTGGGTACTCAGACCTACATTTGCAACGTGAACCACAAACCTTCCAACACCAAGGTCGACAAGAAAGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGTCCCCCGTGTCCCGCCCCTGAGCTGCTGGGCGGCCCCAGCGTGTTCCTCTTCCCGCCTAAGCCGAAGGACACTCTGATGATCTCGAGAACCCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGATCCGGAAGTGAAGTTCAATTGGTACGTGGACGGAGTGGAAGTCCATAACGCCAAGACCAAGCCCCGCGAGGAACAGTACAACTCAACTTACCGGGTGGTGTCAGTGCTGACCGTGCTGCACCAAGATTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTCTCCAACAAGGCGCTGCCGGCCCCCATTGAAAAGACCATCAGCAAGGCTAAGGGCCAGCCCCGGGAACCACAGGTCTACACCTTGCCCCCTTCCCGGGAGGAAATGACCAAGAACCAAGTGTCGCTGACGTGCCTGGTCAAGGGCTTTTATCCATCTGACATCGCCGTGGAGTGGGAAAGCAACGGCCAGCCGGAAAACAACTACAAGACTACCCCGCCTGTGCTGGACTCCGACGGCTCGTTCTTCCTGTATTCCAAGCTCACCGTGGATAAGTCCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCATGAGGCCCTGCACAACCACTACACTCAGAAATCACTGTCCCTTTCCCCCGGAAAGTAA

[0525] SEQ ID NO: 87: Nucleotide sequence of the huκ constant region

[0526] CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA

[0527] SEQ ID NO: 101: Complete heavy chain amino acid sequence of h2731

[0528] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGSGRTYY

[0529] SDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSSA

[0530] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

[0531] LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP

[0532] SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

[0533] TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM

[0534] TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ

[0535] QGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0536] SEQ ID NO: 102: h2731 complete light chain amino acid sequence

[0537] DVVMTQSPLSLPVTLGEPASISCKSSQSLLDYDGKTYLNWLLQKPGQSPQRLIYRVTNRD

[0538] TGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRSFGQGTKVEIKRTVAAPSV

[0539] FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0540] SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0541] SEQ ID NO: 103: h2731 complete heavy chain nucleotide sequence

[0542]

[0543] SEQ ID NO: 104: h2731 complete light chain nucleotide sequence

[0544] GATGTTGTGATGACCCAGTCCCCACTCTCTTTGCCCGTTACCCTTGGAGAACCTGCCTCCATCTCTTGCAAGTCAAGTCAGAGCTCTTAGATTACGATGGAAAGACATATTTGAATTGGTTGCTGCAGAAGCCAGGCCAGTCTCCACAGCGCCTAATCTATCG GGTGACCAACCGGGACACTGGAGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTGGGAGTTTATTGCTGGCAAGGCACACATTTTCCGCGCTCTTTCGGACAGGGGACCAAGGTGG AAATAAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGCTTAAGTCCGGAACTGCTAGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGAAACTCC CAGGAGAGCGTCACAGAGCAGGACAGCAAAGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0545] Example

[0546] The following embodiments are included to illustrate the patterns disclosed herein. Certain aspects of the following embodiments are described in accordance with techniques and procedures that have been discovered or conceived by the co-inventors of the invention and work well in the practice disclosed herein. Given this disclosure and the general level of skill in the art, those skilled in the art will understand that the following embodiments are intended to be exemplary only and that many variations, modifications, and alterations may be made without departing from the scope of this disclosure.

[0547] As used in these experiments, “Aducanumab” or “Adu” refers to an antibody having the heavy chain of SEQ ID NO: 70 and the light chain of SEQ ID NO: 71, and as described in US Patent Publication No. US 2015 / 0315267 and PCT Publication No. WO 2014 / 089500.

[0548] As used in these experiments, “BAN-2401” and “gantenerumab” refer to antibodies having the heavy chain of SEQ ID NO: 82 and the light chain of SEQ ID NO: 83, as described, for example, in European Patent No. EP 1960428B1.

[0549] In the following methods, the binding profile of antibodies to aggregated or fibrillary Aβ was characterized by ELISA, surface plasmon resonance (SPR), and immunohistochemistry (IHC). The ability to mediate phagocytic plaque clearance was assessed in vitro in APP / PS1 transgenic mouse brains and AD brains with primary rat microglia using immunofluorescence, ELISA, and MSD quantification. Neutralization of Aβ oligomer neuronal binding was assessed in rat primary hippocampal cultures.

[0550] The results presented herein demonstrate that, compared to other N-terminal Aβ antibody therapies (bavizumab, adunarumab), the mAb described herein exhibits greater apparent affinity for aggregated and fibrillary Aβ in competitive or standard binding ELISA. The enhanced affinity of the disclosed mAb for fibrillary Aβ was confirmed by SPR equilibrium binding kinetics, indicating a 5 to 11-fold higher affinity than adunarumab due to a slower dissociation rate kinetic. IHC dose-response assessment of frozen human AD brain slices showed greater apparent affinity and plaque area binding than adunarumab, regardless of the individual AD donor tissue tested. In in vitro activity assays, the disclosed mAb significantly promoted Aβ plaque reduction via microglia phagocytosis in APP / PS1 mouse tissue and blocked the binding of soluble Aβ oligomers to primary rat neurons in a concentration-dependent manner. In in vitro functional assays of human AD brain, the mAb from this specification significantly promoted the clearance of pyroglutamylated Aβ (a post-translational modification component of senile plaques).

[0551] Example 1. Aβ antibody design

[0552] The Aβ antibody bapizumab (hBP) is a humanized antibody developed from the parental mouse antibody 3D6. Here, a multi-pronged approach is employed to construct a superior antibody against hBP. The humanization of hBP is analyzed, and decisions are made to optimize the humanization of the light chain.

[0553] The protein sequences in the PDB database [Deshpande et al., 2005] were searched to find structures that would provide a rough structural model of hBP. The crystal structure of hBP fab PDB code 4HIX [Miles et al., 2013] was used for both Vh and Vk structures because it has acceptable resolution (2.2 Å) and an exact sequence that matches hBP Vh and Vk, preserving the same canonical structure for the loop.

[0554] IMGT / DomainGapAlignment was performed on hBP VL as the input sequence. The human germline VK gene sequence IGHV2-30*02 best matched hBP VL. The frame region of hBP VL shared high sequence similarity with the corresponding frame region of IGHV2-30*02. Therefore, the frame region of IGHV2-30*02 VL was selected as the guide sequence for further optimization of the hBP frame region. In addition, based on the hBP 3D structure, three residues in CDR-L2 that do not have any direct contact with the antigen were also changed to germline sequences, resulting in the following changes: L50K, K53N, and L54R (Kabat).

[0555] Three different formats of the VL were designed by incorporating human germline framework residues into the hBP VL sequence. Canonical or interface residues remained unchanged. The alignment of the designed VK formats is shown in [image / image / etc.]. Figure 1 middle.

[0556] Based on structural observations that P15 is located at a corner and that the germline gene has Leu at that position, P15L was tested in a format of the variable light chain.

[0557] Based on 3D structural observations, substitutions were designed at numerous residues in the light and heavy chain CDRs and framework. In the first round of rational design, a total of thirty-one light chain and thirty-two heavy chain mutants (VL and VH formats) were generated and their binding was tested. In the second round of rational design, mutants that improved binding were combined to demonstrate the results. Furthermore, new structure-guided mutations will be incorporated into the design through further analysis.

[0558] The following positions within CDR-H1, T28, S30, N31, Y32, and G33 (Kabat) were subjected to mutagenesis based on a rational design. Mutagenesis was also performed at positions I51, G53, G54, T57, S60, D61, and N62 in CDR-H2 (Kabat). Rational mutagenesis was also performed at positions D96, H97, S99, S100a, and Y102 in CDR-H3 (Kabat).

[0559] For the variable light chain, multiple substitutions were attempted at positions K24, L27c, D27d, and S27e (Kabat) on CDR-L1. Directional and finite mutagenesis (Kabat) was performed at positions K53 and L54 on CDR-L2 of the light chain. No substitution was performed at position CDR-L3.

[0560] For both heavy and light chains, several selected locations within the frame region are also subjected to appropriate mutations.

[0561] Fifty-seven additional heavy chain and thirty-three light chain variants were designed and analyzed using Atum GPSpro software, which analyzes a database of human variable heavy and light chains and suggests query sequence-specific variations based on computer learning.

[0562] For variable repetition domains, multiple substitutions (Kabat) were designed and analyzed at positions A24, S25, G26, F27, T28, F29, S30, N31, Y32, G33, and M34. Most of these positions are within CDR-H1. Similarly, many CDR-H2 residues were mutagenized, such as positions A49, S50, I51, R52, S52a, G53, G54, G55, R56, T57, Y58, Y59, S60, D61, N62, V63, and K64 (Kabat). Furthermore, multiple substitutions of amino acids within CDR-H3 were performed, such as positions V93, R94, Y95, D96, H97, Y98, S99, G100, S100a, S100b, D101, and Y102 (Kabat).

[0563] Multiple substitutions were also designed at positions K24, S25, S26, Q27, S27a, L27b, L27c, D27d, S27e, D28, G29, K30, T31, Y32, L33, and N34 (Kabat) in the variable light chain CDR-L1. For CDR-L2, mutagenesis was performed at positions L50, V51, S52, K53, L54, D55, and S56 (Kabat). Most CDR-L3 positions (such as Q90, G91, T92, H93, F94, P95, R96, and T97) were also rationally substituted with multiple amino acids (Kabat).

[0564] The expression, melting point (Tm), affinity, and co-occurrence of all variant antibodies generated by both rational design and GPSpro design were analyzed. Based on the analysis mentioned above, eight antibodies from rational design and six antibodies from computer learning activities were selected for further analysis.

[0565] Example 2. IC determined by competitive ELISA 50 Ratio determination.

[0566] The IC50 of the antibodies of this disclosure is determined using a competitive (inhibitory) assay based on the binding of the labeled antibody to the antigen-coated plate. 50 .

[0567] To generate fibrils, the Aβ1-42 peptide, pre-treated with HFIP (hexafluoroisopropanol) and dried, was resuspended in DMSO to 5 mM, and then further diluted to 100 uM with 10 mM HCl. The sample was incubated at 37°C for 24 hours and then centrifuged to separate soluble and fibril types. The precipitate was resuspended in 1x D-PBS to the initial volume and acoustic-treated before use.

[0568] The plate was coated with 0.5 mg / ml fibrils Aβ 42 and blocked, for example, with 1% BSA / PBS. Seven 3-fold dilutions of hBP prepared in 0.1% BSA / PBS (starting at 150 μg / ml (final concentration 75 μg / ml)) and four 3-fold dilutions of the test antibody (starting at 20 μg / ml (final concentration 10 μg / ml)) were added to each well in triplicate, 50 μl per well. 50 μl of 0.75 μg / ml hBP-biotin prepared in 0.1% BSA / PBS (final concentration 0.35 μg / ml) was added to all wells, and the plate was incubated at room temperature for 2 hours, followed by washing 3x with TTBS. Then, 100 μl of GE streptavidin HRP diluted 1 / 10,000 was added and incubated for 30 minutes. The plate was then washed 6x with TTBS. Prepare ThermoFisher o-phenylenediamine dihydrochloride (OPD) substrate fresh according to the manufacturer's instructions, adding 100 μL per well. Incubate the reaction for 15 minutes and terminate with 50 μL of 2N H₂SO₄. Read the sample at 490 nM on a Spectromax instrument. Figure 2 , Figure 3 and Figure 4 Explanation of 4918, 4917, 4921, 3818, 493, 2931 and bapizumab control ( Figure 2 ), 2926, 2831, 2927, 2726, 2731, 2826 and bapizumab control ( Figure 3 ) and 2727, 2929 and bapizumab control ( Figure 4 Competitive ELISA assay graph. IC50 of each test antibody. 50 IC divided by hBP 50 To obtain the half-maximal inhibitory concentration (IC50) 50 Ratio. A ratio less than one indicates better performance than hBP. See Table 3A.

[0569] Table 3A

[0570]

[0571] Example 3. Determination of monoclonal antibody potency by competitive ELISA

[0572] The binding potency of certain monoclonal antibodies and hBP disclosed herein was determined by their competitive binding ability to biotinylated bapizumab bound to aggregated Aβ42, which was assessed by competitive ELISA. 1 mg of Aβ42 was added to 1 ml diH2O and vortexed vigorously and placed on a nutator at room temperature for 48 hours. The plate was coated with a mixture of 0.5 mg / ml heterogeneous Aβ42 aggregates and blocked, for example, with 1% BSA / PBS. Seven 3-fold dilutions of hBP (starting at 150 μg / ml (75 μg / ml after dilution with hBP-biotin)) and four 3-fold dilutions of the test antibody (starting at 20 μg / ml (10 μg / ml after dilution with hBP-biotin)) were added in triplicate to each well, 50 μL per well. Add 50 μL of 0.75 μg / mL hBP-biotin (0.35 μg / mL after dilution) to all wells and incubate the plate at room temperature for 2 hours, then wash 3x with TTBS. Next, add 100 μL of 1 / 10,000 diluted GE streptavidin (HRP) and incubate for 30 minutes. Wash the plate six times with TTBS. Prepare Thermo Fisher o-phenylenediamine dihydrochloride (OPD) substrate fresh according to the manufacturer's instructions, adding 100 μL to each well. Incubate the reaction for 15 minutes and terminate the reaction with 50 μL of 2N H₂SO₄. Read the samples at 490 nM on a Spectromax plate. Figure 5A Showing competitive ELISA assays of 2931, 2731 and bavizumab control; Figure 5B Showing competitive ELISA assays of 2726, 2831, and bavizumab control. Figure 20A The competitive ELISA assays of 2931, 2731 and the bavicilumab control are shown (data is displayed in rows 1-2 of Table 3B); Figure 20B Competitive ELISA assays for 2831, 2726, and bapizumab control are shown (data displayed in columns 4 and 5 of Table 3B). For Figure 20A and Figure 20B The curve and the obtained IC50 estimate represent a nonlinear three-parameter least squares fit of the data. A single point is the average of three samples (coefficient of variation <20%).

[0573] Table 3B

[0574]

[0575] The results showed that antibodies 2931, 2731, 2726, and 2831 had higher titers than hBP; and IC50 values ​​were approximately 2 to 4 lower than hBP. 50 value.

[0576] Example 4. Characterization of humanized mAbs or Fabs using BIAcore

[0577] To compare the effects of humanized antibodies or humanized antigen-binding fragments (Fab) on recombinant Aβ 1-42 The binding characteristics of the fibrils were analyzed using a BIAcore T200 (GE Life Sciences).

[0578] To generate fibrils, Aβ that has been pretreated with HFIP (hexafluoroisopropanol) and dried is used. 1-42 The peptides were resuspended in DMSO to 5 mM, and then further diluted to 100 μM with 10 mM HCl. The samples were incubated at 37°C for 24 hours and then centrifuged to separate soluble and fibrillary types. The precipitate was resuspended in D-PBS to the initial volume and sonicated before use.

[0579] Fibrous bonds were immobilized on a CM5 sensor chip (GE Healthcare Life Sciences) via amine coupling to ensure maximum binding of approximately 100 RU of analyte. Different concentrations of antibody or Fab (ranging from 1 nM to 100 nM) were passed through the coupling ligand at a rate of 30 μL / min in electrophoresis buffer (HBS + 0.05% P-20, 1 mg / mL BSA) for 300 seconds of binding and 1200 seconds of dissociation. Regeneration of the chip surface was accomplished by two short injections of 10 mM glycine-HCl (pH 1.7). The data blank was subtracted from the sensor concentration without ligand and the 0 nM analyte concentration. Analysis was performed using BIAcore Insight evaluation software (v2.0) with the bulk refractive index set to 0 RU, using a global 1:1 fit. Dissociation data (k... diss The results (kd) are shown in Table 4 (Fab) and Table 6 (Antibody).

[0580] Compared to adunatumab, it can be seen that h2726, h2731, h2831 and h2931 Fab and the antibody have similar, smaller dissociation constants, while adunatumab shows a significantly larger dissociation constant.

[0581] Table 4

[0582]

[0583] Example 5. Characterizing the epigenetic affinity of humanized mAbs using BIAcore

[0584] Using Biacore T200 to test anti-Aβ candidates against Aβ 1-28 Determination of binding affinity for (Bachem, Torrance, CA). Anti-human Fc antibodies were immobilized onto a CM3 sensor chip (GE Healthcare LifeSciences) via amine coupling and used to capture Aβ antibodies.

[0585] Various concentrations of Aβ 1-28 (Analytes, ranging from 100 nM to 0.39 nM, each dilution step a 2-fold serial dilution) were passed through the captured ligands in electrophoresis buffer (HBS + 0.05% P-20, 1 mg / mL BSA) at a rate of 50 µl / min for 240 seconds of binding time and 900 seconds of dissociation time. Data blanks were subtracted from irrelevant sensors containing ligands and electrophoresis buffer containing 0 nM analyte. Analysis was performed using a global 1:1 fit with Biacore evaluation software (v3.0).

[0586] The apparent dissociation constant (KD) is shown in Table 5, where mAb of this disclosure shows the effect on Aβ. 1-28 The monomer binding affinity is 4 to 7 nM. Sensing patterns of binding at concentrations from 0.39 nM to 100 nM are shown in [data missing]. Figure 6A (h2726) Figure 6B (h2731) Figure 6C (h2831) and Figure 6D (h2931)

[0587] Table 5

[0588]

[0589] Example 6. Characterizing the epigenetic affinity of humanized mAbs using BIAcore

[0590] To compare humanized antibodies with recombinant Aβ 1-42 The binding characteristics of the fibrils were analyzed using a BIAcore T200.

[0591] To generate fibrils, Aβ that has been pretreated with HFIP (hexafluoroisopropanol) and dried is used. 1-42 The peptides were resuspended in DMSO to 5 mM, and then further diluted to 100 μM with 10 mM HCl. The samples were incubated at 37°C for 24 hours and then centrifuged to separate soluble and fibrillary types. The precipitate was resuspended in 1x D-PBS to the initial volume and sonicated before use.

[0592] Protofibrils were immobilized on the CM5 sensor chip (GE Healthcare Life Sciences) via amine coupling to ensure maximum binding of approximately 50 RU of analyte. Different concentrations of antibody (ranging from 0.411 nM to 100 nM) were in electrophoresis buffer (HBS + 0.05% P-20, 1 mg / mL BSA) at a rate of 30 μL / min through a ligand-coupled ligand for 300 seconds and a dissociation time of 1200 seconds. Regeneration of the chip surface was accomplished by two short injections of 10 mM glycine-HCl (pH 1.7). A blank was calculated by subtracting the sensor without ligand and the 0 nM analyte concentration. Analysis was performed using BIAcore Insight software (v2.0) with the bulk refractive index set to 0 RU and a global 1:1 fit. Apparent dissociation constants (KD) are shown in Table 6, and a comparative sensor plot with 100 nM binding is shown in [Table 6]. Figure 7 middle.

[0593] Table 6

[0594]

[0595] Abeta, amyloid-beta, Aβ; ka, binding rate constant; kd, dissociation rate constant; KD, apparent equilibrium dissociation constant; mAb, monoclonal antibody; R max Maximum response; SPR, surface plasmon resonance.

[0596] The enhanced relative affinity of the monoclonal antibody of this disclosure for fibrillary Aβ, as observed by ELISA, was confirmed by SPR equilibrium binding kinetics (Table 6), and its indicator affinity (apparent KD) was 5 to 11 times greater than that of adunatumumab.

[0597] This is explained by the different dynamical combinations observed in the SPR sensing plots. Figure 7 Although adunatumab binds to Aβ fibrils at a faster binding rate (ka), the much slower dissociation rate (kd) of the monoclonal antibody disclosed herein results in a greater measured affinity (i.e., a lower KD*) than adunatumab.

[0598] Example 7. Aβ fibril binding by ELISA

[0599] The efficacy of certain monoclonal antibodies and adunatumab in this disclosure was evaluated using ELISA. 1-42 and Aβ pE3-42 Direct bonding of fibrils. To generate fibrils, Aβ, which has been pretreated with HFIP (hexafluoroisopropanol) and dried, is... 1-42 or AβpE3-42 The peptides were resuspended in DMSO to 5 mM, and then further diluted to 100 μM with 10 mM HCl. The samples were incubated at 37°C for 24 hours and then centrifuged to separate soluble and fibrillary types. The precipitate was resuspended in 1x D-PBS to the initial volume and sonicated before use.

[0600] Coat the plates overnight at room temperature with 1.0 μg / ml or 2.5 μg / ml Aβ fibrils in PBS. Block the plates with 1% BSA / PBS for 1 hour. Serially dilute the antibodies from 10 μg / ml to 4.8 ng / ml in 0.1% BSA-PBS and 0.1% Tween 20, adding 100 μl of each dilution in duplicate to each well and incubating at room temperature for 2 hours. Wash the plates four times with TBS / Tween 20 and add 100 μl of 1 / 5000 diluted goat anti-human IgG HRP (Jackson Immunno Research Laboratories, Inc., West Grove, PA or Invitrogen, Carlsbad, CA) to each well and incubate at room temperature for 1 hour. Wash the plates six times with TBS / Tween 20 and prepare Thermo Fisher o-phenylenediamine dihydrochloride (OPD) tablets and Thermo Fisher substrate buffer according to the manufacturer's instructions. Add 100 μL of substrate and incubate for 15 minutes. Stop the reaction with 50 μL of H₂SO₄. Read the plate at 490 nm using a Spectromax molecular apparatus. Figure 9 A and Figure 21 .for Figure 21 The curve and the resulting EC 50 The nonlinear three-parameter least squares fit representing the data is estimated (the data is shown in Table 7).

[0601] Table 7

[0602]

[0603] Coat the plate overnight at room temperature with a PBS dilution of 10 μg / ml to 4.8 ng / ml Aβ fibrils. Block the plate with 1% BSA / PBS for 1 hour. Add two copies of antibody at 2 μg / ml in 0.1% BSA / PBS and 0.1% Tween 20 to the appropriate wells and incubate at room temperature for 2 hours. Wash the plate 4x with TBS / Tween 20 and then add 100 μl of Jackson goat anti-human IgG HRP 1 / 5000 dilution to each well and incubate at room temperature for 1 hour. Wash the plate six times with TBS / Tween 20 and prepare Thermo Fisher o-phenylenediamine dihydrochloride (OPD) tablets and Thermo Fisher substrate buffer according to the manufacturer's instructions. Add 100 μl of substrate and incubate for 15 minutes. Stop the reaction with 50 μl of H2SO4. Read the plate at 490 nm on a spectromax molecular device. Figure 9 B, the smaller image on the right.

[0604] Antibodies h2726, h2731, h2831, and h2931 all demonstrated strong affinity for fibrils, with differences between optimal and worst expression within 25%. Furthermore, all four antibodies showed significantly greater affinity than aducunab. Figure 21 The measured signal (OD490) increased by 3 times and the estimated EC 50 A 15- to 20-fold reduction in the overall binding and relative affinity of h2726, h2731, h2831, and h2931 mAb to fibrils Aβ was compared to an increase in aducunamumab.

[0605] Example 8. Binding of Aβ oligomers by h2931 of ELISA

[0606] The direct binding of h2931 to Aβ oligomers was assessed by ELISA. To generate the oligomers, lyophilized biotinylated and unlabeled Aβ (Bachem) was first dissolved at 1 mg / mL in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, Sigma). The HFIP was allowed to evaporate from the sample overnight at room temperature in a fume hood. The aliquots were then centrifuged in a speedvac at room temperature to remove all liquid to obtain 250 µg FIP membrane aliquots, which were stored at -80°C until further use.

[0607] Oligomers were prepared by dissolving 250 µg of biotinylated and unlabeled Aβ HFIP precipitate to a final concentration of 5 mM in anhydrous DMSO (Sigma). For the unlabeled:biotinylated mixture, samples were combined in sterile 1.5 mL low-binding microcentrifuge tubes (Axygen) at a ratio of 9:1 (unlabeled:biotinylated). The DMSO-dissolved samples were then diluted to 100 µM with cold phenol-free neurobasal medium (Invitrogen) and incubated at 4°C for 24 h. After incubation, the oligomers were separated from the large insoluble material by centrifugation at 14,000 g for 15 min. The top 90% of the supernatant was carefully removed and transferred to new sterile low-binding microcentrifuge tubes and stored on ice until use.

[0608] In a Costa ELISA high-binding plate, 100 μL of each preparation in PBS was coated overnight at room temperature and incubated at PBS. The plate was aspirated and then 200 μL of 1% BSA in PBS was added to each well and incubated at room temperature for 1 hour. h2931 mAb was prepared at an initial concentration of 10 μg / mL in 0.1% BSA / PBS 0.1% Tween 20 buffer and serially diluted seven times (1:2 each time). The samples were incubated at room temperature for 2 hours. The plate was washed four times with PBS 0.1% Tween 20. Goat anti-human (H+L) HRP (Jackson Immunoresearch, PA) was diluted 1 / 5000 in 0.1% BSA / PBS 0.1% Tween 20, added at 100 μL / well, and incubated at room temperature for 1 hour. The plate was washed four times and o-phenylenediamine dihydrochloride tablets (ThermoFisher) were prepared according to the manufacturer's instructions. Add 100 µl to each well and incubate at room temperature for 15 minutes. Stop the reaction by adding 50 µl of H₂SO₄ and read the samples at 490 nM on a SpectroMax molecular instrument. The resulting EC₂ curves and EC₂ values ​​are shown. 50 The estimate represents a nonlinear 3-parameter least squares fit of the data using GraphPad Prism software.

[0609] The study showed that mAb h2931 binds to soluble oligomers with high relative affinity, and estimated EC... 50 It is 23 ng / mL or 0.15 nM. Figure 8 .

[0610] Example 9. Anti-Aβ antibody binding in the AD brain

[0611] Tissue samples. Frozen human AD brain samples were obtained from Banner Sun Health Research Institute, Sun City, AZ. Tissues were derived from donors confirmed to have extensive Aβ pathology and graded according to the Braak system of the providing institution (Table 8). In addition, all tissue blocks underwent internal quality control to determine their pathological level and distribution.

[0612] Table 8. AD Donor Information

[0613]

[0614] Tissue sectioning and fixation. Unfixed frozen brain tissue samples were embedded in cryomolds in Tissue-Tek OCT (Sakura Finetek) and immersed in a mixture of 2-methylbutane and dry ice (-60°C), then stored at -80°C until sectioning. Continuous 10 µm thick frozen sections were produced using a Leica 3050S cryostat. Sections were thawed directly, mounted on positively charged slides, and stored at -20°C until use. Prior to the immunohistochemical (IHC) procedure, slides were immersed in 10% neutral buffered formalin at 4°C for 10 minutes, rinsed in PBS, and then incubated at 37°C for one hour in glucose oxidase solution (20 mM βD(+) glucose, 2 mM sodium azide, and 2 units / mL glucose oxidase in 1X PBS). Rinse the slides three times in PBS for 5 minutes each time, then transfer them to a staining rack for processing in an automated staining machine.

[0615] Antibody biotinylation. Humanized IgG antibodies were biotinylated using a non-covalent method by incubating at room temperature for 1 hour with a biotin-conjugated goat anti-human monovalent fab fragment (Jackson ImmunoResearch) at a 1:4 ratio. Unbound excess fab was absorbed by pre-incubation with human serum for 1 hour prior to use. The freshly prepared antibody was then loaded into a staining machine for immediate application to tissue sections.

[0616] Immunostaining. Staining was performed using a BondResearch kit (DS980, Leica Biosystems) and an avidin-biotin amplification immunoperoxidase assay system in an automated Leica Bond Rx staining system (Leica Biosystems). Biotinylated anti-Aβ antibodies or human IgG controls were applied to the sections at specified concentrations for one hour, and staining was observed using the avidin-biotin amplification system (ABC Elite Standard, PK-6100; Vector Laboratories). The sections were then stained with a hematoxylin counter for the cell nuclei, dehydrated in an ascending series of alcohols, cleaned in xylene, covered with coverslips, and air-dried.

[0617] Tissue imaging. Stained slides were digitally imaged using a Hamamatsu NanoZoomer 2.0HT slide scanner (Hamamatsu Corporation), and images were captured in .ndpi file format using NanoZoomer Digital Pathology software (NDP.scan, version 2.7.25). Images included in this report were captured directly from NDP.view without any enhancement transfer. For morphometry, the percentage of stained tissue was measured using Halo software (V2.1.1537), and the results were plotted using GraphPad Prism 8.

[0618] Results of h2726, h2731, h2831, h2931, and adunatumumab. The four humanized anti-Aβ antibodies of this disclosure, h2726, h2731, h2831, and h2931, along with adunatumumab, were applied to all four AD brain regions at escalating concentrations: 0.03, 0.1, 0.3, 1, 3, and 9 µg / ml. Figure 10 As shown in (0.3 µg / mL), AD brain sections incubated with these antibodies exhibited immunopositive structures typical of Aβ pathology in AD. High-density Aβ plaques were observed in AD 13–75 and AD 14–11, while the pathology was relatively sparse in AD 11–97 and AD 15–19. In each brain, staining with four antibodies h2726, h2731, h2831, and h2931 at specific concentrations showed comparable intensity and distribution. Among the samples and concentrations, staining with adunatumumab was the weakest. Figure 11 As illustrated, none of the brain sections incubated with 1 or 9 µg / ml of control human IgG isotype showed pathological staining.

[0619] Figure 12 and Figure 22The figures in the table are quantitative plots of staining with five antibodies in all four AD brains. Measurements of the percentage of tissue surface area occupied by stained pathology confirmed that the four antibodies h2726, h2731, h2831, and h2931 exhibited similar binding levels at all tested concentrations in each AD brain. Accordingly, the data in Table 9 show that the area under the curve and EC50 values ​​of the four antibodies remained comparable for each brain. Values ​​obtained using aducunab were consistently lower in AD brains across the entire tested concentration range.

[0620] Figure 22 Larger plaque area binding (as a percentage of stained positive tissue) was observed compared to aducannamumab, particularly at antibody concentrations estimated at clinically relevant exposure in cerebrospinal fluid with 10 mg / kg aducannamumab. Similar plaque area staining was observed at the highest concentration tested, suggesting saturation of binding at that concentration.

[0621] Table 9

[0622] Area under the curve and half-maximum effective concentration (EC50) 50 )

[0623]

[0624] Results of bavinzizumab (hBP). Sections from brain AD 13 to 75 were incubated with humanized antibody hBP, adunatumab, and BAN2401 at the following increasing concentrations: 0.03, 0.1, 0.3, 1, 3, and 9 µg / ml. As observed with antibodies h2726, h2731, h2831, and h2931, the intensity of hBP staining increased in a dose-dependent manner. Furthermore, at all tested concentrations, hBP staining was stronger than adunatumab and BAN2401. Figure 13 As shown in the image.

[0625] Example 10. Used to determine (Aβ) 1-42 and Aβ pE3-42 In vitro phagocytosis assay for plaque clearance

[0626] In the early stages of Alzheimer's disease (AD), microglia play a neuroprotective role, clearing apoptotic cells and pathological protein aggregates, and forming a barrier around plaques to limit their growth and the spread of synaptic toxic Aβ oligomers. In vitro phagocytosis assays quantify antibody-mediated microglial clearance responses.

[0627] Primary microglia culture generation: For the dissection of neonatal mouse brain tissue, the head of a P1 pup is rapidly removed using sterile scissors. The meninges are removed and the forebrain is immediately immersed in 1 to 5 ml of dissection medium (e.g., high-glucose DMEM with 20% FBS, P / S) on ice until the desired number of pup brains are dissected. The total surgical time is preferably limited to less than 10 minutes to minimize cell damage.

[0628] Using a new sterile pipette and a 22G needle, followed by a 25G needle, carefully aspirate tissue twice consecutively. Centrifuge the sample at 2,500 xg for five minutes at 4°C. Carefully aspirate the supernatant and add 5 ml of fresh growth medium (high glucose DMEM, 10% FBS, P / S, and 25 ng / ml recombinant mouse GM-CSF) to the cell pellet. Use a sterile 10 ml pipette to pipette up and down approximately 10 times to dissociate the pellet.

[0629] Place the cell filter (100 µm pores) onto a new 50 ml conical tube and disperse the material through the filter into the tube. Rinse the cell filter with 4 to 5 ml of fresh culture medium, then centrifuge at 200 x g for 5 minutes at 4°C.

[0630] Cells were seeded at a density of two mouse brains per T-75 plastic culture flask. The supernatant was carefully aspirated, and 3 ml of fresh growth medium (high glucose DMEM, 10% FBS, P / S, and 25 ng / ml recombinant mouse GM-CSF) was added to each cell pellet using a 10 ml sterile pipette. The cells were resuspended by pipetting up and down 10 times with a 10 ml pipette. A sterile T-75 flask was prepared by adding 6 ml of growth medium (high glucose DMEM, 10% FBS, P / S, and 25 ng / ml recombinant mouse granulocyte-monocyte colony-stimulating factor) to each flask. Then, 6 ml of the resuspended cell pellet was added to each flask in a 37°C, 5% CO2 incubator to obtain a final 12 ml volume.

[0631] The flasks were incubated undisturbed for five days to allow cell attachment. On day five, the culture medium in each flask was replaced with 12 ml of fresh growth medium (high glucose DMEM, 10% FBS, P / S, and 25 ng / ml recombinant mouse GM-CSF). Approximately 10% of the plated mixed cells would attach and grow on the plastic surface. The culture medium was changed twice a week (every 3 to 4 days) to allow confluence. These changes were performed very carefully, without touching the bottom of the flask where cells were attached.

[0632] After 7–11 days, these vials were rotated at 200 rpm for 2 hours at 37°C using a Lab-Line oscillator with a 19-mm orbital. The cell suspension was centrifuged at 200 x g and resuspended in assay medium (H-SFM [Life Technologies] medium without hybridoma serum plus 1% FBS, glutamine, P / S and 5 ng / ml recombinant mouse GM-CSF).

[0633] In vitro assay. Cryostat sections (10 μm thick; using a wide blade) of APP / PS1 mouse or human AD brain (postmortem interval, less than 3 hours) were “thawed and mounted” onto polylysine-coated round glass coverslips and placed in the wells of a 24-well tissue culture plate (CT -30°C OT -20°C). Tissue samples could be heated between sections with a thumb or by lowering the OT to -12°C. The coverslips were washed twice with assay medium. (Control or anti-Aβ) antibody was added at a 2X concentration of 250 µl to the assay medium (final 20 μg / ml), and the plate was incubated for 1 hour.

[0634] Microglia were then seeded at a final density of 800,000 cells / ml (1,600,000 cells / ml stock solution) in 250 µl of assay medium. The cultures were incubated in a humidified incubator at 37°C in a 5% CO2 atmosphere for 72 hours.

[0635] Total Aβ (Aβ) 1-42 Quantification of Aβ42 peptide (4G8) was performed by carefully aspirating the culture medium and washing with ice-cold PBS. 100 µl of 8M urea was added, and the tissue was resuspended by pipetting and scraped off with a pipette tip. The suspension was then frozen at -20°C until ready for analysis. The suspension was thawed on ice, centrifuged at 16,000 x g for 20 min at 4°C, and then diluted and analyzed using the V-PLEX Total Aβ42 Peptide (4G8) Kit (Meso Scale Discovery). Results are shown in [image / image / etc.]. Figure 14A , Figure 14B and Figure 24 middle. Figure 14A and Figure 24 Showing the Aβ concentration for each brain slice and Figure 14B Display the same data as the scatter plot for each treatment. Figure 14B The data is shown in Table 10; Figure 24 The data are shown in Table 11. h2731, h2931 and adunatumumab demonstrated a significant reduction in the height of Aβ plaque types compared to the isotype control.

[0636] Table 10

[0637]

[0638] Table 11

[0639]

[0640] Pyroglutamic acid-3 Aβ (Aβ) pE3-42 Quantification of Aβ (e.g., Aβ) truncated at the N-terminus and modified with pyroglutamic acid. pE3-42 Aβ has been described as a component of mature senile plaques in the brain of Alzheimer's disease (Saido et al., Neuron 14, 1995). It is unknown whether N-terminal Aβ pyroglutamate modification affects the binding of N-terminal antibodies (such as h2731) and other antibodies described herein. Similarly, it is unknown whether these antibodies promote the binding of Aβ. pE3-42 The ability of phagocytes to clear [the virus].

[0641] Immunohistochemistry confirmed the presence of pyroglutamate-3 Aβ in AD brain samples used for in vitro experiments and its staining pattern was similar to that of h2931. Figure 25A and 25B To demonstrate the removal of pyroglutamate-3 Aβ, a commercial ELISA method was used to measure its removal during in vitro phagocytosis. Suspensions collected following the above method were thawed on ice, centrifuged at 16,000 x g for 20 min at 4°C, and then diluted and analyzed using a commercial ELISA kit (Amyloid Beta N3pE Aβ, IBLAmerica). When compared with unmodified Aβ... 1-42 In comparison, Aβ pE3-42 ELISA assay for Aβ pE3-42 It exhibits high specificity (data not shown).

[0642] The results are shown in Figure 26A and Figure 26B (Data are shown in Tables 12 and 13 respectively), which show the use of specified antibodies ( Figure 26A h2931 and Figure 26B The concentration of pyroglutamate-3 Aβ in brain slices treated with h2731 was compared with that in healthy controls and AD brains treated with IgG1 isotype control. Slices from different AD brains were used for each treatment. Both h2731 and h2931 showed a highly significant reduction in pyroglutamate-3 Aβ compared with isotype control.

[0643] Figure 24 and Figure 26B Together, the present invention's anti-Aβ antibody (e.g., h2731) promotes Aβ when incubated on brain tissue sections from AD patients containing primary mouse microglia. 1-42 and Aβ pE3-42The antibodies clear both proteins. These results confirm that these antibodies clear Aβ from the human pathological environment. 1-42 and Aβ pE3-42 .

[0644] N-terminal targeting anti-Aβ antibodies promote extensive microglia-mediated clearance of various Aβ plaque types, including pyroglutamate-modified Aβ, in the brain tissue of AD patients. These data support further development of the antibodies of this invention as a subcutaneously administered antibody immunotherapy for Alzheimer's disease.

[0645] Table 12

[0646]

[0647] Table 13

[0648]

[0649] Example 11. Blocking oligomers in hippocampal binding assays

[0650] Aβ binding assay in rat hippocampal neurons

[0651] Primary E18 rat hippocampal neurons were cultured as described by Zago et al. (J. Neurosci, 2012, 22 Feb., 32(8) 2696-2702). Soluble Aβ was pre-incubated on culture DIV14-21 with and without antibody to block neuritis binding to primary neurons.

[0652] Prepare fresh, unlabeled, biotinylated, or (9:1) unlabeled:biotinylated soluble Aβ one day prior and incubate overnight at 4°C. Centrifuge Aβ at 14,000 RPM for 15 minutes before use.

[0653] Prepare half the final treatment volume of Aβ solution and antibody dilutions (2x) at the final treatment concentration using NeuroBasal-Phenol Red-Free (NB-NPR) or NbActiv4-NPR medium. After combining, mix the mixture 3 to 4 times and then incubate at 37°C for 30 minutes.

[0654] Immediately prior to the binding assay, neurons were flushed with preheated NB-NPR at 150 µL / well. Buffer was aspirated and then antibody / Aβ treatment was added to cells at 60 µL / well, followed by incubation at 37°C under normal incubator conditions (5% CO2; 9% O2) for 30 to 40 minutes.

[0655] Neurons were rinsed twice with 150 µL / well of NB-NPR and then fixed for 20 minutes in 4% paraformaldehyde in 1x DPBS at room temperature.

[0656] Cells were permeabilized in 0.1% Triton X-100 in 1x DPBS for 5 minutes and then blocked in 10% normal goat serum (NGS) at room temperature (RT) for 1 hour.

[0657] The samples were incubated overnight at 4°C with primary antibodies against microtubule-associated protein 2 (MAP2) and neuronuclear protein (NeuN) in 100 µL / well of 1x DPBS containing 1% BSA + 1% NGS. The next day, the samples were washed twice with 150 µL / well of 1x DPBS for 5 minutes each time. Secondary antibodies were then added to 100 µL / well of 1x DPBS + 1% BSA + 1% NGS at room temperature for 1 hour.

[0658] High-content imaging (HCI) analysis was performed using an Operetta HCI CLS instrument (Perkin Elmer; modified neuronal spur growth algorithm: 40x H2O objectives; 25 to 40 fields of view per well in microplate format; each condition (n=3)) to quantify soluble Aβ neuritis-binding spots. MAP2 and NeuN neuronal markers were used for each tracked neuronal spur and cell body counts were calculated for each light field (e.g., with microtubule-associated protein 2 (Abcam; Cambridge, UK) and NeuN (EMD Millipore) primary antibodies, followed by AlexaFluor (Thermo Fisher Scientific) secondary detection antibodies). Axonal Aβ spots were detected using various monoclonal and polyclonal Aβ antibodies (e.g., mouse monoclonal anti-Aβ antibody MabN254 (EMD Millipore)), followed by detection of biotinylated Aβ material using AlexaFluor (Thermo Fisher Scientific) secondary detection antibodies or streptoacidin-AF488. Figure 15A and Figure 15B The study showed that increasing the concentration of anti-Aβ antibody reduced the number of spots per neuron, indicating anti-Aβ activity. Figure 23 The results showed that h2731 effectively blocked the binding of soluble Aβ aggregates to rat hippocampal synapses (Aβ42 spots in each neuron) in a concentration-dependent manner. The effect of h2731 was detected at molar mAb:Aβ42 ratios as low as 1:500 (p < 0.05) and achieved >90% binding blockade compared to Aβ42 alone (without mAb pre-incubation) at a molar ratio of 1:50 (p < 0.001). The data are shown in Table 14.

[0659] Table 14

[0660]

[0661] Example 12. Binding of anti-Aβ antibodies to natural and modified Aβ species

[0662] Human Alzheimer's disease (AD) brain sections were thawed, mounted onto poly-D-lysine-coated coverslips, and placed in 24-well tissue culture plates. The plates were incubated with the test antibody at 37°C and 5% CO2 for 1 hour. Primary mouse microglia were then seeded at 800,000 cells / ml, and the culture was maintained at 37°C and 5% CO2 for 72 hours. The culture medium was carefully aspirated, and the sections were washed with PBS. The sections were resuspended in 8M urea for ELISA testing of Aβ. pE3-42 (Immuno-Biological Laboratories, Minneapolis, MN) or via MSD for Aβ 1-42 Quantification was performed at Meso Scale Diagnostics, Rockland, MD. Immuno-Biological Laboratories Aβ pE3-42 The ELISA kit specifically detects pE3-42 species, but no signal was detected for full-length Aβ.

[0663] Figure 27 This demonstrates that h2731 binds with high apparent affinity to the N-terminus of full-length Aβ but not directly to pyroglutamic acid-modified Aβ (Aβ). pE3-42 h2731 was found to have a half-maximal effective concentration (EC50) of 8.1 ng / mL (54 pM). 50 ) binds to Aβ species of fibrils with unmodified N-termini (Aβ) 1-42 h2731 proves that for Aβ pE3-42 Undetectable binding up to 100 ng / ml.

[0664] Example 13. In vitro phagocytic cell-mediated clearance – THP-1 human monocyte-mediated Aβ 1-42 Primary fiber intake

[0665] Generate Aβ containing the S26C mutation 1-42The synthesis of primary fibrils is described, as in Paranjape et al., ACS Chem. Neurosci. 2012, 3, 302-311. Briefly, Aβ peptides were dissolved in 1 mM of 100% hexafluoroisopropanol (HFIP) (Sigma-Aldrich, St. Louis, MO), aliquoted into sterile microcentrifuge tubes, and evaporated overnight uncovered in a fume hood at room temperature. The next day, the aliquots were vacuum centrifuged to remove any residual HFIP and stored in a desiccant at -20°C. Some Aβ peptides were treated with 100% trifluoroacetic acid and vacuum centrifuged before HFIP treatment. Aβ oligomers and fibrils were prepared directly from the lyophilized aliquots by resuspending the lyophilized Aβ peptide aliquots in sterile anhydrous dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO). For oligomer preparation, samples were diluted to 100 μM in sterile, ice-cold, phenol red-free Ham's F-12 cell culture medium (F-12, Bioworld, Dublin, OH) containing L-glutamine and incubated at 4°C for 24 hours. For fibril preparation, samples were diluted to 100 μM in 10 mM HCl and incubated at 37°C for 24 hours. The Aβ concentrations in these formulations are based on peptide dry weight.

[0666] Mature primary fibrils were conjugated to pHrodo RedMaleimide (Thermo Fisher) prior to in vitro phagocytic cell-mediated clearance assays.

[0667] Antibodies at concentrations of 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.098, and 0.049 µg / ml were reacted with pHrodo-Aβ at room temperature. 1-42 Primary protofibrils were pre-incubated together for 30 minutes, followed by the addition of THP-1 phagocytes. After incubation at 37°C and 5% CO2 for 3 hours, antibody-mediated phagocyte-mediated clearance was assessed by measuring cellular pHrodo signaling via flow cytometry.

[0668] like Figure 28A and Figure 28B The results show that anti-Aβ antibodies exhibit a concentration-dependent mode of action against Aβ. 1-42 Primary fibrillary phagocytic activity. These results suggest that the antibody of the present invention may be able to drive Aβ in brain tissue. 1-42 Clear.

[0669] Example 14. Distribution of total Aβ and pyroglutamate-modified Aβ in brain tissue from patients with advanced Alzheimer's disease (AD).

[0670] The ex vivo IHC method described above and in this article was used to determine Aβ in AD brain tissue. 1-XX (Detected using N-terminal anti-Aβ antibody) and anti-Aβ pE3-42 The distribution of .

[0671] For Aβ 1-XX and Aβ pE3-42 Evaluation confirmed that both types were widely distributed in tissues from patients with advanced AD. Aβ 1-XX Percentage of area covered and Aβ pE3-42 Comparative distribution patterns ( Figure 29A (1) and Figure 29A (2) (and each is magnified) Figure 29B (1) and Figure 29B (2))) and quantitative ( Figure 29C Consistent with previous studies, this indicates that Aβ pE3-42 This represents a relatively small library of modified Aβ, a mixture of unmodified Aβ targeted by N-terminal Aβ antibodies. pE3-42 Shown in Figure 29A (2) and Figure 29B (2) and the complete N-terminal Aβ is shown in Figure 29A (1) and Figure 29B (1) In the middle. Anti-Aβ pE3-42 Antibodies do not interact with Aβ 1-42 Cross-reaction (data not shown).

[0672] Figure 29A (1) and Figure 29B The box in (1) shows Aβ with an intact N-terminus and modified Aβ near the blood vessel. pE3-42 Aβ plaques. Table 15 below reports... Figure 29B (1) and Figure 29B (2) Quantitative analysis of staining in plaques, such as... Figure 29C The difference between the means is statistically significant (p=0.007, paired two-tailed t-test).

[0673] Table 15

[0674]

[0675] Example 15. Co-localization of anti-Aβ antibody h2731 and AβpE3-42 in the brain of AD patients

[0676] Immunochromatographic staining of h2731 and Aβ were evaluated using immunofluorescence microscopy. pE3-42 Co-localization. Before tissue application, the N-terminal anti-Aβ antibody (h2731 in this case) was pre-conjugated to the Cy3 secondary anti-human antibody (Jackson Laboratories). Mouse anti-Aβ antibodies were used. pE3-42Antibody conjugated with 488-AlexaFluor anti-mouse secondary antibody to detect Aβ pE3-42 The slides were imaged using a Metamorph-assisted IX81 Olympus microscope connected to a Hamamatsu camera (C10600-10B).

[0677] Figure 30 (Small figure A) shows the localization of h2731 in the Aβ plaque; Figure 30 (Small image B) shows anti-Aβ pE3-42 Antibody signaling localizes Aβ plaques; and Figure 30 (Small figure C) shows h2731 and anti-Aβ. pE3-42 Antibody signals colocalize with Aβ plaques. Overlapping signals are more prominent in the dense core region of the plaques.

[0678] Example 16. The anti-Aβ antibody of the present invention promotes the ex vivo extraction of Aβ from AD brain tissue in a dose-dependent manner and with higher potency than adunatumumab. pE3-42 Clear

[0679] Using the methods described above and elsewhere in this document, the clearance of Aβ from AD brain tissue by adunamoxicillin and the antibodies of the present invention (e.g., h2731) was evaluated. pE3-42 The ability of proteins.

[0680] Physiologically relevant dose-response series of h2731 (3 ng / ml, 10 ng / ml, 30 ng / ml, and 100 ng / ml) were incubated with brain tissue sections from AD patients and primary mouse microglia for 72 hours. h2731 promoted Aβ in a concentration-dependent manner. pE3-42 The removal was completed. The results are presented in Table 16 below. Figure 31A middle.

[0681] Table 16

[0682]

[0683] h2731 promotes Aβ in a concentration-dependent manner via microglia phagocytosis and robustly during a relatively short incubation period (72 hours). pE3-42 Clearance from brain tissue sections of AD patients. Therefore, the antibody of the present invention promotes Aβ at the concentration range expected to be achieved by subcutaneous administration. pE3-42 Ex vivo clearance from the brain of AD patients.

[0684] Another series of experiments were conducted, comparing h2731 at 25 ng / ml and 75 ng / ml with aducanamab at 25 ng / ml and 225 ng / ml. The results are presented in Table 17 and... Figure 31B middle.

[0685] Table 17

[0686]

[0687] h2731 exhibits superior Aβ compared to adunatumab. pE3-42 Eliminates activity, even at concentrations up to 9 times lower.

[0688] Another physiologically relevant dose-response series of h2731 and adunatumab (3 ng / ml, 25 ng / ml, and 225 ng / ml) were incubated with brain tissue sections from AD patients and primary mouse microglia for 72 hours. Both were compared with the IgG1 isotype control. Although both h2731 and adunatumab promoted Aβ in a concentration-dependent manner... pE3-42 Clearance was achieved, but h2731 was again significantly more effective, with a p-value <0.0001 at a concentration 9 times lower than that required for adunatumumab to achieve a p-value of 0.0005. The results are presented in Table 18 below. Figure 32A middle.

[0689] Table 18

[0690]

[0691] To verify that h2731-mediated in vitro phagocytosis activity is microglia-dependent, + / - microglia experiments were performed. Although microglia alone drive some Aβ... pE3-42 Clearance was achieved from tissue sections of AD patients, but the combination of h2731 and microglia resulted in significantly more robust clearance. The clearance activity of h2731 appears to require the presence of microglia, as h2731 alone showed no activity in the absence of microglia. Results are presented in Table 19 and... Figure 32B middle.

[0692] Table 19

[0693]

[0694] The antibody concentrations tested were based on CNS ranges estimated from modeled pharmacokinetic values ​​at minimum and maximum concentrations in 0.1% steady-state plasma following monthly administration of 3 mg / kg subcutaneous h2731 (25 to 75 ng / ml) or 10 mg / kg intravenous adunamoba (25 to 225 ng / ml) to humans. Figure 33 ).

[0695] The antibody of this invention promotes Aβ at concentrations expected to be achieved via subcutaneous administration. pE3-42 It can be ex vivo cleared from the brain of AD patients and has greater biological activity than adunatumab.

[0696] Antibody h2731 reduces Aβ in the brain of Alzheimer's disease (AD). pE3-42 dyeing. Figure 34 The results showed that the human IgG isotype control antibody ( Figure 34 A and Figure 34 B) Aβ was observed in plaques (white triangles) in the treated AD brain. pE3-42 (Staining is indicated by white arrows) and with blood vessels ( Figure 34 A and Figure 34 (The circle in C) is associated. As demonstrated by the reduction of plaques, treatment with h2731 enhances microglia-mediated Aβ. pE3-42 Concentration decreased ( Figure 34 C and Figure 34 D). The antibodies of the present invention (as exemplified by h2731) reduce the content of Aβ in tissues. pE3-42 plaques.

[0697] Example 17. h2731 target bonding

[0698] Female APPxPS1 mice expressing mutant human amyloid precursor protein (hAPP[V717I]) and mutant human presenilin 1 (hPS1[A246E]) were used to evaluate the ability of h2731 and adunatumab to cross the blood-brain barrier and bind to amyloid β (Aβ) plaques in the brain after peripheral administration. The mean age of the animals at the start of the study was 6.7 months. All animals received an injection of anti-CD4 antibody (20 mg / kg, intravenously) the day before drug administration to prevent the formation of anti-drug antibodies in mice receiving h2731 or adunatumab, both of which were fully humanized antibodies. h2731 (3 or 10 mg / kg, subcutaneously, SC) or adunatumab (10 mg / kg, intravenously) was administered weekly for three consecutive weeks, and the animals were euthanized one week later. After heart perfusion with ice-cold saline, the brains were extracted from the mice and rapidly frozen in 2-methylbutane on dry ice and stored at -80°C.

[0699] Serial sagittal 10 µm thick frozen sections were produced using a Leica 3050S cryostat. Sections were thawed directly, mounted on positively charged slides, and stored at -20°C until use. Prior to IHC, slides were immersed in 10% neutral buffered formalin at 4°C for 10 minutes, rinsed in PBS, and then incubated at 37°C for one hour in glucose oxidase solution (20 mM βD(+) glucose, 2 mM sodium azide, and 2 units / mL glucose oxidase in 1X PBS). Slides were rinsed three times for 5 minutes each in PBS and then transferred to a staining rack for processing in an automated staining machine. Biotin-SP conjugated goat anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories #109-065-088) was used to detect h2731 or adunaza in APPxPS1 brain tissue. Staining was performed using the Bond Research kit (DS980, Leica Biosystems) in an automated Leica Bond Rx staining system (Leica Biosystems). The sections were then stained with a hematoxylin counter for the cell nuclei, followed by dehydration in a series of increasing alcohols, cleaning in xylene, coverslipping, and air-drying. The entire section was imaged using a NanoZoomer 2.0HT slide scanner (Hamamatsu Corporation, Japan). Morphometry analysis was performed on the digital images using Halo software (V2.1.1537). The percentage of stained tissue area was determined after delineating the cerebral cortex as the region of interest. The data are presented in Table 20.

[0700] Table 20

[0701]

[0702] ROI = Region of Analysis. All data represent the mean ± SD for each group of n = 5 animals.

[0703] A reduction in the number or size of Aβ plaques in Alzheimer's disease can be associated with a slowing or reversal of disease progression. The ability of the anti-Aβ antibodies of the present invention to bind to and clear Aβ in vivo after peripheral administration supports the potential utility of these antibodies as therapeutic agents.

[0704] Therefore, the antibody of the present invention promotes microglia-mediated Aβ in the brain tissue of AD patients. 1-42 Clearance. Although the antibody of the present invention may not directly target pyroglutamate modification, it can effectively clear Aβ at concentrations predicted to be clinically relevant. pE3-42They also exhibit higher potency and greater biological activity than adunatumab, as exemplified by h2731. The clearance of pyroglutamate species by these antibodies may be attributed to microglia recognizing opsonized plaques and engulfing large particles with varying concentrations. The antibodies of this invention may therefore clear other neurotoxic components co-deposited in plaques via the same mechanism.

[0705] Example 18. Reducing amyloid plaques in patients

[0706] To reduce amyloid plaques (which has been associated with inhibiting, reducing, and / or reversing Alzheimer's disease symptoms), patients are given a pharmaceutically effective amount of an anti-Aβ antibody (or its antigen-binding fragment), such as one or more antibodies described in the examples above.

[0707] Patients: Individuals suspected of having or already diagnosed with amyloid plaque-related diseases such as Alzheimer's disease were selected for treatment with anti-Aβ antibodies.

[0708] Treatment: Administer 70 mg of anti-Aβ antibody h2931 subcutaneously to the patient approximately every 4 weeks. Administer 200 mg of anti-Aβ antibody h2931 subcutaneously to the patient approximately every 4 weeks.

[0709] As described in this article, plaque reduction and improvement in Alzheimer's disease symptoms can be measured.

[0710] Patients with Alzheimer's disease who receive subcutaneous administration of 70 mg or 200 mg of anti-Aβ antibodies h2731, h2726, h2831, or h2931 approximately every 4 weeks are treated by a reduction in amyloid plaque burden as measured by PET imaging.

[0711] Example 19. Phase 1 single-dose escalation study evaluating the safety, tolerability, immunogenicity, and pharmacokinetics of h2731.

[0712] This embodiment describes a phase 1, randomized, double-blind, placebo-controlled single-escalation-dose (SAD) study evaluating the safety, tolerability, and immunogenicity of h2731 at 70 mg and 200 mg doses. The aim of this study was to characterize the plasma pharmacokinetic (PK) profile of h2731 in healthy volunteers (HV) and patients with Alzheimer's disease (AD), particularly those with AD confirmed by molecular imaging to have substantial amyloid load. Participants with AD will be required to meet the study criteria and guidelines of the National Institute on Aging and Alzheimer's Association (NIA-AA) for AD (McKhann, 2011) or mild cognitive impairment (MCI) caused by AD (Albert, 2011).

[0713] Research rationality

[0714] Preclinical studies in transgenic mice that produce excessive Aβ have shown that antibodies targeting the N-terminus of Aβ can enter the brain and reduce amyloid deposition in brain tissue and the cerebral vascular system (Bard, 2000). Clinical evidence suggests that monoclonal antibodies targeting N-terminal amyloid can remove and reduce Aβ aggregate deposition from the brain and mitigate cognitive decline (Sevigny, 2016; Swanson, 2021; Aduhelm USPI, 2021).

[0715] As illustrated in the examples above, preclinical studies have shown that h2731 rapidly and robustly removes Aβ plaques by enhancing microglia-mediated clearance mechanisms. These data suggest that h2731 has the potential to slow the clinical decline in patients with AD. The 70 mg and 200 mg doses used in the Phase 1 clinical trial were based on fractional occupancy in the CNS, modeled from predictive clinical exposure at these doses.

[0716] Research Objective

[0717] The primary objective of this study was to evaluate the safety and tolerability of h2731 as a single dose, including (1) general safety, tolerability and immunogenicity in all subjects, and (2) target-related safety and tolerability in subjects with confirmed presence of amyloid.

[0718] The secondary objective of this study was to characterize the PK profile of h2731 and the PK profile of cerebrospinal fluid (CSF) following a single dose of h2731 administered as SC.

[0719] Research Design

[0720] Figure 35 This is a schematic diagram of the study plan. The study will include at least two dose cohorts of subjects with biologically confirmed AD. The study will further include two cohorts of healthy volunteers (“HV”). The four cohorts are as follows: AD cohort 1 (70 mg), AD cohort 2 (200 mg), HV cohort 1 (70 mg), and AD cohort 2 (200 mg), each of which will be administered subcutaneously (“SC”) at their respective doses.

[0721] Following initial field observation, subjects will return for four follow-up visits over approximately 12 weeks, during which safety assessments and pharmacokinetic (PK) collection will be completed. Selected cohorts may have additional CSF collection via lumbar puncture on days 3 and 29.

[0722] end

[0723] The primary endpoints will include:

[0724] • Safety and tolerability based on adverse event (“AE”) reports (incidence of AE, SAE, and h2731-related AEs), ECG, clinical laboratory tests, vital signs, and physical examinations.

[0725] • Immunogenicity was measured by confirming the presence of ADA in plasma.

[0726] • Amyloid-related imaging abnormalities (ARIA-H and ARIA-E) and other urgent radiological findings

[0727] Secondary endpoints may include

[0728] · h2731's blood plasma PK

[0729] · H2731's CSF PK

[0730] • C for h2731 at each sampling time obs

[0731] Inclusion criteria

[0732] Each cohort will contain approximately eight subjects with a body mass index (BMI) between 18.0 and 32.0 kg / m2.

[0733] AD subjects will be selected based on inclusion criteria including the following:

[0734] (a) Possible AD based on evidence of AD pathophysiological processes according to the criteria of the National Institute on Aging and Alzheimer's Association (NIA-AA) (McKhann et al., Alzheimers Dement., 7(3):264-9, 2011), or a high probability of having AD according to the NIA-AA criteria (Albert et al., Alzheimers Dement., 7(3):270-79, 2011), having confirmed AD or a suspected diagnosis of AD;

[0735] (b) Gradual and progressive changes in memory function reported by the subject or research peers over a period of ≥6 months;

[0736] (c) A Mini-Mental State Examination (MMSE) score ≥18; and

[0737] (d) Evidence of the pathological process of AD, such as amyloid PET scans.

[0738] Exclusion criteria

[0739] Subjects will be selected based on the following exclusion criteria. Subjects must not meet any exclusion criteria, including the following:

[0740] (a) Impaired coagulation (prothrombin time 1.2 × ULN) or other coagulation disorders

[0741] (b) A history of severe, clinically significant central nervous system (CNS) trauma (e.g., brain contusion) with persistent neurological deficits or structural brain injury, or a history of epilepsy.

[0742] (c) Any contraindications to MRI studies, including claustrophobia, the presence of contraindicated metallic (ferromagnetic) implants or pacemakers.

[0743] (d) Patients who have been taking anticoagulants for 3 months prior to screening, have no planned initiation of anticoagulants before randomization, or have a history of prolonged bleeding following minor trauma. Note: Low-dose aspirin (up to 162 mg / day) is permitted.

[0744] (e) A history of or presence of reversible posterior encephalopathy syndrome (PRES) (Fugate et al., Posterior reversible encephalopathy syndrome: clinical and radiological manifestations, pathophysiology, and outstanding questions. Lancet Neurol. 2015;14(9):914-25.)

[0745] Clinical laboratory evaluation

[0746] Laboratory analyses will be conducted on hematology, clinical biochemistry, coagulation, urine, plasma, biomarkers, and CSF. Pregnancy testing will be performed at both the center and locally.

[0747] Brain MRI scans will be read locally, and scans will be submitted to a centralized MRI provider for final MRI eligibility and central assessment of baseline ARIA findings. MRI should be performed using a 1.5 T or 3.0 T scanner, and the same scanner should be used for each individual subject throughout the study duration. The first MRI will be performed during the screening period as a baseline measurement confirming eligibility based on structural brain imaging. MRI scans will include, but are not limited to, the following sequence: T2-weighted FLAIR, two-dimensional (2D) T2*-weighted gradient echo (GRE), or susceptibility-weighted imaging (SWI); diffusion-weighted three-dimensional (3D) T1-weighted GRE. MRI scans will be evaluated and read by a central MRI reader who will provide diagnostic readings and assessments of MRI outcome measurements. MRI data (day 29 MRI data for all subjects, as well as any other available MRI and safety data) will be made available to the DSMB to confirm the h2731 dose.

[0748] If CSF analysis is performed, the cohort will undergo two LPs: the first LP on day 3 (48 hours after study drug administration) and the second LP on day 29. CSF will be analyzed to determine h2731 levels. CSF samples with clear evidence of blood contamination will not be used for PK assessment. CSF analysis will also include, but is not limited to, standard analyses, including pressure, color, glucose, protein, lactate, red blood cells, and white blood cells.

[0749] Amyloid PET imaging will be used for biological confirmation of the diagnosis of AD, providing evidence of its presence as a pathological marker of neuritis plaques composed of β-amyloid. Isotopically labeled compounds (tracers) showing high affinity for aggregated forms of β-amyloid can provide evidence of β-amyloid in vivo. Three radioligands are used for screening purposes: [18F]flubetaprazole / AV45 (Amyvid), [18F]flumethoxazole (Vizamyl), and [18F]flubetabenzene (Neuraceq). Subjects in cohorts 1–4 will undergo amyloid PET collection to biologically confirm the diagnosis of AD. Positive PET scans using [18F]flubetaprazole / AV45, [18F]flumethoxazole, or [18F]flubetabenzene obtained outside of this trial protocol within 18 months prior to the first screening visit may be allowed for confirmation via central readout to confirm patient inclusion.

[0750] APOE4 status (e.g., APOE4 / APOE4, APOE4 / APOE3, APOE3 / APOE3, APOE4 / APOE2, APOE3 / APOE2) will be determined by a central assessment of subjects enrolled in cohorts 1 through 4.

[0751] Additional evaluation

[0752] The Cogstate CBB (Maruff, 2013) is administered to subjects during the first screening visit (days -72 to -8). The CBB is a short (approximately 15 minutes), computer-based cognitive test suite designed to measure memory, working memory, psychomotor function, and attention. The CBB has been shown to be a sensitive tool for detecting cognitive decline associated with AD in healthy older adults and adults with amnestic mild cognitive impairment (Darby, 2002; Lim, 2013) and cognitive improvement resulting from cognitive enhancement drug therapy (Davison, 2011; Jaeger, 2011; Nathan, 2013).

[0753] Prior to the Cogstate CBB assessment, subjects will be administered MMSE (Folstein, 1975) during the first screening visit (days -72 to -8) to determine whether they meet the entry criteria for cognitive impairment.

[0754] Subjects will undergo plasma sampling for biomarkers of AD pathology (including but not limited to Aβ42 / 40) and biomarkers associated with tau pathology (including but not limited to total tau, p181-tau, and p217-tau).

[0755] Subjects will undergo PK sampling of plasma and CSF h2731.

[0756] Plasma anti-h2731 antibody levels will be measured (antibody detection will be performed using electrochemiluminescence immunoassay (ECLIA)).

[0757] ARIA Assessment: Screening MRI scans will be used to exclude subjects with pre-existing angioedema (ARIA-E), >4 microbleeds, or >1 area of ​​superficial siderosis (ARIA H). In addition to scheduled MRIs, investigators may decide to perform unscheduled MRIs if ARIA is suspected based on the presence of symptoms. All subjects will have MRIs scheduled before study drug administration (baseline), and subjects with AD will have visits on day 29 and day 85 (days 28 and 84 post-drug administration, respectively). Radiographic evidence of ARIA will be assessed, categorized, and recorded.

[0758] Example 20: A Phase I multiple-escalation dose study evaluating the safety, tolerability, immunogenicity, pharmacokinetics, and pharmacodynamics of h2721 in subjects with Alzheimer's disease.

[0759] This embodiment describes a phase 1, randomized, double-blind, placebo-controlled multiple escalation dose (MAD) study evaluating the safety, tolerability, immunogenicity, PK, and pharmacodynamic (PD) effects of h2731 in patients with Alzheimer's disease (AD). Figure 36 This is a schematic diagram of the research plan.

[0760] Research Objective

[0761] As discussed in further detail below, the primary objective of this study was to evaluate the safety, tolerability, and immunogenicity of h2731 after multiple SC doses. Secondary objectives included characterizing the pharmacokinetic profile of h2731 after multiple SC doses, characterizing the plasma and CSF pharmacokinetic profiles of h2731 after multiple SC doses, and assessing the PD effect of h2731 on cerebral amyloid plaque deposition after multiple SC doses. An exploratory objective of this study was to evaluate the PD effect of h2731 on blood and CSF biomarkers after multiple CS administrations and to assess ARIA findings using apolipoprotein E4 (APOE4) status.

[0762] Study population

[0763] The study consisted of two parts, each studying a different group of subjects: Group A consisted of AD subjects who were heterozygous for the apolipoprotein E4 (APOE4) allele or were non-carriers (referred to as the non-homozygous population), and Group B consisted of AD subjects who were homozygous for APOE4 (referred to as the homozygous population). The h2731 dose level to be evaluated was the same for both the non-homozygous (Group A) and homozygous (Group B) populations.

[0764] Apolipoprotein E (APOE) genotype status has been shown to influence the onset of Alzheimer's disease (AD) (Corder, 1993; van Duijn, 1994) and the incidence of acute rheumatic arterial disease (ARIA) following anti-Aβ antibody therapy (Arrighi, 2016; Ketter, 2017; Muralidharan, 2022). The development of ARIA is dose-dependent, and most events occur within the first few months after the initiation of anti-Aβ therapy (Muralidharan, 2022). AD patients with one copy of the APOE4 allele may have an elevated risk of anti-Aβ antibody-mediated ARIA compared to non-carriers, although the risk demonstrated in clinical trials is somewhat inconsistent. However, homozygous APOE4 patients (with two alleles) consistently show a higher incidence of ARIA events compared to both heterozygous and non-carrier patients.

[0765] Basic principles of dose selection

[0766] The proposed doses of 45 mg, 70 mg, and 200 mg are based on an analysis of estimated target engagement (relative occupancy [fOcc]) levels resulting from simulated clinical drug exposure at these doses, balancing predictions of effective exposure and the likelihood of inducing ARIA. These doses are further supported by results from completed non-clinical toxicity studies with repeated-dose administration over up to 3 months, and confirm certain information from the single-dose escalation (SAD) study based on Example 19, including:

[0767] • All safety and tolerability data up to day 15 from selected cohorts administered 70 mg and / or 200 mg;

[0768] • PK data up to day 29 from selected cohorts administered 70 mg and / or 200 mg (approximately 8 subjects per cohort; 6 receiving h2731 and 2 receiving placebo); and

[0769] • MRI results on day 29 from a selected cohort (approximately 12 subjects; 9 received h2731 and 3 received placebo) who were administered 70 mg and / or 200 mg.

[0770] Study Design

[0771] This phase 1, randomized, double-blind, placebo-controlled, escalation-dose study will be conducted in two dose cohorts in subjects with biologically confirmed AD to evaluate the safety, tolerability, immunogenicity, PK, and PD of h2731.

[0772] The study consisted of two parts, each evaluating a different group of participants: Group A consisted of AD participants who were heterozygous for the APOE4 allele or were non-carriers, and Group B consisted of AD participants who were homozygous for the APOE4 allele. The three dose cohorts for each group are described in Table 21.

[0773] Table 21

[0774]

[0775] AD subjects who are heterozygous for APOE4 or who are non-carriers of the APOE4 allele will be assigned to group A; subjects who are homozygous for APOE4 will be assigned to group B.

[0776] For each Group A cohort, approximately 32 participants will be randomly assigned in a 3:1 ratio to either the h2731 or placebo group: approximately 24 participants will receive h2731 and approximately 8 participants will receive placebo. Randomization will be stratified by APOE4 carrier status (APOE4 heterozygous or APOE4 non-carrier).

[0777] For each B cohort, approximately 12 participants will be randomly assigned in a 3:1 ratio to either the h2731 or placebo group: approximately 9 participants will receive h2731 and approximately 3 participa...

Claims

1. A method for treating Alzheimer's disease in a subject, comprising administering to the subject approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof approximately every 3-5 weeks.

2. A method for reducing amyloid plaques in a subject, comprising administering to the subject approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof approximately every 3-5 weeks.

3. A method for converting a subject from amyloid-positive to amyloid-negative, comprising administering to the subject approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody or its antigen-binding fragment every 3 to 5 weeks.

4. The method according to any one of claims 1 to 3, wherein the anti-amyloid β antibody or its antigen-binding fragment binds to an epitope located at the N-terminus of the Aβ peptide, and the epitope comprises at least one amino acid selected from amino acids 1-10 of the Aβ peptide.

5. The method of claim 4, wherein the anti-amyloid β antibody or its antigen-binding fragment binds to an epitope comprising at least one amino acid selected from amino acids 1-7 of the Aβ peptide.

6. The method according to any one of claims 1 to 5, wherein the anti-amyloid β antibody or its antigen-binding fragment binds to amyloid β at an apparent KD of about 5 nM or less. 1-42 Primary fibers are bound together.

7. The method according to any one of claims 1 to 6, wherein the anti-amyloid β antibody or its antigen-binding fragment binds to amyloid β at an apparent KD of about 1 nM or less. 1-42 Primary fibers are bound together.

8. The method according to any one of claims 1 to 7, wherein the anti-amyloid β antibody or its antigen-binding fragment binds to amyloid β at an apparent KD of about 10 nM or less. 1-28 Monomer combination.

9. The method according to any one of claims 1 to 8, wherein the method comprises administering about 20 mg to about 100 mg of the anti-amyloid β antibody or its antigen-binding fragment.

10. The method according to any one of claims 1 to 8, wherein the method comprises administering about 100 mg to about 200 mg of the anti-amyloid β antibody or its antigen-binding fragment.

11. The method according to any one of claims 1 to 8, wherein the method comprises administering about 45 mg of the anti-amyloid β antibody or its antigen-binding fragment.

12. The method according to any one of claims 1 to 8, wherein the method comprises administering about 70 mg of the anti-amyloid β antibody or its antigen-binding fragment.

13. The method according to any one of claims 1 to 8, wherein the method comprises administering about 200 mg of the anti-amyloid β antibody or its antigen-binding fragment.

14. The method according to any one of claims 1 to 13, wherein the anti-amyloid β antibody or its antigen-binding fragment is administered as a pharmaceutical composition comprising the anti-amyloid β antibody or its antigen-binding fragment and a pharmaceutically acceptable diluent.

15. The method according to any one of claims 1 to 14, wherein the anti-amyloid β antibody is administered approximately every 4 weeks.

16. The method according to any one of claims 1 to 15, wherein the administration is intravenous or subcutaneous.

17. The method of claim 16, wherein the application is subcutaneous.

18. The method according to any one of claims 1 to 17, wherein the anti-amyloid β antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3, and the light chain variable region comprising light chain CDR1, CDR2, and CDR3, wherein The heavy chain CDR1 contains an amino acid sequence of one of SEQ ID NO: 16, 19, or 20. The heavy chain CDR2 contains an amino acid sequence of one of SEQ ID NO: 20, 21, 22, or 23. The heavy chain CDR3 contains an amino acid sequence of one of SEQ ID NO: 18, 24, or 25. The light chain CDR1 contains an amino acid sequence of one of SEQ ID NO: 26, 29, 31, or 32. The light chain CDR2 contains an amino acid sequence of one of SEQ ID NO: 33, 34, 35, or 36, and The light chain CDR3 contains an amino acid sequence of one of SEQ ID NO: 28, 38 or 39.

19. The method of claim 18, wherein the anti-amyloid β antibody or its antigen-binding fragment comprises The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO:

16. The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO:

20. The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO:

18. The light chain CDR1 contains the amino acid sequence of SEQ ID NO:

29. The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 34, and The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

38.

20. The method of claim 19, wherein the heavy chain variable region excluding the CDR is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, and the light chain variable region excluding the CDR is at least 95% identical to the amino acid sequence of SEQ ID NO:

9.

21. The method of claim 19, wherein the heavy chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, and the light chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO:

9.

22. The method of claim 20, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

9.

23. The method of claim 22, wherein the heavy chain variable region is composed of SEQ ID NO: 3, and wherein the light chain variable region is composed of the amino acid sequence of SEQ ID NO:

9.

24. The method according to any one of claims 1 to 23, wherein the anti-amyloid β antibody is humanized IgG1.

25. The method according to any one of claims 1 to 24, wherein the anti-amyloid β antibody is a whole antibody, a chimeric antibody, a CDR transplantation antibody, or a recombinant antibody.

26. The method according to any one of claims 1 to 25, wherein the anti-amyloid β antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, the heavy chain constant region comprising at least 95% of the same amino acid sequence as SEQ ID NO: 40, and the light chain constant region comprising at least 95% of the same amino acid sequence as SEQ ID NO:

41.

27. The method according to any one of claims 1 to 26, wherein the anti-amyloid β antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, the heavy chain constant region comprising at least 98% of the same amino acid sequence as SEQ ID NO: 40, and the light chain constant region comprising at least 98% of the same amino acid sequence as SEQ ID NO:

41.

28. The method according to any one of claims 1 to 27, wherein the anti-amyloid β antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 40 with or without a C-terminal lysine, and the light chain constant region comprising the amino acid sequence of SEQ ID NO:

41.

29. The method according to any one of claims 1 to 28, wherein the anti-amyloid β antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region consisting substantially of the amino acid sequence of SEQ ID NO: 40 having or not having a C-terminal lysine, and the light chain constant region consisting substantially of the amino acid sequence of SEQ ID NO:

41.

30. The method according to any one of claims 1 to 29, wherein the anti-amyloid β antibody comprises a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

31. The method according to any one of claims 1 to 30, wherein the anti-amyloid β antibody is h2731.

32. A method for treating Alzheimer's disease in a subject, the method comprising administering, approximately every 4 weeks, a subcutaneous dose of approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody; said anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO:

102.

33. A method for treating Alzheimer's disease in a subject, the method comprising administering approximately 45 mg of an anti-amyloid β antibody subcutaneously to the subject approximately every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

34. A method for treating Alzheimer's disease in a subject, the method comprising administering approximately 70 mg of an anti-amyloid β antibody subcutaneously to the subject approximately every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

35. A method for treating Alzheimer's disease in a subject, the method comprising administering approximately 200 mg of an anti-amyloid β antibody subcutaneously to the subject approximately every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

36. A method for reducing amyloid plaques in a subject, the method comprising administering, approximately every 4 weeks, a subcutaneous dose of approximately 20 mg to approximately 200 mg of an anti-amyloid β antibody to the subject; said anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO:

102.

37. A method for reducing amyloid plaques in a subject, the method comprising administering approximately 45 mg of an anti-amyloid β antibody subcutaneously to the subject approximately every 4 weeks; said anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO:

102.

38. A method for reducing amyloid plaques in a subject, the method comprising administering approximately 70 mg of an anti-amyloid β antibody subcutaneously to the subject approximately every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

39. A method for reducing amyloid plaques in a subject, the method comprising administering approximately 200 mg of an anti-amyloid β antibody subcutaneously to the subject approximately every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

40. A method for treating Alzheimer's disease in a subject, comprising administering subcutaneous C to the subject sufficient to achieve a concentration of about 20 μg / mL to about 40 μg / mL. ave A dose of anti-Aβ antibody, the anti-Aβ antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

41. A method for treating Alzheimer's disease in a subject, comprising subcutaneously administering to the subject an AUC sufficient to achieve approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL. 0-tau A dose of anti-Aβ antibody, the anti-Aβ antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

42. The method according to any one of claims 1 to 41, wherein the maximum concentration (C0) of the anti-amyloid β antibody or its antigen-binding fragment in the subject during the dosing interval. max The concentration is approximately 30 µg / mL to approximately 60 µg / mL.

43. The method according to any one of claims 1 to 42, wherein the C-cell of the anti-amyloid β antibody or its antigen-binding fragment is present in the subject. max The value is approximately 50 µg / mL to approximately 60 µg / mL.

44. The method according to any one of claims 1 to 43, wherein the C-cell of the anti-amyloid β antibody or its antigen-binding fragment is present in the subject. max The value does not exceed approximately 60 µg / mL.

45. The method according to any one of claims 42 to 44, wherein C max The value is serum C max value.

46. ​​The method according to any one of claims 42 to 44, wherein C max The value is plasma C max value.

47. The method according to any one of claims 1 to 46, wherein the average concentration (C0) of the anti-amyloid β antibody or its antigen-binding fragment during the dosing interval. ave The value is approximately 20 µg / mL to approximately 40 µg / mL.

48. The method according to any one of claims 1 to 47, wherein the anti-amyloid β antibody or its antigen-binding fragment is present in the C of the subject. ave The value is approximately 30 µg / mL to approximately 40 µg / mL.

49. The method according to any one of claims 1 to 48, wherein the anti-amyloid β antibody or its antigen-binding fragment is present in the C of the subject. ave The value does not exceed approximately 40 µg / mL.

50. The method according to any one of claims 40 and 47 to 49, wherein the C ave The value is serum C ave value.

51. The method according to any one of claims 40 and 47 to 49, wherein C ave The value is plasma C ave value.

52. The method according to any one of claims 1 to 51, wherein the area under the concentration-time curve (AUC) of the dosing interval of the anti-amyloid β antibody or its antigen-binding fragment in the subject 0-tau The value is approximately 15,000 hr*ug / mL to approximately 30,000 hr*ug / mL.

53. The method according to any one of claims 1 to 52, wherein the AUC of the anti-amyloid β antibody or its antigen-binding fragment in the subject is... 0-tau The value is approximately 20,000 hr*ug / mL to approximately 30,000 hr*ug / mL.

54. The method according to any one of claims 1 to 53, wherein the AUC of the anti-amyloid β antibody or its antigen-binding fragment in the subject is... 0-tau The value does not exceed approximately 30,000 hr*ug / mL.

55. The method according to any one of claims 41 and 52 to 54, wherein the AUC 0-tau The value is the serum AUC. 0-tau value.

56. The method according to any one of claims 41 and 52 to 54, wherein the AUC 0-tau The value is the plasma AUC. 0-tau value.

57. The method according to any one of claims 1 to 56, wherein amyloid plaques in the subject are reduced.

58. The method according to any one of claims 2, 3 and 57, wherein the reduction of cerebral amyloid β plaques comprises a reduction of at least about 30 centiloids to about 70 centiloids.

59. The method according to any one of claims 3, 3 and 57, wherein the reduction of cerebral amyloid β plaques comprises a reduction of about 45 centiloids to about 80 centiloids.

60. The method according to any one of claims 2, 3 and 57, wherein the reduction of cerebral amyloid β plaques comprises a reduction of about 50 centiloids to about 85 centiloids.

61. The method according to any one of claims 2, 3 and 57, wherein the reduction of cerebral amyloid β plaques comprises a reduction of at least about 40% to about 90%.

62. The method according to any one of claims 2, 3 and 57, wherein the reduction of cerebral amyloid β plaques comprises a reduction of about 60% to about 100%.

63. The method according to any one of claims 2, 3 and 57, wherein the reduction of cerebral amyloid β plaques comprises a reduction of about 65% to about 100%.

64. The method according to any one of claims 2, 3 and 57 to 63, wherein the reduction of cerebral amyloid plaques is a reduction compared to baseline.

65. The method according to any one of claims 2, 3 and 57 to 64, wherein the reduction in cerebral amyloid β plaques is a reduction compared to the subject before administration of the anti-amyloid β antibody.

66. The method according to any one of claims 2, 3 and 57 to 65, wherein the reduction of cerebral amyloid β plaques is achieved after 6 months of treatment.

67. The method according to any one of claims 2, 3 and 57 to 65, wherein the reduction of cerebral amyloid β plaques is achieved after about 12 months of treatment.

68. The method according to any one of claims 2, 3 and 57 to 65, wherein the reduction of cerebral amyloid β plaques is achieved after about 18 months of treatment.

69. The method according to any one of claims 2, 3 and 57 to 68, wherein the reduction of cerebral amyloid β plaques is assessed by positron emission tomography (PET).

70. The method according to any one of claims 1 to 69, wherein the subject is switched from amyloid-positive to amyloid-negative.

71. The method according to any one of claims 1 to 70, wherein the treatment comprises increasing the probability of converting the subject from amyloid-positive to amyloid-negative.

72. The method according to any one of claims 1 to 71, wherein the treatment comprises making the probability of converting the subject from amyloid positive to amyloid negative about 10% to about 40%.

73. The method according to any one of claims 1 to 71, wherein the treatment comprises making the probability of converting the subject from amyloid positive to amyloid negative about 30% to about 60%.

74. The method according to any one of claims 1 to 71, wherein the treatment comprises making the probability of converting the subject from amyloid positive to amyloid negative about 40% to about 80%.

75. The method according to any one of claims 72 to 74, wherein the probability of the subject changing from amyloid-positive to amyloid-negative is the probability after about 6 months of treatment.

76. The method according to any one of claims 72 to 74, wherein the probability of the subject changing from amyloid-positive to amyloid-negative is the probability after about 12 months of treatment.

77. The method according to any one of claims 72 to 74, wherein the probability of the subject changing from amyloid-positive to amyloid-negative is the probability after about 18 months of treatment.

78. The method according to any one of claims 1 to 77, wherein the treatment comprises slowing, stopping, or reversing the decline in cognitive function.

79. The method of claim 78, wherein the treatment comprises slowing the decline in cognitive function.

80. The method of claim 78 or 80, wherein cognitive function is measured by at least one of the following: CRD-SB, ADAS-Cog14, ADCOMS, and ADCS MCI-ADL.

81. The method of claim 80, wherein cognitive function is measured by ADCOMS.

82. A method for modulating biomarkers in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody about every 3-5 weeks, said anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO:

102.

83. A method for increasing the Aβ 42 / 40 ratio in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody about every 3-5 weeks, said anti-amyloid β antibody comprising a heavy chain having or not having a C-terminal lysine of SEQ ID NO: 101, and a light chain of SEQ ID NO:

102.

84. A method for reducing the amount of phosphorylated-tau in a subject, comprising administering to the subject about 20 mg to about 200 mg of an anti-amyloid β antibody about every 3 to 5 weeks, said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

85. The method according to any one of claims 82 to 84, wherein the application comprises subcutaneous injection.

86. The method according to any one of claims 1 to 85, wherein the biomarkers in the subject are modulated.

87. The method of claim 86, wherein the biomarker in the subject is adjusted compared to baseline.

88. The method according to any one of claims 1 to 87, wherein the method further comprises detecting biomarkers in samples collected from the subject.

89. The method according to any one of claims 1 to 88, wherein the method further comprises quantifying biomarkers in samples collected from the subject.

90. The method according to any one of claims 82 and 86 to 89, wherein the biomarker comprises the ratio of Aβ 42 / 40 in the subject.

91. The method according to any one of claims 83 and 90, wherein the ratio of Aβ 42 / 40 in the subject is [missing information].

92. The method according to any one of claims 83 and 90, wherein the ratio of Aβ 42 / 40 in the subject is increased by at least 25%.

93. The method according to any one of claims 83 and 90, wherein the ratio of Aβ 42 / 40 in the subject is increased by at least 50%.

94. The method according to any one of claims 83 and 90, wherein the ratio of Aβ 42 / 40 in the subject is increased by about 25% to about 100%.

95. The method according to any one of claims 83 and 90, wherein the ratio of Aβ 42 / 40 in the subject is increased by about 50% to about 100%.

96. The method according to any one of claims 82 and 86 to 89, wherein the biomarker comprises a phosphorylated tau value.

97. The method according to any one of claims 84 and 96, wherein the phosphorylation-tau value comprises at least one of the following: p181-tau value, p212-tau value, p217-tau value, p231-tau value, and p235-tau value.

98. The method according to any one of claims 84 and 96, wherein the phosphorylation-tau value includes the p181-tau value.

99. The method according to any one of claims 84 and 96, wherein the phosphorylation-tau value includes the p212-tau value.

100. The method according to any one of claims 84 and 96, wherein the phosphorylation-tau value includes the p217-tau value.

101. The method according to any one of claims 84 and 96, wherein the phosphorylation-tau value includes the p231-tau value.

102. The method according to any one of claims 84 and 96, wherein the phosphorylation-tau value includes the p235-tau value.

103. The method according to any one of claims 84 and 96 to 102, wherein the phosphorylation-tau value is reduced.

104. The method of claim 103, wherein the phosphorylation-tau value is reduced by at least about 10%.

105. The method according to any one of claims 84 and 103, wherein the phosphorylation-tau value is reduced by about 10% to about 30%.

106. The method according to any one of claims 84 and 103, wherein the phosphorylation-tau value is reduced by about 20% to about 30%.

107. The method according to any one of claims 89 and 90, wherein the sample comprises blood or a portion thereof collected from the subject.

108. The method according to any one of claims 89 and 90, wherein the sample comprises plasma collected from the subject.

109. The method according to any one of claims 89 and 90, wherein the sample comprises serum collected from the subject.

110. The method of any one of claims 89 and 90, wherein the sample comprises cerebrospinal fluid ("CSF") collected from the subject.

111. The method according to any one of claims 1 to 110, wherein the method includes a risk of less than about 45% ARIA-E.

112. The method according to any one of claims 1 to 110, wherein the method includes about 25% to about 45% of the risk of ARIA-E.

113. The method according to any one of claims 1 to 110, wherein the method includes a risk of less than about 75% ARIA-E.

114. The method according to any one of claims 1 to 110, wherein the method includes about 50% to about 75% of the risk of ARIA-E.

115. The method according to any one of claims 1 to 114, wherein the method includes a risk of less than about 15% for symptomatic ARIA-E.

116. The method according to any one of claims 1 to 114, wherein the method includes a risk of less than about 30% for symptomatic ARIA-E.

117. The method according to any one of claims 111 to 116, wherein the risk of said ARIA-E is the risk of severe ARIA-E.

118. The method according to any one of claims 111 to 117, wherein the risk of ARIA-E is the risk following approximately 6 months of treatment.

119. The method according to any one of claims 111 to 117, wherein the risk of ARIA-E is the risk following treatment for about 12 months.

120. The method according to any one of claims 111 to 1117, wherein the risk of ARIA-E is the risk following treatment for approximately 18 months.

121. The method according to any one of claims 1 to 117, wherein the subject does not experience symptomatic ARIA-E during treatment.

122. The method according to any one of claims 1 to 121, wherein the method includes a risk of less than about 35% ARIA-H.

123. The method according to any one of claims 1 to 121, wherein the method includes a risk of about 10% to about 35% of ARIA-H.

124. The method of claim 112 or 123, wherein the risk of ARIA-H is the risk of severe ARIA-H.

125. The method according to any one of claims 123 to 124, wherein the risk of ARIA-H is the risk following approximately 6 months of treatment.

126. The method according to any one of claims 1 to 125, wherein the subject does not experience symptomatic ARIA-H during treatment.

127. The method of any one of claims 111 to 126, wherein ARIA is evaluated by magnetic resonance imaging ("MRI").

128. The method according to any one of claims 1 to 127, wherein the subject is an APOE4 homozygous subject.

129. The method according to any one of claims 1 to 127, wherein the subject is an APOE4 heterozygous subject or an APOE4 non-carrier.

130. The method according to any one of claims 1 to 129, wherein the method further comprises determining the subject's APOE4 status prior to administration.

131. The method according to any one of claims 1 to 130, wherein the duration of the treatment is at least 6 months.

132. The method according to any one of claims 1 to 130, wherein the duration of said treatment is at least 12 months.

133. The method according to any one of claims 1 to 130, wherein the duration of the treatment is at least 18 months.

134. The method according to any one of claims 1 to 133, wherein the application is performed using a syringe.

135. The method according to any one of claims 1 to 133, wherein the application is performed using an autoinjector.

136. The method according to any one of claims 1 to 135, wherein the subject is a mammal.

137. The method according to any one of claims 1 to 136, wherein the subject is a human.

138. A pharmaceutical composition comprising an anti-amyloid β antibody or an antigen-binding fragment thereof, for treating a subject with Alzheimer's disease by administering about 20 mg to about 200 mg of the antibody or an antigen-binding fragment thereof to the subject once every 3-5 weeks.

139. A pharmaceutical composition comprising an anti-amyloid β antibody or an antigen-binding fragment thereof, for use in reducing amyloid plaques in a subject by administering about 20 mg to about 200 mg of an anti-amyloid β antibody or an antigen-binding fragment thereof to the subject once every 3 to 5 weeks.

140. A pharmaceutical composition comprising an anti-amyloid β antibody or an antigen-binding fragment thereof, for converting a subject from amyloid-positive to amyloid-negative by administering about 20 mg to about 200 mg of the anti-amyloid β antibody or an antigen-binding fragment thereof to the subject once every 3-5 weeks.

141. Use of an anti-amyloid β antibody or antigen-binding fragment in the preparation of a medicament for treating a subject with Alzheimer's disease, wherein the medicament is administered to the subject at a dose of about 20 mg to about 200 mg of the anti-amyloid β antibody or antigen-binding fragment approximately every 3-5 weeks.

142. Use of an anti-amyloid β antibody or antigen-binding fragment in the preparation of a medicament for reducing amyloid plaques in a subject by administering about 20 mg to about 200 mg of an anti-amyloid β antibody or antigen-binding fragment thereof to the subject once every 3-5 weeks.

143. Use of an anti-amyloid β antibody or antigen-binding fragment in the preparation of a medicament for converting a subject from amyloid-positive to amyloid-negative by administering about 20 mg to about 200 mg of an anti-amyloid β antibody or antigen-binding fragment thereof to the subject once every 3-5 weeks.

144. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the anti-amyloid β antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3, and the light chain variable region comprising light chain CDR1, CDR2, and CDR3, wherein The heavy chain CDR1 contains an amino acid sequence of one of SEQ ID NO: 16, 19, or 20. The heavy chain CDR2 contains an amino acid sequence of one of SEQ ID NO: 20, 21, 22, or 23. The heavy chain CDR3 contains an amino acid sequence of one of SEQ ID NO: 18, 24, or 25. The light chain CDR1 contains an amino acid sequence of one of SEQ ID NO: 26, 29, 31, or 32. The light chain CDR2 contains an amino acid sequence of one of SEQ ID NO: 33, 34, 35, or 36, and The light chain CDR3 contains an amino acid sequence of one of SEQ ID NO: 28, 38 or 39.

145. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the anti-amyloid β antibody or its antigen-binding fragment comprises The heavy chain CDR1 contains the amino acid sequence of SEQ ID NO:

16. The heavy chain CDR2 contains the amino acid sequence of SEQ ID NO:

20. The heavy chain CDR3 contains the amino acid sequence of SEQ ID NO:

18. The light chain CDR1 contains the amino acid sequence of SEQ ID NO:

29. The light chain CDR2 contains the amino acid sequence of SEQ ID NO: 34, and The light chain CDR3 contains the amino acid sequence of SEQ ID NO:

38.

146. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the heavy chain variable region excluding the CDR is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, and the light chain variable region excluding the CDR is at least 95% identical to the amino acid sequence of SEQ ID NO:

9.

147. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the heavy chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, and the light chain variable region excluding the CDR is at least 98% identical to the amino acid sequence of SEQ ID NO:

9.

148. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

9.

149. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the heavy chain variable region is composed of SEQ ID NO: 3, and wherein the light chain variable region is composed of the amino acid sequence of SEQ ID NO:

9.

150. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the anti-amyloid β antibody is humanized IgG1.

151. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the anti-amyloid β antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region, the heavy chain constant region comprising at least 98% of the same amino acid sequence as SEQ ID NO: 40, and the light chain constant region comprising at least 98% of the same amino acid sequence as SEQ ID NO:

41.

152. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the anti-amyloid β antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 40 with or without a C-terminal lysine, and the light chain constant region comprising the amino acid sequence of SEQ ID NO:

41.

153. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the anti-amyloid β antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region consisting substantially of the amino acid sequence of SEQ ID NO: 40 having or not having a C-terminal lysine, and the light chain constant region consisting substantially of the amino acid sequence of SEQ ID NO:

41.

154. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the anti-amyloid β antibody comprises a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

155. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the anti-amyloid β antibody is h2731.

156. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the administration comprises subcutaneous administration of about 20 mg to about 200 mg of an anti-amyloid β antibody to the subject about once every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

157. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the administration comprises subcutaneous administration of about 45 mg of an anti-amyloid β antibody to the subject about once every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

158. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the administration comprises subcutaneous administration of about 70 mg of an anti-amyloid β antibody to the subject about once every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

159. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the administration comprises subcutaneously administering about 200 mg of an anti-amyloid β antibody to the subject about once every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

160. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the administration comprises subcutaneous administration of about 20 mg to about 200 mg of an anti-amyloid β antibody to the subject about once every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

161. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the administration comprises subcutaneously administering about 45 mg of an anti-amyloid β antibody to the subject about once every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

162. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the administration comprises subcutaneously administering about 70 mg of an anti-amyloid β antibody to the subject about once every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO:

102.

163. The pharmaceutical composition according to any one of claims 138-140 or the use according to any one of claims 141-143, wherein the administration comprises subcutaneously administering about 200 mg of an anti-amyloid β antibody to the subject about once every 4 weeks; said anti-amyloid β antibody comprising a heavy chain of SEQ ID NO: 101 having or not having a C-terminal lysine, and a light chain of SEQ ID NO: 102.

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