Neutralizing anti-amyloid beta antibodies for the treatment of Alzheimer's disease

Abstract

Novel binding polypeptides (e.g., antibodies and antigen-binding fragments thereof) are provided that specifically bind to one or more soluble, synaptotoxic amyloid beta (Aβ) derived from AD brains without binding to classical monomeric, prefibrillar, or fibrillar Aβ. Pharmaceutical compositions comprising binding polypeptides that specifically bind to one or more soluble, synaptotoxic Aβ are provided. Methods for producing binding polypeptides that specifically bind to one or more soluble, synaptotoxic Aβ are provided. Methods for treating Alzheimer's disease using binding polypeptides that specifically bind to one or more soluble, synaptotoxic Aβ are provided. Methods for reducing one or more symptoms of Alzheimer's disease using binding polypeptides that specifically bind to one or more soluble, synaptotoxic Aβ are provided.

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 874,724, filed Jul. 16, 2019, the contents of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Field of the Invention The present invention relates to novel binding polypeptides that specifically bind to soluble amyloid beta. The present invention also relates to methods of treating Alzheimer's disease using the novel binding polypeptides provided herein.

Background Art

[0003] Background Alzheimer's disease is the most common form of dementia. Tens of millions of people worldwide are affected, and this number is increasing dramatically. The amyloid hypothesis (Non-Patent Document 1; Non-Patent Document 2; and Non-Patent Document 3) proposes amyloid beta (Aβ) as the main cause of this disease, and the misfolding of extracellular Aβ protein (Non-Patent Document 4) accumulated in senile plaques and the intracellular deposition of misfolded tau protein in neurofibrillary tangles suggest that memory loss and abnormal executive function are caused, and cognitive and behavioral decline occur over time. The accumulated Aβ peptide is the main component of senile (amyloid) plaques and is derived from proteolytic cleavage of a larger glycoprotein called amyloid precursor protein (APP) (Non-Patent Document 5; Non-Patent Document 6).

[0004] Aβ monomers aggregate to form various types of aggregates, including oligomers, protofibrils, and amyloid fibrils. Amyloid fibrils are large and insoluble, and may further aggregate to form amyloid plaques, while amyloid oligomers are soluble in water and can spread throughout the brain. Aβ encompasses a group of peptides ranging in size from 37 to 49 residues. Amyloid plaques containing Aβ as the main component are most commonly found in the marginal cortex and neocortex in the brains of Alzheimer's disease patients. Aβ(1-42) is the major proteinaceous component of amyloid deposition in Alzheimer's disease.

[0005] Amyloid fibrils are larger and insoluble, and aggregate to form fibrous amyloid plaques that are characteristic histological lesions in Alzheimer's disease, while Aβ oligomers are soluble and can diffuse throughout the brain. The size distribution of Aβ oligomers is heterogeneous. There is a broad consensus on the preferential accumulation of soluble high molecular weight chemical species of approximately 100 - 200 kDa under relatively physiological conditions in vitro (Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11). Aβ monomers can form higher-order aggregates ranging from low molecular weight oligomers including dimers, trimers, and tetramers, to medium molecular weight oligomers including hexamers and decamers, to soluble protofibrils and insoluble fibrils (Chen et al., supra).

[0006] Approaches using monoclonal antibodies targeting amyloid β-protein (Aβ) constitute the largest and most advanced therapeutic efforts to treat Alzheimer's disease (AD) (Non-Patent Document 12; Non-Patent Document 13; Non-Patent Document 14). Despite generally good outcomes in preclinical mouse models, anti-Aβ immunotherapy has had limited success in humans (Golde, van Dyck, supra). Explanations offered to account for the inadequate translation of preclinical lead antibodies to human therapy include complete trial design, intervention at the disease stage when significant neurological deficits are already present, and inappropriate target selectivity of the antibodies used (Golde, supra; Non-Patent Document 15; Selkoe and Hardy, supra).

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

[0008] Therefore, there remains a need for alternative therapeutic monoclonal antibodies for the treatment of Alzheimer's disease. [[Means for Solving the Problems]]

[0009] [[Abstract]] The present invention is based on the discovery of a novel anti - amyloid beta (Aβ) binding protein (e.g., an antibody) that specifically binds to one or more epitopes of water - soluble AD brain - derived synaptotoxic Aβ.

[0010] Accordingly, in certain embodiments, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ, wherein the binding polypeptide comprises three heavy - chain complementarity - determining region (HCDR) sequences and three light - chain complementarity - determining region (LCDR) sequences. In certain embodiments, the three HCDR sequences are selected from the group consisting of SEQ ID NOs: 20, 21, 22, 26, 27, 28, 32, 33, 34, 38, 39, 40, 44, 45, 46, 50, 51, 52, 56, 57, 58, 62, 63, and 64, and the three LCDR sequences are selected from the group consisting of SEQ ID NOs: 17, 18, 19, 23, 24, 25, 29, 30, 31, 35, 36, 37, 41, 42, 43, 47, 48, 49, 53, 54, 55, 59, 60, and 61.

[0011] In certain exemplary embodiments, soluble Aβ is synaptotoxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptotoxicity. In certain exemplary embodiments, soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0012] In certain exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, protofibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptotoxicity of soluble Aβ.

[0013] In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ, wherein the binding polypeptide comprises a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence. In certain embodiments, the HCVR sequence is selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, and the LCVR sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15.

[0014] In certain exemplary embodiments, soluble Aβ is synaptotoxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptotoxicity. In certain exemplary embodiments, soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0015] In certain exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, pro-fibrillar Aβ or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0016] In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ, wherein the binding polypeptide comprises an HCVR / LCVR sequence pair. In certain embodiments, the HCVR / LCVR pair is selected from the group consisting of SEQ ID NO: 2 / 1, 4 / 3, 6 / 5, 8 / 7, 10 / 9, 12 / 11, 14 / 13 and 16 / 15.

[0017] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0018] In certain exemplary embodiments, the binding polypeptide does not specifically bind monomeric Aβ, pro-fibrillar Aβ or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunoadsorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0019] In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the HCDR sequences of SEQ ID NOs: 20, 21, and 22, and the LCDR sequences of SEQ ID NOs: 17, 18, and 19. In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the HCVR / LCVR pair of SEQ ID NOs: 2 and 1. In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the complete heavy chain / light chain sequence pair of SEQ ID NOs: 66 and 65.

[0020] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0021] In certain exemplary embodiments, the binding polypeptide does not specifically bind monomeric Aβ, pro-fibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds soluble Aβ from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0022] In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and that comprises the HCDR sequences of SEQ ID NOs: 26, 27, and 28 and the LCDR sequences of SEQ ID NOs: 23, 24, and 25. In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and that comprises the HCVR / LCVR pair of SEQ ID NOs: 4 and 3. In certain aspects, there is provided an isolated polypeptide that specifically binds soluble Aβ and that comprises the full heavy / light chain sequences of SEQ ID NOs: 68 and 67.

[0023] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0024] In certain exemplary embodiments, the binding polypeptide does not specifically bind monomeric Aβ, pro-fibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0025] In certain aspects, provided is a binding polypeptide that specifically binds to soluble Aβ and comprises the HCDR sequences of SEQ ID NOs: 32, 33, and 34, and the LCDR sequences of SEQ ID NOs: 29, 30, and 31. In certain aspects, provided is an isolated binding polypeptide that specifically binds to soluble Aβ and comprises the HCVR / LCVR pair of SEQ ID NOs: 6 and 5. In certain aspects, provided is an isolated binding polypeptide that specifically binds to soluble Aβ and comprises the complete heavy chain / light chain sequence pair of SEQ ID NOs: 70 and 69.

[0026] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0027] In certain exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, pro-fibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0028] In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the HCDR sequences of SEQ ID NOs: 38, 39, and 40 and the LCDR sequences of SEQ ID NOs: 35, 36, and 37. In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the HCVR / LCVR pair of SEQ ID NOs: 8 and 7. In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the complete heavy / light chain sequences of SEQ ID NOs: 72 and 71.

[0029] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0030] In certain exemplary embodiments, the binding polypeptide does not specifically bind monomeric Aβ, pro-fibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds soluble Aβ from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0031] In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and comprises the HCDR sequences of SEQ ID NOs: 44, 45, and 46 and the LCDR sequences of SEQ ID NOs: 41, 42, and 43. In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and comprises the HCVR / LCVR pair of SEQ ID NOs: 10 and 9. In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and comprises the complete heavy chain / light chain sequence pair of SEQ ID NOs: 74 and 73.

[0032] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0033] In certain exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, pro-fibrillar Aβ or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunoadsorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0034] In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the HCDR sequences of SEQ ID NOs: 50, 51 and 52, and the LCDR sequences of SEQ ID NOs: 47, 48 and 49. In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the HCVR / LCVR pair of SEQ ID NOs: 12 and 11. In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble Aβ and that comprises the full heavy chain / light chain sequence pair of SEQ ID NOs: 76 and 75.

[0035] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0036] In certain exemplary embodiments, the binding polypeptide does not specifically bind monomeric Aβ, pro-fibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds soluble Aβ from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0037] In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and that comprises the HCDR sequences of SEQ ID NOs: 56, 57, and 58, and the LCDR sequences of SEQ ID NOs: 53, 54, and 55. In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and that comprises the HCVR / LCVR pair of SEQ ID NOs: 14 and 13. In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and that comprises the complete heavy chain / light chain sequence pair of SEQ ID NOs: 78 and 77.

[0038] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0039] In certain exemplary embodiments, the binding polypeptide does not specifically bind monomeric Aβ, pro-fibrillar Aβ or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0040] In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and that comprises the HCDR sequences of SEQ ID NOs: 62, 63 and 64, and the LCDR sequences of SEQ ID NOs: 59, 60 and 61. In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and that comprises the HCVR / LCVR pair of SEQ ID NOs: 16 and 15. In certain aspects, there is provided an isolated binding polypeptide that specifically binds soluble Aβ and that comprises the full heavy chain / light chain sequence pair of SEQ ID NOs: 80 and 79.

[0041] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0042] In certain exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, pro-fibrillar Aβ or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0043] In certain aspects, there is provided an isolated binding polypeptide that specifically binds to soluble amyloid beta (Aβ), wherein the binding polypeptide comprises three heavy chain complementarity determining region (HCDR) sequences and light chain complementarity determining region (LCDR) sequences, and the three HCDR sequences are selected from the group consisting of SEQ ID NOs: 20, 21, 22, 50, 51, 52, 44, 45, 46, 26, 27, 28, 62, 63, and 64, and the three LCDR sequences are selected from the group consisting of SEQ ID NOs: 17, 18, 19, 47, 48, 49, 41, 42, 43, 23, 24, 25, 59, 60 and 61.

[0044] In certain exemplary embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof, which is optionally a human antibody and / or optionally IgG1.

[0045] In certain exemplary embodiments, the binding polypeptide comprises a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the group consisting of SEQ ID NOs: 2 and 1; SEQ ID NOs: 10 and 9; SEQ ID NOs: 4 and 3; SEQ ID NOs: 12 and 11; and SEQ ID NOs: 16 and 15.

[0046] In certain exemplary embodiments, the three HCDR sequences comprise SEQ ID NOs: 20, 21, and 22, and the three LCDR sequences comprise SEQ ID NOs: 17, 18, and 19. In certain exemplary embodiments, the three HCDR sequences comprise SEQ ID NOs: 44, 45, and 46, and the three LCDR sequences comprise SEQ ID NOs: 41, 42, and 43. In certain exemplary embodiments, the three HCDR sequences comprise SEQ ID NOs: 26, 27, and 28, and the three LCDR sequences comprise SEQ ID NOs: 23, 24, and 25. In certain exemplary embodiments, the three HCDR sequences comprise SEQ ID NOs: 50, 51, and 52, and the three LCDR sequences comprise SEQ ID NOs: 47, 48, and 49. In certain exemplary embodiments, the three HCDR sequences comprise SEQ ID NOs: 62, 63, and 64, and the three LCDR sequences comprise SEQ ID NOs: 59, 60, and 61.

[0047] In certain exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 2 and SEQ ID NO: 1. In certain exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 10 and SEQ ID NO: 9. In certain exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 4 and SEQ ID NO: 3. In certain exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 12 and SEQ ID NO: 11. In certain exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 16 and SEQ ID NO: 15.

[0048] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0049] In certain exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, pro-fibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0050] In certain aspects, there is provided a pharmaceutical composition comprising an isolated binding polypeptide that specifically binds to soluble amyloid beta (Aβ) [wherein the binding polypeptide comprises three heavy chain complementarity determining region (HCDR) sequences and three light chain complementarity determining region (LCDR) sequences, wherein the three HCDR sequences are selected from the group consisting of SEQ ID NOs: 20, 21, 22, 50, 51, 52, 44, 45, 46, 26, 27, 28, 62, 63, and 64, and the three LCDR sequences are selected from the group consisting of SEQ ID NOs: 17, 18, 19, 47, 48, 49, 41, 42, 43, 23, 24, 25, 59, 60, and 61], and a pharmaceutically acceptable carrier.

[0051] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0052] In certain exemplary embodiments, the binding polypeptide does not specifically bind monomeric Aβ, pro-fibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0053] In certain exemplary embodiments, provided is a method of treating Alzheimer's disease in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition.

[0054] In certain aspects, provided is an isolated binding polypeptide that specifically binds soluble amyloid beta (Aβ) [wherein the binding polypeptide comprises a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the group consisting of SEQ ID NO: 2 and 1; SEQ ID NO: 10 and 9; SEQ ID NO: 4 and 3; SEQ ID NO: 12 and 11; and SEQ ID NO: 16 and 15]; and a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0055] In certain exemplary embodiments, the soluble Aβ is synaptically toxic. In certain exemplary embodiments, the binding polypeptide neutralizes Aβ synaptic toxicity. In certain exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0056] In certain exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, pro-fibrillar Aβ, or fibrillar Aβ. In certain exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In certain exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease. In certain exemplary embodiments, the binding is immunosorption. In certain exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject having Alzheimer's disease. In certain exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of soluble Aβ.

[0057] In certain exemplary embodiments, provided is a method of treating Alzheimer's disease in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition.

[0058] In certain aspects, provided is an isolated polynucleotide encoding a binding polypeptide that specifically binds to soluble amyloid beta (Aβ), wherein the binding polypeptide comprises three heavy chain complementarity determining region (HCDR) sequences and three light chain complementarity determining region (LCDR) sequences, the three HCDR sequences are selected from the group consisting of SEQ ID NOs: 20, 21, 22, 50, 51, 52, 44, 45, 46, 26, 27, 28, 62, 63, and 64, and the three LCDR sequences are selected from the group consisting of SEQ ID NOs: 17, 18, 19, 47, 48, 49, 41, 42, 43, 23, 24, 25, 59, 60, and 61, and a pharmaceutically acceptable carrier.

[0059] In certain exemplary embodiments, provided is a vector encoding the polynucleotide.

[0060] In certain exemplary embodiments, provided is a host cell comprising the polynucleotide or the vector.

[0061] Brief Description of the Drawings The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of exemplary embodiments when interpreted in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0062]

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[0063] Detailed Description The present disclosure provides novel binding polypeptides (e.g., antibodies) that bind to epitopes present in one or more of soluble synaptic toxic Aβ. Importantly, the novel binding polypeptides described herein have reduced binding to Aβ, pro-fibrillar Aβ, and / or fibrillar Aβ compared to antibodies known in the art, or do not bind to monomeric Aβ, pro-fibrillar Aβ, and / or fibrillar Aβ.

[0064] In certain embodiments, the novel binding polypeptides described herein prevent or reduce the formation of higher-order synaptic toxic forms of Aβ, such as monomeric Aβ, pro-fibrillar Aβ, and fibrillar Aβ. In certain embodiments, the novel binding polypeptides described herein prevent or reduce soluble synaptic toxic Aβ from forming higher-order synaptic toxic forms of Aβ, such as monomeric Aβ, pro-fibrillar Aβ, and / or fibrillar Aβ.

[0065] Of course, the methods described in the present disclosure are not limited to the specific methods and experimental conditions disclosed herein, as the methods and experimental conditions can vary. Of course, the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0066] Furthermore, the experiments described herein use conventional molecular and cell biological and immunological techniques within the skill of the art, unless otherwise indicated. Such techniques are well known to those of skill in the art and are described in sufficient detail in the literature. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008) (including all supplements), Molecular Cloning: A Laboratory Manual (Fourth Edition) by M.R. Green and J. Sambrook, and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (Second Edition, 2013).

[0067] Unless defined otherwise, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In the case of any potential ambiguity, the definitions provided herein will prevail over any dictionary or external definition. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Unless otherwise stated, "or" means "and / or". The use of other forms such as "including" and "included" in addition to the term "comprising" is not limiting.

[0068] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, described herein is well known and widely used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more detailed references cited and considered throughout this specification unless otherwise indicated. Enzyme reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry, as well as their laboratory procedures and techniques, are well known in the art and are commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceuticals, formulation, and delivery, and for the treatment of patients.

[0069] Thus, the present disclosure may be more readily understood, and selected terms are defined below.

[0070] The term "polypeptide" refers to any polymer chain of amino acids and includes, where the context is not inconsistent therewith, natural or artificial proteins, polypeptide analogs or variants of protein sequences, or fragments thereof. A polypeptide may be monomeric or multimeric. A polypeptide fragment includes, for example, at least about 5 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, or at least about 20 contiguous amino acids.

[0071] The terms "isolated protein" or "isolated polypeptide" refer to a protein or polypeptide that, by virtue of its origin or source of derivation, is without the natural associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by cells from a different species; or does not occur naturally. Thus, a protein or polypeptide that is chemically synthesized or synthesized in a cell line different from the cell from which it is naturally derived will be "isolated" from its natural associated components. A protein or polypeptide can be rendered substantially free of its natural associated components by isolation using protein purification techniques well known in the art.

[0072] As used herein, the terms "binding protein" or "binding polypeptide" shall refer to a protein or polypeptide (e.g., an antibody or an immunoadhesin) that contains at least one binding site responsible for selective binding to a target antigen of interest (e.g., a human target antigen). Exemplary binding sites include antibody variable domains, receptor ligand-binding sites, or ligand receptor-binding sites. In certain embodiments, the binding protein or binding polypeptide comprises multiple (e.g., two, three, four or more) binding sites. In certain embodiments, the binding protein or binding polypeptide is not a therapeutic enzyme.

[0073] The term "ligand" refers to any substance that can bind or be bound to another substance. Similarly, the term "antigen" refers to any substance against which an antibody can be generated. "Antigen" is generally used with respect to antibody-binding substances, and "ligand" is often used when referring to receptor-binding substances, but these terms are not distinguished from each other and encompass a broad range of overlapping chemical entities. To avoid doubt, antigens and ligands are used interchangeably throughout this specification. Antigen / ligand can be a peptide, polypeptide, protein, aptamer, polysaccharide, sugar molecule, carbohydrate, lipid, oligonucleotide, polynucleotide, synthetic molecule, inorganic molecule, organic molecule, and any combination thereof.

[0074] As used herein, the term "specifically binds" refers to the ability of an antibody or immunoadhesin to bind an antigen with a dissociation constant (Kd) of at most about 1x10 -6 M, about 1x10 -7 M, about 1x10 -8 M, about 1x10 -9 M, about 1x10 -10 M, about 1x10 -11 M, about 1x10 -12 M or less, and / or the ability of an antibody or immunoadhesin to bind an antigen with an affinity that is at least about 2-fold higher than its affinity for a non-specific antigen.

[0075] As used herein, the term "antibody" refers to an assembly (e.g., an intact antibody molecule, an immunoadhesin, or a variant thereof) having significant known specific immunoreactive activity against an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins include light and heavy chains that may or may not include interchain covalent linkages between them. The basic immunoglobulin structure in the vertebrate system is relatively well understood.

[0076] As used herein, the term "multiselective antibody" refers to an antibody that includes at least two different binding specificities. In one embodiment, the multiselective antibodies described herein are specific for two different antigens, e.g., specific for a blood-brain barrier (BBB) receptor and soluble amyloid beta (Aβ).

[0077] As used herein, the term "monospecific antibody" refers to an antibody having one or more binding sites, each of which has the same binding specificity, i.e., a monospecific antibody that binds to a single antigen, e.g., soluble amyloid beta (Aβ).

[0078] As will be discussed in more detail below, the general term "antibody" includes five different antibody classes that can be biochemically distinguished. Although all five antibody classes are clearly within the scope of the present disclosure, the following discussion generally relates to the IgG class of immunoglobulin molecules. With respect to IgG, an immunoglobulin comprises two identical light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000 - 70,000. The four chains are linked by disulfide bonds in a "Y" configuration, where the light chains begin at the mouth of the "Y" and continue to surround the heavy chains up to the variable regions.

[0079] The light chains of immunoglobulins are classified as either kappa (κ) or lambda (λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, the light and heavy chains are covalently bound to each other, and the "tail" portions of the two heavy chains are bound to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulin is produced by any of a hybridoma, B cell, or genetically engineered host cell. In the heavy chains, the amino acid sequence extends from the N-terminus at the bifurcated ends of the "Y" configuration to the lower C-terminus of each chain. As will be readily appreciated by those skilled in the art, the heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), with several subclasses therebetween (e.g., γ1 - γ4). Determining the "class" of an antibody as IgG, IgM, IgA, IgG, or IgE, respectively, is a property of this chain. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to confer functional properties. Modified versions of each of these classes and isotypes are readily recognizable by those skilled in the art in view of the present disclosure and are thus within the scope of the present disclosure.

[0080] Both the light and heavy chains are divided into regions of structural and functional homology. The term "region" refers to a portion or part of an immunoglobulin or antibody chain and includes the constant or variable regions, as well as more discrete portions or parts of such regions. For example, the light chain variable region includes "complementary determining regions" or "CDRs" incorporated between "framework regions" or "FRs", as defined herein.

[0081] Regions of an immunoglobulin heavy or light chain can be defined as "constant" (C) regions or "variable" (V) regions based on the relative lack of sequence variability within the regions of various class members in the case of "constant regions", or based on significant variability within the regions of various class members in the case of "variable regions". The terms "constant region" and "variable region" can also be used functionally. In this context, it is of course understood that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. Conversely, the constant region of an immunoglobulin or antibody confers important effector functions such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. The subunit structure and three-dimensional arrangement of the constant regions of the various immunoglobulin classes are well known.

[0082] The constant and variable regions of immunoglobulin heavy and light chains fold into domains. The term "domain" refers to a globular region of a heavy or light chain that includes, for example, β-pleated sheets and / or peptide loops (e.g., containing 3 to 4 peptide loops) stabilized by intrachain disulfide bonds. The constant region domain on the light chain of an immunoglobulin is interchangeably referred to as the "light chain constant region domain", the "CL region" or the "CL domain". The constant domains on the heavy chain (e.g., the hinge, CH1, CH2 or CH3 domains) are interchangeably referred to as the "heavy chain constant region domains", the "CH" region domains or the "CH domains". The variable domain on the light chain is interchangeably referred to as the "light chain variable region domain", the "VL region domain" or the "VL domain". The variable domain on the heavy chain is interchangeably referred to as the "heavy chain variable region domain", the "VH region domain" or the "VH domain".

[0083] By convention, the amino acid numbering of the variable constant region domain increases as it gets further from the antigen-binding site or amino terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light immunoglobulin chain is the variable region, and the C-terminus is the constant region. The CH3 and CL domains include the carboxy termini of the heavy and light chains, respectively. Thus, the domains of the light chain immunoglobulin are arranged in a VL-CL orientation, while the domains of the heavy chain are arranged in a VH-CH1-hinge-CH2-CH3 orientation.

[0084] The amino acid assignment to each variable region domain follows the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or different light chain variable regions are assigned the same number. CDR1, 2, and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively. CDR1, 2, and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively. When referred to as such, the CDR assignment may follow IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003) instead of Kabat. The numbering of the heavy chain constant region is by the EU index as shown in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0085] As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino-terminal variable domain of an immunoglobulin light chain.

[0086] As used herein, the term "CH1 domain" includes, for example, the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain that extends from positions approximately 114-223 (EU positions 118-215) in the Kabat numbering system. The CH1 domain is adjacent to the VH domain, is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule, and does not form part of the Fc region of the immunoglobulin heavy chain.

[0087] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upstream, middle, and downstream hinge domains (Roux et al. J. Immunol. 1998, 161:4083).

[0088] As used herein, the term "CH2 domain" includes, for example, the portion of the heavy chain immunoglobulin molecule that spans positions approximately 244-360 (EU positions 231-340) in the Kabat numbering system. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact native IgG molecule. In one embodiment, the binding polypeptide of the present disclosure includes a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0089] As used herein, the term "CH3 domain" includes the portion of the heavy chain immunoglobulin molecule that extends approximately 110 residues from the N-terminus of the CH2 domain, e.g., from positions approximately 361-476 (EU positions 341-445) of the Kabat numbering system. The CH3 domain typically forms the C-terminal portion of the antibody. However, in some immunoglobulins, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the ε chain of IgE). In one embodiment, the binding polypeptide of the present disclosure includes a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0090] As used herein, the term "CL domain" includes the constant region domain of the immunoglobulin light chain that extends from, e.g., Kabat position approximately 107A to Kabat position approximately 216. The CL domain is adjacent to the VL domain. In one embodiment, the binding polypeptide of the present disclosure includes a CL domain derived from a kappa light chain (e.g., a human kappa light chain).

[0091] As used herein, the term "Fc region" is defined as the portion of the heavy chain constant region that begins immediately above the papain cleavage site (i.e., residue 216 in IgG, interpreting the first residue of the heavy chain constant region as 114) and ends at the C-terminus of the antibody. Thus, the complete Fc region includes at least the hinge domain, CH2 domain, and CH3 domain.

[0092] As used herein, the terms "native Fc" or "wild-type Fc" refer to a molecule, whether in monomeric or multimeric form, that contains the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, and may contain a hinge region. The original immunoglobulin source of native Fc is typically of human origin and can be any of the immunoglobulins such as IgG1 and IgG2. Native Fc molecules are composed of monomeric polypeptides that can be linked by covalent (i.e., disulfide bonds) and non-covalent bonds to form dimeric or multimeric forms. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of native Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. As used herein, the term "native Fc" is a general term for monomeric, dimeric, and multimeric forms.

[0093] As used herein, the terms "Fc variant" or "modified Fc" refer to a molecule or sequence that has been modified from native / wild-type Fc but still contains the binding site for FcRn. Thus, the term "Fc variant" can include molecules or sequences that have been humanized from non-human native Fc. Furthermore, native Fc contains regions that can be removed because they confer structural features or biological activities that are not required for the antibody-like binding polypeptides described herein. Thus, the term "Fc variant" includes: (1) molecules or sequences lacking one or more native Fc sites or residues, or having one or more Fc sites or residues modified, that affect or are involved in disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cell cytotoxicity (ADCC).

[0094] As used herein, the term "Fc domain" encompasses native / wild-type Fc as well as the Fc variants and sequences defined above. Similar to Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form whether digested from whole antibodies or produced by other means.

[0095] As noted above, the variable regions of an antibody enable it to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains of an antibody combine to form the variable region (Fv) that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites present at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by the complementarity-determining regions (CDRs) on each of the heavy and light chain variable regions. As used herein, the term "antigen-binding site" includes the site that specifically binds (immunoreacts with) an antigen (e.g., a cell surface or soluble antigen). The antigen-binding site includes the immunoglobulin heavy and light chain variable regions, and the binding site formed by these variable regions determines the specificity of the antibody. The antigen-binding site is formed by different variable regions for each antibody. The modified antibodies of the present disclosure include at least one antigen-binding site.

[0096] In certain embodiments, the binding polypeptide of the present disclosure includes at least two antigen-binding domains that effect binding of the binding polypeptide to a selected antigen. The antigen-binding domains need not be from the same immunoglobulin molecule. In this regard, the variable regions can be from any type of animal that can initiate a humoral response and be induced to generate immunoglobulins against a desired antigen. As such, the variable regions of the binding polypeptide can be of mammalian origin, for example, human, mouse, rat, goat, sheep, non-human primates (e.g., cynomolgus monkey, macaque, etc.), wolf, or camelids (e.g., camel, llama, and related species).

[0097] In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short discontinuous sequences of amino acids that are specifically positioned to form the antigen-binding site as the antibody adopts its three-dimensional configuration in an aqueous environment. The remaining portions of the heavy and light variable domains exhibit less intermolecular variability in amino acid sequence and are referred to as framework regions. The framework regions predominantly adopt a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Thus, these framework regions act to form a scaffold that positions the six CDRs in the correct orientation by intermolecular non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines the surface complementarity to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to the immunoreactive antigen epitope.

[0098] Exemplary binding polypeptides include antibody variants. As used herein, the term "antibody variant" includes antibodies that have been altered such that they do not occur naturally, e.g., antibodies that contain at least two heavy chain portions but do not contain two complete heavy chains (e.g., domain-deleted antibodies or minibodies); multispecific antibodies that have been altered to bind to two or more different antigens or to different epitopes on a single antigen (e.g., bispecific, trispecific, etc.); synthetic and engineered forms such as heavy chain molecules conjugated to scFv molecules, etc. Further, the term "antibody variant" includes multivalent antibodies (e.g., trivalent antibodies, tetravalent antibodies, etc. that bind to three, four or more copies of the same antigen. An "antibody variant" may be multispecific and / or multivalent.

[0099] As used herein, the term "valency" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or a specific site on a target molecule. If a polypeptide contains more than one target binding site, each target binding site may specifically bind to the same molecule or to different molecules (e.g., bind to different ligands or different antigens, or bind to different epitopes on the same antigen). The binding polypeptides of the subject typically have at least one binding site specific for a human antigen molecule.

[0100] The term "specificity" refers to the ability to specifically bind (e.g., immunoreact) to a given target antigen (e.g., a human target antigen). A binding polypeptide may be monospecific and contain one or more binding sites that specifically bind to the target, or the polypeptide may be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In certain embodiments, the binding polypeptide is specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, the binding polypeptide is specific for more than one target.

[0101] In certain exemplary embodiments, the binding peptides (e.g., antibodies) described herein specifically bind to one or more synaptically toxic forms of Aβ. In certain exemplary embodiments, the binding peptides (e.g., antibodies) described herein that specifically bind to one or more synaptically toxic forms of Aβ do not have specificity for monomeric Aβ, fibrillar Aβ, or any combination of protofibrillar Aβ. In certain exemplary embodiments, the binding peptides (e.g., antibodies) described herein that specifically bind to one or more synaptically toxic forms of Aβ do not have specificity for one or more non-Aβ aggregates such as SOD-1 and / or aggregated synuclein.

[0102] In certain exemplary embodiments, the binding peptides (e.g., antibodies) described herein that specifically bind to one or more synaptically toxic forms of Aβ prevent and / or reduce one or more symptoms associated with AD. As used herein, "symptoms associated with Alzheimer's disease" or "symptoms associated with AD" refer to signs associated with any of the pre-onset, mild, moderate, or severe stages of AD. Symptoms associated with AD include one or more physical changes, such as changes in the brain, including, but not limited to: for example, neurotoxicity and / or synaptotoxicity in the olfactory cortex, hippocampus, and cerebral cortex; accumulation of glial cells; accumulation of Aβ; accumulation of tau; formation of Aβ oligomers; formation of Aβ paranuclei; formation of Aβ preprotofibrils; formation of mature Aβ fibrils; formation of amyloid plaques; formation of neurofibrillary tangles; chronic inflammation; reduced blood flow to the brain; breakdown of the BBB; brain atrophy, and the like.

[0103] Symptoms associated with mild AD include, but are not limited to: memory loss; decreased judgment leading to incorrect decisions; loss of initiative and spontaneity; taking longer to complete normal daily tasks; repeating questions; difficulty handling money or making payments; getting lost while wandering; misplacing or forgetting things in strange places; mood and personality changes; increased anxiety and / or aggression, and the like.

[0104] Symptoms associated with moderate AD include, but are not limited to: increased memory loss and confusion; inability to learn new things; language impairment and difficulty working with reading, writing, and numbers; difficulty organizing thoughts and thinking logically; shortened attention span; difficulty understanding new situations; difficulty performing multi-step tasks such as getting dressed; difficulty recognizing family and friends; hallucinations, delusions, and delusional disorders; impulsive behavior such as undressing at inappropriate times or using vulgar language; inappropriate outbursts of anger; restlessness, agitation, anxiety, tearfulness, wandering - especially before evening or at night; repeating statements or movements, occasional muscle contractions, and the like.

[0105] Symptoms associated with severe AD include, but are not limited to: memory loss; impaired judgment resulting in incorrect decisions; loss of initiative and spontaneity; taking longer to complete normal daily tasks; repeating questions; difficulty handling money or making payments; wandering and getting lost; misplacing or forgetting things in strange places; mood and personality changes; increased anxiety and / or aggression, etc.

[0106] As used herein, the "synaptically toxic form of amyloid beta", "synaptically toxic form of Aβ", or "synaptically toxic Aβ" refers to the form of Aβ present in soluble brain extracts from AD brains and associated with synaptic loss. Synaptically toxic Aβ has a molecular weight between about 8 kD and about 100 kD and is unstable.

[0107] According to certain embodiments, binding polypeptides that specifically bind to synaptically toxic Aβ are provided herein. The full-length heavy and light chain sequences of exemplary binding peptides described herein are shown in Table 1.

[0108] [Table 1] [Table 2] [Table 3] [Table 4]

[0109] The light chain variable region (LCVR) sequences and heavy chain variable region (HCVR) sequences of exemplary binding peptides described herein are shown in Table 2.

[0110] [Table 5] [Table 6]

[0111] Table 3 shows the light chain framework (LFW) sequences and heavy chain framework (HFW) regions of the exemplary binding peptides described herein.

[0112]

Table 7

Table 8

[0113] Table 4 shows the complementarity determining region (CDR) sequences of the heavy and light chains of the exemplary binding peptides described herein.

[0114]

Table 9

Table 10

[0115] The term "antigen" or "target antigen", as used herein, refers to a molecule or a part of a molecule that can be bound by the binding site of a binding polypeptide. The target antigen can have one or more epitopes.

[0116] The term "about" or "approximately" means within about 20% of a given value or range, such as within about 10%, within about 5%, or within about 1% or less.

[0117] As used herein, "administering" or "administration" refers to the act of physically delivering a substance, as it exists outside the body (e.g., an isolated binding polypeptide provided herein), to a patient by injection or other means, e.g., but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or by any other means of physical delivery described herein or known in the art. When a disease or its symptoms are managed or treated, administration of the substance typically occurs after the onset of the disease or its symptoms. When a disease or its symptoms are prevented, administration of the substance typically occurs prior to the onset of the disease or its symptoms and may be continued chronically to delay or reduce the appearance or magnitude of disease-related symptoms.

[0118] As used herein, the term "composition" encompasses a product that contains specific ingredients (e.g., an isolated binding polypeptide provided herein), in optionally specified amounts, and further any product that results directly or indirectly from a combination of optionally specified amounts of the specific ingredients.

[0119] "Effective amount" means an amount of an active agent (e.g., an isolated binding polypeptide of the present disclosure) sufficient to achieve a desired physiological result in an individual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0120] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a mammal, e.g., a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In certain embodiments, as used herein, the term "subject" refers to a vertebrate, e.g., a mammal. Mammals include, without limitation, humans, non-human primates, wildlife, livestock, sport animals, and pets.

[0121] As used herein, the term "treatment" refers to any protocol, method and / or agent that can be used in the prevention, management, treatment and / or alleviation of a disease or symptoms associated therewith. In some embodiments, the term "treatment" refers to any protocol, method and / or agent that can be used in the modulation of an immune response to an infection in a subject or symptoms associated therewith. In some embodiments, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies, and / or other therapies known to those of ordinary skill in the art, such as medical professionals, that are useful in the prevention, management, treatment and / or alleviation of a disease or symptoms associated therewith. In other embodiments, the term "treatment" refers to biological therapies, supportive therapies, and / or other therapies known to those of ordinary skill in the art, such as medical professionals, that are useful in the modulation of an immune response to an infection in a subject or symptoms associated therewith.

[0122] As used herein, the terms "treating", "treatment" and "treatment thereof" refer to a decrease or alleviation in the progression, severity and / or duration of a disease or symptoms associated therewith that results from the administration of one or more treatments (including, but not limited to, the administration of one or more prophylactic or therapeutic agents such as the isolated binding polypeptides provided herein). As used herein, the term "treatment thereof" may also refer to altering the course of a disease in a subject being treated. Therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, decreasing the rate of disease progression, remission or alleviation of a disease state, and an improved remission or prognosis.

[0123] In certain exemplary embodiments, the binding polypeptides described herein treat one or more symptoms of Alzheimer's disease.

[0124] Binding polypeptide In one aspect, the present disclosure provides a binding polypeptide (e.g., an antibody, an immunoadhesin, an antibody variant, and a fusion protein) that binds to Aβ and treats one or more symptoms of Alzheimer's disease. The binding polypeptides disclosed herein include any binding polypeptide that includes a modified Fc domain. In certain embodiments, the binding polypeptide is an antibody, or an immunoadhesin, or a derivative thereof. Any antibody from any source or species can be used in the binding polypeptides disclosed herein. Suitable antibodies include, without limitation, human antibodies, humanized antibodies, or chimeric antibodies. Suitable antibodies include, without limitation, monoclonal antibodies, polyclonal antibodies, full-length antibodies, or single-chain antibodies.

[0125] Fc domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA, and IgE) and species can be used in the binding polypeptides disclosed herein. Chimeric Fc domains that include portions of Fc domains from various species or Ig classes can also be used. In certain embodiments, the Fc domain is a human Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In other embodiments, the Fc domain is an IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG1 or IgG4 Fc domain. In some embodiments, the Fc domain is a human IgG1 Fc domain. In some embodiments, the Fc domain is a human IgG1 Fc domain containing the "LALA" mutation. For Fc domains of other species and / or Ig classes or isotypes, it will be apparent to those skilled in the art that any of the amino acid substitutions described herein can be appropriately adapted. Certain embodiments include antibodies that have at least one amino acid in one or more of the constant region domains and / or at least one amino acid in one or more of the variable region domains that have been deleted or otherwise modified to produce desired biochemical characteristics such as, for example, decreased or enhanced effector function, the ability to dimerize non-covalently, an increased ability to localize to the site of a tumor, a decreased serum half-life, an increased serum half-life, etc., when compared to the intact antibody with substantially the same immunogenicity.

[0126] In certain other embodiments, the binding polypeptide includes constant regions from different antibody isotypes (e.g., constant regions from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the binding polypeptide includes a chimeric hinge (i.e., a hinge that includes a hinge portion from the hinge domain of a different antibody isotype, e.g., the upstream hinge domain from an IgG4 molecule and the IgG1 middle hinge domain).

[0127] In certain embodiments, the Fc domain can be mutated to increase or decrease effector function using techniques known in the art. In some embodiments, the binding polypeptides of the disclosure comprising a modified Fc domain have an altered binding affinity for Fc receptors. There are several different types of Fc receptors, which are classified based on the type of antibody they recognize. For example, Fc-gamma receptors (FcγR) bind to IgG class antibodies, Fc-alpha receptors (FcαR) bind to IgA class antibodies, and Fc-epsilon receptors (FcεR) bind to IgE class antibodies. FcγR belongs to a family that includes several members, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. In some embodiments, the binding polypeptide comprising a modified Fc domain has an altered FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, the binding polypeptide comprising a modified Fc domain has a decreased FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, the binding polypeptide comprising a modified Fc domain has an enhanced FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain. In some embodiments, the binding polypeptide comprising a modified Fc domain has substantially the same FcγRIIIa binding affinity compared to a binding polypeptide comprising a wild-type Fc domain.

[0128] In other embodiments, the binding polypeptides described herein have a constant region, such as an IgG1 heavy chain constant region, that has been modified to reduce or eliminate glycosylation. For example, a binding polypeptide comprising a modified Fc domain (e.g., an antibody or an immunoadhesin) can further comprise an amino acid substitution that modifies the glycosylation of the antibody Fc. For example, the modified Fc domain described above can have reduced glycosylation (e.g., N-linked or O-linked glycosylation).

[0129] Examples of amino acid substitutions that result in reduced or altered glycosylation are disclosed in International PCT Publication No. WO2005 / 018572, which is incorporated herein by reference in its entirety. In some embodiments, the binding polypeptide is modified to remove glycosylation. Such a binding polypeptide may be referred to as an "agly" binding polypeptide (e.g., an "agly" antibody). Without being bound by theory, it is believed that "agly" binding polypeptides may have an improved safety and stability profile in vivo. Agly binding polypeptides may be of any isotype or subclass thereof, such as those of IgG1, IgG2, IgG3, or IgG4. A number of methods recognized in the art are available for producing "agly" antibodies or antibodies having modified glycans. Such antibodies can be produced using, for example, genetically engineered host cells (e.g., modified yeast, such as Pichia, or CHO cells) having a modified glycosylation pathway (e.g., glycosyltransferase deletion).

[0130] In certain embodiments, the binding polypeptide may include an antibody constant region that mediates one or more effector functions (e.g., an IgG constant region, e.g., a human IgG constant region, e.g., a human IgG1 constant region). For example, binding of the C1-complex to the antibody constant region can activate the complement system. Activation of the complement system is important in the opsonization and lysis of cellular pathogens. Activation of the complement system also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity. Additionally, antibodies bind to receptors on various cells via the Fc domain (the Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell). There are a number of Fc receptors specific for various classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of the antibody to the Fc receptor on the cell surface induces a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental passage, and regulation of immunoglobulin production. In some embodiments, the binding polypeptide (e.g., an antibody or an immunoadhesin) binds to the Fc-gamma receptor. In alternative embodiments, the binding polypeptide may include a constant region that lacks one or more effector functions (e.g., ADCC activity) and / or is unable to bind to the Fcγ receptor.

[0131] In certain embodiments, the binding polypeptide of the present disclosure may include an antigen-binding fragment of an antibody. The term “antigen-binding fragment” refers to an immunoglobulin or polypeptide fragment of an antibody that binds to an antigen or competes with an intact antibody for antigen binding (i.e., specific binding) (i.e., with the intact antibody from which it is derived). Antigen-binding fragments can be produced by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include Fv, Fab, Fab’, and (Fab’)2.

[0132] In some embodiments, the binding polypeptide comprises a single-chain variable region sequence (ScFv). The single-chain variable region sequence comprises a single polypeptide having one or more antigen-binding sites, for example, a VL domain linked to a VH domain by a flexible linker. The ScFv molecule can be constructed in a VH-linker-VL orientation or a VL-linker-VH orientation. The flexible hinge connecting the VL and VH domains that constitute the antigen-binding site contains about 10 to about 50 amino acid residues. Linker peptides are known in the art. The binding polypeptide can comprise at least one scFv and / or at least one constant region. In one embodiment, the binding polypeptide of the present disclosure can comprise at least one scFv linked or fused to a modified Fc domain.

[0133] In some embodiments, the binding polypeptide of the present disclosure is a multivalent (e.g., tetravalent) antibody produced by fusing a DNA sequence encoding an antibody with a ScFv molecule (e.g., a modified ScFv molecule). For example, in one embodiment, these sequences are combined such that the ScFv molecule (e.g., a modified ScFv molecule) is linked to the Fc fragment of the antibody via a flexible linker (e.g., a gly / ser linker) at its N-terminus or C-terminus. In another embodiment, the tetravalent antibody of the present disclosure can be produced by fusing a ScFv molecule to a connecting peptide, which is then fused to a modified Fc domain to construct a ScFv-Fab tetravalent molecule.

[0134] In another embodiment, the binding polypeptide of the present disclosure is a modified minibody. The modified minibody of the present disclosure is a dimeric molecule composed of two polypeptide chains each containing an ScFv fused to a modified Fc domain via a connecting peptide. The minibody can be produced by constructing the ScFv component and connecting it to the peptide component using methods described in the art (e.g., U.S. Patent No. 5,837,821 or WO 1994 / 009817). In another embodiment, a tetravalent minibody can be constructed. The tetravalent minibody can be constructed in the same manner as the minibody except that two ScFv molecules are linked using a flexible linker. The linked scFv-scFv construct is then joined to the modified Fc domain.

[0135] In another embodiment, the binding polypeptide of the present disclosure comprises a diabody. A diabody is a dimeric tetravalent molecule that each has a polypeptide similar to an ScFv molecule but usually has a short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting both variable domains, such that the VL and VH domains on the same polypeptide chain cannot interact. Instead, the VL and VH domains of one polypeptide chain interact (respectively) with the VH and VL domains on the second polypeptide chain (see, e.g., WO02 / 02781). The diabody of the present disclosure comprises an scFv-like molecule fused to a modified Fc domain.

[0136] In other embodiments, the binding polypeptide comprises a multispecific or multivalent antibody that comprises one or more variable domains in series on the same polypeptide chain, such as a tandem variable domain (TVD) polypeptide. Exemplary TVD polypeptides include the "double head" or "dual-Fv" constructs described in U.S. Patent No. 5,989,830. In the dual-Fv construct, the variable domains of two different antibodies are expressed in tandem orientation on two separate chains (one heavy chain and one light chain), where one polypeptide chain has two VH domains in series separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected in series by a peptide linker (VL1-linker-VL2). In the cross double head construct, the variable domains of two different antibodies are expressed in tandem orientation on two separate polypeptide chains (one heavy chain and one light chain), where one polypeptide chain has two VH domains in series separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains connected in series by a peptide linker in the opposite orientation (VL2-linker-VL1). Further antibody variants based on the "dual-Fv" format include dual-variable-domain IgG (DVD-IgG) bispecific antibodies (see U.S. Patent No. 7,612,181) and the TBTI format (see US 2010 / 0226923 A1). In some embodiments, the binding polypeptide comprises a multispecific or multivalent antibody that comprises one or more variable domains in series on the same polypeptide chain fused to a modified Fc domain.

[0137] In another exemplary embodiment, the fusion polypeptide is an immunoadhesin. As used herein, "immunoadhesin" refers to a fusion polypeptide comprising one or more binding domains (e.g., derived from a receptor, ligand, or cell adhesion molecule) linked to an immunoglobulin constant domain (i.e., the Fc region) (see, e.g., Ashkenazi et al. 1995, Methods 8(2):104-115, and Isaacs (1997) Brit. J. Rheum. 36:305, which are incorporated herein by reference in their entirety). Immunoadhesins are identified by the suffix "-cept" in their International Nonproprietary Names (INNs). Like antibodies, immunoadhesins have a long circulatory half-life, are easily purified by affinity-based methods, and have an avidity advantage due to bivalency. Examples of commercially available therapeutic immunoadhesins include etanercept (ENBREL®), abatacept (ORENCIA®), rilonacept (ARCALYST®), aflibercept (ZALTRAP® / EYLEA®), and belatacept (NULOJIX®).

[0138] In certain embodiments, the binding polypeptide comprises an immunoglobulin-like domain. Suitable immunoglobulin-like domains include, without limitation, fibronectin domains (see, e.g., Koide et al. (2007), Methods Mol. Biol. 352:95-109, which is incorporated herein by reference in its entirety), DARPins (see, e.g., Stumpp et al. (2008) Drug Discov. Today 13 (15-16):695-701, which is incorporated herein by reference in its entirety), the Z domain of protein A (see, e.g., Nygren et al. (2008) FEBS J. 275 (11):2668-76, which is incorporated herein by reference in its entirety), lipocalins (see, e.g., Skerra et al. (2008) FEBS J. 275 (11):2677-83, which is incorporated herein by reference in its entirety), Affilins (see, e.g., Ebersbach et al. (2007) J. Mol. Biol. 372 (1):172-85, which is incorporated herein by reference in its entirety), Affitins (see, e.g., Krehenbrink et al. (2008). J. Mol. Biol. 383 (5):1058-68, which is incorporated herein by reference in its entirety), Avimers (see, e.g., Silverman et al. (2005) Nat. Biotechnol. 23 (12):1556-61, which is incorporated herein by reference in its entirety), Fynomers, (see, e.g., Grabulovski et al. (2007) J Biol Chem 282 (5):3196-3204, which is incorporated herein by reference in its entirety), and knotted domain peptides (see, e.g., Nixon et al. (2006) Curr Opin Drug Discov Devel 9 (2):261-8, which is incorporated herein by reference in its entirety).

[0139] Regarding the binding polypeptides and immunoadhesins of the present disclosure, virtually any antigen can be targeted by the binding polypeptide, including, but not limited to, the protein, subunit, domain, motif, and / or epitope of the target antigen, which includes both soluble factors such as cytokines and membrane-bound factors, as well as transmembrane receptors.

[0140] In certain embodiments, the binding polypeptide, such as an antibody, is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. In certain embodiments, the bispecific antibody can be used to cross the blood-brain barrier (BBB). The bispecific antibody can be produced as a full-length antibody or an antibody fragment.

[0141] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain-light-chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and "knob-into-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be made by manipulating the electrostatic steering effect for producing antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); generating bispecific antibodies using leucine zippers (see, e.g., Kostelny, S.A. et al., J. Immunol. 148 (1992) 1547-1553); using "diabody" technology for producing bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and can also be made, for example, by producing trispecific antibodies as described in Tutt, A. et al., J. Immunol. 147 (1991) 60-69.

[0142] In one embodiment, the CH3 domain of the heavy chain of the bispecific antibody is modified by the "knob-into-hole" technology, which is described in detail, for example, using several examples in WO 96 / 027011, WO 98 / 050431, Ridgway J.B., et al., Protein Eng. 9 (1996) 617-621, Merchant, A.M., et al., Nat Biotechnol 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are modified to increase the heterodimerization of both heavy chains containing the two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be a "knob" and the other can be a "hole". The introduction of disulfide bridges may be utilized to stabilize the heterodimer (Merchant, A.M, et al., Nature Biotech 16 (1998) 677-681, Atwell, S., et al. J. Mol. Biol. 270 (1997) 26-35), and further to increase the yield In one embodiment, the bispecific antibody is characterized in that the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain intersect at a boundary that includes the original boundary between the antibody CH3 domains, where the boundary is modified to promote the formation of the bispecific antibody, and the modification is: a) within the original boundary, the CH3 domain of one heavy chain is modified to intersect the original boundary of the CH3 domain of the other heavy chain in the bispecific antibody, and the amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby creating a bulge within the boundary of the CH3 domain of one heavy chain, which can be positioned within the void within the boundary of the CH3 domain of the other heavy chain; and b) within the original boundary of the second CH3 domain, the CH3 domain of the other heavy chain is modified to intersect the original boundary of the first CH3 domain in the bispecific antibody, and the amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby creating a void within the boundary of the second CH3 domain, inside which the bulge within the boundary of the first CH3 domain can be positioned.

[0143] In one embodiment, the amino acid residue having a larger side-chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0144] In one embodiment, the amino acid residue having a smaller side-chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0145] In one embodiment, both CH3 domains are further modified by introducing cysteine (C) as an amino acid at corresponding positions of each CH3 domain such that a disulfide bridge can be formed between the two CH3 domains.

[0146] In an exemplary embodiment, the multispecific antibody comprises the amino acid T366W mutation in the first CH3 domain of the "knob chain" and the amino acid T366S, L368A, Y407V mutations in the second CH3 domain of the "hole chain". Additional inter-chain disulfide bridges between the CH3 domains can also be used (Merchant, A.M., et al., Nature Biotech. 16 (1998) 677-681), for example, by introducing the amino acid Y349C mutation in the CH3 domain of the "hole chain" and the amino acid E356C mutation or the amino acid S354C mutation in the CH3 domain of the "knob chain".

[0147] In one embodiment, the bispecific antibody comprises a Y349C, T366W mutation in one of the two CH3 domains, and an E356C, T366S, L368A, Y407V mutation in the other of the two CH3 domains. In one embodiment, the bispecific antibody comprises a Y349C, T366W mutation in one of the two CH3 domains, and an S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain form an interchain disulfide bond) (numbering according to Kabat's EU index; (Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991))). Additional knob-in-hole technology as described in EP 1 870 459 A1 can be used instead of, or in addition to. Thus, another example of a bispecific antibody is an R409D, K370E mutation in the CH3 domain of the "knob chain" and a D399K, E357K mutation in the CH3 domain of the "hole chain" (numbering according to Kabat's EU index; (Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991))).

[0148] In one embodiment, the bispecific antibody comprises a T366W in the CH3 domain of the "knob chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "hole chain", and further an R409D, K370E mutation in the CH3 domain of the "knob chain" and a D399K, E357K mutation in the CH3 domain of the "hole chain".

[0149] Nucleic acids and expression vectors In one aspect, the present invention provides a polynucleotide encoding a binding polypeptide disclosed herein. Also provided are methods of producing binding polypeptides that include expressing these polynucleotides.

[0150] The polynucleotides encoding the binding polypeptides disclosed herein are typically inserted into an expression vector for introduction into a host cell that can be used to produce the desired amount of the claimed antibody, or immunoadhesin. Accordingly, in certain aspects, the present invention provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0151] The terms "vector" or "expression vector" are used for the purposes of the specification and claims to mean a vector used to introduce a desired gene into a cell and to express the desired gene in the cell. As is known to those of skill in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors include a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0152] A number of expression vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), or SV40 virus. Others include the use of polycistronic systems with internal ribosome binding sites. Further, cells into which the DNA has been integrated into their chromosomes can be selected by introducing one or more markers that enable the selection of transfected host cells. The markers can confer prototrophy to auxotrophic hosts, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Additional elements may also be necessary for optimal synthesis of the mRNA. These elements can include signal sequences, splice signals, as well as transcriptional promoters, enhancers and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human genes) synthesized as discussed above.

[0153] In other embodiments, the binding polypeptides described herein can be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as the heavy and light chains of an antibody, can be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to produce relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference. It will be apparent to those skilled in the art that such expression systems can be used to efficiently produce all of the polypeptides disclosed in this application.

[0154] More generally, when a vector or DNA encoding the binding polypeptide of the present disclosure is produced, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Introduction of the plasmid into the host cell can be achieved by various techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion using envelope DNA, microinjection, and infection using intact virus. See, for example, Ridgway, A.A.G. "Mammalian Expression Vectors", Chapter 24.2, pages 470 - 472 in Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, MA 1988). The transformed cells are grown under conditions suitable for the production of the light and heavy chains, and assayed for heavy chain and / or light chain protein synthesis. Example assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0155] As used herein, the term "transformation" is used in a broad sense to refer to the introduction of DNA into a recipient host cell that changes the genotype and results in a change in the recipient cell.

[0156] Similarly, a "host cell" refers to a cell that is constructed using recombinant DNA technology and transformed with a vector encoding at least one heterologous gene. In the description of the process for isolation of polypeptides from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of the antibody unless another is specifically identified. In other words, the recovery of a polypeptide from a "cell" can mean either from the whole cells that have sedimented or from a cell culture containing both the medium and the sedimented cells.

[0157] In one embodiment, the host cell line used for the expression of the binding polypeptide is of eukaryotic or prokaryotic origin. In one embodiment, the host cell line used for the expression of the binding polypeptide is of bacterial origin. In one embodiment, the host cell line used for the expression of the binding polypeptide is of mammalian origin; one of ordinary skill in the art can determine the specific host cell line most suitable for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR minus), HELA (human cervical cancer), CVI (monkey kidney cell line), COS (a derivative of CVI having SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), 293 (human kidney). In one embodiment, the cell line results in altered glycosylation of the antibody expressed therefrom, e.g., afucosylation (e.g., PER6 TM (Crucell) or FUT8-knockout CHO cell line (POTELLIGENT TM cells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells can be used. Host cell lines are typically available from commercial services, the American Tissue Culture Collection, or published literature.

[0158] In vitro production allows for the upscaling to produce large amounts of the desired binding polypeptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or continuous stirred tank reactor, or cell cultures immobilized or trapped, for example, in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatography methods such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose and / or (immuno-)affinity chromatography.

[0159] One or more genes encoding the binding polypeptide may be expressed in non-mammalian cells such as bacteria, yeast or plant cells. In this regard, of course, various unicellular non-mammalian microorganisms such as bacteria can also be transformed; that is, they can be grown in culture or fermentation. Bacteria that are easily transformable include Enterobacteriaceae such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Streptococcus pneumoniae; members of Streptococcus and Haemophilus influenzae. Furthermore, of course, when expressed in bacteria, the polypeptide may be part of inclusion bodies. The polypeptide must be isolated, purified and then assembled into a functional molecule.

[0160] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or baker's yeast in general, is the most widely used among eukaryotic microorganisms, but a number of other strains are generally available. For expression in yeast, for example, plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene, which provides a selectable marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC number 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). As a result, the presence of a trpl lesion as a characteristic of the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0161] Treatment method In one aspect, the present invention provides a method of treating one or more symptoms of Alzheimer's disease in a patient in need of treatment, the method comprising administering an effective amount of a binding polypeptide disclosed herein. In certain embodiments, the present disclosure provides kits and methods for treating Alzheimer's disease in mammalian subjects in need of such treatment. In certain exemplary embodiments, the subject is human.

[0162] The binding polypeptides disclosed herein can be administered in a number of ways depending on whether local treatment or systemic treatment is desired and on the area to be treated. For example, administration can be parenteral, including but not limited to intravenous infusion, subcutaneous administration, intraperitoneal administration, intramuscular administration, intrathecal administration, or intracerebroventricular (e.g., intraventricular) administration. In certain exemplary embodiments, the binding polypeptide is delivered across the blood-brain barrier (BBB) using various suitable compositions and methods described herein.

[0163] Subjects diagnosed with or suspected of having Alzheimer's disease may have the binding polypeptide administered directly to the brain (e.g., to the globus pallidus or striatum of the basal ganglia and near the medium spiny neurons of the striatum). In addition to the binding polypeptide, the patient may be administered a second treatment, e.g., symptomatic therapy and / or disease-specific therapy. The secondary treatment may be, for example, symptomatic (e.g., to reduce symptoms), neuroprotective (e.g., to slow or stop disease progression), or restorative (e.g., to reverse the disease process). For the treatment of Alzheimer's disease, for example, symptomatic therapies may include the drugs Razadyne® (galantamine), Exelon® (rivastigmine), Aricept® (donepezil), Namenda® (memantine), or Namzaric® (memantine and donepezil). Other treatments may include psychotherapy, physical therapy, speech therapy, communication and memory aids, social support services, and dietary advice.

[0164] The binding polypeptide can be delivered to the nerve cells of the brain. Delivery methods that do not require passage through the BBB of the composition can be utilized. For example, a pharmaceutical composition containing the binding polypeptide can be delivered to a patient by direct injection into the region containing the diseased cells. For example, the pharmaceutical composition can be delivered directly into the brain by injection. The injection can be by stereotactic injection into a specific region of the brain (e.g., the ventricular substantia nigra, cortex, hippocampus, striatum, or globus pallidus). The binding polypeptide can be delivered to multiple regions of the central nervous system (e.g., to multiple regions of the brain and / or to the spinal cord). The binding polypeptide can be delivered to a diffuse region of the brain (e.g., diffuse delivery to the cortex of the brain).

[0165] In one embodiment, the conjugate polypeptide can be delivered using a cannula or other delivery device having one end implanted into tissue, such as the brain, such as the substantia nigra, cortex, hippocampus, striatum or globus pallidus of the brain. The cannula can be connected to a reservoir of the conjugate polypeptide. Flow or delivery can be conveyed by a pump, such as an osmotic pump or a minipump, such as an Alzet pump (Durect, Cupertino, CA). In one embodiment, the pump and reservoir are implanted in a region distal from the tissue, such as the abdomen, and delivery is mediated by a conduit that communicates from the pump or reservoir to the release site. Devices for delivery to the brain are described, for example, in U.S. 6,093,180 and US 5,814,014, which are incorporated herein by reference.

[0166] One of ordinary skill in the art could determine, by routine experimentation, a non-toxic amount of the modified conjugate polypeptide that would be effective for the purpose of treating a pathological lesion. For example, the therapeutically active amount of the conjugate polypeptides of the present disclosure can vary according to factors such as disease stage (e.g., pre-onset Alzheimer's disease, mild cognitive impairment, mild dementia, moderate dementia or severe dementia), age, gender, medical comorbidities (e.g., immunosuppressed state or disease) and the weight of the subject, as well as the ability of the modified antibody to elicit a desired response in the subject. The dosing regimen can be adjusted to provide an optimal therapeutic response. For example, several divided doses can be administered daily, weekly, bi-weekly, every three weeks, every four weeks or the like, and / or the dose can be proportionally decreased as indicated by the exigencies of the therapeutic situation.

[0167] Pharmaceutical composition Methods for preparing and administering the conjugate polypeptides of the present disclosure are well known or can be readily determined by those skilled in the art. The route of administration of the conjugate polypeptides of the present disclosure may be oral, parenteral, inhaled or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. All of these administration forms are intended to be clearly within the scope of the present disclosure, but the form for administration will likely be an injectable solution, particularly an intravenous or intraarterial injection or infusion. Usually, a pharmaceutical composition suitable for injection may contain a buffer (e.g., acetic acid, phosphoric acid or citrate buffer), a surfactant (e.g., polysorbate), and optionally a stabilizer (e.g., human albumin). In some embodiments, the conjugate polypeptide can be delivered directly to the site of the harmful cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0168] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, including saline and buffered media, alcoholic / aqueous solutions, emulsions or suspensions. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01 to 0.1 M, for example, 0.05 M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, dextrose Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluids and nutrient replenishers, electrolyte replenishers, such as those based on dextrose Ringer's solution, etc. For example, preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, and inert gases may also be present. More specifically, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should typically be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyhydric alcohols (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, the use of coatings such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants can maintain appropriate fluidity.

[0169] Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as saccharides, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride, are included in the composition. Prolonged absorption of injectable compositions can occur by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0170] In any case, sterile injectable solutions can be manufactured by incorporating the active compound (e.g., the modified binding polypeptide itself, or in combination with other active agents) in the required amount in a suitable solvent, together with one or a combination of the components listed herein, and subsequently, if necessary, subjecting it to sterile filtration. Generally, a dispersion is produced by incorporating the active compound in a sterile vehicle, which contains a basic dispersion medium and the other necessary components from those listed above. In the case of sterile powders for the manufacture of sterile injectable solutions, exemplary manufacturing methods include vacuum drying and lyophilization, whereby a powder of the active ingredient and any additional desired components is obtained from its solution that has been pre-sterilized by filtration. Preparations for injection are processed, filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations can be packaged and sold in the form of a kit. Such products typically have a label or package insert indicating that the accompanying composition is useful for treating subjects suffering from or predisposed to autoimmune disorders or neoplastic disorders.

[0171] The effective dosage of the compositions of the present disclosure for the treatment of the conditions described above varies depending on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0172] The binding polypeptides of the present disclosure can be administered multiple times. The intervals between single doses can be weekly, monthly, or yearly. The intervals can also be irregular as indicated by measuring the blood levels of the modified binding polypeptide or antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma concentration of the modified binding polypeptide of about 1 to 1000 μg / ml, and in some methods about 25 to 300 μg / ml. Alternatively, the binding polypeptide can be administered as a sustained release formulation, in which case the required dosing frequency is lower. For antibodies, the dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric and non-human antibodies.

[0173] The dosage and dosing frequency can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a composition containing an antibody or a cocktail thereof of the present invention is administered to a patient who is not yet in a diseased state to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dose." Also in this use, the exact amount depends on the health status and the state of the overall immunity of the patient, but generally ranges from about 0.1 to 25 mg per dose, particularly from about 0.5 to about 2.5 mg per dose. Relatively low dosages are administered at relatively low frequency intervals over a long period of time. Some patients continue treatment for the remainder of their lives. In therapeutic applications, relatively high dosages (e.g., about 1 to 400 mg / kg of antibody per dose) at relatively short intervals may be required until the progression of the disease is reduced or arrested, or until the patient shows a partial or complete remission of the disease symptoms. Thereafter, the patient can be administered a prophylactic dosing schedule.

[0174] The pharmaceutical compositions according to the present disclosure may include a pharmaceutically acceptable non-toxic sterile carrier, such as physiological saline, non-toxic buffer solution, preservative, etc. For the purposes of the present application, a pharmaceutically effective amount of the binding polypeptide, immunoadhesin or their recombinants, whether conjugated or not conjugated with a therapeutic agent, achieves effective binding to the antigen and achieves a benefit, for example, continues to mean an amount sufficient to relieve one or more symptoms of Alzheimer's disease. The pharmaceutical compositions of the present disclosure may be administered in a single dose or multiple doses so as to provide a pharmaceutically effective amount of the binding polypeptide.

[0175] Along the scope of the present disclosure, the binding polypeptides of the present disclosure may be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect according to the treatment methods described above. The binding polypeptides of the present disclosure may be administered to humans or other animals as described above in a conventional dosage form produced by combining a conventional pharmaceutically acceptable carrier or diluent and the antibody of the present disclosure according to known techniques. It will be apparent to those skilled in the art that the form and characteristics of the pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient to be combined, the route of administration and other well-known variables. Furthermore, it will be apparent to those skilled in the art that cocktails containing one or more types of the binding polypeptides described in the present disclosure may prove to be particularly effective.

[0176] The content of the articles, patents and patent applications, and all other documents and electronically available information mentioned or cited herein is incorporated herein by reference in its entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents into this application.

[0177] Although the present invention has been described with reference to its particular embodiments, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Further, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or procedure to the objective, spirit and scope of the present invention. It is intended that all such modifications be within the scope of the patent claims appended hereto. Although particular embodiments have been described in detail herein, they will be understood more clearly by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting.

Example

[0178] The present invention is further illustrated by the following examples, which should not be construed as further limiting.

[0179] Example 1: Outline of the Aβ Monoclonal Antibody Discovery Process Trianni mice were immunized with either sonicated human Alzheimer's disease (AD) brain amyloid plaques (FusA), aggregated synthetic Aβ (FusB), or human AD brain amyloid plaque-seeded synthetic Aβ (FusC) (Figure 1). It should be noted that the Fc domain was murine, while in most cases the Fv domain was human.

[0180] Hybridomas with good to moderate binding to synthetic Aβ preprotofibrils (PFs) were selected. Antibodies that bound to monomeric Aβ with high affinity and to other types of aggregated proteins (e.g., synuclein and SOD) with low affinity were counterselected. Finally, mouse IgG antibodies were screened using a "cellular iN assay" (described further below), where neuroprotection against the AD brain synaptotoxic form of oligomeric Aβ (oAβ) was evaluated. Active antibodies with low binding to both Aβ preprotofibrils and fibrillar Aβ were selected. Promising antibodies were cloned and rearranged into the human IgG1 LALA Fc domain.

[0181] The binding properties of the rearranged human IgG1 monoclonal antibodies (mAbs) were evaluated for selectivity to monomeric Aβ and, further, for low binding to Aβ PF and fibrillar Aβ. Two of the identified human mAbs that did not show significant binding to Aβ PF (or other forms) were retested in the iN cell assay and were determined to be neuroprotective.

[0182] Human mAbs (hIgGs) that did not bind to other aggregated proteins were evaluated using the iN assay, an EC50 was obtained, and the results were reproduced. Five hIgGs were tested in a second functional assay, an electrophysiological LTP assay, as further described below in this specification.

[0183] Example 2: Trianni Mouse Immunization and Hybridoma Generation Immunization Transgenic Trianni mice for the human IgG heavy and kappa light chains were immunized as previously reported [please indicate citation] with aggregated synthetic Aβ1-42 peptide seeded on a matrix of aggregated synthetic Aβ1-42 peptide or sonicated human AD brain-derived amyloid plaque fragments. The mice were boost immunized 3 - 5 times every two weeks with these proteins.

[0184] Hybridoma cells were generated by fusing mouse myeloma cells (BALB / c B-lymphoblastoid cell line SP2 / 0 fused with Sendai virus) lacking adenosine phosphoribosyltransferase (APRT) with spleen cells from immunized mice. Hypoxanthine, azaserine, and thymidine (HAT) selection and serial dilutions were performed to achieve single-cell clonality.

[0185] Screening Indirect ELISA was utilized for screening and counter-screening assays. Surface plasmon resonance (SPR) dissociation rate analysis was performed to determine binding affinity. Analysis was performed on a Biacore T100 using HBS-EP and running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20). A Series A Protein A sensor chip was used for analysis. Antibodies were diluted to 5 μg / ml in HBS-EP+. Monomeric Aβ1-42 peptide (SEC purified) was diluted to 1000 μM (4.5 mg / ml) in HBS-EP+ and then serially diluted 2-fold for a total of six concentrations. Aβ1-42 PF was diluted to 100 nM (66 μg / ml) in HBS-EP+ and then serially diluted 2-fold for a total of six concentrations. Antibodies were captured via the Fc domain on the Protein A surface for 60 s at 10 μl / min, then Aβ peptide solution (monomer or PF) was injected over the surface for 180 s at 30 μl / min, followed by dissociation for 360 s. The Protein A surface was regenerated with a 60 s injection of 10 mM glycine-HCl, pH 1.7. The resulting sensorgrams were double referenced and fitted to a 1:1 binding model to obtain k a , k d and K D It was decided.

[0186] Octet dissociation rate and kinetic analysis were performed using an Octet Red96 (Forte Bio), PBS running buffer, and a Protein A sensor chip. Antibodies were diluted to 1 or 5 μg / mL in PBS for binding evaluation against PF or monomer, respectively. Antigens were diluted to 1 μM (Aβ1-40) or 30 nM (PF), and then serially diluted 1:3 for a total of three concentrations. Antibodies were captured onto the sensor to 1 nm load (Aβ1-40) or 0.2 nm load (PF), then these were immersed in antigen for 300 seconds and then dissociated in PBS for 360 seconds. Data were double-referenced and fit using a 1:1 binding model.

[0187] The screening and counter-screening workflows were performed as follows. For analysis of mouse serum titers, indirect ELISA was performed with Aβ pre-fibrils or Aβ-derived diffusible ligands, namely synthetic Aβ oligomers (ADDL) and / or Aβ 1 / 2 for tmax. For primary screening, indirect ELISA was performed against Aβ PF and / or ADDL. For counter-screening, oligomeric vs monomeric Aβ was selected to remove clones that bound strongly to the monomeric form. Dissociation rates were evaluated using Biacore or indirect ELISA (Figure 1A and Figure 1B). Indirect ELISA was used to identify aggregated alpha-synuclein and aggregated SOD to remove clones that also bound to other off-target amyloid structures.

[0188] For secondary screening, an in vitro cell-based assay was used to identify clones that could neutralize the neurite toxicity of AD brain extracts on iN cells.

[0189] For tertiary screening, an in vitro functional LTP assay was used to identify clones that could neutralize the inhibitory effect of AD brain extracts on LTP measured in brain slices.

[0190] Hybridoma Screening Overview Approximately 4,400 clones were subjected to primary ELISA screening. Three different immunization strategies were used and five fusions were performed. The primary ELISA screening was carried out against pre-fibrils derived from Aβ 1-42 and / or ADDLs derived from Aβ 1-42.

[0191] Approximately 80 clones were evaluated by counter-screening to identify clones with minimal or no binding to off-target entities such as Aβ 1-40 monomer and / or Aβ 1-42 monomer, Aβ 1-42 fibrils, synuclein fibrils, and aggregated SOD-1.

[0192] Approximately 50 clones were evaluated by secondary screening using a cell-based functional screening (e.g., iN cell assay). Multiple runs of the assay were performed using the original hybridomas. Additionally, the IgG1 LALA versions of the best candidate clones that were cloned, rearranged, and recombinantly produced were also tested. EC50 data were obtained and replicated for the best clones in order to rank the clones according to their ability to best neutralize oligomeric Aβ synaptic toxicity.

[0193] Four clones were evaluated by tertiary LTP functional screening at one or more concentrations and ranked according to their relative ability to rescue their LTP.

[0194] Lead clones were selected based on good binding selectivity and conservation of functional activity in both the iN and LTP assays.

[0195] Example 3: Fusion B (FusB) Overview Aggregated synthetic Aβ was administered to Trianni mice using Sigma adjuvant. Immunization was carried out using a total of 200 μl (50 μg antigen) per injection intraperitoneally (i.p.). The titers evaluated were 1 / 2Evaluated after 5 immunizations against tmax (4 μg / mL coat) or ADDL (1 μg / mL coat) (Figure 3). Based on these data, mouse M13 was set aside to rest before the final boost and fusion.

[0196] Mouse M13 (titer approximately 1:600,000) Approximately 670 clones were identified for primary screening with ADDL. 90 positive clones were first identified by ELISA against ADDL. The 90 positive clones were advanced to 24-well screening, of which 17 lost binding or did not have sufficient cells. Thus, 73 clones were advanced to the 6-well stage. From the 6-well stage, 15 clones were scaled up for production (Group 1), and 13 slow-growing ones were scaled up later (Group 2). In total, 28 clones were advanced to production and purified by Protein Maker. 4 clones were advanced to functional screening. The FusB screening is summarized in Figure 37. The binding and characterization data of the lead FusB clones are shown in Figures 4 - 18.

[0197] 2 clones were identified as 2 isotypes and recloned. 1 clone, B47-6, was advanced and purified together with the fusion C clones.

[0198] Example 4: Fusion C (FusC) Overview AD brain amyloid plaque seeded synthetic Aβ was administered to Trianni mice using Sigma adjuvant. 1 / 2 The titers were evaluated after 5 immunizations with respect to tmax (4 μg / mL coat) or ADDL (1 μg / mL coat) (Figure 18). Based on these data, mouse M23 was set aside before the final boost and fusion.

[0199] Approximately 750 clones were subjected to a primary screen for ADDL. Twenty-nine weak positives were initially identified and scaled up (96w - 24w - 6w - T150). Six clones maintained very weak positives throughout the scale-up. Two weakly positive clones were advanced to counter-screening. Since the two clones were identical, the remaining clones were reorganized and recombinantly produced as hIgG1 LALA, then re-screened and re-counter-screened. The FusC screening is summarized in Figure 38. The binding and characterization data of the lead FusC clones are shown in Figures 19 - 29.

[0200] One clone was advanced to functional screening.

[0201] Example 5: Cellular iN Assay Production of Aqueous Extract from Human AD Brain Frozen brain tissues were provided by the Manchester Brain Bank at the University of Miami Miller School of Medicine (Miami, FL) and the Medical Research Council at the University of Manchester (Manchester, UK). The brain tissues were obtained from two patients who died with mild to moderate stage AD. Aqueous extracts were prepared as previously described (Jin et al. (2018) Nature Comm. 9:2676). Briefly, 20 grams of temporal cortex gray matter was added to ice-cold artificial cerebrospinal fluid basic buffer (aCSF-B) (124 mM NaCl, 2.8 mM KCl, 1.25 mM NaH 2 PO 4 、26 mM NaHCO 3, homogenized in 5 volumes of pH 7.4 buffer using a Dounce homogenizer. The resulting homogenate was centrifuged at 200,000 g for 110 minutes at 4 °C using a SW41 Ti rotor (Beckman Coulter, Fullerton, CA). The upper 80% of the supernatant was removed, and the extract was dialyzed against fresh aCSF-B with buffer changes every 24 hours for 72 hours. The brain extract was then divided into two parts: one part was subjected to Aβ immunodepletion (ID) by incubating three times with anti-Aβ antibody, S97, and protein A sepharose (PAS) beads at 4 °C for 12 hours each. The second part was treated with pre-immune serum and PAS beads in the same manner. The Aβ-depleted extract was designated ID-AD, and the extract treated with pre-immune serum was designated sham-AD. Beads were removed from the samples, and 0.5 mL aliquots were transferred to low-protein-binding Eppendorf tubes (Eppendorf, Hamburg, Germany) and stored at -80 °C until use. Samples were thawed once and used. Brain extracts from two different cases, AD10 / 27 and AD10 / 14, were used in the functional assays.

[0202] iPSC-Derived Human Neurons (iN) Neurogenin 2 (Ngn2)-induced human neurons (Zhang et al., 2013) were generated as previously described (Hong et al., 2018; Jin et al., 2018). Briefly, YZ1 iPSCs were maintained in medium containing DMEM / F12, knockout serum replacement, penicillin / streptomycin / glutamine, MEM-NEAA, and 2-mercaptoethanol (all from Invitrogen, Carlsbad, CA) and 10 μg / mL bFGF (Millipore, Billerica, MA). The iPSCs were then seeded at 95,000 cells / cm 2Plated at the following concentrations for viral infection at a density of: pTet-O-NGN2-puro: 0.1 μL / 50,000 cells; Tet-O-FUW-eGFP: 0.05 μL / 50,000 cells; Fudelta GW-rtTA: 0.11 μL / 50,000 cells (Alstem, Richmond, CA). To induce neurogenin 2 expression, doxycycline was added at a concentration of 2 μg / mL on “iN day 1”, and puromycin was added at 10 mg / mL on iN day 2 and then maintained in the medium at all subsequent time points. On iN day 4, cells were plated at 5,000 cells / well in Matrigel (BD Biosciences, San Jose, CA)-coated Greiner 96-well microclear plates and maintained in medium consisting of Neurobasal medium (Gibco), Glutamax, 20% dextrose, MEM-NEAA and B27 with BDNF, CNTF, GDNF (PeprpTech, Rocky Hill, NJ) at a concentration of 10 ng / mL each. The number of neurites and the expression of neural markers reached a maximum by iN day 14, and the iNs were fully mature by iN day 21. To examine the effect of AD brain extracts on neurite integrity, cells were used on iN day 21.

[0203] Sample Addition and Live Cell Imaging On the 21st day after induction, the effect of the sample on neurite integrity was examined using neurons with an Incucyte live imaging reader. Approximately 7 hours before adding the sample, images were collected every 2 hours for a total of 6 hours from 4 regions per well, and the baseline neurite length and branching sites were calculated. During the last interval, the brain samples were exchanged using a PD MidiTrap G-25 column (GE Healthcare Life Science, Milwaukee, WI) in neurobasal medium supplemented with B27 / Glutamax. Following 6 hours of baseline imaging, half of the medium was removed from each well (leaving approximately 100 μL), and 50 μL of the exchanged extract or vehicle was added together with 50 μL of fresh medium. Subsequently, images were collected every 2 hours for at least 72 hours from 4 regions per well. Phase contrast image sets were analyzed using IncuCyte Zoom 2016A software (Essen Bioscience, Ann Arbor, MI). Neurites and cell bodies were automatically defined based on phase contrast images using the analysis job Neural Track. Typical settings were: Segmentation mode - Brightness; Segmentation adjustment - 1.2; Cell body cluster filter - minimum 500 μm 2 ; Neurite filtering - Highest; Neurite sensitivity - 0.4; Neurite width - 2 μm. The total neurite length (millimeters) and the number of branching sites were quantified and normalized to the mean value measured during the 6 hours before sample addition. AD brain extracts (+ / - immunodepletion) were added to neurons + / - test mAb.

[0204] The neurite length at the end of the 72-hour period was averaged over the last 3 time points, and the percent protection was calculated at each concentration by comparing to a) no antibody condition (AD) and b) immunodepleted brain extracts (ID-AD). The concentration for 50% neurite toxicity protection (EC50) was calculated.

[0205] Data Analysis and Statistical Tests For live cell imaging experiments, samples and treatments were coded and tested in a blinded fashion. Differences between groups were tested using Bonferroni post - hoc tests or Student's t - tests with two - way analysis of variance (ANOVA). #p < 0.05, ##p < 0.01, and p < 0.001.

[0206] Functional Effects of Hybridoma-Purified mAb in Cell iN Assay Hybridoma - purified mAbs were tested in the cell iN assay to evaluate protection of neurite outgrowth against toxicity induced by soluble AD brain extracts. In human neuron iN cultures, continuous exposure to AD brain extract (AD10 / 27) resulted in a ~50% decrease in human neurons measured after 3 days (see AD samples vs. media or ID samples in Figure 30). Pre - immunodepletion of Aβ from the AD extract (sample ID) prevented neurite toxicity, demonstrating that, as previously shown (Jin et al., 2012; 2018), AD brain extracts contain Aβ - dependent neurite - toxic activity. Further validation of this assay was provided by the positive control Aβ - specific antibody 3D6 at 3 μg / ml, which was able to neutralize the neurite - toxic brain extract, as previously shown (Shankar et al., 2008; Jin et al., 2011) (code C1 in Figure 30). 3D6 binds to the N - terminal free end of the Aβ sequence and recognizes all conformations of Aβ. Another positive control 1C22 (which recognizes multiple oligomeric forms of Aβ but not monomeric Aβ, Jin et al. 2018) was also used in other experiments (see Table 3). Since the exact nature of the toxic Aβ - containing entity in AD brain extracts has not yet been characterized, care was taken to select a large panel of mAbs from immunization campaigns with broad affinity for soluble pre - fibrillar and aggregated Aβ but no binding to monomeric Aβ. This was only possible due to the good capacity / throughput of the unique cell - functional iN assay used (format multiwall MW96). Brain extracts from two different preparations of the same case AD10 / 27 (AD10 / 27 1 st 、2 ndWe used those shown (in Figure 30). Some of the hybridoma-purified mAbs showed neuroprotection similar to positive controls that ranged from mAbs with moderate affinity for PF Aβ forms (such as B73 or B24) to mAbs with high affinity for PF such as B51 or B90. However, the biochemical binding profiles did not predict activity in the iN assay, as B35 and B61 (which have moderate affinity for PF Aβ forms) were inactive in the iN assay, while B8, B30, or B60 had very high affinity for Aβ.

[0207] [[Table 11]]

[0208] Therefore, the iN functional assay using AD brain extracts represents a unique tool for characterizing and selecting novel Aβ mAbs with highly relevant biological activity. Due to the sensitivity of the iN assay, several independent experiments were conducted to ensure the data, including experiments using antibodies generated from additional fusions (see Table 5).

[0209] The neuroprotective effects of the various antibodies varied somewhat between experiments, due in part to the limited stock concentrations of these antibodies, which resulted in limited dilution in the iN cell medium that decreased the modest stability or cell viability of the antibody batches purified from the hybridomas. In some cases, independent antibody production batches had to be generated. For example, hybridoma B28 was not very active in Run #3 and was negative in Runs #4 and 5b, which may be due to stability issues. It was also unexpected that clone B73 was initially negative in the iN runs using the first hybridoma production, but the second preparation yielded very significant activity in Runs #4 and 5b.

[0210] All mAbs can be introduced into the same IgG1 LALA framework, and for further analysis using recombinant versions of hybridoma clones that enabled more controlled production conditions, clones were selected that showed neuroprotection in at least one iN experimental run and had diverse affinities for synthetic Aβ conformers, especially low affinities for prefibrillar and fully aggregated synthetic Aβ preparations (in addition to having no / low affinity for monomeric Aβ).

[0211] Functional Effects of Recombinant mAb in Cell iN Assay Recombinant versions of the selected mAbs were produced at high stock concentrations (>2 mg / ml) and tested in cell iN assays. For each mAb, a first test at one mAb concentration (3 μg / ml in the well) was performed using brain extract AD10 / 27 to confirm the neurite protection previously observed using the hybridoma-purified version (Table 5, runs #6, 8, and 9). Based on the distinctiveness of the biochemical binding profile (lowest possible affinity for classical synthetic forms of Aβ but very significant protection against Aβ Ad brain extracts), a full concentration-response curve was performed for the most interesting mAbs using another brain extract, AD10 / 14. It was important to establish that the neuroprotective activity of the mAbs could also be observed in extracts from other AD cases. The mAbs were tested at concentrations ranging from 0.75 μg / ml to 6 μg / ml or up to 12 μg / ml. As an example, the overall time-course results for mAbs rB24 and rB75 compared to positive control 1C22 are shown in the upper and middle panels of Figure 31, respectively, demonstrating concentration-dependent protection by rB24 and rB75 against neurite loss induced by AD brain extract AD10 / 14. The concentrations (EC50) for 50% neurite toxicity protection were determined to be 2,528 and 2,111 ng / ml for rB24 and rB75, respectively, compared to 1,049 for positive control 1C22 (Figure 31, lower panel). Comparative data are shown in Figure 32 for the pair of rC11 and rB24, and in Figure 33 for the pair of rB73 and rB28.

[0212] Perform a number of experiments using 2 - 3 mAbs and always compare with positive control 1C22, and summarize the EC50 results in Table 6. All selected recombinant mAbs were within 2 - fold the activity of 1C22.

[0213]

Table 12

[0214] Example 6: Long - term potentiation electrophysiological functional assay to demonstrate protection against soluble AD brain extract Preparation of Aqueous Extract from Human AD Brain The preparation of aqueous extracts from human AD brains was performed as above for the functional cell iN assay. Samples were thawed and used only once. Brain extracts from AD case AD10 / 14 were used in the long - term potentiation (LTP) assay.

[0215] Electrophysiological Recording and Testing of Protection by Antibodies Hippocampal LTP recordings were performed using adult mouse (2 - 3 months old, both male and female) brain slices (350 μm thick) in the same way as previous methods (Shankar et al. (2008) Nat. Med. 14(8):837 - 842; Li et al. (2011) J. Neurosci. 31(18):6627 - 6638; Li et al. (2018) Acta Neuropathol. Commun. 6(1):121). Recordings were made on the radiate layer of the hippocampal CA1 region, and the stimulating electrode was placed on the Schaffer collateral using a microelectrode array (MEA). The MED64 recording system (Alpha MED Scientific, Japan) was used for extracellular electric field potential recording. There was an array of 64 planar microelectrodes in the MED64 probe (P515A) arranged in an 8×8 pattern with a 150 - μm inter - electrode distance (Liu et al. (2011) Brain Res. 1382:57). Antibodies (C11, B24, B28, B73, and B75) were added to the perfused artificial cerebrospinal fluid (ACSF) at a concentration of 5 μg / ml. When testing AD extracts, to avoid problems with slice quality, the control group, AD extract group, and antibody + AD extract conditions were randomly selected. Each mouse was checked for daily recordings to ensure that all brain slices were responsive to the antibody or AD extract. If a slice showed only small LTP or no LTP under the test conditions, the data from that mouse was not used. 1) For the antibody - alone condition, the antibody was added to 10 mL of perfused ACSF ensuring a stable baseline in at least 30 minutes of recording, and then high - frequency stimulation (HFS) was applied. After stimulation, recording was continued for an additional 60 minutes. 2) For the antibody plus AD extract experiment, both the antibody and the AD extract (AD10 / 14, diluted 1 / 20) were thawed at room temperature and then gently vortexed. Each antibody was mixed with an AD extract aliquot (0.5 ml) and gently shaken for 60 minutes, then added to 9.5 mL of perfused ACSF (to make a total volume of 10 mL).

[0216] Data Analysis and Statistical Assays Samples and treatments were coded and tested in a blinded fashion. EPSP slope values 60 min after HFS were quantified relative to baseline for each brain slice, and the mean was calculated for each antibody (number of brain slices per antibody / condition in the figure legend). Differences between groups were examined using two-way analysis of variance (ANOVA) with Bonferroni post hoc tests or Student's t-tests. #p < 0.05, ##p < 0.01, and p < 0.001.

[0217] Testing of Selected Human Recombinant mAb in Electrophysiology Assay Hippocampal long-term potentiation (LTP) recordings were performed to analyze synaptic plasticity in brain slices, a model thought to represent memory encoding. It has previously been shown that AD soluble brain extracts contain Aβ-dependent activity that strongly inhibits the induction of LTP by HFS in rodent brain slices (Shankar, G.M. et al. 2008, Hong, W. et al., 2018). First, the effects of the selected antibodies (C11, B24, B28, B73, and B75) on basal LTP were examined. Antibodies were added to the perfusing ACSF at a final concentration of 5 μg / ml in ACSF. At this concentration, C11, B24, B28, and B75 had no effect on basal transmission or LTP induction by HFS (Figs. 34A, 34B), whereas B73 significantly decreased it. B73 had no effect on LTP induction at a concentration lower than 3 μg / ml and was subsequently used.

[0218] A variety of antibodies were then tested (5 μg / ml except for B73 which was 3 μg / ml) to analyze whether they could prevent the inhibitory effect of AD brain extracts on the induction of LTP. As can be seen in FIGS. 35A and 35B, in the presence of the AD brain extract AD10 / 14, the enhancement of EPSP 1 hour after HFS was only 120% of the baseline compared to 150% with ACSF alone (FIG. 34), confirming the inhibitory activity of the AD brain extract. Pre-incubation of the AD brain extract with various antibodies resulted in a very significantly high level of EPSP enhancement that returned to the value of ACSF alone (FIGS. 35A, 35B, p-value lower than 0.05). Most surprisingly, both 5 μg / ml of B75 and 3 μg / ml of B73 resulted in a complete rescue of the inhibitory effect of the AD brain extract on LTP. Thus, it can be concluded that the antibodies described herein can neutralize the LTP inhibitory activity present in AD brain extracts in addition to their protective effects observed in human neuron cultures.

[0219] The most performant antibodies, B73, B75 and C11, were tested at a lower concentration of 2 μg / ml and compared to the reference antibody 1C22 (FIGS. 36A and 36B). A new batch of B73 was used for this experiment. At these low concentrations, it was confirmed that none of the antibodies affected basal transmission or LTP induction by HFS (FIG. 36A). Next, the antibodies were tested for their prevention of the inhibitory effect of AD brain extracts on LTP induction (at 2 μg / ml). At this concentration, B73 retained almost complete prevention of the AD brain extract effect on LTP (FIG. 36B, p-value lower than 0.05), consistent with previous data obtained at 3 μg / ml. C11 also significantly prevented the AD brain extract effect by well over 50% (p-value lower than 0.01), and both were not inferior compared to the reference antibody 1C22. B75 was not significantly effective at this lower concentration. These data support and expand the electrophysiological results discussed above.

[0220] Example 7: Antibodies can capture Aβ from the AD brain but do not bind to amyloid deposits in AD brain sections Biochemical Data Regarding B24, B28, B73, B75 and C11 mAb Using Bead-Based Immunoprecipitation of Human AD Brain Extract: B75 and C11 Immunoprecipitated Aβ x-42 Peptide from AD Brain Immunoprecipitation from human brain extracts was performed using the conventional magnetic bead method, and B24, B28, B73, B75, and C11 mAbs were tested (Figure 39A). Successive elution using 1% SDS followed by 6 M guanidine hydrochloride (GnCl) was measured by Aβ x-42 ELISA. Compared to the negative control (human IgG) and the positive control (1C22), B28 and B73 were able to significantly immunoprecipitate Aβ 1-42-containing species from all four brains tested, consistent with their broad Aβ-binding properties. To a somewhat lesser extent, B75 and C11 were also able to significantly immunoprecipitate Aβ 1-42-containing species from two of the four brains, but B24 did not immunoprecipitate Aβ 1-42-containing species from any of the brains tested (Figure 39B). These data demonstrate that B75 and C11 bind to β 1-42-containing material in the AD brain but do not bind to different synthetic Aβ preparations as described above.

[0221] Biochemical Data Using Column Immunoadsorption of Human AD Brain Extract: C11 and B28 Bound to Aβ-Containing Species in Human AD Brain An alternative method was designed to retest B24, B75, and C11, which showed mild affinity for Aβ-containing species by the conventional magnetic bead method. Instead, a protein-A spin column (Figure 40A) was used, omitting procedures such as rotation or nutation that disrupt weak antibody-antigen binding. This alternative method showed that C11 captured as much Aβ 1-42-containing species from two human extracts as B28 (positive control) (Figure 40B), but emphasized that B75 was less active and that different antibody properties could be revealed by the alternative technique.

[0222] Furthermore, using such a spin column, antigen-antibody interaction was destabilized, and elution was performed using a milder acid washing means on the antigen structure than SDS or guanidine washing performed in the bead immunoprecipitation example. Therefore, the potential structure of the immunocaptured substance was evaluated using a transmission electron microscope (TEM) (Figure 41A). The negatively stained substances purified by B28 and C11 were determined by TEM to be polymorphic (Figure 41B). The B28 purified substance had mainly spherical structures of various sizes. The C11 purified substance had more diverse morphologies.

[0223] Immunohistology: C11 Did Not Bind to Aβ Deposits in Human AD Brain as Evaluated by Light Microscopy Immunohistology Immunohistochemical techniques were used to compare the binding patterns of various antibodies to fixed or freshly frozen human AD brain sections. First, all five mAbs were evaluated by immunohistochemistry on standard paraffin-embedded PFA-fixed AD brain sections. Only B28 produced staining, i.e., was determined to be typical of AD amyloid plaques and cerebral amyloid angiopathy (Figure 42A). The B24, B28, B73, B75, and C11 mAbs were further tested by immunohistochemistry on unfixed brain frozen sections and represented the possibility of a more native conformation of Aβ aggregates in the AD brain. Compared to 1C22 (positive control), B28, B73, and B75 strongly stained amyloid plaques, B24 stained moderately, and C11 did not stain (Figure 42B).

[0224] Materials and Methods Immunoprecipitation (Beads) and Immunoadsorption (Column) of Human Brain Extract Beads: 800 μl of brain extract in TBS was mixed with 10 μg of mAb and protein-A magnetic beads and rotated at 4°C for 12 hours. The magnetic beads were then washed three times with PBS for 30 minutes each at 4°C. The washed magnetic beads were eluted successively with 1% SDS in PBS and then 6 M guanidine hydrochloride.

[0225] Column: 800 μl of TBS brain extract was mixed with 10 μg of mAb and incubated at 4°C for 12 hours. This mixture was applied to a pre-wetted protein A column and rotated at 1,000 g for 1 minute. The flow-through was applied to the same column and rotated at 1,000 g for 1 minute. The column was washed three times with PBS and rotated at 1,000 g for 1 minute. 0.1 M glycine-HCl (pH 2.3) was added to the column and incubated for 5 minutes, followed by rotation at 1,000 g for 1 minute.

[0226] Aβ x-42 ELISA MSD ELISA for Aβ 1-42 was performed by a method known in the art (Liu et al., 2019). Each well of an uncoated 96-well multiarray plate (Meso Scale Discovery, #L15XA-3) was coated with 30 μL of a PBS solution containing 3 μg / mL of the capture antibody (Elan) 266 and incubated overnight at room temperature. The 266 epitope is in the region 13-25 of the Aβ sequence. The detection antibody solution was prepared using a biotinylated monoclonal antibody against the C-terminal residue of Aβ 1-42 (21F12), 100 ng / mL streptavidin sulfo-TAG (Meso Scale Discovery, #R32AD-5), and 1% BSA diluted in wash buffer. After overnight incubation, 50 μL / well of sample, followed by 25 μL / well of the detection antibody solution, was incubated for 2 hours at room temperature with shaking at >300 rpm, and the wells were washed with wash buffer during incubation. The plate was read and analyzed according to the manufacturer's protocol.

[0227] Transmission Electron Microscopy Analysis of Negatively Stained Samples For TEM specimen preparation, 5 μL of the sample solution (glycine eluate of the immunosorbent material (see above)) was placed on a glow-discharged, formvar / carbon-coated grid. The sample was incubated on the grid at room temperature for 20 seconds. Excess solvent was removed using filter paper (Whatman). The grid was washed three times with 10 μL of water and then stained three times with 10 μL of 1% (mass / volume) uranyl acetate in water. The dried grid was examined in a JEM-1200EX TEM (JEOL) equipped with an AMT 2k CCD camera operated at 80 kV.

[0228] Immunohistochemistry Paraffin-embedded PFA-fixed brain sections: Rehydrated paraffin sections were treated with 0.3% H 2 O 2 in PBS containing 0.2% Triton X-100. These sections were incubated overnight at 4 °C with the primary antibody and then incubated for 1 hour with the biotinylated secondary antibody. For visualization, the sections were treated with an avidin-biotin complex (Vector) and then treated with 3,3'-diaminobenzidine containing ammonium nickel sulfate.

[0229] Unfixed brain frozen sections: Fresh or thawed brain tissue was embedded in O.C.T. compound (Sakura) solution and subsequently incubated at -80 °C and -20 °C for 12 hours each. Thin sections were made at 20 - 30 μm intervals using a cryostat (Leica) on the frozen block. The sections were placed directly on adhesive microscope glass slides (Matsunami) for immunohistochemistry. The sections were then treated with 0.3% H 2 O 2 in PBS containing 0.2% Triton X-100. The sections were incubated overnight at 4 °C with the primary antibody and then incubated for 1 hour with the biotinylated secondary antibody. For visualization, the sections were treated with an avidin-biotin complex (Vector) and then treated with 3,3'-diaminobenzidine containing ammonium nickel sulfate. Microscopic photographs were taken with a DMi8 wide-field microscope (Leica).

Claims

**Claim 1** An isolated binding polypeptide comprising an antibody or antigen-binding fragment thereof that specifically binds to soluble amyloid beta (Aβ), wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining region (HCDR) sequences and three light chain complementarity determining region (LCDR) sequences, wherein the three HCDR1, 2, 3 sequences each comprise SEQ ID NO: 50, 51 and 52, and the three LCDR1, 2, 3 sequences each comprise SEQ ID NO: 47, 48 and 49, or the three HCDR1, 2, 3 sequences each comprise SEQ ID NO: 62, 63 and 64, and the three LCDR1, 2, 3 sequences each comprise SEQ ID NO: 59, 60 and 61, said binding polypeptide. **Claim 2** The binding polypeptide according to claim 1, wherein the antibody or antigen-binding fragment thereof is human. **Claim 3** The binding polypeptide according to claim 1, wherein the antibody or antigen-binding fragment thereof is IgG1. **Claim 4** The binding polypeptide according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair comprising SEQ ID NO: 12 and 11; or SEQ ID NO: 16 and 15. **Claim 5** The binding polypeptide according to any one of claims 1 to 4, wherein the soluble Aβ is synaptotoxic, or has a molecular weight between 20 kD and 100 kD, or both. **Claim 6** The binding polypeptide according to any one of claims 1 to 5, which specifically binds to soluble Aβ in the brain from a subject having Alzheimer's disease or neutralizes Aβ synaptotoxicity. **Claim 7** The binding polypeptide according to any one of claims 1 to 6, which does not specifically bind to monomeric Aβ, pro-fibrillar Aβ or fibrillar Aβ, or protein aggregates, or amyloid plaques present in the brain from a subject having Alzheimer's disease. **Claim 8** The binding polypeptide according to claim 6, wherein the binding is immunoadsorption. **Claim 9** The binding polypeptide according to claim 6 or 8, wherein the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject having Alzheimer's disease. **Claim 10** The binding polypeptide according to any one of claims 6, 8 or 9, which neutralizes the synaptotoxicity of soluble Aβ in the brain from a subject having Alzheimer's disease. **Claim 11** A pharmaceutical composition comprising the binding polypeptide according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. A binding polypeptide for use in the treatment of Alzheimer's disease in a subject, comprising administering an effective amount of the pharmaceutical composition according to claim 11 to the subject.

13. An isolated polynucleotide encoding the binding polypeptide according to any one of claims 1 to 4.

14. A vector comprising the polynucleotide according to claim 13.

15. A host cell comprising the polynucleotide according to claim 13 or the vector according to claim 14.

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