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

Abstract

The present invention relates to neutralizing anti-amyloid beta antibodies for use in the treatment of Alzheimer's disease, and specifically provides binding polypeptides (e.g., antibodies and antigen binding fragments thereof) that specifically bind to one or more species of soluble AD brain-derived synaptic toxic amyloid beta (A beta) without binding to classical monomers, protofibrils, or fibril A beta. The present invention provides pharmaceutical compositions comprising a binding polypeptide that specifically binds to one or more species of soluble synaptic-toxic A [beta]. The present invention provides methods of making binding polypeptides that specifically bind to one or more species of soluble synaptic-toxic A [beta]. The present invention provides methods of treating Alzheimer's disease using binding polypeptides that specifically bind to one or more species of soluble synaptic toxic A [beta]. The invention also provides methods of reducing one or more symptoms of Alzheimer's disease using binding polypeptides that specifically bind to one or more species of soluble synaptic toxic A [beta].

Description

[0001] This invention application is a divisional application of the invention patent application filed on July 15, 2020, with application number 202080063538.0 (international application number PCT / US2020 / 042161) entitled "Neutralizing anti-β-amyloid antibody for the treatment of Alzheimer's disease".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 874,724, filed July 16, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0004] This invention relates to novel binding peptides that specifically bind to soluble β-amyloid protein. The invention also relates to methods for treating Alzheimer's disease using the novel binding peptides provided herein. Background Technology

[0005] Alzheimer's disease is the most common type of dementia. It affects tens of millions of people worldwide, and that number is rising rapidly. The amyloid hypothesis (Haass and Selkoe (1993) Cell 75:1039; Glenner and Wong (2012) Biochem Biophys Res Commun 425:534; and Selkoe and Hardy (2016) EMBO Mol Med 8:595) proposed that β-amyloid (Aβ) is the primary cause of the disease and showed that the misfolding of extracellular Aβ protein accumulating in senile plaques (Bloom (2014) JAMA Neurol. 71:505) and the intracellular deposition of misfolded tau protein in neurofibrillary tangles cause memory loss and executive dysfunction, leading to cognitive and behavioral decline over time. Accumulated Aβ peptides are a major component of age-related (amyloid) plaques and are derived from the proteolytic cleavage of a larger glycoprotein called amyloid precursor protein (APP) (Chen et al. (2017) Acta Pharmacologica Sinica 38:1205; Liu et al. (2019) J. Cell Biol. 218:644).

[0006] Aβ monomers aggregate into various types of assemblages, including oligomers, primary protofibrils, and amyloid protofibrils. Amyloid protofibrils are large and insoluble, and they can further assemble into amyloid plaques, while amyloid oligomers are water-soluble and can diffuse throughout the brain. Aβ encompasses a group of peptides ranging in size from 37 to 49 residues. Amyloid plaques with Aβ as a major component are most commonly found in the limbic and neocortical regions of the brains of Alzheimer's disease patients. In Alzheimer's disease, Aβ (1–42) is the major protein component of amyloid deposits.

[0007] Although amyloid fibrils are large, insoluble, and aggregate to form fibrillary amyloid plaques, the histological lesions characteristic of Alzheimer's disease, 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 species of approximately 100–200 kDa under relatively physiological conditions in vitro (Goldsbury et al. (2000) J. Struct. Biol. 130:217; Nichols et al. (2002) Biochemistry 41:6115; Lashuel et al. (2003) J. Mol. Biol. 332:795; Walsh et al. (1997) J. Biol. Chem. 272:22364; Soreghan et al. (1994) J. Biol. Chem. 269:28551). Aβ monomers can form high-order assemblies ranging from low molecular weight oligomers (including dimers, trimers, and tetramers) to medium molecular weight oligomers (including hexamers and dodecamers) to soluble primary fibrils and insoluble fibrils (Chen et al., ibid.).

[0008] The use of monoclonal antibodies targeting β-amyloid (Aβ) constitutes the largest and most advanced therapeutic effort for Alzheimer's disease (AD) (Liu et al. (2016) Drugs Aging 33:685; Golde (2014) Alzheimer's Res. Ther. 6:3; van Dyck (2017) Biol. Psychiatry 83:311). Despite generally favorable outcomes in preclinical mouse models, anti-Aβ immunotherapy has achieved limited success in humans (Golde, van Dyck, ibid.). Explanations for the poor translatability of preclinical lead antibodies into human therapies include imperfect trial design, intervention at disease stages with pre-existing significant neurological damage, and inappropriate target selectivity of the antibodies used (Golde, ibid.; Kohyama and Matsumoto (2015) Immunotargets Ther. 4:27; Selkoe and Hardy, ibid.).

[0009] Therefore, there remains a need for alternative therapeutic monoclonal antibodies for the treatment of Alzheimer's disease. Summary of the Invention

[0010] This invention is based on the discovery of novel anti-β-amyloid (Aβ) binding proteins (e.g., antibodies) that specifically bind to epitopes of one or more types of water-soluble AD brain-derived synaptic toxic Aβ.

[0011] Therefore, in some aspects, a specific binding polypeptide that specifically binds to soluble Aβ is provided, wherein the binding polypeptide comprises three heavy chain complementarity-determining region (HCDR) sequences and three light chain complementarity-determining region (LCDR) sequences. In some embodiments, the three HCDR sequences are selected from SEQ ID NO: 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 SEQ ID NO: 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.

[0012] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0013] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0014] In some aspects, a separate binding polypeptide that specifically binds to soluble Aβ is provided, wherein the binding polypeptide comprises a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence. In some embodiments, the HCVR sequence is selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16, and the LCVR sequence is selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15.

[0015] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0016] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0017] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, wherein the binding polypeptide comprises an HCVR / LCVR sequence pair. In some embodiments, the HCVR / LCVR pair is selected from SEQ ID NO: 2 / 1, 4 / 3, 6 / 5, 8 / 7, 10 / 9, 12 / 11, 14 / 13, and 16 / 15.

[0018] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0019] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0020] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCDR sequences of SEQ ID NO: 20, 21, and 22 and the LCDR sequences of SEQ ID NO: 17, 18, and 19. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCVR / LCVR pair of SEQ ID NO: 2 and 1. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the all-heavy chain / light chain sequence pair of SEQ ID NO: 66 and 65.

[0021] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0022] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0023] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCDR sequences of SEQ ID NO: 26, 27 and 28 and the LCDR sequences of SEQ ID NO: 23, 24 and 25. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCVR / LCVR pair of SEQ ID NO: 4 and 3. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the all-heavy chain / light chain sequence pair of SEQ ID NO: 68 and 67.

[0024] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0025] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0026] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCDR sequences of SEQ ID NO: 32, 33 and 34 and the LCDR sequences of SEQ ID NO: 29, 30 and 31. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCVR / LCVR pair of SEQ ID NO: 6 and 5. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the all-heavy chain / light chain sequence pair of SEQ ID NO: 70 and 69.

[0027] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0028] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0029] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCDR sequences of SEQ ID NO: 38, 39 and 40 and the LCDR sequences of SEQ ID NO: 35, 36 and 37. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCVR / LCVR pair of SEQ ID NO: 8 and 7. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the all-heavy chain / light chain sequence pair of SEQ ID NO: 72 and 71.

[0030] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0031] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0032] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCDR sequences of SEQ ID NO: 44, 45 and 46 and the LCDR sequences of SEQ ID NO: 41, 42 and 43. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCVR / LCVR pair of SEQ ID NO: 10 and 9. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the all-heavy chain / light chain sequence pair of SEQ ID NO: 74 and 73.

[0033] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0034] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0035] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCDR sequences of SEQ ID NO: 50, 51, and 52 and the LCDR sequences of SEQ ID NO: 47, 48, and 49. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCVR / LCVR pair of SEQ ID NO: 12 and 11. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the all-heavy chain / light chain sequence pair of SEQ ID NO: 76 and 75.

[0036] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0037] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0038] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCDR sequences of SEQ ID NO: 56, 57 and 58 and the LCDR sequences of SEQ ID NO: 53, 54 and 55. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCVR / LCVR pair of SEQ ID NO: 14 and 13. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the all-heavy chain / light chain sequence pair of SEQ ID NO: 78 and 77.

[0039] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0040] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0041] In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCDR sequences of SEQ ID NO: 62, 63, and 64 and the LCDR sequences of SEQ ID NO: 59, 60, and 61. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the HCVR / LCVR pair of SEQ ID NO: 16 and 15. In some aspects, a specific binding polypeptide that binds to soluble Aβ is provided, the binding polypeptide comprising the all-heavy chain / light chain sequence pair of SEQ ID NO: 80 and 79.

[0042] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0043] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0044] In some aspects, an isolated binding polypeptide that specifically binds to soluble β-amyloid (Aβ) is provided, 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 SEQ ID NO: 20, 21, 22, 50, 51, 52, 44, 45, 46, 26, 27, 28, 62, 63 and 64, and wherein the three LCDR sequences are selected from SEQ ID NO: 17, 18, 19, 47, 48, 49, 41, 42, 43, 23, 24, 25, 59, 60 and 61.

[0045] In some exemplary embodiments, the binding polypeptide comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is optionally human and / or optionally IgG1.

[0046] In some exemplary embodiments, the binding polypeptide comprises a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the following: 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.

[0047] In some exemplary embodiments, the three HCDR sequences comprise SEQ ID NO: 20, 21, and 22, and the three LCDR sequences comprise SEQ ID NO: 17, 18, and 19. In some exemplary embodiments, the three HCDR sequences comprise SEQ ID NO: 44, 45, and 46, and the three LCDR sequences comprise SEQ ID NO: 41, 42, and 43. In some exemplary embodiments, the three HCDR sequences comprise SEQ ID NO: 26, 27, and 28, and the three LCDR sequences comprise SEQ ID NO: 23, 24, and 25. In some exemplary embodiments, the three HCDR sequences comprise SEQ ID NO: 50, 51, and 52, and the three LCDR sequences comprise SEQ ID NO: 47, 48, and 49. In some exemplary embodiments, the three HCDR sequences comprise SEQ ID NO: 62, 63, and 64, and the three LCDR sequences comprise SEQ ID NO: 59, 60, and 61.

[0048] In some exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 2 and 1. In some exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 10 and 9. In some exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 4 and 3. In some exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 12 and 11. In some exemplary embodiments, the HCVR / LCVR sequence pair is SEQ ID NO: 16 and 15.

[0049] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0050] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0051] In some aspects, a pharmaceutical composition is provided comprising an isolated binding polypeptide that specifically binds to soluble β-amyloid (Aβ) and a pharmaceutically acceptable carrier, 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 SEQ ID NO: 20, 21, 22, 50, 51, 52, 44, 45, 46, 26, 27, 28, 62, 63 and 64, and wherein the three LCDR sequences are selected from SEQ ID NO: 17, 18, 19, 47, 48, 49, 41, 42, 43, 23, 24, 25, 59, 60 and 61.

[0052] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0053] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0054] In some exemplary embodiments, a method for treating a subject with Alzheimer's disease is provided, the method comprising administering an effective amount of the pharmaceutical composition to the subject.

[0055] In some aspects, a pharmaceutical composition is provided comprising an isolated binding polypeptide that specifically binds to soluble β-amyloid (Aβ) and a pharmaceutically acceptable carrier, wherein the binding polypeptide comprises a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the following: 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.

[0056] In some exemplary embodiments, the soluble Aβ has synaptic toxicity. In some exemplary embodiments, the binding polypeptide neutralizes the Aβ synaptic toxicity. In some exemplary embodiments, the soluble Aβ has a molecular weight between about 20 kDa and about 100 kDa.

[0057] In some exemplary embodiments, the binding polypeptide does not specifically bind to monomeric Aβ, primary fibrillary Aβ, or fibrillary Aβ. In some exemplary embodiments, the binding polypeptide does not specifically bind to protein aggregates. In some exemplary embodiments, the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide specifically binds to soluble Aβ from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding is immunoadsorption. In some exemplary embodiments, the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease. In some exemplary embodiments, the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ.

[0058] In some exemplary embodiments, a method for treating a subject with Alzheimer's disease is provided, the method comprising administering an effective amount of the pharmaceutical composition to the subject.

[0059] In some aspects, there is provided an isolated polynucleotide encoding a binding polypeptide that specifically binds to soluble β-amyloid (Aβ), said binding polypeptide comprising three heavy chain complementarity-determining region (HCDR) sequences and three light chain complementarity-determining region (LCDR) sequences, said three HCDR sequences being selected from SEQ ID NO: 20, 21, 22, 50, 51, 52, 44, 45, 46, 26, 27, 28, 62, 63 and 64, and said three LCDR sequences being selected from SEQ ID NO: 17, 18, 19, 47, 48, 49, 41, 42, 43, 23, 24, 25, 59, 60 and 61, and a pharmaceutically acceptable vector.

[0060] In some exemplary embodiments, a vector encoding the polynucleotide is provided.

[0061] In some exemplary embodiments, a host cell comprising the polynucleotide or the vector is provided.

[0062] Specifically, the present invention includes, but is not limited to, the following:

[0063] 1. An isolated binding polypeptide that specifically binds to soluble β-amyloid (Aβ), wherein the binding polypeptide comprises three heavy chain complementarity-determining region (HCDR) sequences and three light chain complementarity-determining region (LCDR) sequences.

[0064] The three HCDR sequences are selected from SEQ ID NO: 20, 21, 22, 50, 51, 52, 44, 45, 46, 26, 27, 28, 62, 63 and 64, and

[0065] The three LCDR sequences are selected from SEQ ID NO: 17, 18, 19, 47, 48, 49, 41, 42, 43, 23, 24, 25, 59, 60 and 61.

[0066] 2. The binding polypeptide according to claim 1, wherein the binding polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0067] 3. The antibody according to claim 2, wherein the antibody or its antigen-binding fragment is human.

[0068] 4. The human antibody according to claim 2, wherein the antibody or its antigen-binding fragment is IgG1.

[0069] 5. The binding polypeptide according to claim 1, wherein the binding polypeptide comprises a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the following: 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.

[0070] 6. The binding polypeptide according to claim 1, wherein the three HCDR sequences comprise SEQ ID NO: 20, 21 and 22, and the three LCDR sequences comprise SEQ ID NO: 17, 18 and 19.

[0071] 7. The binding polypeptide according to claim 1, wherein the three HCDR sequences comprise SEQ ID NO: 44, 45 and 46, and the three LCDR sequences comprise SEQ ID NO: 41, 42 and 43.

[0072] 8. The binding polypeptide according to claim 1, wherein the three HCDR sequences comprise SEQ ID NO: 26, 27 and 28, and the three LCDR sequences comprise SEQ ID NO: 23, 24 and 25.

[0073] 9. The binding polypeptide according to claim 1, wherein the three HCDR sequences comprise SEQ ID NO: 50, 51 and 52, and the three LCDR sequences comprise SEQ ID NO: 47, 48 and 49.

[0074] 10. The binding polypeptide according to claim 1, wherein the three HCDR sequences comprise SEQ ID NO: 62, 63 and 64, and the three LCDR sequences comprise SEQ ID NO: 59, 60 and 61.

[0075] 11. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 2 and 1.

[0076] 12. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 10 and 9.

[0077] 13. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 4 and 3.

[0078] 14. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 12 and 11.

[0079] 15. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 16 and 15.

[0080] 16. The binding polypeptide according to any one of the preceding claims, wherein the soluble Aβ has synaptic toxicity.

[0081] 17. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide neutralizes Aβ synaptic toxicity.

[0082] 18. The binding polypeptide according to any one of the preceding claims, wherein the soluble Aβ has a molecular weight between about 20 kD and about 100 kD.

[0083] 19. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide does not specifically bind monomer Aβ, primary fibril Aβ or fibril Aβ.

[0084] 20. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide does not specifically bind to protein aggregates.

[0085] 21. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease.

[0086] 22. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide specifically binds to soluble Aβ derived from the brain of a subject suffering from Alzheimer's disease.

[0087] 23. The binding polypeptide according to item 22, wherein the binding is immunoadsorption.

[0088] 24. The binding polypeptide according to item 22 or 23, wherein the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease.

[0089] 25. The binding polypeptide according to any one of claims 22-24, wherein the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ derived from the brain of a subject suffering from Alzheimer's disease.

[0090] 26. A pharmaceutical composition comprising a binding polypeptide according to any one of the preceding claims and a pharmaceutically acceptable carrier.

[0091] 27. A method of treating a subject with Alzheimer's disease, the method comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 26.

[0092] 28. An isolated polynucleotide, said isolated polynucleotide encoding a binding polypeptide according to any one of claims 1-15.

[0093] 29. A vector comprising the polynucleotide according to claim 28.

[0094] 30. A host cell comprising the polynucleotide according to claim 28 or the vector according to claim 29. Attached Figure Description

[0095] The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments, taken in conjunction with the accompanying drawings.

[0096] Figure 1 The pre-fusion titers of mice selected for fusions A (FusA), B (FusB), and C (FusC) are depicted graphically. FusA represents sonicated amyloid plaques from human Alzheimer's disease (AD) brains; FusB represents aggregated synthetic β-amyloid (Aβ); and FusC represents AD amyloid plaques inoculated with synthetic Aβ.

[0097] Figures 2A-2B The reverse screening, which demonstrates the selectivity of the monomer Aβ for the oligomer Aβ, is depicted graphically.

[0098] Figure 3 The titer of aggregated synthetic Aβ group 2 after the fifth immunization is depicted graphically, showing the results obtained by ELISA based on synthetic Aβ-derived diffusible ligand (ADDL) (1 μg / ml, overnight at 4ºC). Mouse M13 will be used to generate hybridoma cells (titer, 3X pre-immunization background).

[0099] Figure 4 A table showing a summary of FusB results is depicted.

[0100] Figure 5 Selective absolute size exclusion (aSEC) chromatograms of purified FusB clones were graphically depicted.

[0101] Figure 6 The results of indirect ELISA targeting the FusB clone titrated against primary fibrils (PF) are depicted graphically. Coating: 1 μg / ml PF, incubated overnight at 4ºC.

[0102] Figure 7 BIAcore analysis of reverse screening of monomers was depicted, which showed the dissociation rate assessment of purified FusB clones against Aβ1-42 monomers.

[0103] Figure 8Octet analysis depicts the kinetics of Aβ1-40 monomers relative to PF under selected clonal conditions.

[0104] Figure 9 A table depicting Octet kinetics of Aβ 1-40 monomers compared to PF kinetics under control conditions is presented.

[0105] Figure 10 Reverse screening using fibrils was described in the cases of clones B24, B28, B51, B54, B90, C11 and B73.

[0106] Figure 11 The reverse screening using fibrils in the case of FusB cloning is depicted graphically.

[0107] Figure 12 The reverse screening using aggregated α-synuclein in the case of FusB cloning is depicted graphically.

[0108] Figure 13 The reverse screening using aggregated α-synuclein in the case of FusB cloning is depicted graphically.

[0109] Figure 14 The reverse screening using aggregated α-synuclein in the case of FusB cloning is depicted graphically.

[0110] Figure 15 The reverse screening of SOD-1 by FusB clones at concentrations ranging from 500 ng / ml to 0.5 ng-ml was graphically depicted.

[0111] Figure 16 The reverse screening of SOD-1 for FusB clones at concentrations ranging from 10,000 ng / ml to 100 ng-ml was graphically depicted.

[0112] Figure 17 This is a table showing sequence alignments of the VH region of an exemplary FusB clone.

[0113] Figure 18 The titer of amyloid plaque group 3 after the fifth immunization with Aβ is graphically depicted, showing the results obtained by ELISA based on ADDL (1 μg / ml, overnight at 4ºC). Mouse M23 will be used to generate hybridoma cells (titer, 3X pre-immunization background).

[0114] Figure 19 This is a table illustrating the characteristics of an exemplary FusC clone.

[0115] Figure 20This is a table showing the ELISA screening data for FusC clones.

[0116] Figure 21 The aSEC spectrum of selected purified FusC clones is depicted graphically.

[0117] Figure 22 The ELISA results of FusC clone supernatant for ADDL are depicted graphically. ELISA showed weak to no binding of synthetic Aβ to ADDL.

[0118] Figure 23 The ELISA results of titration of purified FusC clones with PF are depicted graphically. By ELISA, neither the purified clones C10 nor C11 showed significant binding to the synthetic Aβ PF.

[0119] Figure 24 The repeated titration ELISA results of purified C10 and purified C11 clones against synthetic ADDL compared to synthetic PF are graphically depicted. No binding to ADDL or PF was observed with either purified clone C10 or purified clone C11.

[0120] Figure 25 The dissociation rate data of selected purified FusC clones against the Aβ1-42 monomer are graphically depicted.

[0121] Figure 26 The results of reverse screening of purified clone C11 based on protofibrils are depicted graphically.

[0122] Figure 27 The results of reverse screening of protofibrils of purified clone C11 are depicted graphically.

[0123] Figure 28 The results of reverse screening of aggregated α-synuclein of purified clone C11 are depicted graphically.

[0124] Figure 29 The results of SOD-1 reverse screening of fusion clones D and E (10,000 ng / ml - 100 ng / ml) are depicted graphically. Clones C10 and C11 have the same variable region and are referred to as C11 in later experiments.

[0125] Figure 30The assay of hybridoma-purified mAbs in cellular neurite protection iN assays was depicted (run #1). iN cultures (human neurons induced by neural elements derived from iPSCs) were incubated with soluble AD brain extract for 72 h in the presence or absence of the test antibody (hybridoma-purified formulation). Neuron length and branching points were quantified every 2 h under an Incucyte live microscope. Bar graphs show the mean neurite length at the last three time points, normalized relative to the baseline neurite length at each well. The AD extract induced a 50% loss of neurite length, which was protected by pre-immunodepleted Aβ (ID) from the brain extract or by co-incubation with control antibody 3D6 (C1), rather than by an irrelevant antibody (C2), and by some of the novel test antibodies described herein.

[0126] Figures 31A-31C The assays for recombinant rB24 and rB75 in cellular neurite protection were depicted. iN cultures were incubated with AD brain extract for 72 hours with or without the test antibodies (recombinant rB24 and rB75 formulations). Neuron length and number were quantified every 2 hours under an Incucyte live microscope and normalized relative to baseline values ​​for each well. A full-time analysis of normalized neurite length is presented to illustrate the time-dependent neurite toxicity of the AD brain extract and the concentration-dependent protection provided by the recombinant antibodies (rB24 (A) and rB75 (B)). The 50% neurite toxicity protective concentration (EC50) for each antibody was determined using the average of the last three time points, compared to a reference antibody 1C22 (C).

[0127] Figures 32A-32C The assays for recombinant rB24 and rC11 in cellular neurite protection were depicted. iN cultures were incubated with AD brain extract for 72 hours with or without the test antibodies (recombinant rB24 and rC11 formulations). Neuron length and number were quantified every 2 hours under an Incucyte live microscope and normalized relative to baseline values ​​for each well. A full-time analysis of normalized neurite length is presented to illustrate the time-dependent neurite toxicity of the AD brain extract and the concentration-dependent protection provided by the recombinant antibodies (rB24(A), rC11(B)). The 50% neurite toxicity protective concentration (EC50) for each antibody was determined using the average of the last three time points, compared to a reference antibody 1C22 (C).

[0128] Figures 33A-33CThe assays for recombinant rB73 and rB28 in cellular neurite protection were depicted. iN cultures were incubated with soluble AD brain extract for 72 hours in the presence or absence of the test antibodies (recombinant rB73 and rB28 formulations). Neuron length and number were quantified every 2 hours under an Incucyte live microscope and normalized relative to baseline values ​​for each well. A full-time analysis of normalized neurite length is presented to illustrate the time-dependent neurite toxicity of the AD brain extract and the concentration-dependent protection provided by the recombinant antibodies (rB73(A), rB28(B)). The 50% neurite toxicity protective concentration (EC50) for each antibody was determined using the average of the last three time points, compared to a reference antibody 1C22 (C).

[0129] Figures 34A-34B The effects of selected antibodies on electrophysiological recordings in rodent brain slices under basal conditions were depicted. Synaptic transmission (fEPSP slope) was recorded in wild-type brain slices at baseline and after high-frequency stimulation (arrows), with all values ​​normalized relative to baseline transmission. Selected antibodies were added to the brain slices at a final concentration of 5 µg / ml (a second concentration was tested except for B73). (A) Averaged full-time recordings under each condition. The four conditions are shown in both the left and right inset plots. (B) Analysis of synaptic transmission at the endpoint (60 minutes after HFS) under different conditions. The number of slices recorded for each condition was: ACSF (n = 8), C11 (n = 6), B24 (n = 7), B28 (n = 6), B73 (n = 6 at 5 μg / ml and n = 7 at 3 μg / ml), and B75 (n = 6). Differences between groups were tested using two-way ANOVA and Bonferroni post-hoc tests or Student's t-tests. ## p < 0.01.

[0130] Figures 35A-35BThe ability of selected antibodies to neutralize the inhibitory effect of AD brain extract on synaptic plasticity in wild-type mouse brain slices was depicted. Synaptic transmission (fEPSP slope) was recorded in brain slices at baseline and after high-frequency electrical stimulation (arrows), with all values ​​normalized relative to baseline transmission. AD brain extract was added to brain slices either alone or after pre-incubation with selected mAbs at a final concentration of 5 µg / ml (except for B73, which was used at 3 µg / ml). (A) Average full-time recordings under different conditions show the strong inhibitory effect of AD extract on LTP-induced synaptic plasticity. (B) Analysis of synaptic transmission at the endpoint (60 min after HFS) under different conditions. Pre-incubation of AD brain extract with different antibodies resulted in a very significant increase in fEPSP enhancement back to the value under ACSF alone (Fig. x+1, A and B). Notably, both B75 at 5 µg / ml and B73 at 3 µg / ml resulted in full rescue of the inhibitory effect of AD brain extract on LTP. The number of slices recorded for each condition was: AD brain extract (n = 6), AD extract plus C11 (n = 9), plus B24 (n = 7), B28 (n = 8), plus B73 (n = 6 at 3 μg / ml), and plus B75 (n = 6). Differences between groups were tested using two-way ANOVA with Bonferroni post-hoc test or Student's t-test. # p < 0.05, ## p < 0.01, and ### p < 0.001.

[0131] Figures 36A-36B The ability of selected antibodies at lower concentrations to neutralize the inhibitory effect of AD brain extract on synaptic plasticity in brain slices was depicted. Synaptic transmission (fEPSP slope) was recorded in brain slices at baseline and after high-frequency stimulation, and all values ​​were normalized relative to baseline transmission as described above. A) None of the antibodies tested (at 2 µg / ml) had any effect on basal synaptic transmission and LTP induction under basal conditions. B) The antibodies were tested in the presence of AD brain extract (as described above after pre-incubation). B73 almost completely blocked the effect of AD brain extract (p < 0.05). C11 also significantly blocked the effect of AD brain extract by much more than 50% (p < 0.01), and both were comparable to the reference antibody 1C22. B75 had no significant effect at this low concentration.

[0132] Figure 37 The screening of fusion compound B was summarized.

[0133] Figure 38 The screening of fusion compound C was summarized.

[0134] Figures 39A-39BImmunoprecipitation of four human brain extracts using mAbs B24, B28, B73, B75, and C11, as well as protein A beads, is depicted. A) The immunoprecipitation workflow of human brain extracts is schematically illustrated. B) An Aβ x-42 ELISA analysis (n = 3, mean ± SD) is shown, in which the immunoprecipitates of the four brain extracts were sequentially eluted with 1% SDS (top panel) and 6M guanidine hydrochloride (bottom panel). For the immunoprecipitation, 1C22 was used as a positive control, and human IgG was used as a negative control.

[0135] Figures 40A-40B Immunosorbent assays (IMA) of two human brain extracts on a protein A affinity column using mAbs B28, B75, and C11 were depicted. A) The immunosorbent assay workflow for the human brain extracts is schematically illustrated. B) An Aβ x-42 ELISA analysis of the glycine eluent from the immunosorbents of the two brain extracts is depicted (n = 2, mean ± SD). Human IgG was used as a negative control for the immunosorbent assays.

[0136] Figure 41 Figures a and b depict the ultrastructural analysis of immunocapture materials from human brain extracts using mAb B28 and C11. a) Schematic depiction of the immunoadsorption workflow of human brain extracts. b) Negative staining transmission electron microscopy images of immunoadsorption materials from human brain extracts using B28 (top image) or C11 (bottom image).

[0137] Figure 42 Tables a and b depict the immunohistochemistry of brain sections under light microscopy using mAbs B24, B28, B73, B75, and C11. Only B28 showed staining on paraffin-embedded PFA-fixed brain sections. a) Immunohistochemistry of B28 on paraffin-embedded PFA-fixed brain sections. The other four mAbs were negative on such sections (not shown). b) Immunohistochemistry of mAbs B24, B28, B73, B75, and C11 on unfixed frozen brain sections. 1C22 was used as a positive control. Detailed Implementation

[0138] This disclosure provides novel binding peptides (e.g., antibodies) that bind to epitopes present in one or more types of soluble synaptic toxic Aβ. Importantly, the novel binding peptides described herein bind with reduced or no binding to monomeric Aβ, primary fibrillary Aβ, and / or fibrillary Aβ compared to antibodies known in the art.

[0139] In some embodiments, the novel binding peptides described herein prevent or reduce the formation of higher synaptotoxic forms of Aβ, such as monomeric Aβ, primary fibrillary Aβ, and fibrillary Aβ. In some embodiments, the novel binding peptides described herein prevent or reduce the formation of higher synaptotoxic forms of Aβ, such as monomeric Aβ, primary fibrillary Aβ, and / or fibrillary Aβ, from soluble synaptotoxic Aβ.

[0140] It should be understood that the methods described in this disclosure are not limited to the specific methods and experimental conditions disclosed herein, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0141] Furthermore, unless otherwise indicated, the experiments described herein utilize routine molecular and cell biological and immunological techniques within the scope of the art. Such techniques are well known to skilled practitioners and are well explained in the literature. See, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987–2008), including all supplements; Molecular Cloning: A Laboratory Manual (4th edition) (eds.), MR Green and J. Sambrook; and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0142] Unless otherwise defined, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or non-inherent definition. Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Unless otherwise stated, the use of “or” means “and / or”. The use of the term “including” and other forms such as “includes” and “included” is not restrictive.

[0143] Generally, the nomenclature used in combination with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques provided herein are generally performed according to conventional methods well-known in the art and described in the various general and more specific references cited and discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature and laboratory procedures and techniques used in combination with analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0144] To make this disclosure easier to understand, the selected terms are defined below.

[0145] Unless the context otherwise contradicts, the term "peptide" refers to any aggregate chain of amino acids and encompasses natural or artificial proteins, peptide analogs or variants of protein sequences, or fragments thereof. Peptides can be monomers or polymers. For example, a peptide fragment may contain at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, or at least about 20 consecutive amino acids.

[0146] The terms "isolated protein" or "isolated polypeptide" refer to a protein or polypeptide that is non-associated with its naturally occurring associated components due to its origin or derivative; is substantially free of other proteins from the same species; is expressed by cells from a different species; or is not present in nature. Therefore, a protein or polypeptide synthesized chemically or in a cellular system different from its natural source will be "isolated" from its naturally occurring associated components. Isolation can also be achieved by using protein purification techniques well known in the art, making the protein or polypeptide substantially free of its naturally occurring associated components.

[0147] As used herein, the terms "binding protein" or "binding polypeptide" shall refer to a protein or polypeptide (e.g., an antibody or immunoadhesin) containing at least one binding site responsible for selectively binding to a target antigen of interest (e.g., a human target antigen). Exemplary binding sites include antibody variable domains, ligand-binding sites of receptors, or receptor-binding sites of ligands. In some aspects, a binding protein or binding polypeptide contains multiple (e.g., two, three, four, or more) binding sites. In some aspects, a binding protein or binding polypeptide is not a therapeutic enzyme.

[0148] The term "ligand" refers to any substance that can bind to or be bound to another substance. Similarly, the term "antigen" refers to any substance that can produce antibodies against it. Although "antigen" is commonly used to refer to antibody-binding substrates, and "ligand" is frequently used when referring to receptor-binding substrates, these terms are not distinguished from each other and cover a wide range of overlapping chemical entities. For the avoidance of doubt, antigen and ligand are used interchangeably throughout this document. Antigen / ligand can be peptide, polypeptide, protein, aptamer, polysaccharide, sugar molecule, carbohydrate, lipid, oligonucleotide, polynucleotide, synthetic molecule, inorganic molecule, organic molecule, and any combination thereof.

[0149] As used herein, the term "specific binding" refers to antibodies or immunoadhesins with a binding rate of up to approximately 1 x 10⁻⁶. -6 M, approximately 1 x 10 -7 M, approximately 1 x 10 -8 M, approximately 1 x 10 -9 M, approximately 1 x 10 -10 M, approximately 1 x 10 -11 M, approximately 1 x 10 -12 It binds to antigens with a dissociation constant (M) or lower (Kd), and / or has an affinity for antigens that is at least about twice that of its affinity for nonspecific antigens.

[0150] As used herein, the term "antibody" refers to an assembly (e.g., a complete antibody molecule, an immunoadhesin, or a variant thereof) that exhibits significant, known specific immunoreactivity against an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent linkages. The basic immunoglobulin structures in vertebrate systems are relatively well understood.

[0151] As used herein, the term "multispecific antibody" means an antibody containing at least two different binding specificities. In one embodiment, the multispecific antibody described herein is specific to two different antigens, such as the blood-brain barrier (BBB) ​​receptor and soluble β-amyloid (Aβ).

[0152] 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 binds to a single antigen (e.g., soluble β-amyloid protein (Aβ)).

[0153] As will be discussed in more detail below, the general term "antibody" includes five different classes of antibodies that can be distinguished biochemically. While all five classes of antibodies are obviously within the scope of this disclosure, the following discussion will generally refer to immunoglobulin molecules of the IgG class. Regarding IgG, immunoglobulins consist of 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 Daltons. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chain beginning at the opening of the "Y" and continuing to the variable region flanking the heavy chain.

[0154] The light chains of immunoglobulins are classified as kappa (κ) or lambda (λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other via covalent disulfide linkages or non-covalent linkages. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration to the C-terminus at the bottom of each chain. Those skilled in the art will understand that the heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε), among which there are several subclasses (e.g., γ1-γ4). The properties of this chain, respectively, determine the “class” of the antibody as IgG, IgM, IgA, IgG, or IgE. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) have been well characterized, and they are known to confer functional specialization. Given this disclosure, the modifications of each of these categories and types are readily identifiable to a person skilled in the art, and therefore fall within the scope of this disclosure.

[0155] Both light and heavy chains are divided into regions with structural and functional homology. The term "region" refers to a part or portion of an immunoglobulin or antibody chain and includes constant or variable regions as well as more discrete parts or portions of said regions. For example, variable regions of light chains include "complementarity-determining regions" or "CDRs" scattered between "frame regions" or "FRs" as defined herein.

[0156] Regions of the heavy or light chains of immunoglobulins can be defined as "constant" (C) regions or "variable" (V) regions. In the case of "constant regions," the definition is based on the relative lack of sequence variation within the region for each class member; in the case of "variable regions," it is based on significant variation within the region for each class member. The terms "constant region" and "variable region" can also be used in relation to function. In this regard, it will be understood that the variable regions of immunoglobulins or antibodies determine antigen recognition and specificity. Conversely, the constant regions of immunoglobulins or antibodies confer important effector functions such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. The subunit structures and three-dimensional conformations of the constant regions of various immunoglobulin classes are well known.

[0157] The constant and variable regions of the immunoglobulin heavy and light chains are folded into domains. The term "domain" refers to a globular region of the heavy or light chain containing, for example, peptide rings (e.g., containing 3 to 4 peptide rings) stabilized by β-sheets and / or intrachain disulfide bonds. Constant region domains on the immunoglobulin light chain are interchangeably referred to as "light chain constant region domains," "CL regions," or "CL domains." Constant domains on the heavy chain (e.g., hinge, CH1, CH2, or CH3 domains) are interchangeably referred to as "heavy chain constant region domains," "CH" region domains, or "CH domains." Variable domains on the light chain are interchangeably referred to as "light chain variable region domains," "VL region domains," or "VL domains." Variable domains on the heavy chain are interchangeably referred to as "heavy chain variable region domains," "VH region domains," or "VH domains."

[0158] By convention, the amino acid numbers of the variable constant region domains increase with their distance from the antigen-binding site or N-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 contain the C-termini of the heavy and light chains, respectively. Therefore, the domains of the light chain immunoglobulin are aligned in a VL-CL orientation, while the domains of the heavy chain are aligned in a VH-CH1-hinge-CH2-CH3 orientation.

[0159] The amino acid assignment for each variable region domain is as defined in 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 between different light chain variable regions are assigned the same number. The CDRs 1, 2, and 3 for the VL domain are also referred to as CDR-L1, CDR-L2, and CDR-L3, respectively, in this paper. The CDRs 1, 2, and 3 for the VH domain are also referred to as CDR-H1, CDR-H2, and CDR-H3, respectively, in this paper. If indicated otherwise, the CDR assignment may follow IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003), rather than Kabat. The heavy chain constant region is numbered using the EU index proposed by Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0160] As used herein, the term "VH domain" includes the N-terminal variable domain of the immunoglobulin heavy chain, and the term "VL domain" includes the N-terminal variable domain of the immunoglobulin light chain.

[0161] As used herein, the term "CH1 domain" includes the first (most amino-terminal) constant region domain of the immunoglobulin heavy chain, which extends, for example, from approximate positions 114-223 (EU positions 118-215) in the Kabat numbering system. The CH1 domain is adjacent to the amino terminus of the VH domain and the hinge region of the immunoglobulin heavy chain molecule, and does not constitute part of the Fc region of the immunoglobulin heavy chain.

[0162] As used herein, the term "hinge region" refers to the portion of a heavy chain molecule that connects the CH1 and CH2 domains. 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 upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998, 161:4083).

[0163] As used herein, the term "CH2 domain" includes such a portion of a heavy chain immunoglobulin molecule, extending, for example, from approximate positions 244-360 (EU positions 231-340) in the Kabat numbering system. The CH2 domain is unique because it does not pair tightly with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. In one embodiment, the binding polypeptide of this disclosure comprises a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0164] As used herein, the term "CH3 domain" includes a portion of a heavy chain immunoglobulin molecule that extends approximately 110 residues from the N-terminus of the CH2 domain, for example, from approximate positions 361-476 (EU positions 341-445) in the Kabat numbering system. The CH3 domain typically forms the C-terminal portion of an 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 e chain of IgE). In one embodiment, the binding polypeptide of this disclosure comprises a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0165] As used herein, the term "CL domain" includes a constant region domain of an immunoglobulin light chain, which extends, for example, from approximately Kabat position 107A to approximately Kabat position 216. The CL domain is adjacent to the VL domain. In one embodiment, the binding polypeptide of this disclosure comprises a CL domain derived from the κ light chain (e.g., the human κ light chain).

[0166] As used herein, the term "Fc region" is defined as a portion of the heavy chain constant region that begins at the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, which takes the first residue of the heavy chain constant region as 114) and ends at the C-terminus of the antibody. Therefore, a complete Fc region contains at least a hinge domain, a CH2 domain, and a CH3 domain.

[0167] As used herein, the term "natural Fc" or "wild-type Fc" refers to a molecule containing a sequence of a non-antigen-binding fragment produced by antibody digestion or by other means, whether in monomeric or multimeric form, and may contain a hinge region. The original immunoglobulin source of natural Fc is typically human and can be any immunoglobulin, such as IgG1 and IgG2. Natural Fc molecules consist of monomeric polypeptides that can be linked together in dimer or multimeric form via covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between the monomeric subunits of a natural Fc molecule 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 natural Fc is a disulfide-bonded dimer produced by the digestion of IgG with papain. As used herein, the term "natural Fc" is used generally for monomeric, dimer, and multimeric forms.

[0168] As used herein, the term “Fc variant” or “modified Fc” refers to a molecule or sequence derived from natural / wild-type Fc but still containing a binding site for FcRn. Therefore, the term “Fc variant” can include molecules or sequences humanized from non-human natural Fc. Furthermore, natural Fc contains regions that can be removed because they provide structural features or biological activities not required by the antibody-like binding peptides described herein. Therefore, the term “Fc variant” includes molecules or sequences lacking one or more natural Fc sites or residues, or in which one or more Fc sites or residues have been modified, said sites or residues affecting or involved in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity when expressed in selected host cells, (4) glycosylation, (5) complement interaction, (6) binding to Fc receptors other than rescue receptors, or (7) antibody-dependent cytotoxicity (ADCC).

[0169] As used herein, the term "Fc domain" encompasses both natural / wild-type Fc as defined above, as well as Fc variants and sequences. Similar to Fc variants and natural Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether derived from whole antibody digestion or produced by other means.

[0170] As indicated above, the variable regions of an antibody allow it to selectively recognize and specifically bind to epitopes on antigens. That is, the VL and VH domains of the antibody combine to form a variable region (Fv) defining a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) on each heavy and light chain variable region. As used herein, the term "antigen-binding site" includes a site that specifically binds (in an immune response to) an antigen (e.g., a cell surface or soluble antigen). Antigen-binding sites include variable regions of the immunoglobulin heavy and light chains, and the binding sites formed by these variable regions determine the specificity of the antibody. Antigen-binding sites are formed by variable regions that vary between antibodies. The modified antibodies of this disclosure contain at least one antigen-binding site.

[0171] In some embodiments, the binding polypeptide of this disclosure comprises at least two antigen-binding domains that provide association between the binding polypeptide and a selected antigen. The antigen-binding domains need not originate from the same immunoglobulin molecule. In this respect, the variable region may be derived from or derived from any type of animal that can be induced to produce a humoral response and generate immunoglobulins against the desired antigen. Thus, the variable region of the binding polypeptide can be of mammalian origin, for example, from humans, mice, rats, goats, sheep, non-human primates (such as cynomolgus monkeys, macaques, etc.), wolves, or camelids (e.g., from camels, llamas, and related species).

[0172] In naturally occurring antibodies, the six chain-receptor domains (CDRs) present on each monomeric antibody are short, discontinuous amino acid sequences specifically localized to form antigen-binding sites, assuming the antibody exhibits its three-dimensional conformation in an aqueous environment. The remaining heavy and light variable domains exhibit less intermolecular variability in the amino acid sequence and are referred to as framework regions. Framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect to the β-sheet structure and, in some cases, form part of the β-sheet structure. Thus, these framework regions act as a scaffold, providing the six CDRs with the correct orientation through interchain non-covalent interactions. The antigen-binding domain formed by the localized CDRs defines a surface complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to the immunoreactive antigen epitope.

[0173] Exemplary binding peptides include antibody variants. As used herein, the term "antibody variant" includes synthetic and engineered forms of antibodies that are modified such that they are not naturally occurring, such as antibodies containing at least two heavy chain moieties but not two complete heavy chains (e.g., domain-deficient antibodies or microantibodies); multispecific (e.g., bispecific, trispecific, etc.) antibodies modified to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules linked to scFv molecules, etc. Additionally, the term "antibody variant" includes multivalent antibodies (e.g., trivalent, tetravalent, etc. antibodies that bind to three, four, or more copies of the same antigen). "Antibody variants" can be multispecific and / or multivalent.

[0174] As used herein, the term "valence" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to a target molecule or a specific site on a target molecule. When a polypeptide contains more than one target binding site, each target binding site may specifically bind to the same or different molecules (e.g., it may bind to different ligands or different antigens or different epitopes on the same antigen). Subject-binding polypeptides typically have at least one binding site that is specific to human antigen molecules.

[0175] The term "specificity" refers to the ability to specifically bind (e.g., in an immune response) to a given target antigen (e.g., a human target antigen). A binding peptide can be monospecific and contain one or more binding sites that specifically bind to a target, or a peptide can be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In some embodiments, the binding peptide is specific to two different (e.g., non-overlapping) portions of the same target. In some embodiments, the binding peptide is specific to more than one target.

[0176] In some exemplary embodiments, the binding peptides (e.g., antibodies) described herein specifically bind to one or more synaptotoxic forms of Aβ. In some exemplary embodiments, the binding peptides (e.g., antibodies) described herein that specifically bind to one or more synaptotoxic forms of Aβ are not specific to monomeric Aβ, fibrillary Aβ, or protofibrillary Aβ, or any combination thereof. In some exemplary embodiments, the binding peptides (e.g., antibodies) described herein that specifically bind to one or more synaptotoxic forms of Aβ are not specific to one or more non-Aβ aggregates (e.g., like SOD-1 and / or aggregated synuclein).

[0177] In some exemplary embodiments, the binding peptides (e.g., antibodies) that specifically bind to one or more synaptic toxic forms of Aβ described herein prevent and / or alleviate one or more symptoms associated with AD. As used herein, “symptoms associated with Alzheimer’s disease” or “symptoms associated with AD” refers to symptoms associated with any preclinical, mild, moderate, or severe stage of AD. Symptoms associated with AD include one or more bodily changes, such as changes in the brain, including but not limited to: neurotoxicity and / or synaptic toxicity, such as in the entorhinal cortex, hippocampus, and cerebral cortex; accumulation of glial cells; accumulation of Aβ; accumulation of τ protein; formation of Aβ oligomers; formation of Aβ paranuclei; formation of Aβ primary fibrils; formation of mature Aβ fibrils; formation of amyloid plaques; formation of neurofibrillary tangles; chronic inflammation; reduced blood flow to the brain; destruction of the BBB; brain atrophy, etc.

[0178] Symptoms associated with mild Alzheimer's disease include, but are not limited to: memory loss; poor judgment leading to wrong decisions; loss of spontaneity and autonomy; needing longer to complete normal daily tasks; recurring problems; difficulty handling money and paying bills; wandering and getting lost; losing things or misplacing them in strange places; mood and personality changes; increased anxiety and / or aggression, etc.

[0179] Symptoms associated with moderate Alzheimer's disease include, but are not limited to: increased memory loss and confusion; inability to learn new things; language difficulties and problems with reading, writing, and processing numbers; difficulty organizing thoughts and logical thinking; shortened attention span; problems coping with new situations; difficulty performing multi-step tasks, such as dressing; problems recognizing family and friends; hallucinations, delusions, and paranoia; impulsive behaviors, such as undressing at inappropriate times or places or using vulgar language; inappropriate outbursts of anger; agitation, agitation, anxiety, crying, and wandering—especially in the late afternoon or evening; repetitive statements or actions, and occasional muscle twitches.

[0180] Symptoms associated with severe Alzheimer's disease include, but are not limited to: memory loss; poor judgment leading to wrong decisions; loss of spontaneity and autonomy; needing longer to complete normal daily tasks; recurring problems; difficulty handling money and paying bills; wandering and getting lost; losing things or misplacing them in strange places; mood and personality changes; increased anxiety and / or aggression, etc.

[0181] As used herein, “synaptic toxic form of β-amyloid,” “synaptic toxic form of Aβ,” or “synaptic toxic Aβ” refers to the form of Aβ derived from the brain of Alzheimer's disease, present in soluble brain extracts, and associated with synaptic loss. Synaptic toxic Aβ has a molecular weight between approximately 8 kD and approximately 100 kD and is unstable.

[0182] Based on certain aspects, this article provides binding peptides that specifically bind to the synaptic toxic Aβ. The full-length heavy and light chain sequences of the exemplary binding peptides described herein are listed in Table 1.

[0183] clone Full-length light chain sequence Full-length heavy chain sequence B24 EIVMTQSPATLSLSPGERATLSCRASQSVSSSYFSWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQDSNLPLTFGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 65) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGEGLEWVSTISGSGIRTYYADSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAKDGLTGDRRWYFDLWG RGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 66) B28 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPGKAPKLLIYPASSLQSGVPSRFSGSGSDTDFTLTISSLQPEDFATYYCLQDYNFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 67) QVQLVESGGGVVQPGRSVRLSCAATGFTFSSYGMHWVRQAPGKGLEWVAVIWFDGSNEYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGRVGVTRNYYYYNMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 68) B51 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPVKAPKLLIYPASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 69) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSHGMHWVRQAPGKGLEWVAVIWYDGSNKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGRVGVTRNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70) B54 AIQMTQSPSSLSASVGDRVTITCRTSQDIRNDLGWFQQKPGKAPKFLIYPASSLQGGVPSRFSGSGSGTDFILTISSLQPEDFATYYCLQDYNFPWTFGEGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 71) QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVALIWYDGSKKYYADSVQGRFTISRDSSKNTLYLQMNSLRVEDTAVYYCARRGRVGVTRNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 72) B73 QAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGETNNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO: 73) QLQLQMSGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQSPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAMYYCARRSSGRPYYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 74) B75 EIVMTQSPATLSVSPGEKATLSCRASQSFSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 75) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCAREGRTYYDFLTGYFDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 76) B90 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFHQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYVYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 77) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWYDGSKKYYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARRGRVGATRDYYYYSMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 78) C11 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPTLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDFNYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 79) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGSIYYSGTTKYNPSLKSRVTISVGTSKNQFSLKLNSVTAADTAVYYCARDNWGSRFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQID NO: 80)

[0184] Table 1. Full-length heavy chain and light chain sequences of exemplary binding peptides.

[0185] The light chain variable region (LCVR) and heavy chain variable region (HCVR) sequences of the exemplary binding peptides described herein are listed in Table 2.

[0186] Clone Light chain variable region (LCVR) sequence Heavy chain variable region (HCVR) sequence B24 EIVMTQSPATLSLSPGERATLSCRASQSVSSSYFSWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQDSNLPLTFGGGTKVEIK (SEQ ID NO: 1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGEGLEWVSTISGSGIRTYYADSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAKDGLTGDRRWYFDLWGRGTLVTVSS (SEQ ID NO: 2) B28 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPGKAPKLLIYPASSLQSGVPSRFSGSGSDTDFTLTISSLQPEDFATYYCLQDYNFPFTFGPGTKVDIK(SEQ ID NO: 3) QVQLVESGGGVVQPGRSVRLSCAATGFTFSSYGMHWVRQAPGKGLEWVAVIWFDGSNEYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGRVGVTRNYYYYNMDVWGQGTTVTVSS(SEQ ID NO: 4) B51 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPVKAPKLLIYPASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPWTFGQGTKVEIK (SEQ ID NO: 5) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSHGMHWVRQAPGKGLEWVAVIWYDGSNKNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGRVGVTRNYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 6) B54 AIQMTQSPSSLSASVGDRVTITCRTSQDIRNDLGWFQQKPGKAPKFLIYPASSLQGGVPSRFSGSGSGTDFILTISSLQPEDFATYYCLQDYNFPWTFGEGTKVEIK(SEQ ID NO: 7) QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVALIWYDGSKKYYADSVQGRFTISRDSSKNTLYLQMNSLRVEDTAVYYCARRGRVGVTRNYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 8) B73 QAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGETNNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHWVFGGGTKLTVL (SEQ ID NO: 9) QLQLQMSGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQSPGKGLEWIGSIYYSGRTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAMYYCARRSSGRPYYWGQGTLVTVSS (SEQ ID NO: 10) B75 EIVMTQSPATLSVSPGEKATLSCRASQSFSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPYTFGQGTKLEIK (SEQ ID NO: 11) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCAREGRTYYDFLTGYFDFWGQGTLVTVSS (SEQ ID NO: 12) B90 AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWFHQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYVYPWTFGQGTKVEIK (SEQ ID NO: 13) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWYDGSKKYYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARRGRVGATRDYYYYSMDVWGQGTTVTVSS (SEQ ID NO: 14) C11 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPTLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDFNYPYTFGQGTKLEIK (SEQ ID NO: 15) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGSIYYSGTTKYNPSLKSRVTISVGTSKNQFSLKLNSVTAADTAVYYCARDNWGSRFDYWGQGTLVTVSS(SEQ ID NO: 16)

[0187] Table 2. Heavy chain variable region sequences and light chain variable region sequences of exemplary binding peptides.

[0188] The light chain framework (LFW) sequence and heavy chain framework (HFW) region of the exemplary binding peptides described herein are listed in Table 3.

[0189] Clone Light chain framework (LFW) sequence Heavy chain framework (HCF) sequence B24 EIVMTQSPATLSLSPGERATLSCRAS (FW1) (SEQ ID NO: 81)FSWYQQKPGQAPRLLIY (FW2) (SEQ ID NO: 82)TRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYC (FW3) (SEQ ID NO: 83)FGGGTKVEIK (FW4) (SEQ ID NO: 84) EVQLLESGGGLVQPGGSLRLSCAAS (FW1) (SEQ ID NO: 85)MTWVRQAPGEGLEWVST (FW2) (SEQ ID NO: 86)YYADSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYC (FW3) (SEQ ID NO: 87)WGRGTLVTVSS (FW4) (SEQ ID NO: 88) B28 AIQMTQSPSSLSASVGDRVTITCRAS (FW1) (SEQ ID NO: 89)LGWFQQKPGKAPKLLIY (FW2) (SEQ ID NO: 90)SLQSGVPSRFSGSGSDTDFTLTISSLQPEDFATYYC (FW3) (SEQ ID NO: 91)FGPGTKVDIK (FW4) (SEQ ID NO: 92) QVQLVESGGGVVQPGRSVRLSCAAT (FW1) (SEQ ID NO: 93)MHWVRQAPGKGLEWVAV (FW2) (SEQ ID NO: 94)YYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (FW3) (SEQ ID NO: 95)WGQGTTVTVSS (FW4) (SEQ ID NO: 96) B51 AIQMTQSPSSLSASVGDRVTITCRAS (FW1) (SEQ ID NO: 97)LGWFQQKPVKAPKLLIY (FW2) (SEQ ID NO: 98)SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (FW3) (SEQ ID NO: 99)FGQGTKVEIK (FW4) (SEQ ID NO: 100) QVQLVESGGGVVQPGRSLRLSCAAS (FW1) (SEQ ID NO: 101)MHWVRQAPGKGLEWVAV (FW2) (SEQ ID NO: 102)NYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (FW3) (SEQ ID NO: 103)WGQGTTVTVSS (FW4) (SEQ ID NO: 104) B54 AIQMTQSPSSLSASVGDRVTITCRTS (FW1) (SEQ ID NO: 105)LGWFQQKPGKAPKFLIY (FW2) (SEQ ID NO: 106)SLQGGVPSRFSGSGSGTDFILTISSLQPEDFATYYC (FW3) (SEQ ID NO: 107)FGEGTKVEIK (FW4) (SEQ ID NO: 108) QVQLVESGGGVVQPGRSLRLSCAAS (FW1) (SEQ ID NO: 109)MHWVRQAPGKGLEWVAL (FW2) (SEQ ID NO: 110)YYADSVQGRFTISRDSSKNTLYLQMNSLRVEDTAVYYC (FW3) (SEQ ID NO: 111)WGQGTTVTVSS (FW4) (SEQ ID NO: 112) B73 QAVVTQESALTTSPGGTVILTCRSS (FW1) (SEQ ID NO: 113)ANWVQEKPDHLFTGLIG (FW2) (SEQ ID NO: 114)NRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFC (FW3) (SEQ ID NO: 115)FGGGTKLTVL (FW4) (SEQ ID NO: 116) SEQ ID NO: 120) B75 EIVMTQSPATLSVSPGEKATLSCRAS (FW1) (SEQ ID NO: 121)LAWYQQKPGQAPRLLIY (FW2) (SEQ ID NO: 122)TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC (FW3) (SEQ ID NO: 123)FGQGTKLEIK (FW4) (SEQ ID NO: 124) QVQLVESGGGVVQPGRSLRLSCAAS (FW1) (SEQ ID NO: 125)MHWVRQAPGKGLEWVAV (FW2) (SEQ ID NO: 126)YYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYC (FW3) (SEQ ID NO: 127)WGQGTLVTVSS (FW4) (SEQ ID NO: 128) B90 AIQMTQSPSSLSASVGDRVTITCRAS (FW1) (SEQ ID NO: 129)LGWFHQKPGKAPKLLIY (FW2) (SEQ ID NO: 130)SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (FW3) (SEQ ID NO:131)FGQGTKVEIK (FW4) (SEQ ID NO: 132) QVQLVESGGGVVQPGRSLRLSCAAS (FW1) (SEQ ID NO: 133)MHWVRQAPGKGLEWVAI (FW2) (SEQ ID NO: 134)YYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYC (FW3) (SEQ ID NO: 135)WGQGTTVTVSS (FW4) (SEQ ID NO: 136) C11 DIQMTQSPSSLSASVGDRVTITCRAS (FW1) (SEQ ID NO: 137)LGWYQQKPGKAPTLLIY (FW2) (SEQ ID NO: 138)SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (FW3) (SEQ ID NO:139)FGQGTKLEIK (FW4) (SEQ ID NO: 140) QVQLQESGPGLVKPSETLSLTCTVS (FW1) (SEQ ID NO: 141)WSWIRQPPGKGLEWIGS (FW2) (SEQ ID NO: 142)KYNPSLKSRVTISVGTSKNQFSLKLNSVTAADTAVYYC (FW3) (SEQ ID NO: 143)WGQGTLVTVSS (FW4) (SEQ ID NO: 144)

[0190] Table 3. Light chain and heavy chain framework sequences of exemplary binding peptides.

[0191] The complementarity-determining region (CDR) sequences of the heavy and light chains of the exemplary binding peptides described herein are listed in Table 4.

[0192] Clone Light chain CDR (LCDR) sequences Heavy chain CDR (HCDR) sequences B24 QSVSSSY (SEQ ID NO: 17), LCDR1GAS (SEQ ID NO:18), LCDR2QQDSNLPLT (SEQ ID NO: 19), LCDR3 GFTFSSYA (SEQ ID NO: 20), HCDR1ISGSGIRT (SEQ ID NO: 21),HCDR2AKDGLTGDRRWYFDL (SEQ ID NO: 22), HCDR3 B28 QGIRND (SEQ ID NO: 23), LCDR1PAS (SEQ ID NO:24), LCDR2LQDYNFPFT (SEQ ID NO: 25), LCDR3 GFTFSSYG (SEQ ID NO: 26), HCDR1IWFDGSNE (SEQ ID NO: 27),HCDR2ARRGRVGVTRNYYYYNMDV(SEQ ID NO: 28), HCDR3 B51 QGIRND (SEQ ID NO: 29), LCDR1PAS (SEQ ID NO:30), LCDR2LQDYNYPWT(SEQ ID NO: 31), LCDR3 GFTFSSHG (SEQ ID NO: 32), HCDR1IWYDGSNK (SEQ ID NO: 33),HCDR2ARRGRVGVTRNYYYYGMDV(SEQ ID NO: 34), HCDR3 B54 QDIRND (SEQ ID NO: 35), LCDR1PAS (SEQ ID NO:36), LCDR2LQDYNFPWT(SEQ ID NO: 37), LCDR3 GFTFSTYG (SEQ ID NO: 38), HCDR1IWYDGSKK (SEQ ID NO: 39),HCDR2ARRGRVGVTRNYYYYGMDV(SEQ ID NO: 40), HCDR3 B73 TGAVTTSNY (SEQ ID NO: 41), LCDR1ETN (SEQ ID NO:42), LCDR2ALWYSTHWV (SEQ ID NO: 43), LCDR3 GGSISSSSYY (SEQ ID NO: 44), HCDR1IYYSGRT (SEQ ID NO: 45),HCDR2ARRSSGRPYY(SEQ ID NO: 46), HCDR3 B75 QSFSSN (SEQ ID NO: 47), LCDR1GAS (SEQ ID NO:48), LCDR2QQYNNWPYT (SEQ ID NO: 49), LCDR3 GFTFSSYG (SEQ ID NO: 50), HCDR1IWYDGSYK (SEQ ID NO: 51), HCDR2AREGRTYYDFLTGYFDF (SEQ ID NO: 52), HCDR3 B90 QGIRND (SEQ ID NO: 53), LCDR1AAS (SEQ ID NO:54), LCDR2LQDYVYPWT (SEQ ID NO: 55), LCDR3 GFTFSSYG (SEQ ID NO: 56), HCDR1IWYDGSKK (SEQ ID NO: 57), HCDR2ARRGRVGATRDYYYYSMDV (SEQ ID NO: 58), HCDR3 C11 QGIRND (SEQ ID NO: 59), LCDR1AAS (SEQ ID NO:60), LCDR2LQDFNYPYT (SEQ ID NO: 61), LCDR3 GGSISSYY (SEQ ID NO: 62), HCDR1IYYSGTT (SEQ ID NO: 63), HCDR2ARDNWGSRFDY (SEQ ID NO: 64), HCDR3

[0193] Table 4. Heavy chain CDR sequences and light chain CDR sequences of exemplary binding peptides.

[0194] As used herein, the term "antigen" or "target antigen" refers to a molecule or part of a molecule that can be bound to a binding site of a binding polypeptide. A target antigen may have one or more epitopes.

[0195] The term “about” or “approximately” means about 20% of a given value or range, such as about 10%, about 5%, or about 1% or less.

[0196] As used herein, “administer” or “administration” means the act of injecting or otherwise physically delivering a substance present outside the body (e.g., the isolated binding peptides provided herein) to a patient, such as via, but not limited to, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When managing or treating a disease or its symptoms, the administration of a substance is typically performed after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the administration of a substance is typically performed before the onset of the disease or its symptoms and may be prolonged to delay or reduce the onset or severity of disease-related symptoms.

[0197] As used herein, the term "composition" is intended to cover products containing optional specified amounts of specified ingredients (e.g., isolated bound polypeptides provided herein), and any product produced directly or indirectly from a combination of optional specified amounts of specified ingredients.

[0198] "Effective amount" means an amount of active pharmaceutical agent (e.g., the isolated binding peptide of this disclosure) sufficient to achieve the desired physiological outcome in an individual requiring the agent. Effective amounts can vary between individuals depending on the health and physical condition of the individual being treated, the individual's taxonomy, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0199] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey and a human). In some embodiments, as used herein, the term “subject” refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, untamed animals, farm animals, sporting animals, and pets.

[0200] As used herein, the term "therapy" means any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease or related symptoms. In some embodiments, the term "therapy" means any protocol, method, and / or agent that can be used to modulate a subject's immune response to an infection or related symptoms. In some embodiments, the terms "therapies" and "therapy" mean biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (such as medical personnel) for the prevention, management, treatment, and / or improvement of a disease or related symptoms. In other embodiments, the terms "therapies" and "therapy" mean biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (such as medical personnel) for the modulation of a subject's immune response to an infection or related symptoms.

[0201] As used herein, the terms “treat,” “treatment,” and “treating” refer to a reduction or improvement in the progression, severity, and / or duration of disease or related symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more preventative or therapeutic agents, such as the isolated binding peptides provided herein). As used herein, the term “treatment” may also refer to altering the course of disease in a treated subject. The therapeutic effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating one or more symptoms, reducing the direct or indirect pathological consequences of disease, slowing the rate of disease progression, improving or slowing the disease state, and alleviating or improving prognosis.

[0202] In some exemplary embodiments, the combination peptides described herein are used to treat one or more symptoms of Alzheimer's disease.

[0203] binding peptides

[0204] In one aspect, this disclosure provides binding peptides (e.g., antibodies, immunoadhesins, antibody variants, and fusion proteins) that bind to Aβ and treat one or more symptoms of Alzheimer's disease. The binding peptides disclosed herein encompass any binding peptide containing a modified Fc domain. In some embodiments, the binding peptide is an antibody or immunoadhesin or a derivative thereof. Any antibody from any source or species may be used in the binding peptides disclosed herein. Suitable antibodies include, but are not limited to, human antibodies, humanized antibodies, or chimeric antibodies. Suitable antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, full-length antibodies, or single-chain antibodies.

[0205] Fc domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA, and IgE) and species can be used in the binding peptides disclosed herein. Chimeric Fc domains comprising portions of Fc domains from different species or Ig classes can also be employed. In some 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. In the case of Fc domains from other species and / or Ig classes or isotypes, those skilled in the art will understand that any amino acid substitutions described herein can be adjusted accordingly. Some embodiments include antibodies containing at least one amino acid in one or more constant region domains and / or at least one amino acid in one or more variable region domains, said at least one amino acid being missing or otherwise altered to provide desired biochemical characteristics, such as reduced or enhanced effector function, non-covalent dimerization ability, enhanced ability to localize to tumor sites, reduced serum half-life, increased serum half-life, etc., when compared to an intact, unaltered antibody with substantially the same immunogenicity.

[0206] In some other embodiments, the binding peptide comprises constant regions derived from different antibody isotypes (e.g., constant regions from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the binding peptide comprises a chimeric hinge (i.e., a hinge comprising hinge portions of hinge domains derived from different antibody isotypes, such as the upper hinge domain of the IgG4 molecule and the middle hinge domain of the IgG1 molecule).

[0207] In some embodiments, techniques known in the art can be used to mutate the Fc domain to increase or decrease effector function. In some embodiments, binding peptides comprising modified Fc domains of this disclosure have altered binding affinity for Fc receptors. Several different types of Fc receptors exist, classified based on the types of antibodies they recognize. For example, the Fcγ receptor (FcγR) binds to IgG antibodies, the Fcα receptor (FcαR) binds to IgA antibodies, and the Fcε receptor (FcεR) binds to IgE antibodies. FcγR belongs to a family that includes several members (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb). In some embodiments, binding peptides comprising modified Fc domains have altered FcγRIIIa binding affinity compared to binding peptides comprising wild-type Fc domains. In some embodiments, binding peptides containing modified Fc domains have reduced FcγRIIIa binding affinity compared to binding peptides containing wild-type Fc domains. In some embodiments, binding peptides containing modified Fc domains have enhanced FcγRIIIa binding affinity compared to binding peptides containing wild-type Fc domains. In some embodiments, binding peptides containing modified Fc domains have substantially the same FcγRIIIa binding affinity compared to binding peptides containing wild-type Fc domains.

[0208] In other embodiments, the binding peptides described herein have constant regions (e.g., IgG1 heavy chain constant regions) that are modified to reduce or eliminate glycosylation. For example, binding peptides comprising a modified Fc domain (e.g., antibodies or immunoadhesins) may further comprise amino acid substitutions that alter the glycosylation of the antibody Fc. For example, the modified Fc domain may have reduced glycosylation (e.g., N- or O-linked glycosylation).

[0209] Exemplary amino acid substitutions conferring reduced or altered glycosylation are disclosed in International PCT Publication No. WO 2005 / 018572, which is incorporated herein by reference in its entirety. In some embodiments, the binding peptide is modified to eliminate glycosylation. Such binding peptides may be referred to as “agly” binding peptides (e.g., “agly” antibodies). While not bound by theory, “agly” binding peptides are thought to have improved in vivo safety and stability characteristics. Agly binding peptides may have any of their isotypes or subclasses, such as IgG1, IgG2, IgG3, or IgG4. Many methods recognized in the art can be used to prepare “agly” antibodies or antibodies with modified glycans. For example, genetically engineered host cells (e.g., modified yeast (e.g., Pichia) or CHO cells) with modified glycosylation pathways (e.g., glycosyltransferase deficiency) can be used to generate such antibodies.

[0210] In some embodiments, the binding peptide may include an antibody constant region (e.g., an IgG constant region, such as the human IgG constant region, such as the human IgG1 constant region) that mediates one or more effector functions. 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 inflammatory responses and may also be involved in autoimmune hypersensitivity reactions. Furthermore, antibodies bind to various cellular receptors via their Fc domains (Fc receptor binding sites on the antibody Fc region bind to cellular Fc receptors (FcRs)). Many Fc receptors exist that are specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (μ receptor)). The binding of antibodies to Fc receptors on cell surfaces triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, cytotoxic cell lysis of antibody-coated target cells (a process known as antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the binding peptide (e.g., an antibody or immunoadhesin) binds to the Fcγ receptor. In alternative embodiments, the binding peptide may contain a constant region that lacks one or more effector functions (e.g., ADCC activity) and / or cannot bind to the Fcγ receptor.

[0211] In some embodiments, the binding polypeptides of this disclosure may comprise antigen-binding fragments of antibodies. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes for antigen binding with intact antibodies (i.e., with the intact antibodies from which they are derived) (i.e., specifically binds). Antigen-binding fragments can be generated by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include Fv, Fab, Fab', and (Fab')2.

[0212] In some embodiments, the binding peptide comprises a single-chain variable region sequence (ScFv). The single-chain variable region sequence comprises a single peptide having one or more antigen-binding sites, such as a VL domain linked to a VH domain via 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 constituting the antigen-binding site comprises about 10 to about 50 amino acid residues. Linker peptides are known in the art. The binding peptide may comprise at least one scFv and / or at least one constant region. In one embodiment, the binding peptide of this disclosure may comprise at least one scFv linked to or fused to a modified Fc domain.

[0213] In some embodiments, the binding peptide of this disclosure is a multivalent (e.g., tetravalent) antibody generated by fusing a DNA sequence encoding the antibody with an 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 this disclosure can be prepared by fusing an ScFv molecule with a linker peptide, which is then fused with a modified Fc domain to construct an ScFv-Fab tetravalent molecule.

[0214] In another embodiment, the binding polypeptide of this disclosure is a modified microantibody. The modified microantibody of this disclosure is a dimer molecule composed of two polypeptide chains, each polypeptide chain containing an ScFv molecule fused to a modified Fc domain via a linker peptide. The microantibody can be prepared by constructing the ScFv component and the linker peptide component using methods described in the art (see, for example, U.S. Patent 5,837,821 or WO 1994 / 009817). In another embodiment, a tetravalent microantibody can be constructed. A tetravalent microantibody can be constructed in the same manner as a microantibody, except that two ScFv molecules are linked using a flexible linker. The linked scFv-scFv construct is then linked to the modified Fc domain.

[0215] In another embodiment, the binding peptide of this disclosure comprises a biantibody. A biantibody is a dimeric tetravalent molecule, each having a polypeptide similar to an scFv molecule, but typically having a short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting two variable domains, such that the VL and VH domains on the same polypeptide chain do not interact. Instead, the VL and VH domains of one polypeptide chain interact (respectively) with the VH and VL domains of a second polypeptide chain (see, for example, WO 02 / 02781). The biantibody of this disclosure comprises an scFv-like molecule fused to a modified Fc domain.

[0216] In other embodiments, the binding peptide includes a multispecific or multivalent antibody comprising one or more variable domains tandemly on the same polypeptide chain, such as a tandem variable domain (TVD) peptide. Exemplary TVD peptides include the “double-headed” or “double-Fv” configuration described in U.S. Patent No. 5,989,830. In the double-Fv configuration, the variable domains of two different antibodies are represented in a tandem orientation on two separate chains (one heavy chain and one light chain), wherein one polypeptide chain has two tandem VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains tandemly linked by a peptide linker (VL1-linker-VL2). In a cross-headed configuration, the variable domains of two different antibodies are represented in tandem on two separate polypeptide chains (one heavy chain and one light chain), where one polypeptide chain has two tandem VH domains separated by peptide linkers (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains tandemly linked in opposite directions via peptide linkers (VL2-linker-VL1). Other antibody variants based on the “double Fv” configuration include bispecific antibodies with dual variable domains (DVD-IgG) (see U.S. Patent No. 7,612,181) and TBTI (see US 2010 / 0226923 A1). In some embodiments, the binding polypeptide comprises a multispecific or multivalent antibody containing one or more variable domains fused to a modified Fc domain in tandem on the same polypeptide chain.

[0217] In another exemplary embodiment, the binding peptide is an immunoadhesin. As used herein, “immunoadhesin” means a binding peptide comprising one or more binding domains (e.g., derived from receptors, ligands, or cell adhesion molecules) linked to a constant domain of an immunoglobulin (i.e., the Fc region) (see, for example, 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 Name (INN). Similar to antibodies, immunoadhesins have a long circulating half-life, are readily purified by affinity-based methods, and possess an affinity advantage conferred by divalent. Examples of commercially available therapeutic immunoadhesives include etanercept (ENBREL®), abatacept (ORENCIA®), linalcept (ARCALYST®), aflibercept (ZALTRAP® / EYLEA®), and beracept (NULOJIX®).

[0218] In some implementations, the binding peptide includes an immunoglobulin-like domain. Suitable immunoglobulin-like domains include, but are not limited to, fibronectin domains (see, for example, Koide et al. (2007), Methods Mol. Biol. 352: 95-109, which is incorporated herein by reference in its entirety), DARPin (see, for example, 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, for example, Nygren et al. (2008) FEBS J. 275 (11): 2668-76, which is incorporated herein by reference in its entirety), lipid transport proteins (see, for example, Skerra et al. (2008) FEBS J. 275 (11): 2677-83, which is incorporated herein by reference in its entirety), and affilin (see, for example, Ebersbach et al. (2007) J. Mol. Biol. 372 (1): 172-85, which are incorporated herein by reference in their entirety), Affitin (see, for example, Krehenbrink et al. (2008). J. Mol. Biol. 383 (5): 1058-68, which are incorporated herein by reference in their entirety), Avimer (see, for example, Silverman et al. (2005) Nat. Biotechnol. 23 (12): 1556-61, which are incorporated herein by reference in their entirety), Fynomer (see, for example, Grabulovski et al. (2007) JBiol Chem 282 (5): 3196-3204, which are incorporated herein by reference in their entirety), and Kunitz domain peptide (see, for example, Nixon et al. (2006) Curr Opin Drug Discov Devel 9 (2): 261-8, which are incorporated herein by reference in their entirety).

[0219] Regarding the binding peptides and immunoadhesins of this disclosure, virtually any antigen can be targeted by the binding peptides, including but not limited to the proteins, subunits, domains, motifs and / or epitopes of the target antigen, which includes both soluble factors (such as cytokines and membrane-bound factors) and transmembrane receptors.

[0220] In some embodiments, the binding peptide (e.g., an antibody) is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In some embodiments, bispecific antibodies can be used to cross the blood-brain barrier (BBB). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0221] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO 93 / 08829 and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and engineered “knob-in-hole” structures (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by: engineering electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553); using “dual antibody” techniques to prepare bispecific antibody fragments (see, for example, Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, for example, Gruber, M et al., J. Immunol. 152 (1994)). 5368-5374); and the preparation of trispecific antibodies, as described, for example, in Tutt, A. et al., J. Immunol. 147 (1991) 60-69.

[0222] In one implementation, the CH3 domain of the heavy chain of a bispecific antibody is altered using a "knob-hole" technique, described in detail with several examples, such as WO 96 / 027011, WO 98 / 050431, Ridgway JB et al., Protein Eng. 9 (1996) 617-621, Merchant, AM et al., Nat Biotechnol 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the two heavy chains containing the two CH3 domains. Each of the two CH3 domains can be a "knob," and the other a "hole." The introduction of disulfide bridges can be used to stabilize heterodimers (Merchant, A. M et al., Nature Biotech 16 (1998) 677-681, Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increase yield.

[0223] In one embodiment, the bispecific antibody is characterized in that the CH3 domains of one heavy chain and the CH3 domains of another heavy chain meet each other at an interface (which includes the original interface between the antibody CH3 domains), wherein the interface is modified to facilitate the formation of the bispecific antibody, wherein the modification is characterized by: a) modifying the CH3 domain of one heavy chain such that, within the original interface of the CH3 domain of one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protrusion within the interface of the CH3 domain of one heavy chain, the protrusion being localized within a cavity within the interface of the CH3 domain of the other heavy chain; and b) modifying the CH3 domain of the other heavy chain such that, within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the bispecific antibody, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the interface of the second CH3 domain, the protrusion within the interface of the first CH3 domain being localized within the cavity.

[0224] In one embodiment, the amino acid residues with a large side chain volume are selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0225] In one embodiment, the amino acid residues with smaller side chain volume are selected from alanine (A), serine (S), threonine (T), and valine (V).

[0226] In one implementation, the two CH3 domains are further modified by introducing cysteine ​​(C) as an amino acid at the corresponding position of each CH3 domain, so that a disulfide bridge can be formed between the two CH3 domains.

[0227] In one exemplary embodiment, the multispecific antibody comprises the amino acid T366W mutation in the first CH3 domain of the "pestle chain" and the amino acid T366S, L368A, and Y407V mutations in the second CH3 domain of the "mortar chain". Additional interchain disulfide bridges between the CH3 domains can also be used (Merchant, AM et al., Nature Biotech. 16(1998) 677-681), for example, by introducing the amino acid Y349C mutation into the CH3 domain of the "mortar chain" and introducing the amino acid E356C or amino acid S354C mutation into the CH3 domain of the "pestle chain".

[0228] 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 another 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 (an additional Y349C mutation in one CH3 domain and an additional E356C or S354C mutation in the other CH3 domain form an interchain disulfide bridge) (according to Kabat's EU index number; (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991))). Alternatively or additionally, other pestle-and-mortar structure techniques, such as those described in EP 1 870 459 A1, may be used. Thus, another example of a bispecific antibody is the R409D, K370E mutation in the CH3 domain of the "pepper chain" and the D399K, E357K mutation in the CH3 domain of the "mortar chain" (according to Kabat's EU index number; (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991))).

[0229] In one embodiment, the bispecific antibody comprises the T366W mutation in the CH3 domain of the "pestle chain" and the T366S, L368A, and Y407V mutations in the CH3 domain of the "mortar chain," as well as the R409D and K370E mutations in the CH3 domain of the "pestle chain" and the D399K and E357K mutations in the CH3 domain of the "mortar chain."

[0230] Nucleic acids and expression vectors

[0231] In one aspect, the present invention provides polynucleotides encoding the binding polypeptides disclosed herein. A method for preparing the binding polypeptides is also provided, the method comprising expressing these polynucleotides.

[0232] Typically, a polynucleotide encoding a binding polypeptide disclosed herein is inserted into an expression vector for introduction into a host cell, which can then be used to produce a desired amount of the required protective antibody or immunoadhesin. Therefore, in some aspects, the present invention provides expression vectors comprising the polynucleotides disclosed herein, and host cells comprising these vectors and the polynucleotides.

[0233] For the purposes of this specification and claims, the term "vector" or "expression vector" is used herein to mean a vector for introducing and expressing a desired gene in a cell. As known to those skilled in the art, such vectors can be readily selected from plasmids, bacteriophages, viruses, and retroviruses. Typically, a vector will contain selection markers, appropriate restriction sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0234] Many expression vector systems can be employed. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells that have integrated DNA into their chromosomes can be selected by introducing one or more markers, which allow selection of transfected host cells. Markers can provide resistance to auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals (such as copper). Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splicing signals, and transcription promoters, enhancers, and termination signals. In some embodiments, cloned variable region genes are inserted into the expression vector along with heavy and light chain constant region genes (such as human genes) synthesized as discussed above.

[0235] In other embodiments, the binding peptides described herein can be expressed using polycistronic constructs. In such expression systems, a variety of gene products of interest, such as the heavy and light chains of antibodies, can be generated from a single polycistronic construct. These systems advantageously utilize internal ribosome entry sites (IRES) to provide relatively high levels of the peptide in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference. Those skilled in the art will understand that such expression systems can be used to efficiently generate the full range of peptides disclosed in this application.

[0236] More generally, once a vector or DNA sequence encoding the binding polypeptide of this disclosure has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Plasmids can be introduced into host cells using a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and intact viral infection. See, for example, Ridgway, AAG, “Mammalian Expression Vectors,” Chapter 24.2, pp. 470-472, Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, MA 1988). The transformed cells are grown under conditions suitable for the production of light and heavy chains, and the synthesis of heavy and / or light chain proteins is measured. Exemplary assays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), immunohistochemistry, etc.

[0237] As used in this article, the term “transformation” should be used in a broad sense to refer to the introduction of DNA into a recipient host cell, which alters the genotype and thus causes changes in the recipient cell.

[0238] Following the same line of thought, "host cell" refers to a cell that has been transformed using a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In describing the process for isolating peptides from a recombinant host, unless otherwise explicitly stated, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody. In other words, recovering peptides from "cells" can mean recovering them from whole cells rotated down or from a cell culture containing both culture medium and suspension cells.

[0239] In one embodiment, the host cell line used to express the binding peptide is of eukaryotic or prokaryotic origin. In one embodiment, the host cell line used to express the binding peptide is of bacterial origin. In one embodiment, the host cell line used to express the binding peptide is of mammalian origin; those skilled in the art can determine the specific host cell line most suitable for expressing the desired gene product therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR-), HELA (human cervical cancer), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In one embodiment, the cell line provides altered glycosylation of the antibody expressed therein, such as non-fucosylation (e.g., PER6). TM (Crucell) or FUT8 knockout CHO cell line (POTELLIGENT) TM (Biowa, Princeton, NJ)). In one implementation, NS0 cells may be used. The host cell line is typically available from the American Tissue Culture Collection (ATC) or from publicly available literature.

[0240] In vitro production allows for scale-up to obtain large quantities of the desired bound peptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include homogeneous suspension culture (e.g., in an airlift reactor or a continuous stirred reactor) or cell culture immobilized or embedded in agarose beads or ceramic casks (e.g., in hollow fibers, microcapsules). Solutions of the peptide can be purified by conventional chromatographic methods (e.g., gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno)affinity chromatography) if necessary and / or required.

[0241] One or more genes encoding the binding polypeptide can also be expressed in non-mammalian cells, such as bacteria, yeast, or plant cells. In this regard, it will be understood that various single-celled non-mammalian microorganisms, such as bacteria—those capable of growth in culture or fermentation—can also be transformed. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; Bacillusaceae family, such as *Bacillus subtilis*; *Pneumococcus*; *Streptococcus*; and *Haemophilus influenzae*. It will be further understood that when expressed in bacteria, the polypeptide can become part of an integrity. The polypeptide must be isolated, purified, and then assembled into a functional molecule.

[0242] Besides prokaryotes, eukaryotic microorganisms can also be used. *Saccharomyces cerevisiae* or *Bacillus bakerella* are the most commonly used eukaryotic microorganisms, although many other strains are generally available. For expression in the genus *Saccharomyces*, plasmids such as YRp7 (Stinchcomb et al., *Nature*, 282:39 (1979); Kingsman et al., *Gene*, 7:141 (1979); Tschemper et al., *Gene*, 10:157 (1980)) are commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutant strains lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, *Genetics*, 85:12 (1977)). The presence of *trpl* damage, a characteristic of the yeast host cell genome, then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0243] Treatment

[0244] In one aspect, the present invention provides a method for treating one or more symptoms of Alzheimer's disease in a patient in need, the method comprising administering an effective amount of the binding peptide disclosed herein. In some embodiments, this disclosure provides kits and methods for treating Alzheimer's disease in mammalian subjects in need of such treatment. In some exemplary embodiments, the subject is a human.

[0245] Depending on whether local or systemic treatment is required and the area to be treated, the binding peptides disclosed herein can be administered in a variety of ways. For example, administration can be parenteral, including but not limited to intravenous infusion, subcutaneous administration, intraperitoneal administration, intramuscular administration, intrathecal administration, or intraventricular (e.g., intracerebral) administration. In some exemplary embodiments, the binding peptides are delivered across the blood-brain barrier (BBB) ​​using a variety of suitable compositions and methods described herein.

[0246] The binding peptide can be administered directly to the brain of a subject diagnosed with or suspected of having Alzheimer's disease (e.g., to a medium-sized polyspinous neuron in the globus pallidus of the basal ganglia or striatum, near the striatum). In addition to the binding peptide, secondary therapies, such as palliative and / or disease-specific therapies, can be administered to the patient. Secondary therapies can be, for example, symptomatic (e.g., for symptom relief), neuroprotective (e.g., for slowing or stopping disease progression), or reversible (e.g., for reversing disease progression). For example, for the treatment of Alzheimer's disease, symptomatic therapies may include the drugs Razadyne® (galantamine), Exelon® (levanstigmine), Aricept® (donepezil), Namenda® (memantine), or Namzaric® (memantine and donepezil). Other therapies may include psychotherapy, physical therapy, speech therapy, communication and memory assistance, social support services, and dietary advice.

[0247] The bound peptide can be delivered to nerve cells in the brain. Delivery methods that do not require the composition to pass through the brain via the brain can be utilized. For example, a pharmaceutical composition containing the bound peptide can be delivered to a patient by direct injection into a region containing disease-affected cells. For example, the pharmaceutical composition can be delivered by direct injection into the brain. Injection can be performed via stereotactic injection into specific regions of the brain (e.g., ventricles, substantia nigra, cortex, hippocampus, striatum, or globus pallidus). The bound peptide can be delivered to multiple regions of the central nervous system (e.g., delivery to multiple regions of the brain, and / or delivery to the spinal cord). The bound peptide can be delivered to diffuse areas of the brain (e.g., diffuse delivery into the cortex of the brain).

[0248] In one embodiment, the binding peptide can be delivered via a cannula or other delivery device, one end of which is implanted in tissue (e.g., the brain, such as the substantia nigra, cortex, hippocampus, striatum, or globus pallidus). The cannula can be connected to a reservoir of the binding peptide. Flow or delivery can be mediated by a pump (e.g., an osmotic pump or micropump, such as the Alzet pump (Durect, Cupertino, California)). In one embodiment, the pump and reservoir are implanted in a region remote from the tissue, such as in the abdomen, and delivery is mediated by a catheter leading from the pump or reservoir to the release site. Devices for delivery to the brain are described, for example, in US 6,093,180 and US 5,814,014 (incorporated herein by reference).

[0249] Through routine experiments, those skilled in the art will be able to determine an effective, non-toxic amount of the modified binding peptide for the purpose of treating malignant tumors. For example, the therapeutically active amount of the binding peptide of this disclosure can vary depending on factors such as the subject's disease stage (e.g., preclinical Alzheimer's disease, mild cognitive impairment, mild dementia, moderate dementia, or severe dementia), age, sex, medical complications (e.g., immunosuppressive symptoms or diseases), and weight, as well as the ability of the modified antibody to elicit the desired response in the subject. Dosing regimens can be adjusted to provide optimal therapeutic response. For example, several fractional doses can be administered daily, weekly, every other week, every three weeks, every four weeks, etc., and / or the dose can be proportionally reduced as indicated by an emergency situation in the treatment.

[0250] Pharmaceutical Composition

[0251] Methods for preparing and administering the binding peptides of this disclosure to subjects are well known or readily determined by those skilled in the art. The routes of administration of the binding peptides of this disclosure may be oral, parenteral, inhalation, or topical. As used herein, parenteral administration includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all such forms of administration are clearly considered within the scope of this disclosure, the form of administration will be a solution for injection, specifically for intravenous or intra-arterial injection or infusion. Typically, suitable pharmaceutical compositions for injection may contain buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbate), optional stabilizers (e.g., human albumin), etc. In some embodiments, the binding peptide may be delivered directly to the site of the adverse cell population, thereby increasing the exposure of diseased tissue to the therapeutic agent.

[0252] Formulations intended 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, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media. In the compositions and methods of this disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01–0.1 M (e.g., 0.05 M) phosphate buffer or 0.8% saline. Other common parenteral media include sodium phosphate solutions, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous media include fluids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextran), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be a fluid to the extent that it is easily injectable. It should be stable under manufacturing and storage conditions and will generally be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0253] Antimicrobial activity can be achieved through various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride, will be included in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0254] In any case, a sterile injectable solution can be prepared by incorporating an active compound (e.g., a modified binding polypeptide itself or in combination with other active agents) in a desired amount into a suitable solvent, followed by filtration and sterilization, the solvent having, as needed, one or a combination of the components listed herein. Typically, a dispersion is prepared by incorporating the active compound into a sterile medium containing an alkaline dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, exemplary preparation methods include vacuum drying and freeze-drying, which produce powders of the active ingredient and any other desired components from solutions previously sterile filtered. The formulation for injection is processed, filled into containers (such as ampoules, bags, bottles, syringes, or vials), and sealed under sterile conditions according to methods known in the art. Furthermore, the formulation can be packaged and sold as a kit. Such articles will typically have labels or instructions indicating that the relevant composition is intended for the treatment of subjects with or susceptible to autoimmune or neoplastic disorders.

[0255] The effective dosage of the compositions disclosed herein for treating the aforementioned conditions varies depending on a number of different factors, including the method of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is preventative or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The therapeutic dosage can be gradually adjusted using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0256] The binding peptides of this disclosure can be administered multiple times. The intervals between single doses can be weekly, monthly, or annually. The intervals can also be irregular, as indicated by measuring the blood levels of the modified binding peptide or antigen in the patient. In some methods, the dose is adjusted to achieve a plasma concentration of the modified binding peptide of about 1-1000 μg / ml, and in other methods about 25-300 μg / ml. Alternatively, the binding peptide can be administered as a sustained-release formulation, in which case less frequent administration is required. For antibodies, the dose and frequency vary based on the antibody's half-life in the patient. Typically, humanized antibodies exhibit the longest half-life, followed by chimeric antibodies and non-human antibodies.

[0257] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, a composition containing the antibody or a mixture thereof of the present invention is administered to a patient who is not yet in a disease state to enhance the patient's resistance. Such an amount is defined as a "preventive effective dose." In this use, the precise amount also depends on the patient's health status and overall immunity, but generally ranges from about 0.1 to about 25 mg / dose, particularly from about 0.5 to about 2.5 mg / dose. Relatively low doses are administered at relatively infrequent intervals over a long period. Some patients continue to receive treatment for the rest of their lives. In therapeutic use, it is sometimes necessary to administer relatively high doses (e.g., from about 1 to 400 mg / kg of antibody / dose) at relatively short intervals until disease progression decreases or ceases, or until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient.

[0258] The pharmaceutical compositions according to this disclosure may comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffer, preservative, etc. For the purposes of this application, the pharmaceutically effective amount of the conjugated or unconjugated binding peptide, immunoadhesin, or recombinant thereof, conjugated with a therapeutic agent, shall be an amount sufficient to achieve effective binding to the antigen and sufficient to obtain a benefit (e.g., sufficient to improve one or more symptoms of Alzheimer's disease). The pharmaceutical compositions of this disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the binding peptide.

[0259] To be consistent with the scope of this disclosure, the binding peptides of this disclosure may be administered to humans or other animals in an amount sufficient to produce a therapeutic or preventative effect, as described in the treatment methods described above. The binding peptides of this disclosure may be administered to such humans or other animals in conventional dosage forms prepared by combining the antibody of this disclosure with a conventionally pharmaceutically acceptable carrier or diluent according to known techniques. Those skilled in the art will recognize that the form and characteristics of a pharmaceutically acceptable carrier or diluent depend on the amount of the active ingredient to be combined with, the route of administration, and other well-known variables. Those skilled in the art will further understand that mixtures comprising one or more of the binding peptides described in this disclosure can prove particularly effective.

[0260] The contents of any articles, patents and patent applications, and all other documents and electronically available information mentioned or cited herein are hereby incorporated in their entirety by reference, to the extent that each individual publication is specifically and individually indicated to be incorporated by reference. The applicant reserves the right to actually incorporate any and all material and information from any such articles, patents, patent applications or other physical and electronic documents into this application.

[0261] While the invention has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes and equivalent substitutions can be made without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations can be made to the methods described herein using appropriate equivalents without departing from the scope of the embodiments disclosed herein. Furthermore, many modifications can be made to suit specific circumstances, materials, compositions of matter, processes, process steps, or steps to achieve the objectives, spirit, and scope of the invention. All such modifications are intended to be within the scope of the appended claims. Certain embodiments have now been described in detail and will become clearer from the following examples, which are included for illustrative purposes only and are not intended to be limiting.

[0262] Example

[0263] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations.

[0264] Example 1: A brief overview of the discovery process of Aβ monoclonal antibodies

[0265] Trianni mice were immunized with human Alzheimer's disease (AD) brain amyloid plaques (FusA), aggregated synthetic Aβ (FusB), or human AD brain amyloid plaques (FusC) inoculated with synthetic Aβ. Figure 1 It is worth noting that, in most cases, the Fc domain is mouse-specific, but the Fv domain is human-specific.

[0266] Hybridomas exhibiting good to moderate binding to synthetic Aβ primary fibrils (PF) were selected. Antibodies were reverse-selected that bound to monomeric Aβ with high affinity and to other types of aggregates (e.g., synuclein and SOD) with low affinity. Finally, mouse IgG antibodies were screened using a “cell iN assay” (described further below), in which the neuroprotective effect against the synaptic toxic form of oligomeric Aβ (oAβ) in AD was evaluated. Active antibodies exhibiting low binding to both Aβ primary fibrils and fibrillary Aβ were selected. Promising antibodies were cloned and reformatted to the human IgG1 LALA Fc domain.

[0267] The binding properties of reformulated human IgG1 monoclonal antibodies (mAbs) were evaluated for their selectivity for monomeric Aβ and low binding to Aβ PF and fibrillary Aβ. Two previously identified human mAbs that did not show significant binding to Aβ PF (or any other form) were retested in iN cell assays and their neuroprotective effects were determined.

[0268] Human mAbs (hIgG) that do not bind to other aggregates were evaluated using an iN assay, yielding an EC50 and reproducible results. As further described below, five hIgGs were tested in a second functional assay, the electrophysiological LTP assay.

[0269] Example 2: Immunization and Hybridoma Generation in Trianni Mice

[0270] immunity

[0271] Transgenic Trianni mice, which are genetically modified for human IgG heavy and κ light chains, were immunized with synthetic Aβ1-42 peptides that have been aggregated as previously reported [please cite] or by inoculating and aggregating synthetic Aβ1-42 peptides on scaffolds of sonicated human AD brain-derived amyloid plaque fragments. Mice were boosted with these proteins 3–5 times every two weeks.

[0272] Hybridoma cells were prepared by fusing mouse myeloma cells lacking adenosine phosphoribosyltransferase (APRT) (derived from the BALB / c B lymphoblast line SP2 / 0 fused with Sendai virus) with spleen cells from immunized mice. Selection and serial dilution with hypoxanthine, diazoserine, and thymidine (HAT) were performed to achieve single-cell clonality.

[0273] filter

[0274] Indirect ELISA was used for screening and reverse 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+. The monomeric Aβ1-42 peptide (SEC-purified) was diluted to 1000 μM (4.5 mg / ml) in HBS-EP+, followed by serial 2-fold dilutions for a total of six concentrations. Aβ1-42 PF was diluted to 100 nM (66 μg / ml) in HBS-EP+, followed by serial 2-fold dilutions for a total of six concentrations. The antibody was captured at 10 μl / min via the Fc domain on the surface of protein A for 60 seconds, followed by injection of an Aβ peptide solution (monomer or PF) at 30 μl / min for 180 seconds, and then dissociated for 360 seconds. The protein A surface was regenerated by injection of 10 mM glycine-HCl (pH 1.7) for 60 seconds. The resulting sensor map was double-referenced and fitted to a 1:1 binding model to determine k. a k d and KD .

[0275] Octet dissociation rate and kinetics analyses were performed using OctetRed96 (Forte Bio), PBS run buffer, and a protein A sensor tip. Antibodies were diluted in PBS to 1 or 5 μg / mL to evaluate binding to PF or monomers, respectively. Antigens were diluted to 1 μM (Aβ1-40) or 30 nM (PF), followed by serial dilutions at a 1:3 ratio for a total of three concentrations. Antibodies were captured onto the sensor tip to a 1 nm load (Aβ1-40) or 0.2 nm load (PF), and the sensor tip was then immersed in the antigen for 300 seconds, followed by dissociation in PBS for 360 seconds. Data were double-referenced and fitted using a 1:1 binding model.

[0276] The following is the workflow for screening and reverse screening. To analyze mouse serum titers, Aβ primary fibrils or Aβ-derived diffusing ligands (i.e., synthetic Aβ oligomers (ADDL)) and / or Aβ... 1 / 2 Indirect ELISA was performed on tmax. For primary screening, indirect ELISA was performed on Aβ PF and / or ADDL. For reverse screening, oligomeric Aβ was selected over monomeric Aβ to eliminate clones that strongly bind to the monomeric form. The dissociation rate was assessed using Biacore or indirect ELISA. Figure 1 Indirect ELISA was used to identify aggregated α-synuclein and aggregated SOD in order to eliminate clones that also bind to alternative off-target amyloid protein structures.

[0277] For secondary screening, cell-based in vitro assays will be used to identify clones that can neutralize the neurotoxic effects of AD brain extracts on iN cells.

[0278] For the three screenings, in vitro functional LTP assays were used to identify clones that could neutralize the inhibitory effect of AD brain extracts on LTP (as measured in brain slices).

[0279] Hybridoma Screening Summary

[0280] Approximately 4400 clones passed the initial ELISA screening. Three different immunization strategies were employed, and five fusions were performed. The initial ELISA screening was conducted on Aβ 1-42-derived primary fibrils and / or Aβ 1-42-derived ADDL.

[0281] Approximately 80 clones were evaluated through reverse screening to identify clones with minimal to no binding to off-target entities (e.g., Aβ1-40 monomers and / or Aβ1-42 monomers, Aβ1-42 protofibrils, synuclein protofibrils, and aggregated SOD-1).

[0282] Approximately 50 clones were evaluated through secondary screening using cell-based functional screening (e.g., iN cell assays). Multiple assay runs were performed using the original hybridoma. Additionally, the lead candidate in cloned, reformulated, and recombinant-generated IgG1 LALA form was tested. EC50 data for the lead clone were obtained and replicated to rank clones according to those most capable of neutralizing oligomeric Aβ synaptic toxicity.

[0283] Four clones were evaluated by three LTP functional screenings at one or more concentrations and ranked according to their relative ability to rescue LTP.

[0284] One or more lead clones were selected based on the good binding selectivity and retention of functional activity in both iN and LTP assays.

[0285] Example 3: Summary of Fusion B (FusB)

[0286] Aggregated synthetic Aβ was administered to Trianni mice using Sigma adjuvant. Immunization was performed intraperitoneally with a total dose of 200 μl (50 μg antigen). The assessed titer was evaluated on ½ tmax (4 μg / mL coating) or ADDL (1 μg / mL coating) after the fifth immunization. Figure 3 Based on these data, the mouse M13 mice were set aside to rest before final enhancement and fusion.

[0287] M13 in mice (potency approximately 1:600,000)

[0288] Approximately 670 clones were identified for initial screening on ADDL. Initially, 90 positive clones were identified by an ELISA targeting ADDL. These 90 positive clones were transferred to 24-well selection, where 17 had lost binding or lacked sufficient cells. Therefore, 73 clones were transferred to a 6-well stage. Starting from the 6-well stage, 15 clones were scaled up for production (Group 1), followed by 13 slower-growing clones (Group 2). A total of 28 clones were transferred to production and purified using Protein Maker. Four clones were further subjected to functional screening. FusB screening is summarized in... Figure 37 The binding and characterization data of the lead FusB clone are depicted in... Figures 4-18 middle.

[0289] Two clones were identified as two isotypes and were re-cloned. One clone, B47-6, was moved forward and purified together with the fusion clone C.

[0290] Example 4: Summary of Fusion C (FusC)

[0291] Trianni mice were injected with synthetic Aβ-infused AD amyloid plaques using Sigma adjuvant. Potency was assessed on ½ tmax (4 μg / mL coating) or ADDL (1 μg / mL coating) after the fifth immunization. Figure 18 Based on these data, the M23 mice were set aside to rest before final enhancement and fusion.

[0292] Approximately 750 clones were initially screened on ADDL. 29 weakly positive clones were initially identified and scaled up (96w-24w-6w-T150). Six clones retained very weak positivity after scale-up. Two weakly positive clones were moved to reverse screening. These two clones were identical, so the remaining clones were reformatted and recombined to produce hIgG1 LALA, then rescreened and re-re-re-screened. FusC screening is summarized in... Figure 38 The binding and characterization data of the lead FusC clone are depicted in... Figures 19-29 middle.

[0293] Continue functional screening of a clone.

[0294] Example 5: Cellular iN Measurement

[0295] Aqueous extracts prepared from human Alzheimer's disease brain

[0296] Frozen brain tissue was provided by the University of Miami Miller School of Medicine (Miami, Florida) and the Manchester Brain Bank of the Medical Research Council at the University of Manchester (Manchester, UK). The brain tissue was obtained from two patients who died from mild to moderate stages of Alzheimer's disease (AD). Aqueous extracts were prepared as previously described (Jin et al. (2018) Nature Comm. 9:2676). In short, 20 grams of temporal cortical gray matter were Dounce homogenized in 5 volumes of ice-cold artificial cerebrospinal fluid basal buffer (aCSF-B) (124 mM NaCl, 2.8 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, pH 7.4) supplemented with protease inhibitors (5 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 5 μg / mL leuprolide, 5 μg / mL aprotinin, 2 μg / mL pepsin inhibitor, 120 μg / mL Pefabloc, and 5 mM NaF). The resulting homogenate was centrifuged at 200,000 g for 110 minutes at 4ºC in an SW41 Ti rotor (Beckman Coulter, Fullerton, California), and the upper 80% supernatant was collected and dialyzed against fresh aCSF-B. The buffer was changed three times every 24 hours over a 72-hour period. The brain extracts were then aliquoted into two portions: the first portion was subjected to Aβ immune depletion (ID) by incubating with anti-Aβ antibody S97 and protein A agarose (PAS) beads for 12 hours for three consecutive rounds at 4ºC. The second portion was treated in the same manner with pre-immune serum and PAS beads. The Aβ-depleted extract was designated ID-AD, and the extract treated with pre-immune serum was designated simulated AD. The beads were removed from the sample, and each 0.5 mL aliquot was transferred to a low-protein-binding Eppendorf tube (Eppendorf, Hamburg, Germany) and stored at -80ºC until use. The sample was thawed once and used. Brain extracts from two different cases, AD10 / 27 and AD10 / 14, were used for functional assays.

[0297] iPSC-derived human neurons (iN)

[0298] As previously described (Hong et al., 2018; Jin et al., 2018), neuron 2 (Ngn2)-induced human neurons were prepared (Zhang et al., 2013). Briefly, YZ1 iPSCs were maintained in a medium containing DMEM / F12, knockout serum substitutes, penicillin / streptomycin / glutamine, MEM-NEAA, and 2-mercaptoethanol (all from Invitrogen, Carlsbad, CA) plus 10 μg / mL bFGF (Millipore, Billerica, MA). The iPSCs were then cultured at 95,000 cells / cm². 2 Cells were plated at the following densities and infected with the virus at the following concentrations: 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 neural element 2 expression, doxycycline was added at a concentration of 2 µg / mL on day 1 of in-N and puromycin at a concentration of 10 mg / mL on day 2 of in-N, and maintained in the culture medium thereafter. On day 4 of incubation (iN), cells were seeded at 5,000 cells / well on Greiner 96-well microplates coated with Matrigel (BD Biosciences, San Jose, CA) and maintained in a medium consisting of Neurobasal medium (Gibco), Glutamax, 20% dextrose, MEM-NEAA, and B27 with BDNF, CNTF, and GDNF (PeprpTech, Rockhill, NJ) (each at a concentration of 10 ng / mL). By day 14 of iN, neurite number and expression of neural markers reached their peak, and iN cells were fully mature by day 21. To investigate the effect of AD brain extract on neurite integrity, cells were used on day 21 of iN.

[0299] Sample addition and live-cell imaging

[0300] On day 21 post-induction, neurons were used to investigate the effect of the sample on neurite integrity using an Incucyte live imaging reader. Approximately 7 hours before sample addition, images were collected every 2 hours from four fields of view in each well for a total of 6 hours, and baseline neurite length and branching points were calculated. At the final interval, brain samples were exchanged into basal neuronal medium supplemented with B27 / Glutamax using a PD MidiTrap G-25 column (GE Healthcare Life Science, Milwaukee, Wisconsin). After 6 hours of baseline imaging, half the medium was removed from each well (leaving approximately 100 µL), and 50 µL of the exchanged extract or medium and 50 µL of fresh medium were added. Subsequently, images were collected every 2 hours from four fields of view in each well for at least 72 hours. The phase-contrast image set was analyzed using IncuCyte Zoom 2016A software (EssenBioscience, Ann Arbor, Michigan). The analyte Neural Track was used to automatically define neurite processes and cell bodies based on the phase-contrast images. Typical settings: Segmented mode - Brightness; Segmented adjustment - 1.2; Cell cluster filter - Minimum 500 μm 2 ; neurite filtering - optimal; neurite sensitivity - 0.4; neurite width - 2 μm. Total neurite length (in millimeters) and the number of branching points were quantified and normalized relative to the mean measured over a 6-hour period prior to sample addition. AD brain extract (+ / - immune depletion) was added to the neuronal + / - test mAb.

[0301] The neurite length at the end of the 72-hour period was averaged at the last three time points, and the percentage of protection at each concentration was calculated compared to a) antibody-free condition (AD) and b) immune-depleted brain extract (ID-AD). The 50% protective concentration against neurite toxicity (EC50) was calculated.

[0302] Data analysis and statistical testing

[0303] For live-cell imaging experiments, samples and treatments were coded and tested in a blinded manner. Differences between groups were tested using two-way ANOVA with Bonferroni post-hoc tests or Student's t-tests. # p < 0.05, ## p < 0.01, and ### p < 0.001.

[0304] Functional effect of hybridoma-purified mAbs in cell iN assay

[0305] The hybridoma-purified mAb was tested in cell iN assays to evaluate the protective effect against neurite length against toxicity induced by the soluble AD brain extract. In human neuronal iN cultures, continuous exposure to the AD brain extract (AD10 / 27) resulted in a reduction of approximately 50% of human neurons, as measured after 3 days (see [reference]). Figure 30 The AD sample was compared to the culture medium or ID sample. Pre-immunodepletion of Aβ (sample ID) from the AD extract prevented neurotoxicity, which validates that the AD brain extract contains Aβ-dependent neurotoxic activity, as previously demonstrated (Jin et al. 2012; 2018). A positive control antibody 3D6 against Aβ was used at 3 µg / ml. Figure 30 The code C1 (which can neutralize neurotoxic brain extracts) provided further validation of the assay, as previously demonstrated (Shankar et al., 2008; Jin et al., 2011). 3D6 binds to the N-terminal free end of the Aβ sequence and recognizes Aβ in all conformations. An additional 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 entities containing toxic Aβ in AD brain extracts has not yet been characterized, a large group of mAbs with broad affinity for soluble primary fibrils and aggregated Aβ, but not for monomeric Aβ, was carefully selected from immunomotor activity. This was made possible only by the good volume / throughput of the unique cellular function iN assay used (form multiwalled MW96). Brain extracts from two different formulations from the same case AD10 / 27 (denoted as AD10 / 27 No. 1 and No. 2) were used. Several hybridoma-purified mAbs exhibited neuroprotective effects similar to those of the positive control. Figure 30 The range of mAbs from those with intermediate affinity for the PF Aβ form (such as B73 or B24) to those with high affinity for PF (such as B51 or B90). However, biochemical binding characteristics cannot predict activity in iN assays, as B35 and B61 (with intermediate affinity for the PF Aβ form) are inactive in iN assays, while B8, B30, or B60 have very high affinity for Aβ.

[0306] experiment# Run #1 and #2 Run #3 Run #4 Run #5b Run #6 October 26 Run #811-09-18 Run #911-16-18 Tox max Running #1 Tox reaches approximately 45% completion; running #2 Tox reaches approximately 70% completion. Tox approximately 60% Tox approximately 55% Tox approximately 65% Tox approximately 70% Tox 80% Dilution 1 / 6 Tox: Approximately 75% Combining features The tested mAb brain extract 10 / 27 (1st) 10 / 27 (2nd) 9 / 20 / 18 10 / 27 (2nd) 9 / 20 / 18 10 / 27 (2nd) 9 / 27 / 18 10 / 27 (2nd) 9 / 27 / 18 10 / 27 (2nd) 11 / 01 / 18 10 / 27 (2nd) 11 / 01 / 18 NA Anti-TNP Negative (C2) Negative (C1) Negative (R6) Negative (R2) Negative (A8) Negative (2x) A3A10 M+PF 3D6 Active (C1) Active (C3 and C4) NT NT Active (R1) PF>>M 1C22 NT Active (C2) Active (R2) Active (R8) Active (A9) PF>>M B30 Negative It has some activity (A11). Active (R3) Very moderate (R9) Active (A3) recombinant Weak in PF but >M B75 Negative NT Active (R4) Most active (R7) NT Hybrid with moderate activity (A11) PF>>M B19 reconstituted product (=B24) Not yet available Negative (A1) NT NT NT PF>>M B24 Active 2x (May preparation and July re-preparation) It has some activity (A12). Active (R5) It has very weak activity (R1). Moderately active (A1) recombinant Moderate (A6) recombinant PF>>M B28 Active 2x (May preparation and July re-preparation) It has some activity (A13). Negative* (pSEC) ~Negative (R5) * (pSEC) Moderately active (A2) recombinant Active (A5) recombinant PF>>M B51 It has 2x activity (but is equivalent to B60, which has no activity). It has some activity (A14). NT NT Active (A4) recombinant Active (A4) recombinant PF>>M B54 Active 2x (two preparations) Negative (A15) NT NT Most active recombinant Active recombinants PF>>M B73 Active 2x (two preparations) Active (A16) NT NT Most active recombinant Active recombinants PF>>M B90 Active 2x (two preparations) Active (A17) Partially active (R7) * (pSEC) Partially active (R4) * (pSEC) Very moderate (A6) recombinant Very moderate (A1) recombinant Do not combine with PF, ADDL or M C10 Active Negative (A8) July preparation Negative (R8) July preparation ~Negative (R2) July preparation Most active (A9); *New preparation Do not combine with PF, ADDL or M C11 Most active Negative (A9) July preparation Some active July preparations ~Negative (R3) *Preparation 3 months after July Most active (A10); Newly prepared product Active (A10); newly prepared product Hybridoma: Partial (A4) Recombinant: Negative (A5)

[0307] Table 5. Summary of the activities of different monoclonal antibodies in the iN neurotoxicity protection assay.

[0308] Therefore, the iN function assay using AD brain extract represents a unique tool for characterizing and selecting novel Aβ mAbs with highly relevant biological activities. Due to the sensitivity of the iN assay, several independent experiments were performed to confirm the data, including experiments using antibodies derived from additional fusions (see Table 5).

[0309] The neuroprotective effects of different antibodies varied slightly in experiments, partly due to the moderate stability of antibody batches purified from hybridomas or the limited stock concentrations of these antibodies, resulting in only limited dilution of iN cells in the culture medium and thus impairing cell viability. In some cases, separate antibody production batches had to be generated. For example, hybridoma B28 showed only moderate activity in run #3 and was negative in runs #4 and 5b, likely due to stability issues. Similarly unexpected was clone B73, which was initially negative in iN runs using the first hybridoma production, but provided very significant activity in runs #4 and 5b with the second preparation.

[0310] To enable further analysis using recombinant forms of hybridoma clones that can transfer all mAbs to the same IgG1 LALA framework and allow for more controlled production conditions, clones that provide neuroprotection in at least one iN experimental run but have diverse affinities for synthetic Aβ conformational isomers, particularly those with low affinity for primary protofibrils and fully aggregated synthetic Aβ formulations (aside from those with low / no affinity for monomeric Aβ).

[0311] Functional effects of recombinant mAbs in cell iN assay

[0312] Selected mAbs in recombinant form were generated at high stock concentrations (> 2 mg / ml) and tested in cellular iN assays. For each mAb, a first test was performed using the brain extract AD10 / 27 at a single mAb concentration (3 µg / ml in wells) to confirm the neuroprotective effects previously observed in hybridoma-purified forms (Table 5, runs #6, 8, and 9). Based on the unique biochemical binding characteristics (as low as possible affinity for the classical synthetic form of Aβ, but very significant protection against Aβ Ad brain extract), complete concentration-response curves were performed using the brain extract AD10 / 14 alone for the mAbs of most interest. It was important to determine that the neuroprotective activity of the mAbs could also be observed in the AD case extract alone. The mAbs were tested at concentrations ranging from 0.75 to 6 or up to 12 µg / ml. For example, the full-time results of mAbs rB24 and rB75 compared to the positive control 1C22 are shown in the top and middle subplots of Figure 31, respectively, demonstrating the concentration-dependent protection of rB24 and rB75 against neurite loss induced by the AD brain extract AD10 / 14. The 50% protective concentrations (EC50) for neurite toxicity of rB24 and rB75 were determined to be 2,528 and 2,111 ng / ml, respectively, compared to 1,049 for the positive control 1C22 (Figure 31, bottom subplot). Comparable data are provided for the rC11 and rB24 pair in Figure 32 and the rB73 and rB28 pair in Figure 33.

[0313] Compared to the positive control 1C22, multiple experiments were performed using two to three different mAbs, and the EC50 results are summarized in Table 6. All selected recombinant mAbs showed activity levels within twice that of 1C22.

[0314] Table 6. Neuroprotective activity of selected recombinant antibodies in iN cell assays (EC50 values ​​expressed in ng / ml)

[0315]

[0316] Example 6: Long-term enhanced electrophysiological functional assay demonstrating the protective effect against soluble AD brain extract

[0317] Aqueous extracts prepared from human Alzheimer's disease brain

[0318] Aqueous extracts were prepared from human AD brains as described above for functional cell iN assays. Samples were thawed only once and used. Brain extracts from AD cases AD10 / 14 were used for long-term potentiation (LTP) assays.

[0319] Electrophysiological recordings and antibody protection tests

[0320] Hippocampal LTP recordings were performed using brain slices (350 µm thick) from adult mice (2-3 months old, both sexes) similar to 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). Radiation layers in the CA1 region of the hippocampus were recorded, and stimulating electrodes were placed on the Schaffer lateral branch using a microelectrode array (MEA). The MED64 recording system (Alpha MED Scientific, Japan) was used for extracellular field potential recording. The MED64 probe (P515A) contained an array of 64 planar microelectrodes arranged in an 8 × 8 pattern with an interelectrode distance of 150 μm (Liu et al. (2011) Brain Res. 1382:57). Antibodies (C11, B24, B28, B73, and B75) were added to perfused artificial cerebrospinal fluid (ACSF) at a concentration of 5 µg / ml. When testing AD extract, control, AD extract, and antibody + AD extract conditions were randomly selected to avoid any slide quality issues. Daily records for each mouse were checked to confirm that all brain slides responded well to either the antibody or AD extract. Data from a mouse was not used if a slide showed very little or no LTP under the test conditions. 1) For the antibody-only condition, the antibody was added to 10 mL of perfused ACSF and recorded for at least 30 minutes to ensure baseline stability, followed by high-frequency stimulation (HFS). Recording continued for another 60 minutes after stimulation. 2) For the antibody + AD extract experiment, both the antibody and AD extract (AD10 / 14, dilution 1 / 20) were thawed at room temperature and then gently vortexed. Mix each antibody with an aliquot of AD extract (0.5 ml) and gently shake for 60 minutes, then add to 9.5 mL of perfused ACSF (total volume 10 mL).

[0321] Data analysis and statistical testing

[0322] Samples and treatments were coded and tested in a blinded manner. The EPSP slope value at 60 minutes after HFS was quantified compared to the baseline for each brain slice, and the mean for each antibody was calculated (number of brain slices / condition for each antibody in the legend). Differences between groups were tested using two-way ANOVA with Bonferroni post-hoc test or Student's t-test. #p < 0.05, ##p < 0.01, and ###p < 0.001.

[0323] Test of selected recombinant mAbs in electrophysiological assays

[0324] Hippocampal long-term potentiation (LTP) recordings were performed to analyze synaptic plasticity in brain slices, a model considered representative of memory encryption. It has been previously shown that AD-soluble brain extracts contain Aβ-dependent activity that effectively inhibits HFS-induced LTP induction in rodent brain slices (Shankar, GM et al. 2008; Hong, W. et al. 2018). First, the effects of selected antibodies (C11, B24, B28, B73, and B75) on basal LTP were investigated. Antibodies were added to perfused ACSF at a final concentration of 5 µg / ml in ACSF. At this concentration, C11, B24, B28, and B75 did not affect basal HFS delivery or LTP induction ( Figure 34A , Figure 34B B73 significantly reduced basal delivery or LTP induction of HFS. At lower concentrations of 3 µg / ml, B73 did not affect LTP induction and was subsequently used.

[0325] Next, different antibodies were tested (5 µg / ml, except for B73, tested at 3 µg / ml) to analyze whether they could prevent the inhibition of LTP-induced antibodies by AD brain extract. Figure 35A and Figure 35B As observed, in the presence of AD brain extract AD10 / 14, the enhancement of EPSP 1 hour after HFS was only 120% of baseline, compared to 150% in the case of ACSF alone (Figure 34), confirming the inhibitory activity of AD brain extract. Pre-incubation of AD brain extract with different antibodies resulted in a very significantly higher EPSP enhancement level returning to the value of ACSF alone (Figure 34). Figure 35A , Figure 35B (p-value less than 0.05). More notably, both B75 at 5 µg / ml and B73 at 3 µg / ml resulted in complete rescue of the inhibitory effect of AD brain extract on LTP. Therefore, it can be concluded that, in addition to the protective effect observed in human neuron cultures, the antibodies described herein are also able to neutralize the LTP inhibitory activity present in AD brain extract.

[0326] Next, the best-performing antibodies B73, B75, and C11 were tested at a lower concentration of 2 µg / ml and compared with the reference antibody 1C22. Figure 36A and Figure 36B This experiment used a new batch of B73. It was confirmed that at these low concentrations, no antibody affected basal transfer of HFS or LTP induction. Figure 36ANext, the antibody was tested to prevent the inhibition of LTP-induced activity by AD brain extract (at 2 µg / ml). At this concentration, B73 almost completely blocked the effect of AD brain extract on LTP. Figure 36B (p < 0.05), consistent with previous data obtained at 3 µg / ml. C11 also significantly inhibited the effects of the AD brain extract by well over 50% (p < 0.01), and both were comparable to the reference antibody 1C22. B75 had no significant effect at these lower concentrations. These data confirm and extend the electrophysiological results discussed above.

[0327] Example 7: The antibody can capture Aβ in AD brains, but does not bind to amyloid deposits in AD brain slices.

[0328] Biochemistry of B24, B28, B73, B75 and C11 mAbs based on bead-based immunoprecipitation using human AD brain extract Learning data: B75 and C11 immunoprecipitate Aβ x-42 peptide from AD brain

[0329] Immunoprecipitation of human brain extracts was performed using a conventional magnetic bead method to test for B24, B28, B73, B75, and C11 mAb. Figure 39A The Aβ x-42 ELISA was measured using a sequential eluent of 1% SDS followed by 6 M guanidine hydrochloride (GnCl). Compared to the negative control (human IgG) and positive control (1C22), B28 and B73 significantly immunoprecipitated Aβ 1-42-containing species from all four brains tested, consistent with their broad Aβ binding properties. Although to a slightly lower degree, B75 and C11 also significantly immunoprecipitated Aβ 1-42-containing species from two of the four brains, while B24 failed to immunoprecipitate Aβ 1-42-containing species from any of the brains tested. Figure 39B These data determine that B75 and C11 bind to materials containing Aβ1-42 in the AD brain, but not to the different synthetic Aβ formulations described above.

[0330] Biochemical data from column immunosorbent assay using human AD brain extract: C11 and B28 bind to human AD brain containing... Types of Aβ

[0331] Alternative methods were designed using conventional magnetic bead methods to retest B24, B75, and C11, which showed moderate affinity for species containing Aβ. These methods were used instead of protein A spin columns. Figure 40A This eliminates processes that disrupt weak antibody-antigen association (such as rotation or nutation). Using this alternative method, C11 was shown to capture as many Aβ1-42-containing species from two human brain extracts as B28 (positive control). Figure 40B B75 has lower activity, highlighting that different antibody properties can be revealed through alternative techniques.

[0332] Additionally, for this type of rotating column, elution is performed using acid washing, which is intended to destabilize antigen-antibody interactions and has a milder effect on the antigen structure compared to SDS or guanidine washing performed in the bead immunoprecipitation example. Therefore, the potential structure of the immunocapture material was evaluated using transmission electron microscopy (TEM). Figure 41 a). The negative staining material purified by B28 and C11 was determined to be polymorphic by TEM. Figure 41 b). The material purified by B28 has a predominantly spherical structure of varying sizes. The material purified by C11 exhibits a more diverse range of morphologies.

[0333] Immunohistochemistry: As assessed by immunohistochemistry using light microscopy, C11 does not bind to Aβ deposits in the human AD brain. Accumulation

[0334] Immunohistochemistry was used to compare the binding patterns of different antibodies to fixed or fresh-frozen human AD brain sections. Initially, all five mAbs were evaluated by immunohistochemistry on standard paraffin-embedded PFA-fixed AD brain sections. Only B28 was identified as producing any staining, i.e., typical AD amyloid plaques and cerebral amyloid angiopathy (AD). Figure 42 a). B24, B28, B73, B75, and C11 mAbs were further tested by immunohistochemistry on unfixed frozen brain sections (representing a potentially more native conformation of the Aβ assemblies in AD brains). Compared to 1C22 (positive control), B28, B73, and B75 strongly stained amyloid plaques, B24 moderately stained, while C11 was unstained. Figure 42 b).

[0335] Materials and methods

[0336] Immunoprecipitation (beads) and immunoadsorption (columns) of human brain extracts

[0337] Beads: 800 μl of TBS brain extract was mixed with 10 μg of mAb and protein A magnetic beads and rotated at 4ºC for 12 hours. The magnetic beads were then washed three times with PBS at 4ºC for 30 minutes each time. The washed magnetic beads were eluted sequentially with 1% SDS in PBS and then with 6 M guanidine hydrochloride.

[0338] Column: Mix 800 μl of TBS brain extract with 10 μg of mAb and incubate at 4ºC for 12 h. Add the mixture to a pre-wetted Protein A column and rotate at 1,000 g for 1 min. Add the flow-through to the same column and rotate at 1,000 g for 1 min. Wash the column three times with PBS and rotate at 1,000 g for 1 min. Add 0.1 M glycine-HCl (pH 2.3) to the column and incubate for 5 min, followed by rotation at 1,000 g for 1 min.

[0339] Aβ x-42 ELISA

[0340] MSD ELISA for Aβ 1-42 was performed according to methods 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 mL of PBS solution containing 3 μg / mL of 266 capture antibody (Elan) and incubated overnight at room temperature. The 266 epitope is located in regions 13-25 of the Aβ sequence. Detection antibody solutions were prepared using a biotinylated monoclonal antibody (21F12) targeting the C-terminal residues of Aβ 1-42, 100 ng / mL streptavidin sulfonyl-TAG (Meso Scale Discovery, #R32AD-5), and 1% BSA diluted in wash buffer. After overnight incubation, 50 μL / well of sample was followed by 25 μL / well of detection antibody solution and incubated at room temperature for 2 hours with shaking at >300 rpm. The wells were washed with wash buffer between incubations. Read and analyze the board according to the manufacturer's scheme.

[0341] Transmission EM analysis of negatively stained samples

[0342] For TEM specimen preparation, 5 μL of sample solution (glycine eluent from the immunosorbent material (see above)) was placed on a glow discharge-treated fumwah / carbon-coated grid. The sample was incubated on the grid at room temperature for 20 seconds. Excess solvent was aspirated with filter paper (Whatman). The grid was washed three times with 10 μL of water and stained three times with 10 μL of 1% (w / v) uranyl acetate in water. The dried grid was examined in a JEM-1200EX TEM (JEOL) equipped with an AMT 2k CCD camera operating at 80 kV.

[0343] Immunohistochemistry

[0344] Paraffin-embedded PFA-fixed brain sections: Rehydrated paraffin sections were treated with 0.3% H2O2 in PBS containing 0.2% Triton X-100. Sections were incubated overnight at 4ºC with primary antibody, followed by incubation for 1 hour with biotinylated secondary antibody. For visualization, sections were treated with avidin-biotin complex (Vector) and then with 3,3'-diaminobenzidine containing nickel ammonium sulfate.

[0345] Non-fixed frozen brain sections: Fresh or thawed brain tissue was embedded in an OCT compound (Sakura) solution and then incubated at -80ºC and -20ºC for 12 hours each. The frozen sections were sectioned at 20–30 μm intervals using a cryostat (Leica). Sections were mounted directly onto an adhesion microscope slide (Matsunami) for immunohistochemistry. The sections were then treated with 0.3% H2O2 in PBS containing 0.2% Triton X-100. The sections were incubated overnight at 4ºC with primary antibody, followed by incubation for 1 hour with biotinylated secondary antibody. For visualization, the sections were treated with avidin-biotin complex (Vector) and then with 3,3'-diaminobenzidine containing nickel ammonium sulfate. Photographs were taken using a DMi8 wide-field microscope (Leica).

[0346] The present invention further includes the following:

[0347] 1. An isolated binding polypeptide that specifically binds to soluble β-amyloid (Aβ), wherein the binding polypeptide comprises three heavy chain complementarity-determining region (HCDR) sequences and three light chain complementarity-determining region (LCDR) sequences.

[0348] The three HCDR sequences are selected from the following group: SEQ ID NO: 26, 27, 28, 20, 21, 22, 50, 51, 52, 44, 45, 46, 62, 63, 64, 32, 33, 34, 38, 39, 40, 56, 57 and 58, and

[0349] The three LCDR sequences are selected from the following group: SEQ ID NO: 23, 24, 25, 17, 18, 19, 47, 48, 49, 41, 42, 43, 59, 60, 61, 29, 30, 31, 35, 36, 37, 53, 54 and 55.

[0350] 2. The binding polypeptide according to claim 1, wherein the binding polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0351] 3. The binding polypeptide according to claim 2, wherein the antibody or its antigen-binding fragment is human.

[0352] 4. The binding polypeptide according to item 2 or 3, wherein the antibody or its antigen-binding fragment is IgG1.

[0353] 5. The binding polypeptide according to any one of claims 1 to 4, said binding polypeptide comprising a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the following: SEQ ID NO: 4 and 3; SEQ ID NO: 2 and 1; SEQ ID NO: 10 and 9; SEQ ID NO: 12 and 11; SEQ ID NO: 16 and 15; SEQ ID NO: 6 and 5; SEQ ID NO: 8 and 7; and SEQ ID NO: 14 and 13.

[0354] 6. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 20, 21 and 22, and the three LCDR sequences comprise SEQ ID NO: 17, 18 and 19.

[0355] 7. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 44, 45 and 46, and the three LCDR sequences comprise SEQ ID NO: 41, 42 and 43.

[0356] 8. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 26, 27 and 28, and the three LCDR sequences comprise SEQ ID NO: 23, 24 and 25.

[0357] 9. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 32, 33 and 34, and the three LCDR sequences comprise SEQ ID NO: 29, 30 and 31.

[0358] 10. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 38, 39 and 40, and the three LCDR sequences comprise SEQ ID NO: 35, 36 and 37.

[0359] 11. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 56, 57 and 58, and the three LCDR sequences comprise SEQ ID NO: 53, 54 and 55.

[0360] 12. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 2 and 1.

[0361] 13. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 10 and 9.

[0362] 14. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 4 and 3.

[0363] 15. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 12 and 11.

[0364] 16. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 16 and 15.

[0365] 17. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 6 and 5.

[0366] 18. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 8 and 7.

[0367] 19. The binding polypeptide according to claim 5, wherein the HCVR / LCVR sequence pair is SEQ ID NO: 14 and 13.

[0368] 20. The binding polypeptide according to any one of claims 1 to 19, said binding polypeptide comprising a light chain framework (LFW) sequence and a heavy chain framework (HFW) sequence,

[0369] The LFW sequence comprises sequences selected from the following: SEQ ID NO: 81, 82, 83 and 84; SEQ ID NO: 89, 90, 91 and 92; SEQ ID NO: 97, 98, 99 and 100; SEQ ID NO: 105, 106, 107 and 108; SEQ ID NO: 113, 114, 115 and 116; and SEQ ID NO: 129, 130, 131 and 132; and

[0370] The HFW sequences mentioned therein comprise sequences selected from the following: SEQ ID NO: 85, 86, 87 and 88; SEQ ID NO: 93, 94, 95 and 96; SEQ ID NO: 101, 102, 103 and 104; SEQ ID NO: 109, 110, 111 and 112; SEQ ID NO: 117, 118, 119 and 120; and SEQ ID NO: 133, 134, 135 and 136.

[0371] 21. The binding polypeptide according to claim 18, wherein the LFW sequence comprises SEQ ID NO: 81, 82, 83 and 84; and the HFW sequence comprises SEQ ID NO: 85, 86, 87 and 88.

[0372] 22. The binding polypeptide according to claim 18, wherein the LFW sequence comprises SEQ ID NO: 89, 90, 91 and 92; and the HFW sequence comprises SEQ ID NO: 93, 94, 95 and 96.

[0373] 23. The binding polypeptide according to claim 18, wherein the LFW sequence comprises SEQ ID NO: 97, 98, 99 and 100; and the HFW sequence comprises SEQ ID NO: 101, 102, 103 and 104.

[0374] 24. The binding polypeptide according to claim 18, wherein the LFW sequence comprises SEQ ID NO: 105, 106, 107 and 108; and the HFW sequence comprises SEQ ID NO: 109, 110, 111 and 112.

[0375] 25. The binding polypeptide according to claim 18, wherein the LFW sequence comprises SEQ ID NO: 113, 114, 115 and 116; and the HFW sequence comprises SEQ ID NO: 117, 118, 119 and 120.

[0376] 26. The binding polypeptide according to claim 18, wherein the LFW sequence comprises SEQ ID NO: 129, 130, 131 and 132; and the HFW sequence comprises SEQ ID NO: 133, 134, 135 and 136.

[0377] 27. The binding polypeptide according to any one of the preceding claims, wherein the soluble Aβ has synaptic toxicity.

[0378] 28. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide neutralizes Aβ synaptic toxicity.

[0379] 29. The binding polypeptide according to any one of the preceding claims, wherein the soluble Aβ has a molecular weight of about 20 kD to about 100 kD.

[0380] 30. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide does not specifically bind monomer Aβ, primary fibril Aβ or fibril Aβ.

[0381] 31. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide does not specifically bind to protein aggregates.

[0382] 32. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide does not specifically bind to amyloid plaques present in the brain of a subject with Alzheimer's disease.

[0383] 33. The binding polypeptide according to any one of the preceding claims, wherein the binding polypeptide specifically binds to soluble Aβ derived from the brain of a subject suffering from Alzheimer's disease.

[0384] 34. The binding polypeptide according to item 33, wherein the binding is immunoadsorption.

[0385] 35. The binding polypeptide according to item 33 or 34, wherein the soluble Aβ is present in one or more soluble fractions obtained from the brain of a subject with Alzheimer's disease.

[0386] 36. The binding polypeptide according to any one of claims 33 to 35, wherein the binding polypeptide neutralizes the synaptic toxicity of the soluble Aβ derived from the brain of a subject suffering from Alzheimer's disease.

[0387] 37. A pharmaceutical composition comprising a binding polypeptide according to any one of claims 1-36 and a pharmaceutically acceptable carrier.

[0388] 38. Use of the binding polypeptide according to any one of items 1-36 or the pharmaceutical composition according to item 37 in the preparation of a medicament for treating a subject with Alzheimer's disease.

[0389] 39. An isolated polynucleotide, said isolated polynucleotide encoding a binding polypeptide according to any one of claims 1-36.

[0390] 40. A vector comprising the polynucleotide according to claim 39.

[0391] 41. A host cell comprising the polynucleotide according to claim 39 or the vector according to claim 40.

Claims

1. An isolated binding polypeptide that specifically binds to soluble β-amyloid (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 following group: SEQ ID NO: 26, 27, 28, 20, 21, 22, 50, 51, 52, 44, 45, 46, 62, 63, 64, 32, 33, 34, 38, 39, 40, 56, 57 and 58, and The three LCDR sequences are selected from the following group: SEQ ID NO: 23, 24, 25, 17, 18, 19, 47, 48, 49, 41, 42, 43, 59, 60, 61, 29, 30, 31, 35, 36, 37, 53, 54 and 55.

2. The binding polypeptide according to claim 1, wherein the binding polypeptide comprises an antibody or an antigen-binding fragment thereof.

3. The binding polypeptide according to claim 2, wherein the antibody or its antigen-binding fragment is human.

4. The binding polypeptide according to claim 2 or 3, wherein the antibody or its antigen-binding fragment is IgG1.

5. The binding polypeptide according to any one of claims 1 to 4, wherein the binding polypeptide comprises a heavy chain variable region (HCVR) / light chain variable region (LCVR) sequence pair selected from the following: SEQ ID NO: 4 and 3; SEQ ID NO: 2 and 1; SEQ ID NO: 10 and 9; SEQ ID NO: 12 and 11; SEQ ID NO: 16 and 15; SEQ ID NO: 6 and 5; SEQ ID NO: 8 and 7; and SEQ ID NO: 14 and 13.

6. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 20, 21 and 22, and the three LCDR sequences comprise SEQ ID NO: 17, 18 and 19.

7. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 44, 45 and 46, and the three LCDR sequences comprise SEQ ID NO: 41, 42 and 43.

8. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 26, 27 and 28, and the three LCDR sequences comprise SEQ ID NO: 23, 24 and 25.

9. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 32, 33 and 34, and the three LCDR sequences comprise SEQ ID NO: 29, 30 and 31.

10. The binding polypeptide according to any one of claims 1 to 5, wherein the three HCDR sequences comprise SEQ ID NO: 38, 39 and 40, and the three LCDR sequences comprise SEQ ID NO: 35, 36 and 37.

Images