Catalytic antibodies and their usage

Abstract

This application provides methods, compositions, and kits for determining the level of SHD catalytic antibodies in biological samples and for treating or preventing protein aggregation disorders (PAD) in individuals. It also provides catalytic antibodies that specifically recognize amyloid β (Aβ) peptide and methods for using them.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to provisional patent application No. 62 / 765,150, filed on August 17, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence list submitted as an ASCII text file

[0004] The following content, submitted as an ASCII text file, is incorporated herein by reference in its entirety: Sequence List in Computer-Readable Form (CRF) (filename: 792702000140SEQLIST.txt, record date: August 5, 2019, size: 22KB). Technical Field

[0005] This application relates to catalytic antibodies, methods for determining catalytic antibody levels, and methods for treatment using catalytic antibodies. In particular, this application relates to the specific recognition and cleavage of amyloid β (Aβ) peptide. Background Technology

[0006] Catalytic antibodies are antibodies that specifically bind to target antigens and catalyze the chemical transformation of those target antigens. Catalytic antibodies can hydrolyze and permanently inactivate target peptides. Compared to conventional antibodies that bind stoichiometrically, a single catalytic antibody molecule can hydrolyze thousands of antigen molecules over its biological lifetime, thus achieving enhanced efficacy. Catalytic antibodies have been found naturally in healthy individuals and patients with autoimmune diseases. Catalytic antibodies have also been observed in elevated levels in laboratory animals immunized against synthetic haptens or screened from antibody libraries using transition state analogs. However, due to their relatively low catalytic activity, catalytic antibodies have not yet been widely developed as therapeutic agents.

[0007] Protein aggregation is a biological phenomenon in which misfolded proteins accumulate intracellularly or extracellularly. These aggregations are commonly associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), prions, and other amyloidosis. For example, according to the amyloid hypothesis, the aggregation of soluble and fibrinous amyloid β (Aβ) peptides is causally related to the pathogenesis of AD. Aβ aggregation activates microglial inflammatory processes, directly exerts neurotoxic effects, and disrupts brain anatomy. To treat AD, antibody-mediated immunotherapies have been used to induce the clearance of existing Aβ amyloid and inhibit further Aβ aggregation. However, many of these antibody-based therapies have failed in clinical trials, including Bapineuzumab and Solanezumab.

[0008] All publications, patents, patent applications, and published patent applications mentioned in this document are hereby incorporated in their entirety by reference. Summary of the Invention

[0009] This application provides compositions and methods for diagnosing (including methods for determining risk), treating, and preventing protein aggregation disorders (PADs) in individuals, such as Alzheimer's disease (AD).

[0010] One aspect of this application provides a method for determining the level of one or more SHD catalytic antibodies (i.e., catalytic antibodies containing a catalytic triplet motif of serine, histidine, and aspartic acid, or "SHD motif") in a biological sample, comprising: a) contacting the biological sample with a substrate peptide immobilized on a solid support under conditions allowing the formation of a catalytic antibody-substrate peptide complex, and b) determining the amount of the catalytic antibody-substrate peptide complex, thereby providing the level of one or more SHD catalytic antibodies in the biological sample, wherein the substrate peptide comprises an amino acid sequence (EAR). n (SEQ ID NO:2), and where n is an integer between 1 and 30 (e.g., n is 3). In some embodiments, the biological sample is incubated with the substrate peptide for about 1 hour to about 16 hours, such as about 1 hour to about 3 hours, about 3 hours to about 8 hours, or about 8 hours to about 16 hours.

[0011] In some embodiments of the methods described above, the biological sample is a serum sample. In some embodiments, the serum sample contains at least about 1 μg / mL (e.g., at least about 10 μg / mL, 25 μg / mL, or 100 μg / mL) of immunoglobulin (Ig; e.g., human Ig).

[0012] In some embodiments of the methods described above, the amount of the catalytic antibody-substrate peptide complex is determined using an antibody that specifically binds to total immunoglobulins (Ig, such as total human Ig). In some embodiments, the antibody specifically binds to total IgM, total IgG, total IgA, and / or total IgE. In some embodiments, the antibody is labeled with an enzyme (e.g., horseradish peroxidase or "HRP") or a fluorescent label (e.g., FITC).

[0013] One aspect of this application provides a method for determining the risk of an individual for peptide-associated disease (PAD), wherein PAD is associated with the aggregation of a target protein. The method includes determining the level of one or more SHD-catalyzing antibodies in a biological sample (e.g., a serum sample) of the individual, wherein if the level of one or more SHD-catalyzing antibodies is lower than the level of a control SHD-catalyzing antibody, then the individual is determined to have a risk of PAD. In some embodiments, the level of one or more SHD-catalyzing antibodies is the level of one or more SHD-catalyzing antibodies that specifically bind to the target protein. In some embodiments, the level of one or more SHD-catalyzing antibodies is the level of total SHD-catalyzing antibodies. In some embodiments, the level of total SHD-catalyzing antibodies is determined by contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalytic antibody-substrate peptide complex and determining the amount of the catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises an amino acid sequence (EAR). n (SEQ ID NO:2), where n is an integer between 1 and 30 (e.g., n is 3). In some embodiments, the serum sample contains at least about 1 μg / mL (e.g., at least about 10 μg / mL, 25 μg / mL, or 100 μg / mL) Ig (e.g., human Ig). In some embodiments, the biological sample is incubated with the substrate peptide for about 1 hour to about 16 hours, such as about 1 hour to about 3 hours, about 3 hours to about 8 hours, or about 8 hours to about 16 hours. In some embodiments, the amount of the catalytic antibody-substrate peptide complex is determined using an antibody that specifically binds to total Ig (e.g., total human Ig). In some embodiments, the antibody specifically binds to total IgM, total IgG, total IgA, and / or total IgE. In some embodiments, the antibody is labeled with an enzyme (e.g., horseradish peroxidase or "HRP") or a fluorescent label (e.g., FITC).

[0014] In some embodiments of the methods for determining risk as described above, the methods further include determining the level of autoantibodies against the target protein in an individual's biological sample (e.g., a serum sample), and wherein the risk of the individual having PAD is determined if: (i) the level of one or more SHD-catalyzed antibodies is lower than the level of control SHD-catalyzed antibodies; and (ii) the level of autoantibodies against the target protein is lower than the level of control autoantibodies. In some embodiments, the level of autoantibodies is determined by contacting the individual's serum sample with the target protein under conditions that allow the formation of autoantibody-target protein complexes and determining the amount of autoantibody-target protein complexes. In some embodiments, the level of autoantibodies is determined using an ELISA assay. In some embodiments, the control autoantibody level is the level of autoantibodies against the target protein in healthy individuals (e.g., the same age group). In some embodiments, the control autoantibody level is the median level of autoantibodies against the target protein in a population of individuals (e.g., the same age group).

[0015] In some embodiments of the methods for determining risk as described above, the methods further include determining the level of a target protein in an individual's biological sample (e.g., a serum sample or a cerebrospinal fluid sample), and wherein the individual is determined to have a risk of PAD if: (i) the level of one or more SHD-catalyzing antibodies is lower than the level of a control SHD-catalyzing antibody; and (ii) the level of the target protein is higher than the level of a control target protein. In some embodiments, the level of the target protein is determined by contacting an individual's biological sample with an antibody against the target protein under conditions that allow for the formation of an antibody-target protein complex and determining the amount of the antibody-target protein complex. In some embodiments, an ELISA assay is used to determine the level of the target protein. In some embodiments, the control target protein level is the level of the target protein in healthy individuals (e.g., the same age group). In some embodiments, the control target protein level is the median level of the target protein in a population of individuals (e.g., the same age group).

[0016] In some embodiments of the methods for determining risk as described above, the control SHD catalytic antibody level is the level of one or more SHD catalytic antibodies in healthy individuals (e.g., the same age group). In some embodiments, the control SHD catalytic antibody level is the median level of one or more SHD catalytic antibodies in an individual population (e.g., the same age group).

[0017] In some embodiments of the methods for determining risk as described above, PAD is Alzheimer's disease, and the target protein is amyloid-β (Aβ). In some embodiments, PAD is Parkinson's disease, and the target protein is α-synuclein. In some embodiments, PAD is Alzheimer's disease or dementia, and the target protein is Tau. In some embodiments, PAD is ATTR amyloidosis, and the target protein is transthyretin. In some embodiments, PAD is AL amyloidosis, and the target protein is immunoglobulin light chain. In some embodiments, PAD is frontotemporal degeneration (FTLD) or amyotrophic lateral sclerosis (ALS), and the target protein is TDP43. In some embodiments, PAD is Huntington's disease, and the target protein is Huntington's protein. In some embodiments, PAD is type II diabetes, and the target protein is IAPP. In some embodiments, PAD is amyotrophic lateral sclerosis (ALS), and the target protein is SOD1.

[0018] One aspect of this application provides a method for treating or preventing protein aggregation disorder (PAD) in an individual, wherein PAD is associated with the aggregation of a target protein, the method comprising: a) determining that an individual is at risk of PAD according to any of the methods for determining risk as described above; and b) administering to the individual an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein. In some embodiments, the method is repeated at a frequency not exceeding about three months, for example about every three months, about every six months, or about every year.

[0019] In some embodiments of any of the treatment methods described above, PAD is Alzheimer's disease, and the target protein is amyloid β (Aβ), and the therapeutic catalytic antibody comprises: a light chain variable region comprising the following (V L ): LC-CDR1 containing the light chain complementarity-determining region (LC-CDR) of amino acid sequence SEQ ID NO:12, LC-CDR2 containing the amino acid sequence SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, the therapeutic catalytic antibody comprises: a heavy chain variable region (V) containing the following... H): The heavy chain complementarity-determining region (HC-CDR)1 comprising the amino acid sequence SEQ ID NO:9, the HC-CDR2 comprising the amino acid sequence SEQ ID NO:10, and the HC-CDR3 comprising the amino acid sequence SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, in V L The amino acid residue at position 26 is S, and in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is D or N, and the numbering is based on the Kabat EU index. In some embodiments, the therapeutic catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 95%, 97%, 99%, or 100%) with respect to the amino acid sequence of SEQ ID NO: 4, 6, 19, or 20. H In some embodiments, the therapeutic catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 95%, 97%, 99%, or 100%) with an amino acid sequence having an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21, or 22. L In some embodiments, the therapeutic catalytic antibody comprises: a V amino acid sequence comprising an amino acid sequence selected from SEQ ID NO:4, 6, 19, and 20. H V, and an amino acid sequence comprising an amino acid sequence selected from SEQ ID NO: 5, 7, 8, 21 and 22. L In some embodiments, the therapeutic catalytic antibody comprises: (i) a V comprising the amino acid sequence of SEQ ID NO:4 H and V containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L (iv) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:21 L (v) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:21 L(vi) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:22 L ; or (vii) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:22 L In some embodiments, the therapeutic catalytic antibody is a full-length IgG antibody. In some embodiments, the therapeutic catalytic antibody includes an IgG1 or IgG4 Fc region. In some embodiments, the therapeutic catalytic antibody is a full-length IgM antibody.

[0020] Another aspect of this application provides an isolated anti-Aβ catalytic antibody comprising: V containing the following L LC-CDR1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: V containing the following H HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the isolated anti-Aβ catalytic antibody comprises: V comprising the following H HC-CDR1 containing the amino acid sequence of SEQ ID NO:9, HC-CDR2 containing the amino acid sequence of SEQ ID NO:10, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:11; and HC-CDR3 containing the following V L LC-CDR1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0021] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the following H HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or variants thereof, wherein the HC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, the isolated anti-Aβ catalytic antibody comprises: V containing the following L LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the isolated anti-Aβ catalytic antibody comprises: V comprising the following H HC-CDR1 containing the amino acid sequence of SEQ ID NO:9, HC-CDR2 containing the amino acid sequence of SEQ ID NO:10, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:11; and HC-CDR3 containing the following V L LC-CDR1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0022] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the following H The following are included: HC-CDR1 containing the amino acid sequence of SEQ ID NO:9, HC-CDR2 containing the amino acid sequence of SEQ ID NO:10, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:11; and VL containing the following: LC-CDR1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14, wherein in V... L The amino acid residue at position 1 is D, and in V LThe amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index.

[0023] In some embodiments of any of the anti-Aβ catalytic antibodies described above, the anti-Aβ catalytic antibody cleaves a substrate having the formula EAR-AMC (SEQ ID NO:1).

[0024] In some embodiments of any of the anti-Aβ catalytic antibodies described above, in V L The amino acid residue at position 26 is S, and in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index.

[0025] In some embodiments of the anti-Aβ catalytic antibodies described above, the anti-Aβ catalytic antibody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 95%, 97%, 99%, or 100%) with respect to the amino acid sequence of SEQ ID NO:4, 6, 19, or 20. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%, 95%, 97%, 99%, or 100%) with respect to the amino acid sequence of SEQ ID NO:5, 7, 8, 21, or 22. L In some embodiments, the anti-Aβ catalytic antibody comprises: a V amino acid sequence comprising an amino acid sequence selected from SEQ ID NO:4, 6, 19, and 20. H V, and an amino acid sequence comprising an amino acid sequence selected from SEQ ID NO: 5, 7, 8, 21 and 22. L In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a V-type amino acid sequence comprising SEQ ID NO:4. H and V containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L (iv) V containing the amino acid sequence of SEQ ID NO:19 Hand V containing the amino acid sequence of SEQ ID NO:21 L (v) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:21 L (vi) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:22 L ; or (vii) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:22 L .

[0026] In some embodiments of the anti-Aβ catalytic antibodies described above, the anti-Aβ catalytic antibody is a full-length IgG antibody. In some embodiments, the anti-Aβ catalytic antibody comprises an IgG1 or IgG4 Fc region. In some embodiments, the anti-Aβ catalytic antibody is a full-length IgM antibody.

[0027] In some embodiments, a method of treating or preventing Alzheimer's disease in an individual is provided, comprising administering to the individual an effective amount of any of the anti-Aβ catalytic antibodies described above.

[0028] Methods for preparing and using anti-Aβ catalytic antibodies are also provided, as well as kits and articles applicable to any of the methods described above.

[0029] In some embodiments, a kit is provided for treating or preventing Alzheimer's disease in an individual, comprising: a) an amino acid sequence (EAR). n The kit contains: a) a substrate peptide (SEQ ID NO:2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) an Aβ peptide; and c) an antibody that specifically binds to total Ig (e.g., total human Ig). In some embodiments, the antibody specifically binds to total IgM, total IgG, total IgA, and / or total IgE. In some embodiments, the kit further comprises a solid support, such as an ELISA plate. In some embodiments, the kit further comprises a therapeutic catalytic antibody that specifically binds to Aβ. Attached Figure Description

[0030] Figure 1A The levels of catalytic antibodies in serum samples from young (20–29 years) and older (60–69 years) adults are shown, as determined by EAR-AMC binding assay.

[0031] Figure 1BThe levels of catalytic antibodies in serum samples from young (20–29 years) and older (60–69 years) adults are shown, as determined by EAR3 binding assay.

[0032] Figure 2 The study shows the levels of catalytic antibodies and Aβ-specific autoantibodies in healthy individuals (HS1-HS8) or patients with Alzheimer's disease (ALZ-1-5).

[0033] Figure 3 The image shows two catalytic antibodies (anti-UA15 and anti-VP) and a non-catalytic anti-Aβ antibody 3D6. L Sequence alignment. Amino acid residues corresponding to the SHD motif are marked with an asterisk (*). Amino acid residues that support the catalytic function of the antibody are marked with a hash (#).

[0034] Figure 4 SDS gel images of various purified 3D6-derived catalytic antibodies under non-reducing and reducing electrophoresis conditions are shown. 3D6-D is a catalytic antibody designed based on 3D6 (i.e., 3D6-Y). hu3D6-D H1L1 and hu3D6-D H1L2 are humanized forms of 3D6-D. hu3D6-Y has the same sequence as bepinizumab (i.e., humanized 3D6).

[0035] Figure 5 The catalytic function of 3D6-D, as determined using EAR-AMC substrates, is shown.

[0036] Figure 6 This illustrates the binding of 3D6-D with (EAR)3 (SEQ ID NO:3) and Aβ.

[0037] Figure 7 The binding of the humanized 3D6 catalytic antibody to Aβ is shown.

[0038] Figure 8 This table shows the levels of anti-Aβ autoantibodies and SHD catalytic antibodies (EAR3) in the serum of 30 Alzheimer's disease (AD) patients, measured by ELISA. PBS served as a negative control. The average readings from pooled healthy human serum and serum samples from 8 healthy donors served as controls. "Control %" was calculated as (readings from AD samples) divided by (average readings from pooled human serum and 8 healthy donors). A "+" in the table indicates that the serum levels of anti-Aβ autoantibodies or SHD catalytic antibodies in AD patients were higher than those in healthy donors and pooled serum samples. A "-" in the table indicates that the serum levels of anti-Aβ autoantibodies or SHD catalytic antibodies in AD patients were lower than those in healthy donors and pooled serum samples.

[0039] Figure 9A This paper describes an ELISA design for detecting levels of anti-Aβ autoantibodies and SHD catalytic antibody (recognition (EAR)3) in serum samples from 30 Alzheimer's disease (AD) patients, 8 healthy donors, and a combined healthy donor sample. PBS was used as a negative control. Figure 9B The plate was read at the 5-minute time point for Aβ ELISA binding assay. Figure 9C The plate was depicted at the 1-minute time point (EAR) for 3ELISA binding assay. Detailed Implementation

[0040] In one aspect, this application provides a method for diagnosing and treating protein aggregation disorders (PADs) using a newly developed immunoassay for determining SHD-catalyzing antibody levels in biological samples. This application is based in part on the finding that SHD-catalyzing antibody levels in serum correlate with autoantibody levels against amyloid β (Aβ) peptide in older individuals and patients with Alzheimer's disease (AD). Currently, imaging remains the most reliable diagnostic method for AD. However, by the time the disease shows clear pathological features in imaging analysis, neurological damage has already occurred. At this stage, even if therapeutic agents prevent further disease progression, they are unlikely to reverse the neurological damage. This invention allows for the effective diagnosis and treatment of PADs (such as AD) at an early stage using SHD-catalyzing antibody levels as an early diagnostic biomarker.

[0041] Furthermore, using antibody engineering, catalytic antibodies that specifically recognize and cleave Aβ have been designed based on the non-catalytic anti-Aβ antibody 3D6, a parental mouse antibody of bepinizumab. Bepinizumab has shown safety at low doses; however, two large phase III clinical trials have shown no efficacy in treating patients with advanced AD. Because AD patients have a blood-brain barrier (BBB) ​​due to the accumulation of Aβ1-40 (Aβ40), conventional therapeutic antibodies (such as bepinizumab) can penetrate the BBB and exacerbate neuroinflammation. Compared to bepinizumab, the anti-Aβ catalytic antibody described herein exhibits enhanced efficacy at low doses due to its catalytic activity. In some embodiments, the methods described herein can be used to determine an individual's risk of AD at an early stage and to treat them with the anti-Aβ catalytic antibody described herein to prevent AD.

[0042] Therefore, one aspect of this application provides a method for determining the risk of PAD in an individual, wherein PAD is associated with the aggregation of a target protein, the method comprising determining the level of one or more SHD-catalyzing antibodies in a biological sample (e.g., a serum sample) of the individual, wherein if the level of one or more SHD-catalyzing antibodies is lower than the level of a control SHD-catalyzing antibody, then the individual is determined to have a risk of PAD. In some embodiments, the method comprises: a) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalyzing antibody-substrate peptide complex, and b) determining the amount of the catalyzing antibody-substrate peptide complex, thereby providing the level of one or more SHD-catalyzing antibodies in the biological sample, wherein the substrate peptide comprises an amino acid sequence (EAR). n (SEQ ID NO:2), and where n is an integer between 1 and 30. In some embodiments, the method further includes determining the level of autoantibodies against the target protein in an individual's biological sample (e.g., a serum sample), and wherein the individual is determined to have a risk of PAD if: (i) the level of one or more SHD-catalyzing antibodies is lower than the level of control SHD-catalyzing antibodies; and (ii) the level of autoantibodies against the target protein is lower than the level of control autoantibodies.

[0043] One aspect of this application provides a method for treating or preventing PAD (e.g., AD) in an individual, wherein PAD is associated with the aggregation of a target protein (e.g., Aβ), the method comprising: a) determining the level of one or more SHD catalytic antibodies in a biological sample (e.g., a serum sample) of the individual, wherein if the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody, then the individual is determined to have a risk of PAD; and b) administering to the individual an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein (e.g., Aβ). In some embodiments, step a) comprises: 1) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalytic antibody-substrate peptide complex, and 2) determining the amount of the catalytic antibody-substrate peptide complex, thereby providing the level of one or more SHD catalytic antibodies in the serum, wherein the substrate peptide comprises an amino acid sequence (EAR). n (SEQ ID NO:2), and where n is an integer between 1 and 30. In some embodiments, step a) further includes determining the level of autoantibodies against the target protein in an individual's biological sample (e.g., a serum sample), and wherein if: (i) the level of one or more SHD-catalyzing antibodies is lower than the level of control SHD-catalyzing antibodies; and (ii) the level of autoantibodies against the target protein is lower than the level of control autoantibodies, then the individual is determined to have a risk of PAD.

[0044] Another aspect of this application provides an isolated anti-Aβ catalytic antibody comprising: a light chain variable region (V...L ): Light chain complementarity-determining region (LC-CDR)1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a heavy chain variable region comprising the following (V H ): 1 containing the heavy chain complementarity-determining region (HC-CDR) of amino acid sequence SEQ ID NO:9, 2 containing the amino acid sequence SEQ ID NO:10, and 3 containing the amino acid sequence SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions.

[0045] Compositions (such as pharmaceutical compositions), kits, and articles for the diagnosis, treatment, or prevention of PAD (such as AD) are also provided.

[0046] I. Definition

[0047] As used herein, “treatment” is a method for achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by a disease; reduction of the severity of the disease; stabilization of the disease (e.g., prevention or delay of disease exacerbation); prevention or delay of disease spread; prevention or delay of disease recurrence; delay or slowing of disease progression; improvement of disease status; provision of disease remission (partial or complete remission); reduction of the dosage of one or more other medicines required to treat the disease; delay of disease progression; increase or improvement of quality of life; increase in weight gain; and / or prolongation of survival. The methods of this invention are contemplated for any one or more of these aspects of treatment.

[0048] The term "antibody" includes both full-length antibodies and their antigen-binding fragments. A full-length antibody comprises two heavy chains and two light chains. Variable regions of the light and heavy chains are responsible for antigen binding. These variable regions in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (including the light chain (LC) CDRs of LC-CDR1, LC-CDR2, and LC-CDR3, and the heavy chain (HC) CDRs of HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody and antigen-binding fragments disclosed herein can be defined or identified according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). Three CDRs of the heavy or light chain are inserted between flanking elongations called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0049] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, bifunctional antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain Fv (scFv), scFv dimers (divalent bifunctional antibodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen that binds to a parent antibody or a fragment of a parent antibody (e.g., a parental scFv). In some embodiments, an antigen-binding fragment may contain one or more CDRs from a specific human antibody that are grafted into a frame region from one or more different human antibodies.

[0050] As used herein, an epitope refers to a specific set of atoms or amino acids on an antigen that an antibody binds to. If two antibodies competitively bind to an antigen, they can bind to the same epitopes of that antigen.

[0051] As used herein, the terms “specific binding,” “specific recognition,” or “specific to” refer to a measurable and reproducible interaction, such as the binding between a target and an antibody (e.g., a catalytic antibody), which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically recognizes a target (which may be an epitope) is an antibody that binds to that target with greater affinity, strength, ease, and / or duration than it binds to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with a binding affinity at least about 10 times greater than its binding affinity to other targets.

[0052] As used herein, the term "catalytic antibody" refers to an antibody that has catalytic activity. For example, a catalytic antibody can catalyze the hydrolysis of a target antigen that it specifically recognizes. Exemplary catalytic antibodies include, but are not limited to, proteolytic antibodies. Catabodies are also known as catalytic antibodies, antibody enzymes, and degradative enzymes.

[0053] As used herein, the term "SHD-catalyzed antibody" refers to an antibody that catalyzes the light chain variable region (V0). L This refers to proteolytic antibodies containing an SHD motif. The "SHD motif" refers to serine, histidine, and aspartic acid residues that together act as a catalytic triad to catalyze the cleavage of peptide bonds by the antibody. In some embodiments, the SHD motif is a non-linear motif, wherein the serine, histidine, and aspartic acid residues of the catalytic triad are not adjacent to each other in the amino acid sequence.

[0054] As used in this article, an "isolated" antibody is (1) not related to proteins found in nature, (2) not expressed by other proteins, (3) expressed by cells of a different species, or (4) not found in nature.

[0055] As used herein, the term “isolated nucleic acid” is intended to mean a nucleic acid of genomic, cDNA or synthetic origin or some combination thereof, which, due to its origin, is (1) associated with all or part of the polynucleotides of all “isolated nucleic acids” found in nature, (2) is operatively linked with polynucleotides that are not linked in nature, or (3) is not present in nature as part of a larger sequence.

[0056] As used herein, the term “CDR” or “complementarity-determining region” is intended to refer to discontinuous antigenic combination sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been identified by Kabat et al., *Journal of Biochemistry* 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, “Sequences of Proteins of Immunological Concern” (1991); Chothia et al., *Journal of Molecular Biology* 196:901-917 (1987); Al-Lazikani B. et al., *Journal of Molecular Biology* 273:927-948 (1997); MacCallum et al., *Journal of Molecular Biology* 262:732-745 (1996); Abhinandan and Martin, *Molecular Immunology* 45:3832-3839 (2008); Lefranc MP et al., *Dev. Comp. Immunol.* 27:55-77 (2003); and Honegger and Plückthun, *Journal of Molecular Biology*, 309:657-670 (2001), describe that, when compared with each other, the definitions include overlaps or subsets of amino acid residues. However, the application of any definition of a CDR relating to antibodies or transplanted antibodies or variants thereof is intended to fall within the scope of the terminology defined and used herein. For comparison, Table 1 below outlines the amino acid residues that cover the respective definitions of CDRs in the references cited above. CDR prediction algorithms and interfaces are known in the field, including, for example, Abhinandan and Martin, *Molecular Immunology*, 45:3832-3839 (2008); Ehrenmann F. et al., *Nucleic Acids Res.*, 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., *Nucleic Acids Res.*, 43:D432-D438 (2015). The full text of any reference cited in this paragraph is incorporated herein by reference for the purposes of this invention and may be included in one or more of the claims herein.

[0057] Table 1: CDR Definition

[0058]

[0059] 1 Residue numbering follows the nomenclature of Kabat et al.

[0060] 2 Residue numbering follows the nomenclature of Chothia et al. mentioned above.

[0061] 3 The residue numbering follows the nomenclature of MacCallum et al. mentioned above.

[0062] 4 The residue numbering follows the nomenclature of Lefranc et al. mentioned above.

[0063] 5 The residue numbering follows the nomenclature of Honegger and Plückthun et al.

[0064] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the biological activity of the present invention (see U.S. Patent No. 4,816,567; and Morrison et al., Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA), 81:6851-6855 (1984)).

[0065] "Fv" represents the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer composed of a heavy chain and a light chain variable region domain tightly non-covalently associated. Six hypervariable rings (three from the heavy chain and three from the light chain) emanate from the folding of these two domains, contributing amino acid residues for antigen binding and conferring antigen-antibody binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific to the antigen) can recognize and bind to the antigen, but with lower affinity than the complete binding site.

[0066] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a chain containing V H and V L Antibody fragments are antibody domains linked together to form a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises V H and V L The peptide linkers between the domains enable scFv to form the structures required for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0067] The term "bifunctional antibody" refers to a small antibody fragment prepared by constructing an scFv fragment (see previous paragraph), which is typically V... H and V L Short linkers (e.g., about 5 to about 10 residues) between the domains enable inter-chain but not intra-chain pairing of the V domains, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific bifunctional antibodies are heterodimers of two “cross-linked” scFv fragments, where the V domains of the two antibodies... H Domain and V L The domains are located on different polypeptide chains. Bifunctional antibodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proceedings of the National Academy of Sciences, 90:6444-6448 (1993).

[0068] The “humanized” form of non-human (e.g., rodent) antibodies (e.g., catalytic antibodies) is a chimeric antibody containing a very small sequence derived from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) whose hypervariable region (HVR) residues are replaced by residues from the hypervariable region of a non-human species (donor antibody) with the desired antibody specificity, affinity, and ability, such as mice, rats, rabbits, or non-human primates. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will contain at least one, and typically substantially all, of the two variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin and all or substantially all of the FRs are FRs with a human immunoglobulin sequence. Humanized antibodies optionally also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of human immunoglobulins. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0069] The “percentage of amino acid sequence identity (%)” or “homology” for the peptide and antibody (e.g., catalytic antibody) sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the peptide being compared, after sequence alignment of any conserved substitutions considered to be part of the sequence identity. Alignments performed to determine the percentage of amino acid sequence identity can be performed in various ways within the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this article, the sequence alignment computer program MUSCLE is used to generate sequence identity values ​​(Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0070] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In one embodiment, the FcR of the present invention is an FcR (γ receptor) that binds to an IgG antibody and includes receptors of the FcγRI, FcγRII, FcγRIII, and FcγRIV subclasses, including allelic variants of these receptors and alternative splice forms. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), both having similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain (see M). The term "FcR" is described in the Annals of Immunology, 15:203-234 (1997). The term includes allotypes such as FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA H131. FcRs are described in Ravetch and Kinet, Annals of Immunology, 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed within the term "FcR" as used herein. The term also includes neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., Journal of Immunology 117:587 (1976) and Kim et al., Journal of Immunology 24:249 (1994)).

[0071] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α-chain that binds nonvalently to β2-microglobulin. The various functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000), Annals of Immunology 18, 739-766. FcRn plays a role in the passive delivery of immunoglobulin IgG from the mother to the offspring and in the regulation of serum IgG levels. FcRn can act as a rescue receptor, binding to and transporting intact, pinocytic IgG within and across cells, and rescuing it from the default degradation pathway.

[0072] The "CH1 domain" (also known as the "H1" "C1 domain") of the human IgG Fc region typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0073] The "hinge region" is broadly defined as the elongated segment from Glu216 to Pro230 of human IgG1 (Burton, Molecular Immunology 22:161-206 (1985)). Hinge regions of other IgG isotypes can be compared with the IgG1 sequence by placing the first and last cysteine ​​residues at the same position to form an inter-heavy chain SS bond.

[0074] The "CH2 domain" (also known as the "C2" domain of "H2") in the Fc region of human IgG typically extends from about amino acid 231 to about amino acid 340. The unique feature of the CH2 domain is that it does not pair tightly with another domain. In fact, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact natural IgG molecule. It has been hypothesized that carbohydrates can provide alternatives for domain-domain pairing and contribute to the stabilization of the CH2 domain. (Burton, *Molecular Immunology* 22:161-206 (1985)).

[0075] The “CH3 domain” (also known as the “C2” or “H3” domain) contains an extension of the CH2 domain from the C-terminal residue to the Fc region (i.e., from approximately amino acid residue 341 of the antibody sequence to the C-terminus, typically at amino acid residue 446 or 447 of IgG).

[0076] The "functional Fc fragment" possesses the "effective function" of the native Fc region. Exemplary "effective functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phage activity; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). These effector functions generally require a combination of the Fc region and a binding domain (e.g., antibody variable domain) and can be assessed using various assays well-known in the field.

[0077] Antibodies containing variant IgG Fc with “altered” FcR binding affinity or ADCC activity are antibodies whose FcR binding activity (e.g., FcγR or FcRn) and / or ADCC activity are enhanced or weakened compared to the parent peptide or a peptide containing the native Fc region. Variant Fc exhibiting “increased binding” to FcRs show a higher affinity for at least one FcR than (e.g., a lower apparent Kd or IC50 value) the parent peptide or native IgG Fc. According to some embodiments, the binding improvement is about 3-fold, such as about 5, 10, 25, 50, 60, 100, 150, 200, or up to 500-fold, or a binding improvement of about 25% to 1000% compared to the parent peptide. Variant peptides exhibiting “decreased binding” to FcRs show a lower affinity for at least one FcR than (e.g., a higher apparent Kd or a higher IC50 value). 50 (Value) Parental polypeptide. Compared with the parental polypeptide, the binding reduction can be approximately 40% or more.

[0078] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells using cytotoxins. Antibodies "arm" cytotoxic cells and are essential for this type of killing. Primary cells used to mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Table 3 on page 464 of Ravetch and Kinet, Annu, summarizes FcR expression on hematopoietic cells. Immunol 9:457-92 (1991). To assess the ADCC activity of the molecule of interest, in vitro ADCC assays, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337, can be performed. Useful effector cells for this type of assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in animal models such as those disclosed in Clynes et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS(USA)), 95:652-656 (1998).

[0079] A peptide containing a variant Fc region that “exhibits increased ADCC” or mediates ADCC more effectively than a peptide or parental peptide with wild-type IgG Fc in the presence of human effector cells is a peptide that mediates ADCC more effectively in vitro or in vivo when the amount of the peptide with the variant Fc region is substantially the same as the amount of the peptide (or parental peptide) with the wild-type Fc region in the assay. Generally, any in vitro ADCC assay known in the art, such as assays or methods for determining ADCC activity, will be used to identify such variants, for example in animal models. In some embodiments, the variant mediates ADCC about 5 to about 100 times more effectively than the wild-type Fc (or parental peptide), for example about 25 to about 50 times.

[0080] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. The activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (belonging to an appropriate subclass) to a homologous antigen. To assess complement activation, a CDC assay can be performed, for example, as in Gazzano-Santoro et al., *Journal of Immunology Methods* 202:163 (1996). Peptide variants with altered Fc region amino acid sequences and enhanced or weakened C1q binding are described in U.S. Patent Nos. 6,194,551B1 and WO99 / 51642. The contents of those patents are expressly incorporated herein by reference. See also Idusogie et al., *Journal of Immunology* 164:4178-4184 (2000).

[0081] Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Phrasal nucleotide sequences encoding proteins or RNA may also include introns, to the extent that nucleotide sequences encoding proteins may contain one or more introns in some forms.

[0082] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that leads to the expression of the latter. For example, the first and second nucleic acid sequences are operably linked when they are functionally related. Similarly, if a promoter affects the transcription or expression of a coding sequence, then the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, join two protein-coding regions within the same reading frame.

[0083] "Homology" refers to the sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. Two compared sequences are homologous when positions in both sequences are occupied by the same base or amino acid monomer subunit; for example, if adenine occupies each position in two DNA molecules, then the molecules are homologous at those positions. The percentage of homology between two sequences varies by dividing the number of matching or homologous positions shared by the two sequences by multiplying the number of positions being compared by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, two sequences are compared when the alignment yields the maximum homology.

[0084] The “effective amount” of an antibody or composition as disclosed herein is an amount sufficient to carry out the purpose specifically stated. An “effective amount” can be determined empirically and by known methods relating to the stated purpose.

[0085] As used herein, "pharmaceutical acceptable" or "pharmaceutical compatible" means that the material is not biologically or otherwise undesirable, such as a material that can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a harmful manner with any other component of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicological and manufacturing testing and / or are included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0086] It should be understood that the embodiments of the invention described herein include embodiments that are “composed of” and / or “mainly composed of”.

[0087] In this document, the reference to "about" a value or parameter includes (and describes) the variation with respect to said value or parameter itself. For example, a reference to "about X" includes a description of "X".

[0088] As used in this article, referring to “not” a value or parameter generally means and describes “not” a value or parameter.

[0089] Unless the context clearly specifies otherwise, as used herein and in the appended claims, the singular forms “a,” “or,” and “the” include a plurality of indicators.

[0090] II. Methods of Diagnosis and Treatment

[0091] This application provides methods for determining the levels of one or more catalytic antibodies (e.g., SHD catalytic antibodies) in a sample, and methods for diagnosing (including risk assessment), treating, or preventing protein aggregation disorders (PADs) in an individual. In some embodiments, the levels of one or more catalytic antibodies (e.g., SHD catalytic antibodies) are the protein levels of one or more catalytic antibodies. In some embodiments, the levels of one or more catalytic antibodies are the mRNA levels of one or more catalytic antibodies. In some embodiments, the methods described herein use a substrate peptide in an immunoassay to detect the binding of the substrate peptide to one or more catalytic antibodies (e.g., SHD catalytic antibodies) in a sample, thereby providing the levels of one or more catalytic antibodies in the sample. In some embodiments, the levels of one or more catalytic antibodies are the total catalytic antibody levels, such as the total SHD catalytic antibody levels. In some embodiments, the levels of one or more catalytic antibodies are the levels of one or more (e.g., 1, 2, 3, 4, or more) catalytic antibodies that specifically bind to and cleave PAD-associated target proteins (e.g., Aβ) (e.g., the total SHD catalytic antibody levels).

[0092] In some embodiments, the catalytic antibody is based on an amino acid sequence (EAR). n The binding of the substrate peptide of (SEQ ID NO:2) determines the level of total SHD-catalyzing antibody, where n is an integer between 1 and 30. The substrate peptide may comprise any suitable number of EAR repeat sequences, including, for example, any of about 1-10, 10-20, 20-30, 1-30, 1-5, 5-10, 5-15, or 15-30. In some embodiments, the substrate peptide comprises the amino acid sequence SEQ ID NO:2, where n is any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the amino acid sequence of SEQ ID NO:2 is located at the N-terminus, C-terminus, or internal position of the substrate peptide. In some embodiments, in addition to the amino acid sequence of SEQ ID NO:2, the substrate peptide comprises amino acid residues, for example, at the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO:2. In some embodiments, the substrate peptide comprises, in addition to the amino acid sequence of SEQ ID NO:2, any one of at least about 1, 2, 3, 5, 10, 15, 20, 25, or 30 amino acids. In some embodiments, the total length of the substrate peptide is about 3-100 amino acids, such as any one of about 3-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 3-50, 50-100, 10-30, 30-60, 60-90, 20-40, 40-60, 60-80, or 80-100 amino acids. In some embodiments, the substrate peptide comprises a label, such as a fluorescent label, a peptide tag, or a biotin label. In some embodiments, the substrate peptide comprises the formula EAR-AMC (SEQ ID NO:1).

[0093] Although EAR-AMC, i.e., a peptide having an amino acid sequence that binds to 7-amino-4-methylcoumarin (AMC), is a known proteolytic substrate for SHD-catalyzing antibodies, substrate peptides containing the amino acid sequence of SEQ ID NO:2 have not yet been used to measure binding activity and levels of catalyzing antibodies in biological samples. Whether the binding between the EAR peptide and the SHD-catalyzing antibody is strong and persistent enough to allow accurate determination of SHD-catalyzing antibody levels via binding assays is also unpredictable. This application provides an immunoassay using a substrate peptide containing the amino acid sequence of SEQ ID NO:2, which provides a precise reading of the total SHD-catalyzing antibody levels in biological samples (e.g., serum samples).

[0094] Therefore, in some embodiments, a method is provided for determining the level of one or more SHD catalytic antibodies in a biological sample, comprising: a) contacting the biological sample with a substrate peptide immobilized on a solid support under conditions allowing the formation of a catalytic antibody-substrate peptide complex, and b) determining the amount of the catalytic antibody-substrate peptide complex, thereby providing the level of one or more SHD catalytic antibodies in the biological sample, wherein the substrate peptide comprises an amino acid sequence (EAR). n (SEQ ID NO:2), where n is an integer between 1 and 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the substrate peptide has the formula EAR-AMC (SEQ ID NO:1). In some embodiments, the substrate peptide comprises the amino acid sequence EAREAREAR (SEQ ID NO:3). In some embodiments, the solid support is an ELISA plate.

[0095] In some embodiments, a method is provided for determining the level of one or more SHD-catalyzing antibodies in a biological sample, comprising: a) contacting the biological sample with a substrate peptide immobilized on a solid support under conditions allowing the formation of a catalytic antibody-substrate peptide complex; and b) determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of one or more SHD-catalyzing antibodies in the biological sample, wherein the substrate peptide comprises an amino acid sequence (EAR). n (SEQ ID NO:2), where n is an integer between 1 and 30 (e.g., n is 3). In some embodiments, the antibody is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC). In some embodiments, the solid support is an ELISA plate.

[0096] In some embodiments, a method is provided for determining the level of one or more SHD-catalyzing antibodies in a biological sample, comprising: a) contacting the biological sample with a substrate peptide immobilized on a solid support under conditions allowing the formation of a catalytic antibody-substrate peptide complex; and b) determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of one or more SHD-catalyzing antibodies in the biological sample, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC). In some embodiments, the solid support is an ELISA plate.

[0097] In some embodiments, a method is provided for determining the level of one or more SHD catalytic antibodies in a biological sample, comprising: a) contacting a substrate peptide with an ELISA plate to coat the wells of the ELISA plate with the substrate peptide; b) contacting the biological sample with the coated wells of the ELISA plate under conditions that allow the formation of a catalytic antibody-substrate peptide complex; and b) determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE) labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC), thereby providing the level of one or more SHD catalytic antibodies in the biological sample, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO:3.

[0098] In some embodiments, a method is provided for determining the risk of an individual for a protein aggregation disorder (PAD), wherein PAD is associated with the aggregation of a target protein, the method comprising determining the level of one or more SHD catalytic antibodies in a biological sample (e.g., a serum sample) of the individual, wherein if the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody, then the individual is determined to have a risk of PAD. In some embodiments, the level of one or more SHD catalytic antibodies is the level of one or more SHD catalytic antibodies that specifically bind to the target protein. In some embodiments, the level of one or more SHD catalytic antibodies is the level of total SHD catalytic antibodies. In some embodiments, the level of total SHD catalytic antibodies is determined by contacting the individual's serum sample with a substrate peptide under conditions that allow the formation of a catalytic antibody-substrate peptide complex and determining the amount of the catalytic antibody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support. In some embodiments, the level of one or more SHD catalytic antibodies is determined using any of the methods for determining SHD catalytic antibody levels described herein. In some embodiments, (i) PAD represents Alzheimer's disease and the target protein is Aβ; (ii) PAD represents Parkinson's disease and the target protein is α-synuclein; (iii) PAD represents Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD represents ATTR amyloidosis and the target protein is transthyretin; (v) PAD represents AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD represents FTLD or ALS and the target protein is TDP43; (vii) PAD represents Huntington's disease and the target protein is Huntington's protein; (viii) PAD represents type II diabetes and the target protein is IAPP; and (ix) PAD represents ALS and the target protein is SOD1.

[0099] In some embodiments, a method for determining the risk of an individual for Alzheimer's disease (AD) is provided, comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalytic antibody-substrate peptide complex, and b) determining the amount of one or more SHD catalytic antibodies that specifically bind to Aβ, wherein if the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody, then the individual is determined to have a risk of AD.

[0100] In some embodiments, a method is provided for determining the risk of an individual for PAD, wherein PAD is associated with the aggregation of a target protein, the method comprising a) determining the level of one or more SHD catalytic antibodies in a biological sample (e.g., a serum sample) of the individual, and b) determining the level of an autoantibody against the target protein in a biological sample (e.g., a serum sample) of the individual, and wherein the risk of the individual having PAD is determined if: (i) the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of an autoantibody against the target protein is lower than the level of a control autoantibody. In some embodiments, the level of one or more SHD catalytic antibodies is the level of one or more SHD catalytic antibodies that specifically bind to the target protein. In some embodiments, the level of one or more SHD catalytic antibodies is the level of total SHD catalytic antibodies. In some embodiments, the level of total SHD catalytic antibodies is determined by contacting a serum sample of the individual with a substrate peptide under conditions that allow the formation of a catalytic antibody-substrate peptide complex and determining the amount of the catalytic antibody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support. In some embodiments, the level of one or more SHD catalytic antibodies is determined using any of the methods for determining SHD catalytic antibody levels described herein. In some embodiments, the level of autoantibodies is determined by contacting an individual's serum sample with a target protein (or fragment thereof) under conditions that allow for the formation of autoantibody-target protein complexes and determining the amount of the autoantibody-target protein complex. In some embodiments, an ELISA assay is used to determine the level of autoantibodies. In some embodiments, (i) PAD is Alzheimer's disease and the target protein is Aβ; (ii) PAD is Parkinson's disease and the target protein is α-synuclein; (iii) PAD is Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD is ATTR amyloidosis and the target protein is transthyretin; (v) PAD is AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD is FTLD or ALS and the target protein is TDP43; (vii) PAD is Huntington's disease and the target protein is huntingtin; (viii) PAD is type II diabetes and the target protein is IAPP; and (ix) PAD is ALS and the target protein is SOD1.

[0101] In some embodiments, a method is provided for determining the risk of an individual for PAD, wherein PAD is associated with the aggregation of a target protein, the method comprising a) determining the level of one or more SHD catalytic antibodies in a biological sample (e.g., a serum sample) of the individual, and b) determining the level of the target protein in a biological sample (e.g., a serum sample or a cerebrospinal fluid sample) of the individual, and wherein the risk of the individual having PAD is determined if: (i) the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of the target protein is higher than the level of a control target protein. In some embodiments, the level of one or more SHD catalytic antibodies is the level of one or more SHD catalytic antibodies that specifically bind to the target protein. In some embodiments, the level of one or more SHD catalytic antibodies is the level of total SHD catalytic antibodies. In some embodiments, the level of total SHD catalytic antibodies is determined by contacting a serum sample of the individual with a substrate peptide under conditions that allow the formation of a catalytic antibody-substrate peptide complex and determining the amount of the catalytic antibody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support. In some embodiments, the level of one or more SHD catalytic antibodies is determined using any of the methods for determining SHD catalytic antibody levels described herein. In some embodiments, the level of the target protein is determined by contacting an individual's biological sample with an antibody against the target protein under conditions that allow for the formation of an antibody-target protein complex and determining the amount of the antibody-target protein complex. In some embodiments, an ELISA assay is used to determine the level of the target protein. In some embodiments, (i) PAD is Alzheimer's disease and the target protein is Aβ; (ii) PAD is Parkinson's disease and the target protein is α-synuclein; (iii) PAD is Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD is ATTR amyloidosis and the target protein is transthyretin; (v) PAD is AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD is FTLD or ALS and the target protein is TDP43; (vii) PAD is Huntington's disease and the target protein is huntingtin; (viii) PAD is type II diabetes and the target protein is IAPP; and (ix) PAD is ALS and the target protein is SOD1.

[0102] In some embodiments, a method is provided for determining the risk of an individual's PAD, wherein the PAD is associated with the aggregation of a target protein, the method comprising: a) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises an amino acid sequence (EAR). n(SEQ ID NO:2), where n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catalytic antibody-substrate peptide complex, thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; and c) determining the level of autoantibodies against the target protein in the individual's biological sample (e.g., serum sample), and wherein the individual is determined to have a risk of PAD if: (i) the level of one or more SHD catalytic antibodies is lower than the level of control SHD catalytic antibodies; and (ii) the level of autoantibodies against the target protein is lower than the level of control autoantibodies. In some embodiments, the solid support is an ELISA plate. In some embodiments, the level of autoantibodies is determined by contacting the individual's serum sample with the target protein (or fragments thereof) under conditions that allow the formation of autoantibody-target protein complexes and determining the amount of the autoantibody-target protein complex. In some embodiments, an ELISA assay is used to determine the level of autoantibodies. In some embodiments, (i) PAD represents Alzheimer's disease and the target protein is Aβ; (ii) PAD represents Parkinson's disease and the target protein is α-synuclein; (iii) PAD represents Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD represents ATTR amyloidosis and the target protein is transthyretin; (v) PAD represents AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD represents FTLD or ALS and the target protein is TDP43; (vii) PAD represents Huntington's disease and the target protein is Huntington's protein; (viii) PAD represents type II diabetes and the target protein is IAPP; and (ix) PAD represents ALS and the target protein is SOD1.

[0103] In some embodiments, a method is provided for determining the risk of an individual's PAD, wherein the PAD is associated with the aggregation of a target protein, the method comprising: a) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises an amino acid sequence (EAR). n(SEQ ID NO:2), where n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catalytic antibody-substrate peptide complex, thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; and c) determining the level of the target protein in the individual's biological sample (e.g., a serum sample or a cerebrospinal fluid sample), and wherein if: (i) the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of the target protein is higher than the level of a control target protein, then the individual is determined to have a risk of PAD. In some embodiments, the solid support is an ELISA plate. In some embodiments, the level of the target protein is determined by contacting the individual's biological sample with an antibody against the target protein under conditions that allow the formation of the antibody-target protein complex and determining the amount of the antibody-target protein complex. In some embodiments, an ELISA assay is used to determine the level of the target protein. In some embodiments, (i) PAD represents Alzheimer's disease and the target protein is Aβ; (ii) PAD represents Parkinson's disease and the target protein is α-synuclein; (iii) PAD represents Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD represents ATTR amyloidosis and the target protein is transthyretin; (v) PAD represents AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD represents FTLD or ALS and the target protein is TDP43; (vii) PAD represents Huntington's disease and the target protein is Huntington's protein; (viii) PAD represents type II diabetes and the target protein is IAPP; and (ix) PAD represents ALS and the target protein is SOD1.

[0104] In some embodiments, a method is provided for determining the risk of an individual's PAD, wherein the PAD is associated with the aggregation of a target protein, the method comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow for the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises an amino acid sequence (EAR). n(SEQ ID NO: 2), where n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; and c) determining the level of autoantibodies against the target protein in the individual's biological sample (e.g., serum sample), and wherein the individual is determined to have a risk of PAD if: (i) the level of one or more SHD catalytic antibodies is lower than the level of control SHD catalytic antibodies; and (ii) the level of autoantibodies against the target protein is lower than the level of control autoantibodies. In some embodiments, the Ig-specifically binding antibody is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC). In some embodiments, the solid support is an ELISA plate. In some embodiments, the level of autoantibodies is determined by contacting the individual's serum sample with the target protein (or fragments thereof) under conditions that allow the formation of the autoantibody-target protein complex and determining the amount of the autoantibody-target protein complex. In some embodiments, the level of autoantibodies is determined using an ELISA assay. In some embodiments, (i) PAD represents Alzheimer's disease and the target protein is Aβ; (ii) PAD represents Parkinson's disease and the target protein is α-synuclein; (iii) PAD represents Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD represents ATTR amyloidosis and the target protein is transthyretin; (v) PAD represents AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD represents FTLD or ALS and the target protein is TDP43; (vii) PAD represents Huntington's disease and the target protein is Huntington's protein; (viii) PAD represents type II diabetes and the target protein is IAPP; and (ix) PAD represents ALS and the target protein is SOD1.

[0105] In some embodiments, a method is provided for determining the risk of an individual's PAD, wherein the PAD is associated with the aggregation of a target protein, the method comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow for the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises an amino acid sequence (EAR). n(SEQ ID NO: 2), where n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; and c) determining the level of the target protein in the individual's biological sample (e.g., a serum sample or a cerebrospinal fluid sample), and wherein if: (i) the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of the target protein is higher than the level of a control target protein, then the individual is determined to have a risk of PAD. In some embodiments, the solid support is an ELISA plate. In some embodiments, the level of the target protein is determined by contacting the individual's biological sample with an antibody against the target protein under conditions that allow the formation of the antibody-target protein complex and determining the amount of the antibody-target protein complex. In some embodiments, an ELISA assay is used to determine the level of the target protein. In some embodiments, (i) PAD represents Alzheimer's disease and the target protein is Aβ; (ii) PAD represents Parkinson's disease and the target protein is α-synuclein; (iii) PAD represents Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD represents ATTR amyloidosis and the target protein is transthyretin; (v) PAD represents AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD represents FTLD or ALS and the target protein is TDP43; (vii) PAD represents Huntington's disease and the target protein is Huntington's protein; (viii) PAD represents type II diabetes and the target protein is IAPP; and (ix) PAD represents ALS and the target protein is SOD1.

[0106] In some embodiments, a method for determining the risk of an individual for Alzheimer's disease (AD) is provided, comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions allowing the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO:3; b) determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; c) contacting a serum sample from the individual with Aβ (e.g., Aβ(1-42)) under conditions allowing the formation of an autoantibody-Aβ complex; and d) determining the amount of the autoantibody-Aβ complex, thereby providing the level of an autoantibody against Aβ; and wherein if: (i) the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of an autoantibody against Aβ is lower than the level of a control autoantibody, then the individual is determined to have a risk of AD. In some embodiments, antibodies that specifically bind to Ig are labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC). In some embodiments, the solid support is an ELISA plate. In some embodiments, an ELISA assay is used to determine the level of autoantibodies.

[0107] In some embodiments, a method for determining the risk of an individual for Alzheimer's disease (AD) is provided, comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions allowing the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO:3; b) determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; c) contacting a cerebrospinal fluid sample from the individual with an anti-Aβ antibody under conditions allowing the formation of an antibody-Aβ complex; and d) determining the amount of the antibody-Aβ complex, thereby providing the level of Aβ; and wherein the individual is determined to have a risk of AD if: (i) the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of Aβ is higher than the level of a control Aβ. In some embodiments, the Ig-specifically binding antibody is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC). In some embodiments, the solid support is an ELISA plate. In some embodiments, an ELISA assay is used to determine the level of Aβ.

[0108] Any of the diagnostic methods described herein can be used to inform the use of any known therapeutic agent for treating PAD in the relevant field or any catalytic antibody described herein (such as an anti-Aβ catalytic antibody). The diagnostic methods described herein allow for early detection of an individual's risk of PAD, thereby enabling early intervention and preventative treatment of PAD.

[0109] Therefore, in some embodiments, a method for treating or preventing PAD in an individual is provided, wherein PAD is associated with the aggregation of a target protein, the method comprising: a) determining an individual's risk of having PAD according to any of the risk determination methods described herein; and b) administering an effective amount of a therapeutic agent for treating PAD to the individual.

[0110] In some embodiments, a method is provided for treating or preventing PAD in an individual, wherein PAD is associated with the aggregation of a target protein, the method comprising: a) determining the risk of an individual having PAD according to any of the risk determination methods described herein; and b) administering to the individual an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein. In some embodiments, the method is repeated at a frequency not exceeding about every three months, for example about every three months, about every six months, or about every year. In some embodiments, the method is performed only once.

[0111] In some embodiments, a method for treating or preventing AD in an individual comprises: a) determining that the individual has a risk of AD according to any of the risk determination methods described herein; and b) administering to the individual an effective amount of an anti-Aβ catalytic antibody, such as any of the anti-Aβ catalytic antibodies described in Part III. In some embodiments, the method is repeated at a frequency not exceeding about every three months, for example about every three months, about every six months, or about every year.

[0112] In some embodiments, a method is provided for treating or preventing PAD in an individual, wherein PAD is associated with the aggregation of a target protein, the method comprising: a) determining the level of one or more SHD catalytic antibodies in a biological sample (e.g., a serum sample) of the individual, wherein if the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody, then the individual is determined to have a risk of PAD; and b) administering to the individual an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein. In some embodiments, the level of one or more SHD catalytic antibodies is the level of one or more SHD catalytic antibodies that specifically bind to the target protein. In some embodiments, the level of one or more SHD catalytic antibodies is the level of total SHD catalytic antibodies. In some embodiments, the level of total SHD catalytic antibodies is determined by contacting a serum sample of the individual with a substrate peptide under conditions that allow the formation of a catalytic antibody-substrate peptide complex and determining the amount of the catalytic antibody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support. In some embodiments, (i) the PAD is for Alzheimer's disease and the target protein is Aβ; (ii) the PAD is for Parkinson's disease and the target protein is α-synuclein; (iii) the PAD is for Alzheimer's disease or dementia and the target protein is Tau; (iv) the PAD is for ATTR amyloidosis and the target protein is transthyretin; (v) the PAD is for AL amyloidosis and the target protein is immunoglobulin light chain; (vi) the pad is for FTLD or ALS and the target protein is TDP43; (vii) the PAD is for Huntington's disease and the target protein is huntingtin; (viii) the PAD is for type II diabetes and the target protein is IAPP; and (ix) the PAD is for ALS and the target protein is SOD1. In some embodiments, the method is repeated at a frequency not exceeding approximately every three months, such as approximately every three months, approximately every six months, or approximately every year.

[0113] In some embodiments, a method for treating or preventing Alzheimer's disease (AD) in an individual includes: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow for the formation of a catalytic antibody-substrate peptide complex; b) determining the amount of one or more SHD catalytic antibodies that specifically bind to Aβ; and c) administering an effective amount of anti-Aβ catalytic antibody to the individual if the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody. In some embodiments, the method is repeated at a frequency not exceeding about three months, such as about three months, about six months, or about one year.

[0114] In some embodiments, a method is provided for treating or preventing PAD in an individual, wherein PAD is associated with the aggregation of a target protein, the method comprising a) determining the level of one or more SHD catalytic antibodies in a biological sample (e.g., a serum sample) of the individual, b) determining the level of an autoantibody against the target protein in a biological sample (e.g., a serum sample) of the individual, and c) administering an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein to the individual if: (i) the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of an autoantibody against the target protein is lower than the level of a control autoantibody. In some embodiments, the level of one or more SHD catalytic antibodies is the level of one or more SHD catalytic antibodies that specifically bind to the target protein. In some embodiments, the level of one or more SHD catalytic antibodies is the level of total SHD catalytic antibodies. In some embodiments, the level of total catalytic antibodies is determined by contacting a serum sample of the individual with a substrate peptide under conditions that allow the formation of a catalytic antibody-substrate peptide complex and determining the amount of the catalytic antibody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support. In some embodiments, the level of autoantibodies is determined by contacting an individual's serum sample with the target protein under conditions that allow for the formation of autoantibody-target protein complexes and by determining the amount of the autoantibody-target protein complexes. In some embodiments, an ELISA assay is used to determine the level of autoantibodies. In some embodiments, (i) PAD is Alzheimer's disease and the target protein is Aβ; (ii) PAD is Parkinson's disease and the target protein is α-synuclein; (iii) PAD is Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD is ATTR amyloidosis and the target protein is transthyretin; (v) PAD is AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD is FTLD or ALS and the target protein is TDP43; (vii) PAD is Huntington's disease and the target protein is huntingtin; (viii) PAD is type II diabetes and the target protein is IAPP; and (ix) PAD is ALS and the target protein is SOD1. In some embodiments, the method is repeated at a frequency of no more than about three months, such as about three months, about six months, or about one year.

[0115] In some embodiments, a method is provided for treating or preventing PAD in an individual, wherein PAD is associated with the aggregation of a target protein, the method comprising: a) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises an amino acid sequence (EAR). n(SEQ ID NO:2), where n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catalytic antibody-substrate peptide complex, thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; and c) determining the level of autoantibodies against the target protein in the individual's biological sample (e.g., serum sample); and d) administering an effective amount of the therapeutic catalytic antibody that specifically binds to the target protein to the individual if: (i) the level of one or more SHD catalytic antibodies is lower than the level of control SHD catalytic antibodies; and (ii) the level of autoantibodies against the target protein is lower than the level of control autoantibodies. In some embodiments, the amount of the catalytic antibody-substrate peptide complex is determined using antibodies that specifically bind to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE). In some embodiments, the Ig-specifically binding antibody is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC). In some embodiments, the solid support is an ELISA plate. In some embodiments, the level of autoantibodies is determined by contacting an individual's serum sample with the target protein under conditions that allow for the formation of autoantibody-target protein complexes and by determining the amount of the autoantibody-target protein complexes. In some embodiments, an ELISA assay is used to determine the level of autoantibodies. In some embodiments, (i) PAD is Alzheimer's disease and the target protein is Aβ; (ii) PAD is Parkinson's disease and the target protein is α-synuclein; (iii) PAD is Alzheimer's disease or dementia and the target protein is Tau; (iv) PAD is ATTR amyloidosis and the target protein is transthyretin; (v) PAD is AL amyloidosis and the target protein is immunoglobulin light chain; (vi) PAD is FTLD or ALS and the target protein is TDP43; (vii) PAD is Huntington's disease and the target protein is huntingtin; (viii) PAD is type II diabetes and the target protein is IAPP; and (ix) PAD is ALS and the target protein is SOD1. In some embodiments, the method is repeated at a frequency of no more than about three months, such as about three months, about six months, or about one year.

[0116] In some embodiments, a method of treating or preventing Alzheimer's disease (AD) in an individual is provided, comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises SEQ ID NO. The amino acid sequence of NO:3; b) determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; c) contacting a serum sample of the individual with Aβ (e.g., Aβ(1-42)) under conditions that allow for the formation of an autoantibody-Aβ complex; d) determining the amount of the autoantibody-Aβ complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of autoantibodies against Aβ; and e) administering an effective amount of anti-Aβ catalytic antibody to the individual if: (i) the level of one or more SHD catalytic antibodies is lower than the level of control SHD catalytic antibodies; and (ii) the level of autoantibodies against Aβ is lower than the level of control autoantibodies. In some embodiments, the Ig-specifically binding antibody is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC). In some embodiments, the solid support is an ELISA plate. In some embodiments, the method is repeated no more than about every three months, for example about every three months, about every six months, or about every year.

[0117] In some embodiments, a method of treating or preventing Alzheimer's disease (AD) in an individual is provided, comprising: a) determining the level of one or more SHD-catalyzing antibodies in a biological sample (e.g., a serum sample) of the individual; b) determining the level of an autoantibody against Aβ in a biological sample (e.g., a serum sample) of the individual; and c) administering an effective amount of an anti-Aβ-catalyzing antibody to the individual if: (i) the level of one or more SHD-catalyzing antibodies is lower than the level of a control SHD-catalyzing antibody; and (ii) the level of an autoantibody against Aβ is lower than the level of a control autoantibody, wherein the anti-Aβ-catalyzing antibody comprises: a light chain variable region comprising the following (V L ): Light chain complementarity-determining region (LC-CDR)1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V LThe amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a heavy chain variable region comprising the following (V H ): The heavy chain complementarity-determining region (HC-CDR)1 comprising the amino acid sequence SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions. In some embodiments, in V L The amino acid residue at position 26 is S, and in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19, or 20. H ; and / or V containing an amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22. L In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a V-type amino acid sequence comprising SEQ ID NO:4. H and V containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L (iv) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:21 L (v) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:21 L (vi) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:22 L ; or (vii) V containing the amino acid sequence of SEQ ID NO:20 Hand V containing the amino acid sequence of SEQ ID NO:22 L In some embodiments, the anti-Aβ catalytic antibody is a full-length antibody, such as an IgG1 or IgG4 antibody. In some embodiments, the method is repeated no more than about every three months, for example, about every three months, about every six months, or about every year.

[0118] In some embodiments, a method of treating or preventing AD in an individual includes: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow the formation of a catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises SEQ ID NO. a) The amino acid sequence of NO:3; b) Determining the amount of the catalytic antibody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of one or more SHD catalytic antibodies in the individual's serum; c) Contacting a serum sample of the individual with Aβ (e.g., Aβ(1-42)) under conditions that allow for the formation of an autoantibody-Aβ complex; d) Determining the amount of the autoantibody-Aβ complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE), thereby providing the level of autoantibodies against Aβ; and e) If: (i) the level of one or more SHD catalytic antibodies is lower than the level of control SHD catalytic antibodies; (ii) the level of autoantibodies against Aβ is lower than the level of control autoantibodies, then administering an effective amount of anti-Aβ catalytic antibody to the individual, wherein the anti-Aβ catalytic antibody comprises: V L The light chain complementarity-determining region (LC-CDR) 1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR 2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR 3 containing the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and its numbering is based on Kabat's EU index; and it contains the following V. H HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions. In some embodiments, in V L The amino acid residue at position 26 is S, and in V LThe amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19, or 20. H ; and / or V containing an amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22. L In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a V-type amino acid sequence comprising SEQ ID NO:4. H and V containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L (iv) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:21 L (v) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:21 L (vi) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:22 L ; or (vii) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:22 L In some embodiments, the anti-Aβ catalytic antibody is a full-length antibody, such as an IgG1 or IgG4 antibody. In some embodiments, the antibody that specifically binds to Ig is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC). In some embodiments, the solid support is an ELISA plate. In some embodiments, the method is repeated no more than about every three months, for example, about every three months, about every six months, or about every year.

[0119] The method described herein detects the levels of one or more SHD-catalyzed antibodies, or autoantibody target proteins, including but not limited to protein and mRNA levels. Protein levels can be detected using immunoassay, mass spectrometry, or other molecular biology techniques. mRNA levels can be detected using quantitative PCR or other molecular biology techniques.

[0120] In some embodiments, the method includes detecting the level of one or more SHD-catalyzing antibodies using a substrate peptide comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the level of one or more SHD-catalyzing antibodies is the level of total SHD-catalyzing antibodies. In some embodiments, the level of one or more SHD-catalyzing antibodies is the level of one or more SHD-catalyzing antibodies that specifically bind to a target protein. In some embodiments, the level of one or more SHD-catalyzing antibodies that specifically bind to a target protein is determined by: a) extracting (e.g., immunodip) antibodies that specifically bind to the target protein from a biological sample, and b) contacting the extracted antibodies with a substrate peptide. In some embodiments, the level of one or more SHD catalytic antibodies that specifically bind to the target protein is determined by: a) contacting a biological sample with a target protein (e.g., Aβ) immobilized on a solid support under conditions that allow the formation of a catalytic antibody-target protein complex; b) contacting the catalytic antibody-target protein-substrate peptide complex with a substrate peptide containing a label (e.g., AMC or biotin) under conditions that allow the formation of a catalytic antibody-target protein-substrate peptide complex; c) contacting the catalytic antibody-target protein-substrate peptide with an antibody against the label; and d) determining the amount of the label antibody against the catalytic antibody-target protein-substrate peptide that binds to the label, thereby providing the level of one or more SHD catalytic antibodies that specifically bind to the target protein.

[0121] The substrate peptide or target protein (or fragment thereof, such as Aβ) can be obtained through chemical synthesis. The substrate peptide or target protein (or fragment thereof, such as Aβ) can be immobilized onto a solid support via immobilization motifs, such as biotin, streptavidin, avidin, or peptide tags. In some embodiments, the solid support is functionalized to bind to the substrate peptide. In some embodiments, the solid support is an ELISA plate. The ELISA plate may be a flat-bottomed porous (e.g., 96-well) plate made of polystyrene or polyvinyl chloride. The substrate peptide or target protein (or fragment thereof, such as Aβ) can be passively adsorbed onto the ELISA plate. Adsorption occurs passively due to hydrophobic interactions between the amino acid side chains on the substrate peptide or target protein (or fragment thereof, such as Aβ) and the plastic surface of the ELISA plate. In some embodiments, the substrate peptide is coated onto the surface of the ELISA plate at a density of about 1-2 μg / well.

[0122] Exemplary coating conditions on an ELISA plate involve adding 50-100 μl of coating buffer containing a substrate peptide at a concentration of 1-10 μg / ml and incubating overnight at 4°C or for 1-3 hours at 37°C. Alternative temperatures, times, buffers, and coating agent concentrations may be used and should be tested experimentally. Exemplary coating buffers include bicarbonate buffer and phosphate-buffered saline (PBS) at pH 9.6. In some embodiments, after fixation of the substrate peptide or target protein (or a fragment thereof, such as Aβ), the solid support (e.g., the ELISA plate) is washed (e.g., three times) with a washing buffer, such as PBS or PBST (0.1% TWEEN-20 in PBS). In some embodiments, the solid support (e.g., the ELISA plate) is blocked with a blocking buffer, such as 10% fetal bovine serum (FBS) in PBS, or 1% BSA in PBS. In some embodiments, after blocking, the solid support (e.g., the ELISA plate) is washed (e.g., three times) with a washing buffer, such as PBS or PBST.

[0123] One or more catalytic antibodies, autoantibodies against the target protein, and the level of the target protein are determined using a sample (e.g., a sample from an individual or a reference sample). In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is a biological fluid sample or a biological tissue sample. In some embodiments, the biological fluid sample is a bodily fluid, such as blood, plasma, serum, cerebrospinal fluid (CSF), or interstitial cerebrospinal fluid (ISF). In some embodiments, the biological sample is a biopsy sample. In some embodiments, the biological sample is a tissue or cell sample. In some embodiments, the biological sample is a B cell sample. In some embodiments, the biological sample is, for example, a sample enriched with certain molecules, such as immunoglobulins or target protein binding molecules, through immunoprecipitation.

[0124] In some embodiments, multiple samples are obtained during the course of treatment, such as monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or annually. In some embodiments, samples for determining the levels of one or more catalytic antibodies and samples for determining the levels of autoantibodies or target proteins are obtained from the individual simultaneously, or as aliquots of the same sample. In some embodiments, samples for determining the levels of one or more catalytic antibodies and samples for determining the levels of autoantibodies or target proteins are obtained from the individual at different times and / or from different sources. In some embodiments, the same sample is used to determine the levels of one or more SHD catalytic antibodies and the levels of autoantibodies against a target protein (e.g., Aβ). In some embodiments, serum samples are used to determine the levels of one or more SHD catalytic antibodies, and CSF samples are used to determine the levels of Aβ.

[0125] In some embodiments, the biological sample is a serum sample. In some embodiments, the serum sample contains at least about 1 μg / mL, for example, at least about 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more μg / mL Ig. In some embodiments, the serum sample contains at least about 100 μg / mL Ig. In some embodiments, the serum sample does not contain any greater than about 500, 400, 300, 250, 200 or 150 μg / mL Ig.

[0126] In some embodiments, the biological sample is incubated with a substrate peptide or target protein (or a fragment thereof, such as Aβ) for about 1 hour to about 16 hours, including, for example, any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours. In some embodiments, the biological sample is incubated with a substrate peptide or target protein (or a fragment thereof, such as Aβ) for at least any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours. In some embodiments, the biological sample is incubated with a substrate peptide or target protein (or a fragment thereof, such as Aβ) for no more than any one of about 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour. In some embodiments, the biological sample is incubated with a substrate peptide or target protein (or a fragment thereof, such as Aβ) for about 1 hour to about 3 hours. In some embodiments, the biological sample is incubated overnight with a substrate peptide or target protein (or a fragment thereof, such as Aβ). In some embodiments, the incubation is performed at room temperature. In some embodiments, the incubation is performed at 4°C.

[0127] In some embodiments, after the biological sample is incubated with the substrate peptide or target protein (or a fragment thereof, such as Aβ), the solid support (e.g., an ELISA plate) is washed (e.g., three times) with a washing buffer, such as PBS or PBST. In some embodiments, the solid support (e.g., an ELISA plate) is blocked with a blocking buffer, such as 10% fetal bovine serum (FBS) in PBS, or 1% BSA in PBS. In some embodiments, after blocking, the solid support (e.g., an ELISA plate) is washed (e.g., three times) with a washing buffer, such as PBS or PBST.

[0128] The amount of catalytic antibody-substrate peptide complexes or autoantibody-target protein complexes can be detected using antibodies that specifically bind to species-specific immunoglobulin molecules (such as human Ig). In some embodiments, the antibody specifically binds to total IgM, total IgG, total IgA, and / or total IgE. Exemplary antibodies that specifically bind to human Ig include, but are not limited to, goat anti-human Ig. Ig-specific antibodies can be labeled with enzymes (e.g., HRP) for detection using enhanced chemiluminescence (ECL) substrates. Alternatively, Ig-specific antibodies can be labeled with fluorescent markers (e.g., FITC) for direct detection. A plate reader can be used to detect ECL or fluorescence signals using appropriate excitation, emission, and cutoff wavelength settings.

[0129] Other methods for determining the level of one or more autoantibodies against Aβ are known in the art, and such methods can be used in any of the methods for diagnosing, treating, or preventing AD described herein. See, for example, Weksler ME et al., “Patients with Alzheimer’s disease have lower levels of serum anti-amyloid peptide antibodies than healthy elderly individuals.” *Exp Gerontol.* 37:943-948 (2002); Mrutinti S et al., “Autoimmunity in Alzheimer’s disease: increased levels of circulating IgGs binding Abeta and RAGE peptides.” *Neurobiol. Aging* 25:1023-1032 (2004); Kellner A et al., “Autoantibodies against beta-amyloid are common in Alzheimer’s disease and help control plaque burden.” "Help control plaque burden," *Annals of Neurology* 65:24-31 (2009); Britschgi M, Olin CE, Johns HT, Takeda-Uchimura Y, et al., "Neuroprotective innate antibodies against amyloid-producing peptide assembly decrease with normal aging and the progression of Alzheimer's disease," *Proceedings of the National Academy of Sciences*, 106:12145-12150 (2009), which are incorporated herein by reference. In some embodiments, ELISA assays are used to determine the levels of one or more autoantibodies against target proteins (e.g., Aβ).

[0130] The levels of target proteins (e.g., Aβ) in biological samples (e.g., serum or brain / spinal cord samples) can be determined using ELISA or liquid chromatography / tandem mass spectrometry.

[0131] In some embodiments, the levels of one or more SHD-catalyzing antibodies are compared to the levels of control SHD-catalyzing antibodies. In some embodiments, the levels of one or more SHD-catalyzing antibodies are compared to the levels of one or more SHD-catalyzing antibodies in a control sample. In some embodiments, the levels of one or more SHD-catalyzing antibodies are compared to the levels of one or more SHD-catalyzing antibodies in multiple control samples. In some embodiments, multiple control samples are used to generate a statistical distribution for classifying or ranking the levels of one or more SHD-catalyzing antibodies in individuals of the same age or age group.

[0132] In some embodiments, the level of autoantibodies against a target protein (e.g., Aβ) is compared with the level of control autoantibodies. In some embodiments, the level of autoantibodies against a target protein (e.g., Aβ) is compared with the level of autoantibodies against a target protein (e.g., Aβ) in a control sample. In some embodiments, the level of autoantibodies against a target protein (e.g., Aβ) is compared with the level of autoantibodies against a target protein (e.g., Aβ) in multiple control samples. In some embodiments, multiple control samples are used to generate a statistical distribution for classifying or ranking the levels of autoantibodies against a target protein (e.g., Aβ) in an individual population (e.g., healthy individuals, individuals with PAD (e.g., AD), or individuals of the same age or age group).

[0133] In some embodiments, the level of a target protein (e.g., Aβ) is compared to the level of a control target protein. In some embodiments, the level of a target protein (e.g., Aβ) is compared to the level of a target protein (e.g., Aβ) in a control sample. In some embodiments, the level of a target protein (e.g., Aβ) is compared to the level of a target protein (e.g., Aβ) in multiple control samples. In some embodiments, multiple control samples are used to generate a statistical distribution for classifying or ranking the levels of the target protein (e.g., Aβ) in a population of individuals (e.g., healthy individuals, individuals with PAD (e.g., AD), or individuals of the same age or age group).

[0134] Exemplary age groups include, but are not limited to, 18-30 years old, 30-40 years old, 40-50 years old, 50-60 years old, 60-70 years old, 70-80 years old, 80-90 years old, 18-40 years old, 40-90 years old, 18-60 years old, 60-90 years old, 60 years old or older, 70 years old or older, 75 years old or older, 80 years old or older, 85 years old or older, or 90 years old or older.

[0135] Control samples can be obtained using the same methods as non-control samples. In some embodiments, control samples are obtained from different individuals, such as healthy individuals or individuals without PAD, and / or individuals of similar race, age, and sex. In some embodiments, multiple control samples (e.g., from different individuals) are used to determine the range of levels of one or more SHD-catalyzing antibodies, autoantibodies against target proteins (e.g., Aβ), or target proteins (e.g., Aβ).

[0136] In some embodiments, the control SHD-catalyzing antibody level is the level of one or more SHD-catalyzing antibodies in a healthy individual. In some embodiments, the control SHD-catalyzing antibody level is the average or median level of one or more SHD-catalyzing antibodies in a population of individuals (e.g., individuals in the same age group, or individuals aged approximately 18 to approximately 50 years (e.g., approximately 18 to approximately 40 years, or approximately 18 to approximately 30 years)). In some embodiments, the control autoantibody level is the level of autoantibodies against a target protein (e.g., Aβ) in a healthy individual. In some embodiments, the control autoantibody level is the median level of autoantibodies against a target protein in a population of individuals (e.g., individuals in the same age group). In some embodiments, the control target protein level is the level of the target protein (e.g., Aβ) in a healthy individual. In some embodiments, the control target protein level is the median level of the target protein in a population of individuals (e.g., individuals in the same age group).

[0137] In some embodiments, the level of one or more SHD-catalyzing antibodies, autoantibodies against a target protein (e.g., Aβ), or a target protein (e.g., Aβ) is compared to a control or reference (e.g., the median or average level of an individual's population or the level of a healthy individual). In some embodiments, the control level is a predetermined threshold level. For example, if an individual's level of one or more SHD-catalyzing antibodies is determined to be no greater than approximately 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or lower than the median level of an individual's population at the same age or in the same age group, then the individual is determined to have a low level of one or more SHD-catalyzing antibodies. Alternatively, if an individual's level of one or more SHD-catalyzing antibodies is determined to be greater than approximately 20%, 50%, 75%, 2×, 3×, 5×, 10×, or higher than the median level of an individual's population at the same age group, then the individual is determined to have a high level of one or more SHD-catalyzing antibodies.

[0138] If an individual is determined to have a low level of one or more autoantibodies against a target protein (e.g., Aβ) that is not greater than any of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median level of a population of individuals in the same age group, then the individual is determined to have a low level of one or more autoantibodies against a target protein (e.g., Aβ). Alternatively, if an individual is determined to have a high level of one or more autoantibodies against a target protein (e.g., Aβ) that is greater than any of 20%, 50%, 75%, 2×, 3×, 5×, 10× or higher of the median level of a population of individuals in the same age group, then the individual is determined to have a high level of one or more autoantibodies against a target protein (e.g., Aβ).

[0139] If an individual's target protein (e.g., Aβ) level is determined to be no greater than approximately 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or lower than the median level of a population of individuals in the same age group, then the individual is determined to have a low level of the target protein (e.g., Aβ). Alternatively, if an individual's target protein (e.g., Aβ) level is determined to be greater than approximately 20%, 50%, 75%, 2×, 3×, 5×, 10×, or higher than the median level of a population of individuals in the same age group, then the individual is determined to have a high level of the target protein (e.g., Aβ).

[0140] In some embodiments, an individual is determined to have a risk of PAD (e.g., AD) if: (i) the level of one or more SHD-catalyzing antibodies is lower than the level of control SHD-catalyzing antibodies; and (ii) the level of autoantibodies against a target protein (e.g., Aβ) is lower than the level of control autoantibodies. In some embodiments, the level of one or more SHD-catalyzing antibodies in an individual at risk of PAD (e.g., AD) is no greater than any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of a population of individuals in the same age group. In some embodiments, the level of autoantibodies against a target protein (e.g., Aβ) in an individual at risk of PAD (e.g., AD) is no greater than any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of a population of individuals in the same age group.

[0141] In some embodiments, an individual is determined to have a risk of PAD (e.g., AD) if: (i) the level of one or more SHD-catalyzing antibodies is lower than the level of a control SHD-catalyzing antibody; and (ii) the level of a target protein (e.g., Aβ) is higher than the level of a control target protein. In some embodiments, the level of one or more SHD-catalyzing antibodies in an individual at risk of PAD (e.g., AD) is no greater than any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of a population of individuals in the same age group. In some embodiments, the level of a target protein (e.g., Aβ) in an individual at risk of PAD (e.g., AD) is no greater than any one of 20%, 50%, 75%, 2×, 3×, 5×, 10× or more of the median of a population of individuals in the same age group.

[0142] The diagnostic and treatment methods described herein can be applied to individuals who have PAD or are at risk of developing PAD. In some embodiments, the individual is a mammal and includes, but is not limited to, humans, cattle, horses, cats, dogs, rodents (mice, rats, or hamsters), or non-human primates. In some embodiments, the individual is a human. In some embodiments, the individual is a young human individual, such as a human individual not older than about 60, 50, 40, 30, or 25 years of age. In some embodiments, the individual is an older human individual, such as a human individual older than about 50, 60, 70, or 80 years of age. As used herein, an individual “at risk” is an individual who is at risk of developing PAD (e.g., AD). An individual “at risk” may or may not have a detectable disease prior to the treatment methods described herein, and may or may not have the detectable disease shown. “At risk” means that the individual has one or more so-called risk factors, which are measurable parameters associated with developing the PAD (e.g., AD) described herein. Individuals with one or more of these risk factors have a higher chance of developing PAD (e.g., AD) than individuals without these risk factors.

[0143] Many PADs are known in the field and can be diagnosed, treated, or prevented using the methods described herein. Exemplary PADs include, but are not limited to, Alzheimer's disease associated with the accumulation of Aβ; Parkinson's disease associated with the accumulation of α-synuclein; Alzheimer's disease or dementia associated with the accumulation of Tau; ATTR amyloidosis associated with the accumulation of transthyretin protein; AL amyloidosis associated with the accumulation of immunoglobulin light chains; ubiquitin-positive neurons and glial inclusions (such as FTLD or ALS) associated with the accumulation of TDP43 (a 43 kDa TARDNA-binding protein); Huntington's disease associated with the accumulation of huntingtin protein; type II diabetes associated with the accumulation of IAPP; and ALS associated with the accumulation of SOD1.

[0144] If an individual is determined to have PAD or is at risk of having PAD, for example if: (i) the level of one or more SHD catalytic antibodies is lower than the level of control SHD catalytic antibodies; and (ii) the level of an autoantibody against a target protein (e.g., Aβ) is lower than the level of control autoantibodies, or the level of the target protein (e.g., Aβ) is higher than the level of control target protein, a therapeutic catalytic antibody that specifically binds to and cleaves the target protein (e.g., Aβ) may be administered to the individual. The therapeutic catalytic antibody may be administered to the individual using any suitable dose (including dose and dosing schedule / frequency) and route of administration. The dose (or effective amount of therapeutic catalytic antibody) may be determined based on the size and condition of the individual and in accordance with standard therapeutic practice. The route of administration is based on known and accepted methods, such as by single or multiple bolus or infusion over a period of time in an appropriate manner, for example by injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional, intra-articular, or oral routes. Animal studies provide reliable guidance for determining effective doses for human diagnostic applications. Scaling of effective doses across species may be performed following the principles outlined by Mordenti, J. and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics", Toxicokinetics and New Drug Development, eds. Yacobi et al., Pergamon Press, New York, 1989, pp. 42-46.

[0145] In some embodiments, the effective amount of the therapeutic catalytic antibody (e.g., anti-Aβ catalytic antibody) is about 1 μg / m 2 Up to approximately 100 mg / m 2 Or from about 1 μg / kg to about 100 mg / kg. In some embodiments, the frequency of administration for therapeutic catalytic antibodies (e.g., anti-Aβ catalytic antibodies) is from daily to about once every three months. In some embodiments, the administration of therapeutic catalytic antibodies may be extended over an extended period, such as from about one month to several years.

[0146] In some embodiments, the levels of one or more SHD catalytic antibodies, autoantibodies against a target protein (e.g., Aβ), and / or the target protein (e.g., Aβ) are periodically assessed to adjust the dosage and frequency of administration of the therapeutic catalytic antibody. In some embodiments, the levels of one or more SHD catalytic antibodies, autoantibodies against a target protein (e.g., Aβ), and / or the target protein (e.g., Aβ) are assessed approximately every month, every 2 months, every 3 months, every 4 months, every 6 months, or annually. In some embodiments, if: (i) the level of one or more catalytic antibodies is lower than the level of a control catalytic antibody; and (ii) the level of an autoantibody against a target protein (e.g., Aβ) is lower than the level of a control autoantibody, or the level of a target protein (e.g., Aβ) is higher than the level of a control target protein, then the administration of the therapeutic catalytic antibody (e.g., anti-Aβ catalytic antibody) is repeated.

[0147] III. Anti-Aβ catalytic antibody

[0148] This application provides therapeutic catalytic antibodies that specifically bind to and cleave target proteins associated with PAD. The therapeutic methods described in Part II can utilize any of the therapeutic catalytic antibodies described in this part (e.g., anti-Aβ catalytic antibodies). In some embodiments, the therapeutic catalytic antibody cleaves a substrate having the formula EAR-AMC (SEQ ID NO:1). In some embodiments, the therapeutic catalytic antibody is a catalytic antibody that cleaves a substrate having the formula EAR-AMC (SEQ ID NO:1) in the light chain variable region (V... L Catalytic antibodies containing the SHD motif.

[0149] In some embodiments, a catalytic antibody is provided that specifically binds to and cleaves amyloid β (Aβ) peptide. In some embodiments, the anti-Aβ catalytic antibody cleaves a substrate having the formula EAR-AMC (SEQ ID NO:1). In some embodiments, the anti-Aβ catalytic antibody cleaves a substrate having the formula EAR-AMC (SEQ ID NO:1) in the light chain variable region (V... L It contains the SHD motif.

[0150] In some embodiments, an isolated anti-Aβ catalytic antibody derived from 3D6 is provided. In some embodiments, the V of the anti-Aβ catalytic antibody... L Derived from 3D6, in V L The amino acid at position 1 is Asp(D), and the numbering is based on the Kabat EU index. In some embodiments, at V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, Glu (E), or His (H), and / or in V LThe amino acid residue at position 28 is either D or Gln(N), and the numbering is based on the Kabat EU index. In some embodiments, the heavy chain variable region (V) of the anti-Aβ catalytic antibody H (Derived from 3D6. In some embodiments, based on binding affinity with Aβ, from individuals with human germline V) H Screening for anti-Aβ catalytic antibodies using phage libraries containing sequences H In some embodiments, the anti-Aβ catalytic antibody competitively binds to Aβ specifically in relation to 3D6.

[0151] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing one, two, or three LC-CDRs of 3D6. L Among them, in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V comprising LC-CDR1, LC-CDR2, and LC-CDR3 containing 3D6. L Among them, in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, in V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: V containing one, two, or three HC-CDRs of 3D6. H In some embodiments, the anti-Aβ catalytic antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 of 3D6. In some embodiments, the antibody is derived from a human germline V based on its binding affinity to Aβ. H Screening for anti-Aβ catalytic antibodies using phage libraries containing sequences H .

[0152] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the following LLC-CDR1 containing the amino acid sequence of SEQ ID NO: 12, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 14, or variants thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, at V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: V containing the following H HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, based on binding affinity with Aβ, from a source having human germline V H Screening for anti-Aβ catalytic antibodies using phage libraries containing sequences H .

[0153] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the following L LC-CDR1 containing the amino acid sequence of SEQ ID NO: 12, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 14, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, in V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V LThe amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: V containing the following H The HC-CDR1 comprises the amino acid sequence of SEQ ID NO:9, the HC-CDR2 comprises the amino acid sequence of SEQ ID NO:10, and the HC-CDR3 comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, based on the binding affinity with Aβ, [the HC-CDR3 is derived from a sample of a human germline V]. H Screening for anti-Aβ catalytic antibodies using phage libraries containing sequences H .

[0154] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the following L LC-CDR1 containing the amino acid sequence of SEQ ID NO: 12, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 14; wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and its numbering is based on Kabat's EU index; and it contains the following V. H HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11. In some embodiments, in V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index.

[0155] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the following L LC-CDR1 containing the amino acid sequence of SEQ ID NO: 12, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 14, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, in VL The amino acid residue at position 26 is S, and in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index; and it contains the following V H HC-CDR1 containing the amino acid sequence of SEQ ID NO:9, HC-CDR2 containing the amino acid sequence of SEQ ID NO:10, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:11.

[0156] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO: 12, 13, and 14. L Among them, in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: an amino acid sequence comprising SEQ ID NO: 9, 10, and 11. In some embodiments, in V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index. In some embodiments, the amino acid residue is derived from a human germline V based on its binding affinity to Aβ. H Screening for anti-Aβ catalytic antibodies using phage libraries containing sequences H .

[0157] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a V amino acid sequence having at least about 85% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) of the amino acid sequence of SEQ ID NO: 5, 7, or 8. L Among them, in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, in VL The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a V amino acid sequence having at least about 85% (e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) of the amino acid sequence of SEQ ID NO:4 or 6. H In some embodiments, based on binding affinity with Aβ, from individuals with human germline V... H Screening for anti-Aβ catalytic antibodies using phage libraries containing sequences H .

[0158] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:4. H and V containing the amino acid sequence of SEQ ID NO:5 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:6. H and V containing the amino acid sequence of SEQ ID NO:7 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:6. H and V containing the amino acid sequence of SEQ ID NO:8 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:6. H and V containing the amino acid sequence of SEQ ID NO:21 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:6. H and V containing the amino acid sequence of SEQ ID NO:22 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO: 19. H and V containing the amino acid sequence of SEQ ID NO:7 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:19. Hand V containing the amino acid sequence of SEQ ID NO:8 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:19. H and V containing the amino acid sequence of SEQ ID NO:21 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:19. H and V containing the amino acid sequence of SEQ ID NO:22 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:20. H and V containing the amino acid sequence of SEQ ID NO:7 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:20. H and V containing the amino acid sequence of SEQ ID NO:8 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:20. H and V containing the amino acid sequence of SEQ ID NO:21 L In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: V containing the amino acid sequence of SEQ ID NO:20. H and V containing the amino acid sequence of SEQ ID NO:22 L .

[0159] In some embodiments, an isolated anti-Aβ catalytic antibody is provided that competitively and specifically binds to and cleaves Aβ with any of the anti-Aβ catalytic antibodies described herein.

[0160] In some embodiments, the anti-Aβ catalytic antibody is an antigen-binding fragment, such as scFv or Fab. In some embodiments, the anti-Aβ catalytic antibody comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-Aβ catalytic antibody is a full-length antibody, such as a full-length IgG antibody. In some embodiments, the full-length anti-Aβ catalytic antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the full-length anti-Aβ catalytic antibody comprises an IgG constant domain, such as a constant domain of any one of IgG1, IgG2, IgG3, and IgG4 (including variants thereof). In some embodiments, the anti-Aβ catalytic antibody comprises an IgG1 heavy chain constant region. In some embodiments, the anti-Aβ catalytic antibody comprises an IgG2 heavy chain constant region. In some embodiments, the anti-Aβ catalytic antibody comprises an IgG3 heavy chain constant region. In some embodiments, the anti-Aβ catalytic antibody comprises an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:15. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:16. In some embodiments, the anti-Aβ catalytic antibody comprises a κ light chain constant region. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence of SEQ ID NO:17. In some embodiments, the anti-Aβ catalytic antibody comprises the λ light chain constant region. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence of SEQ ID NO:18. In some embodiments, the anti-Aβ catalytic antibody comprises the κ light chain constant region.

[0161] In some embodiments, the anti-Aβ catalytic antibody comprises an Fc region. In some embodiments, the anti-Aβ catalytic antibody comprises an Fc region of human IgG. In some embodiments, the anti-Aβ catalytic antibody comprises an Fc region having enhanced antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) effector functions. In some embodiments, the anti-Aβ catalytic antibody comprises an Fc region having reduced ADCC and / or CDC effector functions.

[0162] In some embodiments, the anti-Aβ catalytic antibody is mouse, chimeric, humanized, or human.

[0163] In some embodiments, a full-length anti-Aβ catalytic antibody comprising an IgG1 constant domain is provided, wherein the anti-Aβ catalytic antibody comprises: V containing the following L LC-CDR1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein in VL The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, in V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: V containing the following H HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, based on binding affinity with Aβ, from a source having human germline V H Screening for anti-Aβ catalytic antibodies using phage libraries containing sequences H In some embodiments, the anti-Aβ catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22. L In some embodiments, the anti-Aβ catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO:4, 6, 19, or 20. H In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a V-type amino acid sequence comprising SEQ ID NO:4. H and V containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L (iv) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:21 L (v) V containing the amino acid sequence of SEQ ID NO:20 Hand V containing the amino acid sequence of SEQ ID NO:21 L (vi) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:22 L ; or (vii) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:22 L .

[0164] In some embodiments, a full-length anti-Aβ catalytic antibody comprising an IgG4 constant domain is provided, wherein the anti-Aβ catalytic antibody comprises: V containing the following L LC-CDR1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, in V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: V containing the following H HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, based on binding affinity with Aβ, from a source having human germline V H Screening for anti-Aβ catalytic antibodies using phage libraries containing sequences H In some embodiments, the anti-Aβ catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22. LIn some embodiments, the anti-Aβ catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO:4, 6, 19, or 20. H In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a V-type amino acid sequence comprising SEQ ID NO:4. H and V containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L (iv) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:21 L (v) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:21 L (vi) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:22 L ; or (vii) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:22 L .

[0165] Exemplary antibody sequences are shown in Table 2 below. Those skilled in the art will recognize that many algorithms are known for predicting the CDR positions in the variable regions of the antibody heavy and light chains, and that catalytic antibodies comprising CDRs from the catalytic antibodies described herein are also within the scope of this invention, based on alternative prediction algorithms. H or V L Sequences, but based on alternative algorithms, are within the scope of this invention.

[0166] Table 2. Exemplary anti-Aβ catalytic antibody sequences.

[0167]

[0168]

[0169] Methods for treating or preventing Alzheimer's disease in an individual are also provided, comprising administering to the individual an effective amount of any of the anti-Aβ catalytic antibodies (or pharmaceutical compositions thereof) described herein.

[0170] Aβ peptide

[0171] In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves Aβ peptides. In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves human Aβ peptides, such as Aβ(1-40) or Aβ(1-42). In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves Aβ peptides in a helical conformation. In some embodiments, the anti-Aβ catalytic antibody specifically binds to the N-terminus of Aβ, such as the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, or more amino acids at the N-terminus of Aβ(1-40).

[0172] In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves Aβ oligomers. In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves soluble Aβ. In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves Aβ in amyloid plaques. In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves Aβ in the brain. In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves vascular Aβ.

[0173] In some embodiments, the anti-Aβ catalytic antibody cross-reacts with Aβ from species other than humans, such as mice or rats. In some embodiments, the anti-Aβ catalytic antibody is completely specific to human Aβ and does not exhibit species- or other types of non-human cross-reactivity. In some embodiments, the anti-Aβ catalytic antibody cross-reacts with at least one allelic variant of Aβ. In some embodiments, the anti-Aβ catalytic antibody does not cross-react with any allelic variant of Aβ.

[0174] Aβ is a peptide of 36-43 amino acids, which has been suggested as a major component of amyloid plaques found in the brains of Alzheimer's patients. The Aβ peptide is derived from amyloid precursor protein (APP), which is cleaved by β-secretase and γ-secretase to produce Aβ. Aβ molecules can aggregate to form flexible, soluble oligomers, which can exist in several forms and are toxic to neurons.

[0175] Recent studies suggest that the soluble oligomeric form of the peptide may be a contributing factor to Alzheimer's disease. According to the "amyloid hypothesis," Aβ plaques are a pathological cause of Alzheimer's disease. Patients with sporadic Alzheimer's disease have elevated brain Aβ levels. Aβ is a major component of amyloid in brain parenchyma and vascular tissue, and it contributes to cerebrovascular lesions and has neurotoxicity. Aβ circulates primarily in plasma, cerebrospinal fluid (CSF), and interstitial fluid (ISF) as soluble Aβ40. Amyloid plaques in the elderly contain both Aβ40 and Aβ42, while vascular amyloid is primarily composed of the shorter Aβ40. Several Aβ sequences have been found in both lesions. Increased total Aβ levels or the relative concentrations of Aβ40 and Aβ42 have been implicated in the pathogenesis of both familial and sporadic Alzheimer's disease. Due to its greater hydrophobicity, Aβ42 is the peptide with the highest degree of amyloid formation. Aβ42 is also referred to as Aβ(1-42). Aβ40 is also known as Aβ(1-40).

[0176] Bepinizumab is a humanized form of the murine monoclonal antibody 3D6, targeting the N-terminal five residues of the helical Aβ peptide. A large-scale Phase III clinical trial of bepinizumab in patients with mild to moderate Alzheimer's disease was halted in August 2012 when the antibody failed to inhibit cognitive decline. Furthermore, bepinizumab was the first antibody found to cause amyloid-related imaging abnormalities, including flow accumulation in the brain tissue of patients receiving high doses. No health risks were observed in patients receiving 0.5 or 1 mg of bepinizumab.

[0177] 3D6's V H The amino acid sequence containing SEQ ID NO:4. V of 3D6 L The amino acid sequence containing SEQ ID NO:23. Bepinizumab V H The amino acid sequence containing SEQ ID NO:24. Bepinizumab V L The amino acid sequence containing SEQ ID NO:25.

[0178] SEQ ID NO:23 3D6 V L

[0179] YVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRIEAEDLGLYYCWQGTHFPRTFGGGTKLEIKR

[0180] SEQ ID NO:24 Bepipizumab VH

[0181] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCVRYDHYSGSSDYWGQGTLVTVSS

[0182] SEQ ID NO:25 Bepipizumab VL

[0183] YVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKR

[0184] Antibody variants

[0185] This article also provides variants and derivatives of any of the therapeutic catalytic antibodies described above, such as anti-Aβ catalytic antibodies.

[0186] Replacement, Insertion, Deletion, and Variation

[0187] In some embodiments, this encompasses amino acid sequence variants of the therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies) provided herein. For example, it may be necessary to improve the binding affinity and / or other biological properties of the catalytic antibody. Amino acid sequence variants of the catalytic antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the catalytic antibody or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues within the amino acid sequence of the catalytic antibody. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct possesses the desired properties, such as antigen binding and cleavage.

[0188] In some embodiments, catalytic antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutation induction include CDR and FR. Amino acid substitutions can be introduced into the catalytic antibody of interest, and products can be screened for desired activity, such as retained / improved antigen binding and cleavage, reduced immunogenicity, or improved ADCC or CDC.

[0189] Conservative substitutions are shown in Table 3 below.

[0190] Table 3: Conservative Substitution

[0191]

[0192] Amino acids can be classified into different categories based on common side chain characteristics:

[0193] a. Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0194] b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0195] c. Acids: Asp, Glu;

[0196] d. Alkaline: His, Lys, Arg;

[0197] e. Residues that affect chain orientation: Gly, Pro;

[0198] f. Aromatics: Trp, Tyr, Phe.

[0199] Non-conservative substitution would require swapping members of one of these categories with members of another category.

[0200] Exemplary substitution variants are catalytic antibodies for affinity maturation, which can be conveniently generated, for example, using phage display-based affinity maturation techniques. In simple terms, one or more CDR residues are mutated and the variant catalytic antibody is displayed on a phage, and screening is performed for specific biological activities (e.g., binding affinity). Modifications (e.g., substitutions) can be made within the HVR to, for example, improve antibody affinity. Such modifications can be made in HVR “hotspots,” which are residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, Methods in Molecular Biology 207:179-196 (2008)), and / or specifically identified residues (SDRs), where the resulting variant V is tested. H or V L Binding affinity. Affinity maturation by constructing a secondary library and reselecting from it has been described, for example, in Molecular Biology Methods, Hoogenboom et al., 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ (2001)).

[0201] In some embodiments of affinity maturation, diversity is introduced into the variant gene selected for maturation via any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-guided approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomly grouped. Alanine scan mutation induction or modeling can be used, for example, to specifically identify HVR residues involved in antigen binding. CDR-H3 and CDR-L3 are typically targeted in particular.

[0202] In some embodiments, substitution, insertion, or deletion may occur within one or more CDRs, provided that such changes do not significantly reduce the ability of the antibody to bind and cleave the antigen. For example, conserved changes (e.g., conserved substitutions as described herein) may be made in the CDRs without significantly reducing binding affinity or antigen cleavage activity. Such changes may occur outside the HVR “hotspot” or SDR. In the variants V provided above… H and V L In some embodiments of the sequence, each CDR is unchanged, or contains no more than one, two, or three amino acid substitutions.

[0203] One method for identifying target residues or regions for mutation induction is called "alanine scanning mutation induction," as described by Cunningham and Wells (1989) in *Science*, 244:1081-1085. In this method, target residues or groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and substituted with neutral or negatively charged amino acids (e.g., propylamino or polypropylamino) to determine if the antibody-antigen interaction is affected. Other substitutions may be introduced at amino acid positions indicating functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be determined to identify contact points between the antibody and antigen. These contact residues and adjacent residues may be targeted or removed as candidates for substitution. Variants may be screened to determine if they contain the desired properties.

[0204] The catalytic antibody variants described in this article maintain their catalytic activity against target proteins (e.g., Aβ). The catalytic triad, namely the V... L The SHD motif in the antibody cannot be replaced. In some embodiments, the V motif supporting its catalytic activity is present in the therapeutic catalytic antibody. L One or more amino acid residues in V cannot be substituted, including, for example, those in V. L The amino acid residues at positions 26, 27D, and 28 are numbered according to Kabat's EU index.

[0205] Amino acid sequence insertions include fusion of amino and / or carboxyl termini from one residue to peptides containing one hundred or more residues, as well as insertion of one or more amino acid residues within the sequence. Examples of terminal insertions include catalytic antibodies with an N-terminal methionyl residue. Other insertional variants of catalytic antibodies include enzymes that increase the serum half-life of the antibody (e.g., for ADEPT) or fusion of a peptide with the N- or C-terminus of the antibody.

[0206] Catalytic antibody variants also provide an N-terminal leader extension. For example, one or more amino acid residues of the N-terminal leader sequence are present at the N-terminus of any one or more heavy or light chains of the antibody. An exemplary N-terminal leader extension comprises three amino acid residues, VHS, or is composed of them, and is present on one or both light chains of the antibody variant.

[0207] Chimeric and humanized catalytic antibodies

[0208] In some embodiments, the therapeutic catalytic antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., (1984) Proceedings of the National Academy of Sciences, 81:6851-6855 (1984). In one instance, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In another instance, a chimeric antibody is a “class-switching” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0209] In some embodiments, a humanized catalytic antibody is provided. Humanized antibodies are used as therapeutic molecules because they reduce or eliminate the human immune response to non-human antibodies (such as the human anti-mouse antibody (HAMA) response), which can lead to an immune response to antibody therapeutics and reduce the effectiveness of the therapeutic.

[0210] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains wherein the HVR, such as the CDR (or a portion thereof), is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0211] Humanized antibodies and their preparation methods are reviewed in, for example, Almagro and Fransson, (2008) Frontiers in Bioscience 13:1619-1633, and further described in, for example, Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proceedings of the National Academy of Sciences 86:10029-10033; U.S. Patents 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. , (2005) Methods 36:25-34 (describes SDR (a-CDR) transplantation); Padlan, (1991) Molecular Immunology 28:489-498 (describes “surface repair”); Dall'Acqua et al., (2005) Methods 36:43-60 (describes “FR reorganization”); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br.J. Cancer 83:252-260 (describes the “guided selection” approach to FR reorganization).

[0212] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best fit" method (see, for example, Sims et al. (1993) *Journal of Immunology* 151:2296); frame regions of the common sequence of human antibodies derived from specific subgroups of the light or heavy chain variable regions (see, for example, Carter et al. (1992) *Proceedings of the National Academy of Sciences*, 89:4285; and Presta et al. (1993) *Journal of Immunology*, 151:2623); and human mature ( Somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, (2008) Frontiers in Bioscience 13:1619-1633); and framework regions derived from screening FR libraries (see, for example, Baca et al., (1997) Journal of Biochemistry 272:10678-10684 and Rosok et al., (1996) Journal of Biochemistry 271:22611-22618).

[0213] Antibodies from library sources

[0214] Therapeutic catalytic antibodies can be isolated from combinatorial libraries of antibodies with the desired activity. For example, several methods are known in the field for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. These methods are reviewed, for example, in *Methods in Molecular Biology* by Hoogenboom et al., 178:1-37 (edited by O'Brien et al., Human Press, Tetowa, NJ, 2001), and further described in, for example, *Nature* by McCafferty et al., 348:552-554; *Nature* by Clackson et al., 352:624-628 (1991); *Molecular Biology Journal* by Marks et al., 222:581-597 (1992); and *Methods in Molecular Biology* by Marks and Bradbury, 248:161-175 (edited by Lo, Human Press, Human Press, NJ, 2001). Press, Tetowa, NJ, 2003); Sidhu et al., Journal of Molecular Biology 338(2):299-310 (2004); Lee et al., Journal of Molecular Biology 340(5):1073-1093 (2004); Fellouse, Proceedings of the National Academy of Sciences 101(34):12467-12472 (2004); and Lee et al., Journal of Immunological Methods 284(1-2):119-132 (2004).

[0215] In some phage display methods, V H and V LGene lineages are cloned separately via polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phages, as described in Winter et al., *Annalen Ref. Immunol.*, 12:433-455 (1994). Phages typically present antibody fragments as scFv or Fab fragments. Libraries derived from immunogenic sources can provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, native libraries can be cloned to provide a single source of antibodies to a broad range of non-self and self antigens without any immunization, as described by Griffiths et al., *European Journal of Molecular Biology*, 12:725-734 (1993). Finally, native libraries encoding highly variable CDR3 regions can also be prepared by cloning unrearranged V-gene fragments from stem cells and synthesizing them using PCR primers containing random sequences, as described, for example, by Hoogenboom and Winter, *Journal of Molecular Biology*, 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0216] Fc region variants

[0217] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody) provided herein, thereby creating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC effector function, typically associated with binding to an Fc receptor (FcR). In some embodiments, the Fc region variant has reduced ADCC effector function. There are numerous examples of Fc sequence changes or mutations that can alter effector function. For example, WO 00 / 42072 and Shields et al., Journal of Biochemistry 9(2):6591-6604 (2001) describe antibody variants with improved or reduced binding to the FcR. Those disclosures are explicitly incorporated herein by reference.

[0218] In some embodiments, the therapeutic catalytic antibody comprises an Fc region having some, but not all, effector functions, which makes it a desirable candidate for applications where the in vivo half-life of the catalytic antibody is important and certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary cells used to mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, on page 464 below: Ravetch and Kinet, Annals of Immunology 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proceedings of the National Academy of Sciences 83:7059-7063 (1986)) and Hellstrom, I. et al., Proceedings of the National Academy of Sciences 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see, Bruggemann, M. et al., Journal of Experimental Medicine 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assays (Cell Technology Inc., Mountain View, CA); and CytoTox96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Useful effector cells for this type of assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models disclosed in Clynes et al., Proceedings of the National Academy of Sciences, 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., Journal of Immunological Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the field (see, for example, Petkova, SB et al., International Journal of Immunology 18(12):1759-1769 (2006)).

[0219] Antibodies with reduced effector function include those that substitute one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 replaced by alanine (US Patent No. 7,332,581).

[0220] Certain antibody variants with improved or weakened binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., Journal of Biochemistry 9(2):6591-6604(2001).)

[0221] In some embodiments, alterations are made in the Fc region that produces altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), for example as described in U.S. Patent No. 6,194,551, WO 99 / 51642 and Idusogie et al., Journal of Immunology 164:4178-4184 (2000).

[0222] Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, the substitution of Fc region residue 434 (US Patent No. 7,371,826).

[0223] Other examples of Fc region variants can be found in Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821 and WO 94 / 29351.

[0224] Glycosylation variants

[0225] In some embodiments, the degree of glycosylation of the construct of the therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody) provided herein is altered by increasing or decreasing the degree of glycosylation. Adding or deleting glycosylation sites to the antibody can be conveniently achieved by altering the amino acid sequence of the antibody to produce or remove one or more glycosylation sites.

[0226] In cases where the catalytic antibody contains an Fc region, the carbohydrates attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually attached via an N-bond to the Asn297 of the CH2 domain of the Fc region. See example Wright et al., *TIBTECH* 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc linked to the “stem” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the catalytic antibody can be modified to produce catalytic antibody variants with certain improved properties.

[0227] The N-glycans attached to the CH2 domain of Fc are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated via fucosyltransferase activity. See Shoji-Hosaka et al., Journal of Biochemistry 2006, 140:777-83. Typically, small amounts of naturally occurring unfucosylated IgG are detectable in human serum. N-glycosylation of Fc is important for binding to FcγR; and the unfucosylation of N-glycans enhances the binding affinity of Fc to FcγRIIIa. Increased Fc γRIIIa binding may enhance ADCC, which could be advantageous in certain therapeutic applications where cytotoxicity is desired.

[0228] In some embodiments, enhanced effector function can be detrimental when Fc-mediated cytotoxicity is not desired. In some embodiments, the Fc fragment or the CH2 domain is not glycosylated. In some embodiments, the N-glycosylation site in the CH2 domain is mutated to prevent glycosylation.

[0229] In some embodiments, the catalytic antibody variant is provided as a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all glycan structures (e.g., complex, heterozygous, and high-mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU number of the Fe region residue) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucoidylated variants may have improved ADCC function. See, for example, U.S. Patent Publication No. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Publicly disclosed examples associated with “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., Journal of Molecular Biology 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotechnology and Bioengineering 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., *Arch. Biochem. Biophys.* 249:533-545 (1986); US Patent Application No. US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially in Example 11), and gene knockout cell lines, such as α-1,6-fucoside transferase gene, FUT8, gene knockout CHO cells (see, for example, Yamane-Ohnuki et al., *Biotechnology and Bioengineering* 87:614 (2004); Kanda, Y. et al., *Biotechnology and Bioengineering* 94(4):680-688 (2006) and WO2003 / 085107).

[0230] Catalytic antibody variants further possess a bipartite oligosaccharide, such as a bipartite oligosaccharide in which GlcNAc is bipartitely attached to the Fc region of the antibody. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0231] derivative

[0232] In some embodiments, the therapeutic catalytic antibodies provided herein (such as anti-Aβ catalytic antibodies) may be further modified to include additional non-protein moieties known in the art and readily available. Moieties suitable for the derivatization of catalytic antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde is advantageous in manufacturing due to its stability in water. Polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the catalytic antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the catalytic antibody to be improved, whether the catalytic antibody derivative will be used in a therapy under defined conditions, etc.

[0233] IV. Preparation Method

[0234] The therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies) described herein can be prepared using any method known in the field, including those described below and in the examples. Therapeutic metabolites can be obtained by immunizing laboratory animals against a target antigen (such as a transition state analog). See, for example, US2010018361A1 and Taguchi H et al., “Catalytic antibodies to amyloid beta peptidein defense against Alzheimer disease,” *Autoimmune Diseases Review* 7:391-397 (2008), which is incorporated herein by reference. Catalytic antibodies can also be recombinantly expressed.

[0235] Nucleic acid

[0236] This application also provides isolated nucleic acid molecules comprising one or more chains of polynucleotides encoding a therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody) as described herein. In some embodiments, the nucleic acid molecule comprises a heavy chain or a light chain of polynucleotides encoding a therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody). In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of a therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody). In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding a heavy chain, and a second nucleic acid molecule comprises a second polynucleotide encoding a light chain. In some embodiments, the first polynucleotide encoding a heavy chain is operatively linked to a first promoter, and the second polynucleotide encoding a light chain is operatively linked to a second promoter. In some embodiments, the polynucleotides encoding a heavy chain and the polynucleotides encoding a light chain are operatively linked to promoters.

[0237] Additional promoter elements (such as enhancers) regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, allowing promoter function to be preserved when elements are inverted or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline.

[0238] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is extension growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, this invention is not limited to the use of constitutive promoters. Inducible promoters are also considered part of this invention. The use of inducible promoters provides a molecular switch that can activate the expression of a polynucleotide sequence operably linked to it when such expression is needed, or deactivate it when expression is not needed. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0239] In some embodiments, the expression of the therapeutic catalytic antibody is inducible. In some embodiments, the nucleic acid sequence encoding the therapeutic catalytic antibody is operatively linked to an inducible promoter.

[0240] In some embodiments, the polynucleotide encoding the heavy or light chain of a therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody) comprises a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain upon translation. The leader sequence may be a native heavy or light chain leader sequence or another heterologous leader sequence. In some embodiments, the nucleic acid (a set of nucleic acids) encoding the therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody) may also comprise a nucleic acid sequence encoding a peptide tag (such as a protein purification tag, e.g., a His tag, HA tag).

[0241] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize under at least moderately stringent hybridization conditions to any of the nucleic acid sequences encoding the therapeutic catalytic antibodies described herein.

[0242] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the field. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in selected host cells.

[0243] carrier

[0244] A vector is provided comprising a polynucleotide encoding a heavy chain and / or light chain of any of the therapeutic catalytic antibodies described herein (such as anti-Aβ catalytic antibodies). Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide.

[0245] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain, and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first and second vectors are transfected into host cells in similar amounts (e.g., similar molar amounts or similar mass amounts). In some embodiments, the first and second vectors are transfected into host cells in a molar or mass ratio between 5:1 and 1:5. In some embodiments, a mass ratio between 1:1 and 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0246] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0247] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the field and described, for example, in Green and Sambrook (2013, *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Viruses used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site, and one or more optional markers that are functional in at least one organism (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0248] In simple terms, therapeutic catalytic antibodies can be expressed by encoding natural or synthetic nucleic acids through nucleic acids by inserting them into appropriate expression vectors, allowing the nucleic acids to be operatively linked to 5' and 3' regulatory elements, including, for example, promoters (e.g., lymphocyte-specific promoters) and the 3' untranslated region (UTR). The vectors are adapted for replication and integration into host cells. Typical cloning and expression vectors contain transcription and translation terminators, an initial sequence, and a promoter suitable for regulating the expression of the desired nucleic acid sequence.

[0249] In some embodiments, a vector optimized for expressing the peptide in CHO or CHO-derived cells or in NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., *Biotechnol. Prog.* 20:880-889 (2004).

[0250] To assess the expression of a peptide or its fraction, the expression vector to be introduced into cells may also contain a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a target cell population via viral transfection or infection. Alternatively, the selection marker may be carried on a separate DNA fragment and used in co-transfection procedures. Both the selection marker and the reporter gene may be side-linked with appropriate regulatory sequences to enable expression in host cells. Suitable selection markers include, for example, antibiotic resistance genes such as neo.

[0251] Reporter genes are used to identify potentially transfected cells and assess the function of regulatory sequences. Generally, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue and encodes a polypeptide whose expression is indicated by easily detectable properties, such as enzyme activity. Reporter gene expression is measured at an appropriate time after DNA has been introduced into the recipient cells. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000, FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Generally, constructs exhibiting the highest level of reporter gene expression with minimal 5' flanking regions are identified as promoters. These promoter regions can be linked to reporter genes and used to assess the ability of drugs to regulate promoter-driven transcription.

[0252] host cells

[0253] This application provides isolated host cells containing any of the therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies), nucleic acid molecules, or vectors described herein.

[0254] The therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies) described herein can be expressed in prokaryotic cells (such as bacterial cells) or eukaryotic cells (such as fungal cells (yeast), plant cells, insect cells, and mammalian cells). Such expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express peptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44, Lec13CHO cells, and FUT8CHO cells; and PER cells. Crucells and NSO cells are suitable eukaryotic host cells. Suitable non-mammalian host cells include prokaryotes (such as *Escherichia coli* or *Bacillus subtilis*) and yeasts (*Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, or *Kluyveromyces lactis*). In some embodiments, eukaryotic host cells are selected based on the ability of a particular eukaryotic host cell to perform desired post-translational modifications to the heavy and / or light chains of the antibody. For example, in some embodiments, CHO cells produce peptides with higher levels of sialylation than the same peptides produced in 293 cells.

[0255] The introduction of one or more nucleic acids into a desired host cell can be achieved by any method, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, perforation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd ed., Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cell using any suitable method.

[0256] In some embodiments, the therapeutic catalytic antibody is generated in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Molecular Biology Methods 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); and Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0257] purification

[0258] Therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies) can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the antibody's constant region. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind to the constant region and purify antibodies containing the Fc fragment. Hydrophobic interaction chromatography, such as butyl or phenyl columns, is also suitable for purifying certain peptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) is also suitable for purifying certain peptides, such as antibodies. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) is also suitable for purifying certain peptides, such as antibodies. Many methods for purifying peptides are known in the art.

[0259] V. Compositions, reagent kits, and products

[0260] This article also provides compositions (such as pharmaceutical compositions) comprising any of the therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies) described herein, nucleic acids, vectors, or host cells.

[0261] The pharmaceutical compositions of therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies) described herein can be prepared by combining a therapeutic catalytic antibody (such as anti-Aβ catalytic antibody) of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)), in lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl ammonium chloride, hexahydroxyquaternary ammonium chloride; benzalkonium chloride, benzyl chloride). Chloride); phenols, butanol, or benzyl alcohol; p-hydroxybenzoic acid esters, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG). Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted to high protein concentrations with a suitable diluent, and the reconstituted formulations can be subcutaneously administered to the individuals described herein for imaging, diagnosis, or therapy.

[0262] Pharmaceutical compositions intended for internal administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane.

[0263] Kits are also provided for use with any of the methods described herein for determining catalytic antibody levels, diagnosis, and treatment, including kits containing any of the therapeutic catalytic antibodies described herein (such as anti-Aβ catalytic antibodies).

[0264] In some embodiments, a kit is provided for determining the level of a catalytic antibody in a biological sample, comprising: an amino acid sequence (EAR). nThe substrate peptide of (SEQ ID NO:2), wherein n is an integer between 1 and 30 (e.g., n is 3). In some embodiments, the kit further comprises an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE). In some embodiments, the kit comprises a solid support (e.g., an ELISA plate).

[0265] In some embodiments, a kit is provided for diagnosing PAD in an individual or determining the risk of PAD, wherein PAD is associated with a target protein, the kit comprising: a) containing an amino acid sequence (EAR). n The kit contains a substrate peptide (SEQ ID NO:2), wherein n is an integer between 1 and 30 (e.g., n is 3); and b) the target protein (or a fragment thereof) or an antibody against the target protein. In some embodiments, the kit further comprises an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE). In some embodiments, the kit comprises a solid support (e.g., an ELISA plate).

[0266] In some embodiments, a kit is provided for diagnosing AD in an individual or determining the risk of AD, comprising: a) containing an amino acid sequence (EAR). n The kit contains: a) a substrate peptide of (SEQ ID NO:2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) an Aβ peptide (e.g., Aβ(1-42)) or an anti-Aβ antibody; and c) an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE). In some embodiments, the kit comprises a solid support (e.g., an ELISA plate).

[0267] In some embodiments, a kit is provided for treating or preventing PAD in an individual, wherein the PAD is associated with a target protein, the kit comprising: a) containing an amino acid sequence (EAR). n The kit comprises: a) a substrate peptide of (SEQ ID NO:2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) a target protein (or a fragment thereof) or an antibody against the target protein; c) an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA and / or total IgE); and d) a therapeutic catalytic antibody that specifically binds to the target protein. In some embodiments, the kit comprises a solid support (e.g., an ELISA plate).

[0268] In some embodiments, a kit is provided for treating or preventing AD in an individual, comprising: a) containing an amino acid sequence (EAR). nThe kit contains: a) a substrate peptide of (SEQ ID NO:2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) an Aβ peptide (e.g., Aβ(1-42)) or an anti-Aβ antibody; c) an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and / or total IgE); and d) a therapeutic catalytic antibody that specifically binds to Aβ, such as any of the anti-Aβ catalytic antibodies described herein. In some embodiments, the kit comprises a solid support (e.g., an ELISA plate).

[0269] In some embodiments, a kit is provided for treating or preventing AD in an individual, comprising: a pharmaceutical composition comprising an anti-Aβ catalytic antibody and a pharmaceutically acceptable carrier, wherein the anti-Aβ catalytic antibody comprises: V containing the following L LC-CDR1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein in V L The amino acid residue at position 1 is D, and in V L The amino acid residue at position 27A is S, and in V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index. In some embodiments, in V L The amino acid residue at position 26 is Ser(S), in V L The amino acid residue at position 27D is D, E, or H, and / or in V L The amino acid residue at position 28 is either D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: V containing the following H HC-CDR1 containing the amino acid sequence of SEQ ID NO:9, HC-CDR2 containing the amino acid sequence of SEQ ID NO:10, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions.

[0270] The kit described in this application is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags). The kit may optionally include additional components such as reagents (e.g., ECL substrates), buffers (e.g., coating buffer, blocking buffer, washing buffer, antibody dilution buffer, developing buffer, etc.), antibodies (e.g., anti-human Ig antibodies), and explanatory information.

[0271] Therefore, this application also provides articles of manufacture. Articles of manufacture may include a container and markings or packaging inserts on or associated with the container. Suitable containers include vials (such as sealed vials), bottles, jars, flexible packaging, etc. In some embodiments, the container holds the pharmaceutical composition and may have a sterile dispensing port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). In some embodiments, the container holds a substrate peptide or target protein (e.g., Aβ) for immunoassay. The markings or packaging inserts indicate that the composition is used to diagnose (including risk assessment), treat, or prevent PAD (e.g., AD) in an individual. The markings or packaging inserts will also contain instructions for performing immunoassays to determine the levels of one or more SHD-catalyzing antibodies in a biological sample and / or administering the pharmaceutical composition to an individual. The markings may indicate instructions for reconstitution and / or use of the various components. The container holding the pharmaceutical composition may be a reusable vial that allows for repeated administration of the reconstituted formulation (e.g., 2-6 administrations). Packaging inserts refer to the instruction leaflets typically included in the commercial packaging of diagnostic and / or therapeutic products, containing information about indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such products. Additionally, the product may include a second container containing pharmaceutically acceptable buffer solutions, such as bactericidal aqueous solution for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials desired from a commercial and user perspective, including additional buffer solutions, diluents, filters, needles, and syringes.

[0272] The kit may also include multiple unit doses of the pharmaceutical composition and instructions for use, and be packaged in quantities sufficient for storage and use in pharmacies (such as hospital pharmacies and mixed pharmacies).

[0273] Exemplary embodiments

[0274] Example 1. A method for determining the level of one or more SHD-catalyzing antibodies in a biological sample, comprising:

[0275] a) Contact the biological sample with the substrate peptide immobilized on a solid support under conditions that allow for the formation of a catalytic antibody-substrate peptide complex, and

[0276] b) Determine the amount of the catalytic antibody-substrate peptide complex, thereby providing the level of one or more SHD catalytic antibodies in the biological sample.

[0277] The substrate peptide described herein contains an amino acid sequence (EAR). n (SEQ ID NO:2), where n is an integer between 1 and 30.

[0278] Example 2. The method described in Example 1, wherein n is 3.

[0279] Example 3. The method according to Example 1 or 2, wherein the biological sample is a serum sample.

[0280] Example 4. The method according to Example 3, wherein the serum sample contains at least about 1 μg / mL of immunoglobulin (Ig).

[0281] Example 5. The method according to any one of Examples 1 to 4, wherein the biological sample is incubated with the substrate peptide for about 1 hour to about 16 hours.

[0282] Example 6. The method according to any one of Examples 1 to 5, wherein the amount of the catalytic antibody-substrate peptide complex is determined using an antibody that specifically binds to total Ig.

[0283] Example 7. The method according to Example 6, wherein the antibody is labeled with an enzyme or a fluorescent label.

[0284] Example 8. A method for determining the risk of an individual for a protein aggregation disease (PAD), wherein the PAD is associated with the aggregation of a target protein, the method comprising determining the level of one or more SHD catalytic antibodies in a biological sample of the individual, wherein if the level of the one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody, then the individual is determined to have the risk of the PAD.

[0285] Example 9. According to the method of Example 8, the level of one or more SHD catalytic antibodies is the level of one or more SHD catalytic antibodies that specifically bind to the target protein.

[0286] Example 10. According to the method of Example 8, the level of one or more SHD catalytic antibodies is the level of total SHD catalytic antibodies.

[0287] Example 11. The method according to Example 10, wherein the level of total SHD catalytic antibody is determined by contacting an individual's serum sample with a substrate peptide immobilized on a solid support under conditions allowing the formation of a catalytic antibody-substrate peptide complex and determining the amount of the catalytic antibody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence (EAR). n (SEQ ID NO:2), where n is an integer between 1 and 30.

[0288] Example 12. The method according to Example 11, wherein n is 3.

[0289] Example 13. The method according to Example 11 or 12, wherein the serum sample contains at least about 1 μg / mL.

[0290] Example 14. The method according to any one of Examples 11 to 13, wherein the serum sample is incubated with the substrate peptide for about 1 hour to about 16 hours.

[0291] Example 15. The method according to any one of Examples 11 to 14, wherein the amount of the catalytic antibody-substrate peptide complex is determined using an antibody that specifically binds to total Ig.

[0292] Example 16. The method according to Example 15, wherein the antibody is labeled with an enzyme or a fluorescent label.

[0293] Example 17. The method according to any one of claims 8 to 16, further comprising determining the level of autoantibodies against the target protein in a biological sample of the individual, wherein the individual is determined to have the risk of the PAD if: (i) the level of one or more SHD-catalyzing antibodies is lower than the level of control SHD-catalyzing antibodies; and (ii) the level of the autoantibody against the target protein is lower than the level of control autoantibody.

[0294] Example 18. The method according to Example 17, wherein the level of the autoantibody is determined by contacting a serum sample of the individual with the target protein under conditions that allow the formation of an autoantibody-target protein complex and determining the amount of the autoantibody-target protein complex.

[0295] Example 19. The method according to Example 18, wherein the level of the autoantibody is determined using an ELISA assay.

[0296] Example 20. The method according to any one of Examples 17 to 19, wherein the control autoantibody level is the level of the autoantibody against the target protein in a healthy individual.

[0297] Example 21. The method according to any one of Examples 17 to 19, wherein the control autoantibody level is the median level of the autoantibody against the target protein in a population of individuals.

[0298] Example 22. The method according to any one of Examples 8 to 16, further comprising determining the level of the target protein in a biological sample of the individual, wherein if: (i) the level of one or more SHD catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of the target protein is higher than the level of a control target protein, then the individual is determined to have the risk of the PAD.

[0299] Example 23. The method according to any one of Examples 8 to 22, wherein the level of the control SHD catalytic antibody is the level of one or more SHD catalytic antibodies in a healthy individual.

[0300] Example 24. The method according to any one of Examples 8 to 22, wherein the control SHD catalytic antibody level is the median level of one or more SHD catalytic antibodies in an individual.

[0301] Example 25. The method according to any one of Examples 8 to 24, wherein the PAD is Alzheimer's disease, and wherein the target protein is amyloid β (Aβ).

[0302] Example 26. The method according to any one of Examples 8 to 24, wherein:

[0303] (i) The PAD is Parkinson's disease, and the target protein is α-synuclein;

[0304] (ii) The PAD is Alzheimer's disease or dementia, and the target protein is Tau;

[0305] (iii) The PAD is ATTR amyloidosis, and the target protein is transthyretin;

[0306] (iv) The PAD is AL amyloidosis and the target protein is an immunoglobulin light chain;

[0307] (v) The PAD is frontotemporal lobe degeneration or amyotrophic lateral sclerosis, and the target protein is TDP43;

[0308] (vi) The PAD is Huntington's disease, and the target protein is Huntington's protein;

[0309] (vii) The PAD is type II diabetes, and the target protein is IAPP; or

[0310] (viii) The PAD is amyotrophic lateral sclerosis, and the target protein is SOD1.

[0311] Example 27. A method for treating or preventing PAD in an individual, wherein the PAD is associated with the aggregation of a target protein, the method comprising:

[0312] a) Determine the risk of the individual having the PAD according to the method of any one of Examples 1 to 26; and

[0313] b) Administer an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein to the individual.

[0314] Example 28. The method according to Example 27, wherein the method is repeated at a frequency of no more than about every three months.

[0315] Example 29. The method according to Example 27 or 28, wherein the PAD is Alzheimer's disease, wherein the target protein is amyloid β (Aβ), and wherein the therapeutic catalytic antibody comprises: a light chain variable region comprising the following (V L ): a light chain complementarity-determining region (LC-CDR) 1 containing the amino acid sequence of SEQ ID NO: 12, an LC-CDR 2 containing the amino acid sequence of SEQ ID NO: 13, and an LC-CDR 3 containing the amino acid sequence of SEQ ID NO: 14, or variants thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein the V L The amino acid residue at position 1 is D, and in the V L The amino acid residue at position 27A is S, and in the V L The amino acid residue at position 93 is H, and the numbering is based on the EU index of Kabat.

[0316] Example 30. The method according to Example 29, wherein the therapeutic catalytic antibody comprises: a heavy chain variable region (V) comprising the following H ): Heavy chain complementarity-determining region (HC-CDR)1 containing the amino acid sequence of SEQ ID NO:9, HC-CDR2 containing the amino acid sequence of SEQ ID NO:10, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions.

[0317] Example 31. The method according to Example 29 or 30, wherein in the V L The amino acid residue at position 26 is S, in the V L The amino acid residue at position 27D is D, E, or H, and / or in position V L The amino acid residue at position 28 is D or N, and the numbering is based on the Kabat EU index.

[0318] Example 32. The method according to any one of Examples 29 to 31, wherein the therapeutic catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20. H ; and / or an amino acid sequence V having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22.L .

[0319] Example 33. The method according to Example 32, wherein the therapeutic catalytic antibody comprises: (i) a V comprising the amino acid sequence of SEQ ID NO:4 H and V containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L (iv) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:21 L (v) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:21 L (vi) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:22 L ; or (vii) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:22 L .

[0320] Example 34. The method according to any one of Examples 27 to 33, wherein the therapeutic catalytic antibody is a full-length IgG antibody.

[0321] Example 35. The method according to Example 34, wherein the therapeutic catalytic antibody comprises an IgG1 or IgG4 Fc region.

[0322] Example 36. The method according to any one of Examples 27 to 33, wherein the therapeutic antibody is a full-length IgM antibody.

[0323] Example 37. An isolated anti-Aβ catalytic antibody comprising: V containing the following L LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:13, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:14, or variants thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein in the VL The amino acid residue at position 1 is D, and in the V L The amino acid residue at position 27A is S, and in the V L The amino acid residue at position 93 is H, and the numbering is based on the Kabat EU index.

[0324] Example 38. An isolated anti-Aβ catalytic antibody comprising: V containing the following H HC-CDR1 comprising the amino acid sequence of SEQ ID NO:9, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:11, or variants thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions, wherein in the V L The amino acid residue at position 1 is D, and in the V L The amino acid residue at position 27A is S, and in the V L The amino acid residue at position 93 is H, and the numbering is based on the EU index of Kabat.

[0325] Example 39. The anti-Aβ catalytic antibody according to Example 37 or 38, wherein the anti-Aβ catalytic antibody comprises: V containing the following H HC-CDR1 containing the amino acid sequence of SEQ ID NO:9, HC-CDR2 containing the amino acid sequence of SEQ ID NO:10, and HC-CDR3 containing the amino acid sequence of SEQ ID NO:11; and HC-CDR3 containing the following V L LC-CDR1 containing the amino acid sequence of SEQ ID NO:12, LC-CDR2 containing the amino acid sequence of SEQ ID NO:13, and LC-CDR3 containing the amino acid sequence of SEQ ID NO:14.

[0326] Example 40. An anti-Aβ catalytic antibody according to any one of Examples 37 to 39, wherein the anti-Aβ catalytic antibody cleaves a substrate having the formula EAR-AMC (SEQ ID NO:1).

[0327] Example 41. The anti-Aβ catalytic antibody according to any one of Examples 37 to 40, wherein in the V L The amino acid residue at position 26 is S, in the V L The amino acid residue at position 27D is D, E, or H, and / or in position V LThe amino acid residue at position 28 is D or N, and the numbering is based on the Kabat EU index.

[0328] Example 42. An anti-Aβ catalytic antibody according to any one of Examples 37 to 41, wherein the anti-Aβ catalytic antibody comprises: a V amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20. H ; and / or an amino acid sequence V having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22. L .

[0329] Example 43. The anti-Aβ catalytic antibody according to Example 42, wherein the anti-Aβ catalytic antibody comprises: (i) a V amino acid sequence comprising SEQ ID NO:4 H and V containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L (iv) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:21 L (v) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:21 L (vi) V containing the amino acid sequence of SEQ ID NO:19 H and V containing the amino acid sequence of SEQ ID NO:22 L ; or (vii) V containing the amino acid sequence of SEQ ID NO:20 H and V containing the amino acid sequence of SEQ ID NO:22 L .

[0330] Example 44. An anti-Aβ catalytic antibody according to any one of Examples 37 to 43, wherein the anti-Aβ catalytic antibody is a full-length IgG antibody.

[0331] Example 45. The anti-Aβ catalytic antibody according to Example 44, wherein the anti-Aβ catalytic antibody comprises an IgG1 or IgG4 Fc region.

[0332] Example 46. An anti-Aβ catalytic antibody according to any one of Examples 37 to 43, wherein the anti-Aβ catalytic antibody is a full-length IgM antibody.

[0333] Example 47. A method for treating or preventing Alzheimer's disease in an individual, comprising administering to the individual an effective amount of an anti-Aβ catalytic antibody according to any one of Examples 37 to 46.

[0334] Example 48.A. A kit for treating or preventing Alzheimer's disease in an individual, comprising:

[0335] a) Contains the amino acid sequence (EAR) n The substrate peptide of (SEQ ID NO:2), where n is an integer between 1 and 30;

[0336] b) Aβ peptide; and

[0337] c) Antibodies that specifically bind to total Ig.

[0338] Example 49. The kit according to Example 48 further comprises a solid support.

[0339] Example 50. The kit according to Example 48 or 49 further comprises a therapeutic catalytic antibody that specifically binds to Aβ.

[0340] Example

[0341] The following examples are intended to be illustrative of the invention and should therefore not be construed as limiting the invention in any way. The following examples and detailed descriptions are provided for illustration and not for limitation.

[0342] Example 1. Catalytic antibodies as serum biomarkers for Alzheimer's disease

[0343] This example provides experimental data demonstrating that SHD catalytic antibodies (i.e., catalytic antibodies with the “SHD” motif) can be used as serum biomarkers to diagnose or predict protein aggregation disorders (PADs), such as Alzheimer’s disease (AD).

[0344] A. Catalytic antibody levels in serum samples from young and older adults

[0345] Serum SHD-catalyzing antibody levels were measured using two immunoassays in young adults (20 to 29 years old; HS2, HS6, HS7, and HS8) and older adults (60 to 69 years old; HS1, HS3, HS4, and HS5). In the EAR-AMC binding assay, the ELISA plate was coated with the EAR peptide (SEQ ID NO:1) bound to 7-amino-4-methylcoumarin (AMC) (“EAR-AMC”, 100X, Bachem Americas, Cat No. I-1575.0050). In the EAR3 binding assay, the ELISA plate was coated with (EAR)3 (SEQ ID NO:3).

[0346] Briefly, each ELISA plate was coated with the corresponding peptide at a final concentration of approximately 5 μg / mL in coating buffer (0.2 M sodium carbonate-bicarbonate buffer, pH 9.4), 100 μL / well, overnight at 4°C. The plates were washed three times with wash buffer (PBST: 0.1% Tween-20 in phosphate-buffered saline "PBS"). The plates were then blocked for 1 hour at room temperature with blocking buffer (1% BSA in PBS) and washed three times with wash buffer. 100 μL of the corresponding serum sample (25 μg / mL or 100 μg / mL) was added to each well of the plate, with PBS serving as a negative control. After incubation for 1 hour, the plates were then washed three times with wash buffer. 100 μL of a goat polyclonal antibody against human IgG conjugated to horseradish peroxidase (GAH-HRP; Abcam CAT No. ab98605), diluted 1:10,000, was added to each well of the plate, and the plate was incubated at room temperature for 1 hour and protected from light. HRP substrates (Amplex Red and H2O2) were freshly prepared using a developing buffer (3.6 mM Na2HPO4, 1.4 mM NaH2PO4, pH 7.2), and 100 μL of the HRP substrate was added to each well and incubated for 1–5 minutes. The signal was then detected using a plate reader (excitation wavelength = 530 nm, emission wavelength = 590 nm, and cutoff wavelength = 570 nm).

[0347] Figure 1A The results of EAR-AMC binding assays are shown, and Figure 1B The results of the EAR3 binding assay are shown. In both assays, three out of four serum samples from the human serum pool (HS pool) and the young adult group (20-29 years old) showed higher serum SHD catalytic antibody levels compared to three out of four serum samples from the older adult group (60-69 years old). This result suggests that a decrease in serum SHD catalytic antibodies may be an age-related event, and that serum levels of SHD catalytic antibodies can be used as a biomarker to predict protein aggregation disorders.

[0348] B. Correlation between catalytic antibody levels and Aβ-specific autoantibody levels

[0349] Next, two immunoassays were used to determine the levels of catalytic antibodies and Ab-specific autoantibodies in serum samples from healthy individuals (HS1-8) and Alzheimer's disease patients (ALZ1-5). In the EAR3 binding assay, the ELISA plate was coated with (EAR)3 peptide (SEQ ID NO:3) at a final concentration of 5 μg / mL. In the Aβ binding assay, the ELISA plate was coated with biotinylated amyloid (1-42) peptide at a final concentration of 1 μg / mL.

[0350] In short, each ELISA plate is coated with the corresponding peptide in coating buffer (0.2M sodium carbonate-bicarbonate buffer, pH 9.4), 100 μL / well, overnight at 4°C. The plate is washed three times with wash buffer (PBST). Then, the plate is blocked for 1 hour at room temperature with blocking buffer (10% fetal bovine serum), and washed three times with wash buffer. 100 μL of the corresponding serum sample (100 μg / mL) is added to each well of the plate, with PBS serving as a negative control. After incubation for 1 hour, the plate is washed three times with wash buffer and blocked for 1 hour at room temperature with blocking buffer. The plate is then washed three times with wash buffer. 100 μL of GAH-HRP (Abcam CAT No. ab98605), diluted 1:10,000, is added to each well of the plate, and the plate is incubated at room temperature for 1 hour and protected from light. HRP substrates (Amplex Red and H2O2) were freshly prepared using development buffer (3.6 mM Na2HPO4, 1.4 mM NaH2PO4, pH 7.2), and 100 μL of HRP substrate was added to each well and incubated for 1–5 minutes. The signal was then detected using a plate reader (excitation wavelength = 530 nm, emission wavelength = 590 nm, and cutoff wavelength = 570 nm).

[0351] Figure 2 The results of (EAR)3 and Aβ binding assays are shown. Compared to healthy individuals, four out of five AD patients had significantly reduced serum levels of catalytic antibodies and Aβ-specific autoantibodies. This result suggests that the combined reduction in serum SHD catalytic antibody and Aβ-specific autoantibody levels can serve as a biomarker for AD diagnosis.

[0352] Example 2. Design and characterization of anti-Aβ catalytic antibodies

[0353] This example describes the design and characterization of anti-Aβ catalytic antibodies based on 3D6 non-catalytic antibodies that specifically bind to Aβ.

[0354] A. 3D 6-D Design

[0355] Previous studies have shown that human IgM autoantibodies hydrolyze Aβ via a serine protease-like mechanism. Aβ in the brain and surrounding tissues are in equilibrium. Hydrolysis of peripheral Aβ can induce depletion of brain Aβ stores, while IgM does not cross the BBB. However, IgM mediates innate immune responses, typically has lower affinity for target antigens, and is more difficult to manufacture. Therefore, we sought to engineer IgG-catalyzed antibodies capable of hydrolyzing Aβ.

[0356] It is known that SHD catalytic antibodies possess a light chain variable region (V). L The SHD motif of ) . See Gao QS et al., “Site-directed mutagenesis of proteolytic antibody light chain”, Journal of Molecular Biology 253(5):658 (1995). Therefore, we will publish the V of several Aβ-specific IgG non-catalytic antibodies. L The sequence and the published catalytic antibody V L Sequence alignment. We found that, apart from having the "SHY" motif instead of "SHD", the mouse version of bepinizumab, 3D6, is similar to other published catalytic antibodies V. L Sequence, especially with resistance to UA15V L The sequences showed high homology (Planque SA et al., “Physiological IgM Class Catalytic Antibodies Selective for Transthyretin AMyoid”, *Journal of Biochemistry*, 289(19):13243-13258 (2014)). Anti-UA15 (catalytic antibody), anti-VP (catalytic antibody), and 3D6 (anti-Aβ non-catalytic antibody) V L The sequence alignment results are in Figure 3 Shown in.

[0357] By replacing V in 3D6 with D L The Y residue at position 1 was engineered to produce a 3D6-D antibody, which was then recombinantly expressed and purified. Figure 4 The images show the reducing and non-reducing gel electrophoresis of 3D6-D and 3D6 (i.e., 3D6-Y) antibodies and their humanized forms. The sequence of the 3D6-D catalytic antibody is shown in Table 2.

[0358] B. Catalytic activity of 3D6-D

[0359] The catalytic activity of the 3D6-D antibody was evaluated in an EAR-AMC catalytic function assay. In short, each well of the ELISA plate was coated with (100X, Bachem Americas, Cat. I-1575.0050), diluted 1:100. 3D6-D (400 ng / mL), 3D6-Y (400 ng / mL), trypsin (0.25% trypsin-EDTA, diluted 1:10,000), and EAR-AMC (100 μM) were each mixed with enzyme assay buffer (50 mM Tris-HCl, pH 7.7, 0.1 M glycine, 0.025% Tween-20) in PBS buffer. PBS buffer was used as a negative control, and IgG1 was used as an isotype control. The mixture was transferred to different wells of the ELISA plate, which was tightly sealed and incubated at 37°C for 20 or 68 hours. At the end of the incubation period, the signal was detected using a plate reader (excitation wavelength = 360 nm, emission wavelength = 470 nm, and cutoff wavelength = 455 nm). EAR-AMC is the substrate of the SHD-catalyzed antibody, which cleaves EAR-AMC at the covalent binding site between Arg and AMC, thereby releasing AMC.

[0360] The results of EAR-AMC catalytic function determination were in Figure 4 As shown in the figure, 3D6-D exhibits strong cleavage activity, but the wild-type 3D6 antibody (with Y residues) does not cleave the EAR-AMC substrate.

[0361] Aβ-binding of C.3D6-D

[0362] The binding of 3D6-, 3D6, or the allotype control (IgG1) to (EAR)3 and Aβ was determined using the EAR3 binding assay and Aβ binding assay, respectively, as described in Example 1. Each antibody was added to the wells of the ELISA plate at a concentration of 100 μg / mL. Figure 6 As shown, 3D6-D exhibits (EAR)3 and Aβ binding activity comparable to that of the 3D6 antibody, indicating that 3D6-D is a high-affinity IgG1 catalytic antibody against Aβ.

[0363] Humanization of D.3D6-D

[0364] To reduce the immunogenicity of 3D6-D catalytic antibodies, humanized catalytic antibodies were produced by transplanting the CDR of 3D6-D into the human antibody framework sequence. Further, a reversion mutation from the human antibody framework sequence to the original mouse sequence was introduced to maintain the affinity of the humanized antibody and promote further antibody development. In addition, SHD motifs and other residues that support the catalytic function of the SHD motif (e.g., Figure 3Reversion mutations to the original mouse sequence (residues marked with "#") and / or conformational structures that maintain the SHD motif. Exemplary humanized 3D6-D antibody sequences are shown in Table 2.

[0365] The binding affinity of the humanized 3D6-D catalytic antibody to Aβ was determined using an Aβ binding assay. In short, ELISA plates were coated with 1 μg / mL Aβ(1-42) peptide in A buffer (0.2 M sodium carbonate-bicarbonate buffer, pH 9.4), 100 μL / well, overnight at 4°C. The Aβ peptide was removed, and the plates were washed three times with 250 μL / well of wash buffer (PBST). The plates were then blocked for 1 hour at room temperature with 200 μL / well of blocking buffer (1% BSA in PBST), and washed twice with 250 μL / well of wash buffer. 100 μL of antibody sample (3X serial dilutions in the range of 3 ng / mL to 20 μg / mL) was added to each well of the plate and incubated at room temperature for 1 hour. The samples tested included 3D6-D, humanized 3D6-Y (bepinzumab), hu3D6-D H1L1, hu3D6-D H1L2, and 3D6-Y. The plates were then washed three times with 250 μL / well of wash buffer. 100 μL of goat anti-human IgG-HRP (Jackson Immun. Cat. No. 109-035-003), diluted 1:2,000 in assay buffer (0.1% BSA / PBST), was added to each well of the plate, and the plate was incubated at room temperature for 1 hour. The plates were then washed three times with 250 μL / well of wash buffer. Freshly prepared HRP substrate (Amplex Red and H2O2) was prepared using development buffer (3.6 mM Na2HPO4, 1.4 mM NaH2PO4, pH 7.2), and 100 μL of the HRP substrate was added to each well and incubated for 1–60 minutes. The signal was then detected using a board reader (excitation wavelength = 530nm, emission wavelength = 590nm, and cutoff wavelength = 570nm).

[0366] like Figure 7 As shown, humanized 3D6-D catalytic antibodies (hu3D6-D H1L1 and hu3D6-D H1L2) have binding affinity comparable to that of Aβ, such as bepinizumab.

[0367] Phage library panning for E.3D6-D variant

[0368] Aβ-selection of racial V with 3D6-D H and humanized V LA human scFv / Fab phage library was used to select either a humanized anti-Aβ catalytic scFv or Fab. The HC-CDR sequences in the phage library were randomized. scFv or Fab was selected based on high specificity for Aβ. The catalytic activity of the selected scFv and Fab was evaluated using the EAR-AMC catalytic function assay described in Example 2. The selected anti-Aβ scFv and Fab were used to prepare a full-length IgG (e.g., IgG1 or IgG4) catalytic antibody.

[0369] Example 3. Determining the levels of anti-Aβ autoantibodies and SHD catalytic antibodies in the serum of Alzheimer's disease (AD) patients.

[0370] This experiment was conducted to detect the levels of anti-Aβ autoantibodies and SHD catalytic antibody (recognition (EAR) 3-peptide) in the serum of Alzheimer's disease (AD) patients compared to healthy individuals.

[0371] The 30 AD serum samples used in this assay included 25 serum samples from newly diagnosed AD patients and 5 serum samples from older AD patients from Example 1. Eight healthy donor serum (HS) samples were obtained from young adults (20 to 29 years old; HS2, HS6, HS7, and HS8) and older adults (60 to 69 years old; HS1, HS3, HS4, and HS5), as used in Example 1. One pooled healthy donor serum sample served as a positive control (NHS; Innovative Study). PBS served as a negative control. A total of 40 test samples were centrifuged at 16,000 g for 10 minutes, and the supernatant was collected for ELISA assays. Each sample was tested twice.

[0372] In the EAR3 binding assay, the ELISA plate was coated with (EAR)3 (SEQ ID NO:3). The (EAR)3 stock solution (5 mg / mL) was diluted 100-fold with coating buffer (0.2 M sodium carbonate-bicarbonate buffer, pH 9.4) to a final concentration of 50 μg / mL, and 100 μL / well was coated overnight at 4°C. In the Aβ binding assay, the ELISA plate was coated with biotinylated amyloid (1-42) peptide at a final concentration of 2 μg / mL (1 mg / mL stock solution diluted 500-fold) in coating buffer (0.2 M sodium carbonate-bicarbonate buffer, pH 9.4), 100 μL / well, and incubated overnight at 4°C. The plate was washed three times each time with 200 μL of washing buffer (PBST: 0.1% Tween-20 in phosphate-buffered saline "PBS"). The plate was then blocked at room temperature with blocking buffer (1% BSA in PBST) for 2 hours, and washed three times each time with 200 μL of wash buffer (PBST). 100 μL of the corresponding test sample was added to each well of the plate, diluted 1:100 with PBS. All samples were tested in duplicate for each peptide target. See [link to relevant documentation] Figure 9A For loading design. After incubating overnight at 4°C, the plate was then washed three times each with 200 μL of wash buffer (PBST). 100 μL of a goat polyclonal antibody against human IgG conjugated to horseradish peroxidase (GAH-HRP; Abcam CAT No. ab98605), diluted 1:5000 in blocking buffer (1% BSA in PBST), was added to each well of the plate and incubated at room temperature for 1 hour, protected from light exposure. The plate was then washed three times each with 200 μL of wash buffer (PBST). Freshly prepared HRP substrate (Amplex Red and H2O2) was prepared using development buffer (3.6 mM Na2HPO4, 1.4 mM NaH2PO4, pH 7.2): 20 mL development buffer + 26.6 μL Amplex Red + 6.6 μL H2O2. 100 μL of the prepared HRP substrate was added to each well and incubated for 1–5 minutes. The signal was then detected using a plate reader (excitation wavelength = 530 nm, emission wavelength = 590 nm, and cutoff wavelength = 570 nm) at 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, and 60 minutes. The average readings from the pooled human serum and serum samples from 8 healthy donors served as controls. "Control %" was calculated as (readings from AD samples) divided by (average readings from the pooled human serum and serum samples from 8 healthy donors), see [link to relevant documentation]. Figure 8 . Figure 8 The tables and bar charts are based on Figures 9B-9C The readings are generated in the middle.

[0373] As from Figures 9B-9C As can be seen, the binding assays worked very well because the readings of the positive control (combined healthy human serum) were much higher than those of the PBS negative control (more than 80-fold for Aβ binding and more than 100-fold for (EAR)3 binding).

[0374] As from Figure 8 , 9B As can be seen from 9C, approximately 70% of AD patients had lower serum anti-Aβ autoantibody levels compared to controls, and approximately 86.7% of AD patients had lower serum SHD catalytic antibody levels compared to controls, which recognizes the EAR 3-peptide. These data suggest that the combined reduction of serum anti-Aβ autoantibody and SHD catalytic antibody can serve as a good biomarker for the diagnosis and prognosis of AD.

[0375]

[0376] sequence list <110> AB Studio Inc. <120> Catalytic antibodies and their usage <130> 79270-20001.40 <140> Not yet Assigned <141> Concurrently Herewith <150> US 62 / 765,150 <151> 2018-08-17 <160> 25 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Variants <222> 3 <223> Combined with 7-amino-4-methylcoumarin <400> 1 Glu Ala Arg 1 <210> 2 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Variants <222> (1)...(3) <223> It can exist in 1-30 repeating sequences <400> 2 Glu Ala Arg 1 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 3 Glu Ala Arg Glu Ala Arg Glu Ala Arg 1 5 <210> 4 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 4 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Asn Ser Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 5 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Ile Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 6 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 7 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 8 <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 8 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Ile Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 9 Asn Tyr Gly Met Ser 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 10 Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val Lys 1 5 10 15 Gly <210> 11 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 11 Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr 1 5 10 <210> 12 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 12 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 13 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 13 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 14 Trp Gln Gly Thr His Phe Pro Arg Thr 1 5 <210> 15 <211> 330 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 15 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 16 <211> 327 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 16 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 17 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 18 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 18 Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val 35 40 45 Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 19 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Ser Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 20 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Asn Ser Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 21 <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 21 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> twenty two <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty two Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Ile Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg <210> twenty three <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 23 Tyr Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Ile Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg <210> 24 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 24 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 25 <211> 113 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 25 Tyr Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg

Claims

1. An isolated anti-Aβ catalytic antibody, wherein the anti-Aβ catalytic antibody comprises: (i) an immunoglobulin heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:

4. H ) and the immunoglobulin light chain variable region (V) containing the amino acid sequence of SEQ ID NO:5 L (ii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:7 L ; or (iii) V containing the amino acid sequence of SEQ ID NO:6 H and V containing the amino acid sequence of SEQ ID NO:8 L .

2. The anti-Aβ catalytic antibody according to claim 1, wherein the anti-Aβ catalytic antibody cleaves a substrate having the formula EAR-AMC, wherein EAR is a peptide of the amino acid sequence EAR, wherein AMC is 7-amino-4-methylcoumarin, and wherein EAR binds to AMC.

3. The anti-Aβ catalytic antibody according to claim 1, wherein the anti-Aβ catalytic antibody is a full-length IgG antibody.

4. The anti-Aβ catalytic antibody according to claim 3, wherein the anti-Aβ catalytic antibody comprises an IgG1 or IgG4 Fc region.

5. The anti-Aβ catalytic antibody according to claim 1, wherein the anti-Aβ catalytic antibody is a full-length IgM antibody.

Images