Catabodies and methods of use thereof

Abstract

The present application provides methods, compositions and kits for determining SHD-catalyzed antibody levels in a biological sample and for treating or preventing protein aggregation disease (PAD) in an individual. Catalytic antibodies that specifically recognize amyloid beta (A beta) peptides and methods of using the same are also provided.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511451320.3 (22) Application Date 2019.08.16 (30) Priority Data 62 / 765,150 2018.08.17 US (62) Divisional Application Data 201980054508.0 2019.08.16 (71) Applicant AB Studio Co., Ltd. Address USA (72) Inventor Liu Yue (74) Patent Agency Beijing Zhucheng Law Firm 11313 Patent Attorneys Chen Yanjuan and Wang Yanbo (51) Int.Cl. C07K 16 / 18 (2006.01) C12N 9 / 00 (2006.01) G01N 33 / 564 (2006.01) G01N 33 / 68 (2006.01) (54) Title of Invention: Catalytic Antibody and Method of Use Thereof (57) Abstract This application provides methods, compositions, and kits for determining the level of SHD catalytic antibody in biological samples and for treating or preventing protein aggregation disease (PAD) in individuals. It also provides catalytic antibodies that specifically recognize amyloid β (Aβ) peptide and methods of using them. Claims (2 pages), Description (56 pages), Sequence Listing (electronic publication), Drawings (8 pages), CN 121248772 A 2026.01.02 CN 1 21 24 87 72 A 1. A method 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 the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30. 2. The method of claim 1, wherein n is 3. 3. The method of claim 1 or 2, wherein the biological sample is a serum sample. 4. The method of claim 3, wherein the serum sample contains at least about 1 μg / mL immunoglobulin (Ig). 5. The method of any one of claims 1 to 4, wherein the amount of the catalytic antibody-substrate peptide complex is determined using an antibody that specifically binds to total Ig. 6. The method of claim 5, wherein the antibody is labeled with an enzyme or a fluorescent label. 7. Use of a therapeutic catalytic antibody that specifically binds to a target protein in the preparation of a medicament for treating or preventing PAD in an individual, wherein the PAD is associated with the aggregation of the target protein, and the treatment or prevention comprises:a) determining the risk of the individual having the PAD using the method of any one of claims 1 to 6; and b) administering to the individual an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein. 8. Use of an anti-Aβ catalytic antibody in the preparation of a medicament for the treatment or prevention of Alzheimer's disease in an individual, comprising administering to the individual an effective amount of the anti-Aβ catalytic antibody. 9. An isolated anti-Aβ catalytic antibody comprising: a VL comprising the following: 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 a variant thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions; and / or a VH comprising the following: 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 a variant thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions; wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the Kabat EU index; or a VH comprising the following: comprising SEQ ID NO: 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; or VH containing the amino acid sequence of SEQ ID NO: 4 and VL containing the amino acid sequence of SEQ ID NO: 5; or VH containing the amino acid sequence of SEQ ID NO: 6 and VL containing the amino acid sequence of SEQ ID NO: 7; or VH containing the amino acid sequence of SEQ ID NO: 6 and VL containing the amino acid sequence of SEQ ID NO: 8; or VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 21; or VL containing the amino acid sequence of SEQ ID NO: 12. VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 21; or VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 21.VL containing the amino acid sequence of SEQ ID NO: 22; or VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 22. Claims 1 / 2 page 2 CN 121248772 A 10. A method for panning a phage library of a 3D6-D variant, the method comprising: panning a human scFv / Fab phage library having human lineage VH and humanized VL sequences of 3D6-D against Aβ to select a humanized anti-Aβ catalytic scFv or Fab; the HC-CDR sequences in the phage library are randomized; scFv or Fab is selected based on high specificity for Aβ; the catalytic activity of the selected scFv and Fab is evaluated using EAR-AMC catalytic function assay; and the selected anti-Aβ scFv and Fab are used to prepare a full-length IgG catalytic antibody. Claims 2 / 2 Page 3 CN 121248772 A Catalytic Antibody and Method of Using Thereof

[0001] This application is a divisional application of Chinese Patent Application No. 201980054508.0, filed on August 16, 2019, entitled “Catalytic Antibody and Method of Using Thereof”.

[0002] Cross-Reference to Related Applications

[0003] 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.

[0004] Sequence List Submitted in ASCII Text File

[0005] The following content, submitted in ASCII text file, is incorporated herein by reference in its entirety: a sequence list in computer-readable form (CRF) (filename: 792702000140SEQLIST .txt, record date: August 5, 2019, size: 22KB). Technical Field

[0006] 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) peptides. Background Art

[0007] Catalytic antibodies are antibodies that specifically bind to a target antigen and catalyze the chemical transformation of that target antigen. Proteolytic catalytic antibodies can hydrolyze and permanently inactivate the target peptide. Compared to conventional antibodies that bind stoichiometrically, a single catalytic antibody molecule can hydrolyze thousands of antigen molecules over its biological lifetime, thereby achieving enhanced efficacy. Catalytic antibodies have been found naturally in normal individuals and patients with autoimmune diseases. Catalytic antibodies have also been elevated 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 been widely developed as therapeutic agents.

[0008] 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.

[0009] All disclosures, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety. Summary of the Invention

[0010] This application provides compositions and methods for diagnosing (including methods for determining risk), treating, and preventing protein aggregation diseases (PADs) such as Alzheimer's disease (AD) in individuals.

[0011] 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 wherein 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.

[0012] In some embodiments of any 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 immunoglobulins (Ig; e.g., human Ig).

[0013] In some embodiments according to any of the methods described above, total immunoglobulins (Ig, ...) are used.The amount of antibody-substrate peptide complex catalyzed by an antibody that specifically binds to total human IgM is determined. 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] 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 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 the 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 a 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 antibody is determined by contacting an individual's serum sample 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 the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein 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).

[0015] In some embodiments of the methods for determining risk as described above, the method further includes determining the level of an autoantibody against a 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 a control SHD-catalyzed antibody; and (ii) the level of the autoantibody against the target protein is lower than the level of a control autoantibody. In some embodiments, the risk of PAD is determined by contacting the individual's serum sample with the target protein under conditions that allow the formation of an autoantibody-target protein complex and determining...The level of autoantibody is determined by the amount of the autoantibody-target protein complex. In some embodiments, an ELISA assay is used to determine the level of autoantibody. In some embodiments, the control autoantibody level is the level of autoantibody 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 autoantibody against the target protein in a population of individuals (e.g., the same age group).

[0016] In some embodiments of any of the methods for determining risk as described above, the method further includes determining the level of the target protein in an individual's biological sample (e.g., a serum sample or a cerebrospinal fluid sample), and wherein, as described on page 2 / 56 of the specification, if (i) the level of one or more SHD-catalyzing antibodies is lower than the control SHD-catalyzing antibody level; and (ii) the level of the target protein is higher than the control target protein level, then the individual is determined to have a PAD risk. 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 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 an individual population (e.g., the same age group).

[0017] 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).

[0018] 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 lobe 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...In the example, 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.

[0019] One aspect of this application provides a method for treating or preventing protein aggregation disease (PAD) in an individual, wherein PAD is associated with the aggregation of a target protein, the method comprising: a) determining that an individual has a 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.

[0020] 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 (VL) comprising: a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence SEQ ID NO: 12, an LC-CDR 2 comprising the amino acid sequence SEQ ID NO: 13, and an LC-CDR 3 comprising the amino acid sequence SEQ ID NO: 14, or a variant thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein the amino acid residue at position 1 of VL is D, the amino acid residue at position 27A of VL is S, and the amino acid residue at position 93 of VL is H, and wherein the numbering is based on the EU index of Kabat. In some embodiments, the therapeutic catalytic antibody comprises a heavy chain variable region (VH) comprising: a heavy chain complementarity-determining region (HC-CDR) 1 comprising the amino acid sequence SEQ ID NO: 9, an HC-CDR 2 comprising the amino acid sequence SEQ ID NO: 10, and an HC-CDR 3 comprising the amino acid sequence SEQ ID NO: 11, or a variant 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 amino acid residue at position 26 of the VL is S, the amino acid residue at position 27D of the VL is D, E, or H, and / or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is based on the Kabat EU index. In some embodiments, the therapeutic 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 therapeutic catalytic antibody comprises: an amino acid sequence having at least about 85% sequence identity (e.g., at least about 90%) with respect to SEQ ID NO: 5, 7, 8, 21, or 22.Page 3 / 56 of the manual, CN 121248772 A, describes a VL containing an amino acid sequence of 95%, 97%, 99%, or 100%. In some embodiments, the therapeutic catalytic antibody comprises: a VH containing an amino acid sequence selected from SEQ ID NO: 4, 6, 19, and 20, and a VL containing an amino acid sequence selected from SEQ ID NO: 5, 7, 8, 21, and 22. In some embodiments, the therapeutic catalytic antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 21; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 22; or (vii) a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the therapeutic catalytic antibody is a full-length IgG antibody. In some embodiments, the therapeutic catalytic antibody comprises an IgG1 or IgG4 Fc region. In some embodiments, the therapeutic catalytic antibody is a full-length IgM antibody.

[0021] Another aspect of this application provides an isolated anti-Aβ catalytic antibody comprising: a VL comprising: 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, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the EU index of Kabat. In some embodiments, the anti-Aβ catalytic antibody comprises: VH comprising the following: 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 VH comprising the amino acid sequence of SEQ ID NO: 9.HC-CDR3, or a variant thereof, having an amino acid sequence of SEQ ID NO: 9, comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR. In some embodiments, the isolated anti-Aβ catalytic antibody comprises: a VH comprising: 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; and a VL comprising: 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.

[0022] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: VH comprising: 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 the amino acid residue at position 1 of VL is D, the amino acid residue at position 27A of VL is S, and the amino acid residue at position 93 of VL is H, and wherein the numbering is based on the EU index of Kabat. In some embodiments, the isolated anti-Aβ catalytic antibody comprises: a VL comprising: 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 a variant 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: a VH comprising: 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; and a VL comprising: 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.

[0023] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: VH comprising the following: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, comprising SEQ ID NO: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 the amino acid residue at position 1 of VL is D, the amino acid residue at position 27A of VL is S, and the amino acid residue at position 93 of VL is H, and wherein the numbering is based on the EU index of Kabat.

[0024] 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).

[0025] In some embodiments of any of the anti-Aβ catalytic antibodies described above, the amino acid residue at position 26 of VL is S, the amino acid residue at position 27D of VL is D, E, or H, and / or the amino acid residue at position 28 of VL is D or N, and wherein the numbering is based on the EU index of Kabat.

[0026] In some embodiments of any 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 VL 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. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising an amino acid sequence selected from SEQ ID NO: 4, 6, 19, and 20, and a VL comprising an amino acid sequence selected from SEQ ID NO: 5, 7, 8, 21, and 22. In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 4;(v) VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 21; (vi) VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 21; (vi) VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 22; or (vii) VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 22.

[0027] In some embodiments of any 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 contains an IgG1 or IgG4 Fc region. In some embodiments, the anti-Aβ catalytic antibody is a full-length IgM antibody.

[0028] 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 an anti-Aβ catalytic antibody according to any of the anti-Aβ catalytic antibodies described above.

[0029] Methods for preparing and using anti-Aβ catalytic antibodies are also provided, as well as kits and articles suitable for any of the methods described above.

[0030] In some embodiments, a kit for treating or preventing Alzheimer's disease in an individual is provided, comprising: a) a substrate peptide comprising the amino acid sequence (EAR)n (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. Brief Description of the Drawings

[0031] Figure 1A illustrates the levels of catalytic antibodies in serum samples from young (20–29 years) and older (60–69 years) adults as determined by EAR-AMC binding assay.

[0032] Figure 1B shows the levels of catalytic antibodies in serum samples from young (20–29 years) and older (60–69 years) adults, as determined by EAR3 binding assay, on page 5 / 56 of the instruction manual, 8 CN 121248772 A.

[0033] Figure 2 shows the levels of catalytic antibodies and Aβ-specific autoantibodies in healthy (HS1–HS8) individuals or patients with Alzheimer’s disease (ALZ-1–5).

[0034] Figure 3 shows the alignment of the VL sequences of two catalytic antibodies (anti-UA15 and anti-VP) and the non-catalytic anti-Aβ antibody 3D6.Amino acid residues corresponding to the SHD motif are marked with an asterisk (*). Amino acid residues that support the catalytic function of the catalytic antibody are marked with a hash (#).

[0035] Figure 4 shows SDS gel images of various purified 3D6-derived catalytic antibodies under non-reducing and reducing electrophoresis conditions. 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).

[0036] Figure 5 shows the catalytic function of 3D6-D as determined using EAR-AMC substrates.

[0037] Figure 6 shows the binding of 3D6-D to (EAR)3 (SEQ ID NO: 3) and Aβ.

[0038] Figure 7 shows the binding of the humanized 3D6 catalytic antibody to Aβ.

[0039] Figure 8 shows the levels of anti-Aβ autoantibodies and SHD catalytic antibodies (EAR3) in the serum of 30 Alzheimer's disease (AD) patients as 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 of AD samples) divided by (average readings of pooled human serum and 8 healthy donor serum). A "+" in the table indicates that the serum level of anti-Aβ autoantibodies or SHD catalytic antibodies in the serum of AD patients is higher than that in the serum of healthy donors and pooled serum samples. A "-" in the table indicates that the serum level of anti-Aβ autoantibodies or SHD catalytic antibodies in the serum of AD patients is lower than that in the serum of healthy donors and pooled serum samples.

[0040] Figure 9A depicts an ELISA design for detecting levels of anti-Aβ autoantibodies and SHD catalytic antibodies (recognition (EAR)3) in serum samples from 30 Alzheimer's disease (AD) patients, 8 healthy donors, and combined healthy donor serum samples. PBS served as a negative control. Figure 9B depicts a plate reading the Aβ ELISA binding assay at the 5-minute time point. Figure 9C depicts a plate reading the (EAR)3 ELISA binding assay at the 1-minute time point. Detailed Description

[0041] In one aspect, this application provides a method for diagnosing and treating protein aggregation diseases (PAD) using a newly developed immunoassay for determining SHD catalytic antibody levels in biological samples. This application is based in part on the finding that SHD catalytic 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, when the disease shows obvious pathological features in imaging analysis, neurological damage has already occurred. At this stage, even if therapeutic agents can prevent the disease...Further developments are needed, but the therapeutic agents are unlikely to reverse nerve damage. This invention allows for the effective diagnosis and treatment of PAD (such as AD) at an early stage using SHD catalytic antibody levels as an early diagnostic biomarker.

[0042] Furthermore, using antibody engineering, catalytic antibodies that specifically recognize and cleave Aβ have been designed based on the parent mouse antibody of the non-catalytic anti-Aβ antibody 3D6, bepinizumab. Bepinizumab has shown safety at low doses, but two large phase III clinical trials showed 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 (e.g., bepinizumab) can penetrate the BBB and exacerbate neuroinflammation. Compared to bepinizumab, the anti-Aβ catalytic antibodies described herein have enhanced efficacy at low doses due to their 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 antibodies described herein to prevent AD. Specification 6 / 56 pages 9 CN 121248772 A

[0043] 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 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 the 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 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 wherein n is an integer between 1 and 30. In some embodiments, the method further includes determining the level of an autoantibody against a 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 a control SHD-catalyzing antibody; and (ii) the level of an autoantibody against the target protein is lower than the level of a control autoantibody, then the individual is determined to have a risk of PAD.

[0044] 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-catalyzing antibodies in an individual's biological sample (e.g., a serum sample), wherein if the level of one or more SHD-catalyzing antibodies is lower than the level of a control SHD-catalyzing antibody.The steps include: (i) determining the level of an individual's autoantibody against the target protein; and (ii) administering an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein (e.g., Aβ) to the individual. 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 wherein n is an integer between 1 and 30. In some embodiments, step a) further comprises determining the level of an autoantibody against the target protein in the individual's biological sample (e.g., serum 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 an autoantibody against the target protein is lower than the level of a control autoantibody, then the individual is determined to have a risk of PAD.

[0045] Another aspect of this application provides an isolated anti-Aβ catalytic antibody comprising: a light chain variable region (VL) comprising: a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR 2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR 3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the EU index of Kabat. In some embodiments, the anti-Aβ catalytic antibody comprises: a heavy chain variable region (VH) comprising: a heavy chain complementarity-determining region (HC-CDR) 1 comprising the amino acid sequence SEQ ID NO: 9, an HC-CDR 2 comprising the amino acid sequence SEQ ID NO: 10, and an HC-CDR 3 comprising the amino acid sequence SEQ ID NO: 11, or a variant thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions.

[0046] Compositions (such as pharmaceutical compositions), kits, and articles thereof for the diagnosis, treatment, or prevention of PAD (e.g., AD) are also provided.

[0047] I. Definitions

[0048] As used herein, “treatment or treating” is a method for obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, a beneficial or desired clinical outcome includes, but is 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 prolongation of disease).Delaying the worsening of the disease); preventing or delaying the spread of the disease; preventing or delaying the recurrence of the disease; delaying or slowing the progression of the disease; improving the disease state; providing disease remission (partial or complete remission); reducing the dosage of one or more other drugs required to treat the disease; delaying the progression of the disease; increasing or improving the quality of life; increasing weight gain; and / or prolonging survival. Specification 7 / 56 pages 10 CN 121248772 A The method of the present invention contemplates any one or more of these aspects of treatment.

[0049] The term "antibody" includes full-length antibodies and their antigen-binding fragments. Full-length antibodies comprise two heavy chains and two light chains. Variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains generally contain three highly variable loops, referred to as 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-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 divided 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).

[0050] 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 a complete antibody structure. Antigen-binding fragments can bind to parental antibodies or parental antibody fragments.(e.g., parental scFv) binding to the same antigen. In some embodiments, the antigen-binding fragment may comprise one or more CDRs from a particular human antibody that are transplanted into a frame region from one or more different human antibodies.

[0051] As used herein, the term “epitope” refers to a specific set of atoms or amino acids on an antigen to which an antibody binds. If two antibodies competitively bind to an antigen, then they may bind to the same epitope of said antigen.

[0052] 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 this target with greater affinity, affinity, ease, and / or a longer 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 that of its binding affinity to other targets.

[0053] 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 degrading enzymes.

[0054] As used herein, the term “SHD catalytic antibody” refers to a proteolytic catalytic antibody having an SHD motif in the variable region (VL) of the light chain. An “SHD motif” refers to serine, histidine, and aspartic acid residues that together act as a catalytic triplet to catalyze the cleavage of peptide bonds by the antibody. In some embodiments, the SHD motif is a nonlinear motif in which the serine, histidine, and aspartic acid residues of the catalytic triplet are not adjacent to each other in the amino acid sequence.

[0055] As used herein, an “isolated” antibody means (1) that is not associated with a protein found in nature, (2) that has no other protein, (3) that is expressed by cells of a different species, or (4) that does not exist in nature.

[0056] 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, in which, due to its origin, “isolated nucleic acid” (1) is associated with all or part of the polynucleotides of all “isolated nucleic acids” found in nature, (2) is operatively linked with polynucleotides not linked in nature, or (3) does not exist in nature as part of a larger sequence.

[0057] As used herein, the term “CDR” or “complementarity-determining region” is intended to mean within the variable region of the heavy and light chain polypeptides.Discontinuous antigen combination sites were discovered. 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), where the definitions include overlaps or subsets of amino acid residues when compared with each other. However, the application of any definition of a CDR relating to an antibody or a transplanted antibody or a variant 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.

[0058]

[0059] 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 they exhibit the biological activity of the present invention (see U.S. Patent No. 4,816,567; and Morrison et al., *The United States...*).Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 81:6851-6855 (1984).

[0060] "Fv" is 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 rings each from the heavy chain and light chain) emanate from the folding of these two domains, which contribute amino acid residues for antigen binding and confer antigen-antibody binding specificity. However, even a single variable domain (or half of the specification of Fv containing only three CDRs specific to the antigen, page 9 / 56, 12 CN 121248772 A) has the ability to recognize and bind antigens, but the affinity is lower than that of a complete binding site.

[0061] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment containing VH and VL antibody domains linked together to form a single polypeptide chain. In some embodiments, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure 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).

[0062] The term “bifunctional antibody” refers to a small antibody fragment prepared by constructing an scFv fragment (see preceding paragraph), which typically has a short linker (e.g., about 5 to about 10 residues) between the VH and VL domains, enabling inter-chain but not intra-chain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific bifunctional antibodies are heterodimers of two “crossed” scFv fragments, wherein the VH and VL domains of the two antibodies 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).

[0063] 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, humanized antibodies are residues from the acceptor hypervariable region (HVR) derived from a non-human species (donor antibody) with the desired antibody specificity, affinity, and ability, such as mice, rats,Human immunoglobulins (receptor antibodies) are made by replacing residues of the hypervariable region of a rabbit or non-human primate. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced with 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, humanized antibodies will contain at least one, and typically substantially all, of the two variable domains, wherein 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 having the human immunoglobulin sequence. Humanized antibodies optionally also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. 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).

[0064] The “percentage of amino acid sequence identity (%)” or “homology” of the polypeptide 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 compared polypeptide 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 the appropriate parameters to be used for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this document, sequence consistency values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0065] 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, FcγRIIII, and FcγRIIV subclasses, including allelic variants of these receptors and alternative splicing forms. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibitory receptor”), which have 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. Daëron, Annals of Immunology 15:203-234 (1997) for a review.) The terminology includes allotypes such as Fcγ RIIIA allotypes: Fcγ RIIIA-Phe158, Fcγ RIIIA-Val158, Fcγ RIIA-R131, and / or Fcγ RIIA 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" herein. The term also includes the 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)).

[0066] 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 is non-covalently bound 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 be used as a rescue receptor to bind and transport intact IgG intracellularly and transcellularly and to rescue it from the default degradation pathway.

[0067] The "CH1 domain" (also known as the "C1" domain of "H1") in the human IgG Fc region typically extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0068] The “hinge region” is generally defined as the elongation 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 S-S bond.

[0069] The “CH2 domain” (also known as the “C2” domain of “H2”) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in 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 speculated that carbohydrates can provide alternatives for domain-domain pairing and contribute to the stability of the CH2 domain. Burton, Molecular Immunology 22:161-206 (1985).

[0070] The “CH3 domain” (also referred to as the “C2” or “H3” domain) comprises an elongation from the C-terminal residue to the CH2 domain in 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).

[0071] The “functional Fc fragment” has an “effector function” of the native sequence Fc region. Exemplary “effector functions” include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phage activity; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region in combination with a binding domain (e.g., antibody variable domain) and can be evaluated using a variety of assays well known in the art.

[0072] 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 sequence Fc region. Variant Fc exhibiting “increased binding” to FcRs binds to at least one FcR with a higher affinity (e.g., lower apparent Kd or IC50 value) than the parent peptide or native sequence 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. Peptide variants exhibiting “decreased binding” to FcRs bind to at least one FcR with a lower affinity (e.g., higher apparent Kd or higher IC50 value) than the parent peptide. Compared to the parent peptide, the binding reduction can be approximately 40% or more.

[0073] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which the secretion of Ig (as described in the manual, page 11 / 56, 14 CN 121248772 A) binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages). This allows 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 FcγRIIII, while monocytes express FcγRI, FcγRII, and FcγRIIII. Ravetch and Kinet, Annu, page 464, Table 3 summarizes FcR expression on hematopoietic cells. Immunol 9: 457-92 (1991). To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as the assay described in U.S. Patent Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays 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 (PNAS (USA)), 95:652-656 (1998).

[0074] A peptide containing a variant Fc region that “exhibits increased ADCC” or mediates ADCC more effectively than a peptide or parent peptide having wild-type IgG Fc in the presence of human effector cells is a peptide that mediates ADCC substantially more effectively in vitro or in vivo when the amount of the peptide having the variant Fc region is substantially the same as the amount of the peptide (or parent peptide) having 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, variants mediate ADCC about 5 to about 100 times more effectively than wild-type Fc (or parental peptide), for example about 25 to about 50 times.

[0075] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. The binding of the first component (C1q) of the complement system to an antibody (belonging to the appropriate subclass) bound to a homologous antigen initiates activation of the classical complement pathway. To assess complement activation, a CDC assay can be performed, for example, as in Gazzano-Santoro et al., *Journal of Immunological Methods* 202:163 (1996). Changes in the amino acid sequence of the Fc region and increased or decreased C1q binding capacity...The polypeptide variants 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).

[0076] 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.

[0077] The term “operably linked” refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence that causes the latter to be expressed. For example, the first nucleic acid sequence is operably linked to the second nucleic acid sequence when they are in a functional relationship. For example, the promoter is operably linked to the coding sequence if it affects the transcription or expression of the coding sequence. Generally, operatively ligated DNA sequences are contiguous and, if necessary, two protein-coding regions are joined in the same reading frame.

[0078] “Homology” refers to sequence similarity or sequence consistency between two polypeptides or two nucleic acid molecules. The molecules are homologous at said positions when positions in both compared sequences are occupied by the same base or amino acid monomer subunit, for example, if adenine occupies each position in two DNA molecules. 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 compared positions by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, two sequences are compared when aligned to obtain maximum homology.

[0079] The “effective amount” of an antibody or composition as disclosed herein is an amount sufficient to carry out the purpose specifically stated. The “effective amount” can be determined empirically and by known methods associated with said purpose. Instructions for Use, Page 12 / 56, 15 CN 121248772 A

[0080] As used herein, “pharmaceutical acceptable” or “pharmaceutical compatible” means not a biologically or otherwise undesirable material, 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 formulations approved by the U.S. Food and Drug Administration (FDA).In the Inactive Ingredient Guide prepared by the administration.

[0081] It should be understood that the embodiments of the invention described herein include embodiments that are “composed of” and / or “composed primarily of”.

[0082] A reference to “about” a value or parameter herein includes (and describes) a variation of said value or parameter itself. For example, a description of “about X” includes a description of “X”.

[0083] As used herein, a reference to “not” a value or parameter generally means and describes “not” a value or parameter.

[0084] Unless the context otherwise clearly specifies, the singular forms “a,” “or,” and “the” as used herein and in the appended claims include a plurality of indicators.

[0085] II. Methods for Diagnosis and Treatment

[0086] This application provides methods for determining the level of one or more catalytic antibodies (e.g., SHD catalytic antibodies) in a sample, and methods for diagnosing (including determining risk), treating, or preventing protein aggregation disease (PAD) in an individual. In some embodiments, the level of one or more catalytic antibodies (e.g., SHD catalytic antibodies) is the protein level of one or more catalytic antibodies. In some embodiments, the level of one or more catalytic antibodies is the mRNA level of one or more catalytic antibodies. In some embodiments, the methods described herein use substrate peptides 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 level of one or more catalytic antibodies in the sample. In some embodiments, the level of one or more catalytic antibodies is the level of total catalytic antibodies, such as the level of total SHD catalytic antibodies. In some embodiments, the level of one or more catalytic antibodies is the level of one or more (e.g., 1, 2, 3, 4 or more) catalytic antibodies that specifically bind and cleave PAD-associated target proteins (e.g., Aβ) (e.g., the level of total SHD catalytic antibodies).

[0087] In some embodiments, the level of total SHD catalytic antibodies is determined based on the binding of the catalytic antibody to a substrate peptide comprising the amino acid sequence (EAR)n (SEQ ID NO: 2), 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 SEQ ID NO: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, in addition to the amino acid sequence of SEQ ID NO: 2, the substrate peptide comprises 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 EAR-AMC (SEQ ID NO: 1).

[0088] 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 catalytic antibodies, a substrate peptide comprising the amino acid sequence of SEQ ID NO: 2 has not been used to measure binding activity and the level of catalytic antibodies in biological samples. Whether the binding between the EAR peptide and the SHD catalytic antibody is strong and durable enough to allow accurate determination of the level of SHD catalytic antibodies via binding assay is also unpredictable. This application provides an immunoassay using a substrate peptide comprising the amino acid sequence of SEQ ID NO: 2, which provides an accurate reading of the level of total SHD catalytic antibodies in a biological sample (e.g., a serum sample).

[0089] 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 the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein 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.

[0090] 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 (EAR)n (SEQ ID NO: 2), and wherein 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.

[0091] 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 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 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.

[0092] 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 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) 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.

[0093] In some embodiments, a method is provided for determining the risk of an individual for protein aggregation disease (PAD), wherein PAD is associated with the aggregation of target proteins, the method comprising determining the level of one or more SHD catalytic antibodies in an individual's biological sample (e.g., a serum sample), wherein if the level of one or more SHD catalytic antibodies is lower than that of a control SHD catalytic antibody.The level of one or more SHD catalytic antibodies determines the risk of an individual having 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 an 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 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 Huntington's protein; (viii) PAD is type II diabetes and the target protein is IAPP; and (ix) PAD is ALS and the target protein is SOD1.

[0094] In some embodiments, a method for determining the risk of 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 allowing 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, and 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.

[0095] In some embodiments, a method for determining the risk of PAD in an individual is provided, 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 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, then the individual is determined to have a risk of PAD. In some embodiments, the level of one or more SHD catalytic antibodies is one or more SHD catalytic antibodies that specifically bind to the target protein.The level of one or more SHD catalytic antibodies is defined as the level of total SHD catalytic antibodies. In some embodiments, the level of total SHD catalytic antibodies is determined by contacting an individual's serum sample with a substrate peptide under conditions allowing the formation of an autoantibody-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 described herein for determining SHD catalytic antibody levels. In some embodiments, the level of autoantibodies is determined by contacting an individual's serum sample with a target protein (or fragments thereof) under conditions allowing the formation of an autoantibody-target protein complex 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.

[0096] 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, any of the descriptions of the methods for determining SHD catalytic antibody levels described herein are used.Page 15 / 56, CN 121248772 A: Determining the level of one or more SHD-catalyzed antibodies. 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 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 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.

[0097] In some embodiments, a method is provided for determining the risk of 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 allowing 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 an autoantibody against the target protein in a biological sample (e.g., a serum sample) of the individual, 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 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 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 (or fragments thereof) under conditions that allow for the formation of autoantibody-target protein complexes and by 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 Huntington's protein; (viii) PAD is type II diabetes and the target protein is IAPP; and (ix) PAD is ALS and the target protein is SOD1.

[0098] In some embodiments, a method is provided for determining the risk of 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 allowing 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 a biological sample of the individual (e.g., a serum sample or a cerebrospinal fluid sample), 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 solid support is an ELISA plate. In some embodiments, the level of the target protein is determined by contacting a biological sample of the individual with an antibody against the target protein under conditions allowing 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.

[0099] In some embodiments, a method is provided for determining the risk of PAD in an individual, wherein PAD is associated with the aggregation of a target protein, the method comprising: a) subjecting serum from the individual to conditions that allow the formation of a catalytic antibody-substrate peptide complex.The sample is contacted with a substrate peptide immobilized on a solid support, 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) 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, 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) the level of autoantibodies against the target protein is determined in the 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 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 an individual's serum sample with a target protein (or fragment thereof) under conditions that allow for the formation of an autoantibody-target protein complex 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.

[0100] 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 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 providingThe levels of one or more SHD-catalyzing antibodies in an individual's serum; and c) determining the levels of the target protein in an individual's biological sample (e.g., a serum sample or a cerebrospinal fluid sample), wherein the risk of the individual having PAD is determined if: (i) the levels of one or more SHD-catalyzing antibodies are lower than the levels of control SHD-catalyzing antibodies; and (ii) the levels of the target protein are higher than the levels of control target protein. In some embodiments, the solid support is an ELISA plate. 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 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 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.

[0101] In some embodiments, a method for determining an individual's risk of Alzheimer's disease (AD) is provided, comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO: 3, under conditions allowing the formation of a catalytic antibody-substrate peptide complex; 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 autoantibody-Aβ. The amount of the complex, thereby providing the level of autoantibodies against Aβ; and wherein 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 Aβ is lower than the level of control autoantibodies, then an individual is determined to have the risk of AD. In some embodiments, antibodies that specifically bind to Ig are used with an enzyme (e.g.,HRP (hydrolyzable plasma) or fluorescent labeling (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.

[0102] 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, 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 that allow 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 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, 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 Aβ.

[0103] Any of the diagnostic methods described herein can be used to inform the use of any known therapeutic agent for treating PAD in the art or any catalytic antibody (e.g., anti-Aβ catalytic antibody) described herein to treat PAD. The diagnostic methods described herein allow for early detection of an individual's risk of PAD, thereby allowing for early intervention and preventative treatment of PAD.

[0104] Thus, 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 that an individual has a risk of 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.

[0105] In some embodiments, a method for treating or preventing PAD in an individual, 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 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.

[0106] In some embodiments, a method of treating or preventing AD in an individual includes: a) determining the risk of an individual having AD according to any of the risk determination methods described herein; and b) administering 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.

[0107] In some embodiments, a method of treating or preventing PAD in an individual, wherein PAD is associated with the aggregation of a target protein, the method includes: 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 risk of the individual having PAD is determined; and b) administering an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein to the individual. 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 an individual's serum sample with the 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) 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 no more than about every three months, for example about every three months, about every six months, or about every year.

[0108] In some embodiments, a method of providing treatment or prevention of AD in an individual comprises: 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; and c) if one or more SHD catalytic antibodies...If the level of the anti-Aβ catalytic antibody is lower than the level of the control SHD catalytic antibody, then an effective amount of the anti-Aβ catalytic antibody is administered to the individual. 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.

[0109] 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 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) if: (i) the level of one or more SHD catalytic antibodies is lower than the level of the control SHD catalytic antibody; and (ii) the level of the autoantibody against the target protein is lower than the level of the control autoantibody, then an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein is administered to the individual. 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 antibody is determined by contacting an individual's serum sample with a substrate peptide under conditions that allow for the formation of a catalytic antibody-substrate peptide complex and by 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 autoantibody is determined by contacting an individual's serum sample with a target protein under conditions that allow for the formation of an autoantibody-target protein complex and by determining the amount of the autoantibody-target protein complex. In some embodiments, an ELISA assay is used to determine the level of autoantibody. 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.

[0110] 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) subjecting a serum sample of the individual to conditions that allow the formation of a catalytic antibody-substrate peptide complex.The method involves contacting a substrate peptide immobilized on a solid support, 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, page 19 / 56, CN 121248772 A); and d) 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 autoantibodies against the target protein is lower than the level of a control autoantibody. 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, such as total IgM, total IgG, total IgA, and / or total IgE). 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, the level of autoantibodies is determined by contacting an individual's serum sample 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. In some embodiments, an ELISA assay is used to determine the level of autoantibodies. 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.

[0111] 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 the amino acid sequence of SEQ ID NO: 3; b) using a serum sample with total Ig (e.g., total human Ig)The amount of the catalytic antibody-substrate peptide complex is determined using an antibody that specifically binds to Ig (e.g., 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) the individual's serum sample is contacted with Aβ (e.g., Aβ(1-42)) under conditions that allow for the formation of an autoantibody-Aβ complex; d) the amount of the autoantibody-Aβ complex is determined 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 a control SHD catalytic antibody; and (ii) the level of autoantibodies against Aβ is lower than the level of a control autoantibody, then an effective amount of anti-Aβ catalytic antibody is administered to the individual. 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 at a frequency of no more than about three months, such as about three months, about six months, or about one year.

[0112] 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 (VL) comprising: a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR 2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR 3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein the amino acid residue at position 1 of VL is D, and ... The amino acid residue at position 27A is S, and the amino acid residue at position 93 of VL is H, wherein the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a heavy chain variable region (VH) comprising: a heavy chain complementarity-determining region (HC-CDR) 1 comprising the amino acid sequence SEQ ID NO: 9, an HC-CDR 2 comprising the amino acid sequence SEQ ID NO: 10, and an HC-CDR 3 comprising the amino acid sequence SEQ ID NO: 11, or a variant thereof, wherein the HC-CDR contains at most 10 amino acids.Page 23 of document CN 121248772 A, approximately 5 amino acid substitutions. In some embodiments, the amino acid residue at position 26 of VL is S, the amino acid residue at position 27D of VL is D, E, or H, and / or the amino acid residue at position 28 of VL is D or N, and wherein the numbering is based on the EU index of Kabat. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19, or 20; and / or a VL comprising 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. In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 21; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 22; or (vii) a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 22. 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 at a frequency not exceeding about three months, for example about three months, about six months, or about one year.

[0113] In some embodiments, a method of providing treatment or prevention of AD in an individual comprises: 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 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 the individual with a serum sample with a substrate peptide immobilized on a solid support under conditions that allow the formation of an autoantibody-Aβ complex.The individual's serum sample is contacted with Aβ (e.g., Aβ(1-42)), d) the amount of autoantibody-Aβ complex is determined 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 autoantibody against Aβ; and e) if: (i) the level of one or more SHD-catalyzing antibodies is lower than the level of control SHD-catalyzing antibodies; (ii) the level of autoantibody against Aβ is lower than the level of control autoantibody, then an effective amount of anti-Aβ-catalyzing antibody is administered to the individual, wherein the anti-Aβ-catalyzing antibody comprises: a VL comprising: a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR 2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR 3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof, which comprises up to about 5 amino acid substitutions in the LC-CDR, wherein at position 1 of the VL The amino acid residue at position 26 of VL is D, the amino acid residue at position 27A of VL is S, and the amino acid residue at position 93 of VL is H, wherein the numbering is based on the Kabat EU index; and VH comprising: 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, the amino acid residue at position 26 of VL is S, the amino acid residue at position 27D of VL is D, E, or H, and / or the amino acid residue at position 28 of VL is D or N, wherein the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19, or 20; and / or a VL comprising 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. In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 19.(v) VL containing the amino acid sequence of SEQ ID NO: 21; (vi) VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 22; or (vii) VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 22. 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 at a frequency of no more than about three months, such as about three months, about six months, or about one year.

[0114] The methods described herein detect the levels of one or more SHD-catalyzing antibodies, autoantibodies against target proteins, including but not limited to protein levels and mRNA levels. Protein levels can be detected using immunoassays, mass spectrometry, or other molecular biology techniques. mRNA levels can be detected using quantitative PCR or other molecular biology techniques.

[0115] In some embodiments, the method includes detecting the levels 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 levels of one or more SHD-catalyzing antibodies are the levels of total SHD-catalyzing antibodies. In some embodiments, the levels of one or more SHD-catalyzing antibodies are the levels of one or more SHD-catalyzing antibodies that specifically bind to the target protein. In some embodiments, the levels of one or more SHD-catalyzing antibodies that specifically bind to the target protein are 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.

[0116] 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.

[0117] 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.

[0118] The levels of one or more catalytic antibodies, autoantibodies against the target protein, and 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 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.

[0119] 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 are 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, a serum sample is used to determine the levels of one or more SHD catalytic antibodies, and a CSF sample is used to determine the levels of Aβ.

[0120] 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 of the values ​​greater than about 500, 400, 300, 250, 200, or 150 μg / mL Ig.

[0121] 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 of the values ​​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 of the values ​​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 the substrate peptide or target protein (or a fragment thereof, such as Aβ) for no more than any 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 the 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 with the substrate peptide or target protein (or a fragment thereof, such as Aβ) overnight. In some embodiments, the incubation is performed at room temperature. In some embodiments, the incubation is performed at 4°C.

[0122] In some embodiments, the biological sample is incubated with the substrate peptide or target protein (or a fragment thereof, such as Aβ) for...Afterward, the solid support (e.g., an ELISA plate) is washed with a washing buffer, such as PBS or PBST (e.g., three times). 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 with a washing buffer, such as PBS or PBST (e.g., three times).

[0123] The amount of catalytic antibody-substrate peptide complex or autoantibody-target protein complex can be detected using antibodies that specifically bind to species-specific immunoglobulin molecules (e.g., 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. Antibodies that specifically bind to Ig can be labeled with an enzyme (e.g., HRP) for detection using enhanced chemiluminescence (ECL) substrates. Alternatively, antibodies that specifically bind to Ig can be labeled with a fluorescent label (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.

[0124] 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); Mruthinti 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., “Targets β-amyloid peptides…”Autoantibodies against beta-amyloid are common in Alzheimer's disease and help control plaque burden, Ann. Neurol. 65:24-31 (2009); Britschgi M, Olin CE, Johns HT, Takeda-Uchimura Y et al. "Neuroprotective innate antibodies against peptide assembly of amyloid protein 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 a target protein (e.g., Aβ).

[0125] The levels of a target protein (e.g., Aβ) in a biological sample (e.g., a serum sample or a brain or spinal cord sample) can be determined using ELISA assays or liquid chromatography / tandem mass spectrometry.

[0126] In some embodiments, the levels of one or more SHD-catalyzing antibodies are compared with the levels of control SHD-catalyzing antibodies. In some embodiments, the levels of one or more SHD-catalyzing antibodies are compared with 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 with 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.

[0127] In some embodiments, the levels of autoantibodies against a target protein (e.g., Aβ) are compared with the levels of control autoantibodies. In some embodiments, the levels of autoantibodies against a target protein (e.g., Aβ) are compared with the levels of autoantibodies against a target protein (e.g., Aβ) in a control sample. In some embodiments, the levels of autoantibodies against a target protein (e.g., Aβ) are compared with the levels 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 one or more SHD-catalyzing antibodies against a target protein (e.g., Aβ) in a group of individuals (e.g., healthy individuals, etc.). The levels of autoantibodies against target proteins (e.g., Aβ) in individuals with PAD (e.g., AD), or individuals of the same age or age group, are classified or sorted.

[0128] In some embodiments, the levels of target proteins (e.g., Aβ) are compared with control target protein levels. In some embodimentsIn this process, the level of the target protein (e.g., Aβ) is compared with the level of the target protein (e.g., Aβ) in a control sample. In some embodiments, the level of the target protein (e.g., Aβ) is compared with the level of the 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).

[0129] 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.

[0130] Control samples may 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, as described on pages 24 / 56 of the specification (CN 121248772 A), 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 a target protein (e.g., Aβ), or a target protein (e.g., Aβ).

[0131] 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 about 18 to about 50 years (e.g., about 18 to about 40 years, or about 18 to about 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 a 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 an individual's population (e.g., the same age group).

[0132] 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 one or more of an individual's...The individual is determined to have a low level of one or more SHD-catalyzing antibodies if the level of the SHD-catalyzing antibody is not greater than any of approximately 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median level of a population of individuals of the same age or age group. Alternatively, if the individual is determined to have a high level of one or more SHD-catalyzing antibodies if the level of the SHD-catalyzing antibody is greater than any of approximately 20%, 50%, 75%, 2×, 3×, 5×, 10× or higher of the median level of a population of individuals of the same age group, the individual is determined to have a high level of one or more SHD-catalyzing antibodies.

[0133] If the individual is determined to have a low level of one or more autoantibodies against a target protein (e.g., Aβ) if the level of the autoantibody against a target protein (e.g., Aβ) is not greater than any of approximately 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median level of a population of individuals of the same age group, 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 found to have a level of one or more autoantibodies against a target protein (e.g., Aβ) greater than approximately 20%, 50%, 75%, 2×, 3×, 5×, 10× or more 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).

[0134] If an individual is found to have a level of a target protein (e.g., Aβ) that is not greater than approximately 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less 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 a target protein (e.g., Aβ). Alternatively, if an individual is found to have a level of a target protein (e.g., Aβ) greater than approximately 20%, 50%, 75%, 2×, 3×, 5×, 10× or more 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 a target protein (e.g., Aβ).

[0135] 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 an autoantibody against a target protein (e.g., Aβ) is lower than the level of control autoantibody. In some embodiments, the level of one or more SHD-catalyzing antibodies in an individual at risk of PAD (e.g., AD) is not greater than any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of the population of individuals in the same age group. In some embodiments, an individual at risk of PAD (e.g., AD)The level of autoantibodies against the target protein (e.g., Aβ) is not greater than any of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of the population of individuals in the same age group.

[0136] In some embodiments, 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 target protein (e.g., Aβ) is higher than the level of control target protein, then an individual is determined to have a risk of PAD (e.g., AD). In some embodiments, the level of one or more SHD-catalyzing antibodies in an individual at risk of PAD (e.g., AD) is not greater than any of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of the 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 20%, 50%, 75%, 2×, 3×, 5×, 10× or more of the median of a population of individuals in the same age group.

[0137] The diagnostic and treatment methods described herein can be applied to individuals who have PAD or are at risk of 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 at risk of developing PAD (e.g., AD). Individuals “at risk” may or may not have a detectable disease prior to the treatments described herein, and may or may not have the detectable disease shown. “At risk” means that an individual has one or more so-called risk factors, which are measurable parameters associated with developing the PADs (e.g., AD) described herein. Individuals with one or more of these risk factors have a higher probability of developing a PAD (e.g., AD) than individuals without these risk factors.

[0138] Many PADs are known in the art 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; and PADs associated with the accumulation of transthyretin.ATTR amyloidosis; 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 TAR DNA-binding protein); Huntington's disease associated with the accumulation of Huntington's protein; type II diabetes associated with the accumulation of IAPP; and ALS associated with the accumulation of SOD1.

[0139] 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 autoantibody, 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 dosage (or effective amount of therapeutic catalytic antibody) can be determined based on individual size and condition and according to standard therapeutic practice. The route of administration follows known and accepted methods, such as by single or multiple boluses or infusions over a period of time in an appropriate manner, for example, via subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional, intra-articular, or oral routes. Animal studies provide reliable guidance for determining effective dosages for human diagnostic applications. Scaling of effective doses between species can 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.)

[0140] In some embodiments, the effective amount of the therapeutic catalytic antibody (e.g., anti-Aβ catalytic antibody) is from about 1 μg / m2 to about 100 mg / m2, or from about 1 μg / kg to about 100 mg / kg. In some embodiments, the frequency of administration of the therapeutic catalytic antibody (e.g., anti-Aβ catalytic antibody) is from once daily to about once every three months. In some embodiments, the administration of the therapeutic catalytic antibody may be extended over an extended period of time, such as from about one month to several years.

[0141] In some embodiments, the levels of one or more SHD catalytic antibodies, autoantibodies against target proteins (e.g., Aβ), and / or target proteins (e.g., Aβ) are periodically assessed to adjust the dose and frequency of administration of the therapeutic catalytic antibody. (See some specifications, pages 26 / 56, 29 CN 121248772 A)In the embodiments, the levels of one or more SHD catalytic antibodies, autoantibodies against target proteins (e.g., Aβ), and / or target proteins (e.g., Aβ) are assessed approximately every month, every two months, every three months, every four months, every six months, or annually. In some embodiments, if: (i) the level of one or more catalytic antibodies is lower than the level of control catalytic antibodies; and (ii) the level of autoantibodies against target proteins (e.g., Aβ) is lower than the level of control autoantibodies, or the level of target proteins (e.g., Aβ) is higher than the level of control target proteins, then the administration of a therapeutic catalytic antibody (e.g., anti-Aβ catalytic antibody) is repeated.

[0142] III. Anti-Aβ Catalytic Antibody

[0143] This application provides therapeutic catalytic antibodies that specifically bind to and cleave target proteins associated with PAD. The treatment methods described in Part II may use any of the therapeutic catalytic antibodies (e.g., anti-Aβ catalytic antibodies) described in this part. 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 having an SHD motif in the light chain variable region (VL).

[0144] In some embodiments, a catalytic antibody that specifically binds to and cleaves amyloid β (Aβ) peptide is provided. 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 contains an SHD motif in the light chain variable region (VL).

[0145] In some embodiments, an isolated anti-Aβ catalytic antibody derived from 3D6 is provided. In some embodiments, the VL of the anti-Aβ catalytic antibody is derived from 3D6, wherein the amino acid at position 1 of the VL is Asp (D), and wherein the numbering is based on the Kabat EU index. In some embodiments, the amino acid residue at position 26 of the VL is Ser (S), the amino acid residue at position 27D of the VL is D, Glu (E), or His (H), and / or the amino acid residue at position 28 of the VL is D or Gln (N), and wherein the numbering is based on the Kabat EU index. In some embodiments, the heavy chain variable region (VH) of the anti-Aβ catalytic antibody is derived from 3D6. In some embodiments, VHs of anti-Aβ catalytic antibodies are screened from a phage library having a human germline VH sequence based on Aβ binding affinity. In some embodiments, the anti-Aβ catalytic antibody competitively binds specifically to Aβ with 3D6.

[0146] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VL containing one, two, or three LC-CDRs of 3D6, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and...Furthermore, the amino acid residue at position 93 of VL is H, and the numbering is based on the Kabat EU index. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VL containing LC-CDR1, LC-CDR2, and LC-CDR3 of 3D6, wherein the amino acid residue at position 1 of VL is D, the amino acid residue at position 27A of VL is S, and the amino acid residue at position 93 of VL is H, and the numbering is based on the Kabat EU index. In some embodiments, the amino acid residue at position 26 of VL is Ser(S), the amino acid residue at position 27D of VL is D, E, or H, and / or the amino acid residue at position 28 of VL is D or N, and the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH containing one, two, or three HC-CDRs of 3D6. In some embodiments, the anti-Aβ catalytic antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 of 3D6. In some embodiments, VHs of anti-Aβ catalytic antibodies are screened from a phage library having human germline VH sequences based on binding affinity to Aβ.

[0147] In some embodiments, isolated anti-Aβ catalytic antibodies are provided, comprising: VLs comprising: 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-CDRs contain up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the EU index of Kabat. In some embodiments, the amino acid residue at position 26 of VL is Ser(S), the amino acid residue at position 27D of VL is D, E, or H, and / or the amino acid residue at position 28 of VL is D or N, wherein the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: VH comprising: 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, which contain up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR. In some embodimentsFor example, VH of anti-Aβ catalytic antibodies is screened from a phage library having a human germline VH sequence based on binding affinity to Aβ.

[0148] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VL comprising: 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, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the Kabat EU index. In some embodiments, the amino acid residue at position 26 of the VL is Ser(S), the amino acid residue at position 27D of the VL is D, E, or H, and / or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising the following: 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, VHs of the anti-Aβ catalytic antibody are screened from a phage library having human germline VH sequences based on their binding affinity to Aβ.

[0149] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: VL comprising: 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; wherein the amino acid residue at position 1 of VL is D, the amino acid residue at position 27A of VL is S, and the amino acid residue at position 93 of VL is H, and wherein the numbering is based on the EU index of Kabat; and VH comprising: 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, the amino acid residue at position 26 of VL is Ser(S), the amino acid residue at position 27D of VL is D, E, or H, and / or the amino acid residue at position 28 of VL is D or N, and wherein the numbering is based on the Kabat EU index.

[0150] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VL containing the following: SEQ ID NO: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 the amino acid residue at position 1 of VL is D, the amino acid residue at position 27A of VL is S, the amino acid residue at position 93 of VL is H, the amino acid residue at position 26 of VL is S, the amino acid residue at position 27D of VL is D, E, or H, and / or the amino acid residue at position 28 of VL is D or N, and wherein the numbering is based on the EU index of Kabat; and VH containing the following: 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.

[0151] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VL containing the amino acid sequences of SEQ ID NO: 12, 13, and 14, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: an amino acid sequence containing SEQ ID NO: 9, 10, and 11. In some embodiments, the amino acid residue at position 26 of the VL is Ser(S), the amino acid residue at position 27D of the VL is D, E, or H, and / or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is based on the Kabat EU index. In some embodiments, the VH of the anti-Aβ catalytic antibody is screened from a phage library having a human germline VH sequence based on binding affinity to Aβ.

[0152] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VL containing an 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 identical to the amino acid sequence of SEQ ID NO: 5, 7, or 8, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the Kabat EU index. In some embodiments, the amino acid residue at position 26 of the VL is D.The base is Ser(S), the amino acid residue at position 27D of VL is D, E or H, and / or the amino acid residue at position 28 of VL is D or N, and wherein the numbering is based on the EU index of Kabat. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising an 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 with the amino acid sequence of SEQ ID NO: 4 or 6. In some embodiments, the VH of the anti-Aβ catalytic antibody is screened from a phage library having a human germline VH sequence based on binding affinity to Aβ.

[0153] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH containing the amino acid sequence of SEQ ID NO: 6 and a VL containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH containing the amino acid sequence of SEQ ID NO: 6 and a VL containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH containing the amino acid sequence of SEQ ID NO: 6 and a VL containing the amino acid sequence of SEQ ID NO: 21. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH containing the amino acid sequence of SEQ ID NO: 6 and a VL containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH containing the amino acid sequence of SEQ ID NO: 19 and a VL containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 22.The VH comprising the amino acid sequence of SEQ ID NO: 20 and the VL comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 20 and the VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 20 and the VL comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, an isolated anti-Aβ catalytic antibody is provided, comprising: a VH comprising the amino acid sequence of SEQ ID NO: 20 and the VL comprising the amino acid sequence of SEQ ID NO: 22.

[0154] In some embodiments, an isolated anti-Aβ catalytic antibody is provided, which competitively and specifically binds to and cleaves Aβ with any of the anti-Aβ catalytic antibodies described herein.

[0155] 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 includes 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 includes an IgG1 heavy chain constant region. In some embodiments, the anti-Aβ catalytic antibody includes an IgG2 heavy chain constant region. In some embodiments, the anti-Aβ catalytic antibody includes an IgG3 heavy chain constant region. In some embodiments, the anti-Aβ catalytic antibody includes an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region includes or consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain constant region includes or consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-Aβ catalytic antibody package contains 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.

[0156] In some embodiments, the anti-Aβ catalytic antibody comprises an Fc region. In some embodiments, the anti-Aβ catalytic antibody comprises human...The Fc region of IgG. In some embodiments, the anti-Aβ catalytic antibody includes 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 includes an Fc region having reduced ADCC and / or CDC effector functions.

[0157] In some embodiments, the anti-Aβ catalytic antibody is mouse, chimeric, humanized, or human.

[0158] In some embodiments, a full-length anti-Aβ catalytic antibody comprising a constant IgG1 domain is provided, wherein the anti-Aβ catalytic antibody comprises: a VL comprising: 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 a variant thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the Kabat EU index. In some embodiments, the amino acid residue at position 26 of the VL is Ser(S), the amino acid residue at position 27D of the VL is D, E, or H, and / or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising: 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 a variant 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 VH of the anti-Aβ catalytic antibody is screened from a phage library having a human germline VH sequence based on binding affinity to Aβ. In some embodiments, the anti-Aβ catalytic antibody comprises: a VL comprising 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. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19, or 20. In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 5.(iii) VH containing the amino acid sequence of SEQ ID NO: 6 and VL containing the amino acid sequence of SEQ ID NO: 8; (iv) VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 21; (v) VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 21; (vi) VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 22; or (vii) VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 22.

[0159] In some embodiments, a full-length anti-Aβ catalytic antibody comprising a constant IgG4 domain is provided, wherein the anti-Aβ catalytic antibody comprises: a VL comprising: 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 a variant thereof, wherein the LC-CDR contains up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the Kabat EU index. In some embodiments, the amino acid residue at position 26 of the VL is Ser(S), the amino acid residue at position 27D of the VL is D, E, or H, and / or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: a VH comprising the following: 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 at most about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions. In some embodiments, the VH of the anti-Aβ catalytic antibody is screened from a phage library having a human germline VH sequence based on binding affinity to Aβ. In some embodiments, the anti-Aβ catalytic antibody comprises: a VL comprising 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. In some embodiments, the anti-Aβ catalytic antibody comprises: a VL comprising 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.NO: VH has an amino acid sequence of at least about 85% sequence identity for amino acid sequences of amino acid sequences of amino acid sequence 4, 6, 19 or 20. In some embodiments, the anti-Aβ catalytic antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 21; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 22; or (vii) a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 22.

[0160] 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 position in the variable regions of the heavy and light chains of antibodies, and that catalytic antibodies containing CDRs from the catalytic antibodies described herein are also within the scope of the invention based on alternative prediction algorithms. Those skilled in the art will also recognize that catalytic antibodies contain VH or VL sequences from the catalytic antibodies described herein, but are within the scope of the invention based on alternative algorithms.

[0161] Table 2. Exemplary anti-Aβ catalytic antibody sequences.

[0162] Specification 31 / 56 pages 34 CN 121248772 A

[0163] 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.

[0164] Aβ peptide

[0165] In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves the Aβ peptide. 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 N-terminus of Aβ(1-40) containing 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0166] 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 Aβ oligomers. In some embodiments, the anti-Aβ catalytic antibody specifically binds to and cleaves Aβ oligomers.Page 35 CN 121248772 A 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.

[0167] 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.

[0168] 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. Aβ peptides are 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 neurotoxic.

[0169] Recent studies have shown that the soluble oligomeric form of the peptide may be a cause of 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 the brain parenchyma and blood vessels, and it contributes to cerebrovascular lesions and is neurotoxic. Aβ mainly circulates in plasma, cerebrospinal fluid (CSF), and interstitial fluid (ISF) as soluble Aβ40. Amyloid plaques in the elderly contain Aβ40 and Aβ42, while blood vessel amyloid is mainly composed of the shorter Aβ40. Several Aβ sequences have been found in both lesions. Increased total Aβ levels, or relative concentrations of Aβ40 and Aβ42, have been implicated in the pathogenesis of familial and sporadic Alzheimer's disease. Aβ42 is the peptide with the highest degree of amyloid formation due to its greater hydrophobicity. Aβ42 is also referred to as Aβ(1-42). Aβ40 is also referred to as Aβ(1-40).

[0170] Bepinizumab is a humanized form of the murine monoclonal antibody 3D6 that targets the N-terminal 5 residues of the helical conformation of the 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 brain tissue of patients receiving high doses. No health risks were observed in patients receiving 0.5 or 1 mg of bepinizumab.

[0171] The VH of 3D6 contains the amino acid sequence of SEQ ID NO: 4. The VL of 3D6 contains the amino acid sequence of SEQ ID NO: 23. The VH of bepinizumab contains the amino acid sequence of SEQ ID NO: 24. The VL of bepinizumab contains the amino acid sequence of SEQ ID NO: 25. SEQ ID NO:23 3D6 VL EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFT ISRDNAKNSLYLQMNSLRAEDTALYYCVRYDHYSGSSDYWGQGTLVTVSS

[0176] SEQ ID NO: 25 Bepinzumab VL

[0177] YVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKR

[0178] Antibody Variants

[0179] This document also provides variants and derivatives of any of the therapeutic catalytic antibodies described above, such as anti-Aβ catalytic antibodies. Specification 33 / 56 pages 36 CN 121248772 A

[0180] Substitution, Insertion, Deletion and Variants

[0181] In some embodiments, amino acid sequence variants of the therapeutic catalytic antibodies provided herein, such as anti-Aβ catalytic antibodies, are covered. 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 can be performed to obtain the final construct, provided that the final construct possesses the desired properties, such as antigen binding and cleavage.

[0182] 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 targeted...For products with desired activity, such as retained / improved antigen binding and cleavage, reduced immunogenicity, or improved ADCC or CDC.

[0183] Conserved substitutions are shown in Table 3 below.

[0184]

[0185] Amino acids can be classified into different categories based on common side chain characteristics:

[0186] a. Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0187] b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0188] c. Acidic: Asp, Glu; Specification 34 / 56 pages 37 CN 121248772 A

[0189] d. Basic: His, Lys, Arg;

[0190] e. Residues affecting chain orientation: Gly, Pro;

[0191] f. Aromatic: Trp, Tyr, Phe.

[0192] Non-conservative substitution would require an exchange of a member of one of these categories with another.

[0193] Exemplary substituted 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, such as 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 in specifically identified residues (SDRs), where the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by constructing a secondary library and reselecting from it has been described, for example, in Molecular Biology Methods 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ (2001)).

[0194] In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by 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.

[0195] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such changes do not significantly reduce the ability of the antibody to bind to and cleave the antigen. For example, conserved changes (e.g., conserved substitutions as presented 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 some embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0196] A method suitable for identifying residues or regions that can target antibodies for mutation induction is called “alanine scanning mutation induction,” as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, residues or groups of target residues (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 whether the interaction between the antibody and the antigen 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 the antigen. Such contact residues and adjacent residues may be targeted or removed as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0197] The catalytic antibody variants described herein maintain their catalytic activity against a target protein (e.g., Aβ). The catalytic triplet, i.e., the SHD motif in the VL of the therapeutic catalytic antibody, cannot be substituted. In some embodiments, one or more amino acid residues in the VL of the therapeutic catalytic antibody that support its catalytic activity cannot be substituted, including, for example, amino acid residues at positions 26, 27D, and 28 of the VL, where the numbering is based on the Kabat EU index.

[0198] Amino acid sequence insertions include amino and / or carboxyl terminus fusions from one residue to a polypeptide containing one hundred or more residues, as well as the insertion of one or more amino acid residues within the sequence. Examples of terminus insertions include catalytic antibodies having an N-terminal methionine residue. Other insert variants of the catalytic antibody include enzymes (e.g., for ADEPT) that increase the serum half-life of the antibody, or the fusion of a polypeptide with the N or C terminus of the antibody.

[0199] 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 consists of them, which are present on one or both light chains of the antibody variant.

[0200] Chimeric and Humanized Catalytic Antibodies

[0201] 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 the class or subclass of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0202] In some embodiments, humanized catalytic antibodies are provided. Humanized antibodies are used as therapeutic molecules because they reduce or eliminate human immune responses to non-human antibodies (such as human anti-mouse antibody (HAMA) responses), which can lead to immune responses to antibody therapeutics and reduce the effectiveness of the therapeutics.

[0203] In some embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains of the HVR, such as the CDR (or a portion thereof), derived from the non-human antibody, and FR (or a portion thereof) derived from the human antibody sequence. Optionally, the humanized antibody will also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., antibodies from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0204] Humanized antibodies and their preparation methods are reviewed, for example, in Almagro and Fransson, (2008) Frontiers in Bioscience 13: 1619-1633, and further described, for example, in 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 (describing SDR (a-CDR) transplantation); Padlan, (1991) Molecular Immunology 28:489–498 (description of “surface rework”); Dall'Acqua et al., (2005) Methods 36:43–60 (description of “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 for FR reorganization).

[0205] 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 derived from the common sequences of human antibodies from specific subgroups of 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); human maturation (somatic mutation) frame regions or human germline frame regions (see, for example, Almagro and Fransson, (2008) *Frontiers in Bioscience* 13:1619-1633); and frame 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).

[0206] Antibodies derived from libraries

[0207] Therapeutic catalytic antibodies can be isolated by screening combinatorial libraries of antibodies with the desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed in, for example, Hoogenboom et al., *Methods in Molecular Biology* 178:1-37 (edited by O'Brien et al., Human Press, Tetowa, NJ, 2001), and further described in, for example, McCafferty et al., *Nature* 348:552-554; Clackson et al., *Nature* 352:624-628 (1991); Marks et al., *Molecular Manual* 36 / 56 pp. 39 CN 121248772 A *Journal of Biology* 222:581-597 (1992); Marks and Bradbury, *Methods in Molecular Biology* 248:161-175 (edited by Lo, Human 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).

[0208] In some phage display methods, lineages of the VH and VL genes are cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments as scFv fragments 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 wide range of non-self and self antigens without any immunization, as described by Griffiths et al., *European Journal of Molecular Biology (EMBO J)*, 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.

[0209] Fc Region Variants

[0210] 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 producing Fc region variants. In some embodiments, the Fc region variants have enhanced ADCC effector function, typically associated with binding to an Fc receptor (FcR). In some embodiments, the Fc region variants have reduced ADCC effector function. There are many instances of Fc sequence variations 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 FcR. Those disclosures are explicitly incorporated herein by reference.

[0211] In some embodiments, the therapeutic catalytic antibody comprises an Fc region having some, but not all, effector functions.The Fc region makes it a desirable candidate for applications where the in vivo half-life of the antibody is important and certain effector functions (such as CDC and ADCC) are unnecessary or harmful. 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 evaluating 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, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology Inc., Mountain View, CA); and the CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example in animal models disclosed in, such as, Clynes et al., Proceedings of the National Academy of Sciences, 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, WO 2006 / 029879 and WO C1q and C3c binding ELISA in 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); et al., 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 art (see, for example, Petkova, SB et al., International Immunology 18 (12):1759-1769 (2006)).

[0212] Antibodies with reduced effector function include antibodies that substitute one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Patent No. 6,737,056). These Fc mutants include Fc mutants having substitutions at two or more of the amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant (US Patent No. 7,332,581) with residues 265 and 297 replaced by alanine.

[0213] Certain antibody variants with improved or weakened binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., Journal of Biochemistry 9(2): 6591-6604 (2001).)

[0214] In some embodiments, modifications are made in the Fc region that produces altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., Journal of Immunology 164: 4178-4184 (2000).

[0215] Antibodies with increased half-life and improved FcRn binding are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having 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 (U.S. Patent No. 7,371,826).

[0216] Other examples of Fc region variants can also 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.

[0217] Glycosylation Variants

[0218] 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. Adding or deleting glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence of the antibody to produce or remove one or more glycosylation sites.

[0219] In the case where the catalytic antibody contains an Fc region, the carbohydrates attached thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are typically attached by 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 may 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 dual-antenna oligosaccharide structure. In some embodiments, oligosaccharides in catalytic antibodies may be modified to produce catalytic antibody variants with certain improved properties.

[0220] N-glycans attached to the CH2 domain of Fc are heterogeneous. Antibodies or Fc fusion proteins generated 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 unfucosylation of N-glycans improves the binding affinity of Fc to FcγRIIIa. Fc Specification 38 / 56 pages 41 CN 121248772 A Increased γRIIIa binding can enhance ADCC, which may be advantageous in certain therapeutic applications where cytotoxicity is desired.

[0221] In some embodiments, enhanced effector function may be disadvantageous when Fc-mediated cytotoxicity is not desired. In some embodiments, the Fc fragment or CH2 domain is not glycosylated. In some embodiments, the N-glycosylation site in the CH2 domain is mutated to prevent glycosylation.

[0222] In some embodiments, catalytic antibody variants are provided as having a carbohydrate structure lacking fucose attached (directly or indirectly) 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 ratio at Asn297 to Asn, as measured by MALDI-TOF mass spectrometry.The average amount of fucose within the glycan chain of the sum of all sugar structures attached to 297 (e.g., complex, heterozygous, and high-mannose structures), as described, for example, as in WO 2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 (EU number of the Fe region residue) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants may possess 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 “fucosylated” 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; WO 2003 / 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 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially in Example 11), and gene knockout cell lines, such as those with the α-1,6-fucoside transferase gene, FUT8, and 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).

[0223] Catalytic antibody variants further possess a bipartite oligosaccharide, for example, 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.).

[0224] Derivatives

[0225] In some embodiments, the therapeutic catalytic antibodies provided herein (such as anti-Aβ catalytic antibodies) may be further modified to contain 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 and 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 can have any molecular weight and can 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 quantity 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.

[0226] IV. Preparation Methods

[0227] The therapeutic catalytic antibodies described herein (such as anti-Aβ catalytic antibodies) can be prepared using any method known in the art, including those described below and in examples. Immunization against a target antigen (such as a transition state analog) can be performed.Laboratory animals were used to obtain therapeutic metabolites. See, for example, US2010018361A1 and Taguchi H et al., “Catalytic antibodies to amyloid beta peptide in defense against Alzheimer disease,” Autoimmune. Rev. 7: 391-397 (2008), which is incorporated herein by reference. Catalytic antibodies may also be recombinantly expressed.

[0228] Nucleic Acids

[0229] 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) described herein. In some embodiments, the nucleic acid molecule comprises a heavy chain or a light chain of polynucleotide encoding a therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody). In some embodiments, the nucleic acid molecule comprises a heavy chain of polynucleotide and a light chain of polynucleotide encoding a therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody). In some embodiments, a first nucleic acid molecule contains a first polynucleotide encoding a heavy chain, and a second nucleic acid molecule contains a second polynucleotide encoding a light chain. In some embodiments, the first polynucleotide encoding the heavy chain is operatively linked to a first promoter, and the second polynucleotide encoding the light chain is operatively linked to a second promoter. In some embodiments, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are operatively linked to promoters.

[0230] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, such that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline.

[0231] 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 levels of expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences can 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, and Rous sarcoma virus.Virus promoters, and human gene promoters, such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, the invention is not limited to the use of constitutive promoters. Inducible promoters are also considered part of the invention. The use of inducible promoters provides a molecular switch capable of enabling the expression of a polynucleotide sequence operably linked to it when such expression is needed, or shutting off the expression when expression is not needed. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. Specification 40 / 56 pages 43 CN 121248772 A

[0232] In some embodiments, the expression of a therapeutic catalytic antibody is inducible. In some embodiments, a nucleic acid sequence encoding a therapeutic catalytic antibody is operably linked to an inducible promoter.

[0233] In some embodiments, the polynucleotide encoding a 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 natural heavy chain or light chain leader sequence, or it may be another heterologous leader sequence. In some embodiments, the nucleic acid (a set of nucleic acids) encoding a therapeutic catalytic antibody (such as an anti-Aβ catalytic antibody) may also include a nucleic acid sequence encoding a peptide tag (such as a protein purification tag, e.g., a His tag, a HA tag).

[0234] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize to a nucleic acid sequence encoding any of the therapeutic catalytic antibodies described herein under at least moderately stringent hybridization conditions.

[0235] Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0236] Vector

[0237] A vector is provided comprising a polynucleotide encoding a heavy chain and / or a light chain of any of the therapeutic catalytic antibodies described herein (such as an anti-Aβ catalytic antibody). 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.

[0238] In some embodiments, the first vector contains a polynucleotide encoding the heavy chain, and the second vector contains a polynucleotide encoding the light chain. In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (e.g., similar molar amounts or similar mass amounts). In some embodiments, the first vector and the second vector are transfected in a molar or mass ratio between 5:1 and 1:5.The second vector is transfected into the host cell. 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.

[0239] Nucleic acids can be cloned into a variety of 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 granules. Vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0240] 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 is 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, a suitable vector contains a replication start point, a promoter sequence, a convenient restriction endonuclease site, and one or more optional markers (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0241] In simple terms, therapeutic catalytic antibodies can be expressed by encoding natural or synthetic nucleic acids that encode catalytic antibodies by inserting nucleic acids into a suitable expression vector, such that the nucleic acids are operatively linked to 5' and 3' regulatory elements, including, for example, promoters (e.g., lymphocyte-specific promoters) and a 3' untranslated region (UTR). The vector can be adapted for replication and integration in 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.

[0242] In some embodiments, vectors preferred for expressing peptides in CHO or CHO-derived cells or in NSO cells are selected. Exemplary vectors of this type are described, for example, in Running Deer et al., *Biotechnol. Prog.* 20:880-889 (2004).

[0243] To assess the expression of the peptide or a portion thereof, 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 vector transfection.Or infection. In other respects, selection markers may be carried on separate DNA fragments and used in co-transfection procedures. Both selection markers and reporter genes 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.

[0244] 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 source and encodes a polypeptide whose expression is manifested by some easily detectable property, such as enzyme activity. The expression of the reporter gene is determined at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared or commercially available using known techniques. Generally, constructs exhibiting the highest level of reporter gene expression with a minimum 5' flanking region are identified as promoters. Such promoter regions can be linked to reporter genes and used to assess the ability of pharmaceutical agents to regulate promoter-driven transcription.

[0245] Host Cells

[0246] This application provides isolated host cells comprising any of the therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies) described herein, nucleic acid molecules, or vectors.

[0247] The therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies) described herein can be expressed in prokaryotic cells (such as bacterial cells); or in 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; and CHO cells, including CHO-S and DG44. Lec13CHO cells and FUT8CHO cells; PER.C6® cells (Crucell); and NSO cells. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli or Bacillus subtilis) and yeasts (Saccharomyces cerevisiae, Schizosoma spp.; or Kluyveromyces lactis). In some embodiments, eukaryotic host cells are selected based on the ability of a particular eukaryotic host cell to perform the 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 peptide produced in 293 cells.

[0248] The introduction of one or more nucleic acids into a desired host cell can be achieved by any method, including but not limited toThis is 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 Edition, Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected in the desired host cells according to any suitable method.

[0249] In some embodiments, therapeutic catalytic antibodies are generated in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods in Molecular Biology 498: 229-44 (2009); Spirin, Trends in Biotechnology 22: 538-45 (2004); Endo et al., Advances in Biotechnology 21: 695-713 (2003).

[0250] Purification

[0251] 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 those that bind to the constant region of the antibody. 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.

[0252] V. Compositions, Kits, and Articles

[0253] This document also provides compositions (such as pharmaceutical compositions) comprising any of the therapeutic catalytic antibodies (such as anti-Aβ catalytic antibodies), nucleic acids, vectors, or host cells described herein.

[0254] Pharmaceutical compositions of the 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 formulation or aqueous solution form. Acceptable carriers, excipients, or stabilizers at the dosage usedNon-toxic to recipients at certain concentrations and including 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); phenols, butanol, or benzyl alcohol; alkyl p-hydroxybenzoates, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; 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™, PLURONICS™, 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 individuals described herein for imaging, diagnosis, or treatment.

[0255] The pharmaceutical composition intended for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through a sterile filter membrane.

[0256] Kits suitable for any of the methods described herein for determining catalytic antibody levels, diagnosis, and treatment are also provided, including kits comprising any of the therapeutic catalytic antibodies described herein (such as anti-Aβ catalytic antibodies).

[0257] In some embodiments, a kit for determining catalytic antibody levels in a biological sample is provided, comprising: a substrate peptide comprising the amino acid sequence (EAR)n (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).

[0258] In some embodiments, a kit is provided for diagnosing PAD in an individual or determining the risk of PAD, wherein the PAD is associated with a target protein, the kit comprising: a) a substrate peptide comprising the amino acid sequence (EAR)n (SEQ ID NO: 2), and 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 also includes 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 includes a solid support (e.g., an ELISA plate).

[0259] In some embodiments, a kit is provided for diagnosing AD in an individual or determining the risk of AD, comprising: a) a substrate peptide comprising the amino acid sequence (EAR)n (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 includes a solid support (e.g., an ELISA plate).

[0260] In some embodiments, a kit is provided for treating or preventing a PAD in an individual, wherein the PAD is associated with a target protein, the kit comprising: a) a substrate peptide comprising the amino acid sequence (EAR)n (SEQ ID NO: 2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) the 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).

[0261] In some embodiments, a kit for treating or preventing AD in an individual is provided, comprising: a) a substrate peptide comprising the amino acid sequence (EAR)n (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).

[0262] In some embodiments, a kit for treating or preventing AD in an individual is provided, comprising: a pharmaceutical composition comprising an anti-Aβ catalytic antibody and a pharmaceutically acceptable carrier, wherein the anti-Aβ catalytic antibody comprises: VL comprising: 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 VL comprising:LC-CDR3, or a variant thereof, having an amino acid sequence of 14, comprising up to about 5 amino acid substitutions in the LC-CDR, wherein the amino acid residue at position 1 of VL is D, the amino acid residue at position 27A of VL is S, and the amino acid residue at position 93 of VL is H, and wherein the numbering is based on the Kabat EU index. In some embodiments, the amino acid residue at position 26 of VL is Ser(S), the amino acid residue at position 27D of VL is D, E, or H, and / or the amino acid residue at position 28 of VL is D or N, and wherein the numbering is based on the Kabat EU index. In some embodiments, the anti-Aβ catalytic antibody comprises: VH comprising: 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 a variant thereof, comprising up to about 5 amino acid substitutions in the HC-CDR.

[0263] The kit of this application is in a suitable package. 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 buffers, blocking buffers, washing buffers, antibody dilution buffers, development buffers, etc.), antibodies (e.g., anti-human Ig antibodies), and interpretive information.

[0264] Therefore, this application also provides articles of manufacture. Articles of manufacture may include containers and markings or packaging inserts on or associated with the containers. Suitable containers include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc. In some embodiments, the container holds the pharmaceutical composition and may have a sterile access 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 intended for the diagnosis (including risk assessment), treatment, or prevention of PAD (e.g., AD) in an individual. The labeling or packaging insert 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 labeling 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 doses). The packaging insert refers to the instruction leaflet, 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 article may contain a second container containing a pharmaceutically acceptable buffer solution, such as bacteriostatic water for injection (BWFI).Phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials desired from the perspective of the trade description (pages 44 / 56, CN 121248772 A) and the user, including other buffers, diluents, filters, needles, and syringes.

[0265] The kit may also include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in a pharmacy (e.g., hospital pharmacies and general pharmacies).

[0266] Exemplary Examples

[0267] Example 1. A method for determining the level of one or more SHD catalytic antibodies in a biological sample, comprising:

[0268] 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

[0269] 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,

[0270] wherein the substrate peptide comprises an amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30.

[0271] Example 2. The method according to Example 1, wherein n is 3.

[0272] Example 3. The method according to Example 1 or 2, wherein the biological sample is a serum sample.

[0273] Example 4. The method according to Example 3, wherein the serum sample contains at least about 1 μg / mL of immunoglobulin (Ig).

[0274] 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.

[0275] 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.

[0276] Example 7. The method according to Example 6, wherein the antibody is labeled with an enzyme or a fluorescent label.

[0277] 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.

[0278] Example 9. The method according to Example 8, wherein 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.

[0279] Example 10. According to the method described in Example 8, the level of one or more SHD catalytic antibodies is totalThe level of SHD catalytic antibody.

[0280] 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 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 the amino acid sequence (EAR)n (SEQ ID NO: 2), where n is an integer between 1 and 30.

[0281] Example 12. The method according to Example 11, wherein n is 3.

[0282] Example 13. The method according to Example 11 or 12, wherein the serum sample contains at least about 1 μg / mL 1g.

[0283] 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.

[0284] 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 (pages 45 / 56, CN 121248772 A).

[0285] Example 16. The method according to Example 15, wherein the antibody is labeled with an enzyme or fluorescent label.

[0286] 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 catalytic antibodies is lower than the level of a control SHD catalytic antibody; and (ii) the level of the autoantibody against the target protein is lower than the level of a control autoantibody.

[0287] 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.

[0288] Example 19. The method according to Example 18, wherein the level of the autoantibody is determined using an ELISA assay.

[0289] 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.

[0290] 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.

[0291] Example 22. The method according to any one of Examples 8 to 16, further comprising determining the level of the autoantibody against the target protein in the individual.The level of the target protein in the sample, wherein if: (i) the level of the one or more SHD-catalyzing antibodies is lower than the level of the control SHD-catalyzing antibodies; and (ii) the level of the target protein is higher than the level of the control target protein, then the individual is determined to have the risk of the PAD.

[0292] Example 23. The method according to any one of Examples 8 to 22, wherein the level of the control SHD-catalyzing antibody is the level of one or more SHD-catalyzing antibodies in a healthy individual.

[0293] Example 24. The method according to any one of Examples 8 to 22, wherein the level of the control SHD-catalyzing antibody is the median level of one or more SHD-catalyzing antibodies in an individual.

[0294] 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).

[0295] Example 26. The method according to any one of Examples 8 to 24, wherein:

[0296] (i) the PAD is Parkinson's disease, and the target protein is α-synuclein;

[0297] (ii) the PAD is Alzheimer's disease or dementia, and the target protein is Tau;

[0298] (iii) the PAD is ATTR amyloidosis, and the target protein is transthyretin;

[0299] (iv) the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;

[0300] (v) the PAD is frontotemporal lobe degeneration or amyotrophic lateral sclerosis, and the target protein is TDP43;

[0301] (vi) the PAD is Huntington's disease, and the target protein is huntingtin protein;

[0302] (vii) The PAD is type II diabetes, and the target protein is IAPP; or

[0303] (viii) The PAD is amyotrophic lateral sclerosis, and the target protein is SOD1.

[0304] 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:

[0305] a) determining the risk of the individual having the PAD according to any one of Examples 1 to 26; and

[0306] b) administering to the individual an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein.

[0307] Example 28. The method according to Example 27, wherein the method described in the specification, pages 46 / 56, CN 121248772 A, is repeated at a frequency of no more than about every three months.

[0308] Example 29. The method according to Example 27 or 28, wherein the PAD is Alzheimer's disease, whereinThe target protein is amyloid β (Aβ), and the therapeutic catalytic antibody comprises: a light chain variable region (VL) comprising: a light chain complementarity-determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR 2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR 3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the Kabat EU index.

[0309] Example 30. The method according to Example 29, wherein the therapeutic catalytic antibody comprises: a heavy chain variable region (VH) comprising: a heavy chain complementarity-determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR 2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR 3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof, wherein the HC-CDR contains up to about 5 amino acid substitutions.

[0310] Example 31. The method according to Example 29 or 30, wherein the amino acid residue at position 26 of the VL is S, the amino acid residue at position 27D of the VL is D, E, or H, and / or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is based on the Kabat EU index.

[0311] Example 32. The method according to any one of Examples 29 to 31, wherein the therapeutic catalytic antibody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20; and / or a VL comprising 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.

[0312] Example 33. The method according to Example 32, wherein the therapeutic catalytic antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 19.VL containing the amino acid sequence of SEQ ID NO: 21; (v) VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 21; (vi) VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 22; or (vii) VH containing the amino acid sequence of SEQ ID NO: 20 and VL containing the amino acid sequence of SEQ ID NO: 22.

[0313] Example 34. The method according to any one of Examples 27 to 33, wherein the therapeutic catalytic antibody is a full-length IgG antibody.

[0314] Example 35. The method according to Example 34, wherein the therapeutic catalytic antibody contains an IgG1 or IgG4 Fc region.

[0315] Example 36. The method according to any one of Examples 27 to 33, wherein the therapeutic antibody is a full-length IgM antibody.

[0316] Example 37. An isolated anti-Aβ catalytic antibody comprising: a VL comprising the following: 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 a variant thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the EU index of Kabat. Specification 47 / 56 pages 50 CN 121248772 A

[0317] Example 38. An isolated anti-Aβ catalytic antibody comprising: VH comprising the following: 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 a variant thereof, wherein the HC-CDR contains at most about 5 amino acid substitutions, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is based on the EU index of Kabat.

[0318] Example 39. An anti-Aβ catalytic antibody according to Example 37 or 38, wherein the anti-Aβ catalytic antibody comprises: VH comprising the following: 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: 10, and a variant thereof, wherein the amino acid sequence of SEQ ID NO: 11 is D, the amino acid sequence of SEQ ID NO: 10 is S, and the amino acid sequence of SEQ ID NO: 11 is H, and wherein the numbering is based on the EU index of Kabat.HC-CDR2 containing the amino acid sequence of SEQ ID NO: 11, 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.

[0319] 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).

[0320] Example 41. An anti-Aβ catalytic antibody according to any one of Examples 37 to 40, wherein the amino acid residue at position 26 of the VL is S, the amino acid residue at position 27D of the VL is D, E or H, and / or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is based on the EU index of Kabat.

[0321] Example 42. An anti-Aβ catalytic antibody according to any one of Examples 37 to 41, wherein the anti-Aβ catalytic antibody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20; and / or a VL comprising 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.

[0322] Example 43. The anti-Aβ catalytic antibody according to Example 42, wherein the anti-Aβ catalytic antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6 and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 21; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 22; or (vii) a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 22. VL of the amino acid sequence of ID NO: 22.

[0323] Example 44.The 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.

[0324] Example 45. The anti-Aβ catalytic antibody according to Example 44, wherein the anti-Aβ catalytic antibody comprises an IgG1 or IgG4 Fc region.

[0325] Example 46. The 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.

[0326] Example 47. A method of treating or preventing Alzheimer's disease in an individual, comprising administering an effective amount of the anti-Aβ catalytic antibody according to any one of Examples 37 to 46 to the individual.

[0327] Example 48. A kit for treating or preventing Alzheimer's disease in an individual, comprising:

[0328] a) a substrate peptide comprising the amino acid sequence (EAR)n (SEQ ID NO: 2), wherein n is an integer between 1 and 30;

[0329] b) an Aβ peptide; and specification 48 / 56 pages 51 CN 121248772 A

[0330] c) an antibody that specifically binds to total Ig.

[0331] Example 49. The kit according to Example 48, further comprising a solid support.

[0332] Example 50. The kit according to Example 48 or 49, further comprising a therapeutic catalytic antibody that specifically binds to Aβ.

[0333] Examples

[0334] The following examples are intended only as examples 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.

[0335] Example 1. Catalytic antibodies as serum biomarkers for Alzheimer's disease

[0336] 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).

[0337] A. Catalytic antibody levels in serum samples from young and older adults

[0338] Serum SHD catalytic antibody levels were measured 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) using two immunoassays. In the EAR-AMC binding assay, ELISA plates were coated with an 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).

[0339] 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), followed by three washes 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 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 development buffer (3.6 mM Na2HPO4, 1.4 mM NaH2PO4, at 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).

[0340] Figure 1A shows the results of the EAR-AMC binding assay, and Figure 1B shows the results of the EAR3 binding assay. In both assays, the human serum pool (HS pool) and the three out of four serum samples from the young adult group (20–29 years) showed higher serum SHD catalytic antibody levels compared to three out of four serum samples from the older adult group (60–69 years). 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 diseases.

[0341] B. Correlation between Catalytic Antibody Levels and Aβ-Specific Autoantibody Levels

[0342] Next, two immunoassays were used to determine the levels of catalytic antibodies and Aβ-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.

[0343] Briefly, each ELISA plate was coated with a coating buffer (0.2 M sodium carbonate-bicarbonate buffer, pH 9.4).The plate was coated with the corresponding peptide, 100 μL / well, and incubated overnight at 4°C. The plate was washed three times with wash buffer (PBST). Then, at room temperature (pages 49 / 56, CN 121248772 A), the plate was blocked with blocking buffer (10% fetal bovine serum) for 1 hour, and washed three times with wash buffer. 100 μL of the corresponding serum sample (100 μg / mL) was added to each well of the plate, with PBS serving as a negative control. After incubation for 1 hour, the plate was washed three times with wash buffer and blocked with blocking buffer for 1 hour at room temperature. The plate was then washed three times with wash buffer. 100 μL of 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 development buffer (3.6 mM Na2HPO4, 1.4 mM NaH2PO4, at 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).

[0344] Figure 2 shows the results of the (EAR)3 and Aβ binding assay. Compared to healthy individuals, 4 out of 5 AD patients had significantly lower serum levels of catalytic antibodies and Aβ-specific autoantibodies. This result suggests that the combined reduction in serum SHD catalytic antibody levels and Aβ-specific autoantibody levels can be used as a biomarker for AD diagnosis.

[0345] Example 2. Design and characterization of anti-Aβ catalytic antibodies

[0346] This example describes the design and characterization of anti-Aβ catalytic antibodies based on 3D6, a non-catalytic antibody that specifically binds to Aβ.

[0347] A. Design of 3D6-D

[0348] Previous studies have found that human IgM autoantibodies hydrolyze Aβ via a serine protease-like mechanism. Brain and peripheral Aβ are in equilibrium with each other. Peripheral Aβ hydrolysis can induce depletion of brain Aβ stores, while IgM does not cross the BBB. However, IgM mediates innate immune responses, generally has a lower affinity for target antigens, and is more difficult to manufacture. Therefore, we seek to engineer IgG-catalyzed antibodies that can hydrolyze Aβ.

[0349] SHD-catalyzed antibodies are known to have an SHD motif as a light chain variable region (VL). See, Gao QS et al., “Site-directed mutagenesis of proteolytic antibody light chain”, Journal of Molecular Biology 253(5): 658 (1995). Therefore, we will present several Aβ-specific...The VL sequences of the non-catalytic IgG antibody were compared with those of the published catalytic antibodies. We found that, except for having the "SHY" motif instead of the "SHD" motif, the mouse version of bepinizumab, 3D6, had high homology with other published catalytic antibody VL sequences, especially with the anti-UA15 VL sequence (Planque SA et al. "Physiological IgM Class Catalytic Antibodies Selective for Transthyretin AMyoid" Journal of Biochemistry, 289(19): 13243-13258 (2014)). The sequence alignment results of the VL of anti-UA15 (catalytic antibody), anti-VP (catalytic antibody) and 3D6 (anti-Aβ non-catalytic antibody) are shown in Figure 3.

[0350] The 3D6-D antibody was engineered by replacing the Y residue at position 1 of the VL of 3D6 with D, and then recombinantly expressed and purified. Figure 4 shows the reducing and non-reducing gel electrophoresis of 3D6-D and 3D6 (i.e., 3D6-Y) antibodies and their humanized forms. The sequences of the 3D6-D catalytic antibody are shown in Table 2.

[0351] B. Catalytic activity of 3D6-D

[0352] The catalytic activity of the 3D6-D antibody was evaluated in an EAR-AMC catalytic function assay. Briefly, each well of the ELISA plate was coated with (100X, Bachem Americas Cat No. I-1575.0050) diluted 1:100. 3D6-D (400 ng / mL), 3D6-Y (400 ng / mL), and trypsin (0.25% trypsin-EDTA, diluted 1:10,000) were each mixed with enzyme assay buffer (50 mM Tris-HCl, pH 7.7, 0.1 M glycine, 0.025% Tween-20) and EAR-AMC (100 μM) in PBS buffer. PBS buffer was used as a negative control, and IgG1 was used as an isotype control. The mixtures were transferred to different wells of an ELISA plate, which was tightly sealed and incubated at 37°C for 20 or 68 hours. At the end of incubation, 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 catalytic antibody, which cleaves EAR-AMC at the covalent binding site between Arg and AMC, thereby releasing AMC as described on pages 50 / 56 of the specification, CN 121248772 A.

[0353] The results of the EAR-AMC catalytic function assay are shown in Figure 4. 3D6-D shows strong cleavage activity, but wild-type...The 3D6 antibody (with Y residues) did not cleave the EAR-AMC substrate.

[0354] C. Aβ-binding of 3D6-D

[0355] 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 as described in Example 1, respectively. Each antibody was added to the wells of an ELISA plate at a concentration of 100 μg / mL. As shown in Figure 6, 3D6-D exhibited (EAR)3 and Aβ binding activity comparable to that of the 3D6 antibody, indicating that 3D6-D is a high-affinity IgG1 catalytic antibody for Aβ.

[0356] D. Humanization of 3D6-D

[0357] To reduce the immunogenicity of the 3D6-D catalytic antibody, a humanized catalytic antibody was produced by transplanting the CDR of 3D6-D into a human antibody framework sequence. Further reversion mutations were introduced from the human antibody framework sequence to the original mouse sequence to maintain the affinity of the humanized antibody and facilitate further antibody development. Additionally, reversion mutations were introduced to the SHD motif and other residues (e.g., residues marked "#" in Figure 3) and / or to maintain the conformational structure of the SHD motif to the original mouse sequence. Exemplary humanized 3D6-D antibody sequences are shown in Table 2.

[0358] 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 of Aβ (1-42) peptide in A buffer (0.2 M sodium carbonate-bicarbonate buffer, pH 9.4) at 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 blocking buffer (1% BSA in PBST) and washed twice with 250 μL / well washing buffer. 100 μL of antibody sample (3X serial dilution 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. 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 washing 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 incubated at room temperature for 1 hour. The plate was then washed three times with 250 μL / well of washing buffer. It was then washed with developing buffer (3.6 mM Na₂HPO₄, 1.4 mM NaH₂PO₄, at pH...).7.2 (below) Freshly prepared HRP substrates (Amplex Red and H2O2) were added to each well and incubated for 1–60 minutes. The signal was then detected using a plate reader (excitation wavelength = 530 nm, emission wavelength = 590 nm, and cutoff wavelength = 570 nm).

[0359] As shown in Figure 7, the humanized 3D6-D catalytic antibodies (hu3D6-D H1L1 and hu3D6-D H1L2) have binding affinity comparable to that of Aβ, such as bepinizumab.

[0360] E. Phage library panning for 3D6-D variants

[0361] Human scFv / Fab phage libraries with human line VH and humanized VL sequences of 3D6-D were panned for Aβ to select humanized anti-Aβ catalytic scFv or Fab. The HC-CDR sequences in the phage libraries were randomized. Based on the high specificity for Aβ, scFv or Fab was selected. 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 full-length IgG (e.g., IgG1 or IgG4) catalytic antibodies.

[0362] Example 3. Determination of anti-Aβ autoantibody and SHD catalytic antibody levels in human serum of Alzheimer's disease (AD) patients

[0363] This experiment was performed to detect the levels of anti-Aβ autoantibody and SHD catalytic antibody (recognition (EAR)3 peptide) in the serum of Alzheimer's disease (AD) patients compared with healthy individuals.

[0364] The 30 AD serum samples used in this assay included 25 new AD patient serum samples and 5 older AD patient serum samples from page 54 of the specification of Example 1, 51 / 56, CN 121248772 A. 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. A 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.

[0365] 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 coating buffer (0.2 M sodium carbonate-bicarbonate buffer, pH 9.4) to a final concentration of 50 µg / mL.Biotinylated amyloid (1-42) peptides were coated at a final concentration of 2 μg / mL (1 mg / mL stock solution diluted 500-fold) in sodium-bicarbonate buffer (pH 9.4), 100 μL / well, and incubated overnight at 4°C. The plates were washed three times each with 200 µL of wash buffer (PBST: 0.1% Tween-20 in phosphate-buffered saline "PBS"). The plates were then blocked for 2 hours at room temperature with blocking buffer (1% BSA in PBST), and washed three times each 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 in PBS. All samples were tested in duplicate for each peptide target. See Figure 9A for the loading design. After incubation overnight at 4°C, the plates were then washed three times each with 200 µL of wash buffer (PBST). Add 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), to each well of the plate and incubate at room temperature for 1 hour, protected from light exposure. Then wash the plate three times each with 200 µL of washing buffer (PBST). Freshly prepare the HRP substrate (Amplex Red and H2O2) 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. Add 100 μL of the prepared HRP substrate to each well and incubate 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, respectively. The average readings from the combined human serum and serum samples from 8 healthy donors served as controls. “Control %” was calculated as (the reading of the AD sample) divided by (the average reading of the combined human serum and serum from 8 healthy donors), see Figure 8. The tables and bar graphs in Figure 8 are based on the readings in Figures 9B-9C.

[0366] As can be seen from Figures 9B-9C, the binding assays worked very well, as 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).

[0367] As can be seen from Figures 8, 9B, and 9C, approximately 70% of AD patients had lower serum anti-Aβ autoantibody levels compared to controls, and approximately 86% had lower levels of these levels.0.7% of AD patients had lower serum SHD catalytic antibody levels compared to controls, which recognizes the EAR 3-peptide. These data suggest that the co-reduction of serum anti-Aβ autoantibodies and SHD catalytic antibodies can serve as a good biomarker for the diagnosis and prognosis of AD.

[0368] Sequence Listing

[0369] SEQ ID NO: 1 (Substrate peptide amino acid sequence; AMC = 7-amino-4-methylcoumarin)

[0370] EAR-AMC

[0371] SEQ ID NO: 2 (Substrate peptide amino acid sequence; n = 1-30)

[0372] (EAR)n

[0373] SEQ ID NO: 3 (Substrate peptide amino acid sequence)

[0374] EAREAREAR

[0375] SEQ ID NO: 4 (Anti-Aβ catalytic antibody 3D6 VH amino acid sequence; CDR is underlined)

[0376] EVKLVESGGGLVKPGASLKLSCAASGFTFSNYGMSWVRQNSDKRLEWVASIRSGGGRTYYSDNVKGRFT ISRENAKNTLYLQMSSLKSEDTALYYCVRYDHYSGSSDYWGQGTTVTVSS Instructions 52 / 56 Page 55 CN 121248772 A

[0377] SEQ ID NO: 5 (Anti-Aβ catalytic antibody 3D6-D VL amino acid sequence; CDR is underlined)

[0378] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTG SGSGTDFTLKISRIEAEDLGLYYCWQGTHFPRTFGGGTKLEIKR

[0379] SEQ ID NO: 6 (Anti-Aβ catalytic antibody hu3D6-D VHv1 “H1” amino acid sequence; CDR is underlined)

[0380] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPDKRLEWVASIRSGGGRTYYSDNVKGRFT ISRDNAKNTLYLQMNSLRAEDTALYYCVRYDHYSGSSDYWGQGTLVTVSS

[0381] SEQ ID NO: 7 (Anti-Aβ catalytic antibody hu3D6-D VLv1 "L1" amino acid sequence; CDR is underlined)

[0382] DVVMTQSPLSLPVTLGEPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGGGTKLEIKR

[0383] SEQ ID NO: 8 (Anti-Aβ catalytic antibody hu3D6-D VLv2 "L2" amino acid sequence; CDR is underlined)

[0384] DVVMTQSPLSLPVTLGEPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSG SGSGTDFTLKISRIEAEDVGLYYCWQGTHFPRTFGGGTKLEIKR

[0385] SEQ ID NO: 9 (Anti-Aβ catalytic antibody HC-CDR1 amino acid sequence)

[0386] NYGMS

[0387] SEQ ID NO: 10 (Anti-Aβ catalytic antibody HC-CDR2 amino acid sequence)

[0388] SIRSGGGRTYYSDNVKG

[0389] SEQ ID NO: 11 (Anti-Aβ catalytic antibody HC-CDR3 amino acid sequence)

[0390] YDHYSGSSDY

[0391] SEQ ID NO: 12 (Anti-Aβ catalytic antibody LC-CDR1 amino acid sequence)

[0392] KSSQSLLDSDGKTYLN

[0393] SEQ ID NO: 13 (Anti-Aβ catalytic antibody LC-CDR2 amino acid sequence)

[0394] LVSKLDS

[0395] SEQ ID NO: 14 (Anti-Aβ catalytic antibody LC-CDR3 amino acid sequence)

[0396] WQGTHFPRT

[0397] SEQ ID NO: 15 (IgG1 heavy chain constant region amino acid sequence)

[0398] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0399] SEQ ID NO:16 (IgG4 heavy chain constant region amino acid sequence)

[0400] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK

[0401] SEQ ID NO: 17 (Light chain κ constant region amino acid sequence)

[0402] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Specification 53 / 56 pages 56 CN 121248772 A

[0403] SEQ ID NO: 18 (Amino acid sequence of light chain λ constant region)

[0404] QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0405] SEQ ID NO: 19 (Amino acid sequence of anti-Aβ catalytic antibody hu3D6-D VHv2 “H2”; CDR is underlined)

[0406] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQASDKRLEWVASIRSGGGRTYYSDNVKGRFT ISRDNSKNTLYLQMNSLRAEDTALYYCVRYDHYSGSSDYWGQGTLVTVSS

[0407] SEQ ID NO: 20 (Anti-Aβ catalytic antibody hu3D6-D VHv3 "H3" amino acid sequence; CDR is underlined)

[0408] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQNSDKRLEWVASIRSGGGRTYYSDNVKGRFT ISRDNSKNTLYLQMNSLRAEDTALYYCVRYDHYSGSSDYWGQGTLVTVSS

[0409] SEQ ID NO: 21 (Anti-Aβ catalytic antibody hu3D6-D VLv3 "L3" amino acid sequence; CDR is underlined)

[0410] DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGGGTKVEIKR

[0411] SEQ ID NO: 22 (Anti-Aβ catalytic antibody hu3D6-D VLv4 "L4" amino acid sequence; CDR is underlined)

[0412] DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPDRFSG SGSGTDFTLKISRIEAEDVGVYYCWQGTHFPRTFGGGTKVEIKR

[0413] SEQ ID NO: 23 (Anti-Aβ catalytic antibody 3D6 VL amino acid sequence; CDR is underlined)

[0414] YVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTG SGSGTDFTLKISRIEAEDLGLYYCWQGTHFPRTFGGGTKLEIKR

[0415] SEQ ID NO: 24 (Bepinizumab VH amino acid sequence; CDR is underlined)

[0416] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFT ISRDNAKNSLYLQMNSLRAEDTALYYCVRYDHYSGSSDYWGQGTLVTVSS

[0417] SEQ ID NO: 25 (Bepipinzamab VL amino acid sequence; CDR is underlined)

[0418] YVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIKR

[0419] The relevant information (English version) for SEQ ID NO: 1 and SEQ ID NO: 2 is as follows:

[0420] <210> 1

[0421] <211> 3

[0422] <212> PRT

[0423] <213> Artificial Sequence

[0424] <220>

[0425] <223> Synthetic Construct

[0426] <220>

[0427] <221> VARIANT

[0428] <222> 3

[0429] <223> Conjugated to 7‑amino‑4‑methylcoumarin

[0430] <400> 1

[0431] Glu Ala Arg

[0432] 1

[0433] <210> 2. Instruction manual, pages 54 / 56, 57 CN 121248772 A

[0434] <211> 3

[0435] <212> PRT

[0436] <213> Artificial Sequence

[0437] <220>

[0438] <223> Synthetic Construct

[0439] <220>

[0440] <221> VARIANT

[0441] <222> (1) ...(3)

[0442] <223> Can be present in repeats of 1‑30

[0443] <400> 2

[0444] Glu Ala Arg

[0445] 1

[0446] The relevant information of SEQ ID NO: 1 and SEQ ID NO: 2 (corresponding Chinese versions; as disclosed in the parent application of this application) is as follows:

[0447] <210> 1

[0448] <211> 3

[0449] <212> PRT

[0450] <213> Artificial sequence

[0451] <220>

[0452] <223> Synthetic construct

[0453] <220>

[0454] <221> Variant

[0455] <222> 3

[0456] <223> Conjugated with 7-amino-4-methylcoumarin

[0457] <400> 1

[0458] Glu Ala Arg

[0459] 1

[0460] <210> 2

[0461] <211> 3

[0462] <212> PRT

[0463] <213> Artificial sequence

[0464] <220>

[0465] <223> Synthetic constructs

[0466] <220>

[0467] <221> Variant

[0468] <222> (1) ...(3)

[0469] <223> It can exist in 1-30 repeating sequences

[0470] <400> 2

[0471] Glu Ala Arg Specification 55 / 56 pages 58 CN 121248772 A

[0472] 1. Instruction manual, pages 56-56, 59 CN 121248772 A, Figure 1A, Figure 1B; Instruction manual, Figure 1 / 8, page 60 CN 121248772 A, Figure 2; Instruction manual, Figure 2 / 8, page 61 CN 121248772 A, Figure 3; Instruction manual, Figure 3 / 8, page 62 CN 121248772 A, Figure 4, Figure 5; Instruction manual, Figure 4 / 8, page 63 CN 121248772 A, Figure 6, Figure 7; Instruction manual, Figure 5 / 8, page 64 CN 121248772 A, Figure 8; Instruction manual, Figure 6 / 8, page 65 CN 121248772 A, Figure 9A, Figure 9B; Instruction manual, Figure 7 / 8, page 66 CN 121248772 A, Figure 9C; Instruction manual, Figure 8 / 8, page 67 CN 121248772 A. Abstract: The present application provides methods, compositions, and kits for determining SHD catalytic antibody levels in biological samples and for treating... or preventing protein aggregation disease (PAD) in an individual Catalytic antibodies that specifically recognize amyloid β(A β) peptide and methods of using the same are also provided.

Claims

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

2. The method according to claim 1, wherein n is 3.

3. The method according to claim 1 or 2, wherein the biological sample is a serum sample.

4. The method of claim 3, wherein the serum sample contains at least about 1 μg / mL of immunoglobulin (Ig).

5. The method according to any one of claims 1 to 4, wherein the amount of the catalytic antibody-substrate peptide complex is determined using an antibody that specifically binds to total Ig.

6. The method of claim 5, wherein the antibody is labeled with an enzyme or a fluorescent label.

7. Use of a therapeutically catalytic antibody that specifically binds to a target protein in the preparation of a medicament for the treatment or prevention of PAD in an individual, wherein the PAD is associated with the aggregation of the target protein, and the treatment or prevention comprises: a) Determining the risk of the individual having the PAD using the method of any one of claims 1 to 6; and b) Administer an effective amount of a therapeutic catalytic antibody that specifically binds to the target protein to the individual.

8. Use of anti-Aβ catalytic antibody in the preparation of a medicament for the treatment or prevention of Alzheimer's disease in an individual, including administering an effective amount of anti-Aβ catalytic antibody to said individual.

9. An isolated anti-Aβ catalytic antibody, comprising: Includes 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, or variants thereof, wherein the LC-CDR contains up to about 5 amino acid substitutions; and / or 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; 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 Kabat EU index; or Includes 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; 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; or 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 ;or 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 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 ;or 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 ;or 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 ;or 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 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 .

10. A method for panning a phage library of 3D6-D variants, the method comprising: Aβ-selection of racial V with 3D6-D H and humanized V L A 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 EAR-AMC catalytic function assays; and the selected anti-Aβ scFv and Fab were used to prepare full-length IgG catalytic antibodies.

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