Methods to inhibit fibril growth

Abstract

This disclosure relates, in some embodiments, to methods and compositions for reducing or inhibiting the growth of amyloid fibrils.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 520,007, filed on 16 August 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Submission of the sequence list in ASCII text file. The following submission in ASCII text file is incorporated herein by reference in its entirety: a computer-readable format (CRF) sequence listing (filename: 165992001340seqlist.xml, date: August 2, 2024, size: 82KB).

[0003] This disclosure relates, in some aspects, to methods for preventing or reducing fibril growth. [Background technology]

[0004] Amyloidosis is a fatal protein folding disorder characterized by the aggregation and deposition of protein fibrils and heparan sulfate proteoglycans in essential organs and tissues, including the kidneys, pancreas, liver, spleen, nerve cells, and heart. Amyloid accumulation leads to organ failure and severe morbidity or death. Transthyretin-related (ATTR) amyloidosis and light chain-related (AL) amyloidosis are the two most common systemic amyloidosis diseases, with a median survival time of less than two years. The median survival time for patients diagnosed with severe cardiac AL amyloidosis is less than five months. This is due to the inability to diagnose the disease before rapid organ destruction and organ failure occur, and the lack of effective anti-amyloid therapies.

[0005] To date, no therapies that remove amyloid are available clinically. However, numerous amyloid-reactive antibodies, including anceramimab (CAEL101), virtamimab (NEOD001), dezamizumab (GSK2398852), solanezumab, PRX004, NI006, bapinuzumab, and aducumumab, have been evaluated in clinical trials. Each of these therapies has limitations and / or failed to meet primary outcomes in late-stage clinical trials (Phase 2 / 3). Furthermore, these therapies are specific to a single type of amyloid (e.g., AL or ATTR) and provide limited efficacy in patients with many other types of amyloidosis. Therefore, there remains an urgent need for effective and clinically approved therapies that can address amyloidosis and amyloid-related diseases. [Overview of the project]

[0006] In some embodiments, provided herein is a method for reducing or preventing fibril growth in an individual at risk of developing an amyloid-related disease, comprising administering to the individual a therapeutically effective amount of an antibody-peptide fusion protein, wherein the antibody-peptide fusion protein comprises (i) an amyloid-reactive peptide and (ii) an antibody that binds to amyloid fibrils, the antibody comprising a heavy chain and a light chain, the amyloid-reactive peptide and the antibody linked at the C-terminus of the light chain, and the amyloid-reactive peptide linked to the antibody via a spacer.

[0007] In other embodiments, the foregoing provides a method for reducing or delaying the progression of amyloid-related disease in an individual diagnosed with amyloid disease, comprising administering to the individual a therapeutically effective amount of an antibody-peptide fusion protein, the antibody-peptide fusion protein comprising (i) an amyloid-reactive peptide and (ii) an antibody that binds to amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

[0008] In some embodiments, the amyloid fibril comprises amyloid-forming λ6 variable domain protein (Vλ6Wil) or amyloid-forming immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibril or amyloid-forming Aβ precursor protein, or serum amyloid protein A (AA).

[0009] In some embodiments, amyloid fibrils include amyloid-forming immunoglobulin heavy chains (AH), β2-microglobulin (Aβ2M), trans tyretin (ATTR wild-type; ATTP variant), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemochemoattractant (ALect2), fibrinogen A variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP); α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALβ4, or ALβ1.

[0010] In some embodiments, fibril growth is measured via an in vitro rVλ6WIL(WIL) fibril elongation assay.

[0011] In some embodiments, the antibody-peptide fusion protein reduces the growth of rVλ6WIL fibrils by at least about 20%. In some embodiments, the antibody-peptide fusion protein reduces the growth of rVλ6WIL fibrils by about 33%. In some embodiments, the antibody-peptide fusion protein reduces the growth rate of rVλ6WIL fibrils by at least about 20%. In some embodiments, the antibody-peptide fusion protein reduces the growth rate of rVλ6WIL fibrils in rVλ6WIL fibril elongation assays by about 33%. In some embodiments, the antibody-peptide fusion protein reduces the total amount of rVλ6WIL fibrils by at least about 20%. In some embodiments, the antibody-peptide fusion protein reduces the total amount of rVλ6WIL fibrils by about 33%.

[0012] In some embodiments, amyloid-related disease is systemic or focal amyloidosis. In some embodiments, amyloid-related disease is selected from the group consisting of AL, AH, Aβ2M, ATTRv, AATRwt, AA, AApoAI, AApoAII, AGel, Alys, ALECT2, AFib, ACys, ACa, AMed, AIAPP, APro, AIns, APrP, or Aβ amyloidosis.

[0013] In some embodiments, the individual has a genetic predisposition to amyloid-related disease. In some embodiments, the individual has a family history of amyloid-related disease. In some embodiments, the individual has early-stage amyloid-related disease. In some embodiments, the individual has early-stage AL amyloidosis. In some embodiments, early-stage AL amyloidosis is diagnosed according to the Mayo Clinic system. In some embodiments, early-stage AL amyloidosis is stage 1 AL amyloidosis.

[0014] In some embodiments, the individual is in the early stages of ATTR amyloidosis. In some embodiments, the early stages of ATTR amyloidosis include stage 1 ATTR amyloidosis.

[0015] In some embodiments, the antibody light chain includes a light chain constant region, and the antibody heavy chain includes a heavy chain constant region.

[0016] In some embodiments, the spacer is selected from the group consisting of sequence numbers 23-24, 27, and 83-86. In some embodiments, the spacer is selected from the group consisting of sequence number 83 and sequence number 86.

[0017] In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 13, which includes 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions.

[0018] In some embodiments, the antibody-peptide fusion protein comprises two heavy chains and two light chains, each light chain being linked to an amyloid-reactive peptide at its C-terminus.

[0019] In some embodiments, the antibody is a chimeric antibody or a humanized antibody.

[0020] In some embodiments, the antibody light chain comprises a light chain variable region (VL) comprising CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and the antibody heavy chain comprises a heavy chain variable region (VH) comprising CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19.

[0021] In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 36, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 55.

[0022] In some embodiments, the antibody is a full-length antibody comprising an Fc region. In some embodiments, the Fc region is of an IgG1 isotype.

[0023] In some embodiments, the antibody-peptide fusion protein comprises (i) an amyloid-reactive peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) an antibody that binds to human amyloid fibrils, the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprises a heavy chain variable region (VH), and the light chain of the antibody comprises a light chain variable region (VL), the VH comprises CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 19, the VL comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 22, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 24, 27, and 83 to 86.

[0024] In some embodiments, the antibody-peptide fusion protein comprises (i) an amyloid-reactive peptide comprising the amino acid sequence shown in SEQ ID NO: 2, and (ii) an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH), and the light chain of the antibody comprising a light chain variable region (VL), the VH comprising CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 19, the VL comprising CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 22, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer comprising the amino acid sequence described in SEQ ID NO: 83.

[0025] In some embodiments, the antibody-peptide fusion protein comprises (i) an amyloid-reactive peptide having the amino acid sequence shown in SEQ ID NO: 2, and (ii) an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH) having VH CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 55, and the light chain of the antibody comprising a light chain variable region (VL) having VL CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 36, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer having the amino acid sequence shown in SEQ ID NO: 83.

[0026] In some embodiments, the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide, each containing the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each containing the heavy chain of the antibody, wherein each of the first polypeptide and the second polypeptide contains the amino acids shown in SEQ ID NO: 89, and each of the third polypeptide and the fourth polypeptide contains the amino acid sequence shown in SEQ ID NO: 91.

[0027] In some embodiments, the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide each comprising an antibody light chain, and a third polypeptide and a fourth polypeptide each comprising an antibody heavy chain, wherein each of the first polypeptide and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 89, and each of the third polypeptide and the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 93.

[0028] In some embodiments, the individual is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings illustrate certain features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. [Figure 1] A shows an exemplary amyloid-reactive peptide. B shows an exemplary amyloid-binding antibody-peptide fusion. C shows an exemplary amyloid-binding antibody. [Figure 2] A shows the results of an rVλ6WIL fibril elongation assay using biotinylated rVλ6WIL monomer mixed with p5R (SEQ ID NO: 2), an amyloid-binding peptide. Quantification of bound rVλ6WIL (fmoles europium) is shown for a positive control sample containing only rVλ6WIL fibrils and biotinylated rVλ6WIL monomers, and for test wells containing fibrils, monomers, and various concentrations of the amyloid-binding peptide (2, 0.5, 0.05, 0.01, 0.005 and 0.001 μM). B shows the results of an rVλ6WIL fibril elongation assay using biotinylated rVλ6WIL monomer mixed with p5R (SEQ ID NO: 2), an amyloid-binding peptide. The rate of change of rVλ6WIL fibril elongation relative to a control sample is shown for each concentration of amyloid-binding peptide tested in Figure 2A. [Figure 3]A shows the results of an rVλ6WIL fibril elongation assay using biotinylated rVλ6WIL monomers mixed with rVλ6WIL fibrils in the presence or absence of amyloid-binding antibody-peptide fusion protein. It shows the quantification of bound rVλ6WIL (fmoles europium) in a positive control sample without amyloid-binding antibody-peptide fusion protein, and in test wells containing various concentrations of amyloid-binding antibody-peptide fusion protein (2, 0.5, 0.05, 0.01, 0.005, and 0.001 μM). B shows the results of an rVλ6WIL fibril elongation assay using biotinylated rVλ6WIL monomers mixed with rVλ6WIL fibrils in the presence or absence of amyloid-binding antibody-peptide fusion protein. It shows the percentage change in rVλ6WIL fibril elongation compared to the control sample. [Figure 4] A shows the results of an rVλ6WIL fibril elongation assay using biotinylated rVλ6WIL monomers mixed with rVλ6WIL fibrils in the presence or absence of amyloid-binding antibody. It shows the quantification of bound rVλ6WIL (fmoles europium) in a positive control sample without amyloid-binding antibody, and in test wells containing various concentrations of amyloid-binding antibody (2, 0.5, 0.05, 0.01, 0.005, and 0.001 μM). B shows the results of an rVλ6WIL fibril elongation assay using biotinylated rVλ6WIL monomers mixed with rVλ6WIL fibrils in the presence or absence of amyloid-binding antibody. It shows the percentage change in rVλ6WIL fibril elongation compared to the control sample. [Modes for carrying out the invention]

[0030] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated in their entirety for all purposes, just as each individual publication is incorporated herein by reference individually. If any definition described herein contradicts or otherwise conflicts with any definition described in any patent, application, publication, or other publication incorporated herein by reference, the definition described herein shall prevail over the definition incorporated herein by reference.

[0031] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described.

[0032] I. Overview Provided herein are methods for reducing or preventing fibril growth in an individual at risk of developing amyloid-related disease, comprising administering an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein comprises an amyloid-reactive peptide and an antibody that binds to amyloid fibrils. In some embodiments, the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked, for example, at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

[0033] In some embodiments, provided herein is a method for reducing or preventing fibril growth in an individual at risk of developing an amyloid-related disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein comprises (i) an amyloid-reactive peptide and (ii) an antibody that binds to amyloid fibrils, the antibody comprising a heavy chain and a light chain, the amyloid-reactive peptide and the antibody linked at the C-terminus of the light chain, and the amyloid-reactive peptide linked to the antibody via a spacer.

[0034] In some embodiments, provided herein are methods for reducing or delaying the progression of amyloid-related disease in an individual diagnosed with amyloid disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein comprises (i) an amyloid-reactive peptide and (ii) an antibody that binds to amyloid fibrils, the antibody comprising a heavy chain and a light chain, the amyloid-reactive peptide and the antibody linked at the C-terminus of the light chain, and the amyloid-reactive peptide linked to the antibody via a spacer.

[0035] II. Definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and plural forms unless the context explicitly indicates otherwise. The abbreviation "eg" derives from the Latin "exempli gratia" and is used herein to indicate non-restrictive examples. Thus, the abbreviation "eg" is synonymous with the term "for example." As used herein, the term "comprises" means "includes."

[0036] In this specification, a range can be expressed as a range from one specific value that is "about" and / or from another specific value that is "about". Where such a range is expressed, another aspect includes from one specific value of the range and / or from the other specific value of the range. Furthermore, it is understood that each endpoint of a range has meaning both in relation to the other endpoints and independently of the other endpoints. Similarly, where a value is expressed as an approximation using the preceding "about", it is understood that a specific value forms another aspect. In certain exemplary embodiments, the term "about" is understood to mean within the normal tolerance range in the art, for example, within two standard deviations of the mean. About may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the value being stated. Unless otherwise specified in the context, all numerical values ​​provided herein may be modified by the term "about". Furthermore, terms used herein such as “example,” “exemplary,” or “illustrated” do not imply preference, but rather are intended to indicate that the embodiments discussed thereafter are merely examples of the embodiments shown.

[0037] Furthermore, it should be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximations and are provided for illustrative purposes only. Similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are described below. In case of any inconsistency, this specification shall prevail, including the explanation of terms. In addition, the materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0038] To facilitate a review of the various embodiments of this disclosure, explanations of specific terms are provided below.

[0039] The terms "amyloid," "amyloid deposits," "amyloid fibrils," and "amyloid fibers" refer to insoluble fibrous protein aggregates that share specific structural characteristics. Protein aggregates are formed, for example, by the aggregation of any one of several different proteins and have a tertiary structure consisting of an ordered arrangement of β-sheets stacked perpendicular to the fiber axis. See Sunde et al., J.Mol.Biol.(1997)273:729-39. Abnormal accumulation of amyloid in organs can lead to amyloidosis. Although their development is diverse, all amyloids share common morphological characteristics in that they stain with certain dyes, such as Congo red, and after staining, have a characteristic red-green birefringent appearance under polarized light. Amyloids also share common suprastrumatic features, as well as common X-ray diffraction and infrared spectra.

[0040] The term "amyloidosis" refers to a pathological condition or disease characterized by the presence of amyloid, for example, the presence of amyloid deposits. "Amyloid disease" or "amyloidosis" is a disease associated with the formation, deposition, accumulation, or persistence of amyloid fibrils. Such diseases include, but are not limited to, AA amyloidosis, AL amyloidosis, ATTR amyloidosis, ALECT2 amyloidosis, and AIAPP amyloidosis in type II diabetes.

[0041] The term "amyloidogenicity" refers to the production or tendency to produce amyloid deposits. For example, certain soluble monomeric proteins can undergo extensive conformational changes, aggregating into aligned, unbranched fibrils 8-10 nm wide, leading to the formation of amyloid aggregates. For instance, more than 30 proteins have been found to form amyloid deposits (or amyloid) in humans. Not all proteins in a diverse class of proteins, such as immunoglobulin light chains, are capable of forming amyloid. That is, some immunoglobulin light chains are non-amyloidogenic, meaning they have no tendency to form amyloid. However, other proteins in this class are amyloidogenic because they can form amyloid deposits. Furthermore, within the class of light chain proteins, some may be considered more "amyloidogenic" than others based on their ease of forming amyloid fibrils. Certain light chain proteins are considered non-amyloidogenic or hypoamyloidogenic because they do not readily form amyloid fibrils in patients or in vitro.

[0042] The terms “reduce (remove)” or “reduce (remove)” refer to reducing or removing to a measurable degree. For example, the clearance of amyloid deposits as described herein refers to reducing or removing the deposit to a measurable or identifiable degree. Clearance may result in 100% removal, but is not required. Rather, clearance may result in less than 100% removal, such as approximately 10%, 20%, 30%, 40%, 50%, 60% or more.

[0043] As used herein, the term “conjugate” refers to a coupling or linking product of two or more substances, the resulting product having at least two distinguishable elements, e.g., at least two domains. The linked materials may be the same or different. Such coupling may be via one or more linking groups. For example, a “protein conjugate” results from the coupling of two or more amino acid sequences. A conjugate of two proteins results in a single protein having domains corresponding to each of the individually linked proteins.

[0044] In this specification, the term “antibody” is used in its broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit the desired biological activity.

[0045] An “isolated” antibody is one that has been identified, separated, and / or recovered from components of its natural environment. Contaminations from that natural environment are materials that would interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to (1) more than 95% by weight of the antibody, and in some embodiments more than 99% by weight, as determined, e.g., by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, e.g., using a spinning cup sequencer; or (3) to a degree until homogeneity is obtained by SDS-PAGE under reducing or non-reducing conditions, e.g., using Coomassie blue or silver staining. An isolated antibody includes antibodies in their in-vivo location within recombinant cells, because at least one component of the antibody's natural environment is absent. However, typically, isolated antibodies are prepared by at least one purification step.

[0046] "Natural antibodies" are typically heterotetrameric glycoproteins with approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide links differs between heavy chains of different immunoglobulin isotypes. Each heavy chain, and the light chains, also have intrachain disulfide bridges at regularly spaced intervals. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the variable domain of the light chain aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain.

[0047] The term "constant region" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the variable region, which contains the antigen-binding site, and is the other part of the immunoglobulin. The constant region contains the CR1, CR2, and CR3 domains of the heavy chain (also referred to as CH1, CH2, and CH3; collectively CH) and the CHL (or CL or CL1) domain of the light chain.

[0048] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody's heavy or light chain. The variable domain of the heavy chain may be denoted as "VH," and the variable domain of the light chain may be denoted as "VL." These domains are typically the most variable parts of the antibody and contain the antigen-binding site.

[0049] The term "variable" refers to the fact that the sequence of a particular portion of the variable domain differs significantly between antibodies, and this is used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The native heavy and light chain variable domains each contain four FR regions that largely employ a beta-sheet configuration, connected by three CDRs that link beta-sheet structures and, in some cases, form loops that form part of the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md, (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0050] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domains.

[0051] The terms IgG “isotype” or “subclass,” as used herein, mean any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

[0052] Depending on the amino acid sequence of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional arrangements of various classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed. (WBSaunders, Co., 2000). Antibodies may also be part of a larger fusion molecule formed by the covalent or noncovalent association of an antibody with one or more other proteins or peptides.

[0053] The terms “full-length antibody,” “intact antibody,” and “complete antibody” are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody-fragment as defined below. These terms specifically refer to antibodies having a heavy chain containing an Fc region.

[0054] As used herein, "exposed antibody" for the purposes of this specification is an antibody that is not conjugated to a cytotoxic site or radiolabel.

[0055] An "antibody fragment" comprises a portion of an intact antibody, preferably including its antigen-binding region. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0056] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments (each with a single antigen-binding site) and the remaining "Fc" fragment (its name reflects its ability to easily crystallize). Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of crosslinking antigens.

[0057] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy chain and one light chain variable domain in a tight non-covalent association. In a single-chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain may be covalently linked by a soft peptide linker so that the light and heavy chains can relate in a "dimer" structure similar to that in a two-chain Fv species. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In total, six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0058] The Fab fragment contains light chain and heavy chain variable domains, as well as the constant domain of the light chain and the first constant domain (CHI.) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is a designation used herein for Fab' fragments in which the cysteine ​​residue(s) of the constant domain possess a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0059] "Single-stranded Fv" or "scFv" antibody fragments contain the VH and VL domains of the antibody, and these domains are located within a single polypeptide chain. Typically, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see, for example, Pluckfhun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0060] When used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, where, for example, the individual antibodies constituting the population are identical except for any mutations that may exist in small amounts, such as naturally occurring mutations. Thus, the modifier “monoclonal” indicates a characteristic of the antibody that it is not a mixture of distinct antibodies. In certain embodiments, such a monoclonal antibody typically comprises an antibody containing a polypeptide sequence that binds to a target, and the target-binding polypeptide sequence is obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be the selection of a unique clone from a plurality of clones, such as a hybridoma clone, a phage clone, or a pool of recombinant DNA clones. It should be understood that the selected target-binding sequence may be further modified, for example, to improve affinity to the target, humanize the target-binding sequence, improve its generation in cell culture, reduce its immunogenicity in vivo, or produce a multispecific antibody, and that an antibody containing a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to its specificity, monoclonal antibody preparations are typically uncontaminated with other immunoglobulins.

[0061] The term "amyloid-reactive antibody" refers to an antibody that binds to amyloid. In this specification, the terms "amyloid-reactive antibody" and "antibody that binds to amyloid fibril" are used interchangeably.

[0062] The term "amyloid-reactive peptide" refers to peptides that bind to any of the following amyloid fibrils, for example, but not limited to, amyloid-forming λ6 variable domain protein (Vλ6Wil) or amyloid-forming immunoglobulin light chain (AL), A13(1-40) amyloid-like fibrils or amyloid-forming AP precursor protein, or serum amyloid protein A (AA). In other embodiments, the amyloid to which the antibody-peptide fusion protein binds includes amyloid-forming forms of immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), trans tyretin variant (ATTR), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemochemoattractant (ALect2), fibrinogen A variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP); α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALκ4, Alλ1, and other amyloid-forming peptides.

[0063] The modifier "monoclonal" indicates a characteristic of antibodies that are obtained from a population of substantially identical antibodies, and should not be interpreted as requiring the production of antibodies by any specific method. For example, monoclonal antibodies used in accordance with the present invention include, for example, hybridoma methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 1.4(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, U.S. Patent No. 4,8.16,567), and phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al. al,J.Mol.Biol.222:581-597(1992);Sidhu et al,J.Mol.Biol.338(2):299-310(2004);Lee et al. al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004);and Lee et al.,J.Immunol.Methods See 284(1-2):119-132(2004), and techniques for generating human or human-like antibodies in animals having a human immunoglobulin locus or part or all of a human immunoglobulin gene encoding a human immunoglobulin sequence (e.g., WO1998 / 24893;WO1996 / 34096;WO1996 / 33735;WO1991 / 10741;Jakobovits 11 et al, Proc. Natl. Acad. Sci.USA 90:2551 (1993); Jakobovits et al, Nature 362:255-258 (1993); Bruggemann et al, Year in Immunol 7:33 (1993); US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425 and 5,661,016; Marks et al, Bio / Technology 10:779-783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al, Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature It can be produced by a variety of technologies, including Biotechnol 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol 13:65-93 (1995).

[0064] Monoclonal antibodies, as used herein, specifically include “chimeric” antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies insofar as they exhibit the desired biological activity (see, for example, U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include primate-like antibodies in which the antigen-binding region of the antibody is derived, for example, from an antibody produced by immunizing a macaque monkey with the target antigen.

[0065] Humanized non-human (e.g., mouse) antibodies are chimeric antibodies containing the smallest sequence derived from non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's CDR are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capabilities. In some examples, the FR residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications may be made to further improve antibody performance. Typically, the humanized antibody will contain substantially all of at least one, usually two, variable domains, in which case all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the FRs are from the human immunoglobulin sequence. Humanized antibodies will also optionally include the constant region (Fc) of immunoglobulins, typically at least a portion of the Fc of human immunoglobulins. For further details, see, for example, Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0066] A “human antibody” is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or one produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. (Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991); Marks et al, J.Mol.Biol., 222:581 (1991)). For the preparation of human monoclonal antibodies, the method described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al, J.Immunol., 147(l):86-95 (1991) is also available. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to transgenic animals, such as immunized xenomous mice, which have been modified to produce such antibodies in response to antigen loading, but whose endogenous gene locus has been deactivated (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 relating to XENOMOUSE® technology). See also, for example, Li et al, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0067] The term “complementarity-determining region” or “CDR,” as used herein, refers to the region of the antibody-variable domain that binds to an epitope, e.g., human amyloid fibril. Generally, antibodies contain six CDRs, three located in VH (H1, H2, H3) and three in VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the highest diversity among the six CDRs, and H3, in particular, is thought to play a unique role in conferring superior specificity to the antibody. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. For example, see Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Structs. Biol. 3:733-736 (1996).

[0068] Numerous CDR descriptions are used and incorporated herein. In some embodiments, the CDR may be a Kabat CDR, which is most commonly used based on sequence variability (Kabat et al. (cited above)). In some embodiments, the CDR may be a Chothia CDR, where Chothia instead refers to the location of a structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some embodiments, the CDR may be an AbM CDR, representing a compromise between the Kabat CDR and the Chothia structural loop and used by Oxford Molecular's AbM antibody modeling software. In some embodiments, the CDR may be a "contact" CDR, based on the analysis of available complex crystal structures. Residues from each of these CDRs are described below. [Table 1]

[0069] The CDR may include “extended CDRs” as follows: in the VL, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3); and in the VH, 26-35 (H1), 50-65 or 49-65 (preferred embodiment) (H2), and 93-102, 94-102, or 95-102 (H3). The variable domain residues are numbered for each of these extended CDR definitions according to Kabat et al., (cited above).

[0070] "Framework" or "FR" residues are variable domain residues other than the CDR residues as defined herein.

[0071] As used herein, the terms “specifically bind to” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, that determine the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more easily, and / or for a longer duration than when it binds to other molecules. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of its binding to the target, for example, as measured by radioimmunoassay (RIA). In a given embodiment, an antibody that specifically binds to a target has a dissociation constant (Kd) of <1 μM, <100 μM, <10 nM, <1 nM, or <0.1 nM. In a given embodiment, the antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In another embodiment, specific binding may include, but is not required, exclusive binding.

[0072] The terms “effective dose” or “therapeutic effective dose,” as used herein, refer to an amount of an agent sufficient to prevent, treat (including prevention), alleviate and / or improve any symptom and / or underlying cause of any disorder or disease, for example, an amount of an agent sufficient to prevent, inhibit and / or improve amyloidosis. In some embodiments, an “effective dose” is sufficient to reduce or resolve the symptoms of a disease. An effective dose may be administered in one or more doses. For example, an effective dose of a peptide is an amount sufficient to bind to amyloid. A peptide may be effective when administered parenterally, for example, in amounts greater than about 1 μg and up to about 30 mg / kg of body weight.

[0073] As used herein, the term “inhibit” means to reduce to a measurable degree. Inhibition does not require, for example, a complete loss or complete cessation of the function of the measured embodiment. For example, inhibiting fibril formation may mean stopping further growth of fibrils, slowing further proliferation of fibrils, or reducing the size of fibrils.

[0074] With respect to amyloid fibril formation, "reducing fibril growth" refers to a reduction in the rate of fibril formation or a reduction in the total amount of amyloid fibrils. For example, a reduction in fibril growth may result in a reduction of approximately 10%, 20%, 30%, 40%, 50%, or 60% or more of amyloid deposits compared to a control. Furthermore, a reduction in fibril growth may result in a reduction of approximately 10%, 20%, 30%, 40%, 50%, or 60% or more of the rate of fibril formation compared to a control. As used herein, "preventing fibril growth" refers to preventing further fibril growth after administration of the antibody-peptide fusion protein disclosed herein.

[0075] The term "labeled" refers to any detectable compound or composition that is directly or indirectly conjugated to another molecule to facilitate its detection. Specific, non-limiting examples of labeling include fluorescent tags, chemiluminescent tags, haptens, enzyme conjugates, and radioisotopes. For example, a protein that is "labeled for detectability" means that the presence of the protein can be determined by the label associated with it.

[0076] "Isolated" biological components, such as peptides (e.g., one or more of the peptides disclosed herein), cells, nucleic acids, or serum samples, are substantially separated, separately produced, or purified from other biological components within the cells of organisms in which they naturally occur, such as other chromosomal DNA and extrachromosomal DNA and RNA, as well as proteins. Therefore, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. This term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression within cells, as well as chemically synthesized peptides and nucleic acids. The terms "isolated" or "purified" do not require absolute purity and are intended as relative terms. Therefore, for example, an isolated peptide preparation means a preparation in which the peptide or protein is more concentrated than in its natural environment within the cell. Preferably, the preparation is purified to a peptide or protein concentration of, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even more than 99%, such that the protein or peptide accounts for at least 50% of the total peptide or total protein content in the preparation.

[0077] As used herein, the terms “to conjugate,” “conjugated,” “linked,” or “linked” refer to any method known in the art for functionally connecting proteins and / or protein domains. For example, one protein domain may be linked to another protein domain via a covalent bond, with or without intervening sequences or domains, such as in an antibody-peptide fusion protein. “Conjugated” also includes incorporating two sequences together, for example, by placing two nucleic acid sequences together on the same nucleic acid chain so that they are expressed together.

[0078] The term “nucleic acid” refers to polymers composed of nucleotide units linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-natural analogs, which include ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-natural analogs. Therefore, the term includes nucleotide polymers, including, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA), and synthetic analogs in which nucleotides and their linkages do not exist naturally. Such polynucleotides can be synthesized, for example, using automated DNA synthesizers. The term “oligonucleotide” typically refers to short polynucleotides, usually about 50 nucleotides or less. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it is understood that this also includes an RNA sequence (i.e., A, U, G, C) where “U” is replaced by “T”.

[0079] The term "nucleotide" includes, but is not limited to, monomers containing a base linked to a sugar, such as pyrimidines, purines or their synthetic analogs, or a base linked to an amino acid, such as peptide nucleic acids (PNAs). A nucleotide is a single monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0080] Conventional notation is used herein to describe nucleotide sequences: the left end of a single-stranded nucleotide sequence is the 5' end; the left direction of a double-stranded nucleotide sequence is referred to as the 5' direction. The direction of nucleotide addition from 5' to 3' to the RNA transcript being formed is referred to as the transcription direction. The DNA strand having the same sequence as mRNA is referred to as the "coding strand"; the sequence on the DNA strand having the same sequence as the mRNA transcribed from that DNA, and located 5' relative to the 5' end of the RNA transcript, is referred to as the "upstream sequence"; the sequence on the DNA strand having the same sequence as RNA, and located 3' relative to the 3' end of the coding RNA transcript, is referred to as the "downstream sequence".

[0081] The term "cDNA" refers to DNA that is complementary to or identical to mRNA in either single-stranded or double-stranded form.

[0082] The term “coding” refers to the inherent and resulting biological properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene codes for a protein if the transcription and translation of the mRNA produced by that gene produces a protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand used as a template for the transcription of a gene or cDNA can be said to code for a protein or other product of that gene or cDNA. Unless otherwise specified, “nucleotide sequence coding an amino acid sequence” includes all nucleotide sequences that denature each other's versions and code for the same amino acid sequence. Nucleotide sequences coding for proteins and RNA may contain introns.

[0083] The term "pharmaceutically acceptable carrier" refers to conventionally used pharmaceutically acceptable carriers. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995) describes compositions and formulations suitable for the pharmaceutically acceptable delivery of the fusion proteins disclosed herein.

[0084] Generally, the properties of the carrier depend on the specific mode of administration used. For example, parenteral formulations typically contain an injectable fluid with a pharmaceutically and physiologically acceptable fluid as the vehicle, such as water, saline, equilibrium salt solution, dextrose aqueous solution, or glycerol. In the case of solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the pharmaceutical composition to be administered may contain small amounts of non-toxic adjuncts, such as humectants or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0085] The term "polypeptide" refers to a polymer in which monomers are amino acid residues linked together by an amide bond. When the amino acids are α-amino acids, either L-optical isomers or D-optical isomers may be used, with L-isomers being preferred. As used herein, the terms "polypeptide" or "protein" are intended to encompass any amino acid sequence, including modified sequences such as glycoproteins. The term "polypeptide" is particularly intended to encompass naturally occurring proteins as well as proteins produced by recombinant or synthetic means.

[0086] The term "purified" does not require absolute purity; rather, it is intended as a relative term. Therefore, for example, a purified protein preparation is one in which the protein in question is purer than the protein in its natural environment, either intracellular or within a reaction chamber (where appropriate).

[0087] Regarding recombinant nucleic acids, the term "recombinant" refers to the fact that recombinant nucleic acids have sequences that do not exist in nature, or sequences created by the artificial combination of two sequence segments that are originally distinct. This artificial combination is often achieved by chemical synthesis, or more commonly, by artificially manipulating isolated segments of nucleic acids, for example, by genetic engineering techniques.

[0088] The term "sequence identity" refers to the similarity between two nucleic acid sequences or two amino acid sequences, and is also sometimes called sequence identity. Sequence identity is frequently measured in terms of the degree of identity (or similarity or homology), with a higher degree indicating greater similarity between the two sequences.

[0089] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman Adv. Appl. Math. 2:482,1981; Needleman & Wunsch J.Mol.Biol.48:443,1970; Pearson & Lipman Proc.Natl.Acad.Sci.USA 85:2444,1988; Higgins & Sharp Gene 73:237-244,1988; Higgins & Sharp CABIOS 5:151-153,1989; Corpet et al. Nuc. Acids Res.16,10881-90,1988; Huang et al. Computer Appls. In the Biosciences 8,155-65,1992; and Pearson et al. Meth.Mol.Bio.24,307-31,1994. Altschul et al. (J.Mol.Biol.215:403-410,1990) present detailed considerations for sequence alignment methods and homology calculations.

[0090] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol.Biol.215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), and on the internet for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0091] As used herein, a first nucleic acid sequence is "operably ligated" to a second nucleic acid sequence when the first nucleic acid sequence is positioned to have a functional relationship with the second nucleic acid sequence. For example, a promoter is operably ligated to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably ligated DNA sequences are adjacent to each other in the same reading frame and, if necessary, are used to link two protein-coding regions.

[0092] The term "vector" refers to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A recombinant DNA vector is a vector containing recombinant DNA. A vector may contain nucleic acid sequences that enable replication in the host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. A viral vector is a recombinant DNA vector containing at least several nucleic acid sequences derived from one or more viruses. The term vector includes plasmids, linear nucleic acid molecules, and, as described everywhere, adenovirus vectors and adenoviruses.

[0093] "Subject" or "individual" refers to a mammal, such as a human. A subject may be a human patient. A subject may be a patient who has or is suspected of having an amyloid-related disease or condition in its early stages and who may require treatment or diagnosis, or who may need monitoring for the progression of the disease or condition. The patient may also be receiving treatment that needs to be monitored for its effectiveness. In some exemplary embodiments, a subject includes an individual with systemic amyloidosis.

[0094] Preferably, non-identical residue positions are distinguished by conserved amino acid substitutions. The term “conserved amino acid substitution” refers to the interchangeability of residues having similar side chains. For example, the group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains are serine and threonine; the group of amino acids with amide-containing side chains are asparagine and glutamine; the group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains are lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. Preferred conserved amino acid substitutions include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0095] As discussed herein, small variations in the amino acid sequence of antibody or immunoglobulin molecules are intended to be included by the present invention and provide that the variation in the amino acid sequence is maintained at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid substitutions are intended. Conservative substitutions occur within a family of related amino acids in their side chains. Genetically encoded amino acids are typically divided into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) non-charged amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, independent substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similar substitutions of amino acids structurally related to an amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a framework site. Whether amino acid changes result in functional peptides can be determined by assaying the specific activity of polypeptide derivatives, assays are described in detail herein.Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino and carboxyl termini of the fragment or analog arise near the boundaries of functional domains. Structural and functional domains can be identified by comparison of nucleotide and / or amino acid sequence data against public or private sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that arise in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known (Bowie et al. Science 253:164 (1991)). Thus, the above examples demonstrate that those skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains according to the present invention.

[0096] Preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for protein complex formation, (4) alter binding affinity, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs may include various mutant proteins of sequences other than naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conserved amino acid substitutions) may occur in naturally occurring sequences (preferably a portion of a polypeptide outside of the domain(s) that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural features of the parent sequence (e.g., the substituted amino acid should not tend to break helices resulting in the parent sequence, or disrupt other types of secondary structures that characterize the parent sequence). Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0097] Except for CDR1 in VH, CDRs typically contain amino acid residues that form a hypervariable loop. CDRs also contain "specificity-determining residues" or "SDRs," which are residues that contact the antigen. SDRs are contained within a region of the CDR, called a shortened CDR or a-CDR. Exemplary a-CDRs (a-CDR-Ll, a-CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) arise from amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).)

[0098] III. Treatment Methods Provided herein are methods for reducing or preventing fibril growth in an individual at risk of developing amyloid-related disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual. Alternatively, in some embodiments, provided herein are methods for reducing or delaying the progression of amyloid-related disease in an individual diagnosed with amyloid disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual. In some embodiments, the antibody-peptide fusion protein comprises (i) an amyloid-reactive peptide and (ii) an antibody that binds to amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

[0099] This specification also provides a method for reducing or preventing fibril growth in an individual having amyloid-related disease, comprising administering the individual a therapeutically effective amount of antibody-peptide fusion protein. This specification also provides a method for reducing or preventing fibril growth in an individual in which amyloid fibrils have been identified, comprising administering the individual a therapeutically effective amount of antibody-peptide fusion protein. This specification also provides a method for reducing or preventing fibril growth in an individual in which amyloid fibrils have been identified, comprising administering the individual a therapeutically effective amount of antibody-peptide fusion protein. This specification also provides a method for reducing or preventing fibril growth in an individual diagnosed with amyloid disease, comprising administering the individual a therapeutically effective amount of antibody-peptide fusion protein.

[0100] In some embodiments, the antibody-peptide fusion protein reduces or prevents amyloid fibril growth in vitro and / or in vivo. In some embodiments, an in vitro rVλ6WIL fibril elongation assay is used to measure the effect of the antibody-peptide fusion protein on amyloid fibril growth. In some embodiments, the rVλ6WIL fibril elongation assay measures rVλ6WIL fibril elongation in the presence of the antibody-peptide fusion protein described herein. In some embodiments, the antibody-peptide fusion protein reduces rVλ6WIL fibril elongation by 10% to 50%. In some embodiments, the antibody-peptide fusion protein reduces rVλ6WIL fibril elongation by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the antibody-peptide fusion protein reduces rVλ6WIL fibril elongation by about 33%.

[0101] In some embodiments, a method for reducing or preventing fibril growth in an individual at risk of developing amyloid-related disease includes administering a therapeutically effective amount of amyloid-reactive antibody to the individual. Alternatively, in some embodiments, a method for reducing or delaying the progression of amyloid-related disease in an individual diagnosed with amyloid disease includes administering a therapeutically effective amount of amyloid-reactive antibody to the individual. In some embodiments, the amyloid-reactive antibody binds to amyloid fibrils, and the antibody comprises a heavy chain and a light chain.

[0102] In some embodiments, amyloid-reactive antibodies reduce or prevent amyloid fibril growth in vitro and / or in vivo. In some embodiments, amyloid-reactive antibodies reduce rVλ6WIL fibril elongation by 10% to 95%. In some embodiments, amyloid-reactive antibodies reduce rVλ6WIL fibril elongation by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, amyloid-reactive antibodies reduce rVλ6WIL fibril elongation by approximately 89%.

[0103] In some embodiments, fibril growth can occur by recruiting fibril monomers either at the fibril ends or along the long axis. In some embodiments, the antibody-peptide fusion protein inhibits amyloid fibril growth by sequestering fibril monomers or misfolded fibril monomers. In some embodiments, the antibody-peptide fusion protein inhibits amyloid fibril growth by blocking access to elongation sites at either end of the amyloid fibril. In some embodiments, the antibody-peptide fusion protein inhibits amyloid fibril growth by blocking access to elongation sites along the long axis of the amyloid fibril. In some embodiments, the antibody-peptide fusion protein inhibits amyloid fibril growth through a combination of these mechanisms.

[0104] In some embodiments, individuals are at risk of developing amyloid-related disease. In some embodiments, individuals have a family history of amyloid disease. In some embodiments, individuals have a genetic predisposition to amyloid disease. In some embodiments, individuals have a genetic predisposition to AA amyloidosis. In some embodiments, the genetic predisposition to AA amyloidosis includes mutations in the amyloid-forming precursor protein SAA1. In some embodiments, the genetic predisposition to AA amyloidosis includes polymorphisms in the mannose-binding lectin 2 (MBL-2) gene. In some embodiments, amyloidosis is genotype amyloidosis. In some embodiments, genotype amyloidosis includes mutant transthyretin (TTR)-associated cardiac amyloidosis. In some embodiments, TTR-associated cardiac amyloidosis disproportionately affects African Americans. In some embodiments, the TTR mutant includes a point mutation resulting in an amino acid substitution of isoleucine instead of valine at position 122 (Val122Ile). Those skilled in the art will be able to easily identify individuals susceptible to amyloid-related diseases, for whom the method of the present invention is particularly useful.

[0105] Furthermore, the present invention is particularly useful for improving or preventing the progression of symptoms of amyloid disease. In some embodiments, the individual has an early stage of amyloid disease. In some embodiments, the individual has one or more symptoms of amyloid disease. In some embodiments, the symptoms of amyloid disease include swelling of the ankles and legs, severe fatigue, shortness of breath, significant weight loss, difficulty swallowing, stinging, numbness or pain in the wrists or feet, tongue enlargement, arrhythmia, diarrhea, and / or bruising. In some embodiments, treatment with a therapeutically effective amount of antibody-peptide fusion protein slows the progression of the disease in an individual with diagnosed amyloid-related disease. In some embodiments, one or more of these symptoms are improved and / or stabilized after treatment with antibody-peptide fusion protein.

[0106] In some embodiments, the individual has an early stage of amyloid-related disease. In some embodiments, the individual has an early stage of amyloid light chain amyloidosis (AL). In some embodiments, AL amyloidosis is staged according to the Mayo Clinic system (Kumar S. et al., J. Clin. Oncol. 2012;30:989-995).

[0107] In some embodiments, the individual has early-stage hereditary or wild-type ATTR amyloidosis. In some embodiments, hereditary or wild-type ATTR amyloidosis is staged according to an ATTR staging system using the cardiac biomarker NT-proBNP and the renal biomarker eGFR (Gillmore JDet al., Eur Heart J. 2018;39(30):2799-2806).

[0108] In some embodiments, amyloid deposits may contribute to the pathology of a disease. In other embodiments, amyloid deposits may indicate amyloidosis or amyloid-related disease in an individual. In some embodiments, an amyloid-reactive antibody-peptide fusion protein or an amyloid-reactive antibody binds to amyloid in an individual having amyloidosis. In some embodiments, amyloidosis is localized to a specific tissue or organ system, such as the pancreas, liver, heart, or central nervous system.

[0109] In other embodiments, amyloidosis is systemic amyloidosis. In some embodiments, amyloidosis is familial amyloidosis. In other embodiments, amyloidosis is sporadic amyloidosis. In some embodiments, amyloidosis or amyloid-related disease is AA amyloidosis, AL amyloidosis, AH amyloidosis, Aβ amyloidosis, ATTRv amyloidosis, ATTRwt amyloidosis, ALECT2 amyloidosis, and IAPP amyloidosis in type II diabetes, Alzheimer's disease, Down syndrome, hereditary cerebral hemorrhage with Dutch type amyloidosis, cerebral beta-amyloid angiopathy, cavernous encephalopathy, thyroid tumors, Parkinson's disease, Lewy body dementia, tauopathy, Huntington's disease, senile systemic amyloidosis, familial hemodialysis, senile systemic aging, age-related pituitary disorders, iatrogenic syndromes, cavernous encephalopathy, reactive chronic inflammation, thyroid tumors, myeloma or other forms of cancer. In some embodiments, amyloid-related diseases are selected from the group consisting of AL, AH, Aβ2M, ATTRv, ATTRwt, AA, AApoAI, AApoAII, AApoAIV, AApoCII, AApoCII, AGel, Alys, ALECT2, AFib, ACys, ACa, AMed, AIAPP, APro, AIns, APrP, ASPC, ACa7, ACor, Aker, ALac, AOAPP, ASem1, AEnf, or Aβ amyloidosis. In some embodiments, treatment with an amyloid-reactive antibody-peptide fusion protein or an amyloid-reactive antibody results in amyloid clearance. In some embodiments, the amyloid-reactive antibody-peptide fusion protein or the amyloid-reactive antibody binds to amyloid associated with normal aging. In other embodiments, the amyloid-reactive antibody-peptide fusion protein or the amyloid-reactive antibody is used for the diagnosis, treatment, or prognosis of amyloidosis or amyloid-related diseases in an individual.

[0110] In some embodiments, amyloid-related diseases are localized amyloidosis.

[0111] In some embodiments, the amyloid-reactive antibody-peptide fusion protein or amyloid-reactive antibody is administered via intradermal, subcutaneous, intramuscular, intracardiac, intravascular, intravenous, intraocular, intraarterial, epidural, intrathecal, extracorporeal, intraperitoneal, intrapleural, intratubular, intravitreous, intracavernosal, intraventricular, intraosseous, intraarticular, intracellular, or pulmonary pathways.

[0112] In some embodiments, the individual is a mammal such as a primate, a cattle, a rodent, or a pig. In some embodiments, the individual is a human.

[0113] IV. Antibody-peptide fusion proteins In some embodiments, the methods provided herein include reducing or preventing the growth of amyloid fibrils by administering an antibody-peptide fusion protein. Alternatively, in some embodiments, the methods provided herein include reducing or delaying the progression of amyloid-related disease in an individual by administering an antibody-peptide fusion protein. Examples of such antibody-peptide fusion proteins include amyloid-reactive peptides that are linked to an antibody by elongation of the N-terminus or C-terminus of the light chain of the antibody or a fragment thereof, or via the N-terminus or C-terminus of the heavy chain of the antibody or a fragment thereof, thereby forming a peptide-antibody fusion. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer. In some embodiments, the spacer includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer including an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86. Antibody-peptide fusion proteins can be used, for example, to slow or prevent amyloid fibril growth and to reduce or delay the progression of amyloid-related diseases by administering the antibody-peptide fusion protein to subjects who have or are suspected of having amyloidosis.

[0114] In some embodiments, provided herein are antibody-peptide fusion proteins comprising an amyloid-reactive peptide and an antibody that reduces or prevents amyloid fibril growth or delays the progression of amyloid-related disease. In some embodiments, the antibody comprises a heavy chain containing a heavy chain variable region (VH) and a light chain containing a light chain variable region (VL). In some embodiments, the amyloid-reactive peptide and the antibody are linked at the N and / or C-terminus of the light chain and / or the N and / or C-terminus of the heavy chain. In some embodiments, the antibody-peptide fusion protein comprises a plurality of amyloid-reactive peptides linked to the antibody. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86. In some embodiments, the amyloid-reactive peptide and antibody are linked at the C-terminus of the light chain. In some embodiments, the amyloid-reactive peptide and antibody are linked at the C-terminus of the heavy chain. In some embodiments, the antibody is a full-length antibody. In some embodiments, the amyloid-reactive peptide contains the amino acid sequence shown in Table 1 below.

[0115] In some embodiments, the amyloid-reactive peptide antibody fusion comprises a heavy chain containing amyloid-reactive peptide, a spacer, VH, CH1, CH2, and CH3 in order from N to C-terminus. In some embodiments, the amyloid-reactive peptide antibody fusion comprises a heavy chain containing VH, CH1, CH2, CH3, a spacer, and amyloid-reactive peptide in order from N to C-terminus. In some embodiments, the amyloid-reactive peptide antibody fusion comprises a light chain containing amyloid-reactive peptide, a spacer, VL, and CL in order from N to C-terminus. In some embodiments, the amyloid-reactive peptide antibody fusion comprises a light chain containing VL, CL, a spacer, and amyloid-reactive peptide in order from N to C-terminus. [Table 2]

[0116] While we do not wish to be bound by any particular theory, it is believed that the amyloid-reactive peptide in an antibody-peptide fusion protein, when administered to a target, targets the antibody-peptide fusion protein to amyloid deposits. The Fc domain then triggers an immune response at the amyloid deposition site, thereby potentially inhibiting or delaying amyloid formation by removing amyloid through opsonization or other means. In addition, antibody-peptide fusion proteins are thought to have a longer half-life than the amyloid-reactive peptide alone. For example, the circulating half-life of IgG in humans is approximately 21 days, compared to approximately 11 hours for the amyloid-reactive peptide alone in humans. Therefore, Ig enhances the half-life of the antibody-peptide fusion protein in circulation. In some embodiments, the half-life of the antibody-peptide fusion protein is increased by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more compared to the amyloid-reactive peptide alone. Therefore, when administered to a target, antibody-peptide fusion proteins can exert their immunostimulatory effects for a longer period at amyloid deposition sites, thereby enhancing the immune response at those sites. In some embodiments, the amyloid-reactive peptide binds to heparan sulfate glycosaminoglycans. In some embodiments, the amyloid-reactive peptide can bind to multiple types of amyloid fibrils. In some embodiments, the amyloid-reactive peptide has pan-amyloid specificity.

[0117] In some embodiments, the amyloid-reactive peptide of the antibody-peptide fusion protein described herein includes an amino acid sequence that is at least 80%, 85%, 90%, or more identical to the amino acid sequence shown as any one of SEQ ID NOs: 1 to 13, for example, an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence shown as any one of SEQ ID NOs: 1 to 13. In some embodiments, the amyloid-reactive peptide linked to the antibody or its functional fragment may contain or consist of about 10 to about 55 amino acids. The amyloid-reactive peptides of the present invention may, for example, contain or consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids. Such peptides are described, for example, in international patent application WO2016032949 and Wall et al. (PLoS One. 2013 Jun 4;8(6):e66181), which are incorporated herein by reference in their entirety. In some embodiments, the amyloid-reactive peptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, or more sequence identity with any one of the amino acid sequences shown as SEQ ID NOs: 1 to 13. In some embodiments, the amyloid-reactive peptide includes an amino acid sequence having at least 80%, 85%, 90%, 95%, or more sequence identity with any one of the amino acid sequences shown as SEQ ID NOs: 1 to 13. In some embodiments, the amyloid-reactive peptide includes an amino acid sequence shown as SEQ ID NOs: 1 to 13, which includes one or more amino acid substitutions. In some embodiments, the amyloid-reactive peptide includes the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the amyloid-reactive peptide includes the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the amyloid-reactive peptide includes the amino acid sequence shown in SEQ ID NO: 12.In some embodiments, the amyloid-reactive peptide comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the amyloid-reactive peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13. [Table 3]

[0118] In certain embodiments, the antibody-peptide fusion protein comprises an antibody comprising VH comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19, (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, wherein the antibody is linked to an amyloid-reactive peptide via a spacer comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. m11-1F4 VH Sequence ID 15 QVQLKESGPGLVAPSQSLSITCTVSGFSLSSYGVSWVRQPPGKGLEWLGVIWGDGSTNYHPNLMSRLSISKDISKSQVLFKLNSLQTDDTATYYCVTLDYWGQGTSVTVSS m11-1F4 VL Sequence ID 16 DVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCFQTTYVPNTFGGGTKLEIK

[0119] This specification also provides antibody-peptide fusion proteins comprising a humanized antibody that binds to human amyloid fibrils fused to an amyloid-reactive peptide. In some embodiments, the antibody-peptide fusion protein comprises the humanized antibody described herein. In some embodiments, the humanized antibody comprises the humanized VH and / or VL sequence derived from m11-1F4. In some embodiments, the antibody-peptide fusion protein comprises the humanized antibody described in International Application No. PCT / US2020 / 060596 (which is incorporated herein by reference in its entirety). Exemplary amino acid sequences of the humanized VH and VL regions are provided in Tables 3-4 below. In Tables 3-4, the CDR sequence is underlined, and reverse mutant residues and further mutations introduced into the humanized variants VL4 and VH9 are in bold and italics. Further mutations introduced into VL4 and VH9 are listed in the IgG column of Tables 3-4; these mutations are numbered relative to the N-terminus of VL or VH. The CDR amino acid sequences for variants of VL4 and VH9 with modified CDRs are shown in Tables 5 and 6, respectively, in comparison to VL4 and VH9 below. [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 6] [Table 7-1] [Table 7-2]

[0120] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody including a light chain variable region (VL) and a heavy chain variable region (VH), where the VL includes CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 20, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and the VH includes CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 18, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the humanized antibody includes 1, 2, 3, 4, 5, or 6 CDRs of the antibody shown in Table 2. In some embodiments, the humanized antibody comprises CDR-H1, CDR-H2, and CDR-H3, each containing the amino acid sequences of VH CDR-H1, CDR-H2, and CDR-H3 having the sequence shown in SEQ ID NO: 15; and CDR-L1, CDR-L2, and CDR-L3, each containing the amino acid sequences of VL CDR-L1, CDR-L2, and CDR-L3 having the sequence shown in SEQ ID NO: 16. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86.

[0121] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL, which comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NOs. 64-70, CDR-L2 containing the amino acid sequence shown in SEQ ID NOs. 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NOs. 22, and VH, which comprises CDR-H1 containing the amino acid sequence shown in SEQ ID NOs. 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NOs. 19. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, which comprises VL, which comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NOs. 20, CDR-L2 containing the amino acid sequence shown in SEQ ID NOs. 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NOs. 22, and VH, which comprises CDR-H1 containing the amino acid sequence shown in SEQ ID NOs. 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NOs. 71-81, and CDR-H3 containing the amino acid sequence shown in SEQ ID NOs. 19. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 83-86.

[0122] In some embodiments, the antibody-peptide fusion protein comprises humanized antibodies comprising CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-70, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and VH containing CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 71, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86.

[0123] In some embodiments, the antibody-peptide fusion protein includes humanized antibodies comprising CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-70, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, as well as VH containing CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 72, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the antibody-peptide fusion protein includes an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86.

[0124] In some embodiments, the antibody-peptide fusion protein includes humanized antibodies comprising CDR-L1 containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 64-70, CDR-L2 containing the amino acid sequence shown in SEQ ID NOs. 21, and VL containing CDR-L3 containing the amino acid sequence shown in SEQ ID NOs. 22, and VH containing CDR-H1 containing the amino acid sequence shown in SEQ ID NOs. 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NOs. 73, and VH containing CDR-H3 containing the amino acid sequence shown in SEQ ID NOs. In some embodiments, the antibody-peptide fusion protein includes an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 83-86.

[0125] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL, which includes CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and VH, which includes CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 81, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19.

[0126] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL, which includes CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 65, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and VH, which includes CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 81, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19.

[0127] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL, which includes CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 66, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and VH, which includes CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 81, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19.

[0128] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL, which includes CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 67, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and VH, which includes CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 81, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19.

[0129] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL, which includes CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 68, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and VH, which includes CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 81, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19.

[0130] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL, which includes CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 69, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and VH, which includes CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 81, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19.

[0131] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL, which includes CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 70, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22, and VH, which includes CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 71 to 81, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19.

[0132] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL comprising CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 22, and VH comprising CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the antibody-peptide fusion protein is a full-length antibody. In some embodiments, the antibody-peptide fusion protein has an IgG1 isotype. In some embodiments, the antibody-peptide fusion protein comprises a light chain containing VL, CL, a spacer, and an amyloid-reactive peptide in order from N to the C-terminus. In some embodiments, the amyloid-reactive peptide is fused to the C-terminus of the light chain via a spacer.

[0133] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody containing the amino acid sequence of VL shown in Table 3. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, the humanized antibody comprising a VL selected from the group consisting of VL2, VL3, VL4, VL4-N33S, VL4-N33Q, VL4-N33E, VL4-N33A, VL4-N33H, VL4-G34A, or VL4-G34V shown in Table 3. In some embodiments, the VL contains the amino acid sequence shown in the group consisting of SEQ ID NOs. 32-42. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 83-86. In some embodiments, the amyloid-reactive peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 13.

[0134] In some embodiments, the antibody-peptide fusion protein includes a humanized antibody containing the amino acid sequence of VH shown in Table 4. In some embodiments, the antibody-peptide fusion protein includes a humanized antibody, the humanized antibody containing VH selected from the group consisting of VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH9-D54S, VH9-D54Q, VH9-D54E, VH9-D54A, VH9-D54H, VH9-G55A, VH9-G55V, VH9-M64V, VH9-M64I, VH9-M64L, or VH9-M64A shown in Table 4. In some embodiments, VH contains the amino acid sequence shown in the group consisting of SEQ ID NOs: 43 to 63. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 83-86. In some embodiments, the amyloid-reactive peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-13.

[0135] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL containing the amino acid sequence shown in SEQ ID NO: 34 and VH containing the amino acid sequence shown in SEQ ID NO: 48.

[0136] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL containing the amino acid sequence shown in SEQ ID NO: 35 and VH containing the amino acid sequence shown in SEQ ID NO: 51.

[0137] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL containing the amino acid sequence shown in SEQ ID NO: 35 and VH containing the amino acid sequence shown in SEQ ID NO: 52.

[0138] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL containing the amino acid sequence shown in SEQ ID NO: 35 and VH containing the amino acid sequence shown in SEQ ID NO: 50.

[0139] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising VL containing the amino acid sequence shown in SEQ ID NO: 35 and VH containing the amino acid sequence shown in SEQ ID NO: 49.

[0140] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody comprising a VL containing the amino acid sequence shown in SEQ ID NO: 36 and a VH containing the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer. In some embodiments, the amyloid-reactive peptide is fused to the C-terminus of the light chain via a spacer. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83 and 86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing the amino acid sequence shown in SEQ ID NO: 83. In some embodiments, the amyloid-reactive peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13. In some embodiments, the amyloid-reactive peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the amyloid-reactive peptide comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to the amyloid-reactive peptide shown in SEQ ID NO: 2 via a spacer comprising the amino acid sequence shown in SEQ ID NO: 83.

[0141] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody containing VL of VL4 shown in Table 3 and VH of VH9 shown in Table 4. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody containing VL containing the amino acid sequence shown in SEQ ID NO: 35 and VH containing the amino acid sequence shown in SEQ ID NO: 51. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86. In some embodiments, the amyloid-reactive peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13.

[0142] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody containing VL of VL4-N33S shown in Table 3 and VH of VH9-D54E shown in Table 4. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody containing VL containing the amino acid sequence shown in SEQ ID NO: 36 and VH containing the amino acid sequence shown in SEQ ID NO: 55. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86. In some embodiments, the amyloid-reactive peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13.

[0143] In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, the humanized antibody comprising a light chain. In some embodiments, an amyloid-reactive peptide is fused to the C-terminus of the light chain. In some embodiments, the antibody-peptide fusion protein comprises a humanized antibody, the humanized antibody comprising a light chain, and the amyloid-reactive peptide is fused to the C-terminus of the light chain by a spacer. In some embodiments, the spacer is a peptide spacer. In some embodiments, the spacer is a soft spacer. In some embodiments, the spacer comprises glycine and serine residues. In some embodiments, the spacer comprises the amino acid sequence GGGYS. In some embodiments, the spacer comprises the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the spacer is a hard spacer. In some embodiments, the spacer is uncharged. In some embodiments, the spacer comprises the amino acid sequences shown in SEQ ID NOs: 83-86.

[0144] In certain embodiments, the antibody-peptide fusion protein may include an amino acid spacer sequence between the C-terminus of the light chain and the amyloid-reactive peptide. In certain embodiments, the peptide-Ig conjugate may include an amino acid spacer sequence between the N-terminus of the peptide and the leader sequence required for the secretion of the Ig peptide from the reagent-expressing cell. In some embodiments, the spacer is a soft spacer. In some embodiments, the spacer is a hard spacer peptide. In some embodiments, the spacer is uncharged. In some embodiments, the spacer peptide may contain or consist of about 3 to about 55 amino acids. The spacer peptide of the present invention may contain or consist of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids. As used herein, a nucleic acid sequence or amino acid sequence is "adjacent" to another nucleic acid sequence or amino acid sequence if such nucleic acid sequences or amino acid sequences are close to each other in sequence. For example, two nucleic acid sequences may be adjacent to each other as described herein, but still contain an intervening spacer sequence. In some embodiments, the spacer peptide includes the amino acid sequence shown in Table 7 below. In some embodiments, the spacer includes the amino acid sequence shown in SEQ ID NOs. 23-24, 27, and 83-86. [Table 8]

[0145] In some embodiments, one or more of the peptides shown in Table 1 may be ligated to a humanized antibody or its functional fragment via the C-terminus or N-terminus of the light chain protein or the C-terminus or N-terminus of the heavy chain protein, thereby forming an antibody-peptide fusion protein containing the humanized antibody. That is, any of the sequences identified in Table 1 may be ligated independently or simultaneously to the heavy chain or light chain of a humanized antibody or its functional fragment, forming an antibody-peptide fusion protein. For example, two of the amyloid-reactive peptides may be ligated to a single antibody by ligating the amyloid-reactive peptide amino acid sequence to the N-terminus of the light chain of the humanized antibody, or by ligating the amyloid-reactive peptide amino acid sequence to the C-terminus of the light chain of the humanized antibody, or by ligating the amyloid-reactive peptide amino acid sequence to the C-terminus of the heavy chain of the humanized antibody.

[0146] In some embodiments, the antibody-peptide fusion protein comprises a light chain further comprising a light chain constant region (e.g., comprising CL1) and a heavy chain comprising a heavy chain constant region (e.g., comprising CH1, CH2, and CH3). In some embodiments, the antibody-peptide fusion protein comprises two light chains and two heavy chains.

[0147] In some embodiments, the antibody-peptide fusion protein comprises a light chain containing a light chain and an amyloid-reactive peptide from N to C-terminus. In some embodiments, the light chain contains VL and CL1 from N to C-terminus. In some embodiments, VL is any one of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain containing VH, CH1, CH2, and CH3 from N to C-terminus. In some embodiments, VH is any one of the VHs described herein. In some embodiments, the antibody-peptide fusion protein comprises first and second light chains containing a variable light chain region, a constant light chain region, a spacer, and an amyloid-reactive peptide from N-terminus to C-terminus, and first and second heavy chains containing VH, CH1, CH2, and CH3 from N to C-terminus, wherein CH2 and CH3 of the first and second heavy chains form a dimer.

[0148] In some embodiments, the antibody-peptide fusion protein comprises a light chain from N to the C-terminus, a spacer peptide, and a light chain containing an amyloid-reactive peptide. In some embodiments, the spacer peptide contains the amino acid sequence of SEQ ID NO: 23. In some embodiments, the spacer peptide contains the amino acid sequence of SEQ ID NO: 27. In some embodiments, the spacer peptide contains the amino acid sequences of SEQ ID NOs: 83-86. In some embodiments, the light chain comprises VL and CL1 from N to the C-terminus. In some embodiments, VL is any one of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain from N to the C-terminus, containing VH, CH1, CH2, and CH3. In some embodiments, VH is any one of the VHs described herein. In some embodiments, the antibody-peptide fusion protein comprises first and second light chains containing VL, CL1, a spacer, and an amyloid-reactive peptide from N to the C-terminus, and first and second heavy chains containing VH, CH1, CH2, and CH3 from N to the C-terminus, wherein the CH2 and CH3 of the first and second heavy chains form a dimer.

[0149] In some embodiments, the antibody-peptide fusion protein comprises, from N to C-terminus, a secretion leader peptide, a first spacer peptide, an amyloid-reactive peptide, a second spacer peptide, and a light chain. In some embodiments, the first spacer peptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the first spacer peptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the second spacer peptide comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first and / or second spacer peptides comprises the amino acid sequences of SEQ ID NOs: 83-86. In some embodiments, the light chain comprises, from N to C-terminus, VL and CL1. In some embodiments, VL is any one of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises, from N to C-terminus, a heavy chain comprising VH, CH1, CH2, and CH3. In some embodiments, VH is any one of the VHs described herein. In some embodiments, VH is any one of the VHs described herein.

[0150] In some embodiments, the antibody-peptide fusion protein comprises, from N to C-terminus, a secretion leader peptide, a first spacer peptide, a light chain, a second spacer peptide, and an amyloid-reactive peptide. In some embodiments, the first spacer peptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the first spacer peptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the second spacer peptide comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the first and / or second spacer peptides comprises the amino acid sequences of SEQ ID NOs: 83-86. In some embodiments, the light chain comprises, from N to C-terminus, VL and CL1. In some embodiments, VL is any one of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises, from N to C-terminus, a heavy chain comprising VH, CH1, CH2, and CH3. In some embodiments, VH is any one of the VHs described herein. In some embodiments, VH is any one of the VHs described herein.

[0151] In some embodiments, the antibody-peptide fusion protein comprises a light chain from N to the C-terminus containing VL and CL1. In some embodiments, VL is any one of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain from N to the C-terminus containing an amyloid-reactive peptide, VH, CH1, CH2, and CH3. In some embodiments, VH is any one of the VHs described herein. In some embodiments, the antibody-peptide fusion protein comprises first and second light chains from N to the C-terminus containing VL and CL1, and first and second heavy chains from N to the C-terminus containing an amyloid-reactive peptide, VH, CH1, CH2, and CH3, wherein CH2 and CH3 of the first and second heavy chains form a dimer.

[0152] In some embodiments, the antibody-peptide fusion protein comprises a light chain from N to the C-terminus containing VL and CL1. In some embodiments, VL is any one of the VLs described herein. In some embodiments, the antibody-peptide fusion protein comprises a heavy chain from N to the C-terminus containing VH, CH1, CH2, CH3, and an amyloid-reactive peptide. In some embodiments, VH is any one of the VHs described herein. In some embodiments, the antibody-peptide fusion protein comprises first and second light chains from N to the C-terminus containing VL and CL1, and first and second heavy chains from N to the C-terminus containing VH, CH1, CH2, CH3, and an amyloid-reactive peptide, wherein CH2 and CH3 of the first and second heavy chains form a dimer.

[0153] In some embodiments, the antibody-peptide fusion comprises a light chain containing a variable light chain region, a constant light chain region, a spacer, and an amyloid-reactive peptide in the N-terminus to C-terminus direction. In some embodiments, the amyloid-reactive peptide contains the amino acid sequence of SEQ ID NO: 2. In some embodiments, the spacer contains the amino acid sequence of SEQ ID NO: 83. In some embodiments, the light chain comprises a VL containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, the heavy chain comprises a VH containing the amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibody-peptide fusion protein comprises a light chain containing the amino acids shown in SEQ ID NO: 89 and a heavy chain containing the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide containing an amyloid-reactive peptide linked to the C-terminus of the light chain of an antibody that binds to human amyloid fibril, and third and fourth polypeptides containing the heavy chain of an antibody that binds to human amyloid fibril, wherein the first and second polypeptides contain the amino acids shown in SEQ ID NO: 89, and the third and fourth polypeptides contain the amino acid sequences shown in SEQ ID NO: 91. In some embodiments, the antibody-peptide fusion protein includes the structure shown in Figure 1C.

[0154] In some embodiments, the antibody-peptide fusion protein comprises a light chain containing a light chain, a spacer peptide, and an amyloid-reactive peptide from N to the C terminus. In some embodiments, the amyloid-reactive peptide contains the amino acid sequence of SEQ ID NO: 2. In some embodiments, the spacer contains the amino acid sequence of SEQ ID NO: 86. In some embodiments, the light chain contains VL containing the amino acid sequence of SEQ ID NO: 36. In some embodiments, the heavy chain contains VH containing the amino acid sequence of SEQ ID NO: 55. In some embodiments, the antibody-peptide fusion protein comprises a light chain containing the amino acids shown in SEQ ID NO: 90 and a heavy chain containing the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide containing an amyloid-reactive peptide linked to the C terminus of the light chain of an antibody that binds to human amyloid fibril, and third and fourth polypeptides containing the heavy chain of an antibody that binds to human amyloid fibril, wherein the first and second polypeptides contain the amino acids shown in SEQ ID NO: 90, and the third and fourth polypeptides contain the amino acid sequences shown in SEQ ID NO: 91. In some embodiments, the antibody-peptide fusion protein includes the structure shown in Figure 1B.

[0155] In some embodiments, the antibody-peptide fusion protein comprises an antibody that binds to amyloid fibril, comprising a first polypeptide and a second polypeptide each comprising the light chain of the antibody, and a third and a fourth polypeptide each comprising the heavy chain of the antibody. In some embodiments, the antibody-peptide fusion protein comprises an amyloid-reactive peptide linked to the N-terminus or C-terminus of the light chain or heavy chain. In some embodiments, the first and second polypeptides comprise the amino acids shown in SEQ ID NO: 87, and the third and fourth polypeptides comprise the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the first and second polypeptides comprise the amino acids shown in SEQ ID NO: 88, and the third and fourth polypeptides comprise the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the first and second polypeptides comprise the amino acids shown in SEQ ID NO: 89, and the third and fourth polypeptides comprise the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the first and second polypeptides comprise the amino acids shown in SEQ ID NO: 90, and the third and fourth polypeptides comprise the amino acid sequence shown in SEQ ID NO: 91. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 23-24, 27, and 83-86. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs. 83-86. In some embodiments, the amyloid-reactive peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 1-13. In some embodiments, the amyloid-reactive peptide contains the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2. In some embodiments, the amyloid-reactive peptide contains the amino acid sequence shown in SEQ ID NO. 2. In some embodiments, the antibody-peptide fusion protein comprises an antibody linked to an amyloid-reactive peptide shown in SEQ ID NO. 2 via a spacer containing the amino acid sequence shown in SEQ ID NO. 83.

[0156] In some embodiments, the antibody-peptide fusion protein includes an amyloid-reactive peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the antibody-peptide fusion protein includes an antibody that binds to human amyloid fibril. In some embodiments, the antibody comprises a variable heavy chain (VH) and a variable light chain (VL), where the VH includes CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 19, and the VL includes CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain. In some embodiments, the amyloid-reactive peptide is linked to the antibody via a spacer. In some embodiments, the antibody-peptide fusion includes a variable light chain region, a constant light chain region, a spacer, and a light chain containing the amyloid-reactive peptide, in the direction from the N-terminus to the C-terminus. In some embodiments, the spacer includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86. In some embodiments, the spacer includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-86. In some embodiments, the spacer includes the amino acid sequence shown in SEQ ID NO: 83.

[0157] Exemplary amyloid-reactive peptide-antibody fusion protein amino acid sequences are shown in Tables 8 and 9. In Table 8, the amino acid sequence of the amyloid-reactive peptide p5R (SEQ ID NO: 2) is shown in bold, and the spacer sequence is in underlined italics. [Table 9-1] [Table 9-2] [Table 9-3] [Table 10]

[0158] In some embodiments, the antibody-peptide fusion protein comprises an amyloid-reactive peptide having the amino acid sequence shown in SEQ ID NO: 2, and an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH), and the light chain of the antibody comprising a light chain variable region (VL), the VH comprising CDR-H1 having the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 19, the VL comprising CDR-L1 having the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 22, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer having the amino acid sequence described in SEQ ID NO: 83.

[0159] In some embodiments, the antibody-peptide fusion protein comprises an amyloid-reactive peptide having the amino acid sequence shown in SEQ ID NO: 2, and an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH) comprising VH CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 55, and the light chain of the antibody comprising a light chain variable region (VL) comprising VL CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 36, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer comprising the amino acid sequence shown in SEQ ID NO: 83.

[0160] In some embodiments, the antibody that binds to amyloid fibril comprises a first polypeptide and a second polypeptide, each comprising the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each comprising the heavy chain of the antibody, wherein the first polypeptide and the second polypeptide comprise the amino acids shown in SEQ ID NO: 89, and the third polypeptide and the fourth polypeptide comprise the amino acid sequence shown in SEQ ID NO: 91.

[0161] In some embodiments, the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide, each containing the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each containing the heavy chain of the antibody, wherein the first polypeptide and the second polypeptide contain the amino acids shown in SEQ ID NO: 89, and each of the third polypeptide and the fourth polypeptide contains the amino acid sequence shown in SEQ ID NO: 91.

[0162] In some embodiments of the methods provided herein, the antibody-peptide fusion protein binds to amyloid deposits or fibrils. In some embodiments, the antibody-peptide fusion protein binds to one or more amyloid-forming peptides in the amyloid. In some embodiments, the amyloid to which the antibody-peptide fusion protein binds includes amyloid-forming λ6 variable domain protein (Vλ6Wil) or amyloid-forming immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloid-forming Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, the amyloid to which the antibody-peptide fusion protein binds includes amyloid-forming forms of immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), trans tyretin (ATTR wild-type; ATTR variant), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemochemoattractant (ALect2), fibrinogen A variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP); α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALκ4, Alλ1, or other amyloid-forming peptides. The amyloid-forming peptide to which the antibody-peptide fusion protein binds may be a protein, a protein fragment, or a protein domain. In some embodiments, the amyloid deposit or amyloid fibril comprises a recombinant amyloid-forming protein. In some embodiments, amyloid is part of the pathology of a disease.

[0163] The amino acids forming all or part of the amyloid-reactive peptide bound to the antibody or a fragment thereof may be naturally occurring amino acids, naturally occurring amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, stereoisomers and modifiers such as constructs or structures designed to mimic amino acids. The amino acids forming the peptide of the present invention may be one or more of the 20 common amino acids found in naturally occurring proteins, or one or more of modified and aberrant amino acids. Antibody-peptide fusion proteins can be produced by any technique well known to those skilled in the art, including chemical synthesis or recombinant means using standard molecular biological techniques.

[0164] In some embodiments, the antibody of the antibody-peptide fusion protein provided herein specifically binds to amyloid light chain fibrils. In some embodiments, the amyloid-reactive peptide binds to various amyloid fibrils, such as amyloid-forming λ6 variable domain protein (Vλ6Wil) or amyloid-forming immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloid-forming Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, the amyloid to which the antibody-peptide fusion protein binds includes amyloid-forming forms of immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), trans tyretin (ATTR wild-type; ATTR variant), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemochemoattractant (ALect2), fibrinogen A variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP); α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALκ4, Alλ1, or other amyloid-forming peptides. In some embodiments, the amyloid-reactive peptide binds to heparan sulfate glycosaminoglycans. In some embodiments, the amyloid-reactive peptide can bind to multiple forms of amyloid. In some embodiments, the amyloid-reactive peptide has panamyloid binding.

[0165] In some embodiments, the amyloid deposits or fibrils to which the antibody-peptide fusion protein binds are located in one or more organs. In some embodiments, the amyloid deposits are located in one or more tissue types. In some embodiments, the amyloid deposits or fibrils are located in one or more of the liver, spleen, heart, kidneys, brain, muscle, pancreas, stomach, upper intestine, lower intestine, and blood. In some embodiments, the antibody-peptide fusion protein binds to amyloid deposits or fibrils located in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different organs and / or tissue types. In some embodiments, the antibody-peptide fusion protein binds to amyloid deposits or fibrils located in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different organs. In some embodiments, the antibody-peptide fusion protein binds to amyloid deposits or fibrils located in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different tissue types. In some embodiments, the antibody-peptide fusion protein exhibits pan-amyloid reactivity. In some embodiments, the antibody-peptide fusion protein exhibits reactivity to amyloid deposits or fibrils located in the liver, spleen, heart, kidneys, brain, muscles, pancreas, stomach, upper intestine, lower intestine, and / or blood.

[0166] In some embodiments, the antibody-peptide fusion protein comprises an amyloid-reactive peptide having the amino acid sequence shown in SEQ ID NO: 2, and an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH), and the light chain of the antibody comprising a light chain variable region (VL), the VH comprising CDR-H1 having the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 19, the VL comprising CDR-L1 having the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 22, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, the amyloid-reactive peptide is linked to the antibody via a spacer having the amino acid sequence described in SEQ ID NO: 83, and the antibody-peptide fusion protein exhibits pan-amyloid reactivity. In some embodiments, the antibody-peptide fusion protein exhibits reactivity to amyloid deposits or fibrils located in the liver, spleen, heart, kidneys, brain, muscles, pancreas, stomach, upper intestine, lower intestine, and / or blood.

[0167] In some embodiments, the antibody-peptide fusion protein comprises an amyloid-reactive peptide having the amino acid sequence shown in SEQ ID NO: 2, and an antibody that binds to human amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH) comprising VH CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 55, and the light chain of the antibody comprising a light chain variable region (VL) comprising VL CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 36, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, the amyloid-reactive peptide is linked to the antibody via a spacer comprising the amino acid sequence shown in SEQ ID NO: 83, and the antibody-peptide fusion protein exhibits pan-amyloid reactivity. In some embodiments, the antibody-peptide fusion protein exhibits reactivity to amyloid deposits or fibrils located in the liver, spleen, heart, kidney, brain, muscle, pancreas, stomach, upper intestine, lower intestine, and / or blood.

[0168] In some embodiments, the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide, each containing the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each containing the heavy chain of the antibody, wherein the first and second polypeptides contain the amino acids shown in SEQ ID NO: 89, and each of the third and fourth polypeptides contains the amino acid sequence shown in SEQ ID NO: 91, and the antibody-peptide fusion protein exhibits panamyloid reactivity. In some embodiments, the antibody-peptide fusion protein exhibits reactivity to amyloid deposits or fibrils located in the liver, spleen, heart, kidneys, brain, muscle, pancreas, stomach, upper intestine, lower intestine, and / or blood.

[0169] In some embodiments, the antibody-peptide fusion protein described herein binds to amyloid deposits or fibrils with high binding affinity. In some embodiments, the antibody-peptide fusion protein described herein binds to amyloid substrates with high binding affinity. In some embodiments, the binding affinity is less than 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1.5 nM. In some embodiments, the binding affinity is less than 500 nM. In some embodiments, the binding affinity is less than 100 nM. In some embodiments, the binding affinity is less than 10 nM. In some embodiments, the binding affinity is less than 1.5 nM. In some embodiments, the binding affinity is 0.05 nM to 100 nM, 0.1 nM to 50 nM, 0.2 nM to 25 nM, 0.3 nM to 10 nM, 0.4 nM to 5 nM, 0.5 nM to 2 nM, 0.6 nM to 1 nM, or 0.2 nM to 1.5 nM. In some embodiments, the binding affinity is the same or different for different amyloid substrates. In some embodiments, the binding affinity is the same or different for human amyloid substrates. In some embodiments, the binding affinity is the same or different for synthetic amyloid substrates.

[0170] In some embodiments, the antibody-peptide fusion protein comprises an amyloid-reactive peptide having the amino acid sequence shown in SEQ ID NO: 2, and an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH), and the light chain of the antibody comprising a light chain variable region (VL), the VH comprising CDR-H1 having the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 19, the VL comprising CDR-L1 having the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 22, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, the amyloid-reactive peptide is linked to the antibody via a spacer having the amino acid sequence described in SEQ ID NO: 83, and the EC50 binding affinity is less than 10 nM, less than 5 nM, or less than 1.5 nM. In some embodiments, the EC50 binding affinity is less than 10 nM. In some embodiments, the EC50 binding affinity is less than 1.5 nM. In some embodiments, the EC50 binding affinity is the same or different for different amyloid substrates. In some embodiments, the EC50 binding affinity is the same or different for human amyloid substrates.

[0171] In some embodiments, the antibody-peptide fusion protein comprises an amyloid-reactive peptide having the amino acid sequence shown in SEQ ID NO: 2, and an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH) comprising VH CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 55, and the light chain of the antibody comprising a light chain variable region (VL) comprising VL CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 36, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, the amyloid-reactive peptide is linked to the antibody via a spacer comprising the amino acid sequence shown in SEQ ID NO: 83, and the EC50 binding affinity is less than 500 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 1.5 nM. In some embodiments, the EC50 binding affinity is less than 10 nM. In some embodiments, the EC50 binding affinity is less than 1.5 nM. In some embodiments, the EC50 binding affinity is the same or different for different amyloid substrates. In some embodiments, the EC50 binding affinity is the same or different for human amyloid substrates.

[0172] In some embodiments, the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide, each containing the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each containing the heavy chain of the antibody, wherein the first and second polypeptides contain the amino acids shown in SEQ ID NO: 89, and each of the third and fourth polypeptides contains the amino acid sequence shown in SEQ ID NO: 91, and the EC50 binding affinity is less than 500 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 1.5 nM. In some embodiments, the EC50 binding affinity is less than 10 nM. In some embodiments, the EC50 binding affinity is less than 1.5 nM. In some embodiments, the EC50 binding affinity is the same or different for different amyloid substrates. In some embodiments, the EC50 binding affinity is the same or different for human amyloid substrates.

[0173] As those skilled in the art will understand, the fragment antigen binding (or Fab region) is the head of the antibody that naturally interacts with the target antigen. Components of the Fab region allow the antibody to bind to a specific ligand, for example, and through that interaction, further activate the immune system. For the IgG, IgA, IgD, IgE, and IgM antibody isotypes, Ig consists of two proteins, a heavy chain and a light chain, which interact in pairs to form intact Ig containing two heavy chains and two light chains. Both the heavy and light chains are further divided into variable and constant domains, with the variable domains of the light and heavy chains containing the Fab functional region, and the heavy chain forming a fragment crystallizable (Fc) domain that interacts with cell receptors and complement. The Fc region of Ig has highly conserved N-glycosylation sites.

[0174] In certain exemplary embodiments, one or more of the peptides shown in Table 1 below may be ligated to an antibody or its functional fragment via the C-terminus or N-terminus of a light chain protein or a heavy chain protein, thereby forming an antibody-peptide fusion protein. That is, any of the sequences identified in Table 1 below may be ligated independently or simultaneously to the heavy chain or light chain of an antibody or its functional fragment, forming a peptide-antibody conjugate. For example, two of the amyloid-reactive peptides may be ligated to a single antibody by, for example, ligating the amino acid sequence of the amyloid-reactive peptide to the C-terminus of an Ig light chain protein.

[0175] In some embodiments, the antibody is a full-length antibody containing an Fc region. In some embodiments, the Fc is of the IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody-peptide fusion protein containing the humanized antibody promotes Fc-mediated antibody effector function. In some embodiments, the antibody-peptide fusion protein containing the humanized antibody promotes antibody-dependent cellular phagocytosis and inhibits or restricts amyloid fibril growth.

[0176] In some embodiments, the antibody-peptide fusion protein comprising the humanized antibody of this disclosure comprises an Fc region. In some embodiments, Fc is of the IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody-peptide fusion protein comprising the humanized antibody facilitates Fc-mediated antibody effector function. In some embodiments, the antibody-peptide fusion protein comprising the humanized antibody reduces or prevents amyloid fibril growth.

[0177] In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a dissociation constant (Kd) of approximately 100, 10, 1, 0.1, 0.01, 0.001, or less than 0.0001 μM. In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a Kd of approximately 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 μM (including any value or range between these values). In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a Kd of less than 500, 100, 10, or 1 nM. In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a Kd of less than approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000, 2000, or 2200 nM. In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a Kd of approximately 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000, 2000, or 2200 nM (including any value or range between these values). In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a Kd of approximately 40-50 nM. In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a Kd of 40-50 nM. In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a Kd of less than 50 nM. In some embodiments, the antibody-peptide fusion protein binds to human amyloid fibrils with a Kd of less than the Kd of c11-1F4 that binds to human amyloid fibrils.

[0178] In some embodiments, the antibody-peptide fusion protein is present at an antibody concentration (EC2) of about 0.01, 0.1, or less than 1 μM. 50) binds to human amyloid fibrils with half-maximal binding. In some embodiments, the antibody-peptide fusion protein has an antibody concentration (EC 50 ) binds to human amyloid fibrils with half-maximal binding. In some embodiments, the antibody-peptide fusion protein has an antibody concentration (EC that is less than about 1, 10, 100, or 1000 nM 50 ) binds to human amyloid fibrils with half-maximal binding. In some embodiments, the antibody-peptide fusion protein has an antibody concentration (EC that is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 250, 500, 750, or 1000 nM, including any value or range between any of these values 50 ) binds to human amyloid fibrils with half-maximal binding. In some embodiments, the antibody-peptide fusion protein has an antibody concentration (EC that is about 17 nM, 7 nM, 16 nM, 75 nM, or 95 nM 50 ) binds to human amyloid fibrils with half-maximal binding. In some embodiments, the antibody-peptide fusion protein has an antibody concentration (EC that is less than about 10 nM, 20 nM, 80 nM, or 100 nM 50 ) binds to human amyloid fibrils with half-maximal binding. In some embodiments, the antibody-peptide fusion protein has an EC of chimeric 11-1F4 that binds to human amyloid fibrils 50 that is less than the antibody concentration (EC 50 ) binds to human amyloid fibrils with half-maximal binding.

[0179] Dissociation constant and EC 50Methods for calculating EC are known in the art and include, for example, surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), and europium-linked immunosorbent assay (EuLISA). In some embodiments, the dissociation constant is determined, for example, by measuring the binding to Len(1-22) monomer peptides using surface plasmon resonance. In some embodiments, EC 50 This is determined using EuLISA. In some embodiments, EC 50 The binding level to rVλ6WIL fibril, ATTRwt human extract, ATTRv human extract, ALλ human extract, or ALκ human extract is determined using EuLISA to measure the binding level.

[0180] In some embodiments, the antibody-peptide fusion protein binds to rVλ6WIL fibrils, tATTRwt human extract, ATTRv human extract, ALλ human extract, and / or ALκ human extract. In some embodiments, the antibody-peptide fusion protein described herein binds to amyloid deposits or fibrils. In some embodiments, the antibody-peptide fusion protein binds to one or more amyloid-forming peptides in amyloid. In some embodiments, the amyloid to which the antibody-peptide fusion protein binds includes amyloid-forming λ6 variable domain protein (Vλ6WIL) or amyloid-forming immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibrils or amyloid-forming Aβ precursor protein, or serum amyloid protein A (AA). In other embodiments, the amyloid to which the antibody-peptide fusion protein binds includes amyloid-forming forms of immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), trans tyretin (ATTR wild-type; ATTR variant), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemochemoattractant (ALECT2), fibrinogen A variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP); α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALκ4, Alλ1, or other amyloid-forming peptides. The amyloid-forming peptide to which the antibody-peptide fusion protein binds may be a protein, a protein fragment, or a protein domain. In some embodiments, the amyloid deposit or amyloid fibril comprises a recombinant amyloid-forming protein. In some embodiments, amyloid is part of the pathology of a disease.

[0181] In some embodiments, the antibody-peptide fusion protein exhibits one or more in vivo features selected from improved in vivo distribution compared to a reference antibody, pan-amyloid reactivity, and inhibition of fibril growth. In some embodiments, the antibody-peptide fusion protein exhibits improved in vivo distribution compared to a reference antibody, and the antibody-peptide fusion protein is detectable in organs throughout the body. In some embodiments, the antibody-peptide fusion protein exhibits improved in vivo distribution, and the antibody-peptide fusion protein is detectable in one or more of the liver, spleen, heart, kidney, brain, muscle, pancreas, stomach, upper intestine, lower intestine, and blood. In some embodiments, the antibody-peptide fusion protein exhibits pan-amyloid reactivity compared to a reference antibody, and the antibody-peptide fusion protein exhibits reactivity to one or more different amyloid substrates in vivo. In some embodiments, the antibody-peptide fusion protein is reactive to amyloid substrates in the liver, spleen, heart, kidney, brain, muscle, pancreas, stomach, upper intestine, lower intestine, and blood. In some embodiments, the antibody-peptide fusion protein exhibits inhibition of fibril growth compared to a reference antibody, and contact of the amyloid substrate with the antibody-peptide fusion protein in vivo reduces amyloid fibril growth. In some embodiments, contact of the amyloid substrate with the antibody-peptide fusion protein in vivo reduces amyloid fibril growth. In some embodiments, contact of the amyloid substrate with the antibody-peptide fusion protein in vivo reduces amyloid fibril growth, resulting in a reduction or delay in the progression of amyloid-related disease. In some embodiments, contact of the amyloid substrate with the antibody-peptide fusion protein in vivo provides therapeutic benefits for individuals with amyloid-related disorders. In some embodiments, the reference antibody is not engineered to bind to the amyloid substrate. In some embodiments, the reference antibody does not contain an amyloid-reactive peptide. In some embodiments, the reference antibody is not fused to an amyloid-reactive peptide. In some embodiments, the reference antibody functions as a negative control.In some embodiments, the reference antibody is an IgG antibody. In some embodiments, the reference antibody is an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the reference antibody is an IgG1 isotype.

[0182] To produce the fusion protein, an expression vector for the fusion protein can be introduced into one or more suitable producing cell lines known in the art. The introduction of the expression vector can be carried out by simultaneous transfection by electroporation or by any other suitable transformation method available in the art. Then, a cell line producing the fusion protein is selected and grown, and the antibody is purified. The purified fusion protein can then be analyzed by standard techniques such as SDS-PAGE or size exclusion chromatography (SEC).

[0183] Antibody-peptide fusion proteins can be produced by any technique well known to those skilled in the art, including chemical synthesis or recombinant methods using standard molecular biological techniques (e.g., WO2022 / 246433).

[0184] Antibodies that bind to amyloid fibrils (amyloid-reactive antibodies) In some embodiments, the methods provided herein include reducing or preventing the growth of amyloid fibrils by administering an amyloid-reactive antibody. Alternatively, in some embodiments, the methods provided herein include reducing or delaying the progression of amyloid-related disease in an individual by administering an amyloid-reactive antibody.

[0185] In some embodiments, the amyloid-reactive antibody binds to amyloid. In some embodiments, the amyloid-reactive antibody binds to amyloid in immunoglobulin light chains (ALs). In some embodiments, the amyloid-reactive antibody binds to the same amyloid type as the amyloid-reactive peptide. In some embodiments, the amyloid-reactive antibody binds to a different amyloid type than the amyloid-reactive peptide. In some embodiments, the amyloid-reactive peptide has the pan-amyloid specificity described herein. Therefore, although not bound by this theory, the amyloid-reactive peptide of the fusion protein described herein targets the fusion protein to any amyloid deposit, and the Fc domain of the amyloid-reactive antibody induces an immune response against any amyloid type.

[0186] In some embodiments, the antibody comprises a heavy chain containing a heavy chain variable region (VH) and a light chain containing a light chain variable region (VL). In some embodiments, the amyloid-reactive antibody comprises a humanized antibody containing the VL amino acid sequence shown in Table 3. In some embodiments, the amyloid-reactive antibody comprises a humanized antibody containing a VL selected from the group consisting of VL2, VL3, VL4, VL4-N33S, VL4-N33Q, VL4-N33E, VL4-N33A, VL4-N33H, VL4-G34A, or VL4-G34V shown in Table 3. In some embodiments, the VL contains the amino acid sequence shown in the group consisting of SEQ ID NOs. 32 to 42. In some embodiments, the amyloid-reactive antibody contains the VL CDR amino acid sequence shown in Table 5.

[0187] In some embodiments, the amyloid-reactive antibody comprises a humanized antibody containing the amino acid sequence of VH shown in Table 4. In some embodiments, the amyloid-reactive antibody comprises a humanized antibody containing VH selected from the group consisting of VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VH10, VH9-D54S, VH9-D54Q, VH9-D54E, VH9-D54A, VH9-D54H, VH9-G55A, VH9-G55V, VH9-M64V, VH9-M64I, VH9-M64L, or VH9-M64A shown in Table 4. In some embodiments, VH contains the amino acid sequence shown in the group consisting of SEQ ID NOs: 43 to 63. In some embodiments, the amyloid-reactive peptide contains the VH CDR amino acid sequence shown in Table 6.

[0188] In some embodiments, the amyloid-reactive antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH), and the light chain of the antibody comprising a light chain variable region (VL), wherein VH comprises CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 19, and VL comprises CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 22.

[0189] In some embodiments, the amyloid-reactive antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprising a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 of VH containing the amino acid sequence shown in SEQ ID NO: 55, and the light chain of the antibody comprising a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 of VL containing the amino acid sequence shown in SEQ ID NO: 36.

[0190] In some embodiments, the antibody that binds to amyloid fibril comprises a first polypeptide and a second polypeptide, each comprising the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each comprising the heavy chain of the antibody, wherein the first polypeptide and the second polypeptide comprise the amino acids shown in SEQ ID NO: 88, and the third polypeptide and the fourth polypeptide comprise the amino acid sequence shown in SEQ ID NO: 91.

[0191] In some embodiments, the antibody that binds to amyloid fibril comprises a first polypeptide and a second polypeptide, each comprising the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each comprising the heavy chain of the antibody, wherein the first polypeptide and the second polypeptide comprise the amino acids shown in SEQ ID NO: 88, and the third polypeptide and the fourth polypeptide comprise the amino acid sequence shown in SEQ ID NO: 93. [Examples]

[0192] The following examples are included for illustrative purposes only and are not intended to limit the scope of this disclosure. Example 1: Method for rVλ6WIL fibril elongation using biotinylated rVλ6WIL monomers mixed with amyloid-reactive peptide, amyloid-reactive antibody, or amyloid-reactive antibody-peptide fusion protein.

[0193] This example describes a method for analyzing the elongation of rVλ6WIL(WIL) fibrils in the presence of amyloid-reactive peptides, amyloid-reactive immunoglobulins, and amyloid-reactive antibody-peptide fusion proteins.

[0194] A WIL fibril elongation assay was developed to analyze amyloid fibril formation in vitro using biotinylated WIL monomers.

[0195] First, a highly bound 96-well ELISA plate was coated with 0.83 μM sonicated rVλ6WIL synthetic fibrils (50 mL per well). Next, the fibrils were dried in an oven at 37°C. Subsequently, the plate was washed twice with washing buffer (PBS + 0.05% tween 20). Then, the plate was blocked at 37°C for 1 hour with 1% BSA in PBS (200 mL / well). Next, the blocking buffer was removed, and the plate was washed twice with washing buffer. 100 mL of 20 nM biotinylated rVλ6WIL monomer (biotinylated according to the EZ-link Sulfo-NHS-biotin (ThermoFisher Scientific) protocol) in 1% BSA + 0.05% tween 20 (BSAT) in PBS was added per positive control well. Each test well was treated with 100 mL of a mixture of 20 nM biotinylated rVλ6WIL monomer in BSAT and amyloid-reactive peptide, amyloid-reactive antibody, or amyloid-reactive antibody-peptide fusion protein (2, 0.5, 0.05, 0.01, 0.005, and 0.001 mM). A background control well (fibril-free) was treated with 100 mL of 20 nM biotinylated rVλ6WIL monomer in BSAT. The microplate was then incubated at 37°C for 1 hour. The plate was then washed twice with wash buffer. Next, 100 mL of a 1:1000 dilution of europium-labeled streptavidin (Perkin Elmer) in BSAT was added to each well and incubated at 37°C for 1 hour. Finally, the plate was washed three times with wash buffer. To quantify amyloid fibril formation, 100 mL of enhancement solution (Perkin Elmer) was added to each well, and time-resolved fluorescence emission was measured in each well.

[0196] Example 2: rVλ6WIL fibril elongation in the presence of amyloid-reactive peptide, amyloid-reactive antibody-peptide fusion protein, or amyloid-reactive antibody. This example describes the level of rVλ6WIL fibril elongation in the presence of amyloid-reactive peptide p5R, amyloid-reactive antibody-peptide fusion protein, or amyloid-reactive antibody.

[0197] The method of Example 1 was carried out in the presence of amyloid-reactive peptide p5R, amyloid-reactive antibody-peptide fusion protein, amyloid-reactive humanized antibody, or a control.

[0198] The amyloid-reactive peptide used in this example includes peptide p5R (SEQ ID NO: 2).

[0199] The antibody-peptide fusion protein used in this embodiment comprises an amyloid-reactive peptide containing the amino acid sequence shown in SEQ ID NO: 2, and an antibody that binds to human amyloid fibril. The antibody comprises a heavy chain and a light chain. The heavy chain of the antibody comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO: 55, and the light chain of the antibody comprises a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 36. VH comprises CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19. VL comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22. The amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer containing the amino acid sequence shown in SEQ ID NO: 83.

[0200] The amyloid-reactive humanized antibody used in this embodiment comprises an antibody that binds to human amyloid fibrils, the antibody comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 55, the light chain comprising a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO: 36, the VH comprising CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 19, and the VL comprising CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 22.

[0201] The assay conditions included: 20 nM biotinylated WIL monomer, a 1-hour incubation with BSA as a blocking agent, and 10 μg / mL fibril (0.83 μg) in 50 μL / well.

[0202] As shown in Figures 2A and 2B, the rVλ6WIL fibrils continued to elongate in the presence of the amyloid-reactive peptide p5R (SEQ ID NO: 2). These results suggest that the amyloid-reactive peptide alone is insufficient to inhibit rVλ6WIL fibril elongation at concentrations of 0.001 μM to 2.0 μM (Figures 2A and 2B).

[0203] As shown in Figures 3A and 3B, rVλ6WIL fibril elongation was significantly attenuated in the presence of amyloid-reactive antibody-peptide fusion proteins at concentrations of 0.5 μM or 2.0 μM. The rVλ6WIL fibril elongation rate was similar between 0.5 μM and 2.0 μM of antibody-peptide fusion proteins (Figures 3A and 3B).

[0204] As shown in Figures 4A and 4B, rVλ6WIL fibril elongation was significantly attenuated in the presence of amyloid-reactive antibody, starting from a concentration of 0.01 μM. The rVλ6WIL fibril elongation rate decreased as the concentration of amyloid-reactive antibody increased. The maximum level of rVλ6WIL fibril elongation inhibition was observed at a concentration of 2.0 μM amyloid-reactive antibody (Figures 4A and 4B).

[0205] Table 10 summarizes the changes in rVλ6WIL fibril elongation rate in the presence of amyloid-reactive peptide p5R, amyloid-reactive antibody-peptide fusion protein, or amyloid-reactive antibody. [Table 11]

[0206] While we do not wish to be bound by theory, these results suggest that antibody-peptide fusion proteins inhibit the growth of existing fibrils (e.g., recruitment of additional monomeric WILs) by binding to amyloid fibrils (long axis) and blocking recruitment sites. Amyloid-reactive antibodies (metabolite substitutes) either sequester misfolded WIL VLs, or WIL VL amyloid-producing high molecular weight aggregates, preventing recruitment by fibrils, or bind to recruitment sites on fibrils at a different or higher density than those to which antibody-peptide fusion proteins bind.

[0207] This disclosure is not intended to be limited in scope to any particular embodiment disclosed, but is provided, for example, to illustrate various aspects of the disclosure. Various modifications to the compositions and methods described herein will become apparent from the description and teachings herein. Such modifications may be made without departing from the true scope and spirit of the disclosure and are intended to be included within the scope of the disclosure.

[0208] Exemplary Embodiments Embodiments disclosed herein may include: 1. A method for reducing or preventing fibril growth in an individual at risk of developing amyloid-related disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein is (i) Amyloid-reactive peptides, and (ii) The method comprising an antibody that binds to amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

[0209] 2. A method for reducing or delaying the progression of amyloid-related disease in an individual diagnosed with amyloid disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein is (i) Amyloid-reactive peptides, and (ii) The method comprising an antibody that binds to amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

[0210] 3. Embodiment 1 is the method according to 2, wherein the amyloid fibril comprises amyloid-forming λ6 variable domain protein (Vλ6Wil) or amyloid-forming immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibril or amyloid-forming Aβ precursor protein, or serum amyloid protein A (AA).

[0211] 4. The method according to Embodiment 1 or 2, wherein the amyloid fibril comprises an amyloid-forming immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), trans tyretin (ATTR wild-type; ATTP variant), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemochemoattractant (ALect2), fibrinogen A variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP); α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALβ4, or ALβ1.

[0212] 5. The method according to any one of Embodiments 1 to 4, wherein fibril growth is measured via an in vitro rVλ6WIL(WIL) fibril elongation assay.

[0213] 6. The method according to any one of Embodiments 1 to 5, wherein the antibody-peptide fusion protein reduces the growth of rVλ6WIL fibrils by at least about 20%.

[0214] 7. The method according to any one of Embodiments 1 to 5, wherein the antibody-peptide fusion protein reduces the growth rate of rVλ6WIL fibrils by at least about 20%.

[0215] 8. The method according to any one of Embodiments 1 to 5, wherein the antibody-peptide fusion protein reduces the total amount of rVλ6WIL fibrils by at least about 20%.

[0216] 9. The method according to Embodiment 6, wherein the antibody-peptide fusion protein reduces the growth of rVλ6WIL fibrils by approximately 33%.

[0217] 10. The method according to Embodiment 7, wherein the antibody-peptide fusion protein reduces the growth rate of rVλ6WIL fibrils by approximately 33%.

[0218] 11. The method according to Embodiment 8, wherein the antibody-peptide fusion protein reduces the total amount of rVλ6WIL fibrils by approximately 33%.

[0219] 12. The method according to any one of Embodiments 1 to 11, wherein the amyloid-related disease is systemic or localized amyloidosis.

[0220] 13. The method according to any one of Embodiments 1 to 12, wherein the amyloid-related disease is selected from the group consisting of AL, AH, Aβ2M, ATTRv, ATTRwt, AA, AApoAI, AApoAII, AGel, Alys, ALECT2, AFib, ACys, ACa, AMed, AIAPP, APro, AIns, APrP, or Aβ amyloidosis.

[0221] 14. The method according to any one of Embodiments 1 to 13, wherein the individual has a genetic predisposition to amyloid-related disease.

[0222] 15. The method according to any one of Embodiments 1 to 14, wherein the individual has a family history of amyloid-related disease.

[0223] 16. The method according to any one of Embodiments 2 to 15, wherein the individual is suffering from an early-stage amyloid-related disease.

[0224] 17. The method according to any one of Embodiments 2 to 16, wherein the individual is in the early stage of AL amyloidosis.

[0225] 18. The method according to Embodiment 17, wherein the initial stage of AL amyloidosis is diagnosed according to the Mayo Clinic system.

[0226] 19. The method according to Embodiment 18, wherein the initial stage AL amyloidosis is stage 1 AL amyloidosis.

[0227] 20. The method according to any one of Embodiments 2 to 16, wherein the individual is in the early stage of ATTR amyloidosis.

[0228] 21. The method according to Embodiment 20, wherein the initial stage ATTR amyloidosis includes stage 1 ATTR amyloidosis.

[0229] 22. The method according to any one of Embodiments 1 to 21, wherein the light chain of the antibody includes a light chain constant region, and the heavy chain of the antibody includes a heavy chain constant region.

[0230] 23. The method according to any one of embodiments 1 to 22, wherein the spacer is selected from the group consisting of sequence numbers 23 to 24, 27, and 83 to 86.

[0231] 24. The method according to any one of embodiments 1 to 23, wherein the spacer is selected from the group consisting of sequence numbers 83 and 86.

[0232] 25. The method according to any one of Embodiments 1 to 24, wherein the amyloid-reactive peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 13, which includes 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions.

[0233] 26. The method according to any one of Embodiments 1 to 25, wherein the amyloid-reactive peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 13.

[0234] 27. The method according to any one of Embodiments 1 to 26, wherein the antibody-peptide fusion protein comprises two heavy chains and two light chains, each light chain being linked to the amyloid-reactive peptide at its C-terminus.

[0235] 28. The method according to any one of Embodiments 1 to 27, wherein the antibody is a chimeric antibody or a humanized antibody.

[0236] 29. A method according to any one of Embodiments 1 to 28, The light chain of the antibody comprises a light chain variable domain (VL) comprising CDR-L1 containing the amino acid sequence described in SEQ ID NO: 64, CDR-L2 containing the amino acid sequence described in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence described in SEQ ID NO: 22. The method wherein the heavy chain of the antibody comprises a heavy chain variable domain (VH) comprising CDR-H1 containing the amino acid sequence described in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence described in SEQ ID NO: 73, and CDR-H3 containing the amino acid sequence described in SEQ ID NO: 19.

[0237] 30. The method according to Embodiment 29, wherein VL comprises the amino acid sequence shown in SEQ ID NO: 36, and VH comprises the amino acid sequence shown in SEQ ID NO: 55.

[0238] 31. The method according to any one of Embodiments 1 to 30, wherein the antibody is a full-length antibody containing an Fc region.

[0239] 32. The method according to Embodiment 31, wherein the Fc region is of the IgG1 isotype.

[0240] 33. The antibody-peptide fusion protein is (i) the amyloid-reactive peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprises a heavy chain variable region (VH), the light chain of the antibody comprises a light chain variable region (VL), the VH comprises CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19, and the VL comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 22. The method according to any one of Embodiments 1 to 32, wherein the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86.

[0241] 34. The antibody-peptide fusion protein is (i) the amyloid-reactive peptide comprising the amino acid sequence shown in Sequence ID No. 2, and (ii) The method according to any one of Embodiments 1 to 33, comprising the antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprises a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 of VH comprising the amino acid sequence shown in SEQ ID NO: 55, the light chain of the antibody comprises a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 of VL comprising the amino acid sequence shown in SEQ ID NO: 36, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer comprising the amino acid sequence shown in SEQ ID NO: 83.

[0242] 35. The antibody-peptide fusion protein, (i) the amyloid-reactive peptide comprising the amino acid sequence shown in Sequence ID No. 2, and (ii) The method according to any one of Embodiments 1 to 34, comprising the antibody that binds to human amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprises a heavy chain variable region (VH), the light chain of the antibody comprises a light chain variable region (VL), the VH comprises CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 19, the VL comprises CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 22, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer comprising the amino acid sequence described in SEQ ID NO: 83.

[0243] 36. The method according to any one of Embodiments 1 to 35, wherein the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide, each comprising the light chain of the antibody and an amyloid-reactive peptide, and a third polypeptide and a fourth polypeptide, each comprising the heavy chain of the antibody, wherein each of the first polypeptide and the second polypeptide comprises the amino acid shown in SEQ ID NO: 89, and each of the third polypeptide and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO: 91.

[0244] 37. The method according to any one of Embodiments 1 to 35, wherein the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide, each comprising the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each comprising the heavy chain of the antibody, each of the first polypeptide and the second polypeptide comprising the amino acid shown in SEQ ID NO: 89, and each of the third polypeptide and the fourth polypeptide comprising the amino acid sequence shown in SEQ ID NO: 93.

[0245] 38. The method according to any one of Embodiments 1 to 37, wherein the individual is a human.

[0246] 39. A method for reducing or preventing fibril growth in an individual having an amyloid-related disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein is (i) Amyloid-reactive peptides, and (ii) The method comprising an antibody that binds to amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

[0247] 40. A method for reducing or preventing fibril growth in an individual in which amyloid fibrils have been identified, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein is (i) Amyloid-reactive peptides, and (ii) The method comprising an antibody that binds to amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

[0248] 41. A method for reducing or preventing fibril growth in an individual diagnosed with amyloid disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein is (i) Amyloid-reactive peptides, and (ii) The method comprising an antibody that binds to amyloid fibrils, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

Claims

1. A method for reducing or preventing fibril growth in an individual at risk of developing amyloid-related disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein is (i) Amyloid-reactive peptides, and (ii) The method comprising an antibody that binds to amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

2. A method for reducing or delaying the progression of amyloid-related disease in an individual diagnosed with amyloid disease, comprising administering a therapeutically effective amount of an antibody-peptide fusion protein to the individual, wherein the antibody-peptide fusion protein is (i) Amyloid-reactive peptides, and (ii) The method comprising an antibody that binds to amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer.

3. Claim 1 is the method according to claim 2, wherein the amyloid fibril comprises amyloid-forming λ6 variable domain protein (Vλ6Wil) or amyloid-forming immunoglobulin light chain (AL), Aβ(1-40) amyloid-like fibril or amyloid-forming Aβ precursor protein, or serum amyloid protein A (AA).

4. The method according to claim 1 or 2, wherein the amyloid fibril comprises an amyloid-forming immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), trans tyretin (ATTR wild-type; ATTP variant), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemochemoattractant (ALect2), fibrinogen A variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactoadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (AIns), prion protein (APrP); α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, ALβ4, or ALβ1.

5. The method according to any one of claims 1 to 4, wherein fibril growth is measured via an in vitro rVλ6WIL (WIL) fibril elongation assay.

6. The method according to any one of claims 1 to 5, wherein the antibody-peptide fusion protein reduces the growth of rVλ6WIL fibrils by at least about 20%.

7. The method according to any one of claims 1 to 5, wherein the antibody-peptide fusion protein reduces the growth rate of rVλ6WIL fibrils by at least about 20%.

8. The method according to any one of claims 1 to 5, wherein the antibody-peptide fusion protein reduces the total amount of rVλ6WIL fibrils by at least about 20%.

9. The method according to claim 6, wherein the antibody-peptide fusion protein reduces the growth of rVλ6WIL fibrils by approximately 33%.

10. The method according to claim 7, wherein the antibody-peptide fusion protein reduces the growth rate of rVλ6WIL fibrils by approximately 33%.

11. The method according to claim 8, wherein the antibody-peptide fusion protein reduces the total amount of rVλ6WIL fibrils by about 33%.

12. The method according to any one of claims 1 to 11, wherein the amyloid-related disease is systemic or localized amyloidosis.

13. The method according to any one of claims 1 to 12, wherein the amyloid-related disease is selected from the group consisting of AL, AH, Aβ2M, ATTRv, ATTRwt, AA, AApoAI, AApoAII, AGel, ALys, ALECT2, AFib, ACYs, ACaal, AMed, AIAPP, APro, AIns, APrP, or Aβ amyloidosis.

14. The method according to any one of claims 1 to 13, wherein the individual has a genetic predisposition to amyloid-related disease.

15. The method according to any one of claims 1 to 14, wherein the individual has a family history of amyloid-related disease.

16. The method according to any one of claims 2 to 15, wherein the individual is suffering from an early-stage amyloid-related disease.

17. The method according to any one of claims 2 to 16, wherein the individual is in the early stage of AL amyloidosis.

18. The method according to claim 17, wherein the initial stage of AL amyloidosis is diagnosed according to the Mayo Clinic system.

19. The method according to claim 18, wherein the initial stage of AL amyloidosis is stage 1 AL amyloidosis.

20. The method according to any one of claims 2 to 16, wherein the individual is in the early stage of ATTR amyloidosis.

21. The method according to claim 20, wherein the initial stage of ATTR amyloidosis includes stage 1 ATTR amyloidosis.

22. The method according to any one of claims 1 to 21, wherein the light chain of the antibody includes a light chain constant region, and the heavy chain of the antibody includes a heavy chain constant region.

23. The method according to any one of claims 1 to 22, wherein the spacer is selected from the group consisting of sequence numbers 23 to 24, 27, and 83 to 86.

24. The method according to any one of claims 1 to 23, wherein the spacer is selected from the group consisting of sequence number 83 and sequence number 86.

25. The method according to any one of claims 1 to 24, wherein the amyloid-reactive peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 13, which includes 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions.

26. The method according to any one of claims 1 to 25, wherein the amyloid-reactive peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 13.

27. The method according to any one of claims 1 to 26, wherein the antibody-peptide fusion protein comprises two heavy chains and two light chains, each light chain being linked to the amyloid-reactive peptide at its C-terminus.

28. The method according to any one of claims 1 to 27, wherein the antibody is a chimeric antibody or a humanized antibody.

29. A method according to any one of claims 1 to 28, The light chain of the antibody comprises a light chain variable domain (VL) comprising CDR-L1 containing the amino acid sequence described in SEQ ID NO: 64, CDR-L2 containing the amino acid sequence described in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence described in SEQ ID NO:

22. The method wherein the heavy chain of the antibody comprises a heavy chain variable domain (VH) comprising CDR-H1 containing the amino acid sequence described in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence described in SEQ ID NO: 73, and CDR-H3 containing the amino acid sequence described in SEQ ID NO:

19.

30. The method according to claim 29, wherein VL comprises the amino acid sequence shown in SEQ ID NO: 36, and VH comprises the amino acid sequence shown in SEQ ID NO:

55.

31. The method according to any one of claims 1 to 30, wherein the antibody is a full-length antibody including an Fc region.

32. The method according to claim 31, wherein the Fc region is of the IgG1 isotype.

33. The antibody-peptide fusion protein, (i) the amyloid-reactive peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and (ii) an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprises a heavy chain variable region (VH), the light chain of the antibody comprises a light chain variable region (VL), the VH comprises CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 19, the VL comprises CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

22. The method according to any one of claims 1 to 32, wherein the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-24, 27, and 83-86.

34. The antibody-peptide fusion protein, (i) the amyloid-reactive peptide comprising the amino acid sequence shown in Sequence ID No. 2, and (ii) The method according to any one of claims 1 to 33, comprising an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprises a heavy chain variable region (VH) comprising CDR-H1, CDR-H2, and CDR-H3 of VH comprising the amino acid sequence shown in SEQ ID NO: 55, the light chain of the antibody comprises a light chain variable region (VL) comprising CDR-L1, CDR-L2, and CDR-L3 of VL comprising the amino acid sequence shown in SEQ ID NO: 36, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer comprising the amino acid sequence shown in SEQ ID NO:

83.

35. The antibody-peptide fusion protein, (i) the amyloid-reactive peptide comprising the amino acid sequence shown in Sequence ID No. 2, and (ii) The method according to any one of claims 1 to 34, comprising an antibody that binds to human amyloid fibril, wherein the antibody comprises a heavy chain and a light chain, the heavy chain of the antibody comprises a heavy chain variable region (VH), the light chain of the antibody comprises a light chain variable region (VL), the VH comprises CDR-H1 comprising the amino acid sequence shown in SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence shown in SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence shown in SEQ ID NO: 19, the VL comprises CDR-L1 comprising the amino acid sequence shown in SEQ ID NO: 64, CDR-L2 comprising the amino acid sequence shown in SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence shown in SEQ ID NO: 22, the amyloid-reactive peptide and the antibody are linked at the C-terminus of the light chain, and the amyloid-reactive peptide is linked to the antibody via a spacer comprising the amino acid sequence described in SEQ ID NO:

83.

36. The method according to any one of claims 1 to 35, wherein the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide, each comprising the light chain of the antibody and an amyloid-reactive peptide, and a third polypeptide and a fourth polypeptide, each comprising the heavy chain of the antibody, wherein each of the first polypeptide and the second polypeptide comprises the amino acid shown in SEQ ID NO: 89, and each of the third polypeptide and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:

91.

37. The method according to any one of claims 1 to 35, wherein the antibody-peptide fusion protein comprises a first polypeptide and a second polypeptide, each comprising the light chain of the antibody, and a third polypeptide and a fourth polypeptide, each comprising the heavy chain of the antibody, wherein each of the first polypeptide and the second polypeptide comprises the amino acid shown in SEQ ID NO: 89, and each of the third polypeptide and the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO:

93.

38. The method according to any one of claims 1 to 37, wherein the individual is a human.

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